Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures.

Gja1(Jrt)/+ mice carry a mutation in one allele of the gap junction protein α1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1(Jrt)/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1(Jrt)/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1(Jrt)/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1(Jrt)/+ osteoblasts and increased Pparg2 expression in bone marrow-derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1(Jrt)/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1(Jrt)/+ mouse model.

The "connexin" between bone cells and skeletal functions.

The processes of bone modeling and remodeling are crucial in the skeleton's functions as a supportive and protective structure, a mineral reservoir, and an endocrine organ. The coordination between bone cell activities (bone formation and bone resorption), necessary to maintain the integrity of the skeleton during these processes, is mediated at least in part by cell-cell and cell-environment interactions across gap junctions and hemichannels. The increasing number of genetically engineered Connexin 43 (Cx43) knockout and missense mouse models have provided insight into the complex and critical roles of Cx43-containing gap junctions and hemichannels in the development and turnover of the skeleton, in differentiation, activity and survival of the bone cell lineages, and in the cellular and molecular mechanisms by which Cx43 functions and assists in mediating cellular responses to stimuli in bone. Cx43 may be an important potential therapeutic target, making it crucial that we continue to gain understanding of the multiple and complex roles of Cx43 in bone.

First mouse model for combined osteogenesis imperfecta and Ehlers-Danlos syndrome.

By using a genome-wide N-ethyl-N-nitrosourea (ENU)-induced dominant mutagenesis screen in mice, a founder with low bone mineral density (BMD) was identified. Mapping and sequencing revealed a T to C transition in a splice donor of the collagen alpha1 type I (Col1a1) gene, resulting in the skipping of exon 9 and a predicted 18-amino acid deletion within the N-terminal region of the triple helical domain of Col1a1. Col1a1(Jrt) /+ mice were smaller in size, had lower BMD associated with decreased bone volume/tissue volume (BV/TV) and reduced trabecular number, and furthermore exhibited mechanically weak, brittle, fracture-prone bones, a hallmark of osteogenesis imperfecta (OI). Several markers of osteoblast differentiation were upregulated in mutant bone, and histomorphometry showed that the proportion of trabecular bone surfaces covered by activated osteoblasts (Ob.S/BS and N.Ob/BS) was elevated, but bone surfaces undergoing resorption (Oc.S/BS and N.Oc/BS) were not. The number of bone marrow stromal osteoprogenitors (CFU-ALP) was unaffected, but mineralization was decreased in cultures from young Col1a1(Jrt) /+ versus +/+ mice. Total collagen and type I collagen content of matrices deposited by Col1a1(Jrt) /+ dermal fibroblasts in culture was ∼40% and 30%, respectively, that of +/+ cells, suggesting that mutant collagen chains exerted a dominant negative effect on type I collagen biosynthesis. Mutant collagen fibrils were also markedly smaller in diameter than +/+ fibrils in bone, tendon, and extracellular matrices deposited by dermal fibroblasts in vitro. Col1a1(Jrt) /+ mice also exhibited traits associated with Ehlers-Danlos syndrome (EDS): Their skin had reduced tensile properties, tail tendon appeared more frayed, and a third of the young adult mice had noticeable curvature of the spine. Col1a1(Jrt) /+ is the first reported model of combined OI/EDS and will be useful for exploring aspects of OI and EDS pathophysiology and treatment.

EB1 levels are elevated in ascorbic Acid (AA)-stimulated osteoblasts and mediate cell-cell adhesion-induced osteoblast differentiation.

Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of ß-catenin distribution at the cell cortex and within the nucleus. Diminished ß-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-ß-catenin and active glycogen synthase kinase 3ß, a kinase known to target ß-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the ß-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with ß-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and ß-catenin. We also determined that cell-cell contacts and cortically associated EB1/ß-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.

The G60S connexin 43 mutation activates the osteoblast lineage and results in a resorption-stimulating bone matrix and abrogation of old-age-related bone loss.

We previously isolated a low bone mass mouse, Gja1(Jrt) / + , with a mutation in the gap junction protein, alpha 1 gene (Gja1), encoding for a dominant negative G60S Connexin 43 (Cx43) mutant protein. Similar to other Cx43 mutant mouse models described, including a global Cx43 deletion, four skeletal cell conditional-deletion mutants, and a Cx43 missense mutant (G138R/ +), a reduction in Cx43 gap junction formation and/or function resulted in mice with early onset osteopenia. In contrast to other Cx43 mutants, however, we found that Gja1(Jrt) /+ mice have both higher bone marrow stromal osteoprogenitor numbers and increased appendicular skeleton osteoblast activity, leading to cell autonomous upregulation of both matrix bone sialoprotein (BSP) and membrane-bound receptor activator of nuclear factor-κB ligand (mbRANKL). In younger Gja1(Jrt) /+ mice, these contributed to increased osteoclast number and activity resulting in early onset osteopenia. In older animals, however, this effect was abrogated by increased osteoprotegerin (OPG) levels and serum alkaline phosphatase (ALP) so that differences in mutant and wild-type (WT) bone parameters and mechanical properties lessened or disappeared with age. Our study is the first to describe a Cx43 mutation in which osteopenia is caused by increased rather than decreased osteoblast function and where activation of osteoclasts occurs not only through increased mbRANKL but an increase in a matrix protein that affects bone resorption, which together abrogate age-related bone loss in older animals.