Abstract: Endometriosis, defined as the presence of endometrial tissue outside the uterus, is a common gynecological disease with poorly understood pathogenesis. MicroRNAs are members of a class of small noncoding RNA molecules that have a critical role in posttranscriptional regulation of gene expression by repression of target mRNAs translation. We assessed differentially expressed microRNAs in ectopic endometrium compared with eutopic endometrium in 3 patients through microarray analysis. We identified 50 microRNAs differentially expressed and the differential expression of five microRNAs was validated by real-time RT-PCR in other 13 patients. We identified in silico their predicted targets, several of which match the genes that have been identified to be differentially expressed in ectopic versus eutopic endometrium in studies of gene expression. A functional analysis of the predicted targets indicates that several of these are involved in molecular pathways implicated in endometriosis, thus strengthening the hypothesis of the role of microRNAs in this pathology.
Abstract: In liver ischemic preconditioning (IP), stimulation of adenosine A2a receptors (A2aR) prevents ischemia/reperfusion injury by promoting diacylglycerol-mediated activation of protein kinase C (PKC). By concerting diacylglycerol to phosphatidic acid, diacylglycerol kinases (DGKs) act as terminator of diacylglycerol signalling. This study investigates the role of DGK in the development of hepatocyte IP. DGK activity and cell viability were evaluated in isolated rat hepatocytes preconditioned by 10 min hypoxia followed by 10 min re-oxygenation or by the treatment with the A2aR agonist, CGS21680, and subsequently exposed to prolonged hypoxia. We observed that after IP or A2aR activation, a decrease in DGK activity was associated with the onset of hepatocyte tolerance to hypoxia. CGS21680-induced stimulation of A2aR specifically inhibited DGK isoform theta by activating RhoA-GTPase. Consistently, both siRNA-mediated downregulation of DGK theta and hepatocyte pretreatment with the DGK inhibitor R59949 induced cell tolerance to hypoxia. The pharmacological inhibition of DGK was associated with the diacylglycerol-dependent activation of PKC delta and epsilon and of their downstream target p38 MAPK. In conclusion, we unveil a novel signalling pathway contributing to the onset of hepatocyte preconditioning, which through RhoA-GTPase, couples A2aR to the downregulation of DGK. Such an inhibition is essential for the sustained accumulation of diacylglycerol required for triggering PKC-mediated survival signals.
Abstract: Diacylglycerol kinases (DGKs) convert diacylglycerol (DAG) into phosphatidic acid (PA), acting as molecular switches between DAG- and PA-mediated signaling. We previously showed that Src-dependent activation and plasma membrane recruitment of DGKalpha are required for growth-factor-induced cell migration and ruffling, through the control of Rac small-GTPase activation and plasma membrane localization. Herein we unveil a signaling pathway through which DGKalpha coordinates the localization of Rac. We show that upon hepatocyte growth-factor stimulation, DGKalpha, by producing PA, provides a key signal to recruit atypical PKCzeta/iota (aPKCzeta/iota) in complex with RhoGDI and Rac at ruffling sites of colony-growing epithelial cells. Then, DGKalpha-dependent activation of aPKCzeta/iota mediates the release of Rac from the inhibitory complex with RhoGDI, allowing its activation and leading to formation of membrane ruffles, which constitute essential requirements for cell migration. These findings highlight DGKalpha as the central element of a lipid signaling pathway linking tyrosine kinase growth-factor receptors to regulation of aPKCs and RhoGDI, and providing a positional signal regulating Rac association to the plasma membrane.
Abstract: Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys(3)-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca(2+)](i) followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca(2+) entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys(3)-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca(2+) activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys(3)-GHRP-6 ghrelin antagonist features ghrelin independent activities.
Abstract: 8-Prenylnaringenin (8PN), one of the strongest plant-derived oestrogen receptors (ERs) ligand, has been suggested to have potential cancer chemo-preventive activities and anti-angiogenic properties. Because published data suggest that ERs serve as nodal point that allows interactions between hormones and growth factors mediated pathways, we decided to investigate the effects exerted by 8PN on Epidermal growth factor (EGF)-elicited pathways in breast cancer cells. Here we show that in ER positive MCF-7 cells, 8PN interferes with EGF induced cell proliferation by strongly inhibiting activation of PI(3)K/Akt pathway, without affecting EGFR expression or tyrosine phosphorylation, and exerting a synergistic activation of Erk1/2 phosphorylation. Moreover, we demonstrate that 8PN is a direct inhibitor of PI(3)K activity as it is shown by in vitro experiments with the purified enzyme and by its inability to impair serine phosphorylation of a constitutive active form of Akt. These findings suggest that inhibition of PI(3)K is a novel mechanism which contributes to 8PN activity to inhibit cancer cell survival and EGF induced proliferation.
Abstract: Cell transplantation therapy has raised a great interest in the perspective of its employment for nerve tissue repair. Among the various cell populations proposed, olfactory ensheathing glial cells have raised great interest over recent years, especially in the perspective of their employment for neural repair because of their homing capacity in both central and peripheral nervous system. This paper is aimed to provide an in vitro characterization of the NOBEC (neonatal olfactory bulb ensheathing cell) line that was obtained from primary cells dissociated from rat neonatal olfactory bulb (OB) and immortalized by retroviral transduction of SV40 large T antigen. Light and electron microscopy investigation showed that NOBECs are a homogeneous cell population both at structural and ultrastructural level. RT-PCR, Western blotting and immunocytochemistry showed that NOBECs express the glial markers S100, GFAP (Glial Fibrillar Acid Protein) and p75NGFR as well as NRG1 (neuregulin-1) and ErbB1-2-3 receptors; while they are negative for ErbB4. Yet, NOBECs exhibit a high proliferation and migration basal activity and can be transducted with vectors carrying GFP (green fluorescent protein) and NRG1 cDNA. Functional stimulation by means of NRG1-III-beta3 overexpression through viral transduction induced a significant increase in cell proliferation rate while it had no effect on cell migration. Altogether, these results show that NOBEC cell line retain glial features both morphologically and functionally, responding to the NRG1/ErbB-mediated gliotrophic stimulus, and represents thus a good tool for in vitro assays of glial cell manipulation and for in vivo experimental studies of glial cell transplantation in the central and peripheral nervous system.
