Strain-induced mechanotransduction through primary cilia, extracellular ATP, purinergic calcium signaling, and ERK1/2 transactivates CITED2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes.

OBJECTIVE: To determine the strain-induced signaling pathways involved in regulating the transactivation of the transcription regulator Cbp/p300 Interacting Transactivator with ED-rich tail 2 (CITED2) and downstream targets in chondrocytes. METHODS: Primary human chondrocytes or C28/I2 chondrocytic cells were subjected to various strain regimes. C57BL/6 mice were subjected to treadmill running. Loss-of-function was carried out using siRNA or inhibitors specific for targeted molecules. mRNA levels were assayed by RT-qPCR, and proteins by western blotting, immunofluorescence, and/or immunohistochemical staining. CITED2 promoter activity was assayed in chondrocytes using wild-type or mutant constructs. RESULTS: Cyclic strain at 5%, 1 Hz induced CITED2 expression and suppressed expression of matrix metalloproteinase (MMP)-1 and -13 at the messenger RNA (mRNA) and protein levels in human chondrocytes. Abolishing primary cilia through knockdown of intraflagellar transport protein (IFT88) attenuated CITED2 gene expression and decreased protein levels. Similar effects were observed with inhibitors of extracellular adenosine triphosphate (ATP) or P2 purinergic receptors, or antagonists of Ca(2+) signaling. Knockdown of IFT88 in articular chondrocytes in vivo diminished treadmill induced-CITED2 expression and upregulated MMPs. Knockdown of hypoxia-inducible factor (HIF)1α, specificity protein 1 (Sp1), or deletion of the shear stress response element (SSRE) in the CITED2 promoter limited cyclic strain-induced transactivation of CITED2. However, the strain induced-transactivation of CITED2 was abolished only on knockdown of HIF1α, Sp1, and SSRE or by loss-of-function of IFT88 or extracellular-signal-regulated kinases (ERK)1/2. CONCLUSIONS: CITED2 transactivation is a critical event in signaling generated by strain and transduced by primary cilia, extracellular ATP, P2 purinergic receptors, and Ca(2+) signaling. Strain-induced CITED2 transactivation requires HIF1α, Sp1, and an intact SSRE and leads to the downregulation of MMPs such as MMP-1 and MMP-13.

Transcriptome profiling of hippocampal CA1 after early-life seizure-induced preconditioning may elucidate new genetic therapies for epilepsy.

Injury of the CA1 subregion induced by a single injection of kainic acid (1 × KA) in juvenile animals (P20) is attenuated in animals with two prior sustained neonatal seizures on P6 and P9. To identify gene candidates involved in the spatially protective effects produced by early-life conditioning seizures we profiled and compared the transcriptomes of CA1 subregions from control, 1 × KA- and 3 × KA-treated animals. More genes were regulated following 3 × KA (9.6%) than after 1 × KA (7.1%). Following 1 × KA, genes supporting oxidative stress, growth, development, inflammation and neurotransmission were upregulated (e.g. Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12 and Grik1). After 3 × KA, protective genes were differentially over-expressed [e.g. Cat, Gpx7, Gad1, Hspa12A, Foxn1, adenosine A1 receptor, Ca(2+) adaptor and homeostasis proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp and suppressor of cytokine signaling (Socs3)]. Distinct anti-inflammatory interleukins (ILs) not observed in adult tissues [e.g. IL-6 transducer, IL-23 and IL-33 or their receptors (IL-F2 )] were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1 × KA but reduced after 3 × KA; the pro-inflammatory gene Cox1 was either upregulated or unchanged after 1 × KA but reduced by ~70% after 3 × KA. Enhanced GFAP immunostaining following 1 × KA was selectively attenuated in the CA1 subregion after 3 × KA. The observed differential transcriptional responses may contribute to early-life seizure-induced pre-conditioning and neuroprotection by reducing glutamate receptor-mediated Ca(2+) permeability of the hippocampus and redirecting inflammatory and apoptotic pathways. These changes could lead to new genetic therapies for epilepsy.

The connexin43-dependent transcriptome during brain development: importance of genetic background.

