90 TGFβ in mesenchymal cells (Grotendorst 1997; Leask et al. 2004). CCN2 may exert its stimulating effect on TGFβ signaling by decreasing Smad7 availability and increasing Smad2 (Qi et al. 2007). However, CCN2-deficient fibro- blasts show only a partial impairment of TGFβ responses and no defect in Smad3-dependent responses (Shi-wen et al. 2006). In this review, we summarize recent key observations concerning the regulation of the expression of the CCN2 gene, insights derived from these observations into the potential contribution that CCN2 may make to cell biology, and evaluate the potential use of CCN2 as a fibrogenic bio-marker. CCN2 gene expression in health and disease CCN2 is principally regulated at the level of transcription (Grotendorst et al. 1996). The CCN2 proximal promoter is induced by a number of specific factors such as endothelin-1 (ET-1) and TGFβ in addition to changes such as hypoxia (Holmes et al. 2001; Leask et al. 2001, 2003; Shi-wen et al. 2004; Higgins et al. 2004). TGFβ induction of CCN2 mRNA in fibroblasts is immediate-early, occurring within 30 min of TGFβ treatment, in a fashion that does not involve de novo protein synthesis (Grotendorst et al. 1996). This induction is severely impaired in the fibro- blasts deficient in Smad3 (Holmes et al. 2001). Consistent with this observation, a functional Smad element resides within the CCN2 promoter (Holmes et al. 2001; Fig. 1). However, the ability of TGFβ to fully induce the CCN2 promoter and protein also requires protein kinase C and the ras/MEK/ERK MAP kinase cascade (Chen et al. 2002; Leask et al. 2003; Fig. 1). As for all Smad-responsive promoters, additional basal transcription factors (co-activators/co-repressors) are required for complete coordinated regulation of CCN2 expression by TGFβ. The Smad element of the CCN2 promoter is not sufficient to confer TGFβ-responsiveness to a heterologous promoter (Leask et al. 2001), but rather acts in concert with a tandem repeat of an element similar to an Ets/TEF recognition motif (Leask et al. 2003). The protein(s) which bind to this element are enriched in fibroblasts relative to keratinocytes, suggesting that this protein directly contributes to the specific induction of CCN2 in different cell types (Leask et al. 2003). Recently, we have shown this motif to be a functional ETS binding element; Ets-1 is required for the ability of TGFβ to induce CCN2 expression. siRNA recognizing Ets-1 ablates the ability of TGFβ to induce CCN2 (van Beek et al. 2006; Fig. 1). Moreover, the oncogenic transcriptional co-activator YAP also operates through this site (14). Additional elements, including a BCE- 1 (basal control element-1) site and a Sp1 site (Holmes et al. 2001, 2003), have been shown to play a role in basal activity Wnt3a ? TGFβ ET-1 HIF Smad BCE-1 PKC ERK Ets-1 Sp1 CCN2 A. Leask et al. hypoxia CAESAR Fig. 1 Elements in the CCN2 promoter involved with its expression in normal mesenchymal cells. HIF-1=hypoxia inducible factor-1; BCE- 1=basal control element-1; TEF/ets=transcription enhancer factor; CAESAR=cis-acting element of structure-anchored repression of the CCN2 promoter (Fig. 1) and hence are indirectly required for TGFβ induced CCN2 gene expression. Sp1, similar to factors binding to a TATA box, though required for formation of a functional transcriptional complex is not directly required for TGFβ induced CCN2 expression (Holmes et al. 2003). The BCE-1 site was originally thought to be involved with the TGFβ-induction of CCN2, but has subsequently been shown to be involved with basal promoter activity (Grotendorst et al. 1996; Leask et al. 2001; Holmes et al. 2001) and in response of the CCN2 promoter to ET-1, through ras/MEK/ERK signal transduction cascade (Shi-wen et al. 2004). Since TGFβ induces ET-1 and ET-1 is essential for the ability of TGFβ to induce CCN2 (Shi-wen et al. 2007b), this element appears to be, albeit indirectly, a TGFβ response element (Grotendorst et al. 1996). Finally, Wnt3a induces CCN2 through a β-catenin-dependent mechanism, although the promoter sequences mediating this action are unknown, they reside within in the first 805 base pairs of the CCN2 proximal promoter (Luo et al. 2004; Chen et al. 2007). It is now appreciated that post-transcriptional controls also contribute to CCN2 gene regulation. The chicken ccn2 gene is regulated not only at the transcriptional level, but also by the interaction between a post-transcriptional element in the 3′-untranslated region (3′-UTR) and a nucleophosmin (NPM/B23) cofactor (Mukudai et al. 2008). The 3′-untranslated region (3′-UTR) has a suppressive effect on CCN2 gene expression via a minimal RNA element in the 3′-UTR, which acts as a cis-acting element of structure- anchored repression (CAESAR) (Kubota et al. 2000). Moreover, hypoxia increases stability of CCN2 mRNA in chondrosarcoma cells (Kondo et al. 2006). Finally, the unusually long 5′UTRs of CCNs 1, 2, and 4 harbor cryptic promoters that show varying degrees of activity in different cell types (Huang et al. 2007). In fibrotic fibroblasts isolated from the involved skin area of scleroderma patients, CCN2 is over-expressed independent of TGFβ and Smads but is dependent on BCE-1, Sp1 and endothelin-1 (Holmes et al. 2001, 2003; Chen et al. 2006; Shi-wen et al. 2007a). Finally, a functional polymorphism has been found in some sclero-