Abstract: Diacylglycerol (DAG) kinases (Dgk), which phosphorylate DAG to generate phosphatidic acid, act as either positive or negative key regulators of cell signaling. We previously showed that Src mediates growth factors-induced activation of Dgk-alpha, whose activity is required for cell motility, proliferation and angiogenesis. Here, we demonstrate that both hepatocytes growth factor (HGF) stimulation and v-Src transformation induce tyrosine phosphorylation of Dgk-alpha on Y335, through a mechanism requiring its proline-rich C-terminal sequence. Moreover, we show that both proline-rich sequence and phosphorylation of Y335 of Dgk-alpha mediate: (i) its enzymatic activation, (ii) its ability to interact respectively with SH3 and SH2 domains of Src, (iii) its recruitment to the membrane. In addition, we show that phosphorylation of Dgk-alpha on Y335 is required for HGF-induced motility, while its constitutive recruitment at the membrane by myristylation is sufficient to trigger spontaneous motility in absence of HGF. Providing the first evidence that tyrosine phosphorylation of Dgk-alpha is required for growth-factors-induced activation and membrane recruitment, these findings underscore its relevance as a rheostat, whose activation is a threshold to elicit growth factors-induced migratory signaling.
Abstract: The experimental investigation of nerve regeneration after microsurgical repair is usually carried out in rats, rather than mice, because of the larger sized peripheral nerves. Today however, the availability of genetically modified mice makes the use of this laboratory animal very intriguing for investigating nerve regeneration at a molecular level. In this study we aimed to provide a standardization of the experimental model based on microsurgical direct repair, by 12/0 suture, of the left median nerve in adult male mice. Postoperative recovery was regularly assessed by the grasping test. At day-75 postoperative, regenerated median nerve fibers were analyzed by design-based quantitative morphology and electron microscopy. Yet, sections were immuno-labelled using two axonal antibodies commonly employed for rat nerve fibers. Results indicated that functional recovery begun at day-15 and progressively increased reaching values not significantly different from normal by day-50. Quantitative morphology showed that, at day-75, the number of regenerated nerve fibers was not significantly different in comparison to controls. In contrast, differences were detected in fiber density, mean axon and fiber diameter and myelin thickness which were all significantly lower than controls. Immunohistochemistry showed that axonal markers commonly used for rat nerves studies are effective also for mouse nerves. Similar to the rat, the mouse median nerve model is superior to sciatic nerve model for the minimal impact on animal well-being and the effectiveness of the grasping test for motor function evaluation. The main limitation is the small nerve size which requires advanced microsurgical skills for performing 12/0 epineurial suturing.
Abstract: Proteins essential for vesicle formation by the Coat Protein I (COPI) complex are being identified, but less is known about the role of specific lipids. Brefeldin-A ADP-ribosylated substrate (BARS) functions in the fission step of COPI vesicle formation. Here, we show that BARS induces membrane curvature in cooperation with phosphatidic acid. This finding has allowed us to further delineate COPI vesicle fission into two sub-stages: 1) an earlier stage of bud-neck constriction, in which BARS and other COPI components are required, and 2) a later stage of bud-neck scission, in which phosphatidic acid generated by phospholipase D2 (PLD2) is also required. Moreover, in contrast to the disruption of the Golgi seen on perturbing the core COPI components (such as coatomer), inhibition of PLD2 causes milder disruptions, suggesting that such COPI components have additional roles in maintaining Golgi structure other than through COPI vesicle formation.
Abstract: Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G(q) stimulation by the thromboxane A(2) analogue U46619. Importantly, activation of phospholipase C and intracellular Ca(2+) mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G(i)-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.
Abstract: BACKGROUND/AIMS: Glycine hepatoprotection is well known. However, the mechanisms involved are still poorly characterized. METHODS: Glycine protection was investigated in isolated rat hepatocytes pretreated with 2 mmol/L glycine 15 min before incubation under hypoxic conditions. RESULTS: Glycine significantly reduced Na+ overload and hepatocyte death caused by hypoxia. Glycine protection required the activation of a signal pathway involving Src, Pyk2 and p38 MAP kinases. Glycine treatment also induced a 11% increase of hepatocyte volume and transient ATP release. The prevention of cell swelling by hepatocyte incubation in a hypertonic medium as well as the degradation of extracellular ATP with apyrase or the block P2 purinergic receptors with suramin reverted glycine-induced cytoprotection and inhibited Src, Pyk2 and p38 MAPK activation. Glycine down-modulated Na+/H+ exchanger (NHE) activity, without affecting the development of intracellular acidosis during hypoxia. Such an effect was reverted by inhibiting p38 MAPK that also abolished glycine protection against Na+ overload caused by hypoxia. CONCLUSIONS: Glycine-induced ATP release in response to a moderate hepatocyte swelling led to the autocrine stimulation of P2 receptors and to the activation of Src, Pyk2 and p38 MAPK that increased hepatocyte resistance to hypoxia by preventing Na+ influx through NHE.
Abstract: Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.
