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

Takashi Kojima1, Alfredo Fort1, Mingyuan Tao1, Masao Yamamoto2, and David C. Spray1

1 Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461; and 2 Department of Anatomy, Hiroshima University School of Medicine, Hiroshima 737-0023, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

connexin32; connexin26; knockout mice


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GAP JUNCTION CHANNELS, COMPOSED of proteins termed connexins (Cx), mediate reciprocal intercellular exchange of ions and molecules with molecular masses <1,000 Da between adjacent cells, including second messengers such as cyclic AMP, inositol 1,4,5-triphosphate and Ca2+ (18, 19). Hepatocytes, the parenchymal cells that comprise the bulk of the liver, are connected by large gap junctions formed of two gap junction proteins, Cx32 and Cx26; the approximate relative ratio of these connexins (Cx32/Cx26) in isolated hepatocyte gap junctions is 2:1 in mouse, 10:1 in rat, and 1:3 in guinea pig (17, 23). Gap junctional intercellular communication is thought to play a crucial role in development, cell growth, and cell differentiation (2, 4, 14, 30), and Cx26 has even been proposed to be a tumor suppressor gene (13).

It has recently been reported that a human genetic disease, the X-linked form of Charcot-Marie-Tooth disease (CMTX), involves mutations of Cx32 (3). In an attempt to mimic CMTX in a rodent model, Cx32 null [Cx32 knockout (KO)] mice were generated through homologous recombination (15). Although these mice did demonstrate progressive demyelination reminiscent of the human disease (1), they also showed two deficiencies that have not been observed in CMTX patients. First, liver function was compromised, as evidenced by reduced glucose release from intact liver in response to sympathetic nerve stimulation (15). Second, proliferation rate of Cx32 KO hepatocytes in vivo was high, and spontaneous and chemically induced liver tumors were more prevalent in Cx32 KO than in wild-type livers (24). The Cx32 KO mouse thus potentially provides an extraordinarily promising model by which to understand various functions of liver gap junctions.

We have previously reported that DMSO/glucagon treatment could induce Cx32 and Cx26 expression in primary rat hepatocytes and inhibit cell growth (8, 9). In the present study, we have shown that similar DMSO/glucagon treatment leads to differentiation of primary cultures of mouse hepatocytes, and we have used this model to examine the changes in expression and function of gap junctions in hepatocytes from wild-type and Cx32 KO mice. Results of these studies provide further support for a functional role of gap junctions in the process of hepatocyte growth and suggest that gap junctions formed of these connexins may perform different functions and be both independently and coordinately regulated.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Animals

Breeding pairs of mice deficient in Cx32 (generated by targeted homologus recombination) (15) were generously provided by K. Willecke (Bonn, Germany); all procedures followed protocols approved by the Albert Einstein College of Medicine Animal Institute. Cx32 is located on the X chromosome; to avoid the complication of chromosome inactivation, we have used male Cx32 KO mice exclusively for these studies; Cx32 KO mice at 12-20 wk age were compared with male C57BL/6 mice at 12-15 wk age as wild-type controls. To perform genotyping, we examined tail DNA from wild-type and Cx32 KO mice using simultaneously, in the same PCR, three oligonucleotide primers specific for the neo expression cassette (5'-ATCATGCGAAACGATCCTCATCC-3'), sequences 5' upstream of the neo cassette insertion into exon 2 of the Cx32 gene (5'-CCATAAGTCAGGTGTAAAGGAGC-3'), and sequences 3' downstream of the insertion (5'-GAGCATAAAGACAGTGAAGACGG-3'). These primers yield amplicons of 881 bp for wild-type animals and 414 bp for the neo-Cx32 gene of Cx32 KO animals.

Isolation and Culture of Mouse Hepatocytes

Mouse hepatocytes were isolated by a modification of the two-step liver perfusion method of Seglen (21). The liver was perfused in situ through the portal vein with 50 ml Ca2+, Mg2+-free Hanks' balanced salt solution (HBSS) supplemented with 0.5 mM EGTA (Sigma Chemical, St. Louis, MO), 0.5 mg/l insulin from bovine pancreas (Sigma), and antibiotics. The liver was perfused with 100 ml of HBSS containing 20 mg collagenase type IV (Sigma) for 20 min after the initial brief perfusion. The isolated cells were purified by Percoll isodensity centrifugation. Viability of the cells, as judged by the Trypan blue exclusion test was more than 90% in these experiments. The cells were suspended in DMEM medium (GIBCO, Gaithersburg, MD) with 0.2% BSA (Sigma), 20 mM HEPES (Sigma), 0.5 mg/l insulin (Sigma), 10-7 M dexamethasone (Sigma), 1 g/l galactose (Sigma), 30 mg/l proline (Sigma), and antibiotics. The isolated hepatocytes were plated at a density of 5 × 105 cells/ml on 35-mm culture dishes (Corning Glass Works, Corning, NY), which were coated with rat tail collagen (500 µg dried tendon/ml in 0.1% acetic acid), and placed in a 5% CO2-95% air incubator at 37°C. After 96 h of culture, 2% DMSO (Aldrich Chemical, Milwaukee, WI), 10-7 M glucagon from porcine pancreas (Sigma), 10 ng/ml epidermal growth factor (Becton Dickinson Labware, Bedford, MA), and 10 mM nicotinamide (Sigma) were added to the modified DMEM medium. Treated and untreated cultures were then compared at 10 days in culture (after 6 days of treatment).

