TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension

Abstract

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

Collagen Organization Within the Cartilage of Trpv4−/− Mice Studied with Two‐Photon Microscopy and Polarized Second Harmonic Generation

“…Although our measurements point toward more scattered orientations of collagen fibers in Trpv4 −/− mice, the differences were not statistically significant compared with WT samples at this age (4–5 days after birth). We interpret these data in the view of some recent results obtained by Gilchrist et al . Using stem mesenchymal cells grown on micropatterned substrates, the authors demonstrate that continuous exposure to TRPV4 channel inhibitors leads to a marked decrease in the deposition of aligned collagen fibers after 14 days in 2D substrates.…”

“…The impact of TRPV4 signaling is further complicated by the fact that TRPV4 has been implicated in chondrogenic differentiation . In addition, TRPV4 inactivation disrupts collagen deposition in mesenchymal stem cells . These data suggest that TRPV4 may be required for ECM deposition during the development of the cartilage tissue.…”

TRPV4—A Missing Link Between Mechanosensation and Immunity

“…Finally, substrate stiffness is regulated by the composition of the ECM which changes as a function of physiological (development, aging) and pathophysiological (atherosclerosis, hypertension, fibrosis) processes (90)(91)(92)(93). TRPV4 has been identified as a major mechanosensor for substrate stiffness, but so far this function has been exclusively attributed to parenchymal cells (30,31,(94)(95)(96)). Yet, it is fair to speculate that changes in substrate stiffness will similarly affect the mechanical forces that act upon immune cells during the processes of adhesion and transmigration, which accordingly may affect TRPV4-dependent cellular responses.…”

Mechanical sensing protein PIEZO1 regulates bone homeostasis via osteoblast-osteoclast crosstalk

“…It has been reported that osteoblast lineage cells, including bone mesenchymal stem cells (BMSCs), osteoblast progenitor cells, osteoblasts, and osteocytes can respond to mechanical loading in bone 7 . TRPV4 mediates fluid shear stress induced calcium signaling and early osteogenic gene expression in BMSCs 8 . The opening of Connexin43 (Cx43) hemichannel depends on interaction with integrin in response to shear stress in osteocytes 9 .…”