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Keratinocyte Survival, Differentiation, and Death: Many Roads Lead to Mitogen-Activated Protein Kinase

      The epidermis is a dynamic and continually renewing surface that provides and maintains a life-sustaining interface with the environment. The epidermal keratinocyte, the major cell type of the epidermis, undergoes a complex and carefully choreographed program of differentiation. This process requires a balance between keratinocyte proliferation, differentiation, and apoptosis. This overview will concentrate on cascades that regulate the balance between keratinocyte cell proliferation and survival, and apoptosis and cell differentiation, with a particular emphasis on the role of the mitogen-activated protein kinase cascades. A summary of the literature suggests that extracellular regulated kinases function to promote keratinocyte proliferation and survival, whereas p38 mitogen-activated protein kinase functions to promote differentiation and apoptosis.

      Keywords

      In recent decades study of keratinocyte differentiation has yielded substantial new insights. Early efforts identified and cataloged many of the morphologic properties of keratinocytes. Progress was limited, however, by the lack of a suitable in vitro system for the study of keratinocyte differentiation. The 1970s heralded the arrival of various cell culture models that permitted keratinocytes to be grown in mass culture (
      • Rheinwald J.G.
      • Green H.
      Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.
      ;
      • Elgjo K.
      • Hennings H.
      • Michael D.
      • Yuspa S.H.
      Natural synchrony of newborn mouse epidermal cells in vitro.
      ;
      • Bettger W.J.
      • Boyce S.T.
      • Walthall B.J.
      • Ham R.G.
      Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexamethasone.
      ;
      • Boyce S.T.
      • Ham R.G.
      Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.
      ). This, in turn, accelerated progress in identifying major structural proteins that participate in formation of differentiated structures. The evolution of culture technology coupled with the revolution in gene analysis technologies led to the identification of a host of proteins that form differentiated structures in keratinocytes (
      • Green H.
      The keratinocyte as differentiated cell type.
      ;
      • Fuchs E.
      • Byrne C.
      The epidermis: rising to the surface.
      ;
      • Eckert R.L.
      • Crish J.F.
      • Robinson N.A.
      The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation.
      ). The ability to isolate the genes encoding these proteins subsequently identified DNA sequences that regulate gene expression. In recent years, the knowledge built during the 1970s and 1980s has led to a substantial interest in understanding how keratinocyte differentiation is regulated and in elucidating the signaling mechanisms that control gene expression.

      Keratinocyte Survival, Differentiation, and Death – A Complicated Relationship

      Keratinocytes begin as proliferation-competent basal layer cells that are characterized by expression of specific basal-cell-associated marker proteins. These cells, in turn, give rise to daughter cells that terminally differentiate. Cell differentiation results in the stacking of multiple keratinocyte layers above the basal zone, forming the spinous, granular, and cornified layers (
      • Eckert R.L.
      • Crish J.F.
      • Robinson N.A.
      The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation.
      ). Each layer expresses specific gene products that are required for the differentiation process, and expression of each gene product requires specific regulatory inputs. In general, cultured keratinocytes, and keratinocytes in epidermis, undergo differentiation (
      • Gandarillas A.
      Epidermal differentiation, apoptosis, and senescence: common pathways?.
      ), a slow process that involves substantial changes in the intracellular pattern of protein expression. During this process, keratinocytes enlarge, flatten, and are ultimately released from the epithelial surface (
      • Eckert R.L.
      • Crish J.F.
      • Robinson N.A.
      The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation.
      ). In addition to differentiation, however, there is another pathway available –programmed cell death. The programmed cell death (apoptotic) pathway is best characterized as resulting from exposure to harmful stimuli such as ultraviolet (UV) radiation. In contrast to differentiation, apoptosis is executed on a time scale of hours and results in elimination of individual cells from the tissue. In UVB-exposed epidermis, apoptosis is associated with sunburn cell formation. Differentiation and apoptosis are distinct processes that share significant overlap with respect to marker gene expression. Moreover, the selection of a particular pathway depends upon environmental factors (
      • Mitra R.S.
      • Wrone-Smith T.
      • Simonian P.
      • et al.
      Apoptosis in keratinocytes is not dependent on induction of differentiation.
      ;
      • Gandarillas A.
      • Goldsmith L.A.
      • Gschmeissner S.
      • et al.
      Evidence that apoptosis and terminal differentiation of epidermal keratinocytes are distinct processes.
      ;
      • Takahashi H.
      • Aoki N.
      • Nakamura S.
      • et al.
      Cornified cell envelope formation is distinct from apoptosis in epidermal keratinocytes.
      ). For example, treatment of cultured keratinocytes with ionophore results in differentiation that is associated with activation of caspase-14 (
      • Lippens S.
      • Kockx M.
      • Knaapen M.
      • et al.
      Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing.
      ) but not other caspases (
      • Takahashi H.
      • Aoki N.
      • Nakamura S.
      • et al.
      Cornified cell envelope formation is distinct from apoptosis in epidermal keratinocytes.
      ). In contrast, treating keratinocytes with UVB produces annexin V- and propidium iodide-, and caspase 3 positive cells, indicative of an apoptotic process (
      • Takahashi H.
      • Aoki N.
      • Nakamura S.
      • et al.
      Cornified cell envelope formation is distinct from apoptosis in epidermal keratinocytes.
      ). In addition the death receptor signaling pathway, including Fas and Fas ligand, has now been shown to play a role in the UVB-dependent keratinocyte apoptosis (
      • Wehrli P.
      • Viard I.
      • Bullani R.
      • et al.
      Death receptors in cutaneous biology and disease.
      ). The net sum of these findings suggests that apoptosis and differentiation are distinct processes that share many overlapping features and common markers (
      • Gandarillas A.
      Epidermal differentiation, apoptosis, and senescence: common pathways?.
      ).
      There is at present an active search, which will continue in the next decades, to identify signaling pathways that regulate cell survival and death. To achieve a balance between differentiation, apoptosis, and proliferation, keratinocytes utilize multiple countervailing mechanisms – some of which promote survival, whereas others promote extinction. These studies have identified regulatory cascades that play a central role in regulating keratinocyte differentiation. This review will examine the contribution of several regulatory cascades on these processes, with a particular emphasis on the role of the mitogen-activated protein kinase (MAPK) cascades (
      • Davis R.J.
      Transcriptional regulation by MAP kinases.
      ).

