E2F Modulates Keratinocyte Squamous Differentiation

E2F regulation is essential for normal cell cycle progression. Therefore, it is not surprising that squamous cell carcinoma cell lines (SCC) overexpress E2F1 and exhibit deregulated E2F activity when compared with normal keratinocytes. Indeed, deliberate E2F1 deregulation has been shown to induce hyperplasia and skin tumor formation. In this study, we report on a dual role for E2F as a mediator of keratinocyte proliferation and modulator of squamous differentiation. Overexpression of E2F isoforms in confluent primary keratinocyte cultures resulted in suppression of differentiation-associated markers. Moreover, we found that the DNA binding domain and the trans-activation domain of E2F1 are important in mediating suppression of differentiation. Use of a dominant/negative form of E2F1 (E2F d/n) found that E2F inhibition alone is sufficient to suppress the activity of proliferation-associated markers but is not capable of inducing differentiation markers. However, if the E2F d/n is expressed in differentiated keratinocytes, differentiation marker activity is further induced, suggesting that E2F may act as a modulator of squamous differentiation. We therefore examined the effects of E2F d/n in a differentiation-insensitive SCC cell line. We found that treatment with the differentiating agent, 12O-tetradecanoyl-phorbol-13-acetate (TPA), or expression of E2F d/n alone had no effect on differentiation markers. However, a combination of E2F d/n TPA induced the expression of differentiation markers. Combined, these data indicate that E2F may play a key role in keratinocyte differentiation. These data also illustrate the unique potential of anti-E2F therapies in arresting proliferation and inducing differentiation of SCCs. The major function of the skin is to act as a barrier between the internal and external environment. The skin is divided into two layers, the dermis and the epidermis, of which the major cell type in the epidermis is the keratinocyte. During the strictly regulated process of differentiation, keratinocytes undergo morphological and biochemical changes, resulting in dead, enucleated, flat cells that are eventually sloughed from the skin surface. This process of differentiation is initiated by the irreversible growth arrest and suppression of proliferationspecific genes such as p53 (1), E2F1 (2, 3), cdk1 (4), and keratin 14 (5) in proliferative basal cells. Concomitant with the suppression of proliferation-specific genes, there is a corresponding induction of differentiation-specific genes, such as keratin 10 (6), cornifin (7), and transglutaminase type 1 (2). This process of growth suppression and induction of terminal differentiation is predominantly regulated at the transcriptional level by several transcription factor families such as AP1, Sp1, AP2, and E2F (8–10) and disruption of this process frequently accompanies the onset of neoplasia. E2F was first identified as a nuclear factor capable of binding to the adenovirus E2 promoter (11). To date, six members of the E2F transcription factor family have been cloned, E2Fs 1–6 (12–24). E2F exists as a heterodimeric complex in association with a dimeric partner protein, DP1 or DP2 (25–27). This “free” E2F complex acts as a potent trans-activator of E2F-responsive genes. However, the activity of E2F is subject to regulation through inhibitory interactions with hypophosphorylated forms of the pocket proteins, pRb, p107, and p130 (13–18, 21, 28). Specifically, E2Fs 1–3 preferentially bind to pRb, while E2Fs 4–5 bind p107 and E2F5 binds p130. This direct association of E2F isoforms with their cognate pocket protein partner acts to repress E2F-mediated transcriptional activity. In some instances, this repression requires further interactions with specific histone deacetylases (29, 30). The presence of these various E2F-pocket protein complexes act to regulate passage through various phases of the cell cycle. In particular, certain complexes are associated with a specific phase of the cell cycle: the E2F5 p130 complex associates with G0 (31), E2F1–3 pRb with G1 (32) and E2F4 p107 with G0/G1 phase (18). Thus, the coordinated activation/inactivation of these complexes illustrate that cell cycle progression is controlled by complex transcriptional means. Despite clear evidence implicating E2F involvement in cell cycle regulation, there is also compelling evidence for other roles of E2F. For instance, E2Fs 1–3 have been implicated in the initiation of apoptosis (33–36). More recently, E2Fs have also been demonstrated to play a role in the regulation of myocyte, megakaryocyte, and adipocyte differentiation (37–39). We have previously reported that the induction of keratino* This work was supported by grants awarded from The Garnett Passe and Rodney Williams Memorial Foundation, The Australian National Health and Medical Research Council, and the Association for International Cancer Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Sponsored by an Australian Postgraduate Award. ¶ Funded by a Garnett Passe and Rodney Williams Memorial Foundation Postgraduate Scholarship. Supported by a University of Queensland Postdoctoral Fellowship. ‡‡ Sponsored by a Senior Research Fellowship awarded by the Lions Medical Research Foundation. To whom correspondence should be addressed: Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, 4th Floor Bldg. 1 R Wing, Ipswich Rd., Woolloongabba, Queensland, Australia 4102. Tel.: 073240-5936; Fax: 07-3240-5946; E-mail: [email protected]. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 31, Issue of August 1, pp. 28516–28522, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.