Abstract: The mTOR-inhibitor rapamycin is a potent drug used in many immunosuppressive and antiinflammatory therapeutic regimes. In renal transplantation despite its beneficial roles rapamycin in some cases can promote renal fibrosis in the kidney but the underlying mechanisms are unknown. In this study, we tested for possible modulatory effects of rapamycin on the cytokine-triggered matrix metalloproteinase 9 (MMP-9)/tissue inhibitor of metalloproteinase (TIMP)-1 protease-antiprotease system which is critically involved in renal inflammation and fibrosis. Treatment of rat mesangial cells (MC) with rapamycin dose-dependently reduced the interleukin 1β (IL-1β)-triggered increase in gelatinolytic levels as demonstrated by zymography. The reduction in the extracellular MMP-9 content by rapamycin coincided with an attenuation in cytokine-induced steady-state MMP-9 mRNA levels. Conversely, rapamycin caused a dose-dependent increase in cytokine-evoked TIMP-1 expression in a Smad binding element (SBE)-dependent manner. Surprisingly, the attenuation of MMP-9 mRNA levels by rapamycin is accompanied by a potentiation of IL-1β-induced MMP-9 promoter activity in which the stimulatory effects by rapamycin are mainly attributed to a proximal AP-1 binding site. Furthermore, the rapamycin-dependent potentiation of MMP-9 expression is accompanied by an amplification of cytokine-triggered activities of nuclear factor κB (NF-κB) and activator protein 1 (AP-1) transcription factors. Importantly, rapamycin-triggered increase in MMP-9 promoter activity is fully impaired when we used a MMP-9 reporter construct which is under the additional control of the 3' untranslated region (3'-UTR) of MMP-9. Collectively, these data imply that rapamycin inhibits the cytokine-induced MMP-9 mainly through posttranscriptional events and thereby exerts profibrotic activities.
Abstract: Overexpression of the mRNA-binding protein HuR is an important feature of many tumors and in most cases correlates with a high grade malignancy. Since phosphorylation of HuR by protein kinase Cδ (PKCδ) at serine (Ser) 318 implies an important mode in HuR regulation, we studied its functional role in dysregulated HuR and related functions in colon carcinoma cells. Coimmunoprecipitation experiments revealed a high constitutive association of nuclear PKCδ with HuR. Using a phospho-Ser 318-specific HuR antibody we found a strong increase in nuclear HuR phosphorylation in DLD-1 cells when compared with nontransformed CCD 841 colon epithelial cells. Importantly, a strong increase in HuR phosphorylation at Ser 318 was also found in tissue specimen from human colon carcinomas. Employing RNP-immunoprecipitation (IP), we show that DLD-1 cells displayed a strong and constitutive RNA-binding of HuR to cyclooxygenase-2 (COX-2) and cyclin A encoding mRNAs which was strongly impaired by rottlerin an inhibitor of novel PKCs. Accordingly, rottlerin accelerated the decay of COX-2 and cyclin A encoding mRNAs concomitant with a reduced expression of both genes. Functionally, migration and invasion is similarly impaired in PKCδ -or HuR-siRNA-depleted cells and in tumor cells transfected with a nonphosphorylatable serine-to-alanine 318 HuR construct. Conversely, expression of a phosphomimetic Ser 318 aspartic acid (D) HuR caused a significant increase in migration and proliferation of CCD 841 cells. Our data suggest that the increased HuR phosphorylation at Ser 318 by PKCδ reflects an important regulatory paradigm for aberrant HuR functions and emphasize the antitumorigenic potential of PKCδ inhibitory strategies.
Abstract: Thrombin promotes vascular smooth muscle cell (SMC) proliferation and inflammation via protease-activated receptor (PAR)-1. A further thrombin receptor, PAR-3, acts as a PAR-1 cofactor in some cell-types. Unlike PAR-1, PAR-3 is dynamically regulated at the mRNA level in thrombin-stimulated SMC. This study investigated the mechanisms controlling PAR-3 expression. In human vascular SMC, PAR-3 siRNA attenuated thrombin-stimulated interleukin-6 expression and extracellular signal-regulated kinases 1/2 phosphorylation, indicating PAR-3 contributes to net thrombin responses in these cells. Thrombin slowed the decay of PAR-3 but not PAR-1 mRNA in the presence of actinomycin D and induced cytosolic shuttling and PAR-3 mRNA binding of the mRNA-stabilizing protein human antigen R (HuR). HuR siRNA prevented thrombin-induced PAR-3 expression. By contrast, forskolin inhibited HuR shuttling and destabilized PAR-3 mRNA, thus reducing PAR-3 mRNA and protein expression. Other cAMP-elevating agents, including the prostacyclin-mimetic iloprost, also down-regulated PAR-3, accompanied by decreased HuR/PAR-3 mRNA binding. Iloprost-induced suppression of PAR-3 was reversed with a myristoylated inhibitor of protein kinase A and mimicked by phorbol ester, an inducer of cyclooxygenase-2. In separate studies, iloprost attenuated PAR-3 promoter activity and prevented binding of nuclear factor of activated T cells (NFAT2) to the human PAR-3 promoter in a chromatin immunoprecipitation assay. Accordingly, PAR-3 expression was suppressed by the NFAT inhibitor cyclosporine A or NFAT2 siRNA. Thus human PAR-3, unlike PAR-1, is regulated post-transcriptionally via the mRNA-stabilizing factor HuR, whereas transcriptional control involves NFAT2. Through modulation of PAR-3 expression, prostacyclin and NFAT inhibitors may limit proliferative and inflammatory responses to thrombin after vessel injury.
Abstract: BACKGROUND: The importance of the Notch signaling in the development of glomerular diseases has been recently described. Therefore we analyzed in podocytes the expression and activity of ADAM10, one important component of the Notch signaling complex. METHODS: By Western blot, immunofluorescence and immunohistochemistry analysis we characterized the expression of ADAM10 in human podocytes, human urine and human renal tissue. RESULTS: We present evidence, that differentiated human podocytes possessed increased amounts of mature ADAM10 and released elevated levels of L1 adhesion molecule, one well known substrate of ADAM10. By using specific siRNA and metalloproteinase inhibitors we demonstrate that ADAM10 is involved in the cleavage of L1 in human podocytes. Injury of podocytes enhanced the ADAM10 mediated cleavage of L1. In addition, we detected ADAM10 in urinary podocytes from patients with kidney diseases and in tissue sections of normal human kidney. Finally, we found elevated levels of ADAM10 in urinary vesicles of patients with glomerular kidney diseases. CONCLUSIONS: The activity of ADAM10 in human podocytes may play an important role in the development of glomerular kidney diseases.
Abstract: Stabilization of mRNA by the ubiquitous RNA binding protein human antigen R (HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, requires canonical binding to AU-rich element (ARE)-bearing target mRNA and export of nuclear HuR-mRNA complexes to the cytoplasm. In human mesangial cells (HMC) both processes are induced by angiotensin II (AngII) via protein kinase Cdelta (PKCdelta)-triggered serine phosphorylation of HuR. By testing different point-mutated Flag-tagged HuR proteins, we found that Ser 318 within RNA recognition motif 3 (RRM3) is essential for AngII-induced binding to ARE-bearing mRNA but irrelevant for nucleocytoplasmic HuR shuttling. Conversely, mutation at Ser 221 within the HuR hinge region prevents AngII-triggered HuR export without affecting mRNA binding of HuR. Using phosphorylation state-specific antibodies, we found a transient increase in HuR phosphorylation at both serines by AngII. Functionally, PKCdelta mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D(1), and cyclooxygenase-2 (COX-2), and is indispensable for AngII-triggered migration and wound healing of HMC. Our data suggest a regulatory paradigm wherein a simultaneous phosphorylation at different domains by PKCdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR, both of which events are essentially involved in the stabilization of HuR target mRNAs and relevant cell functions.
