Dissecting the roles of endothelin,TGF-β and GM-CSF on myofibroblast differentiation by keratinocytes

Summary

Myofibroblasts are specialized fibroblasts that contribute to wound healing by producing extracellular matrix and by contracting the granulation tissue.They appear in a phase of wound healing when the dermis strongly interacts with activated epidermal keratinocytes. Direct co-culture with keratinocytes upregulates TGF-β???????????activity and also induces fibroblast to differentiate into α-smooth muscle actin (αSMA)-positive myofibroblasts. TGF-βactivity alone cannot completely account for αSMA induction in these co-cultures, and here we analyze mechanical force generation, another potent inducer of myofibroblast differentiation in this model. Using deformable silicone substrates, we show that contractile activity of fibroblasts is already induced after 1-2-days of co-culture, when fibroblasts are generally αSMA negative. Endothelin-1 (ET-1), the most potent inducer of smooth muscle cell contraction, was up-regulated in co-cultures, while blocking ET-1 with the ET receptor inhibitor PD156252 inhibited contraction in these early co-cultures. In 4-5 days of co-culture, however, fibroblast contractile activity correlated with an increased expression of αSMA expression. Stimulation of fibroblast mono-cultures with ET-1 in a low serum medium did not induce αSMA expression; however, ET-1 did synergize with TGF-β. Surprisingly, GM-CSF, another mediatorstimulating myofibroblast differentiation in granulation tissue, inhibited αSMA expression in fibroblasts, costimulated with TGF-β and ET-1. GM-CSF activated NFκB, thus interfering with TGF-β signaling. Blocking TGF-β and ET-1 largely impaired αSMA induction in co-cultures at day 7 and, in combination, almost completely prevented αSMA induction. Our results dissect the roles of TGF-β and ET-1 on mechanical force generation in keratinocyte-fibroblast co-cultures, and identify GM-CSF as an inducer of myofibroblasts acting indirectly.