Use of null mutant mice is a powerful way to evaluate the role of specific proteins in brain function. Studies performed on knockout mice have revealed some unexpected roles of the gap junction proteins (connexins). Thus, analyses of gene expression in connexin43 (Cx43) null brains indicated that deletion of a single gene (Gja1) induced expression level change of numerous other genes located on all chromosomes and involved in a wide diversity of functional pathways. The significant overlap between alterations in gene expression level, control and coordination in Cx43 knockout and knockdown astrocytes raised the possibility that Gja1 represents a transcriptomic node of gene regulatory networks. However, conditional deletion of Gja1 in astrocytes of two mouse strains resulted in remarkably different phenotypes. In order to evaluate the influence of the genetic background on the transcriptome, we performed microarray studies on brains of GFAP-Cre:Cx43(f/f) C57Bl/6 and 129/SvEv mice. The surprisingly low number of Cx43 core genes (regulated in all Cx43 nulls regardless of strain) and the high number of differently regulated genes in the two Cx43 conditional knockouts indicate high influence of mouse strain on brain transcriptome. This article is part of a Special Issue entitled Electrical Synapses.

Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post-ischemic heart failure

After myocardial infarction (MI), activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). The interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression by analyzing hybridization to AECOM 32k mouse microarrays. Ischemic HF significantly increased the levels of total serum IgM (by 5.2-fold) and total IgG (by 3.6-fold) associated with a relatively high content of anti-heart specificity. A comparable increase was observed in the levels of circulating pro-inflammatory cytokines such as IL-1â (3.8X) and TNF-á (6.0X). IFN-ã was also increased by 3.1-fold in the MI group. However, IL-4 and IL-10 were not significantly different between the MI and sham-operated groups. Chemokines such as MCP-1 and IL-8 were 1.4- and 13-fold increased, respectively, in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by post-ischemic HF. Complement activation and immune response were among the most up-regulated processes. Interestingly, 21 of the 101 quantified unigenes involved in the inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune-mediated mechanisms that act both systemically and locally.

Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post-ischemic heart failure.

After myocardial infarction (MI), activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). The interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression by analyzing hybridization to AECOM 32k mouse microarrays. Ischemic HF significantly increased the levels of total serum IgM (by 5.2-fold) and total IgG (by 3.6-fold) associated with a relatively high content of anti-heart specificity. A comparable increase was observed in the levels of circulating pro-inflammatory cytokines such as IL-1beta (3.8X) and TNF-alpha (6.0X). IFN-gamma was also increased by 3.1-fold in the MI group. However, IL-4 and IL-10 were not significantly different between the MI and sham-operated groups. Chemokines such as MCP-1 and IL-8 were 1.4- and 13-fold increased, respectively, in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by post-ischemic HF. Complement activation and immune response were among the most up-regulated processes. Interestingly, 21 of the 101 quantified unigenes involved in the inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune-mediated mechanisms that act both systemically and locally.

Sex-dependent gene regulatory networks of the heart rhythm.

Expression level, control, and intercoordination of 66 selected heart rhythm determinant (HRD) genes were compared in atria and ventricles of four male and four female adult mice. We found that genes encoding various adrenergic receptors, ankyrins, ion channels and transporters, connexins, cadherins, plakophilins, and other components of the intercalated discs form a complex network that is chamber dependent and differs between the two sexes. In addition, most HRD genes in atria had higher expression in males than in females, while in ventricles, expression levels were mostly higher in females than in males. Moreover, significant chamber differences were observed between the sexes, with higher expression in atria than ventricles for males and higher expression in ventricles than atria for females. We have ranked the selected genes according to their prominence (new concept) within the HRD gene web defined as extent of expression coordination with the other web genes and stability of expression. Interestingly, the prominence hierarchy was substantially different between the two sexes. Taken together, these findings indicate that the organizational principles of the heart rhythm transcriptome are sex dependent, with the newly introduced prominence analysis allowing identification of genes that are pivotal for the sexual dichotomy.

P2X7 receptor-Pannexin1 complex: pharmacology and signaling.

Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X(7) receptor (P2X(7)R) activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X(7)R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X(7)R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X(7)R stimulation or by membrane depolarization was blocked by Panx1 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X(7)R antagonist, prevented current activation by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X(7)) peptides, provided evidence that the Src homology 3 death domain of the COOH-terminus of the P2X(7)R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 microM TAT-P2X(7) peptide caused 50% reduction in Src binding to the P2X(7)R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X(7) peptide, and by the Src tyrosine inhibitor PP2 and these compounds prevented both large-conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X(7)R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X(7)R.

Hypertension in connexin40-null mice: a renin disorder.

