Effects of cAMP on intercellular coupling and osteoblast differentiation

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. Direct intercellular communication via gap junctions is an important component of bone homeostasis, coordinating cellular responses to external signals and promoting osteoblast differentiation. The cAMP pathway, a major intercellular signal transduction mechanism, regulates osteoblastic function and metabolism. We investigated the effects of this second messenger on junctional communication and on the expression of differentiation markers in human HOBIT osteoblastic cells. Increased levels of cAMP induce posttranslational modifications (i.e., phosphorylations) of connexin43 and enhancement of gap junction assembly, resulting in an increased junctional permeance to Lucifer yellow and to a positive modulation of intercellular Ca(2+) waves. Increased intercellular communication, however, was accompanied by a parallel decrease of alkaline phosphatase activity and by an increase of osteocalcin expression. cAMP-dependent stimulation of cell-to-cell coupling induces a complex modulation of bone differentiation markers.     

Gap junctions and hemichannels in signal transmission, function and development of bone

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Bone morphogenetic protein-2 modulation of chondrogenic differentiation in vitro involves gap junction-mediated intercellular communication

Undifferentiated mesenchymal cells in the limb bud integrate a complex array of local and systemic signals during the process of cell condensation and chondrogenic differentiation. To address the relationship between bone morphogenetic protein (BMP) signaling and gap junction-mediated intercellular communication, we examined the effects of BMP-2 and a gap junction blocker 18 alpha glycyrrhetinic acid (18alpha-GCA) on mesenchymal cell condensation and chondrogenic differentiation in an in vitro chondrogenic model. We find that connexin43 protein expression significantly correlates with early mesenchymal cellular condensation and chondrogenesis in high-density limb bud cell culture. The level of connexin43 mRNA is maximally upregulated 48 h after treatment with recombinant human BMP-2 with corresponding changes in protein expression. Inhibition of gap junction-mediated intercellular communication with 2.5 microM 18alpha-GCA decreases chondrogenic differentiation by 50% at 96 h without effects on housekeeping genes. Exposure to 18alpha-GCA for only the first 24-48 h after plating does not affect condensation or later chondrogenic differentiation suggesting that gap junction-mediated intercellular communication is not critical for the initial phase of condensation but is important for the onset of differentiation. 18alpha-GCA can also block the chondrogenic effects of BMP-2 without effects on cell number or connexin43 expression. These observations demonstrate 18alpha-GCA-sensitive regulation of intercellular communication in limb mesenchymal cells undergoing chondrogenic differentiation and suggest that BMP-2 induced chondrogenic differentiation may be mediated in part through the modulation of connexin43 expression and gap junction-mediated intercellular communication.     

Regulation of osteoclastogenesis by gap junction communication

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.     

Radiation Response of Connexin43-Transfected Cells in Relation to the “Contact Effect”

Some cell lines grown for only two cell doublings as multicell spheroids develop a form of resistance to killing by ionizing radiation that has been called the “contact” effect. While our previous results have implicated a role for higher order chromatin structure in the contact effect, another possible explanation is the presence of intercellular gap junctions that might facilitate communication between cells grown as spheroids and thereby enhance the ability of cells to resist or recover from radiation damage. To examine the role of gap junctions in the contact effect, rat glioma C6 and mouse EMT6 cell lines were transfected with a gene encoding the gap junctional protein connexin43. While C6 glioma cells are deficient in gap junctional communication, cells from spheroids were nonetheless more resistant than monolayers to killing by ionizing radiation, and the contact effect was present to a similar extent in the three transfected clones. For mouse EMT6 cells, radiosensitivity was similar whether cells were grown as monolayers or spheroids. Transfection of EMT6 cells with connexin43 increased gap junctional communication but did not promote development of a contact effect. Tumor volume doubling time in SCID mice increased significantly for one transfected clone; however, doubling timein vitrowas also increased relative to the EMT6 parent. We conclude that extensive gap junctional communication is not a requirement for the increased radiation resistance observed when some cell lines are grown as spheroids.     

Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes

Innumerable toxic substances present in the environment inhibit gap junctions, intercellular membrane channels that play fundamental roles in coordinated function of cells and tissues. Included are persistent organochlorine compounds, which pose health risks to humans and animals owing to their widespread use, bioaccumulation, and ability to inhibit gap junction channel-mediated intercellular communication in liver, lung, skin, heart, and brain cells. In this study, the organochlorine xenobiotics dieldrin and endosulfan, at micromolar concentrations, were found to inhibit gap junction-mediated intercellular communication and induce hypophosphorylation of connexin 43 in cultured rat astrocytes, the predominant cell type in the brain coupled through gap junctions. This inhibition of gap junctional communication was substantially reduced by preincubation with chaetoglobosin K (ChK), a bioactive natural produce previously shown to have ras tumor suppressor activity. Chaetoglobosin K also prevented dieldrin and endosulfan-induced hypophosphorylation of connexin 43 and prevented dieldrin-induced connexin 43 plaque dissolution in both astrocytes and cultured liver epithelial cells. The results suggest that stabilization of the native, phosphorylated form of connexin 43 by ChK may contribute to its ability to prevent organochlorine-induced inhibition of gap junction-mediated communication and dissolution of gap junction plaques within the plasma membrane. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dominant cataracts result from incongruous mixing of wild-type lens connexins

