Oncogene (2007) 26, 5772–5783 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Id3 is a novel regulator of p27kip1 mRNA in earlyG1 phase and is required for cell-cycle progression A-A Chassot1,4, L Turchi1,4, T Virolle1, G Fitsialos1, M Batoz2, M Deckert2, V Dulic3, G Meneguzzi1, RBusca` 1 and G Ponzio1 1INSERM U634; Faculte´ de Me´decine, Universite´ Nice Sophia Antipolis, Nice cedex, France; 2INSERM U576, Hoˆpital de L’Archet, Universite´ Nice Sophia Antipolis, Nice, France and 3CRBM-CNRS FRE 2593, Montpellier, France P27kip is a keyinhibitoryprotein of the cell-cycle Introduction progression, which is rapidlydownregulated in earlyG1 phase bya post-translational mechanism involving the Most adult tissues are constituted by differentiated proteosomal degradation. In this study, using a wounding cells with reduced proliferative activity. Renewal and/or model that induces cell-cycle entry of human dermal fibro- regeneration of these tissues is supplied by quiescent blasts, we demonstrate that p27mRNA is downregulated cells sensitive to mitogenic signals. During the skin when cells progress into the G1 phase, and then it returns wound healing process, the proliferation of quiescent to its basal level when cells approach the S phase. By cells can be stimulated by signals such as serum afflux using a quantitative polymerase chain reaction screening and/or contact inhibition failure. we identified inhibitors of differentiation (Id3), a bHLH ‘Inhibitors of differentiation’ (Id) proteins are early transcriptional repressor, as a candidate mediator accoun- genes that operate as regulators of cell fate determi- ting for p27 mRNA decrease. Id3 silencing, using an small nation. They play a crucial role in the coordinated interfering RNA approach, reversed the injurymediated regulation of gene expression during cell growth, p27 downregulation demonstrating that Id3 is involved in differentiation and tumorigenesis (Norton, 2000; the transcriptional repression of p27. Reporter gene Benezra et al., 2001). The involvement of Ids was also experiments and a chromatin immunoprecipitation assay recently shown in cellular senescence (Alani et al., 2001; showed that Id3 likelyexerts its repressive action through Ohtani et al., 2001; Zheng et al., 2004) and in the fate of ELK1 inhibition. Byinhibiting earlyp27 downregulation, specialized cells such as lymphocytes, vascular endo- Id3 depletion blocked (i) the G1-phase progression as thelial cells and neurons (Lyden et al., 1999; Benezra assessed bythe inhibition of pRb phosphorylationand et al., 2001; Engel and Murre, 2001). p130 degradation and (ii) the G1/S transition as observed Today, four members of the Id protein family (Id1– bythe inhibition of cyclinA induction, demonstrating that Id4) have been identified. They directly associate with p27 mRNA decrease is required for cell proliferation. and regulate the activity of several families of transcrip- Apart from its effect on the earlyp27 diminution, Id3 tional regulators (Yates et al., 1999; Lasorella et al., appears also involved in the control of the steady-state 2000; Norton, 2000; Roberts et al., 2001). In mammals, level of p27 at the G1/S boundary. In conclusion, this Id proteins exert their biological effects mainly through studyidentifies a novel mechanism of p27 regulation the interaction with bHLH transcription factors by which besides p27 protein degradation also implicates a blocking their DNA-binding activity. Among the Id3 transcriptional mechanism mediated byId3. partners, the members of the E2A family of transcrip- Oncogene (2007) 26, 5772–5783; doi:10.1038/sj.onc.1210386; tion factors (E12, E47) are the most frequently described published online 2 April 2007 (Massari and Murre, 2000; Norton, 2000). However, they are not the exclusive partners of Id3 as it has also Keywords: Ids; cell-cycle; p27mRNA; wound healing been shown that Id3 also interacts with MyoD, Ets, Pax or TCFs Ets-domain transcription factors (Yates et al., 1999; Roberts et al., 2001; Trausch-Azar et al., 2004). Ids proteins lack a basic DNA-binding domain and readily associate with transcription factors of the bHLH family such as E proteins to inhibit their DNA-binding function. Recent data have demonstrated that Ids activity Correspondence: G Ponzio, INSERM U634, Faculte´ de Me´ decine, is inhibited by cell-cycle-dependent kinase activities Universite´ Nice Sophia Antipolis, 28 avenue de Valombrose, 06107 such as cyclin E- and A-CDK2 (Deed et al., 1997). In Nice cedex 02, France. addition, it has also been shown that E2A and Ids E-mail: [email protected] 4 proteins participate to the transcriptional regulation These two authors equally contributed to this work. cip1 Received 13 February 2006; revised 23 January 2007; accepted 