Introduction
CCN2 (connective tissue growth factor) is a member of the CCN family of matricellular proteins that share a similar predicted structure [
1]. It is thought to comprise four protein modules sharing identity with insulin-like growth factor binding proteins, Von Willebrand factor, thrombospondin, and a cysteine knot-containing family of growth regulators [
2]. CCN2 is a secreted protein [
3] and as such promotes cell migration, angiogenesis and fibrotic responses
in vivo and
in vitro [
2] through a unique integrin- and heparin sulfate proteoglycan-dependent mechanism [
4,
5]. CCN2 is expressed in mesenchymal cells during development, and mice possessing a deleted
Ccn2 gene die soon after birth due to an inability to breathe caused by a failure in rib cage ossification, angiogenesis and matrix remodeling [
6]. Embryonic fibroblasts cultured from CCN2-deficient animals show reduced signaling responses to adhesion and impaired stress fiber formation on fibronectin, suggesting that a physiological role of CCN2 is to potentiate interaction of cells with matrix [
5]. Indeed, a principal, if not primary, role of CCN2 is to modulate adhesive signaling [
3‐
5]. Consistent with a role for CCN2 in tissue formation and remodeling, CCN2 is induced during angiogenesis, wound healing and tissue repair [
6], and is constitutively overexpressed in cancer, atherosclerosis, arthritis and fibrosis [
2,
6]. Gaining insight into how CCN2 expression is controlled is likely to improve the understanding of the molecular basis of these pathological conditions, as well as to identify potential new avenues for therapeutic interventions for these disorders.
The cell type in which CCN2 expression has been most extensively studied is the fibroblast. The potent pro-fibrotic protein transforming growth factor (TGF)β induces CCN2 expression in dermal fibroblasts, but not in dermal keratinocytes [
7‐
9]. TGFβ induction of CCN2 mRNA in fibroblasts occurs in an immediate-early fashion, within 30 minutes of TGFβ treatment [
7,
8]. This induction requires Smad3, protein kinase C (PKC) and ras/MEK/ERK [
9‐
11]. In fibroblasts, the TGFβ-mediated induction of CCN2 is antagonized by AP-1/JNK, suggesting that a balance between MEK/ERK and JNK activation is important in controlling CCN2 expression [
9]. The induction of the CCN2 promoter also requires a tandem repeat of the nucleotides GAGGAATGG, which binds factors enriched in fibroblasts relative to keratinocytes, suggesting that this element controls the cell type-restricted response of the CCN2 promoter to TGFβ [
9]. This element has previously been identified and mapped using extensive point mutational analysis [
9]. However, the identities of the factors binding this element have not been elucidated, nor has the potential for control of CCN2 expression by different transcription factors interacting with this element been clarified.
Ets proteins, which bind the promoter element GGAA/T, are a large family of transcription factors of which several members are expressed in a tissue- and cell type-restricted fashion [
12,
13]. Because of this diversity, multiple Ets factors may be able to control the same target genes, albeit to different outcomes. In addition, functional antagonism between different Ets factors and between Ets and other transcription factors has been observed and the combination of Ets proteins and their coactivators expressed in a particular cell type is likely to contribute to the cell-type expression of target genes in normal and pathological states, resulting in distinct pathological consequences. Ets family members regulate the expression of several genes encoding extracellular matrix and adhesive proteins as well as enzymes involved in matrix degradation [
12,
13]. Upon tissue injury, Ets-1 activity is transiently induced in endothelial cells, smooth muscle cells and fibroblasts during the early stages of tissue remodeling (for example, in the early phase of ulcer healing) or immediately after mechanical injury of the vessel wall [
14]. Although Ets-1 DNA binding activity is increased in scleroderma fibroblasts [
15], the Ets family member Fli-1 has reduced expression in this cell type [
16]; however, the consequences of altering the Ets-1/Fli-1 ratios on mesenchymal biology has yet to be fully appreciated. Ets-1 is overexpressed in synovial fibroblasts from arthritis patients [
17] and is induced during physiological and pathological angiogenesis [
13]. The precise target genes, and physiological effect, of Ets family members in remodeling and repair of connective tissue and associated pathologies is still under much scrutiny.
In this study, we evaluate the hypothesis that the expression of CCN2 can be regulated through the activity of Ets-1. Our results reveal new insights into the control of CCN2 expression in fibroblasts, and the role of Ets-1 in fibroblast biology. Our results have implications for the function of CCN2 in physiological tissue repair and in pathologies of the extracellular matrix.
Discussion
CCN2 is induced by TGFβ in adult mesenchymal cells in a Smad-dependent fashion, but is constitutively overexpressed in diseases of excessive matrix production and remodeling, including cancer, fibrosis and arthritis [
6]. The expression of CCN2 can be either dependent or independent of exogenous TGFβ [
6,
19,
24,
25]. Previously, we showed a sequence in the CCN2 promoter, GAGGAATGG, was required for basal and TGFβ-induced CCN2 expression [
9]. In this report, we identify that this element responds to the ETS family of transcription factors, which bind the consensus sequence GGAA [
26,
27]. The TGFβ response element of the CCN2 promoter has several components, including a Smad element and a GAGGAATGG element, that together are capable of conferring TGFβ-responsiveness to a heterologous promoter [
9]. Consistent with the notion that the TGFβ-induction of CCN2 requires Smads, TGFβ does not induce CCN2 protein expression in
Smad3-/- embryonic fibroblasts [
19]. In this report, we show that Ets-1 and Smad3, but not Fli-1 and Smad3, cooperate to activate the CCN2 promoter in the absence of added TGFβ, emphasizing the functional significance of Ets-1 and Smad3 interactions. In addition, we show that Ets-1 is required for the TGFβ induction of CCN2, as dominant negative Ets-1 and siRNA recognizing Ets-1 attenuate the ability of TGFβ to induce the CCN2 promoter activity and protein expression in fibroblasts. Thus, for the first time, our data identify a role for ETS family members, and Ets-1, in the regulation of CCN2 expression.
