OR9,54 IGFBP-6 and IGF-II: novel mediators of myofibroblast differentiation in Dupuytren's Disease?

Abstract

Background: Dupuytren’s Disease (DD), a debilitating contracture of the hand, is typical of other fibrotic diseases in displaying increased levels of extra-cellular matrix (ECM)associated Transforming Growth Factor (TGF)-b and contractile (myo)fibroblasts. TGF-b induces myofibroblast differentiation in DD and regulates the deposition of molecules into the collagenenriched ECM. In addition, TGF-b promotes the accumulation and translocation of b-catenin to the nucleus to trans-activate gene transcription. We have shown that TGF-b signaling and b-catenin accumulation correlate with repressed IGFBP6 transcription and IGFBP-6 secretion in primary cultures of DD cells. In parallel, we have shown that cellular b-catenin levels are increased and ECMassociated IGFBP-6 levels are depleted in DD tissues relative to patient-matched, unaffected tissue controls. Data from a related fibrosis, desmoid tumour, indicate that IGFBP6 transcription can be repressed by b-catenin interactions with the TCF/Lef transcription factor complex. We hypothesize that TGF-b-induced b-catenin interactions with the TCF/Lef transcription factor complex represses IGFBP6 transcription in DD, resulting in decreased levels of IGFBP-6 and increased IGF-II bioavailability. As IGF-II has been shown to act in combination with TGF-b to promote myofibroblast differentiation in other systems, we further hypothesize that IGFBP-6 repression and increased IGF-II bioavailability are previously unrecognized mediators of myofibroblast differentiation in DD. Methods: Cell proliferation (WST-1) assays were performed on collagen-coated substrates with or without IGFBP-6, IGF-II or TGF-b treatments. Myofibroblast differentiation was assessed by stressed Fibroblast Populated Collagen Lattice (sFPCL) contraction assays and SDS-PAGE for a-smooth muscle actin levels. Chromatin Immunoprecipitation (ChIP) assays are underway to assess TGF-binduced b-catenin/TCF/Lef transcription complex interactions with the IGFBP6 promoter in DD and control cells. Results: TGF-b (12.5 ng.ml) or IGFBP-6 (200 and 400ng/ml) did not affect the basal proliferation rates of DD or control cells while IGF-II (100ng/ml) inhibited the proliferation of control cells but not DD cells. TGF-b induced contractility and a-smooth muscle actin levels, indicative of myofibroblast differentiation. IGFBP-6 (400ng/ml) inhibited TGF-b induced contractility and asmooth muscle actin levels in DD and control cells. IGF-II induced the contractility of DD and control cells but did not alter asmooth muscle actin levels. ChIP assays to confirm TGF-b-induced b-catenin/TCF/LEF transcription complex binding to the IGFBP6 promoter are underway. Discussion/Conclusion: TGF-b, IGF-II and IGFBP-6 do not induce DD or control cell proliferation. TGF-b induces myofibroblast differentiation and IGFBP-6 at 400ng/ml, incorporated into the collagen-rich ECM, inhibits this process. IGF-II promotes DD and control cell contractility but not a-smooth muscle actin levels, suggesting that it acts through an alternative mechanism. We are currently investigating the Rho kinase-mediated actin/myosin polymerization in IGF-II-induced cell contraction. Our data suggest that IGFBP-6 acts as an inhibitor of myofibroblast differentiation that is repressed by TGF-b signaling, thereby promoting IGF-IIinduced contractility. IGFBP-6 may have potential as a therapeutic target for the future development of non-surgical treatment interventions in DD and, potentially, other disease conditions where changes in IGF-II bioavailability are primary components of the pathophysiology.