Ablation of Gsα signaling in osteoclast progenitor cells adversely affects skeletal bone maintenance

Abstract

Gsα, the alpha stimulatory subunit of heterotrimeric G proteins that activates downstream signaling through the adenylyl cyclase and cAMP/PKA pathway, plays an important role in bone development and remodeling. The role of Gsα in mesenchymal stem cell (MSC) differentiation to osteoblasts has been demonstrated in several mouse models of Gsα inactivation. Previously, using mice with heterozygous germline deletion of Gsα (Gnas), we identified a novel additional role for Gsα in bone remodeling, and showed the importance of Gnas in maintaining bone quality by regulating osteoclast differentiation and function. In this study, we show that postnatal deletion of Gsα (CreERT2;Gnas) leads to reduction in trabecular bone quality parameters and increased trabecular osteoclast numbers. Furthermore, mice with deletion of Gsα specifically in cells of the macrophage/osteoclast lineage (LysM-Cre;Gnas) showed reduced trabecular bone quality and increased trabecular osteoclasts, but to a reduced extent compared to the CreERT2;Gnas global knockout. This demonstrates that while Gsα has a cell autonomous role in osteclasts in regulating bone quality, Gsα expression in other cell types additionally contribute. In both of these mouse models, cortical bone was more subtly affected than trabecular bone. Our results support that Gsα is required postnatally to maintain trabecular bone quality and that Gsα function to maintain trabecular bone is regulated in part through a specific activity in osteoclasts.

Parental origin of Gsα inactivation differentially affects bone remodeling in a mouse model of Albright hereditary osteodystrophy

“…RT-PCR analysis of mature osteoclast cultures identified that both Gnas E1+/-p and Gnas E1+/-m cultures demonstrated significantly elevated mRNA expression of Rank and Calcr when compared to WT. These findings correlate with previous reports suggesting that Gnas serves as a negative regulator of osteoclastogenesis and osteoclast fusion, (37,44,50) and therefore, it is plausible that Gnas or adenylate cyclase activity may serve a role in directly modulating the surface expression of RANK receptors within preosteoclasts, leading to elevated rates of osteoclastogenesis. (63,64) Despite similar observations within Gnas E1+/-m and Gnas E1+/-p osteoclasts cultures in vitro, only Gnas E1+/-p mice displayed evidence of statistically enhanced bone resorption activity when compared to WT in vivo as measured by histomorphometry and serum CTX-1 testing.…”

“…Collectively, these data correlate with previous reports suggesting Gsα serves as a negative regulator of osteoclastogenesis and that parental inheritance of the Gsα mutation does not selectively influence osteoclastogenesis in vitro. (44,50) In addition to evaluating osteoclastogenesis in vitro, we wanted to assess whether the observed distinctions in bone formation between Gnas E1+/-m and Gnas E1+/-p mice in vivo could be attributed to modifications in osteoclast secreted bone coupling factors. We examined four growth factors that are released by osteoclasts and have been shown to influence bone formation: Semaphorin 4d (Sema4d), Sphingosine Kinase 1 (Sphk1), Sphingosine Kinase 2 (Sphk2) and Wnt10b (Fig 5D).…”

Parental Origin of Gsα Inactivation Differentially Affects Bone Remodeling in a Mouse Model of Albright Hereditary Osteodystrophy

“…RT-PCR analysis of mature osteoclast cultures identified that both Gnas E1+/Àp and Gnas E1+/Àm cultures showed significantly elevated mRNA expression of Rank and Calcr when compared to WT. These findings correlate with previous reports suggesting that Gnas serves as a negative regulator of osteoclastogenesis and osteoclast fusion (37,44,46) ; therefore, it is plausible that Gnas or adenylate cyclase activity may serve a role in directly modulating the surface expression of RANK receptors within preosteoclasts, leading to elevated rates of osteoclastogenesis. (63,64) Despite similar observations within Gnas E1+/Àm and Gnas E1+/Àp osteoclasts cultures in vitro, only Gnas E1+/Àp mice displayed evidence of statistically enhanced osteoclast number and bone resorption activity when compared to WT in vivo as measured by histomorphometry and serum CTX-1 testing.…”

“…Collectively, these data correlate with previous reports suggesting Gsα serves as a negative regulator of osteoclastogenesis and that parental inheritance of the Gsα mutation does not selectively influence osteoclastogenesis in vitro. (44,46) In addition to evaluating osteoclastogenesis in vitro, we wanted to assess whether the observed distinctions in bone formation between Gnas E1+/Àm and Gnas E1+/Àp mice in vivo could be attributed to modifications in osteoclast secreted bone coupling factors. We examined four growth factors that are released by osteoclasts and have been shown to influence bone formation: Semaphorin 4d (Sema4d), Sphingosine Kinase 1 (Sphk1), Sphingosine Kinase 2 (Sphk2), and Wnt10b (Fig.…”

Gnas Inactivation Alters Subcutaneous Tissues in Progression to Heterotopic Ossification

“…Within mesenchymal progenitor cells, such as adipose stromal cells (ASCs) that reside in adipose tissue, Gsα reciprocally regulates adipogenesis and osteogenesis, with high levels of signal associated with adipogenesis and low levels with osteogenesis ( Pignolo et al, 2011 ; Fan et al, 2012 ; Liu et al, 2012 ). Gsα signaling also plays a role in homeostatic mechanisms of the skeleton ( Wu et al, 2011 ; Sinha et al, 2014 , 2016 ; Zhao et al, 2018 ; Ramaswamy et al, 2018 ; Ramaswamy et al, 2017 ), skin and hair follicles ( Iglesias-Bartolome et al, 2015 ).…”