Effects of 18-glycyrrhetinic acid on serine 368 phosphorylation of connexin43 in rat neonatal cardiomyocytes

Cell Biology International 32 (2008) 1371e1379 www.elsevier.com/locate/cellbi

Effects of 18-glycyrrhetinic acid on serine 368 phosphorylation of connexin43 in rat neonatal cardiomyocytes Jyun-Yan Liang a, Seu-Mei Wang a, Tun-Hui Chung a,1, Shih-Hung Yang a,b, Jiahn-Chun Wu a,* a

Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, 1-1 Jen-Ai Road, Taipei 10051, Taiwan b Department of Surgery, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 10002, Taiwan Received 27 September 2007; revised 30 May 2008; accepted 12 August 2008

Abstract 18b-Glycyrrhetinic acid (18b-GA) regulates serine/threonine dephosphorylation of connexin43 (Cx43). Phospho-specific antibodies were used here to determine the effect of 18b-GA on serine 368-phosphorylated Cx43 (pSer368Cx43) in cultured rat neonatal cardiomyocytes by immunofluorescence microscopy and immunoblot analyses. 18b-GA caused a time-dependent increase in pSer368Cx43 levels and induced gap junction disassembly, shown by a change in pSer368Cx43 immunostaining from large aggregates to dispersed punctates at cellecell contact areas. 18b-GA also induced a time-dependent increase in the levels of serine 729-phosphorylated PKC3, the active form of PKC3. The 18b-GA-induced increase in pSer368Cx43 levels and changes in pSer368Cx43 staining pattern were abolished by the PKC inhibitor, chelerythrine. Furthermore, 18b-GA increased the co-immunoprecipitation of Cx43 with PKC3. However, the 18b-GA-induced increase in pSer368Cx43 levels and increased association of Cx43 with PKC3 were inhibited by co-treatment with the protein phosphatase type 1 and type 2A inhibitor, calyculin A. We conclude that 18b-GA induces Ser368 phosphorylation of Cx43 via PKC3. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: 18b-Glycyrrhetinic acid; Gap junction; Connexin43; Protein kinase C; Cardiomyocyte

1. Introduction Gap junctions are aggregates of aqueous channels between contiguous cells that provide a direct route for cytoplasmic diffusion of ions and small molecules (So¨hl and Willecke, 2004). Each gap junction channel is formed by 2 connexons (or hemichannels), one from each of the contiguous cells, which dock with the apposing plasma membrane. Each connexon oligomer is composed of 6 connexin (Cx) subunits. The predominant gap junction protein expressed in ventricular cardiomyocytes is Cx43 (So¨hl and Willecke, 2004). Multiple phosphorylation sites in the C-terminus of Cx43 have been identified, and phosphorylation of Cx43 has been implicated in regulation of its trafficking, the assembly/disassembly of gap * Corresponding author. Fax: þ886 2 23915292. E-mail address: [email protected] (J.-C. Wu). 1 Present address: School of Medicine, Fu-Jen Catholic University, Taipei 24205, Taiwan.

junctions, and channel gating (for review, see Solan and Lampe, 2005). It is well established that serine 368 in the C-terminus of Cx43 is phosphorylated by protein kinase C (PKC) (Sa´ez et al., 1997; Lampe et al., 2000). Phosphorylation of serine 368 results in a change in function by increasing channel permeability and decreasing channel conductance (Lampe et al., 2000; Ek-Vitorin et al., 2006). PKC-mediated serine 368 phosphorylation of recombinant Cx43 reconstituted into liposomes has been demonstrated to lead to hemichannel gating (Bao et al., 2004). In cultured cardiomyocytes, serine 368 phosphorylation is implicated in the 17b-estradiol-mediated attenuation of chemical ischemia-induced dye uncoupling and oleic acid-induced gap junction disassembly (Chung et al., 2004; Huang et al., 2004). 18b-Glycyrrhetinic acid (18b-GA), a hydrolysis product of the triterpene, saponin, isolated from licorice root, has been used as a gap junction uncoupler, which inhibits gap junction intercellular communication between liver epithelial cells and induces electrical uncoupling between endothelial and smooth

