CCN2 Is Required for the TGF-β Induced Activation of Smad1 - Erk1/2 Signaling Network

Abstract

Connective tissue growth factor (CCN2) is a multifunctional matricellular protein, which is frequently overexpressed during organ fibrosis. CCN2 is a mediator of the pro-fibrotic effects of TGF-β in cultured cells, but the specific function of CCN2 in the fibrotic process has not been elucidated. In this study we characterized the CCN2-dependent signaling pathways that are required for the TGF-β induced fibrogenic response. By depleting endogenous CCN2 we show that CCN2 is indispensable for the TGF-β-induced phosphorylation of Smad1 and Erk1/2, but it is unnecessary for the activation of Smad3. TGF-β stimulation triggered formation of the CCN2/β3 integrin protein complexes and activation of Src signaling. Furthermore, we demonstrated that signaling through the αvβ3 integrin receptor and Src was required for the TGF-β induced Smad1 phosphorylation. Recombinant CCN2 activated Src and Erk1/2 signaling, and induced phosphorylation of Fli1, but was unable to stimulate Smad1 or Smad3 phosphorylation. Additional experiments were performed to investigate the role of CCN2 in collagen production. Consistent with the previous studies, blockade of CCN2 abrogated TGF-β-induced collagen mRNA and protein levels. Recombinant CCN2 potently stimulated collagen mRNA levels and upregulated activity of the COL1A2 promoter, however CCN2 was a weak inducer of collagen protein levels. CCN2 stimulation of collagen was dose-dependent with the lower doses (<50 ng/ml) having a stimulatory effect and higher doses having an inhibitory effect on collagen gene expression. In conclusion, our study defines a novel CCN2/αvβ3 integrin/Src/Smad1 axis that contributes to the pro-fibrotic TGF-β signaling and suggests that blockade of this pathway may be beneficial for the treatment of fibrosis.

The matricellular protein CCN1 enhances TGF‐β1/SMAD3‐dependent profibrotic signaling in fibroblasts and contributes to fibrogenic responses to lung injury

“…We speculate that CCN1 binding to these integrins may be necessary for enhanced TGF-b1/SMAD3 signaling. Similar to findings of our study with CCN1, there are reports of crosstalk of the TGF-b/SMAD pathway with other matricellular proteins, specifically secreted protein acidic and rich in cysteine (50), and connective tissue growth factor (connective tissue growth factor; CCN2) (51,52).…”

TGF-β1, but Not Bone Morphogenetic Proteins, Activates Smad1/5 Pathway in Primary Human Macrophages and Induces Expression of Proatherogenic Genes

“…It was shown that TGF-b activates Smad1 and Erk1/2 pathways in fibroblasts (45). Activation of Smad1/5/8 by TGF-b was also reported for C2C12 mouse mesenchymal stem cells and HepG2 human hepatocellular carcinoma cells (46), endothelial cells (47), and smooth muscle cells (48).…”

Effects of CCN3 on fibroblast proliferation, apoptosis and extracellular matrix production

“…6A and B). Previously it was shown that the classical Smad1 pathway plays a role in the TGF-β1 regulation of collagen and α-SMA expression (11). We then determined the activation of Smad1 pathways in response to TGF-β1 stimulation.…”

“…Findings of previous studies provided strong evidence that CCN2 plays a role in scars, particularly as a co-factor of TGF-β1 (12). In addition, CCN2 is likely to mediate the ability of TGF-β1 to stimulate ECM synthesis and is a moderate inducer of collagen synthesis (11,13). CCN2 and CCN3 are opposing factors in regulating collagen promoter activity and the secretion of this ECM protein.…”

“…These proteins are induced by growth factors and cytokines such as TGF-β and endothelin-1, and affect connective tissues, resulting in fibrosis and scarring (9). CCN2 is a member of the CCN family, which plays an important role in a variety of cell processes including angiogenesis, chondrogenesis and wound healing (10,11). Findings of previous studies provided strong evidence that CCN2 plays a role in scars, particularly as a co-factor of TGF-β1 (12).…”