To examine intracellular trafficking pathways used by hepatocyte gap-junction proteins, some cultures were treated at day 10 with 10 µg/ml brefeldin A (Sigma), 10 µg/ml nocodazole (Sigma), 1 µM colchicine (Sigma), or 20 µg/ml cytochalasin B (Sigma), and connexin immunostaining was evaluated 24 h later.

5-Bromo-2'-Deoxyuridine Labeling Index

Immunocytochemical staining for 5-bromo-2'-deoxyuridine (BrdU) was carried out to determine the growth rate of hepatocytes in culture. BrdU (40 µM) was added to each 35-mm dish 24 h before the cells were fixed in cold absolute ethanol. Mouse anti-BrdU (DAKO; Santa Barbara, CA) was used as the primary antibody, and FITC-conjugated anti-rabbit IgG was used as a secondary antibody. Labeled cells with nuclear BrdU staining were counted under a Nikon epifluorescence microscope (magnification approximately ×200). More than 200 cells were counted per dish, and three dishes were examined per experiment.

RNA Isolation and RT-PCR Analysis

RT-PCR was performed on total RNA extracted from cultured mouse hepatocytes and mouse livers. Total RNA was extracted from the cells using the single-step thiocyanate-phenol-chloroform extraction method (6) as modified by Xie and Rothblum (28). One microgram total RNA was reverse transcribed into cDNA using a mixture of oligo(dT) and MuLV RT following recommended conditions (GeneAmp PCR kit, Perkin Elmer, Branchburg, NJ). Each cDNA synthesis was performed in a total volume of 20 µl for 30 min at 42°C and terminated by incubation for 5 min at 99°C. PCR containing 100 pM of primer pairs and 0.5 µl of 20 µl total RT reaction was performed in 20 µl of 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.4 mM dNTPs, and 0.5 U of Taq DNA polymerase (GIBCO), applying 30 cycles with cycle times of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C using a Perkin Elmer/Cetus Thermocycler model 2400. Final elongation time was 7 min at 72°C. Ten microliters of 20 µl total PCR reaction were analyzed in a 1% agarose gel after staining with ethidium bromide. Primers used to detect Cx32, Cx26, Cx43, albumin, and G3PDH by RT-PCR had the following sequences: Cx32 (sense 5'-TCCATCAAACCTTCCCTC-3' and antisense 5'-TTCTCTCTCCATAACTCCCTC-3', amplicon length: 391 bp), Cx26 (sense 5'-AGATGGAGGGAGAGGATGAG-3' and antisense 5'-TCAGAGGAAGAGAAACAATGTG-3', amplicon length: 312 bp), Cx43 (sense 5'-TACCACGCCACCACTGGC-3' and antisense 5'-AATCTCCAGGTCATCATCAGG-3', amplicon length: 407 bp), and albumin (sense 5'-AAGGAGTGCTGCCATGGTGTGA-3' and antisense 5'-GAGGCTGCAAGAAACCTAGG-3', amplicon length: 182 bp).

To provide a qualitative control for reaction efficiency, PCR reactions were performed with primers coding for the housekeeping gene G3PDH (sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'- TCCACCACCCTGTTGCTGTA-3', amplicon length: 452 bp).