      Mapk Cascades

      At least three major MAPK pathways, the ERK1/2, JNK, and p38 MAPK cascades, have been described (Figure 1). Each cascade includes a MEK kinase (MEKK), which activates a MAPK/ERK kinase (MEK), which activates a MAPK (
      • Robinson M.J.
      • Cobb M.H.
      Mitogen-activated protein kinase pathways.
      ;
      • Ichijo H.
      From receptors to stress-activated MAP kinases.
      ). Each pathway, in turn, differentially regulates downstream targets including transcription factors. The activated transcription factors, in turn, bind to DNA elements and modulate gene expression. Figure 1 shows a greatly simplified schematic of these pathways in which each pathway is represented in a linear format. In fact, a great many studies describe extensive crosstalk among these cascades wherein a particular kinase in one pathway influences a kinase in another pathway. For example, a MEKK1, MEK3, p38 MAPK pathway regulates involucrin gene expression (
      • Efimova T.
      • LaCelle P.
      • Welter J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.
      ), and MEKK1 and MEK1 regulate SPRR1B expression (
      • Vuong H.
      • Patterson T.
      • Shapiro P.
      • et al.
      Phorbol ester-induced expression of airway squamous cell differentiation marker, SPRR1B, is regulated by protein kinase cδ/Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway.
      ). These types of observations illustrate that these cascades are wired into highly interdependent regulatory networks, and further suggest that the ultimate regulatory outcome (proliferation, survival, apoptosis, differentiation) is likely to be dependent upon the balance between regulatory inputs.
      Figure thumbnail gr1
      Figure 1The MAPK cascades. Incoming signals that regulate proliferation, differentiation, and apoptosis are shuttled through three major MAPK cascades. These are named based on the target of a three-kinase module that includes a MAPK kinase kinase, a MAPK kinase, and a MAPK. The MAPKs are ERK1/2, JNK1/2, and p38. For simplicity, these pathways are presented as linear cascades. There is substantial interconnection among the cascades at all levels, however.

