Pathways involved in proliferating,senescent and immortalized keratinocyte cell death mediated by two different TRAIL preparations

Properly regulated keratinocyte cell death is fundamentally important to maintain structural integrity and homeostatic function of epidermis. Moreover, from an oncological perspective, therapeutic approaches selectively targeting apoptosis of malignant cell types while sparing normal keratinocytes in surrounding skin is desirable. Apo2Ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been observed to preferentially induce cytopathic effects on transformed/malignant cell types compared with their non-neoplastic counterparts. In this report, two different biologically active preparations of Apo2L/TRAIL, a non-tagged version, NT-Apo2L/TRAIL, and a leucine zipper fusion protein, LZ-Apo2L/TRAIL, were examined for their ability to trigger apoptosis in normal human keratinocytes, and in an immortalized cell line (HaCaT cells). Differences between these preparations were observed, including: NT-Apo2L/TRAIL induced less keratinocyte apoptosis compared with LZ-Apo2L/TRAIL; NT-Apo2L/TRAIL also induced less apoptosis of HaCaT cells compared with LZ-Apo2L/TRAIL; LZ-Apo2L/TRAIL but not NT-Apo2L/TRAIL induced cytotoxic effects when keratinocytes became growth arrested due to undergoing spontaneous replicative senescence--a biological state previously observed to be resistant to UV-light-induced apoptosis. Similarities between preparations included: an enhanced ability for both Apo2L/TRAIL preparations to kill a greater relative percentage of HaCaT cells compared with keratinocytes; enhanced cytotoxicity towards keratinocytes that had their NF-B activity inhibited; a dependence of both Apo2L/TRAIL preparations on FADD and caspase activation; triggering of the same caspase cascades including caspase 8 and 3; and an ability to induce apoptosis even when HaCaT cells and keratinocytes were transduced to overexpress either Bcl-2 or Bcl-x(L) (survival factors that reduce susceptibility to UV-light-induced apoptosis). These results indicate that while both preparations of Apo2L/TRAIL possess biological activity, there are important differences as regards their ability to induce apoptosis in normal and immortalized keratinocytes. Moreover, the death receptor pathway triggered by LZ-Apo2L/TRAIL can overcome the apoptotic resistance normally observed in response to UV-light mediated by Bcl-2/Bcl-x(L), as well as by the state of cellular senescence. Unraveling the molecular basis for these differential biological effects may reveal a new strategic role for these death receptor/ligands linked to apoptosis in maintaining the dynamic balance of keratinocyte proliferation, differentiation, and cell death necessary to achieve a homeostatic thickness and function of normal skin. In addition, it may be possible to utilize these Apo2L/TRAIL preparations for the treatment of various sun-induced skin cancers as they can differentially trigger apoptosis of transformed keratinocytes, or keratinocytes with abnormal NF-kappaB signaling, while sparing adjacent normal keratinocytes.     

Role of NF-kappaB activity in apoptotic response of keratinocytes mediated by interferon-gamma, tumor necrosis factor-alpha, and tumor-necrosis-factor-related apoptosis-inducing ligand.

An important step in tumorigenesis involves loss of sensitivity to various apoptotic signals by malignant cells, imbuing them with an enhanced survival phenotype. NF-kappaB also regulates epidermal thickness, susceptibility to apoptosis, and tumor formation in skin. Keratinocytes were examined for their susceptibility to apoptosis using cytokines produced during an immunologic response to tumor antigens, i.e., interferon-gamma and/or tumor necrosis factor-alpha (TNF-alpha). The role for NF-kappaB in this response was examined using a retroviral vector containing a degradation-resistant form of IkappaBalpha. Whereas interferon-gamma and TNF-alpha either alone or in combination did not induce apoptosis in keratinocytes, after infection with the retrovirus to block NF-kappaB activation they became susceptible to TNF-alpha but not Fas-induced apoptosis. Moreover, when keratinocytes with repressed NF-kappaB activity were simultaneously treated with interferon-gamma, there was a synergistic induction of apoptosis by TNF-alpha that was dependent on FADD, tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL), and caspase activation. Molecular abnormalities accompanying repressed NF-kappaB activity included failure to induce TNF-RII receptor together with enhanced levels of TRAIL death receptor 4. The ability of interferon-gamma when combined with TNF-alpha to mediate keratinocyte apoptosis included induction of TRAIL coupled with diminished capacity of keratinocytes with repressed NF-kappaB activity to increase the TRAIL decoy receptor-1, as well as lower levels of several NF-kappaB-dependent antiapoptotic proteins accompanied by enhanced caspase 8 levels. These results indicate that interferon-gamma and TNF-alpha synergistically induce keratinocyte apoptosis when concomitant induction of NF-kappaB is blocked. Participants in the apoptotic response mediated by NF-kappaB, besides cell-survival proteins, include modulation of TRAIL and both death and decoy receptors. Thus, not only does NF-kappaB signaling influence the intrinsic survival pathway for keratinocytes in normal skin, but it may also play a role in determining the apoptotic response to cytokines generated during an immune response via TRAIL produced by the keratinocytes themselves.     

