Role and regulation of C/EBPalpha in response to DNA damage

C/EBPalpha and C/EBPbeta are members of basic leucine zipper (bZIP) class of transcription factors. They are abundantly expressed in epidermis. C/EBPalpha expression is diminished in mouse and human squamous cell as well as basal cell carcinomas. Recently, C/EBPalpha has been shown to be an epithelial tumor suppressor gene in genetically engineered mouse model. siRNA experiment has suggested a role for C/EBPalpha in DNA damage G 1 checkpoint response. But definitive genetic evidence is lacking. Here, we report that C/EBPalpha is highly inducible in primary dermal fibroblasts by DNA damaging agents that induce strand breaks, alkylate and crosslink DNA as well as those that produce bulky DNA lesions. Fibroblasts deficient in C/EBPalpha (C/EBPalpha-/-) display an impaired G1 checkpoint as evidenced by inappropriate entry into S-phase in response to DNA damage and these cells also display an enhanced G1 to S transition in response to mitogens. The induction of C/EBPalpha by DNA damage in fibroblasts does not require p53. EMSA analysis of nuclear extracts prepared from UVB- and MNNG-treated fibroblasts revealed increased binding of C/EBPbeta to a C/EBP consensus sequence and ChIP analysis revealed increased C/EBPbeta binding to the C/EBPalpha promoter. To determine whether C/EBPbeta has a role in the regulation of C/EBPalpha we treated C/EBPbeta-/- fibroblasts with UVB or MNNG. We observed C/EBPalpha induction was impaired in both UVB- and MNNG-treated C/EBPbeta-/- fibroblasts. Our study reveals a novel role for C/EBPbeta in the regulation of C/EBPalpha in response to DNA damage and provides definitive genetic evidence that C/EBPalpha has a critical role in the DNA damage G1 checkpoint.;Since the evidence for C/EBPalpha as a tumor suppressor gene in human skin is mounting; we decided to further study the signaling pathway of C/EBPalpha induction in response to DNA damage in keratinocytes. C/EBPalpha has been shown to be induced by DNA damage in human and mouse skin, and in primary and immortalized keratinocyte cell lines. In keratinocytes, the induction of C/EBPalpha requires p53; p53 directly binds to C/EBPalpha promoter and is responsible for increases in C/EBPalpha mRNA expression in response to DNA damage. We show that GSK3beta inhibitors block C/EBPalpha protein and message induction in response to DNA damage without altering p53 protein levels. Further, we found that GSK3beta interaction with p53 increased in response to DNA damage. In addition, UVB treatment of keratinocytes resulted in post-translation modification of C/EBPalpha protein. Our results suggest that GSK3beta regulates C/EBPalpha expression, interacts with p53, and that C/EBPalpha protein undergoes post-translational modification in response to DNA damage. Hence, from these two studies we have provided evidence that (i) C/EBPalpha has a role in DNA damage G1 checkpoint response and in mitogen induced G1/S transition. (ii) C/EBPalpha is induced in response to various DNA damage in different cell types and regulation of C/EBPalpha is cell type specific. (iii) GSK3beta might have a role in C/EBPalpha induction in response to DNA damage in keratinocytes.