1 February of the cyclin-dependent kinase inhibitors (CKIs) p21 2007; published online 2 April 2007 (Id1) (Prabhu et al., 1997; Takahashi et al., 2004) and Id3 regulates p27mRNA in early G1 A-A Chassot et al 5773 16ink4a (Id1) (Zheng et al., 2004) in different biological to date has been poorly studied compared to the p27 models including senescence. Recent studies have shown protein fate. that besides p21 and p16, Id1 and Id3 are also involved To perform this analysis, hyperconfluent contact inhi- in the regulation of p27 protein, notably in Xenopus bited (CI) normal HDF were injured to stimulate neural crest progenitors (Kee and Bronner-Fraser, 2005) cell-cycle entry and next p27 mRNA was measured by and in Epstein–Barr virus infected cells (Everly et al., real-time quantitative PCR, before wounding and 1, 3, 2004); however, the molecular basis of this inhibition 6, 15 and 24 h after injury. In parallel we also analysed has never been elucidated. the expression of p27 protein by Western blot. To The CKI p27kip1 is a key regulator of cell-cycle assess the synchronous cell-cycle entry, we measured commitment and progression. P27 is downregulated the expression of cyclin A as a marker of the G1-S when cells are stimulated with mitotic agents and it transition. is induced to play a key role in mediating G1 arrest We observed that p27 mRNA was rapidly down- in response to mitogen starvation, cell confluence or regulated in response to injury reaching its minimal transforming growth factor b (Sherr and Roberts, 1995). levels about 3 h after injury (Figure 1a). About 24 h It is well established that p27 expression is mainly post- later, p27 transcript reached again its basal levels translationally regulated by the ubiquitin-proteasome (Figure 1a) when cells entered in S phase as confir- pathway (Pagano et al., 1995; Montagnoli et al., 1999; med by the measure of cyclin A mRNA expression Malek et al., 2001; Kamura et al., 2004). However, some studies demonstrate that p27 might also be transcrip- tionally regulated (Servant et al., 2000; Sakakibara et al., 2005). Nevertheless, at present, the mechanisms 100 involved in such regulation remain unidentified. Dermal fibroblast proliferation is a major feature of cutaneous wound healing (Martin, 1997), which is altered in several pathologies such as keloids (Calderon 50 Cyclin A et al., 1996) and chronic wounds, which can evoluate (% of max) p27 toward cutaneous carcinoma (Loots et al., 1999; Chraibi Cyc A & p27 mRNA et al., 2004). To date the mechanisms underlying cell 0 proliferation in the cutaneous wound healing context 02030 remain poorly understood. Using a suitable model to Hours after injury study in vitro wound healing (Turchi et al., 2002), we have explored the molecular mechanisms responsible b p27 for the cell-cycle entry in wounded normal human dermal fibroblasts (HDF). We show that this process Conf 3 6 15 24 depends, at least in part, on the rapid downregulation of Hours after wounding p27 mRNA. Using a small interfering RNA (siRNA) approach, we identify Id3 as a mediator of this effect. c 1 The silencing of Id3 abolishes the early downregulation 0.8 of p27 and inhibits the cell progression in G1 as well as the G1/S transition. 0.6 0.4 0.2 Results p27 mRNA level (A.U.) 0 Wounding regulates p27 at the mRNA level and this N WNW requires the synthesis of a mediator protein We have developed an original device that creates cali- brated long size injuries within confluent cell cultures. In Figure 1 Effect of HDF injury on p27 expression: confluent HDF were stimulated to grow by mechanical injury as described in classical conditions, this device wounds about 50% of Materials and methods and harvested at the indicated times. (a) the cell monolayer allowing the detection of a wide Total RNAs were isolated then p27 and cyclin A mRNA were spectrum of molecular events (Turchi et al., 2002). Using analysed by real-time PCRusing the procedure described in our wounding system, hyperconfluent dermal fibroblasts ‘Materials and methods’. The presented experiment corresponds to are synchronously stimulated to enter the cell-cycle a typical one chosen among six different experiments. (b) Cells were lysed and p27 protein levels were measured by Western blot. The (unpublished data). Using this system we have investi- presented experiment was chosen among four independent ones (c) gated the molecular mechanisms involved in the initial Effect of cycloheximide on the downregulation of p27mRNA in step of the cell-cycle entry in HDF in response to injury. wounded HDF. Confluent HDF were incubated 30 min in the We have focused our attention on p27kip1, an inhibitor absence (À) or in the presence of 5 mM cycloheximide (CHX) before injury. Wounded (W) and control (N)