TGF-beta/Smad signaling inhibits IFN-gamma and TNF-alpha-induced TARC (CCL17) production in HaCaT cells

BACKGROUND: A Th2 chemokine, thymus and activation regulated chemokine (TARC/CCL17), produced by keratinocytes, is implicated in the development of atopic dermatitis by recruiting CLA(+)CCR4(+) lymphocytes into lesional skin and its expression was induced by proinflammatory cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). However, it remains unknown how TARC expression is negatively regulated in keratinocytes. OBJECTIVE: We sought to determine whether transforming growth factor-beta 1 (TGF-beta 1) regulated TARC expression in keratinocytes. METHODS: The effect of TGF-beta 1 on mRNA and protein expression of IFN-gamma and TNF-alpha-induced TARC in a human keratinocyte cell line, HaCaT cells, was evaluated by using RT-PCR and ELISA. Adenovector-mediated gene transfer was used to determine the effect of Smad proteins on TARC expression in HaCaT cells. RESULTS: TGF-beta 1 inhibited mRNA and protein expression of IFN-gamma and TNF-alpha-induced TARC in HaCaT cells. The inhibitory effect of TGF-beta 1 on the TARC expression was suppressed by overexpression of Smad7, a major inhibitory regulator of Smad pathway for transforming growth factor-beta (TGF-beta) signaling, but not by PD98059, an inhibitor for ERK/mitogen-activated protein kinase (MAPK) pathway. In addition, overexpression of Smad2 or Smad3, major signal transducing Smads, was sufficient to inhibite the IFN-gamma and TNF-alpha-induced TARC production in HaCaT cells. CONCLUSION: TGF-beta1 inhibited IFN-gamma and TNF-alpha-induced TARC production in HaCaT cells via Smad2/3, suggesting that modulation of TGF-beta/Smad signaling pathway may be beneficial for the treatment of atopic dermatitis.     

Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro

BACKGROUND: thymus and activation-regulated chemokine (TARC)/CCL17 is a CC chemokine that selectively attracts Th2-type lymphocytes. Immunohistochemical analyses have revealed that TARC is expressed in the epidermal keratinocytes of atopic dermatitis (AD), suggesting TARC involvement in the pathogenesis of the disease. However, keratinocyte TARC production has been described only in the transformed keratinocyte cell line HaCaT. OBJECTIVE: to examine TARC production in normal human epidermal keratinocytes (NHEK) in vitro. METHODS: the expression of TARC mRNA and protein were examined in NHEK and HaCaT cells stimulated with various cytokines. RESULTS: stimulation with inflammatory cytokines, including interleukin (IL)-1, IL-4, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha failed to induce TARC mRNA expression in NHEK. However, stimulation with IFN-gamma and TNF-alpha together enhanced expression slightly. ELISA analysis failed to detect TARC protein in NHEK culture supernatant, even following stimulation with IFN-gamma and TNF-alpha. In contrast, HaCaT cells produced TARC protein even without stimulation of cytokines. CONCLUSION: these results indicate that production of TARC by HaCaT cells is a phenomenon specific to the cell line and the observation on TARC in HaCaT cells can not be generalized. NHEK do not produce TARC protein in vitro.     

