Age-Related Changes in Pericellular Hyaluronan Organization Leads to Impaired Dermal Fibroblast to Myofibroblast Differentiation

Abstract

We have previously demonstrated that transforming growth factor-beta1 (TGF-beta1)-mediated fibroblast-myofibroblast differentiation is associated with accumulation of a hyaluronan (HA) pericellular coat. The current study demonstrates failure of fibroblast-myofibroblast differentiation associated with in vitro aging. This is associated with attenuation of numerous TGF-beta1-dependent responses, including HA synthesis and induction of the HA synthase enzyme HAS2 and the hyaladherin tumor necrosis factor-alpha-stimulated gene 6 (TSG-6), which led to an age-related defect in pericellular HA coat assembly. Inhibition of HAS2-dependent HA synthesis by gene silencing, removal of the HA coat by hyaluronidase digestion, or gene silencing of TSG-6 or cell surface receptor CD44 led to abrogation of TGF-beta1-dependent induction of alpha-smooth muscle actin in "young" cells. This result supports the importance of HAS2-dependent HA synthesis and the HA coat during phenotypic activation. Interleukin-1beta stimulation, however, failed to promote phenotypic conversion despite coat formation. A return to basal levels of HA synthesis in aged cells by HAS2 overexpression restored TGF-beta1-dependent induction of TSG-6 and pericellular HA coat assembly. However, this did not lead to the acquisition of a myofibroblast phenotype. Coordinated induction of HAS2 and TSG-6 facilitation of pericellular HA coat assembly is necessary for TGF-beta1-dependent activation of fibroblasts, and both components of this response are impaired with in vitro aging. In conclusion, the HA pericellular coat is integral but not sufficient to correct for the age-dependent defect in phenotypic conversion.

Transforming Growth Factor-β1 (TGF-β1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts

“…We have shown that EGFR is an essential receptor in the differentiation and proliferation of fibroblasts, and its interactions with HA and CD44 are required for both cellular responses (32,33). As fibroblasts age, they display a resistance to phenotypic activation (15,22,23,34,35), and we have shown that this is associated with loss of EGFR expression. This resistance was overcome by overexpression of EGFR with HAS2 (32), confirming that EGFR and HA are necessary components of the differentiation pathway in fibroblasts.…”

“…HAS2 is the enzyme primarily responsible for up-regulation of HA synthesis in fibroblasts following TGF-␤1-induced differentiation to myofibroblasts (22)(23)(24)(25). We have previously shown that HAS2 is an important mediator of differentiation in fibroblasts (32,33).…”

“…The biosynthesis of HA is regulated by three mammalian HA synthase isoenzymes, of which hyaluronan synthase 2 (HAS2) demonstrates the greatest expression in fibroblasts (22)(23)(24)(25). We have previously shown that as a consequence of myofibroblastic differentiation, a pericellular coat of HA accumulated around differentiated cells (26).…”

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

“…Cells were growtharrested in serum-free medium for 48 h before use in experiments, and all experiments were performed under serum-free conditions unless otherwise stated. For the induction of differentiation, cells were exposed to a single treatment of TGF␤1 at the concentrations indicated for times up to 72 h as in previous studies (12)(13)(14).…”

“…We have previously demonstrated that TGF␤1-mediated phenotypic activation is dependent on an increase in the synthesis of the extracellular matrix polysaccharide hyaluronan (HA) (12)(13)(14). TGF␤1, through stimulation of EGF and subsequent activation of EGFR-associated ERK-MAPK activity, stimulates HA synthesis.…”