Abstract: BACKGROUND: Estrogen receptor (ER)-negative breast cancers have a worse prognosis than ER-positive cancers, being more aggressive and overexposed to stimuli leading to their progression. Hepatocyte growth factor (HGF) has been associated with proliferation, migration and invasion of tumor cells, and several tumors, including those of breast cancer, produce HGF and overexpress its receptor. Diacylglycerol kinases (Dgks), which phosphorylate diacylglycerol to phosphatidic acid, are key regulators of cell signaling. Our research was focused on their role in HGF-induced invasion of MDA-MB-231 cells, a model of ER-negative breast cancer. MATERIALS AND METHODS: Dgk activity was evaluated with a kinase assay, MDA-MB-231 cell invasion via culturing of cells in matrigel-coated transwells, and anchorage-independent growth was assessed using a soft agar assay. RESULTS: HGF induces Dgk activation in MDA-MB-231 cells that is required for cell invasiveness. Moreover, Dgks are involved in MDA-MB-231 anchorage-independent growth. CONCLUSION: Dgks could be a target for ER-negative breast cancer therapy.
Abstract: Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkalpha in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkalpha is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkalpha, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkalpha, obtained either pharmacologically by R59949 treatment, or by expression of Dgkalpha dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkalpha, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkalpha, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.
Abstract: We recently showed that Hepatocyte Growth Factor (HGF), known as a survival factor, unexpectedly enhances apoptosis in human ovarian cancer cells treated with the front-line chemotherapeutics cisplatin (CDDP) and paclitaxel (PTX). Here we demonstrate that this effect depends on the p38 mitogen-activated kinase (MAPK). In fact, p38 MAPK activity is stimulated by HGF and further increased by the combined treatment with HGF and either CDDP or PTX. The expression of a dominant negative form of p38 MAPK abrogates apoptosis elicited by drugs, alone or in combination with HGF. HGF and drugs also activate the ERK1/2 MAPKs, the PI3K/AKT and the AKT substrate mTOR. However, activation of these survival pathways does not hinder the ability of HGF to enhance drug-dependent apoptosis. Altogether data show that p38 MAPK is necessary for HGF sensitization of ovarian cancer cells to low-doses of CDDP and PTX and might be sufficient to overcome activation of survival pathways. Therefore, the p38 MAPK pathway might be a suitable target to improve response to conventional chemotherapy in human ovarian cancer.
Abstract: The impact of estrogens on the cardiovascular system and their ability to regulate platelet function are matters of controversy. The recent finding that estrogen receptors are expressed in human platelets renders these cells an excellent model for studying the nongenomic effects of these hormones. In this work, we investigated 17beta-estradiol-dependent signaling in platelets from adult healthy men. 17beta-estradiol caused the rapid phosphorylation of the tyrosine kinases Src and Pyk2 and the formation of a signaling complex, which included Src, Pyk2, and the phosphatidylinositol 3-kinase. Both these events were dependent on estrogen receptor beta engagement. We found that estrogen receptor beta was membrane-associated in platelets. On treatment with 17beta-estradiol, Src and Pyk2 activation occurred in the membrane fraction but not in the cytosol. In contrast, no significant activation of phosphatidylinositol 3-kinase was detected in estrogen-treated platelets. 17beta-estradiol did not induce any platelet response directly, but it strongly potentiated the activation of integrin alpha(IIb)beta3 and the platelet aggregation induced by subthreshold concentrations of thrombin. These effects were dependent on estrogen receptor beta recruitment and were associated with a strong synergistic effect with thrombin on Src activation. Taken together, these results indicate that 17beta-estradiol can modulate platelet function by exercising a proaggregating role.
Abstract: Oncogenic rearrangements of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK), most commonly represented by the nucleophosmin/ALK fusion protein (NPM/ALK), are involved in the pathogenesis of anaplastic large-cell lymphomas (ALCLs). In an effort to identify new intracellular transducers operative in ALK-positive malignancies, we have investigated the potential involvement of diacylglycerol kinase (DGK). Here we show that alphaDGK is constitutively activated in the NPM/ALK-positive ALCL-derived cell line Karpas 299 and in NPM/ALK-infected 32D hematopoietic cells. These results were further validated in fibroblastic NIH-3T3 cells expressing a previously described chimeric epidermal growth factor receptor (EGFR)/ALK molecule that allows dissection of ALK enzymatic function under conditions of controlled ligand-induced activation. In this cell system, we also show that ALK-mediated alphaDGK activation is dependent on p60src tyrosine kinase, with which alphaDGK forms a complex. The specific inhibition of alphaDGK, obtained by cell treatment with R59949, significantly reduced cellular growth in all cell lines. This result was further confirmed in Karpas 299 cells following specific down-regulation of alphaDGK by RNA interference. Overall, our data indicate that alphaDGK activation is involved in the control of ALK-mediated mitogenic properties.
Abstract: Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-alpha (Dgk-alpha) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-alpha is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-alpha: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-alpha forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-alpha, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-alpha dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-alpha by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-alpha in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-alpha generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.
