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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary cultures of
adult mouse hepatocytes are shown here to reexpress differentiated
hepatocyte features following treatment with 2% DMSO and
107 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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), 10To 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 atDye Coupling Assays.
Lucifer yellow [5% (wt/vol); Sigma] was injected into individual cells through sharp microelectrodes (resistances ~20 MElectrical 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 asData 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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).
|
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).
|
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.
|
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.
|
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).
|
|
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.
|
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.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anzini, P,
Neuberg DH,
Schachner M,
Nelles E,
Willecke K,
Zielask J,
Toyka KU,
Stuter U,
and
Martini R.
Structural abnomalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin 32.
J Neurosci
17:
4545-4551,
1997
2.
Bennett, MVL,
Barrio LC,
Bargiello TA,
Spray DC,
Hertzberg E,
and
Sáez JC.
Gap junctions: new tools, new answers, new questions.
Neuron
6:
305-320,
1991[ISI][Medline].
3.
Bergoffen, J,
Scherer SS,
Wang S,
Oronzi Scott M,
Bone LJ,
Paul DL,
Chen K,
Lensch M,
Chance PF,
and
Fishbeck KH.
Connexin mutation in X-linked Charcot-Marie-Tooth disease.
Science
262:
2039-2042,
1993[ISI][Medline].
4.
Berthoud, VM,
Iwanij V,
Garcia AM,
and
Sáez JC.
Connexins and glucagon receptors during development of rat hepatic acinus.
Am J Physiol Gastrointest Liver Physiol
263:
G650-G658,
1992
5.
Chanson, M,
Fanjul M,
Bosco D,
Nelles E,
Suter S,
Willecke K,
and
Meda P.
Enhanced secretion of amylase from exocrine pancreas of connexin32-deficient mice.
J Cell Biol
141:
1267-1275,
1998
6.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
7.
Diez, JA,
Ahmad S,
and
Evans WH.
Assembly of heteromeric connexins in guinea-pig liver en route to the Golgi apparatus, plasma membrane and gap junctions.
Eur J Biochem
262:
142-148,
1999
8.
Kojima, T,
Mitaka T,
Paul DL,
Mori M,
and
Mochizuki Y.
Reappearance and long-term maintenance of connexin 32 in proliferated adult rat hepatocytes: use of serum-free L-15 medium supplemented with EGF and DMSO.
J Cell Sci
108:
1347-1357,
1995
9.
Kojima, T,
Mitaka T,
Shibata Y,
and
Mochizuki Y.
Induction and regulation of connexin26 by glucagon in primary cultures of adult rat hepatocytes.
J Cell Sci
108:
2271-2780,
1995.
10.
Kojima, T,
Sawada N,
Oyamada M,
Chiba H,
Isomura H,
and
Mori M.
Rapid appearance of connexin 26-positive gap junctions in centrilobular hepatocytes without induction of mRNA and protein synthesis in isolated perfused liver of female rat.
J Cell Sci
107:
3579-3590,
1994
11.
Kojima, T,
Yamamoto M,
Mochizuki C,
Mitaka T,
Sawada N,
and
Mochizuki Y.
Different changes in expression and function of connexin 26 and connexin 32 duruing DNA synthesis and redifferentiation in primary rat hepatocytes using a DMSO culture system.
Hepatology
26:
585-597,
1997[ISI][Medline].
12.
Kren, BT,
Kumar NM,
Wang S,
Gilula NB,
and
Steer CJ.
Differential regulation of multiple gap junction transcripts and proteins during rat liver regeneration.
J Cell Biol
123:
707-718,
1993[Abstract].
13.
Lee, SW,
Tomasetto C,
Paul D,
Keyomarsi K,
and
Sager R.
Transcriptional downregulation of gap junction proteins blocks junctional communication in human mammary tumor cell line.
J Cell Biol
118:
1213-1221,
1992[Abstract].
14.
Loewenstein, WR.
Junctional intercellular communication and the control of growth.
Biochim Biophys Acta
560:
1-65,
1979[ISI][Medline].
15.
Nelles, E,
Butzler C,
Jung D,
Temme A,
Gabriel HD,
Dahl U,
Traub O,
Stumpel F,
Jungermann K,
Zielasek J,
Toyka KV,
Dermietzel R,
and
Willecke K.
Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.
Proc Natl Acad Sci USA
93:
9565-9570,
1996
16.
