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The gap junction protein connexin43 (Cx43) has been proposed to play key roles in bone differentiation and mineralization, but underlying cellular mechanisms are not totally understood. To further explore roles of Cx43 in these processes, we immortalized calvarial osteoblasts from wild-type and Cx43-null mice using human telomerase reverse transcriptase (hTERT). Osteoblastic (MOB) cell lines were generated from three individual wild-type and three individual Cx43-null mouse calvaria. Average population doubling times of the cell lines were higher than of the primary osteoblasts but did not greatly differ with regard to genotype. Modest to high level of Cx45 expression was detected in MOBs of both genotypes. Most of the cell lines expressed osteoblastic markers [Type I collagen, osteopontin, osteocalcin, parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP), periostin (OSF-2), osterix (Osx), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP)], and mineralization was comparable to that of primary osteoblasts. Two MOB cell lines from each genotype with most robust maintenance of osteoblast lineage markers were analyzed in greater detail, revealing that the Cx43-null cell lines showed a significant delay in early differentiation (up to 9 days in culture). Matrix mineralization was markedly delayed in one of the Cx43-null lines and slightly delayed in the other. These findings comparing new and very stable wild-type and Cx43-null osteoblastic cell lines define a role for Cx43 in early differentiation and mineralization stages of osteoblasts and further support the concept that Cx43 plays important role in the cellular processes associated with skeleton function. gap junction; alkaline phosphatase; differentiation; mineralization; human telomerase reverse transcriptase doi: 10.1152/ajpcell.00544.2009.

We tested the hypothesis that fluid shear stress ([tau]) modifies the expression, function, and distribution of junctional proteins [connexin (Cx)43, Cx45, and zona occludens (ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1 cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to [tau]-values of 5 or 20 dyn/[cm.sup.2] for 1-3 h. Immunostaining indicated that at 5 dyn/[cm.sup.2], the distribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cell membranes; at 20 dyn/[cm.sup.2], disruption was more severe. Intercellular coupling was significantly decreased at both shear stress levels. Western blots showed the downregulation of membrane-bound Cx43 and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at different levels of shear stress. Similarly, Northern blots revealed that expression of Cx43, Cx45, and ZO-1 was selectively up- and down-regulated in response to different shear stress levels. These results indicate that in cultured bone cells, fluid shear stress disrupts junctional communication, rearranges junctional proteins, and determines de novo synthesis of specific connexins to an extent that depends on the magnitude of the shear stress. Such disconnection from the bone cell network may provide part of the signal whereby the disconnected cells or the remaining network initiate focal bone remodeling. gap junctions; connexin; zona occludens-1; mechanotransduction; bone remodeling

Immunoprecipitation and histochemical staining methods, in conjunction with Northern Blot analysis, enables an assessment of the conductance properties of a hepatoma cell line clone population. A gap junction protein connexin (Cx)45 is present in these clones, which have a voltage dependent-macroscopic junctional conductance of about 1.3 nS. A transjunctional voltage-independent unitary conductance of 32 plus or minus 8 pS is seen in these cells. These human Cx45 gap junction channels and the Xenopus Cx38 channels exhibit similar voltage dependencies.

Cell pairs isolated from crayfish hepatopancreas were subjected to dual whole cell recording voltage-clamp experiments to evaluate the electrical features of their membrane junctions. Membrane potential influence junctional conductance in several cell pair combinations. Unique elementary conductance values were seen in the currents through single gap junction channels observed in these cell combinations, confirming the complexity of gating mechanisms.

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and [10.sup.-7] M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes. connexin32; connexin26; knockout mice

Suadicani, Sylvia O., Monique J. Vink, and David C. Spray. Slow intercellular [Ca.sup.2+] signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes. Am J Physiol Heart Circ Physiol 279: H3076-H3088, 2000.--Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular [Ca.sup.2+] waves that propagated with mean velocities of ~14 [micro]m/s, reaching ~80% of the cells in the field. Deletion of connexin43 (Cx43), the main cardiac gap junction channel protein, did not prevent communication of mechanically induced [Ca.sup.2+] waves, although the velocity and number of cells communicated by the [Ca.sup.2+] signal were significantly reduced. Similar effects were observed in wild-type cardiac myocytes treated with heptanol, a gap junction channel blocker. Fewer cells were involved in intercellular [Ca.sup.2+] signaling in both wild-type and Cx43-null cultures in the presence of suramin, a [P.sub.2]-receptor blocker; blockage was more effective in Cx43-null than in wild-type cells. Thus gap junction channels provide the main pathway for communication of slow intercellular [Ca.sup.2+] signals in wild-type neonatal mouse cardiac myocytes. Activation of P2-receptors induced by ATP release contributes a secondary, extracellular pathway for transmission of [Ca.sup.2+] signals. The importance of such ATP-mediated [Ca.sup.2+] signaling would be expected to be enhanced under ischemic conditions, when release of ATP is increased and gap junction channels conductance is significantly reduced. calcium waves; connexin; gap junctions; purinergic receptors; intercellular communication