Deletion of Tak1 in Fibroblast-specific Protein 1 Positive Cells Reduces Tendon Adhesion Formation Via Reduced Myofibroblast Activation

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2015.The clinical outcomes of flexor tendon injury repairs are frequently complicated by extensive scar formation that impairs hand functions. Fibrotic scars contain myofibroblasts that produce excessive extracellular matrix proteins such as collagen I and collagen III. TGFβ, a potent activator of fibroblasts has been implicated in tendon adhesion formation. Though studies targeting components of the TGFβ signaling pathway have shown effective reduction in adhesion formations, little progress has been made in translating these findings from bench to bedside due to our limited knowledge of the precise role of TGFβ signaling during tendon healing. TGFβ regulates target genes through multiple mechanisms. In particular, TGFβ-Activated Kinase 1 (TAK1), a MAP Kinase Kinase Kinase which is a component of the TGFβ receptor complex, can activate p38 MAP Kinase and JNK. In current study, we employed both in vitro and in vivo approaches to investigate the role of TAK1 in regulation of tendon injury repair. Specifically, we utilized a murine model of primary tendon repair in which Tak1 is conditionally deleted in fibroblasts using the Fsp1-Cre transgene with the Tak1 floxed allele. In vitro, we investigated the role of TAK1 in regulation of fibroblast proliferation, migration, contraction, and collagen production. We found that upon injury, Fsp1-lineage cells populated the reparative tissues at the injury site while native tendon tissue did not have any Fsp1-positive cells. Histology showed reduced scar formation in Fsp1cre;Tak1fx/fx mice during tendon healing beginning at day 7. Consistent with histology, Fsp1cre;Tak1fx/fx mice had improved range of motion in tendon repairs and reduced expression of collagen I and III throughout the repair process. Tendon repairs in Fsp1cre;Tak1fx/fx mice also had reduced expression of Acta2, a marker for myofibroblasts, suggesting that TAK1 is involved in fibroblast activation. Inhibition of TAK1 reduces the transformation of fibroblasts to myofibroblasts following TGFβ1 treatment of fibroblasts in vitro. Tak1 deletion in TGFβ1-treated fibroblasts resulted in decreased expression of Acta2. Several other key functions of activated myofibroblasts, such as proliferation, migration, contraction, and collagen production were also inhibited by the loss of Tak1. Additionally, we found that addition of TGFβ1 to fibroblast cell cultures enhanced the activation and nuclear accumulation of β-catenin. In contrast, Tak1 deletion in MEF cultures resulted in decreased myofibroblast gene expression and reduced activation of Wnt/β-catenin signaling. These analyses demonstrate that TAK1 is a critical mediator in tendon adhesion formation and fibroblast to myofibroblast conversion. Inhibition of Tak1 could serve as novel approach for therapeutic intervention for the treatment of adhesion formation. TAK1 links TGFβ and β-catenin signaling during the development of fibrotic responses after tendon injury