The role of TRPV4 in osteogenesis and cell signaling in MC3T3-E1 ostoeoblast-like cells

Howell, Kristen
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University of Delaware
Bone is a living, dynamic tissue which is constantly being remodeled to maintain the integrity and strength of the skeleton. This process is carried out by three cells types that form the basic multicellular unit (BMU); Osteoclasts which resorb bone, osteoblasts which form new bone and osteocytes which are terminally differentiated osteoblasts that have become trapped in the bone matrix and promote remodeling. Mechanical loading promotes bone remodeling, however it is unclear how bone cells perceive this type of stimulation. Because the skeleton is sensitive to mechanical loads, it is apparent that bone cells must express a mechanism through which these cells can translate a mechanical stimulation into a biochemical response. One of the candidates for this “mechanosensor” is the transient receptor potential vanilloid 4 (TRPV4) channel, a member of the large TRP family of channels. This channel is a cation channel that has been shown to be osmotically sensitive in bone, but has been studied primarily using osteoclasts and chondrocytes. The goal of this thesis is to define how activation of TRPV4 channels affects the normal function of MC3T3-E1 osteoblast-like cells. My overall hypothesis is that MC3T3-E1 osteoblasts express the TRPV4 channel and that this channel acts as the “mechanosensor” to enhance anabolic responses of the cell to mechanical stimuli. I have shown that the TRPV2 and TRPV4 channels are present at both the mRNA and protein levels in MC3T3-E1 osteoblasts and MLO-Y4 osteocytes and performed functional studies to determine its role specifically in osteoblasts. MC3T3-E1 proliferation was significantly reduced by inhibition with the non-specific TRPV blocker ruthenium red, or the TRPV4 specific antagonist, RN-1734. After 96 hours, there was a 63% decrease in cells treated with ruthenium red and a 45% decrease in cellular proliferation in cells treated with RN-1734. A significant decrease in intracellular Ca2+ levels in response to hypotonic swelling was also observed in the presence of the inhibitors as well as a decrease in the number of responding cells, however baseline intracellular Ca2+ levels remained the same. Inhibition of the TRPV4 channel did not alter the response of these cells to fluid shear stress. There was no difference in ATP release or actin stress fiber formation detected when the cells were subjected to fluid shear stress. Finally, the mineralization data collected also did not provide any conclusive results as to if TRPV4 is involved in bone formation however did provide interesting information about the role of TRPV2 during this process. Previous studies have shown conflicting data regarding TRPV4 as a “mechanosensor.” Several studies have shown that the channels are directly activated by mechanical stimuli and others showing they are not. The data I present here suggests that TRPV4 is not directly mechanically sensitive but may help to propagate the mechanotransduction signaling pathways in MC3T3-E1 osteoblasts.