Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction.

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.

Differential regulation of cadherins by dexamethasone in human osteoblastic cells.

Human osteoblasts express a repertoire of cadherins, including N-cadherin (N-cad), cadherin-11 (C11), and cadherin-4 (C4). We have previously shown that direct cell-cell adhesion via cadherins is critical for BMP-2-induced osteoblast differentiation. In this study, we have analyzed the regulation of cadherin expression in normal human trabecular bone osteoblasts (HOB), and osteoprogenitor marrow stromal cells (BMC), during exposure to dexamethasone, another inducer of human bone cell differentiation. Dexamethasone inhibited the expression of both C11 and N-cad mRNA in both BMC and HOB, although the effect was much more pronounced on N-cad than on C11. This action of the steroid was dose dependent, was maximal at 10(-7) M concentration, and occurred as early as after 1 day of incubation. By contrast, expression of C4 mRNA and protein was strongly induced by dexamethasone in BMC and was stimulated in HOB. This stimulatory effect lasted for at least 2 weeks of incubation. A cadherin inhibitor, HAV-containing decapeptide only partially ( approximately 50%) prevented dexamethasone-induced stimulation of alkaline phosphatase activity by BMC, which instead was not altered by incubation with a neutralizing antibody against C4. Therefore, the pattern of cadherin regulation by dexamethasone radically differs form that observed with BMP-2. Dexamethasone effects on certain osteoblast differentiated features, such as induction of alkaline phosphatase activity are not strictly dependent on cadherin function.

Human osteoblasts express a repertoire of cadherins, which are critical for BMP-2-induced osteogenic differentiation.

Direct cell-cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription-polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS-2 and MG-63, expressed mRNA for cadherin-11 (C11) and N-cadherin (N-cad). HOBs and BMCs also expressed low levels of cadherin-4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein-2 (BMP-2) treatment of either BMCs or HOBs. Likewise, N-cad mRNA did not change during BMP-2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down-regulated by BMP-2 treatment of normal cells. Both C11 and C4 were localized to sites of cell-cell contact in both HOBs and BMCs, colocalized with beta-catenin, and bands corresponding to cadherins were coimmunoprecipitated by a beta-catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N-cad partially inhibited Ca2+-dependent cell-cell adhesion and completely prevented BMP-2-induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin-mediated cell-to-cell adhesion is critical for normal human osteoblast differentiation.

Regulation of connexin43 expression and function by prostaglandin E2 (PGE2) and parathyroid hormone (PTH) in osteoblastic cells.

Connexin43 (Cx43) forms gap junctions that mediate intercellular communication between osteoblasts. We have examined the effects of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) on gap junctional communication in the rat osteogenic sarcoma cells UMR 106-01. Incubation with either PGE2 or PTH rapidly (within 30 min) increased transfer of negatively charged dyes between UMR 106-01 cells. This stimulatory effect lasted for at least 4 h. Both PGE2 and PTH increased steady-state levels of Cx43 mRNA, but only after 2-4 h of incubation. Transfection with a Cx43 gene construct linked to luciferase showed that this effect of PTH was the result of transcriptional upregulation of Cx43 promoter. Stimulation of dye coupling and Cx43 gene transcription were reproduced by forskolin and 8Br-cAMP. Exposure to PGE2 for 30 min increased Cx43 abundance at appositional membranes in UMR 106-01, whereas total Cx43 protein levels increased only after 4-6 h of incubation with either PGE2 or PTH. Inhibition of protein synthesis by cycloheximide did not affect this early stimulation of dye coupling, but it significantly inhibited the sustained effect of PTH and forskolin on cell coupling. In summary, both PTH and PGE2, presumably through cAMP production, enhance gap junctional communication in osteoblastic cell cultures via two mechanisms: initial rapid redistribution of Cx43 to the cell membrane, and later stimulation of Cx43 gene expression. Modulation of intercellular communication represents a novel mechanism by which osteotropic factors regulate the activity of bone forming cells.

Stimulation of human osteoblast differentiation and function by ipriflavone and its metabolites.

Ipriflavone (IP), an isoflavone derivative, has been shown to interfere with bone remodeling by inhibiting bone resorption and perhaps stimulating bone formation. In this study, we have analyzed the effect of IP and its metabolites on the differentiation and function of human osteoblastic cells. Bone marrow stromal osteoprogenitor cells (BMC) and trabecular bone osteoblasts (HOB) were isolated from human donors. The former can be induced to differentiate by treatment with dexamethasone, whereas the latter represent a more differentiated osteoblast. Incubation of BMC with metabolite III (10(-5) M) for 1 week induced modest but significant changes of alkaline phosphatase activity. Though both IP and metabolite III stimulated the expression of bone sialoprotein mRNA, a protein involved in cell attachment to the matrix, only metabolite III increased the steady-state level of decorin mRNA, a collagen fibrillogenesis-regulating proteoglycan. Metabolites III and V, but not the other isoflavones, increased the expression of type I collagen mRNA in HOB, whereas no detectable changes were observed in BMC cells with any of the experimental compounds. In HOB, an increased abundance of osteopontin and bone sialoprotein mRNA were also obtained after 1-week treatment with IP or metabolite V. No appreciable effects of IP or its metabolites were seen on osteocalcin expression and synthesis by either cell type. Finally, IP consistently increased the amount of 45Ca incorporated into the cell layer by BMC, and stimulated mineralization of both BMC and HOB, assessed by von Kossa staining. Thus, IP and its metabolites regulate the differentiation and biosynthetic properties of human bone-forming cells by enhancing the expression of some important matrix proteins and facilitating the mineralization process.

Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells.

We examined the expression and function of gap junctions in two rat osteoblastic cell lines, ROS 17/2.8 and UMR 106-01. The pattern of expression of gap junction proteins in these two cell lines was distinct: ROS cells expressed only connexin43 on their cell surface, while UMR expressed predominantly connexin45. Immunoprecipitation and RNA blot analysis confirmed the relative quantitation of these connexins. Microinjected ROS cells passed Lucifer yellow to many neighboring cells, but UMR cells were poorly coupled by this criterion. Nevertheless, both UMR and ROS cells were electrically coupled, as characterized by the double whole cell patch-clamp technique. These studies suggested that Cx43 in ROS cells mediated cell-cell coupling for both small ions and larger molecules, but Cx45 in UMR cells allowed passage only of small ions. To demonstrate that the expression of different connexins alone accounted for the lack of dye coupling in UMR cells, we assessed dye coupling in UMR cells transfected with either Cx43 or Cx45. The UMR/Cx43 transfectants were highly dye coupled compared with the untransfected UMR cells, but the UMR/Cx45 transfectants demonstrated no increase in dye transfer. These data demonstrate that different gap junction proteins create channels with different molecular permeabilities; they suggest that different connexins permit different types of signalling between cells.

Connexin43 mediates direct intercellular communication in human osteoblastic cell networks.

We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.

Heterogeneous intracellular free calcium responses to parathyroid hormone correlate with morphology and receptor distribution in osteogenic sarcoma cells.

The osteogenic sarcoma cell line UMR 106-01 exhibits heterogeneous morphology and hormone response in subconfluent monolayer cultures. In these studies we have explored the correlation between morphological profiles and patterns of cytosolic calcium [Ca2+]i response to PTH and other agonists in single UMR 106-01 cells loaded with the Ca(2+)-sensitive fluorescent indicator, fura-2. Realtime recording of [Ca2+]i revealed that PTH (10(-7) M) produced a transient [Ca2+]i rise in 19% of the cells studied. [Ca2+]i transients were also induced by prostaglandins E2 and F2 alpha, and fetal bovine serum, but with different response frequencies (20%, 12%, and 58%, respectively). Spatial resolution of changes in [Ca2+]i by video image analysis revealed that the response to PTH was more frequent in large polygonal cells with long cytoplasmic processes and less common in smaller cells growing in clusters, whereas there was no clear subtype specificity for the effects of epidermal growth factor and fetal bovine serum on [Ca2+]i. Autoradiographic analysis of cell monolayers demonstrated a higher density of PTH-binding sites on cells with cytoplasmic extensions, whereas epidermal growth factor-binding sites were largely on colony-forming cells. Thus, the [Ca2+]i response to hormonal stimulation is heterogeneous within UMR 106-01 cell populations and within single cells, and it correlates with receptor density. This suggests that osteoblastic cells respond to PTH by activation of changes in [Ca2+]i only at certain specific steps during osteoblast development or stages of the cell cycle.