Western Blot Analysis

Cultures in 35-mm dishes were washed with PBS twice, and 300 µl of buffer [1 mM NaHCO3 and 2 mM phenylmethylsulfonyl fluoride (Sigma)] were added. The cells were scraped from the dishes and collected in Eppendorf tubes and then sonicated for 10 s. Protein concentrations in the suspensions were measured using a protein assay kit (Pierce Chemical, Rockford, IL). Twenty micrograms of protein of each sample per lane were applied and separated by electrophoresis on 12% SDS-polyacrylamide gels (Bio-Rad, Richmond, CA). After the membranes were electrophoretically transferred to nitrocellulose membranes (Bio-Rad), they were saturated overnight at 4°C with a blocking buffer (25 mM Tris, pH 8.0, 125 mM NaCl, 0.1% Tween 20, and 4% skim milk) and incubated with polyclonal anti-Cx32 (ZYMED, San Francisco, CA), polyclonal anti-Cx26 (ZYMED), polyclonal anti-Cx43 (ZYMED), or polyclonal anti-albumin (Sigma) antibodies at room temperature (Tr) for 1 h. Membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at Tr for 1 h, and detection was carried out using an enhanced chemiluminescence Western blotting system (Amersham, Buckinghamshire, UK).

Immunofluorescence Microscopy

Cells grown on coated-glass coverslips were fixed with cold absolute acetone for 10 min. After coverslips were rinsed with PBS, some were incubated with TRITC-phalloidin (Sigma) at Tr for 1 h. Some coverslips were incubated with polyclonal anti-Cx32 (ZYMED), polyclonal anti-Cx26 (ZYMED), or polyclonal anti-Cx43 (ZYMED) antibodies at Tr for 1 h and then were incubated with FITC-conjugated anti-rabbit IgG (Molecular Probes) at Tr for 1 h. Some coverslips were used for double staining for Cx26 and Cx32. Primary antibodies were monoclonal anti-Cx32 (ZYMED) and polyclonal anti-Cx26 (ZYMED) antibodies, and secondary antibodies were FITC-conjugated anti-rabbit IgG (Molecular Probes) and TRITC-conjugated anti-mouse IgG (Molecular Probes). All samples were examined with an epifluorescence microscope (Nikon, Melville, NY) using Hg lamp illumination and fluorescein and rhodamine excitation and emission filter sets.

Freeze-Fracture Analysis

For freeze-fracture experiments (29), liver tissue and hepatocytes cultured on coverslips were immersed in 40% glycerin solution after fixation in 2.5% glutaraldehyde/0.1 M cacodylate buffer (pH 7.3). The specimens were mounted on a copper stage, cooled in liquid nitrogen, and fractured at -170°C to -180°C in a JFD-7000 freeze-fracture device (JEOL, Tokyo, Japan). Platinum-carbon replicas were made without etching. After replicas were thawed, they were floated on filtered 10% sodium hypochlorite solution for 10 min in a Teflon dish. Replicas were washed in distilled water for 30 min, mounted on copper grids, and examined at 80 KV on a JEM transmission electron microscope.

Dye Coupling Assays.

Lucifer yellow [5% (wt/vol); Sigma] was injected into individual cells through sharp microelectrodes (resistances ~20 MOmega if filled with 3 M KCl) until the injected cell glowed brightly, using brief overcompensation of the negative capacitance control on a WPI electrometer. Photographs documenting the extent of dye coupling were taken at 1 or 2 min after the end of the injection using TMAX400 film on a Nikon Diaphot microscope equipped with a xenon arc lamp and FITC filter set.

Electrical Coupling Assay

The dual whole cell voltage-clamp technique (22) with patch pipettes (27) was used to measure macroscopic junctional conductance between cell pairs. Junctional currents (Ij) were measured as the currents recorded in one cell in response to 300-ms 2-mV pulses (Vj) applied to the other cell; junctional conductance (gj) was calculated as -Ij/Vj.

Data Analysis

Scanning densitometry was performed using a Macintosh computer and a Scan Mark II scanner. Signals were quantified by the National Institutes of Health (NIH) Image 1.52 Densimetric analysis program (Wayne Rasband, NIH, Bethesda, MD). Expression of the transcripts is illustrated in histograms as the percentage of values obtained at 0 h (freshly isolated hepatocytes) in the same experiment. Each set of results shown is representative of three separate experiments; error bars represent SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Genotyping Cx32 KO Mice

We examined tail DNA from wild-type and Cx32 KO mice using stimultaneously in the same PCR three oligonucleotide primers specific for the neo expression cassette, sequences 5' upstream of the neo cassette insertion into exon 2 of the Cx32 gene, and sequences 3' downstream of the insertion. As shown in Fig. 1A, amplicons corresponding to wild-type and Cx32 KO alleles were readily identified in wild-type and Cx32 KO mice, respectively. RT-PCR on liver tissue from these animals revealed quantitatively similar levels of Cx26 and Cx43, although the Cx32 transcript was absent from the Cx32 KO liver (Fig. 1B).


