Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation:: a role for CCN2 and low concentration of TGF-β1

The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-beta 1 and CCN2. TGF-beta 1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-beta 1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-beta 1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-beta 1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-beta 1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-beta 1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-beta 1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-beta 1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-beta signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-beta 1 dose and CCN2.