TRAIL-induced apoptosis and gene induction in HaCaT keratinocytes: differential contribution of TRAIL receptors 1 and 2

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) exerts a potent cytotoxic activity especially against many tumor cell types such as transformed keratinocytes. The specific role of the different TRAIL receptors in this process, however, is unknown. In this report we examine the role the TRAIL receptors play in both the apoptotic and nonapoptotic responses of HaCaT keratinocytes to leucine zipper TRAIL (LZ-TRAIL). By employing receptor-specific blocking antibodies we demonstrate that TRAIL receptor 1 plays the primary role in mediating caspase activation and apoptosis in HaCaT cells. Furthermore, we show that this receptor mainly mediates nuclear factor kappaB activation and expression of the pro-inflammatory cytokine interleukin-8 and that nuclear factor kappaB activation is critically required for the induction of pro-inflammatory cytokines in response to LZ-TRAIL. Taken together, our data suggest that beside its potent pro-apoptotic role, LZ-TRAIL leads to pro-inflammatory responses that are mainly mediated by TRAIL receptor 1 in HaCaT keratinocytes.     

TRAIL-induced keratinocyte differentiation requires caspase activation and p63 expression

Cornification, the terminal differentiation of keratinocytes, is a special form of programmed cell death in the skin. In this article, we report that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce the expression of the keratinocyte differentiation markers involucrin and type 1 transglutaminase in normal human epidermal keratinocytes. The induction of differentiation occurs mainly under the activation of caspases 3 and 8, and apoptosis can also be triggered. Inhibition of these apoptotic caspases attenuates both apoptosis and differentiation of keratinocytes caused by TRAIL but barely affects the induction of differentiation caused by calcium and phorbol 12-myristate 13-acetate. Differential regulation of extracellular signal-regulated kinase and p38 activation by TRAIL is also observed. Moreover, the degradation of p63 is induced by TRAIL-elicited caspase activation. However, the existence of p63 is essential for the initiation of keratinocyte differentiation by TRAIL because knockdown of ΔNp63 decreases TRAIL-induced differentiation. Taken together, our results suggest that TRAIL can be an inducer of both differentiation and apoptosis in human keratinocytes, and that caspases critically mediate these processes. This study identifies a new role of apoptotic caspases for terminal differentiation of keratinocytes and further elucidates the molecular pathways involved in this unique model of cell death.     

NF-kappaB inhibition reveals differential mechanisms of TNF versus TRAIL-induced apoptosis upstream or at the level of caspase-8 activation independent of cIAP2

Death ligands not only activate a death program but also regulate inflammatory signalling pathways, for example, through NF-kappaB induction. Although tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF both activate NF-kappaB in human keratinocytes, only TRAIL potently induces apoptosis. However, when induction of NF-kappaB was inhibited with a kinase dead IKK2 mutant (IKK2-KD), TNF- but not TRAIL-induced apoptosis was dramatically enhanced. Acquired susceptibility to TNF-induced apoptosis was due to increased caspase-8 activation. To investigate the mechanism of resistance of HaCaT keratinocytes to TNF-induced apoptosis, we analyzed a panel of NF-kappaB-regulated effector molecules. Interestingly, the inhibitor of apoptosis protein (IAP) family member cIAP2, but not cIAP1, X-linked inhibitor of apoptosis, TNF receptor-associated factor (TRAF)-1, or TRAF2, was downregulated in sensitive but not in resistant HaCaT keratinocytes. Surprisingly, however, stable inducible expression of cIAP2 was not sufficient to render IKK2-KD-sensitized keratinocytes resistant to TNF, and reduction of cIAP2 alone did not increase the sensitivity of HaCaT keratinocytes to TNF. In conclusion, we demonstrate that inhibition of NF-kappaB dramatically sensitizes human keratinocytes to TNF- but not to TRAIL-induced apoptosis and that this sensitization for TNF was largely independent of cIAP2. Our data thus clearly exclude the candidates proposed to date to confer TNF apoptosis resistance and suggest the function of an unanticipated effector of NF-kappaB critical for the survival of HaCaT keratinocytes upstream or at the level of caspase-8 activation.     