1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.08.007

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muscle cells in arterioles (Davidson et al., 1986; Yamamoto et al., 1998). 18b-GA also disassembles gap junctional plaques in liver epithelial and adrenocortical cells, an effect ascribed to phosphatase-mediated dephosphorylation of Cx43 (Guan et al., 1996; Huang et al., 2003). Furthermore, 18b-GA causes dose-dependent inhibition of dye coupling in cardiomyocytes by a mechanism involving tyrosine phosphorylation of Cx43 by Src kinase (Chung et al., 2007). Although 18b-GA has been considered as a PKC inhibitor, O’Brian et al. (1990) have shown that low concentrations of 18b-GA enhance PKC activity. The aims of the present study were therefore to determine whether 18b-GA induces phosphorylation of Cx43 at serine residues, and whether this phosphorylation involves PKC. Our results show that, in cardiomyocytes, 18b-GA induced a selective dephosphorylation of Cx43 concomitant with an increase in the levels of serine 368-phosphorylated Cx43 (pSer368Cx43) and serine 729-phosphorylated PKC3 (pPKC3, the active form of PKC3). 18b-GA also induces a time-dependent change of pSer368Cx43-immunoreactive gap junctional plaques from large aggregates to dispersed punctates. The 18b-GA-induced changes in Ser368Cx43 levels and staining pattern of gap junctions were both attenuated by co-treatment with a PKC inhibitor, chelerythrine, indicating a mechanism involving PKC activation. 2. Materials and methods 2.1. Reagents 18b-Glycyrrhetinic acid, mouse anti-b-actin or antib-tubulin antibodies, rabbit anti-b-tubulin antibody, nitroblue tetrazolium (NBT), and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were purchased from SigmaeAldrich (St. Louis, MO). Calyculin A and chelerythrine chloride were purchased from Calbiochem (San Diego, CA). Affinity purified, mouse monoclonal antibody against non-phosphorylated Ser368 of Cx43 (Cx43-NP, 13-8300) and rabbit polyclonal antibody against total Cx43 (71-0700) were purchased from Zymed (San Francisco, CA). Affinity purified goat polyclonal antibody against serine 729-phosphorylated PKC3 (pPKC3, sc12355), affinity purified rabbit polyclonal antibody against PKC3 (sc-214), horseradish peroxidase-conjugated goat antimouse IgG antibody, and Luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody against pPKC3 (#06-821) and mouse monoclonal antibody against N-cadherin (#05-915) were purchased from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal antibody against total Cx43 (MAB3067), rabbit polyclonal antibody against pSer368Cx43 (AB3841), and FITC-conjugated goat anti-rabbit IgG antibody were purchased from Chemicon (Temecula, CA). Alkaline phosphatase-conjugated goat anti-rabbit IgG and anti-mouse IgG antibodies were purchased from Promega (Madison, WI). Texas red-conjugated horse anti-mouse IgG antibodies were purchased from Vector (Burlingame, CA). Protein G-Sepharose bead slurry was purchased from Pharmacia (Uppsala, Sweden).

