Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43

We have previously shown in cultured rat microvascular endothelial cells (RMEC) that lipopolysaccharide (LPS) stimulates a protein tyrosine kinase (PTK)-dependent reduction in cellular coupling. We hypothesized that connexin 43 (Cx43) becomes phosphorylated following exposure to LPS. Cx43 was immunoprecipitated from control and LPS-treated RMEC monolayers. Tyrosine phosphorylation of Cx43, detected by immunoblot, was found only in the LPS treatment. To verify these results, Cx43 was radiolabeled with [(32)P]-orthophosphate. Radiolabeled Cx43 exhibited a slight increase in phosphorylation in response to LPS; phosphoamino acid analysis displayed equivalent amounts of phosphoserine in control and LPS treatments, but detected phosphotyrosine only in the LPS treatment. The PTK inhibitors PP-2 (10 nM) and geldanamycin (200 nM) were found to block the response to LPS in terms of Cx43 tyrosine phosphorylation and cellular coupling. The phosphatase inhibitor BpV (1 microM) accentuated the effect of LPS, while the putative phosphatase activator C(6)-ceramide prevented it. When measuring cell communication, phosphatase inhibition also blocked the reversal of the LPS response following LPS washout. We conclude that Cx43 is tyrosine phosphorylated following exposure to LPS and suggest that the LPS-induced increase in intercellular resistance may be mediated by tyrosine phosphorylation of this connexin. Altering tyrosine kinase and phosphatase activities can modulate the LPS-induced tyrosine phosphorylation of Cx43 and reductions in cellular coupling.     

Hypoxia/reoxygenation reduces microvascular endothelial cell coupling by a tyrosine and MAP kinase dependent pathway

Communication of electrical signals along the microvascular endothelium plays a key role in integrating microvascular function required for local regulation of blood flow. The aim of the present study was to examine the effect of a short-term hypoxia (0.1% O(2), 1 h) plus reoxygenation (H/R) on electrical coupling in cultured monolayers of microvascular endothelial cells (rat skeletal muscle origin). To assess coupling, we used a current injection technique and a Bessel function model to compute the intercellular resistance (an inverse measure of coupling) and cell membrane resistivity (a measure of resistance to current leakage across the cell membrane). H/R resulted in rapid (within 4 min after reoxygenation) and sustained (up to 100 min) reduction in intercellular coupling, but it did not alter membrane resistivity. H/R did not alter gap junction protein connexin 43 expression nor its tyrosine phosphorylation as determined by immunoblot and immunoprecipitation analyses. Inhibition of mitochondrial respiration (1 mM NaCN) did not mimic the effect of H/R. However, pre-treatment of monolayers with tyrphostin A48 (1.5 microM), PP2 (10 nM) (tyrosine kinase inhibitors), U 0126 (20 microM), and PD 98059 (5 microM) (MEK1/2 inhibitors) inhibited the H/R-induced reduction in coupling. These results indicate that endothelial cell coupling was reduced quickly after reoxygenation, via activation of a tyrosine and MAP kinase dependent pathway. We predict that a short-term H/R can rapidly compromise microvascular function in terms of reduced cellular communication along the vascular wall.     

Lipopolysaccharide reduces electrical coupling in microvascular endothelial cells by targeting connexin40 in a tyrosine-, ERK1/2-, PKA-, and PKC-dependent manner