TNF-alpha impairs the S-G2/M cell cycle checkpoint and cyclobutane pyrimidine dimer repair in premalignant skin cells: role of the PI3K-Akt pathway

Tumor necrosis factor-alpha (TNF-alpha) is induced by UVB radiation and has been implicated in the early stages of skin carcinogenesis. Here, we show that in normal keratinocytes and the transformed keratinocyte cell lines, HaCaT and A431, TNF-alpha stimulates protein kinase B/Akt, which results in activation of the survival complex mTORC1 (mammalian target of rapamycin complex 1) and inhibition of the proapoptotic proteins Bad and FoxO3a. In UVB-irradiated HaCaT cells (10-20 mJ cm(-2)), TNF-alpha increased the proportion of cycling cells and enhanced the rate of apoptosis. A significantly higher proportion of UVB-treated HaCaT cells containing unrepaired cyclobutane pyrimidine dimers (CPDs) escaped the G2/M cell cycle checkpoint in the presence of TNF-alpha (9.5+/-3.3 vs 4.8+/-2.2%). After treatment with the PI3K inhibitor LY294002, only 1.2+/-0.7% of CPD-containing HaCaT cells were actively cycling. TNF-alpha enhanced apoptosis less potently and did not increase the level of CPD or stimulate cell cycle progression in normal keratinocytes. Our data suggest that TNF-alpha overrides the G2/M checkpoint in premalignant skin cells and allows for some cells containing unrepaired CPD to enter the cell cycle. The effect of TNF-alpha seems to be dependent on Akt activation and may constitute a relevant mechanism enhancing mutagenesis and tumor development.     

Both IL-4 and IL-13 inhibit the TNF-alpha and IFN-gamma enhanced MDC production in a human keratinocyte cell line,HaCaT cells

BACKGROUND: Macrophage-derived chemokine (MDC) is a Th2 type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Recent reports demonstrated that MDC is expressed not only by macrophages, dendritic cells and lymphocytes, but also by cultured human keratinocytes (KCs). However, the regulation of MDC production in KCs by various cytokines has not been well documented. OBJECTIVE: In this study, we investigated how Th1/Th2 cytokines regulate MDC production in a human KC cell line, HaCaT cells. METHODS: HaCaT cells were cultured with or without various cytokines for 24 h and RT-PCR was performed using these cells to evaluate MDC mRNA levels. ELISA was carried out using supernatant of HaCaT cells to calculate secreted MDC protein levels. RESULTS: MDC mRNA was weakly expressed in HaCaT cells, and upon stimulation with TNF-alpha or IFN-gamma, MDC expression was strongly upregulated. The supernatant MDC levels when stimulated with TNF-alpha or IFN-gamma were significantly higher than those without stimulation, and were synergistically increased when stimulated with a combination of TNF-alpha and IFN-gamma. Both interleukin-4 (IL-4) and IL-13 inhibited TNF-alpha and IFN-gamma enhanced MDC production in HaCaT cells in a dose-dependent manner. CONCLUSION: Th2-type cytokines IL-4 and IL-13 downregulate the production of MDC, a Th2 type chemokine, by KCs. This may partially contribute to maintaining Th1/Th2 balance in inflammatory skin diseases like atopic dermatitis.     

Possible role of Fas/Fas ligand-mediated apoptosis in the pathogenesis of fixed drug eruption

BACKGROUND: Although epidermal and dermal T cells play roles in the pathogenesis of fixed drug eruption (FDE), not much is known about keratinocyte death and its precise mechanism in FDE. OBJECTIVES: Our aim is to elucidate the mechanism that underlies keratinocyte death in FDE, that is, the role of apoptosis and its signalling pathway. METHODS: We first examined the involvement of apoptosis in the active FDE lesions by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) assay and immunohistochemical analysis of caspase-3. We then examined the expressions of Fas and Fas ligand (FasL) to deduce the possible upstream signalling pathway of apoptosis, if apoptosis were involved. We finally characterized the infiltrated T-cell subpopulations in the active FDE lesions. RESULTS: In the active FDE lesions, TUNEL positivity was strongly observed in the basal keratinocytes, and also weakly observed in the upper dermal infiltrates as well as in a few keratinocytes in the granular layer. The distribution of TUNEL-positive cells was similar to that of the strong staining of active capase-3. Fas was found mainly in the keratinocytes and some infiltrated dermal cells, whereas FasL was identified predominantly in the intraepidermal and dermal infiltrated cells and in some basal keratinocytes. Overlapping expression of Fas and FasL was accompanied by apoptosis in the FDE lesions. Many of the infiltrated mononuclear cells were CD8+. Perforin was rarely observed in the FDE lesions. CONCLUSIONS: These findings suggest that apoptosis of the keratinocyte is highly likely to be involved in the pathogenesis of FDE, and this cytotoxicity might be predominantly mediated by the FasL of the infiltrating CD8+ T cells, possibly also playing an inflammatory role.     

