Delineating the cellular mechanism of osteoblast desensitization to mechanical stimulation

Gangadharan, Vimal
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University of Delaware
Bone is a highly dynamic tissue constantly adapting itself to several physical and biochemical cues. Of these various signals, application of mechanical stimulation has profound anabolic effects on maintaining the integrity and architecture of bone. While physical activity retards bone loss, the lack of physical stimuli results in significant bone loss as observed in immobilized patients and astronauts in microgravity. However, it has been ascertained that bone is habituated to continuous mechanical loading and a similar effect is witnessed at the cellular level in osteoblasts. The goal of the study was to elucidate the cellular mechanism for osteoblast desensitization to mechanical stimulation so the anabolic effects of the physical stimulus on bone can be best utilized. The osteogenic response to mechanical loading is an outcome of an intricate biophysical process. During physical activity, the strain applied on the bone causes interstitial fluid movement across the canaliculi inducing fluid shear stress (FSS) on the bone cells. These cells detect and transmit the physical stimulus to initiate a cascade of downstream signaling that eventually culminates in osteogenesis. This process of converting mechanical stimulus into biochemical response is termed mechanotransduction. Numerous components have been implicated in osteoblast mechanotransduction including the purinergic receptor, P2X 7 (P2X7 R). In vivo studies conducted on P2X7 R KO mice demonstrated that significant percent of mechanically induced bone formation is dependent on this ligand gated ion channel. Interestingly, the P2X7 R were found localized in caveolae of certain cell types. Flask shaped plasmallemmal invaginations, caveolae, are subset of lipid microdomains with caveolin-1 (CAV1) as their primary protein constituent. Caveolae aids in several cellular functions including scaffolding proteins and endocytosis. Furthermore, it has been observed that the CAV1 KO mice have significantly higher bone mass and strength. This led to the hypothesis that caveolae in osteoblasts scaffolds the purinergic receptor, P2X 7 , and the loss of mechanosensitivity is due to the caveolar endocytosis of P2X7 R upon ligand binding. Initial studies carried out demonstrated that CAV1 was internalized when osteoblasts were stimulated with P2X7 R agonists. Sucrose gradient fractionation of MC3T3-E1 pre-osteoblasts showed that CAV1 translocated to the denser cytosolic fractions upon stimulation with ATP. Additionally, both ATP and the more specific P2X7 R agonist, BzATP, induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7 R antagonist, A-839977. The P2X7 R co-fractionated with CAV1 but, using Superresolution Structured Illumination Microscopy (SR-SIM), we found that only a sub-population of the P2X7 R existed in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular [Ca2+ ]i to BzATP, suggesting that caveolae regulates P2X7 R signaling. This proposed mechanism is supported by increased mineralization observed in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X 7 R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatio-temporal signaling of P2X7 R in osteoblasts. Furthermore, it was confirmed that the osteoblasts were desensitized to mechanical stimulus. Consecutive stimulation of the MC3T3-E1 cells with FSS failed to elicit a [Ca2+ ]i response during the 2nd stimulation. By measuring the [Ca2+ ]i response to ATP and BzATP, it was determined that the P2X7 R in osteoblasts were temporarily desensitized. While these results were encouraging, further investigation revealed caveolae has no role in the desensitization of P2X7 R. The suppression of CAV1 had no apparent effect on the loss of sensitivity of P2X7 R to BzATP. Moreover there was no evidence for P2X7 R endocytosis observed when the MC3T3-E1cells were treated with ATP or BzATP. Finally, we ascertained the CAV1 KD cells were unable to retain their mechanosensitivity as postulated. These results indicated the presence of a separate mechanism that led to a different approach involving cytoskeletal reorganization. One of the prominent responses of osteoblasts in response to FSS is the reorganization of actin cytoskeleton to form stress fibers. The increase in stress fibers was found to significantly enhance the cellular stiffness in a purinergic signaling dependent manner. Although the changes in cytoskeleton were postulated to alter cellular mechanosensitivity, there have been no reports demonstrating a detailed mechanism. The purinergic receptor, P2Y2 (P2Y 2 R) has been shown to be essential for cytoskeletal reorganization in certain cell types and interestingly the P2Y2 R KO mice had enhanced bone mineral density. We hypothesized that P2Y2 R induced increase in ASFF is responsible for osteoblast desensitization. MC3T3-E1 cells treated with UTP, a potent agonist of P2Y2 R, displayed strong ASFF. The effect was blocked when the cells were pretreated with Suramin, an antagonist of P2Y2 R. Moreover, when P2Y2 R was suppressed using siRNA, there was significant increase in the number of responding cells to consecutive FSS application. These data validates the hypothesis that the mechanosensitivity of osteoblasts are regulated by P2Y2 R through ASFF. Overall our novel findings provide great insights into better understanding the mechanism of cellular desensitization to physical stimulation in osteoblasts. These findings can be further explored to have significant impact in mechanical load induced osteogenesis to prevent severe bone disorders such as osteoporosis.