Keratinocyte Survival Research Articles

Inhibition of Bcl-xL expression sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli.

The epidermis is continually exposed to harmful mutagens that have the potential to cause DNA damage. To protect the skin from accumulating mutated cells, keratinocytes have developed a highly regulated mechanism of eliminating damaged cells through apoptosis. Bcl-xL is a well-described cell survival protein that when overexpressed in skin can protect keratinocytes from UV radiation-induced apoptosis. To begin to unravel the complex mechanisms that keratinocytes use to survive, we wanted to characterize the role of endogenous Bcl-xL in protecting cells from death. In this study, we describe the development and characterization of an antisense inhibitor to Bcl-xL. We show that this inhibitor reduces Bcl-xL RNA and protein in a concentration-dependent, sequence-specific manner. Furthermore, treatment of keratinocytes and epithelial cells with this inhibitor sensitizes these cells to UV-B radiation and cisplatinum treatment-induced apoptosis. Thus, these results offer direct evidence that Bcl-xL is critical in the protection of skin and epithelial cells from apoptosis and provide a basis for the role of Bcl-xL in keratinocyte and epithelial cell survival.

A Central Role of Bcl-XL in the Regulation of Keratinocyte Survival by Autocrine EGFR Ligands

The epidermal growth factor receptor has multiple roles in epidermal biology relating to growth, migration, and, as shown recently, survival of keratinocytes. In cultured keratinocytes activation of the epidermal growth factor receptor upregulates expression of Bcl-x(L), an anti-apoptotic Bcl-2 homolog. The functional contribution of epidermal growth factor receptor-dependent Bcl-x(L) expression to keratinocyte survival is poorly understood. Here we demonstrate that inhibition of the epidermal growth factor receptor tyrosine kinase activity with either an epidermal growth factor receptor antagonistic monoclonal antibody (MoAb 425) or an epidermal growth factor receptor-selective tyrosine kinase inhibitor (AG 1478) downregulated Bcl-x(L) expression in normal human keratinocytes but had no effect on expression of the pro-apoptotic Bcl-2 homologs Bad, Bak, and Bax. Bovine pituitary extract and insulin partially alleviated both, downregulation of Bcl-x(L) expression and cell death upon epidermal growth factor receptor inhibition. Forced expression of Bcl-x(L) attenuated cell death of immortalized keratinocytes (HaCaT) induced by either forced suspension (anoikis) or by epidermal growth factor receptor blockade. These results demonstrate that epidermal growth factor receptor-dependent signaling pathways control the balance of pro-apoptotic and anti-apoptotic Bcl-2 family members expressed in normal keratinocytes. Inappropriate survival supported by aberrant signaling through the epidermal growth factor receptor may contribute to the pathogenesis of psoriasis and of squamous cell carcinomas.

Activation of the insulin-like growth factor-1 receptor promotes the survival of human keratinocytes following ultraviolet B irradiation

The ultraviolet B (UVB) component of sunlight causes non-melanoma skin cancers due to the damage it inflicts on genomic DNA. The response of epidermal keratinocytes to sunlight depends on the dose of UVB received and the severity of the damage to the DNA. Mild DNA damage typically induces DNA-repair pathways and cell survival, while severe DNA damage provokes apoptosis. Primary human keratinocytes grown in serum-free media respond in a similar manner to UVB irradiation. However, we observed that keratinocytes are exquisitely more susceptible to UVB-induced apoptosis if the growth medium is depleted of exogenous growth factors. Therefore, an exogenous growth factor could provide protection from UVB-induced apoptosis. We found that the only growth factor that provided protection from UVB-induced apoptosis was insulin and that the protective effect elicited by insulin was not due to binding the insulin receptor but, rather, to activation of the insulin-like growth factor-1 (IGF-1) receptor. Additionally, activation of the IGF-1 receptor in combination with UVB irradiation induced keratinocytes to become post-mitotic. This survival function of the IGF-1 receptor in response to UVB irradiation was influenced by activation of phosphatidylinositol-3 kinase and MAP kinase. Prior to UVB irradiation, insulin or IGF-1 had little to no effect on cell growth or viability. Therefore, activation of the IGF-1 receptor in conjunction with UVB irradiation promotes keratinocyte survival at the expense of cell proliferation.