Ultraviolet Light (UV) Regulation of the TNF Family Decoy Receptors DcR2 and DcR3 in Human Keratinocytes

Background: Several additional members of the tumor necrosis factor (TNF) receptor family were recently identified. The existence of such receptors, which may play distinct and unique regulatory roles, suggests that complex regulatory mechanisms are involved in apoptosis. Objective: This study examines the expression of several members of the TNF receptor family in human keratinocytes exposed to ultraviolet B (UVB) irradiation. Methods: Human keratinocytes were exposed to increasing doses of UVB, total RNA was harvested, and a quantitative RNase protection assay was performed. Results: Decoy receptor-3 (DcR3), a nonfunctional receptor that binds to Fas ligand (FasL), was constitutively expressed at high level in keratinocytes but decreased rapidly in cells exposed to UVB. Decoy receptor-2 (DcR2), a nonfunctional receptor that binds to TNF-related apoptosis-inducing ligand (TRAIL)/APO-2L, showed the opposite expression pattern. DcR2 was undetectable in unirradiated keratinocytes and was markedly up-regulated after exposure to UVB. Although the response showed significant delays at higher UVB doses, the patterns observed for DcR3 and DcR2 were consistent in this set of experiments. Conclusion: We conclude that UVB regulates expression of these two TNF decoy receptors in keratinocytes. This pathway may represent a novel mechanism for regulation of apoptosis in the skin.     

TGF-beta1-mediated regulation of thymus and activation-regulated chemokine (TARC/CCL17) synthesis and secretion by HaCaT cells co-stimulated with TNF-alpha and IFN-gamma

Thymus and activation-regulated chemokine (TARC/CCL17) contributes not only to the recruitment of leukocytes, but is also involved in immune disorders, such as atopic dermatitis (AD) and bronchial asthma. We have previously reported that the levels of TARC were high in patients with AD and that lesional epidermis were strongly immunoreactive for TARC. In this paper, the effects of transforming growth factor (TGF)-beta(1) on the expression of TARC/CCL17 were examined in HaCaT cells, a human keratinocytes (KCs) cell line, co-stimulated with TNF-alpha and IFN-gamma. We found that TGF-beta(1) down-regulated the TARC synthesis and secretion of HaCaT cells co-stimulated with TNF-alpha and IFN-gamma in a dose-dependent manner. TGF-beta(1) at a concentration of 10ng/ml maximally inhibited this secretion. Northern blot analysis showed a similar inhibitory effect of TGF-beta(1) on TARC mRNA expression by HaCaT cells. The TGF-beta(1)-induced down-regulation of TARC/CCL17 in HaCaT cells suggests that TGF-beta(1) might regulate the TARC-related inflammatory processes, which may be important for understanding the pathogenesis of allergic diseases.     

Fas ligand reduces viability in primary melanoma short-term cell cultures more than in metastatic melanoma short-term cell cultures

BACKGROUND: Apoptotic pathway aberrations are reported as important tumor progression factors in melanoma. OBJECTIVE: Effect of soluble Fas ligand (sFasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on short-term cultured melanoma cell viability from different stages of melanoma. RESULTS: Recombinant human FasL reduced viability after 18 h in a dose-dependent manner in 4 of 5 cell cultures from primary tumors and 1 of 9 cell cultures from metastatic melanoma (67.5 vs. 96.4%, p = 0.007). DNA fragmentation on flow cytometry confirmed apoptosis. Incubation with TRAIL had no effect on melanoma cell viability. Immunohistochemistry showed Fas in 3 of 4 primary and in 6 of 7 metastatic lesions, no FasL in primary lesions, and FasL in 5 of 7 metastatic lesions. CONCLUSION: Melanoma short-term cell cultures from primary tumors show decreased viability under FasL, but not TRAIL stimulation rather than short-term cell cultures derived from metastases.     