      Pro-Differentiation Pathways

      The MAPKs (Figure 1) are central players in regulating keratinocyte differentiation. Recent studies suggest that a variety of pro-differentiation signaling kinases channel differentiation-promoting input into the MAPK cascades. Apoptosis signaling kinase (ASK1) is a MAPK kinase kinase (MEKK) involved in stress-induced signaling (
      • Matsuzawa A.
      • Ichijo H.
      Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating kinase 1.
      ). ASK1 has been suggested to have a role in regulating keratinocyte differentiation, as it is expressed in the suprabasal epidermal layers (
      • Sayama K.
      • Hanakawa Y.
      • Shirakata Y.
      • et al.
      Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation.
      ). Moreover, expression of constitutively active ASK1 in keratinocytes activates JNK and p38 MAPK-associated signaling (
      • Sayama K.
      • Hanakawa Y.
      • Shirakata Y.
      • et al.
      Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation.
      ). Differentiation-associated marker gene expression (transglutaminase, loricrin, involucrin) is also increased when ASK1 is overexpressed (
      • Sayama K.
      • Hanakawa Y.
      • Shirakata Y.
      • et al.
      Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation.
      ). This kinase appears to be linked to p38 MAPK, as inhibition of p38 activity stops these ASK1-dependent responses.
      Involucrin, a marker of early keratinocyte differentiation, is expressed in the spinous and granular cell layers (
      • Rice R.H.
      • Green H.
      Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.
      ;
      • Eckert R.L.
      • Crish J.F.
      • Robinson N.A.
      The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation.
      ). Studies designed to identify mechanisms that regulate hINV gene expression located two elements in the hINV promoter, the distal- and proximal-regulatory regions, that mediate gene activation (
      • Welter J.F.
      • Gali H.
      • Crish J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by POU domain proteins.
      ;
      • Efimova T.
      • LaCelle P.
      • Welter J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.
      ). Extensive studies of the DNA elements in these regions identify AP1, Sp1, and C/EBP as the transcription factors responsible for basal and differentiation-associated gene expression (
      • Welter J.F.
      • Gali H.
      • Crish J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by POU domain proteins.
      ;
      • Banks E.B.
      • Crish J.F.
      • Welter J.F.
      • Eckert R.L.
      Characterization of human involucrin promoter distal regulatory region transcriptional activator elements - a role for Sp1 and AP1 binding sites.
      ;
      • Banks E.B.
      • Crish J.F.
      • Eckert R.L.
      Transcription factor Sp1 activates involucrin promoter activity in non-epithelial cell types.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK7-dependent activation of p38 MAP kinase in keratinocytes.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK6 regulates human involucrin gene expression via a p38α- and p38δ-dependent mechanism.
      ). Signal transduction analyses, using dominant-negative kinases and kinase inhibitors, show that activation is mediated by a cascade that includes protein kinase c (PKC) (
      • Efimova T.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by novel protein kinase C isoforms.
      ), Ras, MEKK1 (
      • Efimova T.
      • LaCelle P.
      • Welter J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.
      ), various MEK isoforms (
      • Efimova T.
      • LaCelle P.
      • Welter J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK7-dependent activation of p38 MAP kinase in keratinocytes.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK6 regulates human involucrin gene expression via a p38α- and p38δ-dependent mechanism.
      ), and p38 MAPK (
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK7-dependent activation of p38 MAP kinase in keratinocytes.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK6 regulates human involucrin gene expression via a p38α- and p38δ-dependent mechanism.
      ). Induction of gene expression is strictly associated with activation of the novel PKC isoforms (
      • Efimova T.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by novel protein kinase C isoforms.
      ). This pro-differentiation role for the novel PKC isoforms is consistent with other studies showing that nPKC isoforms promote differentiation and/or apoptosis (
      • Ohba M.
      • Ishino K.
      • Kashiwagi M.
      • et al.
      Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the η and δ isoforms of protein kinase C.
      ;
      • Li L.
      • Lorenzo P.S.
      • Bogi K.
      • et al.
      Protein kinase Cδtargets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector.
      ). In contrast, the classical and atypical PKC isoforms do not appear to be involved (
      • Li L.
      • Lorenzo P.S.
      • Bogi K.
      • et al.
      Protein kinase Cδtargets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector.
      ;
      • Efimova T.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by novel protein kinase C isoforms.
      ), and recent studies indicate that the classical PKC isoforms antagonize the calcium-dependent activation of hINV gene expression (