Abstract: Angiotensin (Ang) II-induced fibrosis of the kidney is characterized by the enhanced expression of profibrotic and proinflammatory genes, including the serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) and cyclooxygenase-2 (COX-2). In addition to transcriptional regulation, both genes are subject to post-transcriptional control by AU-rich destabilizing elements that reside within the 3' untranslated region of the mRNA. We demonstrated that the continuous infusion of AngII in rats induced fibrosis concomitant with a significant increase in glomerular PAI-1 and COX-2 expression levels. Using RNA pull-down assays and electromobility shift assays, we demonstrated the increased binding of the ubiquitous RNA-binding protein human-antigen R (HuR) to the mRNAs of both PAI-1 and COX-2 in the cytoplasmic fractions of renal homogenates from AngII-treated rats. Actinomycin D experiments in rat mesangial cells revealed that AngII stabilizes both mRNAs via HuR as proven by small interfering RNA. Mechanistically, AngII promotes an increase in nucleo-cytoplasmic HuR shuttling, which was blocked by the PKC inhibitor rottlerin and the type-I AngII (AT(1)) receptor antagonist valsartan but was unaffected by both AT(2) receptor antagonists PD123319 and CGP42112. Co-immunoprecipitation revealed that AngII treatment caused an increase in nuclear PKC-delta concomitant with binding to nuclear HuR both in vitro and in vivo. The post-transcriptional regulation of PAI-1 and COX-2 by PKC-delta-dependent HuR shuttling may contribute to the pathogenesis of hypertensive nephrosclerosis triggered by AngII.
Abstract: Matrix metalloproteinase-9 (MMP-9) is implicated in the invasion and metastasis of breast cancer cells. We investigated the modulatory effects of nitric oxide (NO) on the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced MMP-9 expression in MCF-7 cells. Different chemical NO donors inhibited the extracellular content of TPA-induced MMP-9 protein and MMP-9 activity as assessed by gelatin-zymography and ELISA, respectively. Concomitant with the reduction in the extracellular MMP-9 content NO strongly decreased the steady-state levels of MMP-9 mRNA which in turn leads to a lower recruitment of MMP-9 transcripts to polysomes and to a diminished MMP-9 translation. Reporter gene assays revealed that the inhibition in MMP-9 expression by NO is mainly attributed to a 0.67 kb fragment of the 5'-promoter region of the MMP-9 gene but independent of the 3'untranslated region thus indicating that MMP-9 suppression by NO mainly results from transcriptional events. Electrophoretic mobility shift assays (EMSA), showed that NO specifically interferes with the TPA-induced DNA binding affinity of c-Jun and c-Fos without affecting the TPA-induced increase in the levels of the transcription factors. Using pharmacological inhibitors and small interfering (si)RNA we found that PKCdelta is indispensably involved in the TPA-triggered MMP-9 expression. Concomitantly, the TPA-evoked increase in total PKC activity was strongly attenuated in the lysates from NO-treated MCF-7 cells, thus suggesting that NO attenuates TPA-triggered MMP-9 mainly through a direct inhibition of PKCdelta. Modulation of MMP-9 by NO highlights the complex roles of NO in the regulation of MMP-9 in breast cancer cells.
Abstract: The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFbeta/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFbeta(1) as shown by ELISA and by the inhibitory effects of a neutralizing TGFbeta antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFbeta type II receptor (TGFbeta-RII) we furthermore demonstrate a functional involvement of both types of TGFbeta receptors. However, rapamycin did not compete with TGFbeta for TGFbeta-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFbeta/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin.
Abstract: Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.
Abstract: The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.
Abstract: The calcineurin inhibitor (CNI)-induced renal fibrosis is attributed to an exaggerated deposition of extracellular matrix, which is mainly due to an increased expression of TGFbeta. Herein we demonstrate that the CNI cyclosporin A and tacrolimus (FK506), independent of TGFbeta synthesis, rapidly activate TGFbeta/Smad signaling in cultured mesangial cells and in whole kidney samples from CNI-treated rats. By EMSA, we demonstrate increased DNA binding of Smad-2, -3, and -4 to a cognate Smad-binding promoter element (SBE) accompanied by CNI-triggered activation of Smad-dependent expression of tissue inhibitor of metalloprotease-1 (TIMP-1) and connective tissue growth factor. Using an activin receptor-like kinase-5 (ALK-5) inhibitor and by small interfering RNA we depict a critical involvement of both types of TGFbeta receptors in CNI-triggered Smad signaling and fibrogenic gene expression, respectively. Mechanistically, CNI cause a rapid activation of latent TGFbeta, which is prevented in the presence of the antioxidant N-acetyl cysteine. A convergent activation of p38 MAPK is indicated by the partial blockade of CNI-induced Smad-2 activation by SB203580; conversely, both TGFbeta-RII and TGFbeta are critically involved in p38 MAPK activation by CNI. Activation of both signaling pathways is similarly triggered by reactive oxygen species. Finally, we show that neutralization of TGFbeta markedly reduced the CNI-dependent Smad activation in vitro and in vivo. Collectively, this study demonstrates that CNI via reactive oxygen species generation activate latent TGFbeta and thereby initiate the canonical Smad pathway by simultaneously activating p38 MAPK, which both synergistically induce Smad-driven gene expression.
Abstract: Lyme borreliosis is a spirochetal infection caused by the Borrelia burgdorferi sensu lato complex that can proceed towards an inflammatory joint manifestation known as Lyme arthritis. Production of chemokines orchestrating neutrophil infiltration is supposed to be key to early arthritic pathogenesis. Using PMA-differentiated macrophage-like THP-1 (mTHP-1) cells we identified by antibody array methodology or mRNA analysis IL-8, GRO-alpha, NAP-2, and SDF-1alpha as being among those chemokines that are upregulated by bacterial lysates obtained from B. burgdorferi. Based on these observations, we set out to characterize in detail mechanisms mediating IL-8 release in this cellular model. TLR2 blocking antibodies, analysis of p65 translocation, and electromobility-shift analysis revealed activation of the TLR2/NF-kappaB axis by B. burgdorferi. The functional importance of this pathway was substantiated by suppression of IL-8 after inhibition of IkappaB kinase. Notably, MAP kinases, specifically the MEK1/2-ERK1/2 pathway, were essential for IL-8 secretion. Those data were confirmed by using freshly isolated adherent peripheral blood mononuclear cells. On the contrary, B. burgdorferi-induced IL-8 in mTHP-1 was unlikely related to flagellin, alpha3beta1-integrin signaling, lipopolysaccharide, bacterial DNA, NOD1/NOD2 agonists, or to intermediate production of IL-1beta and TNF-alpha. Induction of IL-8 by B. burgdorferi was not due to amplification of constitutive AP-1 DNA-binding activity detectable in mTHP-1 cells. Data presented herein validate that TLR2, particularly on mTHP-1 cells, holds a central position in mediating IL-8 secretion associated with extracellular B. burgdorferi and beyond that suggest inhibition of IkappaB kinase and MEK1/2 kinases as promising pharmacological strategies aiming at IL-8 in early Lyme arthritis.
Abstract: Removal of apoptotic cells by phagocytes is considered a pivotal immune regulatory process. Although considerable knowledge has been obtained on the postphagocytic macrophage phenotype, there is little information on molecular mechanisms, which provoke macrophage polarization. In this study, we show that human apoptotic Jurkat cells (AC) or AC-conditioned medium (CM) rapidly induces cyclooxygenase-2 (COX-2) expression in mouse RAW264.7 macrophages via sphingosine-1-phosphate (S1P). Pharmacological inhibition of S1P release from AC or using CM from cells with a knockdown of sphingosine kinase 2 in human MCF-7 cells abrogates this effect. Expression of COX-2 resulted from an increase in mRNA stability via its 3'-untranslated region (UTR), shown by COX-2-3'-UTR and AU-rich element-driven reporter assays. Western analysis corroborated increased nucleocytoplasmic shuttling of the RNA-binding protein HuR after CM treatment. RNA EMSA analysis revealed an S1P- and CM-mediated increase in HuR-RNA binding to a COX-2-specific UTR, whereas HuR knockdown pointed to its importance for S1P in CM-induced COX-2 expression. Immunofluorescence microscopy of phospholipase A2 (PLA2) and ELISA analysis of PGE2 revealed activation of PLA2 and production of PGE2 in response to CM but not S1P. S1P, released from AC, uses HuR to stabilize COX-2 mRNA and thus to increase COX-2 protein expression. However, only CM also activates PLA2 to provide the substrate for COX-2. Our data underscore the importance of S1P in AC-mediated immune regulation, by stabilizing COX-2 mRNA in macrophages, a prerequisite for PGE2 formation.
Abstract: In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.
