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

Yap Ching Chew,Gautam Adhikary,Wen Xu,Gerald M Wilson,Richard L Eckert

doi:10.1074/jbc.m113.477133

Yap Ching Chew, Gautam Adhikary + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m113.477133

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Abstract

KLF4 is a member of the Kruppel-like factor family of transcriptional regulators. KLF4 has been shown to be required for normal terminal differentiation of keratinocytes, but the molecular mechanism whereby KLF4 regulates genes associated with the differentiation process has not been studied. In the present study, we explore the impact of KLF4 on expression of involucrin, a gene that is specifically expressed in differentiated keratinocytes. KLF4 overexpression and knockdown studies show that involucrin mRNA and protein level correlates directly with KLF4 level. Moreover, studies of mutant KLF4 proteins indicate that transcriptionally inactive forms do not increase involucrin expression. PKCδ is a regulator of keratinocyte differentiation that increases expression of differentiation-associated target genes, including involucrin. Overexpression of KLF4 augments the PKCδ-dependent increase in involucrin expression, whereas KLF4 knockdown attenuates this response. The KLF4 induction of human involucrin (hINV) promoter activity is mediated via KLF4 binding to a GC-rich element located in the hINV promoter distal regulatory region, a region of the promoter required for in vivo involucrin expression. Mutation of the GC-rich element, an adjacent AP1 factor binding site, or both sites severely attenuates the response. Moreover, loss of KLF4 in an epidermal equivalent model of differentiation results in loss of hINV expression. These studies suggest that KLF4 is part of a multiprotein complex that interacts that the hINV promoter distal regulatory region to drive differentiation-dependent hINV gene expression in epidermis.

Highlights

KLF4 is an important regulator of terminal differentiation and barrier formation in epidermis, but its mechanism of action is not well understood
The KLF4 induction of human involucrin promoter activity is mediated via KLF4 binding to a GC-rich element located in the hINV promoter distal regulatory region, a region of the promoter required for in vivo involucrin expression
We have demonstrated that PKC␦ activity drives hINV expression via activation of a MEKK1, MEK3/MEK6, and p38␦/ERK signaling cascade that elevates Sp1 and AP1 transcription factor level and binding to the hINV promoter to activate transcription (22, 24 –26, 29, 33–38). 12-O-Tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol analog that activates PKC isoforms, is a pharmacologic agent that activates keratinocyte differentiation via this pathway [23, 24, 39], as do some other naturally occurring agents [40]

Summary

Background

KLF4 is an important regulator of terminal differentiation and barrier formation in epidermis, but its mechanism of action is not well understood. Keratin 5 promoter-targeted overexpression of KLF4 in mouse epidermis stimulates premature barrier formation (9 –11), whereas KLF4 knock-out mice display impaired barrier function [12] This is consistent with a role for this protein in suppressing proliferation and driving differentiation. These findings are consistent with our recent study indicating that KLF4 increases p21Cip expression in normal keratinocytes [13]. Our results show that KLF4 interacts via a GC-rich element in the hINV promoter distal regulatory region (DRR) to drive transcription These findings present the first detailed evidence that KLF4 interacts with response elements on a keratinocyte differentiation-expressed gene to drive transcription

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION