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A2659 - Myofibroblast Differentiation Depends on the Plasma Membrane Translocation of TRPV4/PI3Kg Complexes
Author Block: L. Grove1, M. Mohan1, S. Abraham2, R. G. Scheraga3, B. D. Southern4, J. F. Crish1, S. V. Naga Prasad5, M. A. Olman6; 1Pathobiology, Cleveland Clinic, Cleveland, OH, United States, 2Cleveland Clinic Foundation, Cleveland, OH, United States, 3Respiratory Institute, Cleveland Clinic, Cleveland, OH, United States, 4Pulmonary, Allergy, and Critical Care, Cleveland Clinic, Cleveland, OH, United States, 5Molecular Cardiology, Cleveland Clinic, Cleveland, OH, United States, 6Dept of Pathobiology, Cleveland Clinic, Cleveland, OH, United States.
Introduction/Rationale
Both mechanical and transforming growth factor beta (TGFβ)-initiated signals are necessary for myofibroblast differentiation. While matrix stiffness or internally-generated cell tension can be the source of these mechanical signals, the pathways by which these signals are transmitted are not yet fully understood. Our work has shown that the mechanosensitive, calcium-permeable ion channel, TRPV4, plays a key role in myofibroblast differentiation and in vivo pulmonary fibrosis, while others have shown that the γ isoform of PI3K (PI3Kγ) plays a key role in these processes as well. Initial work in our lab suggests that TRPV4 and PI3Kγ interact to mediate myofibroblast differentiation. Hence, this study aimed to determine the mechanism by which these pathways interact in the context of myofibroblast differentiation.
Methods
Co-IPs of plasma membranes (PMs) ± TGFβ (2 ng/mL, 3-24 h) were used to determine TRPV4/PI3Kγ interactions. PM translocation of TRPV4 and PI3Kγ was assessed by immunofluorescence or Western blot in wild type (WT), PI3Kγ null, or TRPV4 null mouse lung fibroblasts (MLFs). The calcium response to TRPV4 agonist GSK (10-1000 nM) was evaluated using a high-throughput dye-based system. Myofibroblast differentiation was assessed via α-SMA incorporation into stress fibers by immunofluorescence. Interactions between TRPV4 and specific regions of PI3Kγ were evaluated by co-IP in transfected PI3Kγ null MLFs.
Results
For the first time, our data show that PI3Kγ is downstream of TRPV4 activation. Both TRPV4 and PI3Kγ translocate to the PM in WT MLFs with TGFβ stimulation. There is a reciprocal relationship between TRPV4 and PI3Kγ (PI3Kγ translocation requires TRPV4, and TRPV4 translocation requires PI3Kγ). This reciprocal relationship is also functional, as TGFβ-induced PI3Kγ activity is blunted in TRPV4 null MLFs, while TRPV4 activity is nearly absent in PI3Kγ null MLFs. Further, for the first time, we show that PI3Kγ forms a strong complex with TRPV4 (Kd = 320nM via Biacore) that increases at the PM with TGFβ. Interestingly, these complexes depend on TRPV4 interactions with the non-catalytic, amino-terminal region of PI3Kγ. The non-catalytic, amino-terminal region of PI3Kγ is also necessary and sufficient to rescue myofibroblast differentiation in transfected PI3Kγ null MLFs.
Conclusions
For the first time, results of our study reveal that TRPV4 and PI3Kγ form strong complexes that translocate to the PM with TGFβ. These complexes require only the non-catalytic region of PI3Kγ, which is also necessary and sufficient to drive myofibroblast differentiation. These TRPV4/PI3Kγ complexes may be amenable to targeted therapeutics designed to ameliorate pulmonary fibrosis.