Studies described in this issue indicate that the gap junction protein connexin40 (Cx40) appears to play an unexpected role in blood pressure regulation. In mice lacking this gap junction protein, renin secretion is high and not regulated by arteriolar pressure.

Gap junctions remain open during cytochrome c-induced cell death: relationship of conductance to 'bystander' cell killing.

Previous reports have shown that gap junctions relay cell death in many cell types. However, changes in electrical coupling and their dynamics during cell death are poorly understood. We performed comprehensive studies of electrical coupling following induction of cell death by single-cell cytochrome c (cyC) injection in paired Xenopus oocytes. Cell death was rapidly induced by cyC in injected cells, and cell death was also observed in uninjected bystander cells electrically coupled to the cyC-injected oocytes. Gap junction currents either remained at pre-cyC injection levels or increased dramatically as the injected cell died. Nonjunctional currents increased in injected cells immediately following cyC injection; nonjunctional currents increased slowly in uninjected bystander cells. Bystander cell death occurred only when junctional conductance was approximately 6 muS. Both 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N',N'-tetraacetic acid tetraacetoxy-methyl ester and Xestospongin C inhibited bystander cell death in pairs that had reached the death conductance threshold, suggesting that Ca(2+) and inositol 1,4,5 triphosphate are involved in the process.

Human and mouse microglia express connexin36, and functional gap junctions are formed between rodent microglia and neurons.

Microglia, the tissue macrophages of the central nervous system (CNS), intimately interact with neurons physically and through soluble factors that can affect microglial activation state and neuronal survival and physiology. We report here a new mechanism of interaction between these cells, provided by the formation of gap junctions composed of connexin (Cx) 36. Among eight Cxs tested, expression of Cx36 mRNA and protein was found in microglial cultures prepared from human and mouse, and Cx45 mRNA was found in mouse microglial cultures. Electrophysiological measurements found coupling between one-third of human or mouse microglial pairs that averaged below 30 pico-Siemens and displayed electrical properties consistent with Cx36 gap junctions. Importantly, similar frequency of low-strength electrical coupling was also obtained between microglia and neurons in cocultures prepared from neocortical or hippocampal rodent tissue. Lucifer yellow dye coupling between neurons and microglia was observed in 4% of pairs tested, consistent with the low strength and incidence of electrical coupling. Cx36 expression level and/or the degree of coupling between microglia did not significantly change in the presence of activating agents, including lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha, except for some reduction of Cx36 protein when exposed to the latter two agents. Our findings that intercellular coupling occurs between neuronal and microglial populations through Cx36 gap junctions have potentially important implications for normal neural physiology and microglial responses in neuronopathology in the mammalian CNS.

The role of aquaporin-4 in the blood-brain barrier development and integrity: studies in animal and cell culture models.

Aquaporin-4 (AQP4) is the major water channel expressed in brain perivascular astrocyte processes. Although the role of AQP4 in brain edema has been extensively investigated, little information exists regarding its functional role at the blood-brain barrier (BBB). The purpose of this work is to integrate previous and recent data regarding AQP4 expression during BBB formation and depending on BBB integrity, using several experimental models. Results from studies on the chick optic tectum, a well-established model of BBB development, and the effect of lipopolysaccharide on the BBB integrity and on perivascular AQP4 expression have been analyzed and discussed. Moreover, data on the BBB structure and AQP4 expression in murine models of Duchenne muscular dystrophy are reviewed. In particular, published results obtained from mdx(3cv) mice have been analyzed together with new data obtained from mdx mice in which all the dystrophin isoforms including DP71 are strongly reduced. Finally, the role of the endothelial component on AQP4 cellular expression and distribution has been investigated using rat primary astrocytes and brain capillary endothelial cell co-cultures as an in vitro model of BBB.

A novel form of cellular communication among thymic epithelial cells: intercellular calcium wave propagation.