Abstract: BACKGROUND & AIMS: Ischemic preconditioning has been proved effective in reducing ischemia/reperfusion injury during liver surgery. However, the mechanisms involved are still poorly understood. Here, we have investigated the role of phosphatidylinositol 3-kinase (PI3K) in the signal pathway leading to hepatic preconditioning. METHODS: PI3K activation was evaluated in isolated rat hepatocytes preconditioned by 10-minute hypoxia followed by 10-minute reoxygenation. RESULTS: Hypoxic preconditioning stimulated phosphatidylinositol-3,4,5-triphosphate production and the phosphorylation of PKB/Akt, a downstream target of PI3K. Conversely, PI3K inhibition by wortmannin or LY294002 abolished hepatocyte tolerance against hypoxic damage induced by preconditioning. PI3K activation in preconditioned hepatocytes required the stimulation of adenosine A 2A receptors and was mimicked by adenosine A 2A receptors agonist CGS21680. In the cells treated with CGS21680, PI3K activation was prevented either by inhibiting adenylate cyclase and PKA with, respectively, 2,5-dideoxyadenosine and H89 or by blocking Galphai-protein and Src tyrosine kinase with, respectively, pertussis toxin and PP2. H89 also abolished the phosphorylation of adenosine A 2A receptors. However, the direct PKA activation by forskolin failed to stimulate PI3K. This suggested that PKA-phosphorylated adenosine A 2A receptors may activate PI3K by coupling it with Galphai-protein through Src. We also observed that, by impairing PI3K-mediated activation of phospholypase Cgamma (PLCgamma), wortmannin and LY294002 blocked the downstream transduction of preconditioning signals via protein kinase C (PKC) delta/ isozymes. CONCLUSIONS: PI3K is activated following hepatocyte hypoxic preconditioning by the combined stimulation of adenosine A 2A receptors, PKA, Galphai protein, and Src. By regulating PKC-/delta-dependent signals, PI3K can play a key role in the development of hepatic tolerance to hypoxia/reperfusion.
Abstract: Insulin-like growth factor binding protein 3 (IGFBP-3) modulates the activity of IGF-I, which exerts antiapoptotic action upon the myocardiocyte. IGFBP-3 also exerts IGF-independent actions to inhibit cell growth and induce apoptosis, mediating the effects of several antiproliferative agents. We hypothesized that IGFBP-3 mediates cardiomyocyte apoptosis. IGFBP-3 expression was studied in H9c2 rat cardiac cells cultured in serum-deprived medium in the absence or presence of 1 microM doxorubicin during a 72 h time-span. To a greater degree than serum withdrawal, doxorubicin induced IGFBP-3 up-regulation that was time-dependent. IGFBP-3 mRNA levels positively correlated with the degree of apoptosis. Exogenous IGFBP-3 decreased cell viability and induced apoptosis in serum-starved cells exposed to doxorubicin. IGFBP-3 antisense oligonucleotides markedly decreased apoptosis induced by either serum withdrawal or doxorubicin. Binding studies revealed specific high-affinity sites for IGFBP-3 in H9c2 cardiomyocytes, with binding characteristics typical of receptor-ligand interactions. These findings indicate that IGFBP-3 could play proapoptotic action at the myocardial level and suggest a novel role for this protein in cardiovascular dysfunction.
Abstract: Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein-coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal-regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.
Abstract: Despite oxytocin receptors (OTR) being present in human chorio-decidual tissues, their expression and role in placental trophoblast cells in the context of tumor growth or physiological functions related to cell proliferation have never been examined. In the present study we demonstrate the presence and functionality of OTR in normal human trophoblast cell lines (ED77 and ED27) and a choriocarcinoma cell line (BeWo). RT-PCR and immunofluorescence analysis revealed the presence of OTR messenger RNA and protein in these cells. Binding studies using [(125)I]oxytocin ([(125)I]OT) antagonist confirmed the presence of specific binding sites in ED27, ED77, and BeWo cells. OTR functionality was demonstrated by measuring the OT-induced increase in the intracellular calcium concentrations. This effect was dose dependent and was blocked by the selective OT antagonist d(CH(2))(5)[Tyr(Me)(2),Thr(4), Tyr-NH(2)(9)]OVT (OT antagonist). Furthermore, two proteins with apparent molecular masses of 125 and 60 kDa became tyrosine phosphorylated in all of the cell lines after OT stimulation (and an additional protein of 45 kDa in BeWo choriocarcinoma cells), suggesting that this peptide can stimulate tyrosine kinase activity. Finally, we observed a dose-dependent OT stimulation of cell proliferation associated with OTR activation that was completely abolished by the selective OT antagonist. These findings provide the first evidence of the presence of functional OTR in normal trophoblast cell lines as well as in choriocarcinoma cells and show that a specific effect of OT on normal and neoplastic trophoblast is to promote cellular proliferation.
Abstract: The family of GH secretagogues (GHS) includes synthetic peptidyl (hexarelin) and nonpeptidyl (MK-0677) molecules possessing specific receptors in the pituitary and central nervous system as well as in peripheral tissues, including the heart and some endocrine organs. A gastric-derived peptide, named ghrelin, has recently been proposed as the natural ligand of the GHS receptors (GHS-Rs). The presence of specific GHS-Rs has now been investigated in nontumoral and neoplastic human breast tissue using a radioiodinated peptidyl GHS ([(125)I]-Tyr-Ala-hexarelin) as ligand. Specific binding sites for GHS were detected in membranes from several types of breast carcinomas, whereas a negligible binding was found in fibroadenomas and mammary parenchyma. The highest binding activity was found in well-differentiated (G1) invasive breast carcinomas and was progressively reduced in moderately (G2) to poorly (G3) differentiated tumors. [(125)I]-Tyr-Ala-hexarelin bound to tumor membranes was displaced by different unlabeled GHS such as hexarelin, Tyr-Ala-hexarelin, human ghrelin, and MK-0677 as well as by desoctanoyl-ghrelin and hexarelin derivative EP-80317, which are devoid of GH-releasing properties in vivo. In contrast, no competition was seen between radiolabeled Tyr-Ala-hexarelin and some peptides (CRF and insulin-like growth factor I) structurally and functionally unrelated to hexarelin or when GHRH and SRIF were tested in the displacement studies. The presence of specific GHS binding sites was also demonstrated in three different human breast carcinoma cell lines (MCF7, T47D, and MDA-MB231), in which, surprisingly, no messenger RNA for GHS-R1a was demonstrated by RT-PCR. In these cell lines, ghrelin (as well as hexarelin, MK-0677, EP-80317, and even desoctanoyl ghrelin) caused a significant inhibition of cell proliferation at concentrations close to their binding affinity. In conclusion, this study provides the first demonstration of specific GHS binding sites, other than GHS-R1, in breast cancer. These receptors probably mediate growth inhibitory effects on breast carcinoma cells in vitro.