Abstract: BACKGROUND AND PURPOSE: FTY720 is a potent immunomodulatory prodrug that is converted to its active phosphorylated form by a sphingosine kinase. Here we have studied whether FTY720 mimicked the action of sphingosine-1-phosphate (S1P) and exerted an anti-inflammatory potential in renal mesangial cells. EXPERIMENTAL APPROACH: Prostaglandin E(2) (PGE(2)) was quantified by an enzyme-linked immunosorbent-assay. Secretory phospholipase A(2) (sPLA(2)) protein was detected by Western blot analyses. mRNA expression was determined by Northern blot analysis and sPLA(2)-promoter activity was measured by a luciferase-reporter-gene assay. KEY RESULTS: Stimulation of cells for 24 h with interleukin-1beta (IL-1beta) is known to trigger increased PGE(2) formation which coincides with an induction of the mRNA for group-IIA-sPLA(2) and protein expression. FTY720 dose-dependently suppressed IL-1beta-induced IIA-sPLA(2) protein secretion and activity in the supernatant. This effect is due to a suppression of cytokine-induced sPLA(2) mRNA expression which results from a reduced promoter activity. As a consequence of suppressed sPLA(2) activity, PGE(2) formation is also reduced by FTY720. Mechanistically, the FTY720-suppressed sPLA(2) expression results from an activation of the TGFbeta/Smad signalling cascade since inhibition of the TGFbeta receptor type I by a specific kinase inhibitor reverses the FTY720-mediated decrease of sPLA(2) protein expression and sPLA(2) promoter activity. CONCLUSIONS AND IMPLICATIONS: In summary, our data show that FTY720 was able to mimic the anti-inflammatory activity of TGFbeta and blocked cytokine-triggered sPLA(2) expression and subsequent PGE(2) formation. Thus, FTY720 may exert additional in vivo effects besides the well reported immunomodulation and its anti-inflammatory potential should be considered.
Abstract: The effects of the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) on the IL-1beta-induced matrix metalloproteinase-9 (MMP-9) were investigated. Impairment of the protease-antiprotease balance contributes to renal fibrosis, which is observed collectively under long-term treatment with either immunosuppressant. It is demonstrated that CsA, in contrast to FK506, reduced the IL-1beta-induced MMP-9 content in conditioned media of mesangial cells, which coincides with a reduction in the cytokine-induced MMP-9 mRNA level. Similar to FK506, the VIVIT peptide, a specific inhibitor of the nuclear factor of activated T cells, did not affect the cytokine-induced MMP-9 level. Moreover, CsA caused a dose-dependent inhibition on the IL-1beta-induced luciferase activity of a 1.3-kb MMP-9 promoter fragment. Concomitant, electrophoretic mobility shift assay revealed that CsA selectively inhibits the cytokine-induced DNA binding of activator protein-1 and NF-kappaB. The effects on NF-kappaB binding were accompanied by a marked reduction in the nuclear content of the p65 subunit of NF-kappaB. Accordingly, CsA specifically impaired the IL-1beta-triggered degradation of inhibitory NF-kappaB. The suppressive effects by CsA on MMP-9 expression were accompanied by a reduction in the cytokine-induced phosphorylation of p42/p44 and c-Jun N-terminal Kinase (JNK). It is interesting that only the JNK inhibitor SP600125 impaired the cytokine-triggered MMP-9 level, suggesting that CsA, via inhibition of the JNK pathway, negatively interferes with the NF-kappaB-dependent transcriptional control of MMP-9. Interference with MMP-9 transcription may account for the accumulation of extracellular matrix underlying the high fibrotic potential of CsA during anti-inflammatory therapies with calcineurin inhibitors.
Abstract: Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.
Abstract: 1.--The immunomodulating agent FTY720 is a substrate for the sphingosine kinase and the phosphorylated form is able to bind to sphingosine 1-phosphate (S1P) receptors. In this study, we show that exposure of renal mesangial cells to phospho-FTY720 leads to a rapid and transient activation of several protein kinase cascades, including the mitogen- and stress-activated protein kinases. The nonphosphorylated FTY720 also increased MAPK phosphorylation, but with a reduced potency and a more delayed time course. In addition, phospho-FTY720 and FTY720 are able to increase phosphorylation of Smad proteins which are classical members of the transforming growth factor-beta (TGF-beta) signalling device, thus suggesting a crosstalk between FTY720 and TGF-beta signalling. 2.--Pretreatment with the S1P(3) receptor antagonist suramin inhibits FTY720 and phospho-FTY720-induced Smad phosphorylation, whereas pertussis toxin pretreatment, which blocks G(i/0) proteins, has no effect on Smad phosphorylation. 3.--Since TGF-beta is a potent profibrotic cytokine in mesangial cells and upregulates the connective tissue growth factor (CTGF) and collagen as important hallmarks in the fibrotic sequelae, we investigated whether FTY720 and phospho-FTY720 are able to mimic these effects of TGF-beta. Indeed, FTY720 and phospho-FTY720 markedly upregulate CTGF and collagen type IV protein expressions. In addition, the tissue inhibitor of metalloproteinase-1 is transcriptionally activated by FTY720, whereas cytokine-induced matrix metalloproteinase-9 is down-regulated by FTY720. 4.--Depletion of the TGF-beta receptor type II by the siRNA transfection technique blocks not only Smad phosphorylation but also CTGF upregulation. Similarly, Smad-4 depletion by siRNA transfection also abrogates CTGF upregulation induced by FTY720 and phospho-FTY720. 5.--In summary, our data show that FTY720 and phospho-FTY720 not only activate the Smad signalling cascade in mesangial cells, but also upregulate the expression of CTGF and collagen. These findings suggest that FTY720 may have additional effects besides the established immunomodulatory action and, importantly, a profibrotic activity has to be considered in future experimental approaches.
Abstract: Dissociated glucocorticoids are synthetic ligands of the glucocorticoid receptor (GR) and which discriminate between transrepression and transactivation. These compounds were predicted to have large therapeutic benefits when compared to conventional glucocorticoids because of reduced side effects. In this study, we compared the transrepressive properties of different dissociated glucocorticoids on the interleukin-1beta (IL-1beta)-activated metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA) expression in rat mesangial cells (MC). Both proteinases regulate the turnover of extracellular matrix (ECM). We demonstrate that the GR agonist RU 24858, equipotent to dexamethasone (DEX), exhibited strong suppressive effects on the IL-1beta-induced MMP-9 and tPA mRNA levels concomitant with an inhibition of corresponding enzyme activities. In contrast, RU 24782 and RU 40066 exhibited weaker inhibitory activities on both proteinases. Mechanistically, the changes in MMP-9 expression level by different RUs were accompanied by an inhibition of cytokine-induced promoter activity indicating that the inhibition occurs on a transcriptional level. In parallel to the reduction in mRNA levels, we observed an attenuation of cytokine-induced DNA binding of nuclear factor kappa B (NF-kappaB) and reduced contents of the p65 subunit of NF-kappaB within cell nuclei. Along with these transrepressive activities RU 24858, RU 24782 and RU 40066 displayed similar transactivation potentials as indicated by induction of the glucocorticoid-inducible mouse mammary tumor virus (MMTV) reporter gene and by induced expression level of plasminogen activator inhibitor 1 (PAI-1). Interestingly, the different RUs affected the expression of cAMP-induced tPA and inducible NO synthase with the same potency as the IL-1beta-induced protease expression thus indicating that these compounds equipotently modulate cytokine- and cAMP-driven gene expression.
Abstract: Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.
Abstract: Hypoxia evokes a common mechanism of oxygen sensing mediated by hypoxia-inducible transcription factors (HIF) in many mammalian cells. This study investigated the effect of hypoxia on group-IIA secretory phospholipase A(2) (sPLA(2)-IIA) expression in renal mesangial cells. Stimulation of cells with IL-1beta under normoxic conditions (21% O(2)) is known to induce expression and secretion of the group sPLA(2)-IIA. This induction is further enhanced by constantly reducing the O(2) concentration to 1% O(2), and is accompanied by increased sPLA(2) activity. To see whether hypoxia potentiates IL-1beta-induced sPLA(2)-IIA gene expression, a 2.67-kb fragment of the rat sPLA(2)-IIA promoter was fused to a luciferase reporter construct and used to transfect mesangial cells. Hypoxia alone is not able to activate the sPLA(2) promoter, whereas it significantly enhances IL-1beta-stimulated promoter activity. A deletion mutant of the promoter that lacks the two putative hypoxia responsive elements (HRE) is devoid of the potentiating effect of hypoxia. Moreover, site-directed mutagenesis of either of the two HRE is sufficient to abolish the potentiating effect of hypoxia. Electrophoretic mobility shift assays show that HIF-2alpha, which is the only HIF subtype expressed in mesangial cells, binds to both HRE in the sPLA(2)-IIA promoter. In summary, the data show that in an inflammatory setting hypoxia is able to potentiate sPLA(2)-IIA expression and activity in renal mesangial cells, and thereby may critically contribute to enhanced formation of inflammatory lipid mediators seen in a diverse range of kidney diseases.
