Apoptosis: the skin from a new perspective

In this review we present skin biology from the perspective of apoptosis. We stress that apoptosis acts as an important homeostatic and defence mechanism in the developing and mature epidermis. Programmed cell death functions in establishing the architecture of the human epidermis and its appendages during development by deletion of stage-specific cells and in the adult epidermis by elimination of excess and abnormal cells. Arguments are presented to support the hypothesis that known regulators of keratinocyte growth may act as survival factors which suppress the cell death pathway. Surviving cells continue to divide until they encounter anti-proliferative factors. Then, unless cells are severely injured and die of necrosis, they will terminally differentiate to death or will die by apoptosis. The mechanisms controlling keratinocyte maturation are co-ordinated with cell position within the epidermal strata. Inappropriate regulatory signals or response of a cell inappropriate to its state will activate apoptosis. Parallels between terminally differentiating keratinocytes and apoptotic cells imply that terminal differentiation and apoptosis proceed along the same death pathway. For terminally differentiating cells, however, this pathway is more elaborate because it allows expression of tissue- and differentiation-specific genes. A model is presented that integrates apoptosis and keratinocyte growth and differentiation.     

ZNF750 Is Expressed in Differentiated Keratinocytes and Regulates Epidermal Late Differentiation Genes

Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype reminiscent of psoriasis and seborrheic dermatitis. Here we show that ZNF750 is a nuclear protein bearing a functional C-terminal nuclear localization signal. ZNF750 was specifically expressed in the epidermal suprabasal layers and its expression was augmented during differentiation, both in human skin and in-vitro, peaking in the granular layer. Silencing of ZNF750 in Ca2+-induced HaCaT keratinocytes led to morphologically apparent arrest in the progression of late differentiation, as well as diminished apoptosis and sustained proliferation. ZNF750 knockdown cells presented with markedly reduced expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, overexpression of ZNF750 in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation and with its downstream targets can serve in future elucidation of therapeutics for common diseases of skin barrier.     

Functional role of alpha7 nicotinic receptor in physiological control of cutaneous homeostasis

Non-neuronal nicotinic acetylcholine receptors (nAChRs) are abundantly expressed in skin and their function remains to be elucidated. Herein, we report that cutaneous alpha7 nAChR plays a role in the physiological control of cutaneous homeostasis. We studied in vitro effects of functional inactivation of alpha7 receptor on the expression of apoptosis regulators in keratinocytes (KC) lacking alpha7 nAChR, and extracellular matrix regulators in the skin of alpha7 knockout (KO) mice. Elimination of the alpha7 component of nicotinergic signaling in KC decreased relative amounts of the pro-apoptotic Bad and Bax at both the mRNA and the protein levels, suggesting that alpha7 nAChR is coupled to stimulation of keratinocyte apoptosis. The skin of alpha7 KO mice featured decreased amounts of the extracellular matrix proteins collagen 1alpha1 and elastin as well as the metalloproteinase-1. Taken together, these results suggest an important role for alpha7 nAChR in mediating plethoric effects of non-neuronal acetylcholine on cutaneous homeostasis.     

CYLD regulates keratinocyte differentiation and skin cancer progression in humans

CYLD is a gene mutated in familial cylindromatosis and related diseases, leading to the development of skin appendages tumors. Although the deubiquitinase CYLD is a skin tumor suppressor, its role in skin physiology is unknown. Using skin organotypic cultures as experimental model to mimic human skin, we have found that CYLD acts as a regulator of epidermal differentiation in humans through the JNK signaling pathway. We have determined the requirement of CYLD for the maintenance of epidermal polarity, keratinocyte differentiation and apoptosis. We show that CYLD overexpression increases keratinocyte differentiation while CYLD loss of function impairs epidermal differentiation. In addition, we describe the important role of CYLD in the control of human non-melanoma skin cancer progression. Our results show the reversion of the malignancy of human squamous cell carcinomas that express increased levels of CYLD, while its functional inhibition enhances the aggressiveness of these tumors which progress toward spindle cell carcinomas. We have found that the mechanisms through which CYLD regulates skin cancer progression include the control of tumor differentiation, angiogenesis and cell survival. These findings of the role of CYLD in human skin cancer prognosis make our results relevant from a therapeutic point of view, and open new avenues for exploring novel cancer therapies.     

Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis

The skin provides vital protection from infection and dehydration. Maintenance of the skin is through a constant program of proliferation, differentiation and apoptosis of epidermal cells, whereby proliferating cells in the basal layer differentiating to form the keratinized, anucleated stratum corneum. The WNT signalling pathway is known to be important in the skin. WNT signalling has been shown to be important both in epidermal development and in the maintenance and cycling of hair follicles and epidermal stem cells. However, the precise role for this pathway in epidermal differentiation remains unknown.We investigated the role of the WNT signalling inhibitor sFRP4 in epidermal differentiation. sFRP4 is expressed in both normal skin and keratinocytes in culture. Expression of sFRP4 mRNA and protein increases with keratinocyte differentiation and apoptosis, whilst exposure of keratinocytes to exogenous sFRP4 promotes apoptosis and expression of the terminal differentiation marker Involucrin.These data suggest sFRP4 promotes epidermal differentiation.     

Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death

Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of "late" differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.     

Transforming growth factor-beta-activated kinase 1 is essential for differentiation and the prevention of apoptosis in epidermis

Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the mitogen-activated protein (MAP) kinase family and is an upstream signaling molecule of nuclear factor-kappaB (NF-kappaB). Given that NF-kappaB regulates keratinocyte differentiation and apoptosis, TAK1 may be essential for epidermal functions. To test this, we generated keratinocyte-specific TAK1-deficient mice from Map3k7(flox/flox) mice and K5-Cre mice. The keratinocyte-specific TAK1-deficient mice were macroscopically indistinguishable from their littermates until postnatal day 2 or 3, when the skin started to roughen and wrinkle. This phenotype progressed, and the mice died by postnatal day 7. Histological analysis showed thickening of the epidermis with foci of keratinocyte apoptosis and intra-epidermal micro-abscesses. Immunohistochemical analysis showed that the suprabasal keratinocytes of the TAK1-deficient epidermis expressed keratin 5 and keratin 14, which are normally confined to the basal layer. The expression of keratin 1, keratin 10, and loricrin, which are markers for the suprabasal and late phase differentiation of the epidermis, was absent from the TAK1-deficient epidermis. Furthermore, the TAK1-deficient epidermis expressed keratin 16 and had an increased number of Ki67-positive cells. These data indicate that TAK1 deficiency in keratinocytes results in abnormal differentiation, increased proliferation, and apoptosis in the epidermis. However, the keratinocytes from the TAK1-deficient epidermis induced keratin 1 in suspension culture, indicating that the TAK1-deficient keratinocytes retain the ability to differentiate. Moreover, the removal of TAK1 from cultured keratinocytes of Map3k7(flox/flox) mice resulted in apoptosis, indicating that TAK1 is essential for preventing apoptosis. In conclusion, TAK1 is essential in the regulation of keratinocyte growth, differentiation, and apoptosis.     

The role of cathepsin E in terminal differentiation of keratinocytes

Cathepsin E (CatE) is predominantly expressed in the rapidly regenerating gastric mucosal cells and epidermal keratinocytes, in addition to the immune system cells. However, the role of CatE in these cells remains unclear. Here we report a crucial role of CatE in keratinocyte terminal differentiation. CatE deficiency in mice induces abnormal keratinocyte differentiation in the epidermis and hair follicle, characterized by the significant expansion of corium and the reduction of subcutaneous tissue and hair follicle. In a model of skin papillomas formed in three different genotypes of syngeneic mice, CatE deficiency results in significantly reduced expression and altered localization of the keratinocyte differentiation induced proteins, keratin 1 and loricrin. Involvement of CatE in the regulation of the expression of epidermal differentiation specific proteins was corroborated by in vitro studies with primary cultures of keratinocytes from the three different genotypes of mice. In wild-type keratinocytes after differentiation inducing stimuli, the CatE expression profile was compatible to those of the terminal differentiation marker genes tested. Overexpression of CatE in mice enhances the keratinocyte terminal differentiation process, whereas CatE deficiency results in delayed differentiation accompanying the reduced expression or the ectopic localization of the differentiation markers. Our findings suggest that in keratinocytes CatE is functionally linked to the expression of terminal differentiation markers, thereby regulating epidermis formation and homeostasis.     

RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation

The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.     

Reduced TRPC channel expression in psoriatic keratinocytes is associated with impaired differentiation and enhanced proliferation

Psoriasis is a characteristic inflammatory and scaly skin condition with typical histopathological features including increased proliferation and hampered differentiation of keratinocytes. The activation of innate and adaptive inflammatory cellular immune responses is considered to be the main trigger factor of the epidermal changes in psoriatic skin. However, the molecular players that are involved in enhanced proliferation and impaired differentiation of psoriatic keratinocytes are only partly understood. One important factor that regulates differentiation on the cellular level is Ca(2+). In normal epidermis, a Ca(2+) gradient exists that is disturbed in psoriatic plaques, favoring impaired keratinocyte proliferation. Several TRPC channels such as TRPC1, TRPC4, or TRPC6 are key proteins in the regulation of high [Ca(2+)](ex) induced differentiation. Here, we investigated if TRPC channel function is impaired in psoriasis using calcium imaging, RT-PCR, western blot analysis and immunohistochemical staining of skin biopsies. We demonstrated substantial defects in Ca(2+) influx in psoriatic keratinocytes in response to high extracellular Ca(2+) levels, associated with a downregulation of all TRPC channels investigated, including TRPC6 channels. As TRPC6 channel activation can partially overcome this Ca(2+) entry defect, specific TRPC channel activators may be potential new drug candidates for the topical treatment of psoriasis.     

Human Calmodulin-like Protein Is an Epithelial-Specific Protein Regulated during Keratinocyte Differentiation

Human calmodulin-like protein (CLP) is a calcium-binding protein down-regulated in a cell culture model of mammary tumorigenesis as well as in a majority of breast cancers in vivo. CLP down-regulation may be a result of the poorly differentiated state of these cell lines and tumors, or CLP expression may be incompatible with the uncontrolled cell growth associated with tumorigenesis. To learn more about CLP expression and regulation, we determined the distribution of CLP in various human tissues by immunohistochemistry. CLP was expressed exclusively in the epithelium of the tissues surveyed and was most abundant in thyroid, breast, prostate, kidney, and skin. CLP expression appears to increase in stratified epithelium during differentiation, as illustrated in the skin where CLP staining intensified from the basal through the spinous to the granular layers. Using a normal human keratinocyte culture model, we examined CLP expression in response to various agents known to affect keratinocyte differentiation. Agents that inhibit (epidermal growth factor, EGF) or permit (keratinocyte growth factor) terminal differentiation correspondingly regulate CLP expression. Factors modulating the EGF receptor signaling pathway were particularly potent in regulating CLP expression. CLP expression correlated with an agent's ability to promote terminal differentiation regardless of the agent's effect on keratinocyte proliferation. These studies show that CLP expression is coordinately regulated by, and may be involved in, the program of terminal differentiation in human keratinocytes and, likely, other differentiating epithelial cell types.     

Ligand Targeting of EphA2 Enhances Keratinocyte Adhesion and Differentiation via Desmoglein 1

EphA2 is a receptor tyrosine kinase that is engaged and activated by membrane-linked ephrin-A ligands residing on adjacent cell surfaces. Ligand targeting of EphA2 has been implicated in epithelial growth regulation by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2)-mitogen activated protein kinase (MAPK) pathway. Although contact-dependent EphA2 activation was required for dampening Erk1/2-MAPK signaling after a calcium switch in primary human epidermal keratinocytes, the loss of this receptor did not prevent exit from the cell cycle. Incubating keratinocytes with a soluble ephrin-A1-Fc peptide mimetic to target EphA2 further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1–based cues from their neighbors to facilitate entry into a terminal differentiation pathway.     

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca(2+) concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca(2+) regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr(-/-) mice lacking the full-length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca(2+)-signaling properties. The absence of the full-length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down-regulated in the epidermis of Casr(-/-) mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full-length CaR is required for the normal terminal differentiation of keratinocytes.     

Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing

The epidermis is a stratified squamous epithelium in which keratinocytes progressively undergo terminal differentiation towards the skin surface leading to programmed cell death. In this respect we studied the role of caspases. Here, we show that caspase-14 synthesis in the skin is restricted to differentiating keratinocytes and that caspase-14 processing is associated with terminal epidermal differentiation. The pro-apoptotic executioner caspases-3, -6, and -7 are not activated during epidermal differentiation. Caspase-14 does not participate in apoptotic pathways elicited by treatment of differentiated keratinocytes with various death-inducing stimuli, in contrast to caspase-3. In addition, we show that non-cornifying oral keratinocyte epithelium does not express caspase-14 and that the parakeratotic regions of psoriatic skin lesions contain very low levels of caspase-14 as compared to normal stratum corneum. These observations strongly suggest that caspase-14 is involved in the keratinocyte terminal differentiation program leading to normal skin cornification, while the executioner caspases are not implicated. Cell Death and Differentiation (2000) 7, 1218 - 1224     

EGFR regulation of epidermal barrier function

Keratinocyte terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammalian survival. This process is controlled, in part, by signal transduction and gene expression mechanisms, and the epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Using microarray analysis of a confluent cell density-induced model of keratinocyte differentiation, we identified 2,676 genes that are regulated by epidermal growth factor (EGF), a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all of the known essential processes of keratinocyte differentiation by 1) decreasing the expression of lipid matrix biosynthetic enzymes, 2) regulating numerous genes forming the cornified envelope, and 3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, EGF acted to impair epidermal barrier integrity, as shown by increased transepidermal water loss. As defective epidermal differentiation and disruption of barrier function are primary features of many human skin diseases, we used bioinformatic analyses to identify genes that are known to be associated with skin diseases. Compared with non-EGF-regulated genes, EGF-regulated genes were significantly enriched for skin disease genes. These results provide a systems-level understanding of the actions of EGFR signaling to inhibit keratinocyte differentiation, providing new insight into the role of EGFR imbalance in skin pathogenesis.     

UV-induced apoptosis in skin equivalents: inhibition by phorbol ester and Bcl-2 overexpression

To determine the role of apoptosis in epidermal homeostasis and to identify its regulators in skin, we have developed and characterised a physiologically relevant in vitro model of epidermal apoptosis. First, we show that keratinocyte cell death can be induced by ultraviolet irradiation within the stratified epidermis of the skin equivalent in an in vivo-like manner. DNA fragmentation and changes in the patterns of expression of p53 and Bcl-2 suggest that the mechanisms operating in UV-induced apoptosis in the skin equivalent are controlled by these factors. Secondly, we demonstrate that apoptosis in this model is amenable to modulation by exogenous factors present in the culture medium, such as phorbol ester, and by tranfected genes, as shown by overexpression of bcl-2. These studies show that the skin equivalent is a valuable model in which to determine the controllable steps of the apoptotic pathway independently of the immune system and to correlate apoptosis to the physiologic state of the keratinocyte.     

Abnormal epidermal differentiation and impaired epithelial-mesenchymal tissue interactions in mice lacking the retinoblastoma relatives p107 and p130

The functions of p107 and p130, members of the retinoblastoma family, include the control of cell cycle progression and differentiation in several tissues. Our previous studies suggested a role for p107 and p130 in keratinocyte differentiation in vitro. We now extend these data using knockout animal models. We found impaired terminal differentiation in the interfollicular keratinocytes of p107/p130-double-null mice epidermis. In addition, we observed a decreased number of hair follicles and a clear developmental delay in hair, whiskers and tooth germs. Skin grafts of p107/p130-deficient epidermis onto NOD/scid mice showed altered differentiation and hyperproliferation of the interfollicular keratinocytes, thus demonstrating that the absence of p107 and p130 results in the deficient control of differentiation in keratinocytes in a cell-autonomous manner. Besides normal hair formation, follicular cysts, misoriented and dysplastic follicles, together with aberrant hair cycling, were also observed in the p107/p130 skin transplants. Finally, the hair abnormalities in p107/p130-null skin were associated with altered Bmp4-dependent signaling including decreased DeltaNp63 expression. These results indicate an essential role for p107 and p130 in the epithelial-mesenchimal interactions.