Abstract: Integrins regulate adhesive cell-matrix interactions and mediate survival signals. On the other hand, unligated or free cytoplasmic fragments of integrins induce apoptosis in many cell types (integrin-mediated death). We have previously shown that beta(1) integrin expression protects keratinocyte stem cells from anoikis, whereas the role of the beta(1)B integrin isoform has not been clarified. In this study we report that suspended keratinocytes undergo apoptosis through the activation of caspase-8, independently of the Fas/Fas ligand system. Indeed, anti-beta(1) integrin-neutralizing antibodies induced apoptosis in short hairpin RNA Fas-associated death domain-treated cells. Moreover, before and during suspension, caspase-8 directly associated with beta(1) integrin, which in turn internalized and progressively degraded, shedding the cytoplasmic domain. beta(1)B was expressed only in the cytoplasm in a perinuclear manner and remained unaltered during suspension. At 24 hours, as beta(1)A was located close to the nucleus, beta(1)B colocalized with beta(1)A and coimmunoprecipitated with caspase-8. Caspase-8 was activated earlier in beta(1)B integrin-transfected keratinocytes, and these cells underwent a higher rate of apoptosis than mock cells. In contrast, caspase-8 was not activated in small interfering RNA (siRNA) beta(1)B-transfected cells. These results indicate that when beta(1)A is unligated, beta(1)B is responsible for "integrin-mediated death" in human keratinocytes.Journal of Investigative Dermatology advance online publication, 8 July 2010; doi:10.1038/jid.2010.195.
Abstract: Skin and its appendages provide a protective barrier against the assaults of the environment. To perform its role, epidermis undergoes an ongoing renewal through a balance of proliferation and differentiation/apoptosis called homeostasis. Keratinocyte stem cells reside in a special microenvironment called niche in basal epidermis, adult hair follicle, and sebaceous glands. While a definite marker has yet to be detected, data raised part in humans and part in the mouse system point to a critical role of stem and its progeny transit amplifying cells in epidermal homeostasis. Stem cells are protected from apoptosis and are long resident in adult epidermis. This renders them more prone to be the origin of skin cancer. In this review, we will outline the main features of adult stem cells in mouse and humans and discuss their fate in relation to differentiation, apoptosis, and cancer.
Abstract: Neurotrophins (NTs) belong to a family of structurally and functionally related proteins that, depending on the tissue context and the receptors involved, promote neuronal cell survival and differentiation or cell death. NTs also exert important functions in other organs besides the nervous system, including the skin. The presence in the skin of diverse cell types which are able to secrete and/or to respond to stimulation by NTs creates a unique network of molecular signaling in the cutaneous microenvironment. This review summarizes currently available data on the expression and function of NTs and their receptors in several cell types in the skin (namely, keratinocytes, melanocytes and fibroblasts). The role of the skin NT network in the development and maintenance of some relevant skin diseases is presented and the potential implications for therapeutic intervention are discussed.
Abstract: Understanding the acantholytic pathways leading to blistering in pemphigus vulgaris (PV) is a key to development of novel treatments. A novel paradigm of keratinocyte damage in PV, termed apoptolysis, links the suprabasal acantholytic and cell death pathways to basal cell shrinkage rendering a 'tombstone' appearance to PV lesions. In contrast to apoptolysis, the classic keratinocyte apoptosis mediating toxic epidermal necrolysis causes death and subsequent sloughing of the entire epidermis. Apoptolysis includes five consecutive steps. (1) Binding of autoantibodies to PV antigens. (2) Activation of EGF receptor, Src, mTOR, p38 MAPK and other signalling elements downstream of ligated antigens, elevation of intracellular calcium and launching of the cell death cascades. (3) Basal cell shrinkage due to: (i) collapse and retraction of the tonofilaments cleaved by executioner caspases; and (ii) dissociation of interdesmosomal adhesion complexes caused by phosphorylation of adhesion molecules. (4) Massive cleavage of cellular proteins by activated cell death enzymes leading to cell collapse, and tearing off desmosomes from the cell membrane stimulating secondary autoantibody production. (5) Rounding up and death of acantholytic cells. Thus, the structural damage (acantholysis) and death (apoptosis) of keratinocytes are mediated by the same cell death enzymes. Appreciation of the unifying concept of apoptolysis have several important implications: (i) linking together a number of seemingly unrelated events surrounding acantholysis; (ii) opening new avenues of investigation into the pathomechanism of pemphigus; and (iii) creating new approaches to the treatment of pemphigus based on blocking the signalling pathways and enzymatic processes that lead to blistering.