Abstract: Exposure of renal mesangial cells to sphingosine 1-phosphate (S1P) leads to a rapid and transient activation of the mitogen- and stress-activated protein kinases but also the protein kinase B. Here, we show that S1P also induces phosphorylation of Smad proteins, which are members of the transforming growth factor-beta (TGF-beta) signaling device. However, Smad phosphorylation occurred more slowly with a maximal effect after 20-30 min of S1P stimulation when compared with the rapid activation of the MAPKs. Interestingly, Smad phosphorylation is increased by pertussis toxin, which is in contrast to the complete inhibition of S1P-induced MAPK phosphorylation by pertussis toxin. TGF-beta is a potent anti-inflammatory cytokine, which in mesangial cells attenuates the expression of (i) inducible nitricoxide synthase (iNOS) caused by interleukin (IL)-1beta, (ii) secreted phospholipase A(2) (sPLA(2)), and (iii) matrix metalloproteinase-9 (MMP-9). These gene products are also down-regulated by S1P in a concentration-dependent manner. Furthermore, the expression of connective tissue growth factor is enhanced by both TGF-beta(2) and S1P. These effects of S1P are not mediated by the MAPK cascade as neither pertussis toxin nor the MAPK cascade inhibitor U0126 are able to reverse this inhibition. Overexpression of the inhibitory Smad-7 or down-regulation of co-Smad-4 lead to a reversal of the blocking effect of S1P on IL-1beta-induced NO release. Moreover, down-regulating the TGF-beta receptor type II by the siRNA technique or antagonizing the S1P(3) receptor subtype with suramin abrogates S1P-stimulated Smad phosphorylation. In summary, our data show that S1P trans-activates the TGF-beta receptor and triggers activation of Smads followed by activation of connective tissue growth factor gene transcription and inhibition of IL-1beta-induced expression of iNOS, sPLA(2), and MMP-9.
Abstract: OBJECTIVE: Here we investigate the effects of the endogenous prostaglandin D2 metabolite 15-deoxy-Delta(12,14)-prostaglandin J2, on the renal dysfunction and injury caused by ischemia/reperfusion of the kidney. METHODS: Male Wistar rats, subjected to bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h, were administered 15-deoxy-Delta(12,14)-prostaglandin J2 (1 mg/kg, intravenously) 5 min prior to and again after 3 or 12 h reperfusion. RESULTS: 15-deoxy-Delta(12,14)-prostaglandin J2 significantly reduced (i) renal and tubular dysfunction (serum urea and creatinine levels, creatinine clearance, fractional excretion of Na+ (FENA)), (ii) tubular and reperfusion-injury (urinary N-acetyl-beta-D-glucosaminidase, aspartate aminotransferase (ASP) and gamma-glutamyltransferase (gamma-GT)) and (iii) histological evidence of renal injury. 15-deoxy-Delta(12,14)-prostaglandin J2 also improved renal function (plasma creatinine levels) and reduced the histological signs of renal injury (after 48 h reperfusion). Administration of 15-deoxy-Delta(12,14)-prostaglandin J2 markedly reduced the expression of inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 during reperfusion (determined using immunohistochemistry). Immunohistochemical analysis of p65 translocation and Western blot analysis of IkappaB-alpha degradation revealed that 15-deoxy-Delta(12,14)-prostaglandin J2 inhibited the activation of nuclear factor (NF)-kappaB in renal cells. Subsequently, 15d-PGJ2 was able to significantly reduce nitric oxide production during renal ischemia/reperfusion and by primary cultures of rat proximal tubular (PT) cells incubated with interferon-gamma and bacterial lipopolysaccharide (LPS) in combination. CONCLUSIONS: We demonstrate here, for the first time, that 15-deoxy-Delta(12,14)-prostaglandin J2 significantly reduces renal ischemia/reperfusion-injury via reduction of pro-inflammatory gene expression during reperfusion subsequent to the inhibition of the activation of NF-kappaB.
Abstract: The proinflammatory cytokine interleukin (IL)-18 appears to be involved in the pathogenesis of diseases associated with immunoactivation and inflammation. Consequently, blockage of IL-18 bioactivity by use of IL-18 binding protein (IL-18 BP) is likely a promising therapeutic concept. In the present study, we investigated immunomodulatory activities of IL-18 BPa:Fc in human whole blood cultures. We report that IL-18 BPa:Fc (200 ng/mL) significantly inhibited lipopolysaccharide (LPS, 10 ng/mL)/IL-12 (5 ng/mL)-induced release of interferon-gamma (IFNgamma) and matrix metalloproteinase-9 (MMP-9) from whole blood cultures of healthy donors. Notably, IL-18 BPa:Fc (200 ng/mL) further reinforced dexamethasone (5 nM)- or mycophenolic acid (2 microM)-mediated reduction of LPS/IL-12-induced IFNgamma production by an additional 50.5 or 49.9%, respectively. To investigate effects of IL-18 BP:Fc in the context of autoimmune diseases, experiments were performed with whole blood obtained from patients with systemic lupus erythematosus or Wegener's granulomatosis undergoing immunosuppressive therapy. After ex vivo stimulation with LPS (10 ng/mL), production of IFNgamma and MMP-9 was determined. Both mediators likely contribute to renal inflammation frequently seen in these diseases. In accord with the aforementioned data, LPS (10 ng/mL)-induced IFNgamma was significantly reduced by coincubation with IL-18 BPa:Fc at 200 ng/mL. IL-18 BPa:Fc also inhibited production of MMP-9. The present data demonstrate that IL-18 BPa:Fc has the potential to amplify anti-inflammatory actions of immunosuppressive drugs, and thus may prove to be a valuable novel pharmacological component in the treatment of human autoimmune diseases.
Abstract: Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3' untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3' UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.
Abstract: BACKGROUND: Osteopontin (Opn) is highly upregulated in many different animal models of renal disease, where it is suspected to participate in progression of the disease. In some models, angiotensin II (Ang II) seems to induce the elevated Opn production. Therefore, we examined the regulation of Opn in two-kidney, one-clip (2K1C) hypertensive rats, in which Ang II mediates the elevated blood pressure. METHODS: At days 7, 14, and 28, the clipped and nonclipped kidneys of hypertensive or sham-operated rats were analyzed for osteopontin protein, mRNA expression and mononuclear cell infiltration by imumunohistochemistry, in situ hybridization, and Northern blot analysis. Rats were treated with the Ang II type 1 receptor antagonist Valsartan starting 14 days after clipping. RESULTS: In sham-operated rats, Opn was mainly localized to cells of the thin ascending limbs of the outer medulla. No significant Opn staining was observed in cortical tubules. Focally defined tubular cortical Opn staining was observed in clipped and contralateral kidneys of hypertensive animals at days 14 and 28. Osteopontin protein expression correlated with the mRNA expression detected by in situ hybridization and Northern blot. Treatment with Valsartan reduced osteopontin staining by 51%, mRNA by 47%, and mononuclear cell number by 97% in nonclipped kidneys compared to untreated two-kidney, one-clip animals. In clipped kidneys, however, Opn protein and mRNA expression was not reduced, but a 240% increase in interstitial mononuclear cell number was observed. CONCLUSIONS: Osteopontin is involved in the induction of nephrosclerosis in renovascular hypertension, probably by a mechanism augmenting monocyte infiltration. Angiotensin II appears to be an important inducer of Opn in the nonclipped kidney. Ischemic conditions may regulate Opn expression in the clipped kidney.
Abstract: Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.
