RATIONALE: Emerging evidence suggest the macrophage-fibroblast interaction can drive organ fibrosis. Myofibroblast differentiation is a key step in the pathogenesis of pulmonary fibrosis that requires both a soluble (e.g., TGF-β) and a mechanical signal. We have previously implicated the mechanosensitive cation channel, TRPV4, as a mediator of myofibroblast differentiation, experimental pulmonary fibrosis and pro-inflammatory macrophage activation. We undertook this study to determine if TRPV4 regulates crosstalk of macrophages and fibroblasts to drive fibrosis. METHODS: Conditioned media (CM, 1:10 dilution) from bone marrow-derived macrophages (BMDMs) from WT and Trpv4 KO mice that were plated on either polyacrylamide gels of physiologic range stiffnesses (1kPa-normal lung, 8-25kPa fibrotic lung) or tissue culture plastic (polystyrene, 106 kPa) and transferred to WT mouse lung fibroblasts (MLFs, 1-48h) ± immunodepletion of TGF-β (by 80-90%). CM was added to MLFs ± TGF-β receptor kinase inhibitor (SD208, 48h) or SMAD2/SMAD3 siRNA (20nM, 24-48h) ± recombinant TGF-β to test for presence of a TGF- β inhibitor. Myofibroblast differentiation was measured by quantifying α-SMA in stress fibers (phalloidin) and collagen-1 abundance. Active TGF-β was measured using TGF-β-reporter mink lung epithelial cells (MLEC) and phosphorylation of SMAD2/SMAD3, while TGF-β antigen was measured by ELISA. As TGF-β activation requires the cytoskeleton, inhibition of myosin was performed pharmacologically (bleomycin) or with siRNA. Myeloid-specific Trpv4 KO mice (TRPV4LysMcre) and controls (TRPV4fl/fl) were orotracheally given 2U/kg bleomycin and BALF and histology were assessed 7 days later. RESULTS: Conditioned media (CM) from WT BMDMs plated on increasing substrate stiffnesses progressively induced myofibroblast differentiation in WT fibroblasts, an effect that was lost with CM from Trpv4 KO BMDMs. TGF-β receptor kinase inhibitor, or TGF-β immunodepletion from the BMDM CM abrogated the myofibroblast differentiating effect. While the total antigenic TGF-β in WT and Trpv4 KO BMDMs CM was similar, the active fraction of TGF- β was reduced in Trpv4 KO BMDM CM. Spiking of Trpv4 KO CM with recombinant TGF-β revealed evidence for a soluble TGF-β inhibitor, which is being characterized. Interestingly, myeloid-specific TRPV4 KO mice (TRPV4LysMcre) had a reduced inflammatory response (neutrophils, MCP-1) to bleomycin (7 days) as compared to controls (TRPV4fl/fl). CONCLUSIONS: Our study provides a novel mechanistic link between TRPV4-dependent matrix stiffness sensing by macrophages and TGF-β driven pro-fibrotic responses in fibroblasts. Macrophage-fibroblast crosstalk through TRPV4 is a potential therapeutic target to ameliorate pulmonary fibrosis.

Myofibroblasts are key contributors to pathological fibrotic conditions of several major organs. The transdifferentiation of fibroblasts into myofibroblasts requires both a mechanical signal and transforming growth factor-β (TGF-β) signaling. The cation channel transient receptor potential vanilloid 4 (TRPV4) is a critical mediator of myofibroblast transdifferentiation and in vivo fibrosis through its mechanosensitivity to extracellular matrix stiffness. Here, we showed that TRPV4 promoted the transdifferentiation of human and mouse lung fibroblasts through its interaction with phosphoinositide 3-kinase γ (PI3Kγ), forming nanomolar-affinity, intracellular TRPV4-PI3Kγ complexes. TGF-β induced the recruitment of TRPV4-PI3Kγ complexes to the plasma membrane and increased the activities of both TRPV4 and PI3Kγ. Using gain- and loss-of-function approaches, we showed that both TRPV4 and PI3Kγ were required for myofibroblast transdifferentiation as assessed by the increased production of α-smooth muscle actin and its incorporation into stress fibers, cytoskeletal changes, collagen-1 production, and contractile force. Expression of various mutant forms of the PI3Kγ catalytic subunit (p110γ) in cells lacking PI3Kγ revealed that only the noncatalytic, amino-terminal domain of p110γ was necessary and sufficient for TGF-β-induced TRPV4 plasma membrane recruitment and myofibroblast transdifferentiation. These data suggest that TGF-β stimulates a noncanonical scaffolding action of PI3Kγ, which recruits TRPV4-PI3Kγ complexes to the plasma membrane, thereby increasing myofibroblast transdifferentiation. Given that both TRPV4 and PI3Kγ have pleiotropic actions, targeting the interaction between them could provide a specific therapeutic approach for inhibiting myofibroblast transdifferentiation.