Abstract 2737 Examining the role of Arhgef3 in regulating the mechanotransduction of bone

Document Type

Conference Proceeding

Publication Date

5-1-2025

Publication Title

J Biol Chem

Abstract

Genome-wide studies have found ARHGEF3 gene to be situated within a quantitative trait locus for bone mineral density (BMD) and has been identified as a strong positional candidate for the development of osteoporosis. However, the underlying role of Arhgef in regulating bone mass and tissue strength is entirely unknown. Given that Arhgef3 plays a key role in regulating the RhoA signaling pathway, a pathway that limits the mechanotransduction of osteocytes', we hypothesized the absence of Arhgef3 will enhance osteocytes response to loading and the net gain in bone formation under exercise. To test the hypothesis, primary osteocytes were isolated from global Arhgef3 knockout mice (KO) as well as wild-type (WT) mice and then exposed to oscillatory fluid flow (OFF). The response to OFF in KO osteocytes was characterized by a 14-fold increase in PGE2 release that was significantly greater than the 8-fold increase observed in WT cells. As to be expected, the KO cells also displayed a lack of cofilin phosphorylation and actin-stress fiber formation compared to WT cells. Knowing that actin-stress fiber formation in response to loading is mediated through purinergic signaling, we then examined the response to ATP. Following ATP treatment WT cells displayed a 2-fold increase in cofilin phosphorylation and actin stress fiber formation, both of which were completely absent in KO cells. The increased actin-stress fiber formation mediated by ATP through RhoA activation was also shown to increase cell stiffness based on the Brillion shift measured under confocal Brillion microscopy. The increase in cell stiffness was then associated with a loss in mechanosensitivity as evident by a significant shift in ERK1/2 activation, such that activation of RhoA prior to OFF suppresses ERK1/2 by 50%. Increasing the shear stress during OFF to 25 dynes/cm2 was able to restore ERK1/2 activation to the same degree as cells that did not undergo any pre-treatments that increase cell stiffness. Altogether, these findings suggest that RhoA activation through Arhgef3 increases the cell stiffness and thereby reducing the sensitivity of the cell to further mechanical stimulation. Having examined the role of Arhgef3 in regulating osteocytes' response to loading at the cellular level, we then examined the role of Arhgef3 in regulating the response to loading at the tissue level by subjecting male KO and WT mice to an exercise regimen of treadmill running. After 5-weeks of treadmill running, tibia samples from WT mice displayed a significant increase in mineralization (MS) and mineral apposition rate (MAR) compared to sedentary controls. The MAR following exercise was significantly greater in KO mice compared to WT mice subjected to the same exercise regimen (0.92 ± 0.3 μm/day vs. 1.37 ± 0.4 μm/day, p< 0.05, n=7). The KO mice also displayed significant gains in the cross-sectional moment of inertia and overall stiffness of the tibia when compared to WT mice subjected to the same exercise regimen. Altogether these findings demonstrate that the loss of Arhgef3 enhances the anabolic response to loading. Furthermore, Arhgef3 acts as a negative feedback loop in the mechanotransduction pathway by suppressing osteocytes' sensitivity to subsequent loading cycles by increasing the cell stiffness through actin-stress fiber formation. To date, this is the first study to examine the role of Arhgef3 in regulating mechanotransduction and offers a novel target for improving bone formation in response to daily loading. This study was supported by National Institute of Health (RO1 AR076378)

Volume

301

Issue

5

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