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Fig. 1.   Genotyping of wild-type and Connexin (Cx)32 knockout (KO) mice (A) and RT-PCR analysis for Cx32, Cx26, and Cx43 in wild-type (WT) and Cx32 KO livers in vivo (B). A: amplicons corresponding to WT (881 bp) and Cx32 KO (414 bp) alleles are indicated. B: RT-PCR on liver tissues from these animals revealed Cx26 and Cx43 mRNAs in both WT and Cx32 KO livers; although Cx32 mRNA was present in WT liver samples, it was absent from the Cx32 KO liver.

Cellular Morphology and Cell Growth

The overall cellular morphologies of Cx32 KO hepatocytes were found to differ from those of wild-type hepatocytes. Staining of wild-type and Cx32 KO hepatocyte cultures with TRITC-labeled phalloidin revealed that in Cx32 KO hepatocytes at 24 h (Fig. 2B), the intensity and percentage of cells positive for F actin were markedly higher than in wild-type hepatocytes (Fig. 2A). Whereas F actin distribution was diffuse in wild-type hepatocytes (Fig. 2A), in Cx32 KO hepatocytes, phalloidin staining revealed abundant prominent stresslike fibers (Fig. 2B, arrows).


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Fig. 2.   F actin staining of primary mouse hepatocytes at 24 h in culture using FITC phalloidin. A: WT; B: Cx32 KO hepatocytes. In Cx32 KO hepatocytes (B), the intensity and percentage of cells positive for F actin, which appeared to form stress fibers and focal adhesions (arrows in B), were markedly higher than in WT hepatocytes (A). Bars, 20 µm.

To determine whether there were differences in the rates of DNA replication in wild-type and Cx32 KO mouse hepatocytes in vitro, we examined BrdU labeling indexes in cells initially plated at the same density (5 × 105 cells/ml) and cultured in medium lacking serum and growth factors. As illustrated in Fig. 3, A and B, for cultures examined at 96 h, the number of BrdU-labeled nuclei of wild-type and Cx32 KO hepatocytes differed strikingly. The BrdU labeling index as a function of time in culture obtained in three separate experiments is shown in Fig. 3C. This histogram shows that at 48, 72, and 96 h after plating, growth rates of untreated Cx32 KO hepatocytes were markedly higher than those of wild-type hepatocytes. After treatment at 96 h with DMSO and glucagon, growth of both wild-type and Cx32 KO hepatocytes examined at day 10 after plating was markedly decreased (Fig. 3C).


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Fig. 3.   5-bromo-2'-deoxyuridine (BrdU) staining of primary mouse hepatocytes at 96 h (A and B) and BrdU-labeling index with time of culture (C). A: WT; B: Cx32 KO hepatocytes. Many BrdU-positive nuclei were observed in Cx32 KO hepatocytes (B). The BrdU-labeling index was markedly higher in untreated Cx32 KO hepatocytes than in WTs at 72 and 96 h after plating. However, BrdU labeling was very low in both WT and Cx32 KO hepatocytes at 10 days after treatment with DMSO/glucagon (C). Bar, 20 µm.

Expression of Gap Junctions

RT-PCR analysis. We have examined the changes in Cx32, Cx26, and Cx43 mRNA expression in cultured mouse hepatocytes in culture using semiquantitative RT-PCR, in which hybridization signals obtained using Cx-specific probes were standardized with those obtained with probes for the constitutively expressed housekeeping gene G3PDH. Representative results of RT-PCR experiments are shown in Fig. 4A, and densitometric analyses are shown in Fig. 4, B-D. In wild-type hepatocytes (Fig. 4, A and B), Cx32 mRNA decreased to low levels between 24 and 48 h after plating and was reinduced by DMSO/glucagon treatment (treated beginning at 96 h, examined at day 10); by contrast, Cx26 mRNA expression was relatively stable throughout this period (Fig. 4, A and C). Cx43 mRNA gradually decreased over time and was only minimally observed at day 10 in the presence of DMSO/glucagon (Fig. 4D). In Cx32 KO hepatocytes, Cx26 mRNA was stably expressed and Cx43 mRNA gradually decreased, similar to the pattern in wild-type hepatocytes (Fig. 4, A, C, and D). To evaluate the degree of differentiation of the hepatocytes, we also examined albumin mRNA expression as a function of time in culture. Albumin mRNA was found to be decreased at 96 h in wild-type hepatocytes and at both 48 and 96 h in Cx32 KO hepatocytes (Fig. 4, A and E). Notably, however, in both wild-type and Cx32 KO hepatocytes, albumin expression evaluated at day 10 following DMSO/glucagon treatment from 96 h was found to be comparable with that of freshly isolated hepatocytes, demonstrating that this treatment led to the reestablishment of one of the characteristic indicators of hepatocyte differentiation.


