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

Abstract

BackgroundMyofibroblasts, 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 (α-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-β1)-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis.MethodsFibroblasts 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-β1 (2 ng/ml) and/or forskolin (10 μM) (a known stimulator of cAMP). Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis.ResultsThe 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-β1 stimulation of α-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-β1 stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types.ConclusionIn 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.

Anti-fibrotic action of pirfenidone in Dupuytren’s disease-derived fibroblasts

“…The palmar fascia fibrosis seen in DD is associated with excessive deposition of extracellular matrix (ECM) components including fibronectin [19]. DD tissues and cells have been reported to show increased expression of fibronectin type III extra-domain B ( FN 1-ED-B) and “oncofetal” fibronectin levels [34, 35] which prompted us to determine if PFD can play a role in reducing fibronectin levels.…”

“…The inhibition or reversion of the myofibroblast phenotype to normal may mitigate disease progression and severity. Previous studies from our laboratory have shown that increasing cyclic AMP (cAMP) levels via forskolin (an adenylate cyclase (AC) activator) has potential to inhibit TGF-β-induced myofibroblast formation and accumulation of ECM components in DD-derived fibroblasts [19]. Overproduction of cAMP that can provide a means to blunt fibrosis has also been shown in cardiac and pulmonary fibroblasts [20, 21].…”

Characterization of a Novel Dermal Fibrosis Model Induced by Areca Nut Extract that Mimics Oral Submucous Fibrosis

“…Moreover, activated fibroblasts and myofibroblasts, which are characterized by the expression of α-SMA, can be stimulated and observed in fibrotic tissue [30]. Several studies have indicated that the expression of α-SMA may be regulated by different signaling pathways, e.g., the TGF-β, CTGF and cAMP/PKA pathways [31]. The immunoblot analyses revealed that the expression levels of both CTGF and α-SMA were increased following ANE treatment (Fig 4).…”

Whole genome and global expression profiling of Dupuytren's disease: systematic review of current findings and future perspectives

“…A higher level of α 2 integrin (p≤0.05, against internal control fascia) and α 5 integrin chains (p≤0.07, against external control fasica) has been observed in DD cord through fluorescence activated cell sorting 64 . Fibronectin , which is involved in the integrin signalling pathway, is elevated in DD, as shown by multiple studies at both mRNA and protein levels 27 65–67. Isoforms of fibronectin, ED-A and ED-B, are found in the proliferative and involuational stage of DD 67.…”