Tumour necrosis factor-stimulated gene (TSG)6-mediated Interactions with the Inter–alpha-Inhibitor Heavy Chain 5 facilitate TGFβ1-dependent Fibroblast to Myofibroblast Differentiation

Fibroblasts are central to wound healing and fibrosis through Transforming Growth Factor-β1 (TGF-β1)triggered differentiation into contractile, α-smooth muscle actin (α-sma)-positive myofibroblasts. This is mediated by accumulation of a pericellular matrix of hyaluronan (HA) and the HA-dependent colocalisation of CD44 with the Epidermal Growth Factor Receptor (EGFR). Interactions of HA with hyaladherins, such as Inter-alpha-Inhibitor (IaI) and Tumour Necrosis Factor-stimulated gene-6 (TSG-6) are also essential for differentiation. This study investigated the mechanisms involved. TSG-6 and α-sma had different kinetics of induction by TGFβ1, with TSG-6 peaking before α-sma. siCD44 or EGFR inhibition prevented differentiation but had no effect on TSG-6 expression. TSG-6 was essential for differentiation, and mAb A38 (preventing IαI heavy chain [HC] transfer), HA-oligosaccharides, Cobalt, or siBikunin prevented TSG-6 activity, preventing differentiation. A38 also prevented the EGFR/CD44 association. This suggested that TSG6/IαIHC interaction was necessary for the effect of TSG-6 and that HC-stabilisation of HA initiated the CD44/EGF-R association. The newly-described HC5 was shown to be the principal HC expressed and its cell surface expression was prevented by siRNA inhibition of TSG-6 or Bikunin. HC5 was released by hyaluronidase treatment, confirming its association with cell surface HA. Finally, HC5 knock down by siRNA confirmed its role in myofibroblast differentiation. The current study describes a novel mechanism linking the TSG-6 transfer of the newly-described HC5 to the HAdependent control of cell phenotype. The interaction of HC5 with cell surface HA was essential for TGFβ1-dependent differentiation of fibroblasts to myofibroblasts, highlighting its importance as a novel potential therapeutic target. The fibroblast is the most abundant cell type in normal connective tissues. It plays a central role in the synthesis, degradation and remodelling of extracellular matrix both in health and in disease. At sites of tissue damage and in the context of wound healing, activated fibroblasts, termed myofibroblasts, with a contractile phenotype, characterised by the expression of the smooth muscle isoform of α-actin (α-sma), are essential for the synthesis of a collagen-rich scar, providing the force for wound contraction (1). During a healing response, myofibroblasts are a transient cell population (2). Myofibroblasts are also the major effectors of fibrosis, their persistent presence has been established as the best marker of numerous types of progressive organ dysfunction such as that seen in chronic renal failure of various aetiologies (3, 4, 5, 6, 7), liver disease (8) and pulmonary fibrosis (9). http://www.jbc.org/cgi/doi/10.1074/jbc.M115.670521 The latest version is at JBC Papers in Press. Published on May 3, 2016 as Manuscript M115.670521 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. at U N IV W A L E S C O L L O F C A R D IF on M ay 8, 2016 hp://w w w .jb.org/ D ow nladed from IαI heavy chain 5 and myofibroblast differentiation.