      • Deucher A.
      • Efimova T.
      • Eckert R.L.
      Calcium-dependent involucrin expression is inversely regulated by protein kinase C (PKC) α and PKCδ.
      ). These studies also indicate that Ras activity is required for expression and that the Ras signal is conveyed downstream to MEKK1 (
      • Efimova T.
      • LaCelle P.
      • Welter J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.
      ). The MEKK1 signal is then transferred downstream via activation of MEK1, MEK3, MEK6, and/or MEK7 (
      • Efimova T.
      • LaCelle P.
      • Welter J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK7-dependent activation of p38 MAP kinase in keratinocytes.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK6 regulates human involucrin gene expression via a p38α- and p38δ-dependent mechanism.
      ). The specific MEK isoform involved in the regulation may depend upon the source of the incoming differentiation signal, as each MEK isoform differentially regulates MAPK activation. In addition, the differentiation-positive signal is associated with reduced ERK1/2 activity (Efimova and Eckert, unpublished). p38α and p38δ are the major p38 MAPK isoforms that mediate the activation of gene expression (
      • Efimova T.
      • LaCelle P.
      • Welter J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK7-dependent activation of p38 MAP kinase in keratinocytes.
      ;
      • Dashti S.R.
      • Efimova T.
      • Eckert R.L.
      MEK6 regulates human involucrin gene expression via a p38α- and p38δ-dependent mechanism.
      ).
      MAPK cascades have also been reported to regulate expression of other markers of differentiation, including SPRR1B (
      • Vuong H.
      • Patterson T.
      • Shapiro P.
      • et al.
      Phorbol ester-induced expression of airway squamous cell differentiation marker, SPRR1B, is regulated by protein kinase cδ/Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway.
      ). A pathway that includes PKCδ, Ras, MEKK1, MEK, and AP1 regulates this gene. A specific MAPK isoform has not been implicated. The human cystatin A gene has also been studied. Cystatin A expression is positively regulated by a pathway that includes Ras, MEKK1, MEK7, JNK, and negatively regulated by a Ras, Raf1, MEK1, ERK1/2 pathway (
      • Takahashi H.
      • Honma M.
      • Ishida-Yamamoto A.
      • et al.
      Expression of human cystatin A by keratinocytes is positively regulated via the Ras/MEKK1/MKK7/JNK signal transduction pathway but negatively regulated via the Ras/Raf-1/MEK1/ERK pathway.
      ). These findings are particularly interesting, as they imply a role for Ras in promoting differentiation, although Ras can also function in a positive manner to promote cancer progression (
      • Denning M.F.
      • Dlugosz A.A.
      • Howett M.K.
      • Yuspa S.H.
      Expression of an oncogenic rasHa gene in murine keratinocytes induces tyrosine phosphorylation and reduced activity of protein kinase Cδ.
      ). PKC activity is also required for in-creased transglutaminase activity (
      • Dlugosz A.A.
      • Yuspa S.H.
      Protein kinase C regulates keratinocyte transglutaminase (TGK) gene expression in cultured primary mouse epidermal keratinocytes induced to terminally differentiate by calcium.
      ) and this response is mediated by novel PKC isoforms (
      • Ueda E.
      • Ohno S.
      • Kuroki T.
      • et al.
      The η isoform of protein kinase C mediates transcriptional activation of the human transglutaminase 1 gene.
      ).
      PKCη, which is known to promote keratinocyte differentiation via MAPK cascade activation (
      • Efimova T.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by novel protein kinase C isoforms.
      ), also appears to work by a second mechanism – that of forming a complex with cyclinE/cdk2/p21 to reduce cdk2 activity (
      • Ishino K.
      • Ohba M.
      • Kashiwagi M.
      • et al.
      Phorbol ester-induced G1 arrest in BALB/MK-2 mouse keratinocytes is mediated by δ and η isoforms of protein kinase C.
      ;
      • Kashiwagi M.
      • Ohba M.
      • Watanabe H.
      • et al.
      PKCη associates with cyclin E/cdk2/p21 complex, phosphorylates p21 and inhibits cdk2 kinase in keratinocytes.
      ). This may provide a complementary mechanism, permitting PKCη to reduce proliferation while concomitantly enhancing differentiation. In addition, PKCη interacts with Fyn, an Src kinase family member that is required for normal keratinocyte differentiation (
      • Cabodi S.
      • Calautti E.
      • Talora C.
      • et al.
      A PKC-η/Fyn-dependent pathway leading to keratinocyte growth arrest and differentiation.
      ). Fyn appears to reduce keratinocyte proliferation by downregulating epidermal growth factor receptor (EGFR) signaling. PKCη activity is necessary for Fyn activation and the two proteins are associated (
      • Cabodi S.
      • Calautti E.
      • Talora C.
      • et al.
      A PKC-η/Fyn-dependent pathway leading to keratinocyte growth arrest and differentiation.
      ). This result suggests that PKCη suppresses keratinocyte proliferation via a mechanism that involves Fyn-dependent downregulation of EGFR function.
      In general, the above studies suggest that pro-differentiation stimuli act via novel PKCs and various MAPK kinase kinases, including MEKK1 and ASK1, to enhance keratinocyte differentiation. In addition, these stimuli also appear to suppress EGFR activity.

