Ca2+ and BMP-6 signaling regulate E2F during epidermal keratinocyte differentiation

The epidermis consists of a squamous epithelium continuously replenished by committed stem cells, which can either self-renew or differentiate. We demonstrated previously that E2F genes are differentially expressed in developing epidermis (Dagnino, L., Fry, C. J., Bartley, S. M., Farnham, P., Gallie, B. L., and Phillips, R. A. (1997) Cell Growth Differ. 8, 553-563). Thus, we hypothesized that various E2F proteins likely play distinct growth regulatory roles in the undifferentiated stem cells and in terminally differentiated keratinocytes. To further understand the function of E2F genes in epidermal morphogenesis, we have examined the expression, regulation, and protein-protein interactions of E2F factors in undifferentiated cultured murine primary keratinocytes or in cells induced to differentiate with Ca(2+) or BMP-6 (bone morphogenetic protein 6). We find similar patterns of E2F regulation with both differentiating agents and demonstrate a switch in expression from E2F-1, -2, and -3 in undifferentiated, proliferating cells to E2F-5 in terminally differentiated keratinocytes. Inhibition of keratinocyte proliferation by transforming growth factor-beta1 did not enhance E2F-5 protein levels, suggesting that this response is specific to differentiation rather than reversible cell cycle withdrawal. E2F-5 up-regulation is also accompanied by formation of heteromeric nuclear complexes containing E2F5, p130, and histone deacetylase (HDAC) 1. Overexpression of E2F5 specifically inhibited DNA synthesis in undifferentiated keratinocytes in an HDAC-dependent manner, suggesting that E2F-5.p130.HDAC1 complexes are likely involved in the permanent withdrawal from the cell cycle of keratinocytes responding to differentiation stimuli.