Abstract: A significant proportion of patients with skin disease do not respond to treatment and adverse drug reactions are a common problem. Genetic factors are important determinants for both drug efficacy and toxicity. The fields of pharmacogenetics and pharmacogenomics examine inter-individual variations in the DNA sequence that are related to drug efficacy and toxicity. Here, we present pharmacogenomic data relevant to dermatology and explore the role of dermatologists in identifying patients who may respond to treatment or experience adverse drug reactions.
Abstract: Psoriasis is a multigenic disease with a number of susceptibility loci on different chromosomes predisposing to the disease, which is in turn triggered by environmental factors. The pathogenesis of psoriasis is characterized by 2 main components, the dysfunctions of the immune system and the alteration of keratinocyte homeostasis. While the Th1 T cell response has long been considered the sole immune agent in the pathomechanisms of psoriasis, recently, the role of IL-23-driven Th17 has been shown to be predominant. Subsequently, only effector memory T cells expressing alpha1beta1 integrin migrate into the epidermis, and psoriasis development is inhibited by blocking this integrin. Psoriatic epidermis contains high amounts of antimicrobial peptides, which have been shown to be a key mediator of plasmocytoid dendritic cell activation in psoriasis. The keratinocyte component has recently regained the central stage, as the abrogation of JunB/activator protein 1 in mouse keratinocytes induces development of psoriasiform lesions in T cell deficient mice. Moreover, inhibition of nerve growth factor and its receptor in keratinocytes strikingly improves psoriatic lesions.
Abstract: Survivin is a bi-functional member of inhibitor of apoptosis protein family, as it is able to both inhibit apoptosis and to regulate cell cycle. We investigated the role of survivin in human keratinocytes under normal conditions and during UVB irradiation. Survivin siRNA decreases proliferation and induces apoptosis in human keratinocytes, in a mode consistent with the mitotic catastrophe. Low doses UVB increase survivin expression at earlier times, while high doses down-regulate survivin level. Low doses UVB induce cell cycle arrest in G2/M, while high doses UVB cause apoptosis. Moreover, overexpression of survivin protects keratinocytes from UVB-induced apoptosis, and silencing of survivin renders keratinocytes more susceptible to UVB-induced cell death. Finally, survivin siRNA increases UVB-induced reduction of cell proliferation. Taken together, these results indicate that survivin plays a critical role in epidermal homeostasis in normal conditions and during UVB exposure, with possible implication in skin carcinogenesis.
Abstract: Melanoma is a highly aggressive skin tumor that originates in the epidermis from melanocytes. As melanocytes share with the nervous system a common neuroectodermal origin and express all neurotrophins (NTs), we evaluated the expression and function of NTs and their receptors in melanoma. We report that primary and metastatic melanoma cell lines synthesize and secrete all NTs. Moreover, melanoma cells express the low-affinity (p75NTR) and the high-affinity tyrosine kinase NT receptors (Trk). The inhibition of Trk receptors by either K252a or Trk/Fc chimeras prevents proliferation, indicating that autocrine NTs are responsible for this effect. NT-3, NT-4, and nerve growth factor (NGF) induce cell migration, with a stronger effect on metastatic cell lines. Transfection with p75NTR small interfering RNA (p75NTRsiRNA) or treatment with K252a inhibits NT-induced melanoma cell migration, indicating that both the low- and high-affinity NT receptors mediate this effect. All melanoma cell lines express the p75NTR coreceptor sortilin by which proNGF stimulates migration in melanoma cells, but not in cells transfected with p75NTRsiRNA. These results indicate that NTs, through their receptors, play a critical role in the progression of melanoma.