Activation of the insulin‐like growth factor‐1 receptor promotes the survival of human keratinocytes following ultraviolet B irradiation

Activation of the insulin‐like growth factor‐1 receptor promotes the survival of human keratinocytes following ultraviolet B irradiation

Neurohormones and skin diseases

An important constituent of the cellular antioxidant buffering system that controls the redox state of proteins is thioredoxin (TRX), a 13 kDa protein that catalyzes thiol-disulfide exchange reactions, regulates activation of transcription factors, and possesses several other biologic functions similar to cytokines. We have previously reported that TRX released from UVB-irradiated keratinocytes stimulates melanogenesis by upregulating MSH receptor expression and its binding activity in melanocytes. The purpose of this study was to examine the effects of TRX on keratinocytes as an autocrine factor. TRX suppressed the UVB-induced production and secretion of α-melanocyte stimulating hormone (α-MSH) and of adrenocorticotropic hormone (ACTH), and also suppressed proopiomelanocortin (POMC) mRNA expression by normal human keratinocytes; however, TRX upregulated melanocortin 1 receptor (MC1-R) expression synergistically with UVB in normal human keratinocytes. These results suggest that exogenous TRX regulates expression of those genes in different manners. Furthermore, addition of an antibody against TRX induced cell death in keratinocytes, probably due to enhanced signaling of MSH, as it has been shown that MSH suppresses heat shock protein (hsp) 70 expression in differentiated keratinocytes, which express high levels of MC1-R and decreases their survival rate during oxidative stress. Taken together, the results suggest that keratinocyte-derived TRX regulates the expression of stress inducible neuropeptides and their receptor, and is critically involved in the survival of keratinocytes.

DNA repair and cell survival of normal and xeroderma pigmentosum keratinocytes after UV irradiation

A normal level of UV-induced DNA-repair synthesis (UDS) was observed in fibroblasts from a patient affected by trichothiodystrophy (TTD) without photosensitivity. This finding indicates that the hypersensitivity to UV light and the reduced UDS due to the presence of xeroderma pigmentosum complementation group D mutation (XP-D), described in photosensitive TTD patients, are not constantly associated with TTD. Complementation analysis in heterokaryons, obtained by fusion of repair-proficient with repair-deficient TTD cells, demonstrates that cell from the patient showing normal photosensitivity are able to restore UDS in UV-hypertensitive TTD cells.

The photoprotective effect of 1,25-dihydroxyvitamin D 3 on ultraviolet light B-induced damage in keratinocyte and its mechanism of action

We investigated the photoprotective effect of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) both in vivo and in vitro, revealing its relationship with glutathione, a well-known antioxidant. We also probed into the possible mechanism of photoprotection of 1,25(OH) 2D 3 through immunohistochemical study for metallothionein (MT). At the same time, endogenous antioxidant effect of 1,25(OH) 2D 3 was examined. Survival of cultured human keratinocytes was decreased when the cells were irradiated with ultraviolet light-B (UVB) at doses above 30 mJ/cm 2. But in the presence of 1,25(OH) 2D 3 (12 nM), the decrease of survival of keratinocytes by UVB was diminished. The formation of sunburn cells by UVB irradiation in the skin of ICR mice was inhibited by topical application of 1,25(OH) 2D 3, regardless of prior glutathione depletion. Immunohistochemical staining revealed that 1,25(OH) 2D 3 induced the expression of MT, a potent radical scavenger, mainly in the basal layer of ICR mice skin. 1,25(OH) 2D 3 neither inhibited peroxidation of plasma lipids nor interacted with superoxide, nor removed hydrogen peroxide as an antioxidant. These findings suggest that 1,25(OH) 2D 3 has photoprotective effect not related with glutathione or its endogenous antioxidant property. Rather, it could be attributed to 1,25(OH) 2D 3-induced MT and its capacity to prevent radical-related damage in UVB irradiation.

Immunogenicity of cultured keratinocyte allografts deficient in major histocompatibility complex antigens.