      Pro-Apoptosis Pathways

      UVB light is an important apoptotic stimulus that has been extensively studied. UVB exposure produces a number of signaling-related changes in cell function. Efforts to understand the UVB-dependent death process has led to the identification of specific agents that inhibit cell death. Several of these findings are summarized below. UVB exposure on HaCaT cells is associated with sustained activation of p38 MAPK, mitochondrial cytochrome c release, and caspase-3 activation (
      • Assefa Z.
      • Vantieghem A.
      • Garmyn M.
      • et al.
      p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.
      ). Inhibition of p38 MAPK activity inhibits these responses. A related study in HaCaT cells reports UVB-associated p38 MAPK and JNK activation (
      • Shimizu H.
      • Banno Y.
      • Sumi N.
      • et al.
      Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.
      ). Moreover, SB203580 inhibits the apoptotic response, further suggesting a pro-apoptotic role for p38 MAPK (
      • Shimizu H.
      • Banno Y.
      • Sumi N.
      • et al.
      Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.
      ). In normal human keratinocytes, UVB treatment increases reactive oxygen species associated events and activation of p38 and ERK1/2. The ERK1/2 activation is transient, whereas the p38 activation is sustained (
      • Peus D.
      • Vasa R.A.
      • Beyerle A.
      • et al.
      UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.
      ). Inhibition of ERK1/2, using the MEK1/2 inhibitor PD98059, results in enhanced apoptosis (
      • Peus D.
      • Vasa R.A.
      • Beyerle A.
      • et al.
      UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.
      ). UVB treatment also increases p38 phosphorylation in SV40 transformed human keratinocytes (
      • Nakamura S.
      • Takahashi H.
      • Kinouchi M.
      • et al.
      Differential phosphorylation of mitogen-activated protein kinase families by epidermal growth factor and ultraviolet B irradiation in SV40-transformed human keratinocytes.
      ). Taken together, these studies suggest that ERK1/2 is pro-survival and antiapoptotic, and p38 activation is pro-apoptotic.
      Upstream signaling kinases have also been implicated in regulation of cell death. UVB treatment of keratinocytes results in the release of the catalytic fragment of PKCδ into the soluble phase (
      • Denning M.F.
      • Wang Y.
      • Nickoloff B.J.
      • Wrone-Smith T.
      Protein kinase Cδ is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis of human keratinocytes.
      ). This is associated with increased soluble PKCδ catalytic activity and caspase activation. Caspase activation may regulate PKCδ cleavage, as caspase inhibitors block the UVB-dependent formation of soluble PKCδ, and apoptosis (
      • Denning M.F.
      • Wang Y.
      • Nickoloff B.J.
      • Wrone-Smith T.
      Protein kinase Cδ is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis of human keratinocytes.
      ). Moreover, in normal keratinocytes, PKCδ overexpression coupled with phorbol ester treatment results in localization of PKCδ to the mitochondria followed by alteration of mitochondrial membrane potential and cell death. This response requires PKCδ activity, as it is attenuated by PKC inhibitors (
      • Li L.
      • Lorenzo P.S.
      • Bogi K.
      • et al.
      Protein kinase Cδtargets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector.
      ). Vitamin-D-associated apoptosis is also associated with activation of MEKK1 and p38 MAPK, and loss of MEK1 and ERK1/2 activity (
      • McGuire T.F.
      • Trump D.L.
      • Johnson C.S.
      Vitamin D (3)-induced apoptosis of murine squamous cell carcinoma cells. Selective induction of caspase-dependent MEK cleavage and up-regulation of MEKK-1.
      ).