Keywords

Wound healing - myofibroblasts - endothelin,TGF-β - GM-CSF

^§ Current address: Nestlé Research Center, Lausanne, Switzerland

• References

• 1 Martin P. Wound healing-aiming for perfect skin regeneration. Science 1997; 276: 75-81.
• 2 Gabbiani G, Ryan GB, Maino G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 1971; 27: 549-550.
• 3 Tomasek JJ, Gabbiani G, Hinz B. et al. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 2002; 03: 349-363.
• 4 Skalli O, Ropraz P, Trzeciak A. et al. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 1986; 103: 2787-2796.
• 5 Oda D, Gown AM, Vande Berg JS. et al. Instability of the myofibroblast phenotype in culture. Exp Mol Pathol 1990; 52: 221-234.
• 6 Desmouliere A, Geinoz A, Gabbiani F. et al. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 1993; 122: 103-111.
• 7 Serini G, Bochaton-Piallat ML, Ropraz P. et al. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. J Cell Biol 1998; 142: 873-881.
• 8 Hinz B, Gabbiani G. Mechanisms of force generation and transmission by myofibroblasts. Curr Opin Biotechnol 2003; 14: 538-546.
• 9 Arora PD, Narani N, McCulloch CA. The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. Am J Pathol 1999; 154: 871-882.
• 10 Hinz B, Dugina V, Ballestrem C. et al. Alphasmooth muscle actin is crucial for focal adhesion maturation in myofibroblasts. Mol Biol Cell 2003; 14: 2508-2519.
• 11 Vaughan MB, Howard EW, Tomasek JJ. Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast. Exp Cell Res 2000; 257: 180-189.
• 12 Hinz B, Mastrangelo D, Iselin CE. et al. Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am J Pathol 2001; 159: 1009-1020.
• 13 Grinnell F. Fibroblasts, myofibroblasts, and wound contraction. J Cell Biol 1994; 124: 401-404.
• 14 Grinnell F, Zhu M, Carlson MA. et al. Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue. Exp Cell Res 1999; 248: 608-619.
• 15 Niland S, Cremer A, Fluck J. et al. Contraction-dependent apoptosis of normal dermal fibroblasts. J Invest Dermatol 2001; 116: 686-692.
• 16 Desmouliere A, Redard M, Darby I. et al. Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol 1995; 146: 56-66.
• 17 Yang L, Qiu CX, Ludlow A. et al. Active transforming growth factor-beta in wound repair: determination using a new assay. Am J Pathol 1999; 154: 105-111.
• 18 Harris AK, Wild P, Stopak D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 1980; 208: 177-179.
• 19 Yanagisawa M, Kurihara H, Kimura S. et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 411-415.
• 20 Rockey DC, Chung JJ. Endothelin antagonism in experimental hepatic fibrosis Implications for endothelin in the pathogenesis of wound healing. J Clin Invest 1996; 98: 1381-1388.
• 21 Vyalov S, Desmouliere A, Gabbiani G. GMCSF-induced granulation tissue formation: relationships between macrophage and myofibroblast accumulation. Virchows Arch B Cell Pathol Incl Mol Pathol 1993; 63: 231-239.
• 22 Rubbia-Brandt L, Sappino AP, Gabbiani G. Locally applied GM-CSF induces the accumulation of alpha-smooth muscle actin containing myofibroblasts. Virchows Arch B Cell Pathol Incl Mol Pathol 1991; 60: 73-82.
• 23 Pojda Z, Struzyna J. Treatment of non-healing ulcers with rhGM-CSF and skin grafts. Lancet 1994; 343: 1100.
• 24 da Costa RM, Aniceto C, Jesus FM. et al. Quick healing of leg ulcers after molgramostim. Lancet 1994; 344: 481-482.
• 25 Maas-Szabowski N, Starker A, Fusenig NE. Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGFalpha. J Cell Sci 2003; 116: 2937-2948.
• 26 Smola H, Thiekotter G, Fusenig NE. Mutual induction of growth factor gene expression by epidermal-dermal cell interaction. J Cell Biol 1993; 122: 417-429.
• 27 Boukamp P, Petrussevska RT, Breitkreutz D. et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988; 106: 761-771.
• 28 Cody WL, He JX, Reily MD. et al. Design of a potent combined pseudopeptide endothelinA/ endothelin-B receptor antagonist, Ac- DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21 (PD 156252): examination of its pharmacokinetic and spectral properties. J Med Chem 1997; 40: 2228-2240.
• 29 Sanchez-Madrid F, Krensky AM, Ware CF. et al. Three distinct antigens associated with human T-lymphocyte-mediated cytolysis: LFA-1, LFA-2, and LFA-3. Proc Natl Acad Sci USA 1982; 79: 7489-7493.
• 30 Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983; 11: 1475-1489.
• 31 Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 1986; 46: 705-716.
• 32 Thomas JO, Kornberg RD. An octamer of histones in chromatin and free in solution. Proc Natl Acad Sci USA 1975; 72: 2626-2630.
• 33 Hinz B, Celetta G, Tomasek JJ. et al. Alphasmooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 2001; 12: 2730-2741.
• 34 Arora PD, McCulloch CA. Dependence of collagen remodelling on alpha-smooth muscle actin expression by fibroblasts. J Cell Physiol 1994; 159: 161-175.
• 35 Bitzer M, von Gersdorff G, Liang D. et al. A mechanism of suppression of TGFbeta/SMAD signaling by NF-kappa B/RelA. Genes Dev 2000; 14: 187-197.
• 36 Nagarajan RP, Chen F, Li W. et al. Repression of transforming-growth-factor-beta-mediated transcription by nuclear factor kappaB. Biochem J 2000; 348: 591-596.
• 37 Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003; 83: 835-870.
• 38 Maas-Szabowski N, Shimotoyodome A, Fusenig NE. Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism. J Cell Sci 1999; 112: 1843-1853.
• 39 Szabowski A, Maas-Szabowski N, Andrecht S. et al. c-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin. Cell 2000; 103: 745-755.
• 40 Werner S, Smola H. Paracrine regulation of keratinocyte proliferation and differentiation. Trends Cell Biol 2001; 11: 143-146.
• 41 Appleton I, Tomlinson A, Chander CL. et al. Effect of endothelin-1 on croton oil-induced granulation tissue in the rat A pharmacologic and immunohistochemical study. Lab Invest 1992; 67: 703-710.
• 42 Goto T, Yanaga F, Ohtsuki I. Studies on the endothelin-1-induced contraction of rat granulation tissue pouch mediated by myofibroblasts. Biochim Biophys Acta 1998; 1405: 55-66.
• 43 Reinehr RM, Kubitz R, Peters-Regehr T. et al. Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin 1. Hepatology 1998; 28: 1566-1577.
• 44 Waelti ER, Inaebnit SP, Rast HP. et al. Co-culture of human keratinocytes on post-mitotic human dermal fibroblast feeder cells: production of large amounts of interleukin 6. J Invest Dermatol 1992; 98: 805-808.
• 45 Corder R, Carrier M, Khan N. et al. Cytokine regulation of endothelin-1 release from bovine aortic endothelial cells. J Cardiovasc Pharmacol 1995; 26 (Suppl. 03) S56-S58.
• 46 Golden CL, Kohler JP, Nick HS. et al. Effects of vasoactive and inflammatory mediators on endothelin-1 expression in pulmonary endothelial cells. Am J Respir Cell Mol Biol 1995; 12: 503-512.
• 47 Yoshizumi M, Kurihara H, Morita T. et al. Interleukin 1 increases the production of endothelin-1 by cultured endothelial cells. Biochem Biophys Res Commun 1990; 166: 324-329.
• 48 Ziesche R, Petkov V, Williams J. et al. Lipopolysaccharide and interleukin 1 augment the effects of hypoxia and inflammation in human pulmonary arterial tissue. Proc Natl Acad Sci U S A 1996; 93: 12478-83.
• 49 Woods M, Wood EG, Mitchell JA. et al. Signal transduction pathways involved in cytokine stimulation of endothelin-1 release from human vascular smooth muscle cells. J Cardiovasc Pharmacol 2000; 36 (Suppl. 01) S407-S409.
• 50 Ebner K, Bandion A, Binder BR. et al. GMCSF activates NF-kappaB via direct interaction of the GMCSF receptor with IkappaB kinase beta. Blood 2003; 102: 192-199.
• 51 Xing Z, Tremblay GM, Sime PJ. et al. Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-beta 1 and myofibroblast accumulation. Am J Pathol 1997; 150: 59-66.
• 52 Smith CH, Allen MH, Groves RW. et al. Effect of granulocyte macrophage-colony stimulating factor on Langerhans cells in normal and healthy atopic subjects. Br J Dermatol 1998; 139: 239-246.
• 53 Kaplan G, Walsh G, Guido LS. et al. Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colonystimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing. J Exp Med 1992; 175: 1717-1728.