2.2. Primary culture of rat neonatal cardiomyocytes and drug treatment Primary neonatal cardiomyocytes were prepared from postnatal day 3 Wistar rat pups of both sexes as described previously with minor modification (Chung et al., 2004). The hearts were isolated, the atria removed, and the ventricles washed with Ca2þ/Mg2þ-free Hank’s balanced salt solution (HBSS; SigmaeAldrich) to remove excess blood, minced on a watch-glass, and incubated for 5 min at 37  C in HBSS containing 0.125% trypsin/1 mM EDTA (GIBCO, Grand Island, NY) and 0.083% collagenase type II (SigmaeAldrich). The supernatants containing cell debris and connective tissues were discarded and the residual ventricle fragments subjected to 4 more digestion steps as above; in each of these, the dissociated cells in the supernatants were collected and mixed with an equal volume of ice-cold plating medium (10% fetal bovine serum, 100 IU/ml of penicillin, 100 mg/ml of streptomycin, and 2 mM glutamine in Dulbecco’s modified Eagle’s medium (DMEM); SigmaeAldrich). The suspended cells were then pooled, collected by centrifugation at 160g for 10 min, resuspended in 10 ml of plating medium, preplated on a 10 cm culture dish, and placed in a 5% CO2 incubator at 37  C for 2 h to remove contaminating fibroblasts. After fibroblast attachment, the suspended cells were collected, diluted with plating medium, and plated at 37  C at 3e4  104 cells/cm2 on rat tail collagen-coated 35 mm culture dishes or glass coverslips. On the day after plating, the plating medium was replaced with growth medium (10% calf serum, 100 IU/ ml of penicillin, and 100 mg/ml of streptomycin in DMEM) and was then changed every 2 days. The percentage of cardiomyocytes was greater than 90% as determined by the proportion of cells showing spontaneous contraction. The cultures were used 4 days after plating. For the time-course study, cardiomyocytes were washed 3 times in growth medium without serum, incubated for 15, 30, or 60 min at 37  C with 5 mM 18b-GA (Chung et al., 2007). For PKC and protein phosphatase inhibition studies, cardiomyocytes were treated for 30 min at 37  C with 5 mM 18bGA alone or combined with a PKC inhibitor (0.5 or 5 mM chelerythrine) or a protein phosphatase inhibitor (10 nM calyculin A). All compounds were stock solutions in dimethyl sulfoxide (DMSO) in the absence or presence of alkaline phosphatase (8 unit/10 mg) and were added to growth medium in the absence of serum at less than 0.1% of the final volume; 0.1% DMSO was added to the controls. The cardiomyocytes were processed for immunofluorescence microscopy, immunoprecipitation, or immunoblot analysis. 2.3. Immunofluorescence microscopy Cardiomyocyte cultures were fixed in cold acetone for 5 min at 20  C as described previously (Chung et al., 2004). After a brief wash with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4; PBS), they were incubated for 90 min at 37  C with a 1:100 dilution of rabbit antisera against pSer368Cx43, then

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Fig. 1. Immunoblots of cardiomyocyte lysates showing a dose-dependent increase in pSer368Cx43 with 18b-GA treatment. Cardiomyocytes treated for 30 min with 0.1% DMSO (0 mM) or 18b-GA at a concentration of 1, 2.5, 5, or 10 mM were harvested and the cell lysates analyzed by 10% polyacrylamide SDS gel electrophoresis and immunoblotting. The blots were probed with antibody against pSer368Cx43 (pSer368), then stripped and re-probed with anti-b-tubulin antibody (b-tub) as a loading control.

for 90 min at 37  C with a 1:50 dilution of mouse antibody against Cx43-NP. Bound primary antibodies were detected by incubation for 90 min at room temperature with a mixture of FITC-conjugated goat anti-rabbit IgG antibodies and Texas red-conjugated goat anti-mouse IgG antibodies (both diluted 1:50). After washing in PBS, the cultures were mounted using an anti-fading medium (mixture of 2% n-propyl gallate and 60% glycerol in 0.1 M sodium phosphate buffer, pH 8.0) and sealed with nail polish. The labeled cardiomyocytes were examined using a Zeiss Axiophot microscope (Carl Zeiss, Oberkocheu, Germany) equipped with epifluorescence, and images were acquired with an X100 oil-immersed objective and digitized using a Nikon D1X digital camera (Nikon, Tokyo, Japan).

Fig. 3. Chelerythrine prevents the 18b-GA-induced increase in pSer368Cx43 levels. Cardiomyocytes treated for 30 min with 0.1% DMSO (control), 5 mM 18b-GA (GA), or 5 mM chelerythrine (Chel) or co-treated with 5 mM 18b-GA and 5 mM chelerythrine (GA þ Chel) were harvested and the cell lysates analyzed by SDSepolyacrylamide gel electrophoresis and immunoblotting. The blots were probed with antibody against pSer368Cx43 (pSer368), then stripped and re-probed with anti-b-actin antibody (b-act). (A) shows a typical result and (B) shows the densitometric analysis of the pSer368Cx43 blots from 3 experiments. *P < 0.01, compared to the control lane, yP < 0.01, compared to the GA lane by the unpaired Student’s t-test.