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Fig. 4.   RT-PCR analyses for Cx32, Cx26, Cx43, and albumin in primary hepatocytes cultured from WT and Cx32 KO mice with time of culture. A and B: Cx32 mRNA was absent in Cx32 KO cells; in WT hepatocytes, it declined rapidly to low levels at 48 and 96 h and was then upregulated at day 10 with DMSO/glucagon. A and C: Cx26 mRNA was stably expressed throughout this entire period in both WT and Cx32 KO hepatocytes (A and D). Cx43 mRNA levels gradually decreased with time after plating. A and E: albumin mRNA levels underwent a transient decline in Cx32 KO hepatocytes at 48 and 96 h; albumin mRNA expression recovered to levels seen in freshly dissociated hepatocytes cultured in DMSO/glucagon. In the graphs (B-D), expression levels of the mRNAs are shown normalized to 0-h values. Brackets above bars in graphs represent SE from 3 separate experiments in which each of 3 dishes was measured.

Western blot analysis. We examined changes in expression of Cx32, Cx26, Cx43, and albumin proteins in hepatocytes cultured from wild-type and Cx32 KO mice as a function of time in culture (Fig. 5). In wild-type hepatocytes, levels of both Cx32 and Cx26 proteins decreased strongly during the first 24 h in culture and were virtually absent at 96 h; expression of both Cx32 and Cx26 was reinduced by DMSO/glucagon treatment (treated from 96 h, examined at day 10). In Cx32 KO hepatocytes, Cx26 protein transiently decreased, then was detectable at 96 h, and its expression was quite high following DMSO/glucagon treatment. In both wild-type and Cx32 KO hepatocytes, Cx43 expression was very low throughout this period (data not shown). In both wild-type and Cx32 KO hepatocytes, albumin was stably expressed throughout the duration of the experiment.


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Fig. 5.   Western blot analyses for Cx32, Cx26, and albumin in primary hepatocytes from WT and Cx32 KO mice with time in culture. In the graphs, expression of the proteins is shown as the percentage of 0-h values. Levels of both Cx32 and Cx26 decreased from 24 until 96 h in WT cultures and were reinduced by DMSO/glucagon treatment. In Cx32 KO hepatocytes, Cx26 protein decreased even more profoundly but was detected at 96 h and was increased after DMSO/glucagon treatment. Brackets above bars in graphs represent SE from 3 separate experiments.

Immunocytochemistry. To examine the changes in subcellular distribution of Cx32, Cx26, and Cx43 proteins in cultured mouse hepatocytes, we performed immunocyochemistry as a function of time in culture. In untreated wild-type hepatocytes at 24 h, both Cx32 and Cx26 were observed at interfaces between cells, presumably localized to the same junctional domains (Fig. 6, A and B); in untreated Cx32 KO hepatocytes at 24 h, Cx32 was absent (Fig. 6C) and Cx26 was confined to few diffuse and intracellularly located spots (Fig. 6D). Cx26 disappeared from Cx32 KO hepatocytes at 48 h and then partially reappeared at 96 h (Fig. 6, E and F), whereas in wild-type hepatocytes, both Cx32 and Cx26 were maintained at low levels until 96 h (data not shown). In DMSO/glucagon-treated wild-type hepatocytes at day 10, many Cx32- and Cx26-positive spots were seen (Fig. 7, A and B), and these positive spots were colocalized to the same junctional areas (Fig. 7, C and D, arrows). In DMSO/glucagon-treated Cx32 KO hepatocytes at day 10, Cx32 staining was absent (Fig. 7E), but abundant Cx26-positive spots were seen on the membranes and in the cytoplasm (Fig. 7F). Cx43 expression was not observed in any of the samples (data not shown).


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Fig. 6.   Immunocytochemistry for Cx32 (A and C) and Cx26 (B, and D-F) in primary mouse hepatocytes at 24 (A-D), at 48 (E), and at 96 h (F). A and B: WT; C-F: Cx32 KO hepatocytes. Cx32 and Cx26 were observed at interfaces between untreated WT hepatocytes at 24 h (A and B, arrows). Cx32 was absent in untreated Cx32 KO hepatocytes at 24 h (C), and Cx26 was confined to few diffuse and intracellularly located spots (D, arrows). Cx26 in Cx32 KO hepatocytes disappeared at 48 h (E) and then partially reappeared at 96 h (F, arrows). Bar in E, 20 µm.