Abstract: The proinflammatory cytokine interleukin (IL)-18 appears to be involved in the etiology of a variety of pathological conditions, among them rheumatoid arthritis and atherosclerosis as well as tumor growth and metastasis. As biological activity of matrix metalloproteinase-9 (MMP-9) has been identified as a hallmark in the pathogenesis of these diseases, effects of IL-18 on MMP-9 production by human peripheral blood mononuclear cells (PBMC) were investigated. Moreover, effects of immunopharmacological intervention by anti-tumor necrosis factor-alpha (TNFalpha) or IL-10 were evaluated. Here we report that IL-18 augmented production of MMP-9 by PBMC. The potency of IL-18 to induce release of MMP-9 from PBMC was comparable with that of TNFalpha. MMP-9 production was dependent on endogenous production of TNFalpha, as detected by use of neutralizing monoclonal antibodies. Whereas IL-18 and TNFalpha induced the protease, MMP-9 release was not mediated by IFNgamma. IL-18 also induced secretion of MMP-9 from human whole blood cultures. Antiinflammatory IL-10 efficiently downregulated release of MMP-9 from unstimulated and IL-18-activated PBMC. In contrast to MMP-9, secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) was not augmented by IL-18. Addition of IL-10 enhanced release of TIMP-1 from PBMC. The present study broadens the current pattern of IL-18 proinflammatory actions on PBMC, emphasizes the pivotal role of intermediate TNFalpha production in these responses, and relates IL-18 biological functions to the pathological role of MMP-9.
Abstract: BACKGROUND: Glucocorticoids are efficiently used as antiinflammatory and immunosuppressive therapies of renal diseases. However, long-term treatment often is associated with net changes in the turnover of extracellular matrix (ECM) components. METHODS: We examined the impact of glucocorticoids on cAMP-triggered expression of tissue plasminogen activator (tPA), a protease prominently involved in glomerular ECM turnover. RESULTS: By ELISA, the db-cAMP-mediated increase in extracellular tPA activity secreted by mesangial cells (MC) was markedly reduced in the presence of 100 nmol/L dexamethasone. The decrease of enzymatic activity was accompanied by an attenuation of tPA expression, as shown by Northern blot analysis. Furthermore, dexamethasone increased the steady-state mRNA level of the tPA-inhibitor 1 (PAI-1), thereby providing an additional mode of regulation of tPA activity. Mutational analysis revealed that the inhibition of tPA expression was localized within the proximal 2.3 kb of the 5'-flanking region of the rat tPA gene and critically depended on a cAMP response element (CRE) at position -185. EMSA demonstrated that binding to this CRE was affected by dexamethasone, since the db-cAMP-caused DNA binding of CREB and C/EBPbeta-immunopositive complexes was substantially reduced by dexamethasone. In parallel, dexamethasone decreased the nuclear abundance of db-cAMP-induced C/EBPbeta and phosphorylated CREB protein without affecting the total level of either transcription factor. CONCLUSIONS: Suppression of cAMP-stimulated tPA expression by glucocorticoids occurs by interference with CREB and C/EBPbeta, the major transcription factors mediating cAMP responses. These observations may provide the molecular basis for the sclerotic processes within the glomerulus often complicating chronic glucocorticoid treatment.
Abstract: Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.
Abstract: Glucocorticoids and their synthetic analogs exert potent antiinflammatory actions that, in most cases, are due to an inhibition of the expression of inflammatory genes. In this study, we elucidated the mechanisms of dexamethasone-mediated suppression of matrix metalloproteinase-9 (MMP-9) expression triggered by IL-1beta in rat mesangial cells. Treatment of mesangial cells with dexamethasone markedly reduced the gelatinolytic content of conditioned media due to a decrease in MMP-9 expression. Cloning of a 1.3-kb fragment of the rat MMP-9 gene promoter and subsequent site- directed mutagenesis revealed that a nuclear factor kappaB (NF-kappaB) site at -561 to -550 and a region from -511 to -497 bearing a distal activator protein 1 site adjacent to an Ets-binding site are essentially involved in the IL-1beta-mediated transactivation of MMP-9. Inhibition of MMP-9 expression by dexamethasone resides in a promoter region downstream of -597. The IL-1beta-caused increase in DNA binding of both NF-kappaB and Ets-1 immunopositive complexes was substantially suppressed by dexamethasone as shown by EMSA. This was paralleled with a reduced abundance of p65 and Ets-1 proteins in cell nuclei concomitantly with a reduced inhibitor of kappaB (IkappaB) degradation. In addition to NF-kappaB, we suggest a pivotal role for the Ets binding site, in concert with a distal activator protein-1 element, in the transcriptional suppression of cytokine-induced MMP-9 expression by glucocorticoids.
Abstract: BACKGROUND: Dysregulated expression of diverse proteases and their specific inhibitors is critical for the increase in extracellular matrix accumulation that accompanies chronic inflammatory and sclerotic processes within the renal mesangium. Within the activating cascade of several proteases, the plasminogen system plays an important role. METHODS: We tested for modulatory effects of the nitric oxide (NO) donors S-nitroso-N-acetyl-D,L-penicillamine and DETA-NONOate, and the superoxide-generating system hypoxanthine/xanthine oxidase (HXXO) on the expression and activity of tissue plasminogen activator (tPA) by ELISA and Northern blotting. RESULTS: Interleukin-1beta (IL-1beta)-induced tPA and plasminogen activator inhibitor (PAI)-1 mRNA and supernatant tPA antigen were significantly inhibited by both NO donors, which resulted in a net decrease in the IL-1beta-evoked tPA enzyme activity in the conditioned media. Addition of the NO-synthase inhibitor N-monomethyl l-arginine markedly increased the cytokine-triggered tPA- and PAI-1 mRNA levels, respectively. In contrast, HXXO caused a marked amplification of the IL-1beta-induced steady-state tPA mRNA level and tPA enzyme activity that was blocked by catalase. Since MnTBAP, a superoxide dismutase mimetic, had no effects on the amplification of mRNA levels, we suggest that H2O2 is the candidate reactive oxygen species (ROS) responsible for the potentiation of IL-1beta-triggered tPA and PAI-1 expression. CONCLUSIONS: The temporal relationship between NO and ROS generation is a critical step in the modulation of tPA and PAI-1 expression in mesangial cells and may account for a dysregulation of matrix turnover during inflammatory processes in the renal mesangium.
Abstract: One of the greatest biomedical breakthroughs of the twentieth century was the discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO). NO has received special attention ever since: besides its potent vasodilatory and vasoprotective effects, NO was identified as a key player in innate immunity and was found to act as an unconventional type of neurotransmitter. This article focuses on mechanisms of NO signalling that form the basis of functional cell responses to accommodate changes in the cellular microenvironment. Redox-based regulation of signal transduction and, on a more long-term scale, changes in gene expression will be exemplified by NO-modulation of matrix components and matrix-metabolizing enzymes. It seems to be a safe bet that ongoing analyses of NO signalling and gene expression will provide a wealth of promising therapeutic targets in human diseases.
Abstract: Nitric oxide is a crucial mediator of several forms of glomerulonephritis. We examined the effects of NO on the mRNA expression pattern in glomerular mesangial cells by using a low-stringency reverse transcriptase-polymerase chain reaction method and detected a cDNA fragment that was induced by interleukin 1b (IL-1b) and further up-regulated by the NO donor diethylenetriamine-nitric oxide (DETA-NO). Each respective cDNA fragment was found to match with the cDNAs of rat macrophage inflammatory protein 2 (MIP-2) and GRO/cytokine-induced neutrophil chemoattractant 2b (CINC-2b). Further characterization of MIP-2 regulation by Northern blot analysis confirmed an NO- and IL-1b-dependent increase in MIP-2 mRNA levels. Moreover, inhibition of IL-1b-induced endogenous NO formation by the NO-synthase (NOS) inhibitor L-NMMA markedly attenuated MIP-2 protein expression. We cloned 770 bp of the 5'-flanking region of rat MIP-2 and fused this fragment to a luciferase reporter gene. Transfection of the construct into mesangial cells resulted in a 3.5-fold increase in luciferase activity in cells treated with DETA-NO when compared to controls, suggesting a transcriptional mechanism for NO-induced MIP-2 expression. Deletion and mutational analysis identified critical nuclear factor (NF)-kB and NF-IL-6 binding sites required for NO regulation of MIP-2. In vivo, inhibition of NO synthesis in the Thy-1.1 model of mesangioproliferative glomerulonephritis by the specific inducible-NOS inhibitor L-NIL resulted in a marked reduction of MIP-2 mRNA expression. Furthermore, infiltration of neutrophils into the glomerulus was dramatically attenuated in L-NIL-treated rats.
