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Id2 protein is selectively upregulated by UVB in primary, but not in immortalized human keratinocytes and inhibits differentiation

Oncogene volume 24, pages 5443–5458 (2005)Cite this article

Abstract

Solar ultraviolet B (UVB) acts as both an initiator and promoter in models of multistage skin carcinogenesis. We found that, whereas UVB induces apoptosis in human papillomavirus-16 E6/7-immortalized keratinocytes, it inhibits markers of differentiation in human foreskin keratinocytes (HFK). Potential mechanisms for this differential response were examined by DNA microarray, which revealed that UVB alters the expression of three of the four human inhibitor of differentiation/DNA binding (Id) proteins that comprise a class of helix–loop–helix family of transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. These results were verified by RT–PCR and immunoblot analysis of control and UVB-irradiated primary and immortalized keratinocytes. Whereas Id1 was downregulated in both cell types, Id2 expression was upregulated in primary HFK, but not immortalized cells. In contrast, Id3 expression was significantly increased only in immortalized cells. The differential expression pattern of Id2 in response to UVB was recapitulated in reporter constructs containing the 5′ regulatory regions of this gene. Id2 promoter activity increased in response to UVB in HFK, but not in immortalized cells. To identify the regulatory elements in the Id2 promoter that mediate transcriptional activation by UVB in HFK, promoter deletion/mutation analysis was performed. Deletion analysis revealed that transactivation involves a 166 bp region immediately upstream to the Id2 transcriptional start site and is independent of c-Myc. The consensus E twenty-six (ETS) binding site at –120 appears to mediate UVB transcriptional activation of Id2 because point mutations at this site completely abrogated this response. Chromatin immunoprecipitation and electrophoretic mobility-shift assays verified that the Id2 promoter interacts with known Id2 promoter (ETS) binding factors Erg1/2 and Fli1, but not with c-Myc; and this interaction is enhanced after UVB exposure. Similar to the effects of UVB exposure, ectopic expression of Id2 protein in primary HFK resulted in inhibition of differentiation, as shown by decreased levels of the terminal differentiation marker keratin K1 and inhibition of involucrin crosslinking. Reduction of Id2 expression by small interfering RNAs attenuated the UVB-induced inhibition of differentiation in these cells. These results suggest that UVB-induced inhibition of differentiation of primary HFK is at least, in part, due to the upregulation of Id2, and that upregulation of Id2 by UVB might predispose keratinocytes to carcinogenesis by preventing their normal differentiation program.

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Abbreviations

Id:

inhibitor of differentiation/DNA binding

HFK:

human foreskin keratinocytes

UVB:

ultraviolet B

HPV:

human papillomavirus

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Acknowledgements

We thank Danith Ly (Carnegie Mellon University) and Xuefeng Liu (Pathology Deptartment, Georgetown University) for help with the DNA microarray analysis and with the ChIP assay, respectively. This work was supported in part by the National Cancer Institute Grant 5RO1 CA100443-02 and the US Army Medical Research and Materiel Command contract DAMD17-00-C-0026 (to DSR).

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  1. Department of Biochemistry and Molecular Biology, Georgetown University, School of Medicine, Washington, 20007, DC, USA

    Cynthia M Simbulan-Rosenthal, Valerie Trabosh, Ana Velarde, Feng-Pai Chou, Ahmad Daher, Fnu Tenzin & Dean S Rosenthal

  2. Department of Molecular Biology, Sapporo Medical University School of Medicine, Sapporo, 060-8556, Japan

    Takashi Tokino

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  1. Cynthia M Simbulan-Rosenthal
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Simbulan-Rosenthal, C., Trabosh, V., Velarde, A. et al. Id2 protein is selectively upregulated by UVB in primary, but not in immortalized human keratinocytes and inhibits differentiation. Oncogene 24, 5443–5458 (2005). https://doi.org/10.1038/sj.onc.1208709

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