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Fig. 7.   Immunocytochemistry for Cx32 (A, C, E, and G) and Cx26 (B, D, F, H, and I) in DMSO/glucagon-treated hepatocytes at day 10. A-D, G, and H: WT; E, F, and I: Cx32 KO hepatocytes. A-F: without brefeldin A; G-I: with brefeldin A for 24 h. Cx32- and Cx26-positive spots were abundant in DMSO/glucagon-treated WT hepatocytes at day 10 (A and B), where they were colocalized to the same junctional areas (C and D). Cx32 staining was absent (E) in DMSO/glucagon-treated Cx32 KO hepatocytes at day 10, but Cx26-positive spots were abundant on the membranes and in the cytoplasm (F). Cx32-positive spots on the membranes of WT hepatocytes were greatly reduced after brefeldin A treatment (G) completely disappeared. However, no changes were observed in Cx26-positive spots in WT or Cx32 KO hepatocytes after brefeldin A treatment (H and I). Bar in E, 20 µm.

To examine the mechanisms of assembly of gap junction proteins in the cultured hepatocytes, we treated cultures differentiated by DMSO/glucagon treatment at day 10 with an endoplasmic reticulum (ER)-Golgi blocker (brefeldin A), microtubule inhibitors (nocodazole and colchicine), and an actin disrupter (cytochalasin B) for 24 h. For each treatment, Cx-positive spots on appositional membranes were counted from four or five photographs from each of three separate experiments. Almost all Cx32-positive spots on the membranes of wild-type hepatocytes disappeared after brefeldin A treatment (Fig. 7G), whereas Cx26-positive spots were only slightly and not significantly decreased (compare Fig. 7H with 7B: 21 ± 5 vs. 23 ± 4 spots/cell). Similarly, no changes were observed in Cx26-positive spots in Cx32 KO hepatocytes following brefeldin A treatment (compare Fig. 7F with 7I: 18 ± 7 vs. 19 ± 5 spots/cell). After nocodazole, colchicine, and cytochalasin B treatment, no changes in gap junction immunostaining were observed in either wild-type or Cx32 KO hepatocytes (data not shown).

Freeze-fracture. Nelles et al. (15) previously reported that both the number and sizes of gap junction plaques in livers from Cx32 KO mice were lower than in wild-type livers in vivo. To examine gap junction plaques in DMSO/glucagon-treated hepatocytes at day 10, in which immunostaining revealed high levels of gap junction proteins, freeze fracture was carried out on twenty separate dishes of these cultures. In DMSO/glucagon-treated wild-type hepatocytes as illustrated in Fig. 8A, many typical large gap junction plaques were observed in almost all samples (average size 0.2 µm2). In DMSO/glucagon-treated Cx32 KO hepatocytes, the gap junction plaques were small and infrequent (Fig. 8B is representative of the two samples in which small gap junctions were observed; average size of these gap junctions was 0.05 µm2). Thus, as was reported for liver in vivo (15), the number and sizes of gap junction plaques in Cx32 KO hepatocytes were markedly lower than in wild-type hepatocytes.


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Fig. 8.   Freeze-fracture replicas of DMSO/glucagon-treated hepatocytes at day 10. A: WT; B: Cx32 KO hepatocytes. In WT hepatocytes, typical large gap junction plaques were observed (*). In Cx32 KO hepatocytes, small gap junction plaques were commonly observed (arrows), but larger arrays were absent. Bar in B, 100 nm.

Functional Expression of Gap Junctions

Dye transfer. As a test for strength of gap junctional intercellular communication as a function of gap junction expression, we performed dye-transfer measurements using 5% Lucifer yellow injection into primary cultured mouse hepatocytes at 24 h, at 96 h, and at day 10 (following DMSO/glucagon treatment). At 24 and 96 h, the spread of Lucifer yellow was not observed in either wild-type or Cx32 KO hepatocytes (data not shown). Spread of Lucifer yellow in wild-type and Cx32 KO hepatocytes at day 10 is shown in Fig. 9, A and B, respectively, and analyses of the spread are shown in the histogram in Fig. 9. As indicated in the histogram and the representative photomicrographs, dye spread was extensive to an average of six other cells in DMSO/glucagons-treated wild-type hepatocytes at day 10, whereas in DMSO/glucagon-treated Cx32 KO hepatocytes at day 10, Lucifer yellow coupling was almost completely absent.


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Fig. 9.   Dye transfer of intracellularly injected Lucifer yellow in DMSO/glucagon-treated hepatocytes at day 10. Representative photomicrographs of dye spread from injected cell (*) to neighboring cells (arrows). A and B: WT; C and D: Cx32 KO hepatocytes; A and C: phase contrast; B and D: Lucifer yellow. Histogram to the right indicates quantitative evaluation of Lucifer yellow distribution (n = 20). As indicated in the photographs and histogram, dye spread was more extensive in DMSO/glucagon-treated WT than in Cx32 KO hepatocytes at day 10. Bar, 20 µm.