We here describe intercellular calcium waves as a novel form of cellular communication among thymic epithelial cells. We first characterized the mechanical induction of intercellular calcium waves in different thymic epithelial cell preparations: cortical 1-4C18 and medullary 3-10 thymic epithelial cell lines and primary cultures of thymic "nurse" cells. All thymic epithelial preparations responded with intercellular calcium wave propagation after mechanical stimulation. In general, the propagation efficacy of intercellular calcium waves in these cells was high, reaching 80-100% of the cells within a given confocal microscopic field, with a mean velocity of 6-10 microm/s and mean amplitude of 1.4- to 1.7-fold the basal calcium level. As evaluated by heptanol and suramin treatment, our results suggest the participation of both gap junctions and P2 receptors in the propagation of intercellular calcium waves in thymic nurse cells and the more prominent participation of gap junctions in thymic epithelial cell lines. Finally, in cocultures, the transmission of intercellular calcium wave was not observed between the mechanically stimulated thymic epithelial cell and adherent thymocytes, suggesting that intercellular calcium wave propagation is limited to thymic epithelial cells and does not affect the neighboring thymocytes. In conclusion, these data describe for the first time intercellular calcium waves in thymic epithelial cells and the participation of both gap junctions and P2 receptors in their propagation.

[Transcription regulation and coordination of some cell signaling genes in brain and heart of connexin 43 null mouse]. / Reglarea si coordonarea transcrierii unor gene de semnalizare intercelulara în creierul si inima de soarece deficitar de conexina 43.

We performed a cDNA microarray study of the transcription regulation and coordination of four gene families whose products are involved in cell-cell and cell-matrix interaction (ADAM, integrin, MMP, TNF) within brain and hearts of wildtype (WT) and connexin43 null (KO) neonatal C57Bl/6j mice. The study revealed that both WT brain and heart exhibit significant correlations among the transcriptions of cell-signaling genes and that depletion of Cx43 regulates both their expression and coordination. Adam8 was identified as the command gene of the group in WT and Mmp2 in KO, while Tnsf6 plays the dominant role in both WT and KO heart. Our results suggest that the functional coordination of cell-signaling proteins may be related to the expression coordination of the corresponding genes presumably to ensure the efficiency of the functional pathways and that intercellular communication modulates cell-cell and cell-matrix interaction.

Evidence for a role of the N-terminal domain in subcellular localization of the neuronal connexin36 (Cx36).

The expression and functional properties of connexin36 (Cx36) have been investigated in two neuroblastoma cell lines (Neuro2A, RT4-AC) and primary hippocampal neurons transfected with a Cx36-enhanced green fluorescent protein (EGFP) expression vector. Transfected cells express Cx36-EGFP mRNA, and Cx36-EGFP protein is localized in the perinuclear area and cell membrane. Upon differentiation of cell lines, Cx36-EGFP protein was detectable in processes with both axonal and dendritic characteristics. Small gap junction plaques were found between adjacent cells, and electrophysiological recordings demonstrated that the electrical properties of these gap junctions were virtually indistinguishable from those reported for native Cx36. Mutagenesis of Cx36 led to the identification of a structural element that interferes with normal protein localization. In contrast, site directed mutagenesis of putative protein phosphorylation motifs did not alter subcellular localization. This excludes phosphorylation/dephosphorylation as a major regulatory step in Cx36 protein transport.

Control and variability of gene expression in mouse brain and in a neuroblastoma cell line.

We used cDNA microarrays to examine the extent to which the expression of individual genes varies in mouse brain and in cultured N2A neuroblastoma cells mRNA extracted from sixC57B1/6J neonatal mouse brains and from four distinct cultures of N2A neuroblastoma cells was cross-hybridized with ten AECOM cDNA microarray chips to determine the individual gene expression variability. A mathematical algorithm reduced the effect of potential sources of variability not associated to the biological material by about 80%. The interval estimates of the standard deviation of individual gene expressionwere determined through chi-square statistics. The newly introduced relative expression variability, defined as the quotient of the middle of the interval estimate of the standard deviation and the mean expression ratio (and its inverse, gene transcription control), was used to rank the most unstably and the most stably transcribed genes. In brains of different animals and in separate cultures of N2A cells, unique sets ofgenes exhibited exceptional stability or were highly variable. Possible implications for such tight or loose transcriptional control are discussed.

Transcriptomic characterization of four classes of cell-cell/cell-matrix genes in brains and hearts of wild type and connexin43 null mice.