Abstract: OBJECTIVE: To investigate the in vivo reactions and the mechanisms of cell death after photodynamic therapy (PDT) for cutaneous carcinomas. Photodynamic therapy is a new treatment modality for nonmelanoma skin cancers. Its effects on target tissue have been well investigated in vitro, where apoptosis appears to be the main effector mechanism, but its effects remain undefined in vivo. DESIGN: Skin biopsy specimens were obtained sequentially after PDT for basal cell carcinoma and in situ squamous cell carcinoma (Bowen disease). Evidence from routine histologic evaluation was compared with a panel of apoptosis-related (TUNEL [terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling], caspase-3, and Bcl-2) and inflammatory (CD4, CD8, CD20, CD68, and CD56) markers. We used electron microscopy to evaluate cell damage at the ultrastructural level. MAIN OUTCOME MEASURES: Evidence of the mechanisms of tumor cell damage after PDT, detection of histologic and/or immunohistochemical signs of apoptosis, and time course of the tumor destruction and inflammatory reaction. RESULTS: Early epidermal damage and an acute dermal inflammatory response were detected 15 minutes after PDT. In basal cell carcinoma, nodule damage progressed from scant apoptotic cells seen at the dermal-epithelial junction to massive destruction seen after 1 and 2 days. The periphery of the basaloid nodules consistently showed earlier and predominant damage, as demonstrated by the perfect coincidence of histologic and immunohistochemical evidence with apoptotic markers (TUNEL and caspase-3 staining). Fibrosis and lentigolike changes were seen in late biopsy specimens. CONCLUSIONS: This study defines the time course and characteristics of the skin tumor response to PDT. Taken together, our observations suggest that direct damage to cancer cells is the main effector mechanism leading to PDT response. The involvement of apoptosis is demonstrated by the simultaneous appearance of histologic, immunohistochemical, and ultrastructural markers that occur in the early phases of the cutaneous reaction to PDT. These observations could help to develop future refinements of the PDT technique.
Abstract: Survivin belongs to the family of inhibitor of apoptosis proteins and is involved in regulation of cell death as well as cell division. Here, we show that wild-type (WT) survivin is expressed in a subpopulation of basal keratinocytes in normal human skin at the cytoplasmic level. WT survivin is highly expressed in keratinocyte stem cells (KSCs), whereas its mRNA level decreases in transit amplifying (TA) cells and disappears in postmitotic (PM) cells. Likewise, WT survivin protein is expressed in KSCs, almost undetectable in TA cells, and absent in PM cells. Real time polymerase chain reaction demonstrates that the putative antiapoptotic isoforms survivin-2B and survivin-DeltaEx3 are expressed at the highest levels in KSCs, whereas they tend to decrease in TA cells and disappear in PM cells. On the contrary, the putative proapoptotic variants of survivin, survivin-3B, and survivin-2alpha tend to be high in PM and TA cells and are almost absent in KSCs. By confocal microscopy, survivin is predominantly expressed at the nuclear level in KSCs, which proliferate significantly better than TA cells, which, in turn, express mostly cytosolic WT survivin. Blocking beta1 integrin signal downregulates WT survivin mRNA and protein expression and induces apoptosis (anoikis) in KSCs. On the other hand, inhibition of beta1 integrin upregulates mRNA expression of survivin-2alpha. Taken together, these results indicate that survivin identifies human KSCs. Expression of nuclear survivin could reflect the different behavior between KSCs in vitro and in vivo, in terms of proliferation. Finally, survivin could be part of the "niche" protection by preventing anoikis in KSCs.
