Abstract
Fibroblasts are central to wound healing and fibrosis through TGFβ1-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 co-localization of CD44 with the epidermal growth factor receptor (EGFR). Interactions of HA with hyaladherins, such as inter-α-inhibitor (IαI) and tumor 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 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 IαI 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/IαI 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 TGFβ1-dependent differentiation of fibroblasts to myofibroblasts, highlighting its importance as a novel potential therapeutic target.
Highlights
The fibroblast is the most abundant cell type in normal connective tissues
We have examined the role of tumor necrosis factor-stimulated gene 6 (TSG-6) following TGF1 stimulation and highlighted heavy chains (HCs) transfer activity as being key to form a HA matrix that is anchored by CD44, leading to a CD441⁄7EGFR signaling complex
To determine the temporal relationship between TSG-6 expression and induction of the myofibroblast phenotype, growth-arrested fibroblasts were stimulated with TGF1 (10 ng/ml), and mRNA was extracted at times up to 72 h
Summary
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. In the TGF1-triggered generation of the myofibroblast phenotype, we proposed that TSG-6 may play an important part in forming the HA1⁄7CD441⁄7EGFR complex, which signals to induce ERK activation. We have examined the role of TSG-6 following TGF1 stimulation and highlighted HC transfer activity as being key to form a HA matrix that is anchored by CD44, leading to a CD441⁄7EGFR signaling complex.
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