Activation of PKCδ and p38δ MAPK during okadaic acid dependent keratinocyte apoptosis

Abstract

There is substantial interest in identifying agents that differentially activate keratinocyte differentiation versus apoptosis. Okadaic acid (OA) is a tumor promoter in mouse skin that also stimulates apoptosis of murine keratinocytes. OA also enhances human keratinocyte differentiation; however, the impact of OA treatment on apoptosis in these cells has not been examined. We show that OA promotes normal human keratinocyte apoptosis as evidenced by increased accumulation of cells having sub-G1/S DNA content, decreased mitochondrial integrity, increased annexin V binding, increased cytoplasmic cytochrome c level, and increased procaspase 3 and PARP cleavage. Cyclin A, cyclin D1, cdk2, cdk4, p53 and p21 levels are reduced. These changes are associated with release of the PKCdelta catalytic domain and increased phosphorylation of PKCdelta-T(505)-responses consistent with PKCdelta activation. In contrast, phosphorylation of PKCdelta-Y(311) is not increased. The apoptotic response is enhanced in OA treated cells in the presence of p38delta, a PKCdelta target. OA treatment selectively activated p38delta, and OA-dependent apoptosis is not inhibited by treatment with the p38alpha/beta inhibitor, SB203580. These findings are consistent with the idea that the response is mediated by p38delta. Our data indicate that OA is an agent that regulates both keratinocyte differentiation and apoptosis, and that this regulation is mediated via activation of a PKCdelta/p38delta signaling cascade.

Protein Kinase C δ Increases Kruppel-like Factor 4 Protein, which Drives Involucrin Gene Transcription in Differentiating Keratinocytes

“…ester activates this kinase (25,31,47,63,64). Our present studies show that PKC␦ overexpression or treatment with TPA increases KLF4 level in keratinocytes.…”

p38δ mitogen-activated protein kinase regulates skin homeostasis and tumorigenesis

“…52 PKCδ, a member of the novel PKC subfamily, has been implicated in regulating both the differentiation and the apoptosis processes in epidermal keratinocytes. 24,45,[54][55][56][57][58][59] We found that PKCδ potently activates both ectopically expressed and endogenous p38δ in keratinocytes. 45 PKCη and PKCε are additional members of the novel PKC subfamily that are expressed in keratinocytes.…”

“…32 Interestingly, keratinocytes undergoing apoptosis in response to various stimuli, such as H 2 O 2 or OA exposure, or co-expression of PKCδ/p38δ, consistently display concurrent p38δ activation and ERK1/2 inactivation. 9,24,52 We found that reduction in ERK1/2 activity is essential for apoptosis, since forced ERK1/2 activation, by means of expressing constitutively active form of MAP3K Raf1, a specific upstream activator of ERK1/2, inhibits PKCδ/p38δ-induced apoptosis. 52 These data suggest that a balance between prosurvival ERK1/2 signaling and proapoptotic/ prodifferentiation stress-activated protein kinase p38δ signaling is a crucial determinant of cell fate decisions.…”

“…23 The involvement of protein serine/threonine phosphatases PP1 and PP2A in downregulation of endogenous p38δ activity is suggested by finding that PP1/PP2A inhibitor okadaic acid (OA) markedly increases p38δ activity in human epidermal keratinocytes. 9,24 Interestingly, the endogenous p38δ is found in a monophosphorylated, tyrosine-182-phosphorylated, form in macrophages after LPS treatment. 25 Upon treatment with LPS and calyculin A, another inhibitor of PP1 and PP2A, p38δ activity is increased 25-fold versus LPS alone-treated cells, suggesting that threonine-180 may be efficiently dephosphorylated by the calyculin A-sensitive phosphatase(s) in this system.…”

“…Our studies also show that p38δ has a role in regulation of keratinocyte apoptosis. 24,52 This dual role for p38δ in regulation of both epidermal terminal differentiation and apoptosis may not be surprising, considering that both processes are known to share some common regulatory pathways. 53 Our studies show that OA, a potent inhibitor of serine/threonine protein phosphatases PP1 and PP2A, induces both differentiation-and apoptosis-associated responses in keratinocytes.…”