Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's - derived fibroblasts

Background: Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha-smooth muscle actin (alpha-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP (cAMP)/protein kinase A signaling pathway will inhibit transforming growth factor-beta1 (TGF-beta(1))-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis. Methods: Fibroblasts derived from areas of Dupuytren's contracture cord (DC), from adjacent and phenotypically normal palmar fascia (PF), and from palmar fascia from patients undergoing carpal tunnel release (CTR; CT) were treated with TGF-beta(1) (2 ng/ml) and/or forskolin (10 mu M) (a known stimulator of cAMP). Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis. Results: The basal mRNA expression levels of fibronectin- extra domain A (FN1-EDA), type I (COL1A2) and type III collagen (COL3A1), and connective tissue growth factor (CTGF) were all significantly increased in DC-and in PF-derived cells compared to CT-derived fibroblasts. The TGF-beta(1) stimulation of alpha-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-beta(1) stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types. Conclusion: In sum, increasing cAMP levels show potential to inhibit the formation of myofibroblasts and accumulation of ECM components. Molecular agents that increase cAMP may therefore prove useful in mitigating DC progression or recurrence.