Defective bone remodeling is the pathophysiologic basis of most metabolic bone diseases, including postmenopausal and age-dependent osteoporosis. In adults, new bone formation follows an osteoclast mediated resorptive phase, and requires the synchronized activity of bone forming cells. During the tenure of this project, we have demonstrated that osteoblasts are interconnected in a """"""""functional syncytium"""""""" by gap junctions formed primarily by connexin43 (Cx43). We have shown that targeted deletion of Cx43 in mice leads to developmental abnormalities of the skeleton, impaired osteoblast differentiation and reduced mineralization. We have also found that transcriptional regulation of genes pivotal to osteoblast differentiation and function, in particular osteocalcin (OC) is dependent on gap junctional communication, and have identified the -95 to -47 region of the OC promoter as a gap junction sensitive transcriptional unit. Further, we have biochemically purified heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a putative gap junction sensitive transcription factor. The central hypothesis of this proposal is that the type of gap junctional communication provided by Cx43 is critical for osteoblast function and bone formation in the adult skeleton. The proposed experimentation will make use of genetically engineered mice and cell models of disrupted Cx43 expression, in part developed in our laboratory, to test the central hypothesis at 3 levels: organ, cellular and molecular. Accordingly, we propose to; 1: Determine skeletal maturation and response to osteoblast stimulation in mice with targeted deletion of the Cx43 gene in osteoblasts; 2: Determine the mechanisms by which lack of Cx43 alters osteoblast differentiation and function; 3: Identify gap junctional communication sensitive transcriptional factor(s) binding to the proximal region of the OC promoter. We anticipate that deletion of Cx43 in osteoblasts will result in achievement of inadequate peak bone mass, reduced anabolic response to intermittent PTH, impaired osteoblast differentiation and matrix production, and reduced transcriptional activity of hnRNPK or other gap junction sensitive transcription factors. These studies will disclose novel mechanisms by which the activity of bone forming cells is regulated by intercellular communication. Alterations of connexin expression and/or function by hormonal imbalances or aging may lead to osteoblast dysfunction and consequent demineralization.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
7R01AR041255-12
Application #
6783828
Study Section
Special Emphasis Panel (ZRG1-OBM-2 (02))
Program Officer
Sharrock, William J
Project Start
1992-01-01
Project End
2007-03-31
Budget Start
2003-08-01
Budget End
2004-03-31
Support Year
12
Fiscal Year
2003
Total Cost
$257,140
Indirect Cost
Name
Washington University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Stains, Joseph P; Civitelli, Roberto (2016) A Functional Assay to Assess Connexin 43-Mediated Cell-to-Cell Communication of Second Messengers in Cultured Bone Cells. Methods Mol Biol 1437:193-201
Stains, Joseph P; Civitelli, Roberto (2016) Connexins in the skeleton. Semin Cell Dev Biol 50:31-9
Shen, Hua; Grimston, Susan; Civitelli, Roberto et al. (2015) Deletion of connexin43 in osteoblasts/osteocytes leads to impaired muscle formation in mice. J Bone Miner Res 30:596-605
Chang, Kyung Hee; Sengupta, Amitava; Nayak, Ramesh C et al. (2014) p62 is required for stem cell/progenitor retention through inhibition of IKK/NF-?B/Ccl4 signaling at the bone marrow macrophage-osteoblast niche. Cell Rep 9:2084-97
Stains, Joseph P; Watkins, Marcus P; Grimston, Susan K et al. (2014) Molecular mechanisms of osteoblast/osteocyte regulation by connexin43. Calcif Tissue Int 94:55-67
Grimston, Susan K; Watkins, Marcus P; Stains, Joseph P et al. (2013) Connexin43 modulates post-natal cortical bone modeling and mechano-responsiveness. Bonekey Rep 2:446
Watkins, Marcus P; Norris, Jin Yi; Grimston, Susan K et al. (2012) Bisphosphonates improve trabecular bone mass and normalize cortical thickness in ovariectomized, osteoblast connexin43 deficient mice. Bone 51:787-94
Gonzalez-Nieto, Daniel; Li, Lina; Kohler, Anja et al. (2012) Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors. Blood 119:5144-54
Grimston, Susan K; Watkins, Marcus P; Brodt, Michael D et al. (2012) Enhanced periosteal and endocortical responses to axial tibial compression loading in conditional connexin43 deficient mice. PLoS One 7:e44222
Watkins, Marcus; Grimston, Susan K; Norris, Jin Yi et al. (2011) Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling. Mol Biol Cell 22:1240-51

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