      Pro-Survival Pathways

      Several agents that target MAPK cascade function are important in promoting keratinocyte survival. EGFR is a transmembrane tyrosine kinase that activates a variety of intracellular signaling cascades. In keratinocytes, EGFR promotes survival via effects on MAPK cascade activity (
      • Mendelsohn J.
      • Baselga J.
      The EGF receptor family as targets for cancer therapy.
      ;
      • Prenzel N.
      • Fischer O.M.
      • Streit S.
      • et al.
      The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification.
      ). Culturing keratinocytes in suspension medium or treating with UVB radiation results in cell death. Treatment with EGF counters this tendency. EGFR stimulates keratinocyte survival in suspension culture via a mechanism that requires MEK1/2 activity, as shown by inhibition of the survival response by PD98059, an MEK1/2 inhibitor, or by expression of dominant-negative MEK1 (
      • Jost M.
      • Huggett T.M.
      • Kari C.
      • et al.
      Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-xL expression through a MEK-dependent pathway.
      ;
      • Jost M.
      • Huggett T.M.
      • Kari C.
      • Rodeck U.
      Matrix-independent survival of human keratinocytes through an EGF receptor/MAPK-kinase-dependent pathway.
      ). Keratinocyte apoptosis is also stimulated by UVB treatment, and, perhaps paradoxically, is associated with activation of EGFR. EGFR, in turn, activates ERK1/2 and p38 MAPK (
      • Peus D.
      • Vasa R.A.
      • Meves A.
      • et al.
      UVB-induced epidermal growth factor receptor phosphorylation is critical for downstream signaling and keratinocyte survival.
      ). Inhibition of EGFR activation by the EGFR-specific inhibitor PD153035 results in enhanced UVB-dependent apoptosis (
      • Peus D.
      • Vasa R.A.
      • Meves A.
      • et al.
      UVB-induced epidermal growth factor receptor phosphorylation is critical for downstream signaling and keratinocyte survival.
      ). Moreover, treatment with this inhibitor results in reduced ERK1/2 activity, indicating that ERK is required for the survival response (
      • Jost M.
      • Class R.
      • Kari C.
      • et al.
      A central role of Bcl-X (L) in the regulation of keratinocyte survival by autocrine EGFR ligands.
      ;
      • Peus D.
      • Vasa R.A.
      • Meves A.
      • et al.
      UVB-induced epidermal growth factor receptor phosphorylation is critical for downstream signaling and keratinocyte survival.
      ). Thus, EGFR activation may represent an effort by the cell to promote survival in the face of the apoptotic stimulus.
      The nuclear factor κB (NFκB) cascade has also been implicated in keratinocyte survival. NFκB interacts with IκBα, an inhibitor of NFκB activity, to regulate the level of active NFκB (
      • Joyce D.
      • Albanese C.
      • Steer J.
      • et al.
      NF-κB and cell-cycle regulation: th.
      ). Expression of the super-repressor form of IκBα in transgenic mouse epidermis results in hyperplasia and increased sensitivity to UVB-dependent apoptosis (
      • van Hogerlinden M.
      • Rozell B.L.
      • Ahrlund-Richter L.
      • Toftgard R.
      Squamous cell carcinomas and increased apoptosis in skin with inhibited Rel/nuclear factor-κB signaling.
      ). Other survival agents also function via NFκB. Interferon-γ (IFNγ)/phorbol ester cotreatment results in enhanced survival of UVB-challenged keratinocytes (
      • Qin J.Z.
      • Chaturvedi V.
      • Denning M.F.
      • et al.
      Role of NF-κB in the apoptotic-resistant phenotype of keratinocytes.
      ), and this survival is impaired &1QJ;when dominant-negative IκBα is expressed. In addition, IFNγ/phorbol ester treatment inhibits caspase-3, caspase-8, and PARP activation in response to UVB treatment (
      • Chaturvedi V.
      • Qin J.Z.
      • Denning M.F.
      • et al.
      Apoptosis in proliferating, senescent, and immortalized keratinocytes.
      ;
      • Qin J.Z.
      • Chaturvedi V.
      • Denning M.F.
      • et al.
      Role of NF-κB in the apoptotic-resistant phenotype of keratinocytes.
      ).
      Proteins associated with adhesion and adherens junction formation can also function in cell survival via regulation of MAPK function. An example is β1-integrin. Inhibition of β1-integrin function by expression of dominant-negative β1-integrin in cultured keratinocytes results in reduced MAPK activity and cell exit from the stem cell compartment (
      • Zhu A.J.
      • Haase I.
      • Watt F.M.
      Signaling via β1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro.
      ). Overexpression of wild-type β1-integrin restores normal regulation. Expression of dominant-negative MEKK1 decreases adhesiveness and stem cell number similar to β1-integrin knockout, suggesting that the MAPK cascade is downstream of the β1-integrin signal. These studies suggest that β1-integrin and MAPK cooperate to maintain the epidermal stem cell compartment (
      • Zhu A.J.
      • Haase I.
      • Watt F.M.
      Signaling via β1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro.
      ;
      • Haase I.
      • Hobbs R.M.
      • Romero M.R.
      • et al.
      A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis.