Electrical coupling along the endothelium is central in the arteriolar conducted response and in control of vascular resistance. It has been shown that exposure of endothelium to lipopolysaccharide (LPS, an initiating factor in sepsis) reduces intercellular communication in vitro and in vivo. The molecular basis for this reduction is not known. We examined the effect of LPS on electrical coupling in monolayers of cultured mouse microvascular endothelial cells (MMEC) derived from the mouse hindlimb skeletal muscle. To assess coupling, we measured the spread of electrical current injected into the monolayer and computed the monolayer intercellular resistance (inverse measure of coupling). LPS (10 microg/ml, 1 h) reduced coupling (i.e., increased resistance) in MMEC isolated from wild-type, connexin37 (Cx37) null and Cx43(G60S) (nonfunctional mutant) mice, but not in MMEC derived from Cx40 null mice. LPS also activated JNK1/2, p38 and ERK1/2 MAP kinases. Pretreatment of WT monolayers with ERK1/2 inhibitor U0126 (20 microM, 1 h) prevented the LPS-induced decrease in coupling, while inhibition of JNK1/2 with SP600125 (20 microM, 1 h) and p38 with a p38 inhibitor (10 nM, 1 h) had no effect. Furthermore, inhibition of tyrosine kinases with PP-2 (10 nM, 1 h), activation of PKA by 8-bromo-cAMP (1 mM, 5 min), and activation of PKC by bryostatin-2 (10 nM, 1 h) also prevented the reduction in coupling. We propose that LPS reduces inter-endothelial electrical coupling via tyrosine-, ERK1/2-, PKA-, and PKC-dependent signaling that targets Cx40. We suggest that this mechanism contributes to compromised arteriolar function following LPS exposure.     

Lipopolysaccharide reduces intercellular coupling in vitro and arteriolar conducted response in vivo

Our recent in vitro study (Lidington et al. J Cell Physiol 185: 117-125, 2000) suggested that lipopolysaccharide (LPS) reduces communication along blood vessels. The present investigation extended this study to determine whether any effect of LPS and/or inflammatory cytokines [tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6] on endothelial cell coupling in vitro could also be demonstrated for an arteriolar conducted response in vivo. Using an electrophysiological approach in monolayers of microvascular endothelial cells, we found that LPS (10 microg/ml) but not these cytokines reduced intercellular conductance (c(i)) (an index of cell communication) and that LPS together with these cytokines did not further reduce c(i). Also, c(i) was restored after LPS washout, and the LPS-induced reduction was prevented by protein tyrosine kinase (PTK) inhibitors (1.5 microM Tyr A9 and 10 nM PP-2). In our in vivo experiments in arterioles of the mouse cremaster muscle, local electrical stimulation evoked vasoconstriction that conducted along arterioles. LPS in the muscle superfusate did not alter local vasoconstriction but reduced the conducted response. Washout of LPS restored the conducted response, whereas PTK inhibitors prevented the effect of LPS. On the basis of a newly developed mathematical model, the LPS-induced reduction in conducted response was predicted to reduce the arteriolar ability to increase resistance to blood flow. We conclude that LPS can reduce communication in in vitro and in vivo systems comparably in a reversible and tyrosine kinase-dependent manner. Based on literature and present results, we suggest that LPS may compromise microvascular hemodynamics at both the arteriolar responsiveness and the conduction levels.     

Tyrosine phosphorylation of a gap junction protein correlates with inhibition of cell-to-cell communication

Cell-to-cell communication is achieved by passage of small molecules through gap junction membrane channels. The expression of the transforming gene from Rous sarcoma virus, v-src, induces a rapid and dramatic reduction in cell-to-cell communication in cultured cells. To determine whether connexin43, a major gap junction protein expressed in fibroblasts, is a target for the v-src protein tyrosine kinase activity, we examined the phosphorylation state of connexin43 in cells expressing variants of src. Using an antipeptide serum that recognizes connexin43, we demonstrate that this protein is phosphorylated on serine and tyrosine residues in avian and mammalian cells expressing activated src proteins. Connexin43 from control cells and cells expressing nonactivated variants of the src protein was phosphorylated solely on serine residues. In lysates from v-src-transformed cells, all phosphorylated connexin43 molecules were cleared from the lysate by sequential immunoprecipitations using the phosphotyrosine antibodies, suggesting that each molecule of phosphorylated connexin43 contains both phosphoserine and phosphotyrosine. We have also examined junctional permeability in cells expressing src variants and find that loss of cell-to-cell communication correlates with tyrosine phosphorylation of connexin43.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Lipopolysaccharide plus hypoxia and reoxygenation synergistically reduce electrical coupling between microvascular endothelial cells by dephosphorylating connexin40

