Supplementary Materials962957_Supplementary_Materials. in response to DNA damage via the control of

Supplementary Materials962957_Supplementary_Materials. in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21. (gene encoding p21) primarily due to the tumor suppressor protein p53.9-12 p21 functions as a negative regulator of G1/S progression and as a promoter of cell cycle arrest induced by DNA damage through two distinct mechanisms; (1) p21 regulates cell cycle progression by binding to and inhibiting the kinase activity of CDKs, kinases required for cell cycle progression13-16 and (2) p21 binds to proliferating cell nuclear antigen (PCNA) and blocks DNA polymerase progression and DNA replication.17-19 Cells deficient in p21 have a defective G1/S checkpoint and fail to arrest in G1 following UVB exposure.20-22 The basic leucine zipper transcription factor, CCAAT/enhancer binding protein (C/EBP) is abundantly purchase Adriamycin expressed in keratinocytes of the IKBA skin.23,24 Previously, we reported that UVB rays is a potent inducer of C/EBP in individual and mouse keratinocytes, aswell such as mouse epidermis Moreover, C/EBP-deficient keratinocytes in lifestyle or in mouse epidermis neglect to undergo cell routine arrest in G1 in response to UVB-induced DNA damage, thus allowing cells with damaged DNA to inappropriately enter S-phase.25,26 Consistent purchase Adriamycin with these observations, mice containing a skin specific ablation of C/EBP are highly susceptible to skin cancer development following chronic low doses of UVB radiation.25 Despite these critical and novel roles for C/EBP in prevention of UVB-induced skin cancer and the cellular response to DNA damage involving cell cycle arrest, the molecular mechanisms and key players involved downstream of C/EBP in the DNA damage G1/S checkpoint response remain uncharacterized. Our novel findings reveal C/EBP regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 E3 ubiquitin ligase mediated degradation of p21. Results C/EBP is required for UVB-induced up-regulation of p21 protein levels Initially we examined the protein levels of the cyclin-dependent kinase inhibitor, p21, in untreated C/EBP-deficient keratinocytes in culture (siRNA knockdown) and in the epidermis of K5Cretg/+;C/EBPflox/flox mice (epidermal specific knockout). As shown in Fig. 1A, p21 protein levels were normal and not altered by C/EBP deficiency in cultured keratinocytes or in the epidermis of K5Cretg/+;C/EBPflox/flox mice. However, UVB-treated C/EBP-deficient keratinocytes in culture (Fig. 1B) and in the epidermis of K5Cretg/+;C/EBPflox/flox mice (Fig. 1C and D) failed to up-regulate p21 protein levels despite normal purchase Adriamycin upregulation of mRNA levels (Fig. 1E and F). The defect in p21 protein accumulation is usually conserved in UVB-treated human keratinocytes (Fig. 1G), also occurs in response to other types of DNA damaging agents such as the DNA alkylating agent MNNG (Fig. 1H), and occurs despite normal p53 protein levels and activity (Fig. 1I). These data demonstrate that despite the normal transcriptional up-regulation of in response to DNA damage, C/EBP-deficient keratinocytes fail to accumulate p21 protein levels indicating that C/EBP is required for the post-transcriptional regulation of p21 in response to DNA damage. Open in a separate window Physique 1. C/EBP is required for UVB-induced upregulation of p21 protein levels. (A) Immunoblot analysis of untreated control and C/EBP siRNA treated Balb/MK2 keratinocytes and K5Cretg/+ and K5Cretg/+;C/EBPflox/flox epidermal lysates. (B) Immunoblot analysis of lysates from Balb/MK2 keratinocytes treated with C/EBP siRNA or control siRNA and collected at the indicated occasions after exposure to 5 mJ/cm2 UVB. (C) IHC staining for p21 in formalin-fixed paraffin-embedded sections of mouse skin from K5Cretg/+ and K5Cretg/+;C/EBPflox/flox mice 4?h after treatment with 50 mJ/cm2 UVB. (D) Immunoblot analysis of p21, and C/EBP from lysates isolated from K5Cretg/+ and K5Cretg/+;C/EBPflox/flox mouse epidermis collected at the indicated occasions following exposure to 100 mJ/cm2 UVB. (E and F) Relative (p21) mRNA levels in (E) Balb/MK2 cells treated with C/EBP siRNA or control siRNA and (F) K5Cre and K5Cre;C/EBPflox/flox in mouse epidermis collected at the indicated occasions following UVB. Data are expressed as the mean normalized to.

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