Junctional conductance. To quantitatively examine junctional conductance (gj) of wild-type and Cx32 KO hepatocyte pairs as a function of time in culture, cell pairs at each time point were voltage clamped. gj In untreated Cx32 KO hepatocyte pairs markedly and rapidly decreased from 12 h after plating but showed a recovery toward control levels at 96 h (Fig. 10). In untreated wild-type hepatocyte pairs, gj was moderately strong and relatively stably until 96 h. After DMSO/glucagon treatment, however, gj in both wild-type and Cx32 KO hepatocytes was at nearly the same levels observed in freshly isolated cell pairs. In the absence of DMSO/glucagon, gj was very low both at 7 and at 10 days in culture.


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Fig. 10.   Electrophysiological analysis of junctional conductance (gj) recorded between pairs of WT and between pairs of Cx32 KO hepatocytes as a function of time in culture. gj Was largely maintained for 48-96 h in WT hepatocytes, whereas gj in Cx32 KO hepatocyte pairs markedly and rapidly decreased from 12 h after plating and then recovered toward control levels at 96 h. gj In both WT and Cx32 KO hepatocytes was restored to nearly the same levels observed in freshly isolated cell pairs by treatment with DMSO/glucagon. n = 10-20 Cell pairs/point; vertical bars represent SE.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The availability of transgenic animals with the deletion of selected genes allows "negative" physiological studies, in which gene function is inferred by the absence of function in knockouts. Previous studies of Cx32-deficient mice, which lack the major hepatocyte gap junction protein, have found that development and function of liver are grossly normal, although release of glucose from hepatic glycogen stores on electrical stimulation of sympathetic nerves is reduced (15) and Cx32-deficient mice are more susceptible to hepatocarcinogenesis (24). In the present study, we have modified a tissue culture model previously shown to lead to differentiation in rat hepatocytes (8, 9) to compare growth and differentiation of hepatocytes from normal and Cx32-deficient mice. This in vitro approach both supports and extends the previous studies performed on the whole animal.

Expression and Function of Gap Junctions in Primary Cultured Mouse Hepatocytes

We previously reported detailed changes in expression and function of gap junctions in primary cultured normal rat hepatocytes with and without DMSO/glucagon treatment (8, 9, 18). In these studies, expression of Cx32 and Cx26 and their mRNAs, as well as dye coupling and junctional conductance, were markedly depressed within 12 h after plating (8, 9, 18); when treated with DMSO/glucagon, Cx32 and Cx26 and their mRNAs were reinduced (9). In embryonic mouse hepatocytes cultured in defined medium, somewhat similar changes in connexin proteins were also reported: dramatic declines in Cx32 and Cx26 during the first 24 h and then recovery during the next 3 days (26). In the present study, without DMSO/glucagon, expression of protein and mRNA for Cx26, but not Cx32, and junctional conductance in adult wild-type mouse hepatocytes were well maintained for a long time after plating. In Cx32 KO hepatocytes, only Cx26 mRNA was maintained at a high level, whereas Cx26 protein and junctional conductance were markedly reduced. Treatment with DMSO/glucagon led to a strong reinduction of the expression and the function of gap junctions in both wild-type and Cx32 KO hepatocytes, as was previously reported in rat hepatocytes (9). In adult rat liver, the expression of Cx26 has been shown to be influenced by the concentration of intracellular glucagon within the acinar lobules (9). It is possible that Cx26 expression in Cx32 KO hepatocytes in vivo might also be inhibited by hormonal or other factors, because Cx26 expression in Cx32 KO hepatocytes was induced at levels as high as in wild types by DMSO/glucagon treatment.

Cell Growth and Morphology Of Cx32 KO Hepatocytes in Vitro

In Cx32 KO mice, it was reported that the proliferation rate of hepatocytes in vivo was higher and that both spontaneous and chemically induced liver tumors were much more frequently observed in these animals than in wild-type mice (24). In our studies of hepatocytes cultured without serum or growth factors, the growth rates of Cx32 KO hepatocytes, indicated by the BrdU-labeling index, were markedly higher than those of wild types. These results support the in vivo data on hepatocyte proliferation rate and indicate that the high growth rate of hepatocytes may underlie the induction of liver tumors in Cx32 KO mice. Furthermore, the cellular morphology of Cx32 KO hepatocytes at 24 h after plating was generally more stellate, rather than epithelioid, and was accompanied by an intense staining for F actin, which appeared to form stress fibers and focal adhesions. Because gap junction expression was found to be very low in functional, immunostaining, and Western blot analyses of the Cx32 KO hepatocyte cultures during the first 3 days in culture, these results suggest that a novel linkage may exist between gap junctions and cellular morphology via organization of the actin cytoskeleton.

