E2F suppression and Sp1 overexpression are sufficient to induce the differentiation-specific marker, transglutaminase...

Recently, E2F function has expanded to include the regulation of differentiation in human epidermal keratinocytes (HEKs). We extend these findings to report that in HEKs, Sp1 is a differentiation-specific activator and a downstream target of E2F-mediated suppression of the differentiation-specific marker, transglutaminase type 1 (TG-1). Deletion of elements between -0.084 to -0.034 kb of the TG-1 promoter disabled E2F1-induced suppression of promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and Sp3 bound this region. Protein expression analysis suggested that squamous differentiation was accompanied by increased Sp1/Sp3 ratio. Cotransfection of proliferating HEKs or the squamous cell carcinoma (SCC) cell line, KJD-1/SV40, with an E2F inhibitor (E2Fd/n) and Sp1 expression plasmid was sufficient to activate the TG-1 promoter. The suppression of Sp1 activity by E2F in differentiated cells appeared to be indirect since we found no evidence of an Sp1/E2F coassociation on the TG-1 promoter fragment. Moreover, E2F inhibition in the presence of a differentiation stimulus induced Sp1 protein. These data demonstrate that (i) Sp1 can act as a differentiation stimulus, (ii) E2F-mediated suppression of differentiation-specific markers is indirect via Sp1 inhibition and (iii) a combination of E2F inhibition and Sp1 activation could form the basis of a differentiation therapy for SCCs.