GADD45 regulates G2/M arrest, DNA repair, and cell death in keratinocytes following ultraviolet exposure.

GADD45 is a multifunctional protein that is regulated by p53. p53 plays an important role in regulating DNA repair and in the response to ultraviolet light in keratinocytes. This study investigates the role of GADD45 in the response to ultraviolet B. Cell cycle analysis demonstrated that wild-type and Gadd45-deficient cells have transient G2/M arrest, but only in the wild-type cells was arrest sustained. Cdc2 kinase activity in immunoprecipitates from normal and Gadd45-deficient cells decreases after irradiation in normal cells but not in Gadd45-deficient cells. An immunofluorescent study with Cdc2 antibody demonstrated diffuse cellular fluorescence before ultraviolet irradiation in both Gadd45-deficient and wild-type cells, but upon ultraviolet irradiation only Gadd45-proficient cells showed Cdc2 sequestration in the cytoplasm. Gadd45-deficient cells also have a slower rate of nucleotide excision repair. The lack of G2/M arrest coupled with reduced DNA repair leads to a higher ultraviolet sensitivity of Gadd45-deficient cells. These results reveal that GADD45 promotes G2/M arrest via nuclear export and kinase activity of Cdc2, increases global genomic DNA repair, and inhibits cell death in keratinocytes. Thus, GADD45 plays an important role in maintaining genomic integrity in ultraviolet-exposed skin.

UV induces GADD45 in a p53-dependent and -independent manner in human keratinocytes.

BACKGROUND: GADD45 is a multifunctional protein involved in DNA repair and in cell cycle checkpoint control. p53 plays an important role in regulating DNA repair and in response to UVB in keratinocytes. OBJECTIVE: GADD45 and p53 expression was examined and compared at the mRNA and protein level after exposure to UV irradiation. METHODS: Human keratinocytes were exposed to increasing doses of UVB, and an RNA protection assay and a Western blot analysis were performed. RESULTS: The RNase protection assays using human keratinocytes showed that GADD45 mRNA increases after 4 h and remains elevated for 24 h in cells irradiated at 100, 300, or 600 J/m2 UVB. The level of GADD45 protein increases after 8 h and remains elevated for 48 h, with maximal induction at 300 J/m2. p53 mRNA did not rise in concert with GADD45 at any dose used, and p53 protein was not up-regulated at the lower dose of 100 J/m2. CONCLUSION: GADD45 is regulated in both a p53-dependent and a p53-independent manner in keratinocytes after UV exposure.