We showed that lipopolysaccharide (LPS) or hypoxia and reoxygenation (H/R) decreases electrical coupling between microvascular endothelial cells by targeting the gap junction protein connexin40 (Cx40), tyrosine kinase-, ERK1/2-, and PKA-dependently. Since LPS can compromise microvascular blood flow, resulting in micro-regional H/R, the concurrent LPS + H/R could reduce coupling to a much greater extent than LPS or H/R alone. We examined this possibility in a model of cultured microvascular endothelial cells (mouse skeletal muscle origin) in terms of electrical coupling and the phosphorylation status of Cx40. To assess coupling, we measured the spread of electrical current injected into the cell monolayer and computed the intercellular resistance as an inversed measure of coupling. In wild type cells, but not in Cx40 null cells, concurrent LPS + H/R synergistically increased resistance by approximately 270%, well above the level observed for LPS or H/R alone. Cx37 and Cx43 protein expression did not differ between Cx40 null and wild type cells. LPS + H/R increased resistance PKA- and PKC-dependently. By immunoprecipitating Cx40, we found that LPS + H/R reduced serine phosphorylation to a much greater degree than that observed for LPS or H/R alone. Further, PKA-specific, but not PKC-specific serine phosphorylation of Cx40 was also significantly reduced following LPS + H/R. This reduction was prevented by tyrosine kinase and MEK1/2 inhibition, by PKA activation, and mimicked in control cells by PKA inhibition. We conclude that LPS + H/R initiates tyrosine kinase- and ERK1/2-sensitive signaling that synergistically reduces inter-endothelial electrical coupling by dephosphorylating PKA-specific serine residues of Cx40.     

Inhibition of endothelial wound repair by dominant negative connexin inhibitors

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-betaGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ~50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.     

The functional expression of connexin 43 in articular chondrocytes is increased by interleukin 1beta: evidence for a Ca2+-dependent mechanism

Cell-to-cell interactions and gap junctions-dependent communication are crucially involved in chondrogenic differentiation, while in adult articular cartilage direct intercellular communication occurs mainly among chondrocytes facing the outer cartilage layer. Chondrocytes extracted from adult articular cartilage and grown in primary culture express connexin 43 and form functional gap junctions capable of sustaining the propagation of intercellular Ca2+ waves. Degradation of articular cartilage is a characteristic feature of arthritic diseases and is associated to increased levels of interleukin-1 (IL-1) in the synovial fluid. We have examined the effects of IL-1 on gap junctional communication in cultured rabbit articular chondrocytes. Incubation with IL-1 potentiated the transmission of intercellular Ca2+ waves and the intercellular transfer of Lucifer yellow. The stimulatory effect was accompanied by a dose-dependent increase in the expression of connexin 43 and by an enhanced connexin 43 immunostaining at sites of cell-to-cell contact. IL-1 stimulation induced a dose-dependent increase of cytosolic Ca2+ and activates protein tyrosine phosphorylation. IL-1-dependent up-regulation of connexin 43 could be prevented by intracellular Ca2+ chelation, but not by inhibitors of protein tyrosine kinases, suggesting a crucial role of cytosolic Ca2+ in regulating the expression of connexin 43. IL-1 is one of the most potent cytokines that promotes cartilage catabolism: its modulation of intercellular communication represents a novel mechanism by which proinflammatory mediators regulate the activity of cartilage cells.     

The effects of connexin phosphorylation on gap junctional communication

Gap junctions are specialized membrane domains composed of collections of channels that directly connect neighboring cells providing for the cell-to-cell diffusion of small molecules, including ions, amino acids, nucleotides, and second messengers. Vertebrate gap junctions are composed of proteins encoded by the "connexin" gene family. In most cases examined, connexins are modified post-translationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin "lifecycle", such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels. Since connexin43 (Cx43) is widely expressed in tissues and cell lines, we understand the most about how it is regulated, and thus, connexin43 phosphorylation is a major focus of this review. Recent reports utilizing new methodologies combined with the latest genome information have shown that activation of several kinases including protein kinase A, protein kinase C, p34(cdc2)/cyclin B kinase, casein kinase 1, mitogen-activated protein (MAP) kinase and pp60(src) kinase can lead to phosphorylation at 12 of the 21 serine and two of the six tyrosine residues in the C-terminal region of connexin43. In several cases, use of site-directed mutants of these sites have shown that these specific phosphorylation events can be linked to changes in gap junctional communication.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