Full-thickness (FT) and cultured keratinocyte (CK) allografts have been used as temporary skin replacements in patients with massive burns, but these grafts are ultimately rejected after restoration of host immunocompetence. Genetic engineering has permitted the creation of knockout (KO) mice deficient in class I or class II major histocompatibility antigens. This study examines the immunogenicity of such grafts to determine if these genetically modified keratinocytes could be used for permanent wound coverage. Host sensitization to alloantigen was assessed by second-set rejection. CBA mice (n = 111) were primed with flank grafts consisting of FT and CK allografts from normal C57BL/6 donors, FT and CK class I KO allografts, FT and CK class II KO allografts, and CK autografts. Three weeks later, hosts were challenged with normal tail allografts and observed for second-set rejection. Median graft survival was analyzed by chi2 and Wilcoxon rank tests. In the second experiment, cytotoxic T lymphocytes (CTLs) were harvested from CBA mice (n = 28) 3 weeks after flank grafting. CTL effectors were tested on radiolabeled targets at various ratios in a 51Cr release assay. Dilution curves of CTL activity were compared by analysis of variance. Hosts primed with CK or FT allografts demonstrated accelerated rejection of second-set tail grafts compared with hosts covered with CBA autografts. CK knockout grafts were less immunogenic than FT knockout skin; class II KO allografts were considerably less immunogenic than class I KO allografts. CTL activity against the knockout CK allografts was negligible compared with that of hosts primed with normal allografts or FT knockout allografts. Although full-thickness knockout skin retains substantial immunogenicity, cultured keratinocytes deficient in class II antigens fail to prime for accelerated second-set rejection and do not elicit a CTL response in the graft recipient. This lack of immunogenicity may permit the indefinite survival of allogeneic knockout keratinocytes in patients requiring massive wound excision and coverage.

EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL.

Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-2 family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), increased the expression of Bcl-XL in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.

UVB-induced H2O2 activates ERK1/2 but not P38 pathway via EGFR phosphorylation: Effects on keratinocyte growth and survival

UVB-induced H2O2 activates ERK1/2 but not P38 pathway via EGFR phosphorylation: Effects on keratinocyte growth and survival

Survival of human epidermal keratinocytes after short-duration high temperature: synthesis of HSP70 and IL-8.

Thermal injury by short pulses (1-30 s) of relatively high temperature (50-68 degrees C) was investigated in normal human epidermal keratinocytes (NHEK). NHEK were cultured on plastic cover-slips and dipped in medium held at various temperatures. Survival assessed by methylthiazol tetrazolium reduction assay at 6 days postheating demonstrated an inverse time-temperature relationship that indicated that most cells could survive after a 1-s, 60 degrees C exposure or a 30-s, 55 degrees C exposure. Arrhenius plots of the data indicated major transition points for cell injury at 50 and 60 degrees C. Heat shock protein 70 (HSP70) and interleukin-8 (IL-8) were both induced by elevation of temperature between 50 and 60 degrees C for as short a time as 1 s. HSP70 synthesis stimulated by short, high pulses of heat appeared to induce thermotolerance. These results demonstrate that brief exposure to relatively high temperature can induce HSP70 and IL-8 synthesis in keratinocytes.

Regulation of Bcl-xL expression in human keratinocytes by cell-substratum adhesion and the epidermal growth factor receptor.

Cell-substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell-substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-xL. Expression of Bcl-xL protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-xL steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-xL expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-xL represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-xL expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.

EGF-R dependent regulation of keratinocyte survival.

Tissue organization and maintenance within multicellular organisms is in part dependent on the ability of cells to undergo programmed cell death or apoptosis. Conversely, disruption of cell death pathways often is associated with tumor development. At present, the molecular control of apoptosis in epithelial cells is poorly understood. Here we describe evidence linking epidermal growth factor-receptor (EGF-R) activation to survival of normal human keratinocytes in culture. Inhibition of EGF-R activation by an anti-EGF-R antagonistic monoclonal antibody (mAb 425), followed by detachment of keratinocytes from the substratum, induced extensive death with several features of apoptosis in keratinocyte cultures. Other, non-epithelial normal human cells including melanocytes and fibroblasts, did not show this effect. Similar to EGF-R blockade by mAb 425, inhibition of the EGF-R tyrosine kinase activity using tyrphostin AG1478 resulted in lack of attachment and extensive cell death upon passaging. Attachment to keratinocyte-derived ECM partially resuced mAb 425-treated keratinocytes from cell death, indicating that adhesion-dependent and EGF-R-dependent signal transduction pathways serve partially overlapping but not redundant roles in supporting keratinocyte survival.

Delayed expression of lethal mutations and genomic instability in the progeny of human epithelial cells that survived in a bystander-killing environment.