Neveu, MJ,
Hully JB,
Babcock KL,
Vaughan J,
Hertzberg EL,
Nicholson BJ,
Paul D,
and
Pitot HC.
Proliferation-associated differences in the spatial and temporal expression of gap junction genes in rat liver.
Hepatology
22:
202-212,
1995[ISI][Medline].
17.
Nicholson, BJ,
Dermietzel R,
Teplow D,
Traub O,
Willecke K,
and
Revel JP.
Two homologous protein components of hepatic gap junctions.
Nature
329:
732-734,
1987[ISI][Medline].
18.
Sáez, JC,
Connor JA,
Spray DC,
and
Bennet MVL
Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-triphosphate and to calcium ions.
Proc Natl Acad Sci USA
86:
2708-2712,
1989[Abstract].
19.
Sáez, JC,
Spray DC,
Narin AC,
Hertzberg EL,
Greengard P,
and
Bennett MVL
c-AMP increases junctional conductance and stimulates phosphorylation of the 27 KDa principal gap junction polypeptide.
Proc Natl Acad Sci USA
83:
2473-2476,
1986[Abstract].
20.
Sawada, N,
Kojima T,
Obata H,
Saitoh M,
Isomura H,
Kokai Y,
Satoh M,
and
Mori M.
p21waf1/cip1/sdi1 is expressed at G1 phase in primary culture of hepatocytes from old rats, presumably preventing the cells from entering the S phase of the cell cycle.
Biochem Biophys Res Commun
228:
819-824,
1996[ISI][Medline].
21.
Seglen, PO.
Preparation of isolated rat liver cells.
Methods Cell Biol
13:
29-83,
1976[Medline].
22.
Spray, DC,
Harris AL,
and
Bennett MVL
Voltage dependence of junctional conductance in early amphibian embryos.
Science
204:
432-434,
1979[ISI][Medline].
23.
Spray, DC,
Sáez JC,
Hertzberg EL,
and
Dermietzel R.
Gap junctions in liver: composition, function, and regulation.
In: The Liver: Biology and Pathobiology (3rd ed.), edited by Arias IM,
Boyer JL,
Fausto N,
Jakoby WB,
Schachter D,
and Shafriz DA.. New York: Raven, 1994, p. 951-967.
24.
Temme, A,
Buchmann A,
Gabriel HD,
Nelles E,
Schwarz M,
and
Willecke K.
High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.
Curr Biol
7:
713-716,
1997[ISI][Medline].
25.
Traub, O,
Druge PM,
and
Willecke K.
Degradation and resynthesis of gap junction protein in plasma membrane of regenerating liver after partial hepatectomy or cholestasis.
Proc Natl Acad Sci USA
80:
755-759,
1983[Abstract].
26.
Traub, O,
Look J,
Dermietzel R,
Brummer F,
Hulser D,
and
Willecke K.
Comparative characterization of the 21-kDa and 26-kDa gap junction proteins in murine liver and cultured hepatocytes.
J Cell Biol
108:
1039-1051,
1989[Abstract].
27.
White, RL,
Spray DC,
Campos de Carvalho AC,
Wittenberg BA,
and
Bennett MVL
Some physiological and pharmacological properties of gap junctions between cardiac myocytes dissociated from adult rat.
Am J Physiol Cell Physiol
249:
C447-C455,
1985[Abstract].
28.
Xie, W,
and
Rothblum LI.
Rapid, small-scale RNA isolation from tissue culture cells.
Biotechniques
11:
325-327,
1991.
29.
Yamamoto, M,
Toyota T,
and
Kataoka K.
Electron microscope observations on the formation of primitive villi in rat small intestine with special reference to intercellular junctions.
Arch Histol Cytol
55:
551-560,
1992[ISI][Medline].
30.
Yamasaki, H,
and
Naus CCG
Role of connexin genes in growth control.
Carcinogenesis
17:
1199-1213,
1996[ISI][Medline].
31.
Zhang, JT,
Chen M,
Foote CI,
and
Nicholson BJ.
Membrane integration of in vitro-translated gap junctional proteins: co- and post-translational mechanisms.
Mol Biol Cell
7:
471-482,
1996[Abstract].
32.
Zhang, JT,
and
Nicholson BJ.
Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA.
J Cell Biol
109:
3391-3401,
1989[Abstract].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |