Connexin(Cx)43 is the major gap junction protein present in osteoblasts. We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell–cell communication (Koval, M., S.T. Geist, E.M. Westphale, A.E. Kemendy, R. Civitelli, E.C. Beyer, and T.H. Steinberg. 1995. J. Cell Biol. 130:987–995) and transcriptional downregulation of several osteoblastic differentiation markers (Lecanda, F., D.A. Towler, K. Ziambaras, S.-L. Cheng, M. Koval, T.H. Steinberg, and R. Civitelli. 1998. Mol. Biol. Cell 9:2249–2258). Here, using the Cx43-null mouse model, we determined whether genetic deficiency of Cx43 affects skeletal development in vivo. Both intramembranous and endochondral ossification of the cranial vault were delayed in the mutant embryos, and cranial bones originating from migratory neural crest cells were also hypoplastic, leaving an open foramen at birth. Cx43-deficient animals also exhibited retarded ossification of the clavicles, ribs, vertebrae, and limbs, demonstrating that skeletal abnormalities are not restricted to a neural crest defect. However, the axial and appendicular skeleton of Cx43-null animals were essentially normal at birth. Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells. Therefore, in addition to the reported neural crest cell defect, lack of Cx43 also causes a generalized osteoblast dysfunction, leading to delayed mineralization and skull abnormalities. Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.

Mutations of critical components of the Wnt pathway profoundly affect skeletal development and maintenance, probably via modulation of β-catenin signaling. We tested the hypothesis that β-catenin is involved in mesenchymal lineage allocation to osteogenic cells using a β-catenin mutant with constitutive transcriptional activity (ΔN151). Although this stable β-catenin had no effects by itself on osteogenic differentiation of multipotent embryonic cell lines, it synergized with bone morphogenetic protein-2 (BMP-2) resulting in dramatic stimulation of alkaline phosphatase activity, osteocalcin gene expression, and matrix mineralization. Likewise, ΔN151 and BMP-2 synergistically stimulated new bone formation after subperiosteal injection in mouse calvaria in vivo. Conversely, ΔN151 prevented adipogenic differentiation from pre-adipocytic or uncommitted mesenchymal cells in vitro. Intriguingly, the synergism with BMP-2 on gene transcription occurred without altering expression of Cbfa1/Runx2, suggesting actions independent or downstream of this osteoblastspecific transcription factor. Thus, β-catenin directs osteogenic lineage allocation by enhancing mesenchymal cell responsiveness to osteogenic factors, such as BMP-2, in part via Tcf/Lef dependent mechanisms. In vivo, this synergism leads to increased new bone formation. Keywords cell-cell adhesion; mesenchymal differentiation; bone formation; adipogenesisIn adult life, the skeleton is constantly remodeled to replace aging tissue and repair injuries by successive phases of osteoclast bone resorption and osteoblast mediated bone formation. Therefore, a continuous supply of bone forming cells is required to maintain bone homeostasis. Bone marrow mesenchymal stem cells are the source of osteoprogenitors in adult life, although these cells can also differentiate into adipocytes and myocytes in the presence of appropriate stimuli [Pittenger et al., 1999]. Lineage allocation of mesenchymal stem cells to either osteogenic or adipogenic cells must be kept in a critical balance, and shifts that may favor one lineage over the other may have important consequences for an individual's ability to produce sufficient numbers of bone forming cells. An osteogenic to adipogenic shift may explain the pathogenesis of age-dependent bone loss, which is associated with a reduced potential of bone marrow to produce osteogenic cells in the face of an increased number of adipocytes [Jilka et al., 1996;Gimble and Nuttall, 2004]. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe osteogenic differentiation program is under the control of hormonal and local factors converging onto a finite number of transcriptional regulators that ultimately determine the fate of cells committing to the osteogenic lineage [Karsenty and Wagner, 2002]. An emerging body of work demonstrates that the Wnt signaling system is one of the most important local regulators of bone formation, presumably via activation of the β-catenin signaling system. Wnts are a family of secreted protei...

We studied the function of osteoblast cadherins in vivo by transgenic expression of a truncated N-cadherin with dominant-negative action, driven by an osteoblast-specific promoter (OG2-NcadΔC). During the first 3 months of life, bone mineral density was reduced, whereas percent body fat was increased in transgenic animals compared with wild-type littermates, with associated decreased bone formation rate and osteoblast number, but normal osteoclast number. Osteoblast differentiation was delayed in calvaria cells isolated from transgenic mice. Likewise, the number of osteoblast precursors in bone marrow stromal cells from OG2-NcadΔC mice was decreased compared with wild-type cultures, whereas the number of adipogenic precursors was increased. In vitro, a transcriptionally active β-catenin mutant reversed the delay in osteoblast differentiation and the exuberant adipogenesis. Thus, in vivo disruption of cadherin function hinders osteoblast differentiation and favors, indirectly, bone marrow progenitor cell commitment to the alternative adipogenic lineage via interference with β-catenin signaling. This results in decreased bone formation, delayed acquisition of peak bone mass and increased body fat.

Lipopolysaccharides (LPS) of Gram-negative bacteria are important mediators of bacterial virulence that can elicit potent endotoxic effects. Surfactant protein D (SP-D) shows specific interactions with LPS, both in vitro and in vivo. These interactions involve binding of the carbohydrate recognition domain (CRD) to LPS oligosaccharides (OS); however, little is known about the mechanisms of LPS recognition. Recombinant neck+CRDs (NCRDs) provide an opportunity to directly correlate binding interactions with a crystallographic analysis of the binding mechanism. In these studies, we examined the interactions of wild-type and mutant trimeric NCRDs with rough LPS (R-LPS). Although rat NCRDs bound more efficiently than human NCRDs to Escherichia coli J-5 LPS, both proteins exhibited efficient binding to solid-phase Rd2-LPS and to Rd2-LPS aggregates presented in the solution phase. Involvement of residues flanking calcium at the sugar binding site was demonstrated by reciprocal exchange of lysine and arginine at position 343 of rat and human CRDs. The lectin activity of hNCRDs was inhibited by specific heptoses, including l-glycero-α-d-manno-heptose (l,d-heptose), but not by 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo). Crystallographic analysis of the hNCRD demonstrated a novel binding orientation for l,d-heptose, involving the hydroxyl groups of the side chain. Similar binding was observed for a synthetic α1→3-linked heptose disaccharide corresponding to heptoses I and II of the inner core region in many LPS. 7-O-Carbamoyl-l,d-heptose and d-glycero-α-d-manno-heptose were bound via ring hydroxyl groups. Interactions with the side chain of inner core heptoses provide a potential mechanism for the recognition of diverse types of LPS by SP-D.