Knock down of p53 levels in human keratinocytes increases susceptibility to type I and type II interferon-induced apoptosis mediated by a TRAIL dependent pathway

BACKGROUND: Keratinocytes (KCs) in healthy skin only undergo death following differentiation to produce stratum corneum. By contrast, in inflammatory pathological conditions featuring type I (IFN-alpha) and type II (IFN-gamma) interferons KCs undergo premature apoptosis. OBJECTIVE: To define apoptotic susceptibility of KCs, response to interferons was examined. Since molecular cross-talk occurs between interferons and p53, potential mechanistic roles for p53 in KC apoptosis were investigated. METHODS: Knock down of p53 was performed, and apoptotic response to addition of interferons was assessed using FACS and by staining for activated caspase 3 and TUNEL. Elucidation of death pathway was accomplished by using a dominant negative death receptor construct and a neutralizing TRAIL antibody. RESULTS: Reduction in p53 levels in KCs by siRNA treatment enhanced, rather than reduced, apoptotic responses to IFN-alpha plus IFN-gamma. In an immortalized human KC cell line (HaCaT cells with both p53 alleles mutated) enhanced apoptotic susceptibility to interferon exposure was also observed. The mechanism for this enhanced apoptosis involved induction of TRAIL and its interaction with death receptors, as blocking the death receptor pathway using dominant negative FADD, or by addition of neutralizing antibody against TRAIL, reduced the apoptotic response to IFN-alpha and IFN-gamma. CONCLUSION: These results indicate IFN-alpha plus IFN-gamma triggers apoptosis independent of p53 in HaCaT cells, and also demonstrate an unexpected survival role for p53 in human KCs as regards apoptotic responsiveness to cytokines such as IFN-alpha and IFN-gamma involving activation of TRAIL-related death receptors. Strategies enhancing p53 regulated survival proteins in KCs may be of therapeutic benefit in skin disorders characterized by activated immunocytes triggering premature KC apoptosis.     

Evaluation of the potential role of cytokines in toxic epidermal necrolysis

Toxic epidermal necrolysis is a rare disease observed as a consequence of adverse reactions to drugs. It results in the widespread apoptosis of epidermal cells and has a high mortality rate. The mechanisms leading to this apoptosis are not yet elucidated. We investigated whether the cytokines present in the blister fluid, which accumulates under necrotic epidermis, originated from T lymphocytes and may play a role in the propagation of keratinocyte apoptosis. Interferon gamma (IFN-gamma), soluble tumor necrosis factor alpha (TNF-alpha), soluble Fas ligand (sFas-L) were present in much higher concentration in the blister fluids of 13 toxic epidermal necrolysis (TEN) patients than in control fluids from burns. The results of RT-PCR studies, however, indicated that only IFN-gamma and to a lesser extent interleukin (IL)-18 were produced by mononuclear cells present in the fluid. That suggests that the other cytokines also present (TNF-alpha, sFas-L, IL-10) rather originated from activated keratinocytes. Fas-L was indeed overexpressed on the membranes of keratinocytes in lesional skin in situ. The Th1 profile of T lymphocyte activation found in the blister fluid of patients with TEN is consistent with a key role for drug-specific cytotoxic T lymphocytes (CTL) as previously reported, the activation of keratinocytes by IFN-gamma making them sensitive to cell-mediated cytolysis. We propose the hypothesis that the production of Fas-L, TNF-alpha, and IL-10 by keratinocytes could be a defense mechanism against CTL rather than a way of propagating apoptosis among epidermal cells.     

Effects of isocamptothecin, a novel camptothecin analogue, on proliferation, apoptosis and telomerase activity in HaCaT cells

Abstract:  Camptothecin is a topoisomerase I inhibitor with definite anti-psoriatic effect. As it is limited in clinical application because of serious side effects and toxicity, many researchers are striving hard to develop derivatives or analogues of camptothecin with higher effects and less toxicity. To explore the anti-psoriatic potential of isocamptothecin, a novel camptothecin analogue, its effects on proliferation, apoptosis and telomerase activity were investigated in the human keratinocyte cell line HaCaT. Incubation with isocamptothecin resulted in a time- and concentration-dependent inhibition of HaCaT cell proliferation. However, isocamptothecin showed larger inhibitory concentration at 50% than camptothecin, suggesting far less cytotoxicity. In addition, isocamptothecin induced apoptosis in a concentration-dependent manner and induced typical morphologic features of apoptosis in HaCaT cells. Moreover, isocamptothecin downregulated the telomerase activity of HaCaT cells not only at concentrations of apoptosis induction but also at concentration insufficient to induce apoptosis, providing additional mechanisms that further account for its ability to inhibit keratinocytes proliferation and induce apoptosis. These results indicate that isocamptothecin possesses similar effects on keratinocytes with camptothecin, but shows far less cytotoxicity, it may probably become a promising agent for psoriasis therapy.     