Abstract: Neurotrophins regulate cutaneous innervation, act as growth and motility factors on structural skin cells such as keratinocytes and fibroblasts, modulate cutaneous immune function and even serve as stress mediators in skin biology. The multilayered neurotrophin interaction with skin biology through high affinity specific tyrosinekinase receptors and the Janus-faced p75 receptor, which depending on ligand and co-receptor expression can serve as a low-affinity pan-neurotrophin receptor or a high affinity proneurotrophin receptor, guaranties this neuroendocrine peptide family a central position in the control of skin homeostasis in health and disease. It is a challenging task for future research efforts to integrate our knowledge on differential neurotrophin expression patterns and signaling pathways into complex concepts of neuroendocrine tissue remodeling and pathogenetic processes. In addition, we need to improve our understanding of the role of neurotrophin processing enzymes, associated co-receptors and intracellular adaptor molecules in specific cutaneous cell populations to design precise interaction tools for research and treatment. Such tools will allow us to utilize this ancient growth factor family in the management of neurotrophin responsive pathogenetic pathways and cutaneous diseases such as neurogenic inflammation, peripheral nerve degeneration, wound healing, atopic dermatitis or psoriasis.
Abstract: Carboxyfullerenes (CF) act as free radical scavengers in many cell settings and prevent apoptosis in vitro and in vivo. CF protect normal human keratinocytes from UVB-induced apoptosis, although the mechanisms underlying this effect remain to be clarified. Double-staining confocal laser microscopy revealed that CF penetrate the cell and colocalize with cytokeratin-18 within cytoplasm. This localization was confirmed by transmission electron microscopy that showed CF intermingled with keratin filaments. Moreover, double-staining with the mitochondrial marker anti-F1-ATPase antibody demonstrated that CF are expressed in mitochondria. Transmission electron microscopy confirmed that CF actually localize within mitochondria. Then, normal human keratinocytes were UVB-irradiated in the presence or absence of CF at different doses. CF protected keratinocytes from apoptosis induced by reactive oxygen species. CF scavenging effect is associated with a partial blockade of the UVB-induced intrinsic apoptotic pathway by down-modulating caspase-9 activation and cytochrome c release, and by inhibiting the down-regulation of the inhibitor of apoptosis proteins (IAP) survivin, livin, IAP-1 and IAP-2. Finally, CF prevented the cleavage of Bid, up-regulation of Bad and down-regulation of Mcl-1 induced by UVB. Taken together, these results indicate that CF penetrate human keratinocytes, localize within mitochondria where they act both by scavenging free radicals and by protecting cells from apoptosis.
Abstract: A large-scale, pooled analysis of safety data from five Phase III clinical trials (including open-label extensions of two of these studies) and two Phase III open-label clinical trials of efalizumab was conducted to explore whether arthropathy adverse events (AEs) were associated with efalizumab treatment in patients with moderate-to-severe chronic plaque psoriasis. Data from patients who received subcutaneous injections of efalizumab or placebo were stratified for analysis into phases according to the nature and duration of treatment. These included: the 'first treatment' phase (0-12-week data from patients who received either efalizumab, 1 mg/kg once weekly, or placebo in the five placebo-controlled studies); the 'extended treatment' phase (13-24-week data from seven trials for all efalizumab-treated patients); and the 'long-term treatment' phase (data from efalizumab-treated patients who received treatment for up to 36 months in two long-term trials). Descriptive statistics were performed and the incidence of arthropathy AEs per patient-year was calculated using 95% confidence intervals (CIs). During the first treatment phase, a similar proportion of patients had an arthropathy AE in the efalizumab group (3.3%; 58/1740 patients) compared with the placebo group (3.5%; 34/979 patients); the incidence of arthropathy AEs per patient-year was 0.15 in the efalizumab group (95% CI 0.11-0.19) and 0.16 in the placebo group (95% CI 0.11-0.22). Analysis of first treatment phase data from one study (n = 793) showed that the incidence of psoriatic arthropathy per patient-year was lower in efalizumab-treated patients (0.10; 95% CI 0.05-0.18) than in those given placebo (0.17; 95% CI 0.08-0.30). During the extended treatment phase, the incidence of arthropathy remained low (0.17; 95% CI 0.14-0.22). Data from two long-term studies showed that there was no increase in the incidence of arthropathy AEs over time in patients treated with efalizumab for up to 36 months. Patients who had an arthropathy AE during treatment with efalizumab appeared to be more likely to have a history of arthropathy prior to treatment. Efalizumab does not appear to increase the risk of arthropathy AEs compared with placebo.