Down-regulation of connexin43 gap junction by serum deprivation in human endothelial cells was improved by (-)-Epigallocatechin gallate via ERK MAP kinase pathway

Intercellular communication through gap junctions (GJIC) plays an essential role in maintaining the functional integrity of vascular endothelium. Despite emerging evidence suggests that (−)-Epigallocatechin gallate (EGCG) may improve endothelial function. However, its effect on Cx43 gap junction in endothelial cells remains unexplored. Here we investigated the effect of EGCG on connexin43 (Cx43) gap junction in endothelial cells. The levels of Cx43 protein in human umbilical vein endothelial cells (HUVECs) cultured under serum-deprivation 48 h decreased about 50%, accompanied by decreased GJIC. This reduction can be reversed by treatments with EGCG. In addition, EGCG activated ERK, P38, and JNK mitogen-activated protein kinases (MAPKs), which were supposed to participate in the regulation of Cx43. A MEK inhibitor PD98059, but not SB203580 (a p38 kinase inhibitor) or SP600125 (a JNK kinase inhibitor), abolished the effects of EGCG on Cx43 expression and GJIC. Moreover, although both Akt and eNOS phosphorylation were time-dependently augmented by EGCG, neither PI3K inhibitor LY294002 nor eNOS inhibitor L-NAME blocked the effects of EGCG on Cx43 gap junctions. Thus, EGCG attenuated Cx43 down-regulation and impaired GJIC induced by serum deprivation, ERK MAPK Signal transduction pathway appears to be involved in these processes.     

Intracellular calcium regulation of connexin43

The mechanism by which intracellular Ca(2+) concentration ([Ca(2+)](i)) regulates the permeability of gap junctions composed of connexin43 (Cx43) was investigated in HeLa cells stably transfected with this connexin. Extracellular addition of Ca(2+) in the presence of the Ca(2+) ionophore ionomycin produced a sustained elevation in [Ca(2+)](i) that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa fluor 594 (IC(50) of 360 nM Ca(2+)). The Ca(2+) dependency of this inhibition of Cx43 gap junctional permeability is very similar to that described in sheep lens epithelial cell cultures that express the three sheep lens connexins (Cx43, Cx44, and Cx49). The intracellular Ca(2+)-mediated decrease in cell-to-cell dye transfer was prevented by an inhibitor of calmodulin action but not by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II or protein kinase C. In experiments that used HeLa cells transfected with a Cx43 COOH-terminus truncation mutant (Cx43(Delta257)), cell-to-cell coupling was similarly decreased by an elevation of [Ca(2+)](i) (IC(50) of 310 nM Ca(2+)) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca(2+)](i) regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43.     

Cytoplasmic dye transfer between metastatic tumor cells and vascular endothelium

Metastatic colonization of a secondary organ site is initiated by the attachment of blood-borne tumor cells to organ-specific adhesion molecules expressed on the surface of microvascular endothelial cells. Using digital video imaging microscopy and fluorescence activated cell sorting techniques, we show here that highly metastatic cells (B16-F10 murine melanoma and R3230AC-MET rat mammary adenocarcinoma cells) previously labeled with the fluorescent dye BCECF begin to transfer dye to endothelial cell monolayers shortly after adhesion is established. The extent of BCECF transfer to endothelial cell monolayers is dependent upon the number of BCECF-labeled tumor cells seeded onto the endothelial cell monolayer and the time of coculture of the two cell types, as visualized by an increase in the number of BCECF-positive cells among cells stained with an endothelial cell-specific mAb. Dye transfer to BAEC monolayers proceeds with a progressive loss of fluorescence intensity in the BCECF-labeled tumor cell population with time of coculture. The transfer of dye is bidirectional and sensitive to inhibition by 1-heptanol. In contrast, poorly metastatic B16-F0 melanoma cells and non-metastatic R3230AC-LR mammary adenocarcinoma cells do not efficiently couple with vascular endothelial cells. It is inferred from these experiments and from the amounts of connexin43 mRNA expressed by tumor cells that tumor cell/endothelial cell communication is mediated by gap junctional channels and that this interaction may play a critical role in tumor cell extravasation at secondary sites.     

