Tumor Necrosis Factor-stimulated Gene 6 (TSG-6)-mediated Interactions with the Inter-α-inhibitor Heavy Chain 5 Facilitate Tumor Growth Factor β1 (TGFβ1)-dependent Fibroblast to Myofibroblast Differentiation

Abstract

Fibroblasts are central to wound healing and fibrosis through TGF␤1-triggered differentiation into contractile, ␣-smooth muscle actin (␣-SMA)-positive myofibroblasts. This is mediated by accumulation of a pericellular matrix of hyaluronan (HA) and the HA-dependent co-localization of CD44 with the epidermal growth factor receptor (EGFR). Interactions of HA with hyaladherins, such as inter-␣-inhibitor (I␣I) and tumor necrosis factor-stimulated gene-6 (TSG-6), are also essential for differentiation. This study investigated the mechanisms involved. TSG-6 and ␣-SMA had different kinetics of induction by TGF␤1, with TSG-6 peaking before ␣-SMA. Si CD44 or EGFR inhibition prevented differentiation but had no effect on TSG-6 expression. TSG-6 was essential for differentiation, and mAb A38 (preventing I␣I heavy chain (HC) transfer), HA-oligosaccharides, cobalt, or Si bikunin prevented TSG-6 activity, preventing differentiation. A38 also prevented the EGFR/CD44 association. This suggested that TSG-6/I␣I HC interaction was necessary for the effect of TSG-6 and that HC stabilization of HA initiated the CD44/EGFR 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 knockdown 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 HA-dependent control of cell phenotype. The interaction of HC5 with cell surface HA was essential for TGF␤1-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 remodeling 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, characterized by the expression of the smooth muscle isoform of ␣-actin (␣-SMA), 3 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 etiologies (3-7), liver disease (8), and pulmonary fibrosis (9).The cytokine TGF␤1 is a well recognized mediator of progressive tissue fibrosis, and in vitro and in vivo evidence suggests that it is the primary driving force in fibroblast-myofibroblast phenotypic activation (10, 11). The sequence of events leading up to this transformation is being increasingly characterized at a molecular level in an attempt to understan...

ITIH5 induces a shift in TGF‐β superfamily signaling involving Endoglin and reduces risk for breast cancer metastasis and tumor death

“…In case of Id1, ITIH5 ‐ in the absence of ENG in the transient setting ‐ is able to interfere with phosphorylation of Smad1/5/9 which in turn lowers Id1 expression. Since ITIH5 has been shown to control the phenotype of fibroblasts in a TGF‐β1 dependent manner, a general impact of ITIH5 on modulating this pathway can be assumed and the underlying mechanism is currently under investigation.…”

“…Due to linkage of several heavy chains with HA molecules, ITIH‐HA complex formation causes “cable like structures” that are thought to stabilize ECM . As a consequence of ITIH‐mediated stabilization of HA‐oligosaccharides, ECM signaling pathways could be modulated as shown recently in fibroblasts initiating a CD44/EGFR interaction …”

“…Apart from that the role of ITIH5 in metastatic processes, its downstream targets and signaling pathways are still unknown. Interestingly, Martin and colleagues recently reported that interaction of ITIH5 with HA controls differentiation of fibroblasts into myofibroblast in a transforming growth factor (TGF)‐β1‐dependent manner . It is well known that members of the TGF‐β superfamily significantly affect tumorigenesis via regulating processes such as cancer cell adhesion dynamics, migration, proliferation, or the epithelial‐to‐mesenchymal transition (EMT) .…”

“…3,4,10,11 As a consequence of ITIH-mediated stabilization of HA-oligosaccharides, ECM signaling pathways could be modulated as shown recently in fibroblasts initiating a CD44/EGFR interaction. 12 As early as 2008, it has been demonstrated in carcinogenesis that ITIH5 gene expression is inactivated by aberrant DNA hypermethylation in breast cancer. 13,14 .…”

“…Interestingly, Martin and colleagues recently reported that interaction of ITIH5 with HA controls differentiation of fibroblasts into myofibroblast in a transforming growth factor (TGF)-β1-dependent manner. 12 It is well known that members of the TGF-β superfamily significantly affect tumorigenesis via regulating processes such as cancer cell adhesion dynamics, migration, proliferation, 22,23 or the epithelial-to-mesenchymal transition (EMT). [24][25][26] Mechanistically, the prototypical member TGF-β1 binds to membrane receptors type I (ALK5 and ALK1) and type II (TβRII) transferring signals to the cytosol via phosphorylation of Smad proteins.…”