Assembly and Intercellular Transport of Cx26 in Cx32 KO Hepatocytes

Cx26 has the shortest carboxyl terminus of all the Cxs thus far sequenced (32). On the basis of experiments performed in cell-free systems, it has been proposed that Cx26 can insert into membranes posttranslationally, whereas Cx32 is believed to insert cotranslationally (31). We previously reported a rapid specific appearance of Cx26 in the plasma membrane after perfusion of female rat livers without an increase in total Cx26 protein or mRNA levels (10). In the present study, changes of Cx26 proteins of both wild-type and Cx32 KO hepatocytes were observed without changes in their mRNAs, whereas changes of Cx32 protein of wild-type hepatocytes were paralleled with changes in mRNA abundance. These results suggest that Cx26 expression might be controlled posttranscriptionally in mouse hepatocytes. Furthermore, in DMSO/glucagon-treated Cx32 KO hepatocytes, Cx26 was found to be induced without Cx32 expression, and targeting of Cx26 to the plasma membrane was not inhibited by the ER-Golgi blocker brefeldin A. By contrast, brefeldin A treatment of wild-type hepatocytes led to almost complete disappearance of Cx32 and a slight decrease in Cx26. Furthermore, in freeze-fracture replicas of DMSO/glucagon-treated hepatocytes, the number and size of gap junction plaques in Cx32 KO hepatocytes were found to be markedly decreased compared with wild types, as was previously reported in hepatocytes and pancreatic acinar cells of Cx32 KO mice in vivo (5, 15). These results suggest that gap junction plaques formed by Cx26 homotypic channels may be smaller than those formed by Cx32 and Cx26 heteromeric channels, and it is possible that the formation of Cx26 homotypic channels on the membranes may follow an alternative nonclassical trafficking pathway that bypasses the Golgi system (7).

A Role of Liver Gap Junctions During Hepatocyte Growth and Differentiation

During the rapid cell growth that occurs in liver after partial hepatectomy, Cx32 expression is very low (12, 16, 25). However, it is still unclear whether Cx32 actively controls cell growth rate in hepatocytes. In primary cultures of Cx32 KO hepatocytes, the present study clearly revealed higher cell growth than in wild types. It is thought that the process of isolating the hepatocytes leads to entry of most cells from the G0 to the G1 phase of the cell cycle (20). In Cx32 KO hepatocytes, many cells entered into S phase from G1 without stimulation of growth factors, whereas in most wild-type hepatocytes, G1 might be maintained continuously. This suggests that block of the G1/S transition may be weaker in Cx32 KO hepatocytes than in wild types, and it is therefore possible that Cx32 expression may actively inhibit cell growth. Cx26 has been proposed as a tumor suppressor gene (13), and although increase of Cx26 expression during cell growth has been reported (11, 13), it is unclear whether the changes in expression underlie the change in growth. In untreated Cx32 KO hepatocytes at 96 h, increased Cx26 expression was observed during cell growth and was accompanied by moderately high junctional conductances in functional assays. These results suggest that during hepatocyte growth, cell-cell comunication might exist between connected cells via Cx26 channels.

As a test for differentiated function of hepatocytes, we have compared albumin expression in hepatocytes from wild-type and Cx32-deficient mice. Albumin mRNA was decreased at 96 h in wild-type hepatocytes and at both 48 and 96 h in Cx32 KO hepatocytes and recovered to normal levels in hepatocytes of both genotypes after 10 days of treatment with DMSO/glucagon. These results with this marker of the differentiated hepatocyte phenotype indicate that there is minimal impact of Cx32 expression in the process.


    ACKNOWLEDGEMENTS

We thank Dr. K. Willecke (Univ. of Bonn, Germany) for providing breeding pairs of Cx32 KO mice for these studies. We also thank M. Urban for technical support and F. Andrade for secretarial support.


    FOOTNOTES

This work was supported by National Institutes of Health Grant DK-41918.

Address for reprint requests and other correspondence: D. C. Spray, Dept. of Neuroscience, Albert Einstein College of Medicine, Rm. 712 Kennedy Center, 1410 Pelham Parkway South, Bronx, NY 10461 (E-mail: spray{at}aecom.yu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 December 2000; accepted in final form 22 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(4):G1004-G1013
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society




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