Abstract: Growth hormone secretagogues (GHSs) are synthetic peptidyl and nonpeptidyl molecules with strong, dose-dependent, and reproducible growth hormone (GH)-releasing activity even after oral administration. GHSs release GH via actions on specific receptors (GHS-R) at the pituitary and, mainly, at the hypothalamic levels. GHSs likely act as functional somatostatin antagonists and meantime enhance the activity of GH-releasing hormone (GHRH)-secreting neurons. The GH-releasing effect of GHSs is independent of gender but undergoes marked age-related variations. Estrogens play a major role in enhancing the GH response to GHSs at puberty, which GHRH hypoactivity, somatostatinergic hyperactivity and impaired activity of the putative GHS-like ligand and receptors probably explain the reduced GH-releasing effect of GHSs in aging. The activity of GHSs is not fully specific for GH. Their slight prolactin-releasing activity probably comes from direct pituitary action. In physiological conditions, the ACTH-releasing activity of GHSs is dependent on central actions; a direct action on GHS-R in pituitary ACTH-secreting tumors likely explains the peculiar ACTH and cortisol hyperresponsiveness to GHSs in Cushing disease. GHSs have specific receptor subtypes in other central and peripheral endocrine and nonendocrine tissues mediating GH-independent biologic activities. GHSs influence sleep pattern, stimulate food intake, and have cardiovascular activities. GHs have specific binding in normal and neoplastic follicular derived human thyroid tissue and inhibit the proliferation of follicular-derived neoplastic cell lines. The discovery of ghrelin, a 28 amino acid peptide synthesized in the stomach but also in other tissues, has opened new fascinating perspectives of research in this field.
Abstract: Growth hormone secretagogues (GHSs) are synthetic peptidyl and nonpeptidyl molecules that possess strong growth hormone-releasing activity acting on specific pituitary and hypothalamic receptor subtypes. Differently from nonpeptidyl GHSs, peptidyl molecules such as hexarelin, a hexapeptide, possess specific high-affinity binding sites in animal and human heart and, after prolonged treatment, protect rats in vivo from ischemia-induced myocardial damage. To verify the hypothesis that peptidyl GHSs protect heart cells from cell death, we have investigated the cellular effects of hexarelin on H9c2 cardiomyocytes, a fetal cardiomyocyte-derived cell line, and on Hend, an endothelial cell line derived from transformed murine heart endothelium. We show that (i)H9c2 cardiomyocytes show specific binding for 125I-Tyr-Ala-hexarelin, which is inhibited by peptidyl GHSs such as Tyr-Ala-hexarelin and hexarelin but not by the nonpeptidyl GHS MK-0677, (ii) hexarelin promotes survival of H9c2 cardiomyocytes induced to die by doxorubicin, and (iii) that hexarelin inhibits apoptosis, as measured by DNA fragmentation, induced in both H9c2 myocytes and endothelial cells. In conclusion, our findings show that peptidyl GHSs such as hexarelin act as survival factors for cardiomyocytes and endothelium-derived cells in culture. These findings suggest that the inhibitory activity of hexarelin on cardiomyocytes and endothelial cell death could explain, at least partially, its cardioprotective effect against ischemia recorded in rats in vivo.
Abstract: Growth hormone-releasing hormone (GHRH) and somatostatin are the most important hypothalamic neurohormones controlling growth hormone (GH) secretion. Several neurotransmitters and neuropeptides also play an important role in the control of GH secretion, mainly acting via modulation of GHRH and somatostatin. In the past two decades, particular attention has been given to a new family of substances showing a strong GH-releasing effect: GH secretagogues (GHSs). GHSs increase GH secretion in a dose- and age-related manner after iv and even oral administration. The endocrine effects of GHSs, are not fully specific for GH; they show, in fact, prolactin- (PRL), adenocorticotropic hormone- and cortisol-releasing effects. Specific GHS receptors are present in both the central nervous system and peripheral tissues, where they mediate several extraendocrine effects of GHSs. The isolation of these "orphan" receptors suggested the existence of an endogenous GHS-like ligand that could be represented by a recently discovered gastric peptide, named ghrelin. The interaction between GHSs and GHRH at the central level and in the pituitary gland, but not at peripheral level, has clearly been shown. Because GHRH and GHS receptors share the same localization in some peripheral tissues, they may have some interactions even at this level.
Abstract: Growth Hormone (GH)-releasing peptides (GHRPs) and their non peptidyl analogues are synthetic molecules which exhibit strong, dosedependent and reproducible GH-releasing activity but also significant PRL- and ACTH/cortisol-releasing effects. An influence of these compounds on food intake and sleep pattern has been also shown. The neuroendocrine activities of GHRPs are mediated by specific receptors subtypes that have been identified in the pituitary gland, hypothalamus and various extra-hypothalamic brain regions with (125)I-Tyr-Ala-hexarelin, an octapeptide of the GHRP family. In addition, GHRP receptors were also present in different peripheral tissues such as heart, adrenal, ovary, testis, lung and skeletal muscle, with a density significantly higher than that found in the hypothalamo-pituitary -system. A remarkable specific (125)I-Tyr-Ala-hexarelin binding was observed in the human cardiovascular system where the highest binding levels were detected in ventricles, followed by atria, aorta, coronaries, carotid, endocardium and vena cava. The binding of the radioligand to cardiac membranes was inhibited by unlabeled Tyr Ala hexare lin and hexarelin as well as by GHRP-6, GHRP-1 and GHRP-2 but not by MK-677, a non peptidyl GHRP analog. In other experiments on H9c2 myocytes, a fetal cardiomyocytes-derived cell line, specific GHRP binding was found and hexarelin showed an anti-apoptotic activity. On the other hand, in vivo studies in animals and in humans showed that GHRPs possess direct cardiotropic actions. In fact, hexarelin protects from ischemia-induced myocardial damage in aged and GH deficient rats while hexarelin shows a positive inotropic effect in normal subjects as well as in patients with GH deficiency. In conclusion, GHRPs possess extra--neuroendocrine biological activity and, particularly, show direct GH-independent cardiotropic effects.
