The Mechanism Of CIRP In Inhibition Of Keratinocytes Growth Arrest And Apoptosis Following Low Dose UVB Irradiation

There is mounting evidence that skin damaged by ultraviolet light(UV) has an increased risk for formation of skin cancers including basal cell carcinoma, squamous cell carcinoma and cutaneous malignant melanoma. Non-melanoma skin cancers, mainly squamous cell carcinoma(SCC) and basal cell carcinoma(BCC), account for the majority of these skin cancers. Overexposure to ultraviolet B light(UVB) causes DNA damage and induces many cellular responses, such as cell growth arrest and apoptosis, which play vital roles in the regulation of cell fate post-UVB. The molecular mechanism by which UVB, especially chronic low-dose UVB, induces skin carcinogenesis is still under investigation. CIRP(cold inducible RNA binding protein) was first discovered as responding to ultraviolet light(UV)-induced DNA damage in hamster cells. CIRP belongs to a family of cold shock proteins(CSPs) that respond to cold stress and function as an RNA chaperone. CSPs relocate from the nucleus to the cytoplasm to facilitate translation in normal cellular responses to stresses. CIRP has been shown to protect cells from TNF? and genotoxic stress-induced cell growth arrest and apoptosis, and also to inhibit DNA damage-induced apoptosis via the p53 pathway. CIRP has also been reported to be associated with inflammation and multiple kinds of tumorigenesis. In addition, a recent report demonstrated that CIRP expression in the liver was positively correlated with the activation of signal transducer and activator of transcription 3(Stat3), which mediates various cellular processes including the cell cycle, apoptosis and tumor angiogenesis. The activation of Stat3 has been suggested to play a critical role in the development of skin cancer after UV overexposure. UVB induces Tyr705-phosphorylation and thus activation of Stat3, and constitutive activation of Stat3 is associated with a number of human tumors and cancer cell lines. However, the regulatory function(s) of CIRP and Stat3 phosphorylation in UVB-irradiated keratinocytes remains to be elucidated. Here, we characterized the role ofCIRPin the regulation of cell growth arrest, apoptosis and transformation of keratinocytes post-UVB irradiation. In this study, we demonstrate that(1) CIRP is elevated in all tested melanoma and non-melanoma skin cancer cell lines.And the expression of CIRP is upregulated in keratinocytes after being irradiated with relatively low dose(<5 mJ/cm2), but not high dose(50 mJ/cm2), UVB acutely and chronically. The induced expression of CIRP was accompanied with a cytosolic translocation of the protein in HaCaT cells after the UVB(3 mJ/cm2) exposure.(2) The increased expression of CIRP, either induced by UVB or through overexpression, leads to resistance of keratinocytes to UVB-induced growth arrest and death. And reduced expression of CIRP by RNA knockdown sensitizes keratinocyte cells to the low dose UVB irradiation.(3) We also demonstrated that CIRP expression is required for the low dose UVB-induced Tyr705-phosphorylation, but not total amount, of Stat3. The p-Stat3 level is correlated with the expression levels of cyclin D1 and VEGF, two known downstream cell growth regulators of Stat3, as well as Bag-1/S, an apoptosis regulator.(4) Inhibition of Stat3 DNA-binding activity by S3I-201 leads to a reduction of the p-Stat3 and Bag-1/S along with growth and survival of keratinocytes post-UVB; And the effect of S3I-201 on the UVB-irradiated cells can be partially inhibited by overexpression of CIRP or Bag-1/S.(5) Our data showed that while having no effect on PRAR cleavage and caspase 3 activation without UVB treatment, either CIRP or Bag-1/S knockdown increased the ratio of cleaved PARP/PARP and cleaved caspase 3 post-UVB irradiation. Furthermore, the overexpression of Bag-1/S could totally inhibit UVB-induced PARP cleavage andcaspase 3 activation.The results presented above led us to propose that CIRP-p(705)-Stat3 cascade promotes cell proliferation and survival post-UVB via upregulating the expression of cyclin D1 and Bag-1/S respectively. The objective of the proposed study furthered our understanding of the role of CIRP in low dose UVB-induced carcinogenesis. These studies are anticipated to be clinically significant because the results elucidated CIRP as a new point of intervention in low dose UVB-induced skin cancer formation, which could potentially lead to the development of new therapeutics for chemoprevention and treatment of various skin cancers.