Increased Pyridinoline Cross‐Link Levels in Fibrosis is Accomplished by TGFβ Induced Lysyl Hydroxylases 2B Expression

Differentiation of fibroblasts into contractile, α‐smooth muscle actin (α‐SMA) expressing myofibroblasts depends on the action of the cytokine TGFβ in conjunction with mechanical tension. The goal of this study was to assess whether mechanical stress may play a role in activating TGFβ. Previous studies have shown that myofibroblasts secrete TGFβ1 as a large latent complex, consisting of the latency associated protein (LAP), the latent TGFβ1 binding protein (LTBP‐1) and TGFβ1. LTBP‐1 targets the small latent complex of LAP and TGFβ1 to the extracellular matrix (ECM), providing a stock of latent TGFβ1. To reveal the mechanisms triggering the release of active TGFβ1 from LAP and LTBP‐1, myofibroblasts were cultured on flexible silicone membranes for 3d before being subjected to unique 5% uniaxial stretch. After stress application we observed the course of TGFβ1 activation over 24 h by quantifying luciferase synthesis under the control of the TGFβ‐inducible PAI‐1 promoter. TGFβ activation showed two distinct peaks 1 h and 6 h after stretch. TGFβ1 mRNA levels were increased after 6 h as assessed by semi‐quantitative RT‐PCR. To further determine if rapidly activated TGFβ1 was released from ECM stores, we stretched myofibroblast‐derived ECM after cells have been removed by desoxycholate or EDTA treatment. Finally, we assessed the importance of the actin cytoskeleton in TGFβ1 activation by stretching Triton‐X‐100‐ and cytochalasin‐D‐treated myofibroblasts. Our results suggest that mechanical activation of TGFβ in myofibroblast culture requires cell activity and a functional cytoskeleton.