      ). Forced β1-integrin expression in suprabasal epidermal layers results in increased MAPK activity in basal and suprabasal keratinocytes, and enhanced cell proliferation (
      • Haase I.
      • Hobbs R.M.
      • Romero M.R.
      • et al.
      A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis.
      ).
      Other cell adhesion/junction proteins also interface with the MAPK cascades. α-Catenin ablation in mouse epidermis leads to hyperproliferation coupled with the appearance of subrabasal mitoses and multinucleated cells, and is associated with MAPK activation (
      • Vasioukhin V.
      • Bauer C.
      • Degenstein L.
      • et al.
      Hyperproliferation and defects in epithelial polarity upon conditional ablation of α-catenin in skin.
      ). This result suggests that α-catenin expression may be required to maintain cells in the nonproliferative state. In HaCaT cells, E-cadherins stimulate MAPK activity through the ligand-independent activation of EGFR (
      • Pece S.
      • Gutkind J.S.
      Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation.
      ). Inhibition of adherens junction formation by anti-E-cadherin antibody inhibits MAPK activation, as does AG1478, an EGFR inhibitor. PKCδ expression induces serine phosphorylation of α6β4-integrin, promotes its loss from the hemidesmosome complex, and reduces keratinocyte attachment (
      • Alt A.
      • Ohba M.
      • Li L.
      • et al.
      Protein kinase Cδ-mediated phosphorylation of α6β4 is associated with reduced integrin localization to the hemidesmosome and decreased keratinocyte attachment.
      ). These studies point to a close relationship between adhesion protein status and the status of the intracellular signaling cascades.
      In addition to mediating apoptotic responses, MAPKs are also involved in processes that promote cell transformation. As noted above, UVB treatment of keratinocytes induces apoptosis. Persistent UVB exposure, however, leads to DNA damage and tumor formation. MAPK cascades have been implicated in the process. Cyclooxygenase-2 (COX-2) is an important enzyme in the synthesis of prostaglandins, which stimulate inflammation and cell proliferation. UVB treatment of cultured keratinocytes leads to increased COX-2 expression (
      • Tang Q.
      • Chen W.
      • Gonzales M.S.
      • et al.
      Role of cyclic AMP responsive element in the UVB induction of cyclooxygenase-2 transcription in human keratinocytes.
      ). The activation is due to phosphorylation of CREB and ATF1 transcription factors that then bind to the COX-2 promoter. CREB and ATF1 phosphorylation and binding to DNA require p38 MAPK activity, as evidenced by inhibition of these responses by SB202190. Urokinase-type plasminogen activator (uPA) is an enzyme involved in cell mobility and invasiveness (
      • Santibanez J.F.
      • Iglesias M.
      • Frontelo P.
      • et al.
      Involvement of the Ras/MAPK signaling pathway in the modulation of urokinase production and cellular invasiveness by transforming growth factor-β (1) in transformed keratinocytes.
      ). Inhibition of ERK1/2 activity by antisense oligonucleotides or by inhibition of MEK1 using PD98059 reduces basal uPA and eliminates the transforming growth factor β1 induction of uPA, a response that also requires Ras activity (
      • Santibanez J.F.
      • Iglesias M.
      • Frontelo P.
      • et al.
      Involvement of the Ras/MAPK signaling pathway in the modulation of urokinase production and cellular invasiveness by transforming growth factor-β (1) in transformed keratinocytes.
      ). IFNγ also increases COX-2 expression. This response is mediated via activation of EGFR and involves subsequent activation of ERK1/2 to increase COX-2 transcription (
      • Matsuura H.
      • Sakaue M.
      • Subbaramaiah K.
      • et al.
      Regulation of cyclooxygenase-2 by interferon γ and transforming growth factor α in normal human epidermal keratinocytes and squamous carcinoma cells. Role of mitogen-activated protein kinases.
      ).
      Matrix protein breakdown is another feature associated with cancer cell survival and movement. Matrix metalloproteinases (MMP) play an important role in conferring this ability on cells (
      • Stetler-Stevenson W.G.
      • Liotta L.A.
      • Kleiner Jr, D.E.
      Extracellular matrix 6: role of matrix metalloproteinases in tumor invasion and metastasis.
      ). In HaCaT cells, tumor necrosis factor α treatment stimulates ERK1/2, JNK, and p38 activity, which leads to increased MMP-1, MMP-9, and MMP-13 expression (
      • Johansson N.
      • Ala-aho R.
      • Uitto V.
      • et al.
      Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase.
      ). Blocking p38 with SB203580 inhibits these responses. Blocking MEK1 using PD98059 stops the MMP-1 and MMP-9 increase, but not the MMP-13 increase. EGFR activation also increases cell motility and MMP-9 production via an MEK1- and ERK1/2-dependent mechanism (
      • Zeigler M.E.
      • Chi Y.
      • Schmidt T.
      • Varani J.
      Role of ERK and JNK pathways in regulating cell motility and matrix metalloproteinase 9 production in growth factor-stimulated human epidermal keratinocytes.
      ). Thus, ERK1/2 and p38 signaling are important in generating the invasive phenotype.

