Extrinsic factors such as hormones, growth factors, and mechanical strain produce their effects on skeletal growth and remodeling via actions on osteoblasts. Accordingly, there is great interest in defining how specific intracellular signaling pathways mediate the effects of these factors in controlling osteoblast function. G protein signaling occurs in response to many skeletal stimuli, and the consequence of this signaling depends on the nature of the G protein;the temporal delivery of the signal;and the phenotypic state of the osteoblast. A clearer understanding of how calciotropic agents such as PTH elicit their complex effects in bone requires studies that address these issues directly in an in vivo context. In the present proposal, we will assess the role of Gs and Gi signaling in osteoblasts in mediating anabolic skeletal responses. In one approach, novel G protein-coupled receptors termed RASSLs will be targeted to osteoblasts in transgenic mice. RASSLs activate specific G protein pathways in response to administration of synthetic agonists. Activation of Gs- and Gi RASSLs will allow us to dissect the role of these pathways in skeletal responses. In a second approach, Gs and Gi function will be ablated in osteoblasts in vivo by cre-mediated excision of functional Gs-alpha alleles and by targeted expression of the catalytic subunit of pertussis toxin, respectively. We will determine the effects of ablating these signaling pathways on normal skeletal homeostasis and on the anabolic response to PTH. Mechanistic studies will be carried out to assess the effects of G protein signals on osteoblast proliferation and apoptosis in vivo and in bone marrow stromal cells (BMSCs) isolated from the transgenic mice. Convergence of G protein signals with two pathways recently shown to be essential for bone formation - the LRP/canonical wnt pathway and the recently identified RSK2/ATF4 pathway- will be explored in BMSCs. We propose to: 1) assess the role of osteoblast Gs and Gi signaling in the regulation of skeletal homeostasis in mature mice. We will determine the effects of regulated, intermittent Gs and Gi signaling in osteoblasts at different stages of differentiation;determine the skeletal effect of conditional knockout of Gs and Gi signaling;and assess the mechanisms by which manipulation of Gs and Gi signaling elicits these effects;and 2) determine the contribution of Gs and Gi signaling to the anabolic response to PTH and the mechanisms of these effects. These studies will shed new light on the control of bone formation and bone resorption by G protein signaling in osteoblasts at different stages of differentiation. They may also provide new links between G protein signals and anabolic effects in bone, thereby identifying new therapeutic targets for the treatment of osteoporosis. LAY DESCRIPTION: We will explore how activation of specific signals in bone-forming cells can lead to increases in bone mass. The results may lead to new approaches to the treatment of osteoporosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK072071-04
Application #
7564676
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Malozowski, Saul N
Project Start
2006-02-01
Project End
2011-12-31
Budget Start
2009-01-01
Budget End
2011-12-31
Support Year
4
Fiscal Year
2009
Total Cost
$321,872
Indirect Cost
Name
Northern California Institute Research & Education
Department
Type
DUNS #
613338789
City
San Francisco
State
CA
Country
United States
Zip Code
94121
Millard, Susan M; Wang, Liping; Wattanachanya, Lalita et al. (2017) Role of Osteoblast Gi Signaling in Age-Related Bone Loss in Female Mice. Endocrinology 158:1715-1726
Wang, Liping; Hsiao, Edward C; Lieu, Shirley et al. (2015) Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing. J Bone Miner Res 30:1896-904
Wattanachanya, Lalita; Wang, Liping; Millard, Susan M et al. (2015) Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs-G protein signaling in osteoblasts. Exp Cell Res 333:289-302
Wattanachanya, Lalita; Lu, Wei-Dar; Kundu, Ramendra K et al. (2013) Increased bone mass in mice lacking the adipokine apelin. Endocrinology 154:2069-80
Kao, Richard; Lu, Weidar; Louie, Alyssa et al. (2012) Cyclic AMP signaling in bone marrow stromal cells has reciprocal effects on the ability of mesenchymal stem cells to differentiate into mature osteoblasts versus mature adipocytes. Endocrine 42:622-36
Kazakia, G J; Speer, D; Shanbhag, S et al. (2011) Mineral composition is altered by osteoblast expression of an engineered G(s)-coupled receptor. Calcif Tissue Int 89:10-20
Millard, Susan M; Louie, Alyssa M; Wattanachanya, Lalita et al. (2011) Blockade of receptor-activated G(i) signaling in osteoblasts in vivo leads to site-specific increases in cortical and cancellous bone formation. J Bone Miner Res 26:822-32
Hsiao, Edward C; Boudignon, Benjamin M; Halloran, Bernard P et al. (2010) Gs G protein-coupled receptor signaling in osteoblasts elicits age-dependent effects on bone formation. J Bone Miner Res 25:584-93
Hsiao, Edward C; Millard, Susan M; Louie, Alyssa et al. (2010) Ligand-mediated activation of an engineered gs g protein-coupled receptor in osteoblasts increases trabecular bone formation. Mol Endocrinol 24:621-31
Conklin, Bruce R; Hsiao, Edward C; Claeysen, Sylvie et al. (2008) Engineering GPCR signaling pathways with RASSLs. Nat Methods 5:673-8

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