Connexin 43 in LA-25 cells with active v-src is phosphorylated on Y247, Y265, S262, S279/282, and S368 via multiple signaling pathways

Modulation of gap junction structures and gap junctional communication is important in maintaining tissue homeostasis and can be controlled via phosphorylation of connexin 43 (Cx43) through several different signaling pathways. Transformation of cells by v-src has been shown to down-regulate gap junction communication coincident with an increase in tyrosine phosphorylation on Cx43. Activation of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) also lead to down-regulation via phosphorylation on specific serine residues. Using phosphospecific anti-Cx43 antibodies generated by the authors' laboratory to specific tyrosines (src substrates) and serine residues (MAPK and PKC substrates) to probe LA-25 cells (which express temperature-sensitive v-src), the authors show that distinct tyrosine and serines residues are phosphorylated in response to v-src activity. They show that tyrosine phosphorylation appears to occur predominantly in gap junction plaques when src is active. In addition, src activation led to increased phosphorylation of apparent MAPK and PKC sites in Cx43. These results indicate all three signaling pathways could contribute to gap junction down-regulation during src transformation in LA-25 cells.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. Here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Role of connexins in microvascular dysfunction during inflammation

In arterioles, a locally initiated diameter change can propagate rapidly along the vessel length (arteriolar conducted response), thus contributing to arteriolar hemodynamic resistance. The response is underpinned by electrical coupling along the arteriolar endothelial layer. Connexins (Cx; constituents of gap junctions) are required for this coupling. This review addresses the effect of acute systemic inflammation (sepsis) on arteriolar conduction and interendothelial electrical coupling. Lipopolysaccharide (LPS; an initiating factor in sepsis) and polymicrobial sepsis (24 h model) attenuate conducted vasoconstriction in mice. In cultured microvascular endothelial cells harvested from rat and mouse skeletal muscle, LPS reduces both conducted hyperpolarization-depolarization along capillary-like structures and electrical coupling along confluent cell monolayers. LPS also tyrosine-phosphorylates Cx43 and serine-dephosphorylates Cx40. Since LPS-reduced coupling is Cx40- but not Cx43-dependent, only Cx40 dephosphorylation may be consequential. Nitric oxide (NO) overproduction is critical in advanced sepsis, since the removal of this overproduction prevents the attenuated conduction. Consistently, (i) exogenous NO in cultured cells reduces coupling in a Cx37-dependent manner, and (ii) the septic microvasculature in vivo shows no Cx40 phenotype. A complex role emerges for endothelial connexins in sepsis. Initially, LPS may reduce interendothelial coupling and arteriolar conduction by targeting Cx40, whereas NO overproduction in advanced sepsis reduces coupling and conduction by targeting Cx37 instead.     

Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.     

Cloning of a gap junctional protein from vascular smooth muscle and expression in two-cell mouse embryos.

Gap junctional proteins (connexins) form aqueous channels that enable direct cell-cell transfer of ions and small molecules. The distribution and conductance of gap junction channels in cardiac muscle determine the pattern and synchrony of cellular activation. However, the capacity for smooth muscle to restrict contractile events temporally and spatially suggests that cell-cell coupling or its regulation may be decidedly different in this tissue. We isolated a cDNA from vascular smooth muscle which encodes a connexin (Mr 43,187) structurally homologous to cardiac connexin43. Vascular smooth muscle connexin43 mRNA was expressed prominently in smooth muscle tissues, cultured vascular myocytes, and arterial endothelial cells. A model for functional expression of connexins was developed in two-cell B6D2 mouse embryos. Microinjection of in vitro transcribed vascular smooth muscle connexin43 mRNA was shown to be sufficient to induce intercellular coupling in previously uncoupled blastomeres. Through the construction of two deletion mutants of connexin43, we also show that the formation of cell-to-cell connections does not depend upon a predicted cytoplasmic region within 98 residues of the carboxyl terminus. Finally, the identification of connexin43 in smooth muscle and endothelial cells provides supporting evidence for the existence of heterocellular coupling between cells of the vascular intima.