Smads interact with other transcription factors to form an active transcriptional complex on promoters [
23]. That Smad3 and Ets-1 synergize to activate CCN2 expression suggests that Smad3 and Ets-1 functionally interact. Indeed, it has been recently shown that Smad3 and Ets-1 co-immunoprecipitate and act to form a transcriptionally active complex with the transcriptional cofactor p300 [
28]. In this latter report, it was shown that Smad3 and Ets-1 also interact with the basal transcription factor Sp1, and that inhibition of Sp1 with mithramycin blocked the TGFβ induction of tenascin-C [
28]. Consistent with this notion, we have shown that whereas the Sp1 element of the CCN2 promoter is not necessary for the TGFβ response element to act as an enhancer when placed in front of a heterologous promoter [
9,
25], the Sp1 inhibitor mithramycin blocks the TGFβ-mediated induction of CCN2 protein in fibroblasts [
24]. Our studies using an anti-Sp1 antibody revealed that Sp1 was not present in the protein complex binding to the Ets element of the CCN2 promoter, indicating that chromatin looping is likely to be involved in the interaction between Ets and Sp1. It is interesting to note that within the context of the experiments performed in this present study, transfected Smad3 was able to induce the CCN2 promoter to greater effect than TGFβ ligand, emphasizing that endogenous Smad levels are not likely to be saturating.
The different effects of Ets-1 and Fli-1 on controlling CCN2 promoter activity is intriguing in light of the fact that approximately 25 human ETS proteins have been identified, all of which share a highly conserved DNA binding domain that interacts with the core DNA target GGAA/T [
12,
13]. It has been hypothesized that the existence of many different ETS factors suggests that individual Ets members may have unique roles [
12,
13]. Subtle differences in target sites or their own expression in tissues, and differential response to external signals may contribute to distinct functions, activating or repressing target gene expression – either basally or in response to growth factors – depending on a constellation of ETS factors that compete for binding to ETS binding elements [
28‐
38]. Some recent data have shown that ETS family members contribute to the regulation of genes that mediate matrix remodeling, cell migration and cancer progression, including those controlling cell proliferation, adhesion cell survival, invasion, and signaling [
31‐
38]. Several recent studies have focused in particular on the potentially divergent roles of Fli-1 and Ets-1 in providing a balance between tissue homeostasis and repair/remodeling [
22,
30,
34‐
37]. Consistent with this notion, both Ets-1 and Fli-1 activate the promoters of matrix metalloproteinases [
22,
34‐
37], enzymes involved with degrading matrix and promoting cell migration. Similarly, Ets-1 activates tenascin C, an extracellular matrix glycoprotein that promotes cell migration and angiogenesis [
32,
33], and CCN2, encoded by an immediate-early gene that also promotes cell adhesion and migration and angiogenesis [
2,
40,
41]. Conversely, type I collagen is induced by Ets-1 but repressed by Fli-1 [
30,
34,
42]. In the current study, the induction of the CCN2 promoter in response to TGFβ is reduced by Fli-1, and diminished by dominant negative Ets-1, supporting a divergence in the roles of Ets-1 and Fli-1 in gene regulation. As we observed for CCN2, TGFβ induction of tenascin-C is potentiated by Ets-1; however, the TGFβ-induction of type I collagen is impaired by Ets-1 [
30,
34,
42]. Given that Ets-1 is induced during the early phases of tissue repair [
14,
38,
39] and is overexpressed in tumor stroma, [
12,
13,
41], these results, although albeit using principally promoter-based approaches, collectively suggest that Ets-1 could bias the fibroblast population towards a 'pro-migratory' program in that TGFβ and Ets-1 interactions may bias Ets-1 and TGFβ-responsive genes toward a migratory/adhesive/invasive phenotype. Conversely, at later stages of repair when Ets-1 levels decrease, the effects of TGFβ may switch towards matrix rebuilding, with increased type I collagen resulting in wound closure.
Conclusion
Our investigation into the mechanism underlying the control of CCN2 regulation in fibroblasts has revealed a role for an ETS binding element within the CCN2 promoter. In particular, we show that the transcription factor Ets-1 contributes to the TGFβ induction of the CCN2 promoter and protein. Ets-1, but not the related Fli-1, synergize with Smad3 in activating the CCN2 promoter, suggesting that the CCN2 promoter can be differentially regulated by different members of the ETS family. Our results point to the complexity underlying CCN2 expression, and are consistent with the notion that different ETS family members can have distinct influences on gene expression in fibroblasts. As CCN2 plays roles in connective tissue pathologies, targeting Ets-1 may be beneficial in alleviating pathologies of tissue remodeling and repair, including cancer, arthritis and fibrosis.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JvB and LK performed cell culture, transfection, promoter analysis, immunofluorescence and siRNA studies. JR performed the gel shift assay. SB helped write the manuscript. AL performed the gel shift assay, prepared the manuscript and designed the experiments.