Abstract: Neurotrophins (NTs) belong to a family of growth factors, which control the development, maintenance, and apoptotic death of neurons and also fulfill multiple regulatory functions outside the nervous system. Biological effects induced by NTs strongly depend on the pattern of NT receptor/co-receptors expression in target cells, as well as on the set of intracellular adaptor molecules that link NT signalling to distinct biochemical pathways. In this review, we summarize data on the molecular mechanisms underlying the involvement of NTs in the control of non-neuronal functions in normal skin (e.g. keratinocyte proliferation, melanocyte development and apoptosis, hair growth). We also review the data on the role for NTs and their receptors in a number of pathological skin conditions (stress-induced hair loss, psoriasis, atopic dermatitis). Although additional efforts are required to fully understand mechanisms underlying the involvement of NTs and their receptors in controlling functions of normal and pathologically altered skin cells, substantial evidence suggests that modulation of NT signalling by NTs receptor agonists/antagonists may be developed as intervention modalities in distinct skin and hair growth pathologies.
Abstract: Pemphigus vulgaris (PV) is fascinating to dermatologists, epithelial biologists and immunologists alike, as its pathogenesis has been clarified to a much greater extent than that of most other organ-specific autoimmune diseases, and as it has provided abundant novel insights into desmoglein biology and pathology along the way. Historically, the most influential PV pathogenesis concept is that of Stanley and Amagai. This concept holds that autoantibodies against desmogleins are both essential and sufficient for epidermal blister formation (acantholysis) by impeding the normal functioning of these major adhesion proteins. However, as with most good theories, this landmark concept has left a number of intriguing and important questions open (or at least has not managed to answer these to everyone's satisfaction). Moreover, selected dissenting voices in the literature have increasingly called attention to what may or may not be construed as inconsistencies in this dominant PV pathogenesis paradigm of the recent past. The present debate feature therefore bravely rises to the challenge of re-examining the entire currently available evidence, as rationally and as undogmatically as possible, by provocatively asking a carefully selected congregation of experts (who have never before jointly published on this controversial topic!) to discuss how essential anti-desmoglein autoantibodies really are in the immunopathogenesis of PV. Not surprisingly, some of our expert "witnesses" in this animated debate propose diametrically opposed answers to this question. While doing so, incisive additional questions are raised that relate to the central one posed, and our attention is called to facts that may deserve more careful consideration than they have received so far. Together with the intriguing (often still very speculative) complementary or alternative pathogenesis scenarios proposed in the following pages, this offers welcome "food for thought" as well as very specific suggestions for important future research directions--within and beyond the camp of PV aficionados. The editors trust that this attempt at a rational public debate of the full evidence that is currently at hand will constructively contribute to further dissecting the exciting--and clinically very relevant!--immunopathogenesis of PV in all its complexity.
Abstract: Melanocytes and cells of the nervous system are of common ectodermal origin and neurotrophins (NT) have been shown to be released by human keratinocytes. We investigated the expression and function of NT [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/-5] and their receptors in human melanocytes. Human melanocytes produce all NT in different amounts, whereas they only release NT-4. NT-4 release is downregulated, whereas NT-3 is upregulated by ultraviolet (UVB) irradiation. Melanocytes treated with phorbol 12-myristate 13-acetate (PMA) express TrkA and TrkB, but not TrkC. NT fail to stimulate melanocyte proliferation, whereas they stimulate the synthesis of tyrosinase and tyrosinase-related protein-1 (TRP-1). Finally, NT-3, NT-4 and NGF increase melanin production. Taken together, these results demonstrate an intriguing interaction between melanocytes and the nervous system. We speculate that NT could be considered the target of therapy for disorders of skin pigmentation.