Abstract: Inflammatory glomerular diseases are accompanied by changes in the expression patterns of growth factors, mediators and matrix-associated proteins in mesangial cells and by the production of nitric oxide via the cytokine-inducible form of the nitric oxide synthase. Nitric oxide has been shown to act potently on gene transcription. To identify genes that are differentially expressed by endogenously produced nitric oxide, we forced rat mesangial cells to produce high amounts of nitric oxide by exposure to inflammatory cytokines and compared the mRNA expression patterns of cytokine-stimulated mesangial cells with cells that were additionally treated with the nitric oxide synthase inhibitor L-NMMA to block endogenous NO synthesis. We used a modification of a low stringency RT-PCR approach designated as RNA arbitrarily-primed polymerase chain reaction (RAP-PCR). In this way, we identified among others the integrin-linked kinase (ILK) as an NO-regulated gene. The NO-mediated changes in the mRNA and protein expression patterns of ILK were compared to that of "secreted protein acidic and rich in cystein" (SPARC), a gene that was identified as NO-regulated in the same set of experiments (Walpen et al., J. Am. Soc. Nephrol., 11, 468-476). ILK and SPARC mRNA levels by were downregulated by cytokines via endogenously produced nitric oxide in a comparable manner as verified by Northern blot analysis. In contrast, cytokine- induced NO production or administration of exogenous NO-donors strongly reduced SPARC protein levels without altering ILK protein content in mesangial cells over a period up to 72 hours. Blocking de novo protein synthesis showed a short halflife of SPARC (< 2 hours) whereas ILK-protein was stable over a period of 7 hours, indicating that NO-mediated reduction of ILK mRNA levels does not influence protein content of ILK in mesangial cells under the time limitations given under cell culture conditions. However, a role for cytokines/NO in ILK-long-term regulation in chronic inflammatory diseases that may influence phenotypic responses such as apoptosis or cell proliferation remains to be elucidated.
Abstract: Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. BACKGROUND: High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9. CONCLUSION: We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.
Abstract: Nitric oxide (NO) has been implicated in several forms of glomerulonephritis. In this study, a low stringency reversed transcription/PCR protocol was used to evaluate the action of NO on the mRNA expression pattern in rat mesangial cells (MC). To mimic the state of glomerular inflammation, MC were stimulated by exposure to the cytokines interleukin-1beta and tumor necrosis factor-alpha into producing high levels of NO via expression of inducible nitric oxide synthase (NOS). To detect NO-mediated effects, the resulting expression pattern was compared to that of MC stimulated by the cytokines in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA). Computer analysis of a differentially expressed cDNA fragment resulted in a 100% homology to the recently characterized mRNA of SPARC (secreted protein acidic and rich in cysteine). Further characterization of SPARC regulation revealed a cytokine- and cAMP-dependent decrease in SPARC mRNA and protein levels. Blocking NO formation by L-NMMA reversed the effects of cytokines and cAMP on SPARC expression, suggesting an NO-mediated mechanism. The NO donors S-nitroso-N-acetyl-penicillamine and diethylenetriamine/NO further reduced SPARC expression in cytokine-treated MC as well as in controls. Moreover, downregulation of SPARC mRNA and protein expression in whole kidneys obtained from rats treated with endotoxin was observed. This downregulation of SPARC was reversed by treatment with L-N6-l (iminoethyl) lysine dihydrochloride, a potent and highly selective inhibitor of inducible NOS. These data characterize SPARC as an NO-regulated gene. This observation may be important in the context of tissue remodeling in chronic inflammatory kidney diseases.
Abstract: The modulation of cell signaling by free radicals is important for the pathogenesis of inflammatory diseases. Recently, we have shown that NO reduces IL-1beta-induced matrix metalloproteinase (MMP-9) expression in glomerular mesangial cells (MC). Here we report that exogenously administrated superoxide, generated by the hypoxanthine/xanthine oxidase system (HXXO) or by the redox cycler 2, 3-dimethoxy-1,4-naphtoquinone, caused a marked amplification of IL-1beta-primed, steady state, MMP-9 mRNA level and an increase in gelatinolytic activity in the conditioned medium. Superoxide generators alone were ineffective. Cytokine-induced steady state mRNA levels of TIMP-1, an endogenous inhibitor of MMP-9, were affected similarly by HXXO. Transient transfection of rat mesangial cells with 0.6 kb of the 5'-flanking region of the rat MMP-9 gene proved a transcriptional regulation of MMP-9 expression by superoxide. HXXO augmented the IL-1beta-triggered nuclear translocation of p65 and c-Jun and, in parallel, increased DNA binding activities of NF-kappaB and AP-1. Mutation of either response element completely prevented MMP-9 promoter activation by IL-1beta. Moreover, specific inhibitors of the classical extracellular signal-regulated kinase (ERK) pathway and p38 mitogen-activated protein kinase (MAPK) cascade, partially reversed the HXXO-mediated effects on MMP-9 mRNA levels, thus demonstrating involvement of ERKs and p38 MAPKs in MMP-9 expression. Furthermore, IL-1beta-triggered phosphorylation of all three MAPKs, including p38-MAPK, c-Jun N-terminal kinase, and ERK, was substantially enhanced by superoxide. Our data identify superoxide as a costimulatory factor amplifying cytokine-induced MMP-9 expression by interfering with the signaling cascades leading to the activation of AP-1 and NF-kappaB.
Abstract: 1. CGP-43182 has been described as a potent inhibitor of group IIA secreted phospholipase A(2) (group IIA sPLA(2)) activity in vitro. In rat mesangial cells, inhibition of group IIA sPLA(2) activity by CGP-43182 results in a 70% reduction of cytokine-stimulated prostaglandin E(2) biosynthesis, suggesting that group IIA sPLA(2) participates in arachidonic acid release and eicosanoid formation. Under these conditions the cytosolic phospholipase A(2) is not affected. 2. In mesangial cells, in addition to inhibition of catalytic activity, the membrane-permeant CGP-43182 completely blocked interleukin 1beta (IL1beta)-stimulated group IIA sPLA(2) gene expression. 3. A further action of CGP-43182 was a complete inhibition of cyclo-oxygenase-2 gene expression, resulting in a drastic reduction of prostaglandin formation in mesangial cells. 4. Moreover, CGP-43182 completely blocked IL1beta-induced gene expression of the inducible nitric oxide synthase, leading to an inhibition of cytokine-stimulated nitric oxide formation. 5. In contrast, the stimulatory effect of the cell-permeant cyclic AMP-analogue, dibutyryl-cAMP, on the induction of these enzymes was not inhibited by CGP-43182. These data indicate that CGP-43182 interferes with IL1beta- but not cyclic AMP-activated transcriptional regulation. 6. By studying components of the upstream transcription machinery, we observed an inhibition of NFkappaB activation by CGP-43182 in IL1beta-treated cells. Moreover, we observed that CGP-43182 prevented the phosphorylation and proteolytic degradation of the endogenous NFkappaB inhibitor, IkappaB, a process necessary for NFkappaB activation. 7. From our data, we propose that CGP-43182 is a potent anti-inflammatory drug useful for preventing the consequences of a concerted action of cytokine-stimulated pro-inflammatory genes mediated by NFkappaB.
Abstract: To characterize mechanisms which may determine the fate of apoptotic cells, we investigated chemokine expression in apoptotic promonocytic U937 cells or peripheral blood mononuclear cells (PBMC). Exposure of U937 cells to etoposide (VP-16) or the nitric oxide (NO) donor DETA-NO, both inducers of apoptosis in these cells, was associated with increased expression of the chemokines IL-8 and macrophage inflammatory protein-1 alpha. Up-regulation of IL-8 mRNA expression by VP-16 or DETA-NO was observed as early as 4 h or 6 h, respectively, after onset of treatment and was still detectable after 19 h of exposure. A serine protease inhibitor prevented both VP-16-induced apoptosis and release of IL-8, whereas inhibition of p38 MAP kinases reduced IL-8 secretion only. Moreover, we observed that incubation with 2-chlorodeoxyadenosine (CdA) up-regulated release of IL-8 from adherent PBMC in parallel to induction of apoptosis. In these cells a modest but significant induction of TNF-alpha release by CdA was also detected. In addition, CdA augmented release of IL-8 from whole blood cultures. By facilitating adequate recruitment of phagocytes to sites of cell death, stress-induced up-regulation of chemokines associated with apoptosis may contribute to mechanisms aiming at efficient removal of apoptotic cells.