We have used a highly quantifiable cDNA microarray method to determine the stabilities and expression levels within gene families involved in cell-cell and cell-matrix interactions in neonatal mouse brain and heart. In addition, we have characterized the extent to which deletion of the gap junction protein connexin43 (Cx43) affects these characteristics. Our observations for individual genes revealed a range of differences and variabilities in transcription level among family members; calculation of the genomic patholog (a global measure of gene expression alteration) indicates that these cell interaction genes contribute disproportionately to the overall phenotype. We found significant transcriptomic differences between brain and heart, that deletion of Cx43 considerably decreased gene expression variability and that the average contribution to the pathology of the genes whose encoded proteins are involved in cell-cell or cell-matrix interaction in the Cx43-null mice was about twenty times higher than that of other genes. These findings indicate that gap junction gene expression influences the expression of other genes involved in intercellular and cell-substrate interaction and that expression of these genes is under strong regulatory pressure in the Cx43-null mouse, presumably representing a compensatory response to Cx43 deletion.

Gap junctional channels regulate acid secretion in the mammalian gastric gland.

Gap junction channels are regarded as a primary pathway for intercellular message transfer, including calcium wave propagation. Our study identified two gap junctional proteins, connexin26 and connexin32, in rat gastric glands by RT-PCR, Western blot analysis, and immunofluorescence. We demonstrated a potential physiological role of the gap junctional channels in the acid secretory process using the calcium indicator fluo-3, and microinjection of Lucifer Yellow. Application of gastrin (10-7 m) to the basolateral membrane resulted in the induction of uniphasic calcium signals in adjacent parietal cells. In addition, single parietal cell microinjections in intact glands with the cell-impermeant dye Lucifer Yellow resulted in a transfer of dye from the injected cell to the adjacent parietal cell following gastrin stimulation, demonstrating gastrin-induced cell-to-cell communication. Both calcium wave propagation and Lucifer Yellow transfer were blocked by the gap junction inhibitor 18alpha-glycyrrhetinic acid. Our studies demonstrate that functional gap junction channels in gastric glands provide an effective means for rapid cell-to-cell communication and allow for the rapid onset of acid secretion.

Growth-suppressive function of human connexin32 in a conditional immortalized mouse hepatocyte cell line.

Mouse hepatocytes immortalized with a temperature-sensitive allele of the SV40 large T-antigen (CHST8 cells) were found to lack the high expression of the gap junction proteins Cx26 and Cx32 that characterizes normal mouse hepatocytes, expressing instead Cx43 and Cx45 at minimal levels. In order to examine the growth suppressive function of Cx32 on hepatocytes, we transfected these CHST8 cells with human Cx32 complementary deoxyribonucleic acid and measured the growth rates at 33, 37, and 39 degrees C. Expression of human Cx32 and its messenger ribonucleic acid in the stable cell lines was confirmed by immunocytochemistry and by Western and Northern blots analyses. Dye transfer following lucifer yellow injection into the transfectants was extensive; Cx32 channels displayed unitary conductances of about 70 pS and were moderately voltage sensitive. When cultured at 33 and 39 degrees C, growth rates of both parental cells and transfectants were of the same level. When examined at 37 degrees C, growth rate of the transfectant, which highly expressed Cx32 at the membranes, was significantly decreased compared to the parental cells. However, no changes in the expression of Cx32 protein in the transfectants were observed between 33 and 37 degrees C. These results suggest that Cx32 expression could inhibit hepatocyte growth in vitro using the conditional immortalized cells. Cx32 transfectants using a conditional immortalized mouse hepatocyte may be useful for examining the mechanisms of growth and differentiation in hepatocytes by gap junction expression.

Quinine blocks specific gap junction channel subtypes.

We demonstrate that the antimalarial drug quinine specifically reduces currents through gap junctions formed by some connexins (Cx) in transfected mammalian cells, but does not affect other gap junction types. Quinine blocked Cx36 and Cx50 junctional currents in a reversible and concentration-dependent manner with half maximal blocking concentrations of 32 and 73 microM, respectively; Hill coefficients for block by quinine were about 2 for both connexins. In contrast, quinine did not substantially block gap junction channels formed by Cx26, Cx32, Cx40, and Cx43, and only moderately affected Cx45 junctions. To determine the location of the binding site of quinine (pKa = 8.7), we investigated the effect of quinine at various external and internal pH values and the effect of a permanently charged quaternary derivative of quinine. Our results indicate that the binding site for quinine is intracellular, possibly within the pore. Single-channel studies indicated that exposure to quinine induced slow transitions between open and fully closed states that decreased open probability of the channel. Quinine thus offers a potentially useful method to block certain types of gap junction channels, including those between neurons that are formed by Cx36. Moreover, quinine derivatives that are excluded from other types of membrane channels may provide molecules with connexin-specific as well as connexin-selective blocking activity.

Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes.

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10(-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.