E2F and Histone Deacetylase Mediate Transforming Growth Factor β Repression of cdc25A during Keratinocyte Cell Cycle Arrest

Mitogens and antimitogens affect cell proliferation by regulating the activity of G1 cyclin-dependent kinases (Cdk’s) that commit the cell to completing the division cycle (28, 30). G1 Cdk’s, which include the cyclin D-dependent kinases Cdk4 and Cdk6 and the cyclin E-dependent kinase Cdk2, phosphorylate the members of the retinoblastoma protein family pRb, p107, and p130, also known as “pocket proteins” (34, 39). Phosphorylation of pocket proteins regulates their interactions with transcription factors of the E2F family, i.e., E2F1 through E2F5 (3, 11, 16). In mitogenically stimulated cells, Cdk phosphorylation of pocket proteins allows E2F factors to activate the expression of components involved in DNA synthesis, such as dihydrofolate reductase, thymidine kinase, DNA polymerase α, ORC1, and Cdk components such as cyclin E, cyclin A, and cdc2 (10, 11). When the level of G1 Cdk activity in the cell is low, pocket proteins accumulate in a hypophosphorylated state that inhibits these E2F functions. However, recent evidence suggests that the binding of pocket proteins to E2F factors not only silences transcriptional activation but also generates transcriptional repressor complexes (6, 18, 37, 40). Thus, mutation of E2F sites in certain genes can lead to derepression of these genes in quiescent cells, suggesting that these sites are occupied by E2F repressor complexes during G0 (36, 47). The broader role of these repressor complexes and, in particular, their possible involvement in the action of antimitogenic cytokines such as transforming growth factor β (TGF-β) have remained unknown. TGF-β and related family members have a wide range of biological effects, including regulation of cell proliferation (1, 28). TGF-β can promote growth in mesenchymal tissues through effects on extracellular matrix production, cell adhesion receptors, and production of autocrine mitogens. In other cell types, including epithelial, hematopoietic, and certain mesenchymal cells, TGF-β exerts antiproliferative effects through a repertoire of gene responses that varies depending on the cell type (19). TGF-β causes rapid up-regulation of the Cdk inhibitor p15Ink4B in keratinocytes, lung, thyroid, and mammary epithelial cells (7, 15, 32, 33), up-regulation of the Cdk inhibitor p21Cip1 in keratinocytes and ovarian epithelial cells (9, 12, 32), rapid down-regulation of the Cdk-activating tyrosine phosphatase cdc25A in mammary epithelial cells (19), and down-regulation of Cdk4 in lung epithelial cells emerging from serum deprivation (13); furthermore, in most of these cell types, TGF-β rapidly down-regulates c-myc expression (1, 19, 27, 31). Individually or combined, these effects cause a decrease in Cdk activity that compromises further progression through G1 and sets in motion secondary events that place the cell in a quiescent state. One of these secondary events in human keratinocytes is cdc25A down-regulation (19). In contrast to the rapid decrease in cdc25A mRNA caused by TGF-β in mammary epithelial cells, cdc25A mRNA levels in keratinocytes decrease slowly, and this decrease follows the initial fall in Cdk activity induced by TGF-β via Cdk inhibitors (15, 32). Investigating the mechanisms underlying these events, we observed that inhibition of cdc25A promoter activity by TGF-β in human keratinocytes requires the integrity of an E2F site that binds E2F4-p130 and requires histone deacetylase for transcriptional repression.