It has recently been shown that, when irradiated, human epithelial cells produce a factor or signal in the culture medium that can reduce the clonogenic survival of unirradiated cells. The mechanism is unknown, as is the nature of the signal or substance. In this paper, we show that the medium from these irradiated cells is able to induce delayed effects in the progeny of some cell types that survive the initial exposure to the medium. The initial clonogenic survival of normal human keratinocytes exposed to medium from irradiated parallel cultures is reduced by approximately 40%. If the surviving keratinocytes (60%) are grown to confluence and replated for clonogenic assay, than they still show a reduced plating efficiency, this time of approximately 20% less than the parent line. Similarly treated normal human fibroblasts showed no delayed effects either from a direct dose or from receipt of irradiated medium. The progeny of directly irradiated tumourigenic cell lines have previously been shown to have better clonogenic survival (by a factor of at least two) than unirradiated parallel cultures, and this effect was also found, although it varied depending on the cell line used, when the distant progeny of PC-3 cells or SW48 colon carcinoma cells that received medium only from irradiated progenitors were assayed for expression of delayed lethal mutations. These data suggest that the signal/factor produced in medium by irradiated cells is able to induce genomic instability-type effects in distant progeny.

Burn injury impairs second-set rejection and CTL reactivity in mice primed by cultured keratinocyte allografts.

Cultured keratinocyte (CK) allografts have limited antigenicity and have been used as a skin replacement in patients with massive thermal injury. Recent data indicate that CK grafts are more immunogenic than previously believed and could compromise wound healing in the immunocompetent host. The purpose of this study was to determine if the immunosuppression of burn injury might affect the alloantigen response and minimize sensitization to CK allografts. CBA mice received a 0%, 20%, or 40% burn that was partially excised three days later and grafted with a full-thickness (FT) skin allograft, CK allograft, or CK autograft. Two weeks postburn, mice received FT tail skin allografts, which were observed for rejection. We observed that FT and CK allografts primed the unburned host with equal efficacy. However, burn injury selectively minimized priming by CK allografts, resulting in delayed rejection of second-set allografts. With evidence that burn injury inhibits host sensitization to CK allografts, we then examined the effect of burn size on CTL alloreactivity. Additional CBA mice underwent burn injury, excision, and grafting as described above. Host splenocytes were harvested two weeks later and tested on radiolabeled targets for allospecific cytotoxicity. CTLs from unburned mice primed with FT allografts demonstrated the greatest CTL lysis, followed next by CTLs from unburned mice covered with CK allografts. Burn injury inhibited CTL activity as a function of wound size. Activity of CTLs from burned mice primed with CK allografts improved after in vitro allostimulation but remained below that of CTLs from unburned, unprimed mice. We conclude that burn injury selectively inhibits the allospecific response to CK allografts. The decreased immunogenicity of CK allografts, when used for burn wound coverage, may improve the long-term survival of allogeneic keratinocytes, enhancing their potential as a biologic skin replacement.

All-trans retinoic acid (RA) stimulates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to RA while neonatal foreskin responds differently.

Adult human skin from a sun-protected site (hip) and from a sun-exposed site (forearm) was maintained in organ culture for 12 d in the presence of a serum-free, growth factor-free basal medium. Cultures were incubated under conditions optimized for keratinocyte growth (i.e., in 0.15 mM extracellular Ca2+) or for fibroblast growth (i.e., in 1.4 mM extracellular Ca2+). Treatment with all-trans retinoic acid (RA) induced histological changes in the organ-cultured skin under both conditions which were similar to the changes seen in intact skin after topical application. These included expansion of the viable portion of the epidermis and activation of cells in the dermis. In sun-damaged skin samples, which were characterized by destruction of normal connective tissue elements and presence of thick, dark-staining elastotic fibers, a zone of healthy connective tissue could be seen immediately below the dermo-epidermal junction. This zone was more prominent in RA-treated organ cultures than in matched controls. Associated with these histological changes was an increase in overall protein and extracellular matrix synthesis. In concomitant studies, it was found that RA treatment enhanced survival and proliferation of adult keratinocytes and adult dermal fibroblasts under both low- and high-Ca2+ conditions. In all of these assays, responses of sun-protected and sun-exposed skin were identical. In contrast, responses of neonatal foreskin to RA were similar to those of adult skin in the presence of low-Ca2+ culture medium, but under conditions of high extracellular Ca2+ RA provided little or no additional stimulus. Together these studies suggest that the ability of RA to enhance repair of sun-damaged skin (documented in previous studies) may reflect its ability to influence the behavior of skin in a manner that is age dependent but independent of sun-exposure status.

All-trans-retinoic acid preserves viability of fibroblasts and keratinocytes in full-thickness human skin and fibroblasts in isolated dermis in organ culture.

Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue as the criterion, it was observed that no keratinocytes and few fibroblasts survived incubation for 10-12 days in serum-free basal medium containing a low level (0.15 mM) of extracellular Ca2+. Increasing the extracellular Ca2+ concentration to 1.4 mM or treating the tissue with 3 microM retinoic acid (RA) under low Ca2+ conditions resulted in increased keratinocyte and fibroblast survival; the two treatments together were more effective than either treatment alone. The same treatments preserved fibroblast survival when pieces of isolated dermal tissue were incubated in organ culture and also supported fibroblast survival in monolayer culture. These findings indicate that recovery of keratinocytes and fibroblasts from skin after maintenance in organ culture provides a simple but definitive measure of the viability of the major cellular elements present in the tissue. These findings suggest that RA treatment enhances survival of both fibroblasts and keratinocytes and that these effects of RA can be seen at physiological Ca2+ concentrations as well as at suboptimal levels of extracellular Ca2+. Finally, these results indicate that the dermis is a direct target of RA.

Micronuclei induction by dichlorvos in the mouse skin

Micronucleus (MN) induction in cultured keratinocytes was investigated following skin painting of HRA/Skh mice with the pesticide, dichlorvos. Whole skin and partially-purified epidermal cells from 5-6 week old male animals were cultured for 4 days in vitro after single topical applications of various concentrations of dichlorvos in vivo. Appropriate doses, allowing optimum survival of keratinocytes, were selected following an initial range-finding experiment. To evaluate MN induction in dividing cells, the cytokinesis-block method was employed. Results showed statistically significant MN at all dose levels in partially-purified epidermal cells and a positive trend with respect to dose from 51 to 1033 nmol dichlorvos. A significant increase in MN was also detectable in cultured cells from whole skin, dissociated within as little as 1 h after application of dichlorvos. Although a number of technical difficulties are associated with the skin micronucleus method, it has been used successfully in this laboratory to detect several skin carcinogens of both high and low potency. Since dichlorvos is rapidly absorbed through the skin, and can induce MN in skin cells of treated mice, this compound may therefore be considered to pose a contact hazard for exposed humans.

Cultured Skin Keratinocytes from Both Normal Individuals and Basal Cell Naevus Syndrome Patients are More Resistant to γ-rays and UV Light Compared with Cultured Skin Fibroblasts

Measurement of colony-forming ability following exposure to gamma-rays was performed on cultured skin keratinocytes and skin fibroblasts obtained from normal individuals, basal cell naevus syndrome patients (BCNS) and ataxia telangiectasia (A-T) patients. The most striking observation was the radiation resistance of 8/8 keratinocyte strains compared with fibroblasts whether from BCNS patients or normals. The single A-T keratinocyte cell strain showed the same radiosensitivity as A-T fibroblast cell strains. The differential survival of keratinocytes and fibroblasts was also observed following exposure to 254 nm UV light. The survival curves of SV40 immortalized keratinocytes and retinoblasts derived from normal individuals or BCNS patients showed large shoulder regions following exposure to gamma-rays or 254 nm UV light. An increased D37 rather than an increased D0 was therefore the feature of such curves. This contrasted with the SV40 immortalized A-T keratinocytes or fibroblasts which showed a minimal shoulder effect and an increased D0. No difference in survival was observed between BCNS and normal cells following exposure to either UV or gamma-rays.

Role of Solar Conditioning in DNA Repair Response and Survival of Human Epidermal Keratinocytes Following UV Irradiation

We have investigated the cumulative effects of sunlight exposure upon the excision-repair of UV radiation damage to DNA in epidermal keratinocytes from human donors of different ages as well as the possible effect on DNA repair of periodic conditioning of the cultured keratinocytes with sublethal UV radiation exposures. We have also compared the growth properties of UV-irradiated keratinocytes derived from habitually sun-exposed and nonexposed areas from the bodies of young and aged donors. DNA repair replication in keratinocytes from habitually sun-exposed facial skin and the less sun-exposed abdominal skin of middle-aged adults was found to be similar, with respect to both the UV dose response and the time course of repair after 20 J/m2, 254 nm. Growth and survival (after exposure up to 50 J/m2, 254 nm) were greater for keratinocytes from protected areas of the upper arm of young donors (under 18 years) than for cells from their own sun-exposed areas. Growth and survival were markedly reduced for all keratinocyte cultures from aged donors, especially those cultures developed from sun-exposed areas. Nevertheless, the DNA repair response to UV radiation was similar in all cases. The evident uncoupling of UV sensitivity from DNA repair capacity remains to be understood. Our studies confirm that the cumulative effect of sunlight exposure indeed contributes to some skin aging processes. However, we have found no indication that an overall reduction in capacity for excision-repair of UV photoproducts in keratinocyte DNA accompanies senescence in human skin.