Abstract: Diacylglycerol kinases are involved in cell signaling, either as regulators of diacylglycerol levels or as intracellular signal-generating enzymes. However, neither their role in signal transduction nor their biochemical regulation has been elucidated. Hepatocyte growth factor (HGF), upon binding to its tyrosine kinase receptor, activates multiple signaling pathways stimulating cell motility, scattering, proliferation and branching morphogenesis. Herein we demonstrate that: (i) the enzymatic activity of alpha-diacylglycerol kinase (alphaDgk) is stimulated by HGF in epithelial, endothelial and alphaDgk-transfected COS cells; (ii) cellular expression of an alphaDgk kinase-defective mutant inhibits activation of endogenous alphaDgk acting as dominant negative; (iii) specific inhibition of alphaDgk prevents HGF-induced cell movement of endothelial cells; (iv) HGF induces the association of alphaDgk in a complex with Src, whose tyrosine kinase activity is required for alphaDgk activation by HGF; (v) Src wild type stimulates alphaDgk activity in vitro; and (vi) alphaDgk can be tyrosine phosphorylated in intact cells.
Abstract: Growth hormone (GH) secretagogues (GHS) are synthetic peptidyl and non-peptidyl molecules which possess strong, dose-dependent and reproducible GH releasing effects as well as significant prolactin (PRL) and adrenocorticotropic hormone (ACTH) releasing effects. The neuroendocrine activities of GHS are mediated by specific receptors mainly present at the pituitary and hypothalamic level but also elsewhere in the central nervous system. GHS release GH via actions at the pituitary and (mainly) the hypothalamic level, probably acting on GH releasing hormone (GHRH) secreting neurons and/or as functional somatostatin antagonists. GHS release more GH than GHRH and the coadministration of these peptides has a synergistic effect but these effects need the integrity of the hypothalamo-pituitary unit. The GH releasing effect of GHS is generally gender-independent and undergoes marked age-related variations reflecting age-related changes in the neural control of anterior pituitary function. The PRL releasing activity of GHS probably comes from direct pituitary action, which indeed is slight and independent of both age and gender. The acute stimulatory effect of GHS on ACTH/cortisol secretion is similar to that of corticotropin releasing hormone (CRH) and arginine vasopressin (AVP). In physiological conditions, the ACTH releasing activity of GHS is mediated by central mechanisms, at least partially, independent of both CRH and AVP but probably involving GABAergic mechanisms. The ACTH releasing activity of GHS is gender-independent and undergoes peculiar age-related variations showing a trend towards increase in ageing. GHS possess specific receptors also at the peripheral levels in endocrine and non-endocrine human tissues. Cardiac receptors are specific for peptidyl GHS and probably mediate GH-independent cardiotropic activities both in animals and in humans.
Abstract: nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions.
Abstract: Polyoma middle T (PmT)-transformed endothelial cells may represent a unique murine model for human opportunistic vascular tumors. The present study was designed to evaluate the anti-tumor potential of a panel of 13 cytokines against murine PmT-transformed endothelial cells. Interferon gamma (IFNgamma) and transforming growth factor beta 1 (TGFbeta1) substantially decreased in a dose-dependent manner the proliferation of a panel of 6 PmT-transformed cell lines. IFNalpha and tumor necrosis factor alpha(TNFalpha) had marginal anti-proliferative activity, whereas other molecules (interleukins-1, -2, -4, -6 and -13, IFNbeta, leukemia inhibitory factor, oncostatin M, granulocyte-macrophage colony-stimulating factor) caused no growth inhibition. IFNgamma and TGFbeta1 were therefore selected for further analysis of their mechanism of action and in vivo relevance. IFNgamma and TGFbeta1 reduced the activity of phosphatidylinositol-3-kinase and the production of phosphatidylinositol 3,4-biphosphate, without modifying the tyrosine kinase(s) activity associated with PmT. IFNgamma and TGFbeta1 were also tested for their ability to modify the in vivo growth of the PmT-transformed endothelial cells H5V in syngeneic C57B1/6 mice. Treatment with IFNnu and TGFbeta1 significantly delayed tumor growth and increased survival time. In contrast, treatment with IFNalpha and TNFalpha failed to prolong survival. In nude mice, IFNgamma and TGFbeta1 had a transient effect on tumor growth but no effect on survival, suggesting a contribution of T cells to the in vivo anti-tumor activity of these cytokines.