      Summary–Mapk-Dependent Regulation In Keratinocytes

      Although exceptions can be cited, the above results suggest several general regulatory themes. First, there appears to be a consistent role for the ERK1/2 kinases in enhancing keratinocyte proliferation and survival (Figure 2). ERK1/2 appears to mediate pro-proliferation and pro-survival information from a range of different agents, including growth factors and cellular adhesion proteins. Second, agents that promote differentiation and apoptosis appear to signal these processes via a p38 MAPK-dependent mechanism (
      • Efimova T.
      • LaCelle P.
      • Welter J.F.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.
      ). This is a particularly interesting finding, as early studies, in other systems, suggested that p38 MAPK was strictly involved in mediating stress responses. Third, kinases that promote both differentiation and apoptosis appear to share overlapping pathways of regulation. For example, ASK1 is known to promote apoptosis in a variety of cell types, and in keratinocytes it promotes expression of markers of differentiation (
      • Sayama K.
      • Hanakawa Y.
      • Shirakata Y.
      • et al.
      Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation.
      ). Also, the novel PKC isoforms can promote both differentiation and/or apoptosis (
      • Ohba M.
      • Ishino K.
      • Kashiwagi M.
      • et al.
      Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the η and δ isoforms of protein kinase C.
      ;
      • Li L.
      • Lorenzo P.S.
      • Bogi K.
      • et al.
      Protein kinase Cδtargets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector.
      ;
      • Efimova T.
      • Eckert R.L.
      Regulation of human involucrin promoter activity by novel protein kinase C isoforms.
      ;
      • Vuong H.
      • Patterson T.
      • Shapiro P.
      • et al.
      Phorbol ester-induced expression of airway squamous cell differentiation marker, SPRR1B, is regulated by protein kinase cδ/Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway.
      ). The fact that these pathways are regulated by similar input suggests that there may also be common end responses. Thus, it may be difficult to distinguish one process from another. Caspase status may be useful for distinguishing these processes, as recent studies suggest that specific caspase isoforms are expressed during differentiation (caspase-14) versus apoptosis (killer caspases) (
      • Eckhart L.
      • Declercq W.
      • Ban J.
      • et al.
      Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation.
      ;
      • Eckhart L.
      • Ban J.
      • Fischer H.
      • Tschachler E.
      Caspase-14: analysis of gene structure and mRNA expression during keratinocyte differentiation.
      ;
      • Lippens S.
      • Kockx M.
      • Knaapen M.
      • et al.
      Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing.
      ;
      • Kuechle M.K.
      • Predd H.M.
      • Fleckman P.
      • et al.
      Caspase-14, a keratinocyte specific caspase: mRNA splice variants and expression pattern in embryonic and adult mouse.
      ). Fourth, cell fate is likely to depend upon the balance of input from the pro-survival and pro-differentiation/apoptosis cascades. This conclusion is based on studies pitting pro-apoptosis/pro-differentiation agents against pro-survival agents. These studies suggest that the net outcome is a response to the strength of the inputs. In general, these experiments indicate that the ERK cascade is battling the p38 MAPK cascade, suggesting that the balance of flow through the cascades is likely to determine the final fate of the cell.
      Figure thumbnail gr2
      Figure 2MAPK signaling in keratinocytes. Summary of the role of MAPK cascades in regulating keratinocyte function. In general, the ERK1/2 cascade promotes survival/proliferation, whereas the p38 MAPK cascade mediates pro-differentiation and pro-apoptosis responses.

      ACKNOWLEDGMENTS

      This work utilized the facilities of the Skin Diseases Research Center of Northeast Ohio (NIH, AR39750) and was supported by grants from the National Institutes of Health (RLE). Dr. Efimova is a Dermatology Foundation Research Fellow, and Anne Deucher is a Medical Students Training Program Fellow.

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