Abstract: Exposure of mesangial cells to superoxide, generated by the hypoxanthine/xanthine oxidase system or by the redox cycler 2,3-dimethoxy-1,4-naphthoquinone caused a concentration-dependent amplification of interleukin (IL)-1beta-stimulated nitrite production. The effect of superoxide was accompanied by an increase in inducible nitric oxide synthase (iNOS) protein and iNOS mRNA levels. Incubation of mesangial cells with superoxide alone did not induce iNOS expression. To elucidate whether the increase of iNOS expression is due to transcriptional upregulation we fused a 4.5-kb genomic iNOS fragment that contains the transcriptional start site of the rat iNOS gene to a luciferase reporter gene. In transient transfection studies, superoxide caused a 10-fold augmentation of iNOS promoter activity in IL-1beta-challenged mesangial cells. Our data identify superoxide as a co-stimulatory factor amplifying cytokine-induced iNOS gene expression and subsequent nitric oxide (NO) synthesis.
Abstract: Expression of the inducible nitric oxide synthase (iNOS) gene in rat mesangial cells is differentially triggered by IL-1beta and cAMP predominantly at the transcriptional level. The 5'-flanking region of the rat iNOS gene contains several binding sites for transcription factors potentially involved in cytokine and cAMP signaling such as nuclear factor-kappaB/Rel, CCAAT/enhancer-binding protein (C/EBP), and cyclic AMP-responsive element-binding protein/ATF. We tested promoter activities of serial and site-directed deletion mutants of iNOS-chloramphenicol acetyltransferase reporter genes after transient transfection and stimulation of mesangial cells. A region between bp -277 and -111 bearing a CCAAT/enhancer-binding protein-response element was found to be critical for cAMP-mediated gene induction but dispensable for IL-1beta inducibility. Moreover, a minimal promoter ranging from the transcriptional start site up to -111 containing a kappaB site is sufficient to confer IL-1beta-mediated iNOS promoter activation. Consistent with these findings, an electrophoretic mobility shift assay shows the appearance of an IL-1beta-inducible nuclear factor-kappaB p50/p65 heterodimeric complex. Using probes containing C/EBP-binding sites from the iNOS gene revealed further binding of different complexes, all of which were strongly inducible by cAMP and to a lower extent also by IL-1beta. Abs against cyclic AMP-responsive element-binding protein, C/EBPbeta, and C/EBPdelta were able to partially supershift single complexes, suggesting the participation of these transcription factors in the regulation of iNOS gene expression by cAMP and IL-1beta. Finally, we show that both cAMP and IL-1beta strongly induce steady-state levels of C/EBPbeta and C/EBPdelta mRNA levels. These data demonstrate that IL-1beta and cAMP use distinct as well as partially overlapping sets of transcriptional activators to modulate iNOS gene expression in rat mesangial cells.
Abstract: Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) regulate mesangial cell proliferation and matrix production in vitro and in vivo and crucially participate in the pathogenesis of glomerulonephritis. We investigated whether PDGF-BB and bFGF influence nitric oxide (NO) production, another important effector molecule in inflammatory glomerular injury. Inducible NO synthase (iNOS) induction in rat glomerular mesangial cells has been described in response to two principal classes of activating signals comprising inflammatory cytokines such as interleukin 1beta (IL-1beta) or elevation of cyclic AMP (cAMP). Treatment of mesangial cells with IL-1beta induces iNOS activity measured as nitrite levels in cell culture supernatants. Coincubation of mesangial cells with PDGF-BB inhibits production of nitrite by approximately 95%. This effect can be reversed by the simultaneous incubation of PDGF-BB in the presence of calphostin C, a potent and selective inhibitor of protein kinase C. In contrast, incubation of cells in the presence of bFGF potentiates IL-1beta-induced production of NO and is functionally associated with an increased rate of apoptosis of mesangial cells. Western blot analyses reveal that PDGF-BB causes a decrease in the formation of iNOS protein which is preceded by decreases in iNOS mRNA steady state levels. bFGF drastically increases iNOS protein levels as well as the corresponding iNOS mRNA steady state levels. Nuclear run-on experiments reveal that PDGF-BB decreases the IL-1beta-induced transcription rate of the iNOS gene, whereas bFGF potentiates the transcriptional activity of the iNOS gene. Northern blot analyses demonstrate that bFGF strongly potentiates the formation of IL-1beta-induced IL-1 type I receptor mRNA levels, whereas PDGF-BB has no effect. Treatment of mesangial cells with the membrane-permeable cAMP analogue N6, O-2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP) markedly increases the production of nitrite. Whereas PDGF-BB does not affect cAMP-induced nitrite levels, bFGF strongly potentiates them. PDGF-BB alters neither cAMP-induced iNOS protein levels nor the corresponding iNOS mRNA steady state levels. By contrast, bFGF superinduces cAMP-stimulated iNOS protein and iNOS mRNA levels. These changes by bFGF are due to an increase in cAMP-induced transcriptional activity of the iNOS gene which is not affected by PDGF-BB. In summary, the results show that PDGF and bFGF differentially regulate iNOS expression in mesangial cells in a stimulus-specific way. The timely sequence of expression of PDGF and bFGF and of cytokines like IL-1 will crucially determine the amounts of NO produced and the functional consequences thereof in the course of progressive glomerular diseases.
Abstract: Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin 1 beta (IL-1 beta). We have reported that nanomolar concentrations of dexamethasone suppress IL-1 beta-induced iNOS protein expression and production of nitrite, the stable end product of NO formation, without affecting IL-1 beta-triggered increase in iNOS mRNA levels. We now have studied the mechanisms by which dexamethasone suppresses IL-1 beta-stimulated iNOS expression in mesangial cells. Surprisingly, nuclear run-on transcription experiments demonstrate that dexamethasone markedly attenuates IL-1 beta-induced iNOS gene transcription. However, this is counteracted by a prolongation of the half-life of iNOS mRNA from 1 h to 2.5 h by dexamethasone. Moreover, dexamethasone drastically reduces the amount of iNOS protein by reduction of iNOS mRNA translation and increased degradation of iNOS protein. These results indicate that glucocorticoids act at multiple levels to regulate iNOS expression, thus providing important insights into the treatment of inflammatory diseases.
Abstract: The inducible nitric oxide synthase (iNOS) is not constitutively expressed but is induced in many types of mammalian cells by cytokines and bacterial endotoxins. Previously, we have reported the most interesting feature of rat iNOS gene that is up-regulated by cyclic AMP at transcriptional level as demonstrated by nuclear run-on assay. We now have isolated and sequenced the rat iNOS gene promoter and determined the transcription start site. Moreover, we have constructed a rat iNOS promoter-chloramphenicol acetyltransferase (CAT) fusion gene and analyzed its inducibility by interleukin 1 beta and cyclic AMP in transiently transfected Swiss 3T3 fibroblasts.
Abstract: The expression of inducible nitric oxide synthase (iNOS) is triggered in rat renal mesangial cells by exposure to the inflammatory cytokine interleukin 1 beta (IL-1 beta). Here we report that cyclosporin A (CsA) a potent immunosuppressive drug, inhibits IL-1 beta dependent iNOS expression in renal mesangial cells. Addition of CsA dose dependently suppresses IL-1 beta-induced nitrite formation (IC50 = 0.9 microM). Western- and Northern blot analyses of mesangial cell extracts reveal that the inhibition of IL-1 beta-induced nitrite formation by CsA is due to decreased iNOS protein and iNOS mRNA steady state levels. Using nuclear run on experiments we show that the transcription rate of the IL-1 beta-induced iNOS gene is reduced. Furthermore, by electrophoretic mobility shift analysis we demonstrate reduced DNA-binding of the nuclear factor NF kappa B, an essential component of the IL-1 beta-dependent upregulation of iNOS gene transcription. The data presented in this report suggest that the cellular machinery involved in the IL-1 beta dependent transcriptional upregulation of the iNOS gene in mesangial cells is a target for the action of CsA.