Abstract: Signaling by tyrosine kinase receptors is mediated by selective interactions between individual Src homology 2 (SH2) domains of cytoplasmic effectors and specific phosphotyrosine residues in the activated receptor. Here, we report the existence in the hepatocyte growth factor/scatter factor (HGF/SF) receptor of a multifunctional docking site made of the tandemly arranged degenerate sequence YVH/NV. Phosphorylation of this site mediates intermediate- to high-affinity interactions with multiple SH2-containing signal transducers, including phosphatidylinositol 3-kinase, phospholipase C gamma, pp60c-src, and the GRB-2-Sos complex. Mutation of the two tyrosines results in loss of biological function, as shown by abrogation of the transforming activity in the oncogenic counterpart of the receptor. The same bidentate motif is conserved in the evolutionarily related receptors Sea and Ron, suggesting that in all members of the HGF/SF receptor family, signal transduction is channeled through a multifunctional binding site.
Abstract: Oncostatin M (OM) is a polypeptide cytokine that induces autocrine and paracrine effects on AIDS-Kaposi's sarcoma (KS) cells (Nair et al., Science, 255, 1430-1432, 1992; Miles et al., Science, 255, 1432-1434, 1992). The signalling pathways underlying this activation are largely unknown. We have found that OM binding to KS cell lines in vitro identifies a higher affinity binding site (Kd 10-20 pM) with a lower affinity (Kd 1.5 nM), high capacity binding site. The binding of OM to its receptor at the KS cell surface stimulates a rapid tyrosine phosphorylation of multiple proteins, including the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K). In addition OM can stimulate the in vivo activation of PI3K and increases the PI3K activity in anti-phosphotyrosine and anti-src kinase family antibody directed immunoprecipitates. Genistein, an inhibitor of tyrosine kinases, inhibits the synthesis of phosphatidylinositol 3,4-biphosphate and the growth of KS cells. Finally, OM enhances tyrosine kinase activity in immune complex kinase assay performed with antibody anti-src kinase family. These data suggest that in KS cells OM can stimulate formation of tyrosine kinase co-ordinate signalling complexes, containing at least src kinase family and PI3K, which can drive the accumulation of the putative second-messengers D3-phosphorylated phosphoinositides.
Abstract: Hepatocyte growth factor/scatter factor (HGF/SF) induces mitogenesis and cell dissociation upon binding to the protein-tyrosine kinase receptor encoded by the MET proto-oncogene (p190MET). The signal transduction pathways downstream from the receptor activation are largely unknown. We show that HGF/SF activates Ras protein. HGF/SF stimulation of metabolically labeled A549 cells raised the amount of Ras-bound radiolabeled guanine nucleotides by over 5-fold. Furthermore, following HGF/SF stimulation of these cells, 50% of Ras was in the GTP-bound active state. The uptake by Ras of radiolabeled GTP was also increased by 5-fold following HGF/SF stimulation in digitonin-permeabilized A549 cells. Moreover, HGF/SF treatment of A549 cells leads to stimulation of the cytosolic Ras-guanine nucleotide exchange activity, measured as accelerated release of [3H]GDP from purified recombinant Ras protein in vitro, in a dose- and time-dependent manner. Likewise, treatment with the protein-tyrosine kinase inhibitor 3-(1',4'-dihydroxytetralyl)methylene-2-oxindole of GTL-16 cells (featuring a p190MET receptor constitutively active) significantly decreased the cytosolic Ras-guanine nucleotide exchange activity. These data demonstrate that HGF/SF activates Ras protein by shifting the equilibrium toward the GTP-bound state and increases the uptake of guanine nucleotides by Ras, through mechanism(s) including the activation of a Ras-guanine nucleotide exchanger.
Abstract: PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.
Abstract: A monoclonal antibody has been developed against the type II PtdIns 4-kinase from bovine brain. This antibody, 4C5G, causes greater than 90% inhibition of the type II PtdIns 4-kinase from bovine brain, rat brain and human erythrocytes. However, it fails to inhibit type III PtdIns 4-kinase from bovine brain or PtdIns 3-kinase from rat liver. These results suggest that type II and type III PtdIns 4-kinases are distinct gene products, and that 4C5G will be useful in studying the function of the type II PtdIns 4-kinase.
Abstract: The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine kinase encoded by the MET proto-oncogene (p190MET). The signaling pathway(s) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190MET receptor. The autophosphorylated receptor coprecipitated with phosphatidylinositol 3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190MET receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3-kinase activity and with the 85-kDa PI 3-kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca2+] inhibits tyrosine phosphorylation of the p190MET receptor as well as the association with both PI 3-kinase activity and the 85-kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190MET receptor was required for binding of PI 3-kinase from cell lysates. These data strongly suggest that the signaling pathway activated by the HGF/SF receptor includes generation of D-3-phosphorylated inositol phospholipids.
Abstract: Three polyphosphoinositides containing phosphate at the D-3 position of the inositol ring can be generated in vitro by phosphorylation of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate by a phosphatidylinositol-3-kinase (Auger, K. R., Serunian L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175. An alternative pathway for in vivo synthesis of one of these lipids was recently suggested: phosphatidylinositol 3,4-bisphosphate could be produced by phosphorylation of phosphatidylinositol 3-phosphate at the D-4 position of the inositol ring (Yamamoto, K., and Lapetina, E. G. (1990) Biochem. Biophys. Res. Commun. 168, 466-472). Here we demonstrate the presence of an enzyme in human platelets that phosphorylates [32P]phosphatidylinositol 3-phosphate to produce [32P]phosphatidylinositol 3,4-bisphosphate. This enzyme is Mg2(+)-dependent and its apparent molecular mass is approximately 150 kDa as estimated by sucrose gradient centrifugation and gel filtration chromatography. Unlike the major phosphatidylinositol-4-kinase in platelets that is stimulated by the detergent Nonidet P-40, the phosphatidylinositol-3-phosphate-4-kinase is inhibited by Nonidet P-40. Both activities are also differentiated by the action of adenosine. The discovery of this new enzyme raises the possibility that multiple pathways exists for generating D-3 phosphorylated phosphoinositides.