Abstract: Beta1-integrin protects keratinocyte stem cells (KSC) from cell-detachment apoptosis ('anoikis'). Here we show that caspase-8 active protein is detected in both young transit amplifying (TA) cells and TA cells, but not in KSC. On suspension, caspases are activated earlier in young TA than in KSC, whereas anti-beta1-integrin neutralizing antibody accelerates caspase activation in both KSC and young TA. Caspases 8 and 10 are the first caspases to be activated whereas caspase-8 inhibitor zIETD-fmk delays the activation of Bid, caspase-9 and caspase-3. However, the caspase-9 inhibitor zLEDH-fmk does not block the activation of caspase-8, Bid, caspase-10 and caspase-3. Moreover, caspase-8, but not caspase-9 inhibitor partially prevents keratinocyte anoikis. As FLIP inhibits caspase-8 processing, we retrovirally infected HaCaT keratinocytes with c-FLIP(L). Anti-beta1-integrin fails to activate caspase-8, Bid, caspase-9 and to induce the release of cytochrome c in c-FLIP(L) overexpressing keratinocytes. Finally, overexpression of c-FLIP(L) partially prevents anoikis in both suspended and anti-beta1 integrin-treated cells. Taken together, these results indicate that the extrinsic apoptotic pathway triggered by caspase-8 predominates in keratinocyte anoikis. However, the release of cytochrome c and the later activation of caspase-9 seem to suggest that the intrinsic mitochondrial pathway may intervene as a positive feedback loop of caspase activation.
Abstract: This study investigated apoptosis in prostate cancer before and after neoadjuvant treatment with LH-RH analog, demonstrating that this therapy induced high AI and bax and survivin overexpression, thus acting as a proapoptotic agent in prostate cancer. A statistically significant correlation was found between high AI and overexpression of bax protein after therapy. It is probable that this kind of therapy is deeply implicated in promoting the apoptotic intrinsic pathway.
Abstract: Whereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription-polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis.
Abstract: The skin is the most densely innervated organ in the body and there is a close relationship between the skin and the nervous system. Most skin cells express receptors for neuromediators (NM) and skin cells themselves are an important source of NM. In particular, human keratinocytes synthesize neurotrophins and endorphins and express their receptors. In addition to neurotrophic activity, NM are involved in skin homeostasis, trophism and stress responses. NM released from keratinocytes also function in a paracrine fashion on other skin cells, such as Langerhans cells, melanocytes and fibroblasts. We discuss the influence of NM on these cells, which may be involved in major cosmetic problems like ageing, baldness and dyspigmentation. Based on this correlation, it seems reasonable to target neural factors for cosmetic purposes.
Abstract: The Fas/Fas ligand system triggers the extrinsic apoptotic pathway and is involved in several inflammatory conditions, also at the skin level. The Fas/Fas ligand cell death pathway plays a major role in anoikis, a type of apoptosis characterized by cell detachment. As pemphigus is characterized by loss of cell to cell adhesion, we evaluated the role of anoikis and Fas ligand in this bullous disease. We report that, in suprabasal epidermis from perilesional pemphigus skin, most keratinocytes are apoptotic. Moreover, Fas ligand levels are markedly increased in sera from pemphigus patients, whereas they are undetectable in sera from patients undergoing steroid treatment. Sera from untreated patients but not from patients under steroids induce keratinocyte apoptosis. Pemphigus-sera-induced cell death is partially inhibited by pretreatment with anti-Fas ligand antibodies and by incubation with caspase-8 inhibitor Z-IETD-FMK. Finally, caspase-8 is activated in keratinocytes provided with sera from pemphigus patients, whereas cleavage is partially blocked by pretreatment of sera with anti-Fas ligand antibody. These results suggest that increased Fas ligand in pemphigus sera is responsible for keratinocyte apoptosis, which occurs through the activation of a caspase-8-driven extrinsic apoptotic pathway.
