title: Osteopontin expression is required for myofibroblast differentiation.
abstract: Osteopontin (OPN) is a multifunctional cytokine that is strongly expressed in healing wounds and fibrotic lesions, both of which are characterized by the formation of myofibroblasts. We examined the role of OPN in myofibroblast differentiation induced by the profibrotic cytokine transforming growth factor-beta1. In cultured cardiac or dermal fibroblasts treated with transforming growth factor-beta1, there was a 2- to 5-fold increase in the expression of the myofibroblast markers alpha-smooth muscle actin and extradomain A fibronectin but no significant increase of these proteins in OPN-null fibroblasts. Phalloidin staining for actin filaments and immunostaining for alpha-smooth muscle actin and focal adhesion proteins showed reduced stress fibers, focal adhesions, and lamellipodia in OPN-null fibroblasts compared with wild-type cells. OPN-null fibroblasts exhibited 40% to 60% less spreading, 50% less resistance to detachment by shear force, and a approximately 3-fold reduction in collagen gel contraction. These defects were partially rescued by ectopic expression of OPN. Mass spectrometric analysis of proteins in focal adhesions formed on collagen type I beads revealed an enrichment of HMGB1 protein in wild-type cells, whereas HMGB1 was not detected in OPN-null cells. Treatment of wild-type cells with small interfering RNA to knock down OPN reduced transforming growth factor-beta1-induced alpha-smooth muscle actin and HMGB1 to levels observed in OPN-null cells. These studies demonstrate that OPN is required for the differentiation and activity of myofibroblasts formed in response to the profibrotic cytokine transforming growth factor-beta1.
authors: Faculty of Dentistry, University of Toronto, 124 Edward St, Toronto, Ontario M5G 1G6, Canada.
authors: Yair
authors: Y
authors: Lenga
authors: Adeline
authors: A
authors: Koh
authors: Aruni Shamalee
authors: AS
authors: Perera
authors: Christopher A
authors: CA
authors: McCulloch
authors: Jaro
authors: J
authors: Sodek
authors: Ron
authors: R
authors: Zohar
mesh_terms: D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis / Q000235:genetics; D020405:Dermis / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005355:Fibrosis; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D024243:HMGB1 Protein / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D053495:Osteopontin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011554:Pseudopodia / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D022502:Stress Fibers / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology; D014945:Wound Healing / Q000502:physiology
chemical_list: D000199:Actins; D024042:Collagen Type I; D024243:HMGB1 Protein; D034741:RNA, Small Interfering; C506896:Spp1 protein, mouse; D053773:Transforming Growth Factor beta1; D053495:Osteopontin