Abstract: Male C57Bl/10 mice were chronically fed hexachlorobenzene (HCB) (0.02% of the diet) alone or in combination with a single subcutaneous dose of iron (12.5 mg iron per mouse). After eight weeks the group of mice pretreated with the iron overload was highly sensitized to the porphyrogenic effect of HCB, as shown by liver porphyrin accumulation. A synergistic effect of iron was evident on other parameters too, such as HCB-induced hepatic damage, activation of type O of xanthine oxidase, and decreased activity of copper zinc superoxide dismutase and glutathione peroxidase(s). None of these parameters was affected by iron alone. Iron alone and in association with HCB markedly raised the level of lipid peroxides, the increase in the HCB group being smaller. The combined treatment resulted in a significant reduction of HCB's inductive effects on microsomal heme and cytochromes P-450 and b5 and on the activity of aryl hydrocarbon hydroxylase. The content of nonprotein sulfhydryl groups was reduced to the same extent in mice treated with HCB or HCB plus iron. The results suggest that reactive intermediates such as are formed by lipid peroxidation are not sufficient on their own to create the conditions for uroporphyrinogen decarboxylase impairment, as evident in the group of mice receiving iron overload alone. Conversely, HCB administration induced a specific condition of imbalance in the liver between formation and inactivation of reactive intermediates which was associated with hepatic porphyrin accumulation and was potentiated by concomitant administration of iron.
Abstract: Experimental porphyria induced by PHAHs is characterized by a progressive reduction in the activity of UROD. After intoxication with TCDD, the most porphyrogenic compound known to date, the liver was the principal site of action, as regards both porphyrin accumulation (mostly uroporphyrin) and the degree of enzyme impairment; the kidney was the site of the second greatest accumulation; the brain and erythrocytes were unaffected. Additional modifications of the heme pathway involved induction of the activity of ALAS and, at least in HCB-induced porphyria after iron pretreatment, may have involved reduced activity of uroporphyrinogen III cosynthetase. These changes can alter the amount and the isomeric composition of uroporphyrinogens and uroporphyrins present in the liver in a way that is likely to help reduce formation of coproporphyrinogen III in porphyric animals. As in the human syndrome porphyria cutanea tarda, iron administration increased porphyrin accumulation and the degree of reduction of UROD activity in mice fed HCB. Mice fed HCB also presented an activation of the type O form of XO. This activation was independent of tissue injury derived from the lipid peroxidation that was concomitant with iron administration. The increase in activity of the type O form of XO may be a characteristic feature of the liver damage found in PHAH intoxication and, in intoxicated animals, could be a source in the liver of oxidant species involved in the mechanism of UROD inactivation--if this inactivation is in fact due to an oxidative reaction.
Abstract: The effect of hexachlorobenzene (HCB) on microsomal cytochromes P-450 and b5, monooxygenase activity and membrane composition was examined in male and female Fischer rats. Cytochrome P-450 was induced more in male than in female animals while cytochrome b5 was induced only in males. Analysis of patterns of induction of microsomal monooxygenases showed that aminopyrine-N-demethylase activity doubled in both sexes after treatment while aryl hydrocarbon hydroxylase activity was 16 times the control value in the females and 1.5 times in the males. After HCB treatment the phospholipid content of microsomal membranes per gram of liver was increased in both sexes while cholesterol was unchanged. Analysis of the phospholipids (PL) pattern showed that the percentage of sphingomyelin (SPH) decreased significantly (50% of the control value) while phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) did not change. These changes resulted in a reduction of membrane microviscosity and indicate that HCB interferes with the biosynthesis of phospholipids containing choline. Free fatty acid (FFA) content also dropped in both sexes but females were more affected; free arachidonic acid rose in females. HCB induction of microsomal cytochromes and monooxygenases is thus accompanied by marked modifications of membrane composition. Comparing the 2 sexes, HCB showed more pronounced features of 'PB type' inducers in males.
Abstract: Uroporphyrinogen decarboxylase from control mouse liver was markedly inhibited in vitro by addition to the incubation mixture of cytosol fractions from livers of porphyric mice. The animals had been subjected to chronic treatment with 2,3,7,8-tetrachlorodibenzo-para-dioxin (25 micrograms/kg per week, intraperitoneally). The cytosol fractions were deproteinized by heating at 100 degrees C for 5 min and freed of porphyrins by treating the supernatants with Zerolit FF (i.p.) resin. Inhibition was proportional to the amount of fraction added and was not observed if the inhibitor fraction was dialysed before incubation. Increasing the substrate concentration did not reverse enzyme inhibition; the Vmax value calculated for the control enzyme in the presence of the inhibitor fraction was decreased, but Km did not vary. Two alternative hypotheses on the nature of the inhibitor compound(s) in the inhibitor fraction are discussed with relation to hexachlorobenzene (HCB) and 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) porphyria.
Abstract: We investigated the effects of ethinyl estradiol (5 mg/kg body wt daily for 5 days, orally) and/or iron sorbitol (50 mg/kg body wt daily for 5 days, i.m.) on bile flow, bile salt independent fraction (BSIF), hepatic delta-aminolevulinate synthase (ALA-S) and uroporphyrinogen decarboxylase (URO-D) in female rats. Ethinyl estradiol administration was associated with a significant decrease of bile flow and BSIF and an increase in URO-D activity in comparison to control values. Iron alone did not modify biliary parameters, but significantly increased the activity of ALA-S. Combined treatment with ethinyl estradiol plus iron partially corrected the reduction of BSIF and restored the activity of ALA-S and URO-D to control levels. Thus iron appears to exert a partially protective effect against ethinyl estradiol-induced cholestasis. No porphyrinogenic effect was observed.
