Aging Fibroblasts Resist Phenotypic Maturation Because of Impaired Hyaluronan-Dependent CD44/Epidermal Growth Factor Receptor Signaling

Fibroblast differentiation into myofibroblasts is a key event during normal wound repair. We have previously demonstrated an age-related defect in this process associated with impaired synthesis of hyaluronan (HA) synthase (HAS) 2 but failed to prescribe its role in a mechanistic sense. Here we demonstrate that in addition to HAS2, there is loss of EGF receptor (EGF-R) in aged cells, and both are required for normal fibroblast functionality. Analysis of molecular events revealed that in young cells, transforming growth factor (TGF)-β1-dependent phenotypic activation uses two distinct but cooperating pathways that involve TGF-β receptor/Smad2 activation and EGF-mediated EGF-R/extracellular signal-regulated kinase (ERK) 1/2 signaling, and the latter is compromised with in vitro aging. Pharmacological inhibition of any of the five intermediates (TGF-β receptor, Smad2, EGF, EGF-R, and ERK1/2) attenuated TGF-β1 induction of α-smooth muscle actin. We present evidence that the HA receptor CD44 co-immunoprecipitates with EGF-R after activation by TGF-β1. This interaction is HA-dependent because disruption of HA synthesis abrogates this association and inhibits subsequent ERK1/2 signaling. In aged fibroblasts, this association is lost with resultant suppression of ERK1/2 activation. Forced overexpression of EGF-R and HAS2 in aged cells restored TGF-β1-mediated HA-CD44/EGF-R association and α-smooth muscle actin induction. Taken together, these results demonstrate that HA can serve as a signal integrator by facilitating TGF-β1-mediated CD44-EGF-R-ERK interactions and ultimately fibroblast phenotype. We propose a model to explain this novel mechanism and the functional consequence of age-dependent dysregulation.