Cytokine-mediated effects of peripheral blood mononuclear cells from patients with atopic eczema on keratinocytes (HaCaT) in a new coculture system

Summary Interactions between keratinocytes and mononuclear cells via cytokines and adhesion molecules are thought to play a crucial part in inflammatory skin diseases. The cytokine-mediated effects of peripheral blood mononuclear cells (PBMC) from patients with atopic eczema (AE) and healthy individuals on keratinocytes (HaCaT) were investigated in vitro. A new coculture model (Transwell system) which consists of a lower and an upper compartment, which are separated by a polycarbonate-treated membrane, was established. ³[H]thymidine incorporation of keratinocytes and lymphocytes, as well as IL-6. IL-8 and IFN-γ synthesis, were measured. Keratinocyte proliferation was significantly enhanced in the presence of PBMC from patients with AE. In contrast, PBMC from normal donors did not enhance HaCaT cell proliferation when they were cocultttred. Lymphocytes from patients with AH showed a significantly enhanced proliferation after coculture with keratinocytes. However, PBMC from normal donors did not proliferate in the presence of HaCaT cells. Keratinocyte supernalants incubated with PBMC from either atopic or normal volunteers induced a suppression of lymphocyte ³[H]thymidine incorporation. In supernatants from cocultures of PBMC from patients with AE and keratinocytes. significantly enhanced amounts of IL-6 and IL-8. compared with normal donor's lymphocytes and HaCaT cells, were measured. No differences in TFN-γ production were observed. When PBMC were cultured without HaCaT cells, supernatants contained equal levels of IL-6, IL-8 and IFN-γ in normal donors and in patients with AE. Interestingly. HaCaT cells spontaneously secrete measurable amounts of IL-6, IL-8 and IFN-γ. Blocking experiments with neutralizing antibodies against these interleukins showed a complete inhibition of keratinocyte proliferation when PBMC from normal donors were used whereas the proliferative potency of PBMC supernalants from patients with AE on keratinocytes remained. Our data indicate that (i) PBMC from patients with AE stimulate keratinocyte proliferation via soluble factor(s) that are different from IL-6. IL-8 and IEN-γ (ii) probably, HaCaT cells spontaneously produce lymphocyte/monocyle inhibitory soluble factors and IL-6, IL-8 as well as IFN-γ and (iii) secretion and/or activity of keratinocytederived inhibitory mediators is regulated via cytokines of PBMC infiltrating inflammatory skin.     

T‐lymphocyte‐induced,fas‐mediated apoptosis is associated with early keratinocyte differentiation

Please cite this paper as: T‐lymphocyte‐induced, fas‐mediated apoptosis is associated with early keratinocyte differentiation. Experimental Dermatology 2009. Abstract: The development of eczematous lesions is thought to be due in part to a breakdown in skin barrier function as a result of T lymphocytes (T cells) invading the skin causing epidermal keratinocyte apoptosis. In this study, we investigated the interaction of T cells and keratinocytes on apoptosis and terminal differentiation using an in vitro co‐culture system. Experiments were performed using the HaCaT keratinocyte cell line or normal human epidermal keratinocytes. Activated human peripheral blood‐derived T cells were found to induce Fas‐dependent keratinocyte apoptosis by up to sixfold. Increased Fas was associated with increased IFN‐γ. The T‐cell apoptotic signal was found to target preferentially keratinocytes in the very early stages of terminal differentiation, such as those with low levels of α6‐integrin expression, and result in subsequent increased caspase 3 activity. This observation was accompanied by a marked increase in keratinocyte ICAM‐1 expression and its ligand LFA‐1 on T cells. Our data suggest that T cells may initiate the onset of keratinocyte terminal differentiation making them more susceptible to Fas‐dependent cell death signals delivered by the T cells.