Abstract: Because inhibition of integrin signaling induces apoptosis, we investigated whether keratinocytes expressing beta1 and alpha6beta4 integrins (enriched for stem cells) are protected from cell death. Keratinocytes rapidly adhering to type IV collagen expressed highest levels of beta1 and alpha6beta4 and of the anti-apoptotic stem cell marker p63. Apoptotic cells were significantly higher in slowly adhering than in rapidly adhering keratinocytes. Anti-beta1 integrin caused a significant increase in apoptotic cells, while it decreased Bcl-2 levels in stem keratinocytes. Bax and Bad proteins were higher in slowly adhering than in rapidly adhering cells. By contrast, Bcl-2, Bcl-x and Mcl-1 proteins were highest in rapidly adhering keratinocytes and nearly absent in slowly adhering cells. After addition of anti-beta1 integrin, the apoptotic rate was significantly higher in HaCaT cells not expressing Bcl-2 than in controls. These results indicate that keratinocytes enriched for stem cells are protected from apoptosis via beta1 integrin, in a Bcl-2 dependent manner.
Abstract: Nerve growth factor (NGF) is synthesized and released by human keratinocytes. NGF acts as a neurotrophic molecule at the skin level, as it stimulates the sprouting of nerve fibers and regulates the synthesis and expression of neuropeptides. NGF can thus take part in neurogenic inflammation which in turn is involved in the pathogenesis of several inflammatory dermatoses. Human keratinocytes also synthesize and express the low (p75)-and the high-affinity (trk) NGF-receptor (NGF-R). NGF stimulates keratinocyte proliferation which is blocked by the natural alcaloid K252, a specific inhibitor of trk phosphorylation. K252 inhibits keratinocyte proliferation and induces keratinocyte apoptosis, in the absence of exogenous NGF, indicating the existence of an autocrine loop where NGF and trk act as key players. Finally, NGF protein levels are increased in psoriatic as compared to non-lesional and normal skin, and psoriatic keratinocytes express higher amounts of NGF than normal keratinocytes. This review will discuss the above findings in view of a possible involvement of NGF in the pathomechanisms associated with the development of the psoriatic lesion.
Abstract: C60 carboxyfullerene is a novel buckminsterfullerene-derived compound that behaves as a free-radical scavenger. In the present report, we investigated whether this drug exerts a protective activity against oxidative stress-induced apoptosis. Human peripheral blood mononuclear cells (PBMCs) were challenged by 2-deoxy-d-ribose (dRib) or TNF-alpha plus cycloheximide as agents that trigger apoptosis by interfering with the redox status of cell and mitochondrial membrane potential. We found that carboxyfullerene was able to protect quiescent PBMCs from apoptosis caused either by 2-deoxy-d-ribose or TNF-alpha plus cycloheximide by a mechanism partially involving the mitochondrial membrane potential integrity, known to be associated with early stages of apoptosis. These results represent the first indication for a target activity of buckminsterfullerenes on cells of the immune system and their mitochondria.
Abstract: Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2.
Abstract: Biologically active nerve growth factor (NGF) is synthesised and released by proliferating normal human keratinocytes. NGF up-regulates the expression of NGF mRNA in keratinocytes. Keratinocytes express both the low (p75)- and the high-affinity (TrkA) NGF-receptors, which are located in the basal layer of the epidermis. K252, a specific inhibitor of trk phosphorylation, blocks NGF-induced keratinocyte proliferation, in absence of exogenous NGF. Normal keratinocytes over-expressing TrkA proliferate better than control transfectants, while the NGF mimicking anti-Trk antibody induces an increased keratinocyte proliferation in Trk over-expressing cells as compared to mock transfected keratinocytes. In addition, NGF over-expressing keratinocytes proliferate better than mock transfected cells. K252, by blocking TrkA phosphorylation, induces apoptosis in normal keratinocytes, but not in keratinocytes over-expressing bcl-2. Furthermore, NGF transfected keratinocytes are protected from UV-B-induced keratinocyte apoptosis, by maintaining constant levels of Bcl-2 and Bcl-xL . Taken together these results support the concept of an autocrine survival system sustained by NGF and its high-affinity receptor in human keratinocytes. Because NGF and Trk levels are highly expressed in psoriasis. one could speculate that NGF autocrine system plays a role in the mechanisms associated with this and other hyperproliferative skin conditions, including cancer.
