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

Cynthia M Simbulan-Rosenthal¹, Valerie Trabosh, Ana Velarde

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

|July 12, 2005

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.

Insights

Solar UVB radiation inhibits keratinocyte differentiation by upregulating Id2 expression. This Id2 upregulation by UVB may increase skin cancer risk by disrupting normal skin cell development.

Area of Science:

Dermatology
Molecular Biology
Carcinogenesis

Background:

Solar ultraviolet B (UVB) radiation is a known factor in skin carcinogenesis, influencing both initiation and promotion stages.
UVB exhibits differential effects on keratinocytes, inducing apoptosis in immortalized cells while inhibiting differentiation in primary cells.
Inhibitors of differentiation/DNA binding (Id) proteins are key regulators of cell proliferation, differentiation, apoptosis, and carcinogenesis.

Purpose of the Study:

To investigate the differential response of keratinocytes to UVB radiation, focusing on the role of Id proteins.
To elucidate the molecular mechanisms underlying UVB-induced inhibition of keratinocyte differentiation.
To determine the specific role of Id2 in mediating UVB's effects on keratinocyte differentiation and its potential link to skin carcinogenesis.

Main Methods:

DNA microarray analysis to assess global gene expression changes in response to UVB.
RT-PCR and immunoblot analysis to validate changes in Id protein expression.
Reporter construct assays, promoter deletion/mutation analysis, chromatin immunoprecipitation, and electrophoretic mobility-shift assays to identify regulatory elements and protein interactions.
Functional assays involving ectopic Id2 expression and small interfering RNA (siRNA)-mediated knockdown to assess Id2's role in differentiation.

Main Results:

UVB altered the expression of Id1, Id2, and Id3 proteins in keratinocytes differentially.
Id2 expression was upregulated by UVB in primary human foreskin keratinocytes (HFK), correlating with increased Id2 promoter activity via an ETS binding site.
Ectopic Id2 expression inhibited keratinocyte differentiation, while Id2 knockdown attenuated UVB-induced differentiation inhibition, implicating Id2 in this process.

Conclusions:

UVB-induced upregulation of Id2 in primary keratinocytes is a key mechanism for inhibiting differentiation.
The Id2 promoter contains regulatory elements sensitive to UVB, involving ETS transcription factors.
This Id2-mediated inhibition of keratinocyte differentiation by UVB may contribute to skin carcinogenesis by disrupting normal cellular processes.

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