Gap junctions are composed of proteins called connexins (Cx) and facilitate both ionic and biochemical modes of intercellular communication. In the lens, Cx46 and Cx50 provide the gap junctional coupling needed for homeostasis and growth. In mice, deletion of Cx46 produced severe cataracts, whereas knockout of Cx50 resulted in significantly reduced lens growth and milder cataracts. Genetic replacement of Cx50 with Cx46 by knockin rescued clarity but not growth. By mating knockin and knockout mice, we show that heterozygous replacement of Cx50 with Cx46 rescued growth but produced dominant cataracts that resulted from disruption of lens fiber morphology and crystallin precipitation. Impedance measurements revealed normal levels of ionic gap junctional coupling, whereas the passage of fluorescent dyes that mimic biochemical coupling was altered in heterozygous knockin lenses. In addition, double heterozygous knockout lenses retained normal growth and clarity, whereas knockover lenses, where native Cx46 was deleted and homozygously knocked into the Cx50 locus, displayed significantly deficient growth but maintained clarity. Together, these findings suggest that unique biochemical modes of gap junctional communication influence lens clarity and lens growth, and this biochemical coupling is modulated by the connexin composition of the gap junction channels.     

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.     

Zebrafish short fin mutations in connexin43 lead to aberrant gap junctional intercellular communication

Mutations in the zebrafish connexin43 (cx43) gene cause the short fin phenotype, indicating that direct cell-cell communication contributes to bone length. Three independently generated cx43 alleles exhibit short segments of variable sizes, suggesting that gap junctional intercellular communication may regulate bone growth. Dye coupling assays showed that all alleles are capable of forming gap junction channels. However, ionic coupling assays revealed allele-specific differences in coupling efficiency and gating. For instance, oocyte pairs expressing the weakest allele exhibited much higher levels of coupling than either of the strong alleles. Therefore, measurable differences in Cx43 function may be correlated with the severity of defects in bone length.     

Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.     

A Neuroprotective Role for Gap Junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

A neuroprotective role for gap junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

Neurons set the tone of gap junctional communication in astrocytic networks

A number of studies have contributed to demonstrate that neurons and astrocytes tightly and actively interact. Indeed, the presence of astrocytes in neuronal cultures increases the number of synapses and their efficiency, and thanks to enzymatic and uptake processes, astrocytes play a role in neuroprotection. A typical feature of astrocytes is that they establish cell-cell communication in vitro, as well as in situ, through intercellular channels forming specialized membrane areas defined as gap junctions. These channels are composed of junctional proteins termed connexins (Cxs): in astrocytes connexin 43 (Cx43) and 30 (Cx30) have been shown to prevail. Several recent works indicate that gap junctional communication (GJC) and/or connexin expression in astrocytes are controlled by neurons. Altogether, these observations lead to the concept that neuronal and astrocytic networks interact through mutual setting of their respective mode of communication and that astrocyte gap junctions represent a target in neuroglial interaction.     

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.     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication

The mechanism by which gap junction proteins, connexins, act as potent tumor suppressors remains poorly understood. In this study human breast tumor cells were found to exhibit diverse gap junction phenotypes including (a) undetectable Cx43 and no intercellular communication (HBL100); (b) low levels of Cx43 and sparse intercellular communication (MDA-MB-231); and (c) significant levels of Cx43 and moderate intercellular communication (Hs578T). Although retroviral delivery of Cx43 and Cx26 cDNAs to MDA-MB-231 cells did not achieve an expected substantial rescue of intercellular communication, overexpression of connexin genes did result in a dramatic suppression of tumor growth when connexin-expressing MDA-MB-231 cells were implanted into the mammary fat pad of nude mice. Subsequent immunolocalization studies on xenograph sections revealed only cytoplasmic stores of Cx43 and no detectable gap junctions. Moreover, DNA array and Western blot analysis demonstrated that overexpression of Cx43 or Cx26 in MDA-MB-231 cells down-regulated fibroblast growth factor receptor-3. Surprisingly, these results suggest that Cx43 and Cx26 induce their tumor-suppressing properties by a mechanism that is independent of significant gap junctional intercellular communication and possibly through the down-regulation of key genes involved in tumor growth. Moreover, our studies show that retroviruses are effective vehicles for delivering connexins to human breast tumor cells, facilitating potential gene therapy applications.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.