Abstract: Inducible nitric oxide synthase (NOS) is expressed in renal mesangial cells in response to two principal classes of activating signals that interact in a synergistic fashion. These two groups of activators comprise inflammatory cytokines such as interleukin 1 (IL-1) or tumour necrosis factor alpha and agents that elevate cellular levels of cAMP. We have used pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor kappa B (NF kappa B), to determine its role in IL-1 beta- and cAMP-triggered NOS expression. Micromolar amounts of PDTC suppress IL-1 beta-, but not cAMP-stimulated nitrite production, the stable end product of NO formation in mesangial cells. Furthermore, PDTC completely inhibited the increase of NOS mRNA in response to IL-1 beta, while only marginally affecting cAMP-induced NOS mRNA levels. Our data suggest that NF kappa B activation is an essential component of the IL-1 beta signalling pathway responsible for NOS gene activation and that cAMP triggers a separate signalling cascade not involving NF kappa B. These observations may provide a basis for the synergistic stimulation of NOS expression by cytokines and cAMP in mesangial cells.
Abstract: The localization of the extracellular matrix glycoprotein laminin was studied using polyclonal anti-laminin antibodies. The laminin patterns of the basement membranes of the muscular epithelial sheath that envelops the ovariole were conspicuously different from those of the basement membrane of the follicular epithelium. In the latter structure laminin was stained in a pattern of parallel stripes oriented perpendicular to the long axis of the follicle; microfilament bundles at the adjacent basal side of the follicle cells have the same orientation. At late vitellogenic stages the orientation of the microfilaments remained the same while the laminin stripes were no longer visible. The orientation of laminin and F-actin was abnormal in follicles of the egg-shape mutant kugel, which produces shorter and thicker eggs than wild-type flies. This phenotype might result from the disturbance of the normal circular microfilament and/or laminin pattern.
Abstract: During the last decade evidence from numerous studies has been accumulated demonstrating that posttranscriptional gene regulation including mRNA turnover and translation is an important paradigm of eukaryotic gene expression contributing to the vast majority of cellular processes including cell growth and differentiation, metabolism, migration, and cellular senescence. Accordingly, a large number of reports have documented that the Human antigen R (HuR), a ubiquitously expressed member of the ELAV protein family, is one of the major actors in this scenario. Consequently, HuR is implicated in a large variety of pathologies in which deregulated stabilisation of many short-lived key mRNAs is causally linked with the onset and course of disease. Since HuR is most abundantly localised within the cell nucleus, export of HuR to the cytoplasm seems a major prerequisite for its stabilising effects on its cognate target adenylate- and uridylate-rich elements (AREs) containing cargo mRNAs. Although, the list of reports demonstrating a critical involvement of different signalling cascades in HuR-triggered mRNA functions is steadily growing, the mechanisms underlying HuR trafficking are not well understood. For this reason, the review will cover the most recent advances of knowledge of signalling cascades involved in the stimulus-dependent nucleo-cytoplasmic HuR shuttling and a special emphasis will be put on the possible regulatory role of posttranslational HuR modification.
Abstract: During the last decade evidence has accumulated that modulation of mRNA stability plays a central role in cellular homeostasis, including cell differentiation, proliferation and adaptation to external stimuli. The functional relevance of posttranscriptional gene regulation is highlighted by many pathologies, wherein occurrence tightly correlates with a dysregulation in mRNA stability, including chronic inflammation, cardiovascular diseases and cancer. Most commonly, the cis-regulatory elements of mRNA decay are represented by the adenylate- and uridylate (AU)-rich elements (ARE) which are specifically bound by trans-acting RNA binding proteins, which finally determine whether mRNA decay is delayed or facilitated. Regulation of mRNA decay by RNA stabilizing and RNA destabilizing factors is furthermore controlled by different intrinsic and environmental stimuli. The modulation of mRNA binding proteins, therefore, illuminates a promising approach for the pharmacotherapy of those key pathologies mentioned above and characterized by a posttranscriptional dysregulation. Most promisingly, intracellular trafficking of many of the mRNA stability regulating factors is, in turn, regulated by some major signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade, the AMP-activated kinase (AMPK) and the protein kinase (PK) C (PKC) family. In this review, we present timely examples of genes regulated by mRNA stability with a special focus on signaling pathways involved in the ARE-dependent mRNA decay. A better understanding of these processes may form the basis for the development of novel therapeutics to treat major human diseases.
Abstract: Extracellular matrix (ECM) remodeling with successive tissue fibrosis is a key feature of chronic cardiovascular diseases, including atherosclerosis and restenosis. The atherogenic changes underlying these pathologies result from chronification of an acute repair response towards injurious and inflammatory stimuli. Thereby functional tissue is replaced by excessive ECM deposition. In the kidney, impaired remodeling is a major cause of perivascular, interstitial, and glomerular fibrosis but also a common complication of chronic hypertension. Experimental evidence points to the matrix metalloproteases (MMPs) and their intrinsic inhibitors, the tissue inhibitors of MMPs as key mediators of atherogenic and fibrotic pathologies. Mechanistically, a deregulation in ECM turnover tightly correlates with an increased production and release of proinflammatory and profibrotic factors including interleukin-1beta, transforming growth factor beta, angiotensin II, and reactive oxygen species. Unlike these factors the pleiotropic messenger molecule nitric oxide (NO) by acting as the major physiological vasodilator has emerged as one of the most atheroprotective factors. However, under inflammatory conditions NO does acquire proatherogenic and profibrotic properties thereby exacerbating tissue fibrosis. In this review, the mechanisms underlying both opposing properties of NO on perivascular ECM remodeling will exemplarily be discussed for renal fibrosis with a particular focus on the MMPs and intrinsic protease inhibitors.
Abstract: Regulation of signal transduction and gene expression is a multifaceted process involving ligands, receptors, and second messengers that trigger cascades of protein kinases and phosphatases and propagate the signal to the nucleus to alter gene expression. Reduction-oxidation (redox)-based regulatory pathways provide additional means of gating signal transduction, and redox-based regulation of gene expression emerges as a fundamental regulatory mechanism in living cells. The cellular redox state is reflected by the degree of oxidation (or reduction) of various redox-active molecules at a specific cellular location at any given time point. The ratio of oxidized/reduced redox species determines the redox potential, which may vary dramatically in time and in different compartments of a cell and consequently alter in a temporally and spatially dynamic process the activity of signaling enzymes that carry redox-active functional groups. Generation and action of free radicals such as nitric oxide, superoxide, and H(2)O(2) that paradigmatically highlight the impact of redox regulation on cellular signal transduction and gene expression are discussed with a special focus on the renal glomerular response to injury.
Abstract: The glomerular response to injury displays astonishingly uniform features that include infiltration with professional immune cells, activation and proliferation of resident glomerular cells and matrix expansion. Cross-communication of intrinsic mesangial cells with invading immune cells is crucial for the fate of glomerular injury: progression to glomerulosclerosis or resolution and repair. The formation of free radicals, particularly of nitric oxide and superoxide, are key events that initiate redox-based signal transduction and gene expression. The balance between these radicals constitutes redox-operated genetic switches that ensure self-limited inflammatory responses to tissue injury. The aberrant production of the mediators, however, may sustain matrix accumulation and result in irreversible alteration of glomerular structure and function.
Abstract: Nitric oxide (NO) modulates transcription factors that bind specific cis-regulatory DNA responsible for coordinating the spatial and temporal patterns of gene expression that are initiated by a changing microenvironment. In this way NO helps to orchestrate gene transcription and forms the basis of functional cell responses to accommodate metabolic requirements and to coordinate endogenous defense mechanisms against a variety of stress and disease conditions. There is marked overlap between the signalling pathways triggered by NO, superoxide, and hypoxia. Understanding the redox-based regulation of signal transduction and gene expression will provide insights into how cell activities are constantly coordinated and how promising new therapies may be developed.
Abstract: The discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO) was one of the most exciting discoveries of biomedical research in the 1980s. Besides its potent vasodilatory effects, NO was found under certain circumstances to be responsible for the killing of microorganisms and tumour cells by activated macrophages and to act as a novel, unconventional type of neurotransmitter. In 1992, Science picked NO as the 'Molecule of the Year', and over the past years NO has become established as a universal intercellular messenger that acutely affects important signalling pathways and, on a more long-term scale, modulates gene expression in target cells. These actions will form the focus of the present review.
Abstract: In recent years, NO, a gas previously considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell-cell communication throughout the body. In mammals, NO is a recognized mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a non-conventional neurotransmitter and participates in the establishment of long-term plasticity required for memory formation. In addition, NO is responsible for some parts of the host response to sepsis and inflammation and contributes to certain disease states. A number of strategies have emerged with regard to a pharmacological control of pathological NO overproductions. This review will discuss these novel therapeutic approaches that may provide new means for clinical medicine.