2.4. Immunoprecipitation All immunoprecipitation procedures were performed at 4  C unless otherwise stated. Cardiomyocytes were washed 3 times with PBS, then lysed for 30 min with 100 ml of RIPA

Fig. 2. Immunoblots of cardiomyocyte lysates showing a time-dependent increase in pSer368Cx43 with 18b-GA treatment. Cardiomyocytes treated with 0.1% DMSO for 60 min (control) or with 5 mM 18b-GA for 15 min (15 min), 30 min (30 min), or 60 min (60 min) were harvested and the cell lysates analyzed by 10% polyacrylamide SDS gel electrophoresis and immunoblotting. The blots were probed with antibody against pSer368Cx43 (pSer368), then stripped and re-probed with anti-b-actin antibody (b-act). (A) shows a typical result and (B) shows the densitometric analysis of the pSer368Cx43 blots from 3 experiments. *P < 0.05, **P < 0.01 compared to the control lane by the unpaired Student’s t-test.

Fig. 4. 18b-GA increases pPKC3 levels in cultured cardiomyocytes. The details are as in Fig. 3, but the blots were probed with antibody against serine 729-phosphorylated PKC3 (pPKC3), then were stripped at low pH and reprobed with anti-PKC3 antibody (PKC3). (A) shows a typical result and (B) shows the densitometric analysis of the pPKC3 blots from 3 experiments. *P < 0.05, compared to the control lane by the unpaired Student’s t-test.

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buffer (50 mM TriseHCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/ml each of aprotinin, leupeptin, and pepstatin) containing phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4), sonicated for 30 s, and insoluble material removed by centrifugation at 13,000g for 30 min. The lysate was pre-cleared by addition of 10 ml of protein G-Sepharose bead slurry (Pharmacia), incubation for 30 min on a rocker, and removal of the protein G-Sepharose

beads by centrifugation at 13,000g for 10 min. The supernatant was transferred to a microtube and mixed overnight with constant rocking with 10 ml of rabbit polyclonal antibodies against total Cx43 (Zymed), 50 ml of protein G-Sepharose bead slurry was added and the suspension mixed for 1 h. The Sepharose-bound immune complexes were then spun down by centrifugation at 13,000g for 1 min and washed 5 times with 50 mM TriseHCl, pH 7.5, 1% Triton-X 100, 1 mM EDTA,

Fig. 5. Double immunofluorescence staining of cardiomyocytes showing 18b-GA-induced change of Ser368Cx43-immunoreactive spots and Cx43 dephosphorylation. Cardiomyocytes treated with 0.1% DMSO (A, B, C, control) or with 5 mM 18b-GA for 15 min (D, E, F, 150 ), 30 min (G, H, I, 300 ), or 60 min (J, K, L, 600 ) were double-labeled for pSer368Cx43 (pSer368, left panels) and Cx43-NP (NP, center panels). Images were merged to show the colocalization (Merge, right panels). The arrows indicate the junctional distribution of Ser368Cx43 in the left panels and the corresponding labeling of Cx43-NP in the center panels. The arrowheads in H, I, K, and L indicate cytoplasmic staining for Cx43-NP (bar ¼ 10 mm).

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and 1 mM PMSF, the pellets were heated for 5 min at 95  C in 100 ml of Laemmli sample buffer, and the supernatant collected for SDSePAGE. 2.5. Immunoblot analysis The cells were lysed as for immunoprecipitation and the supernatants collected and transferred to an Eppendorf microtube and the protein concentrations determined using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA). The samples were run on a 10% SDSepolyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (PerkinElmer Life Science, Boston, MA). Strips of the membranes were blocked for 1 h at room temperature in Trisebuffered saline (150 mM NaCl and 50 mM TriseHCl, pH 8.2) containing 5% skimmed milk and 0.1% Tween-20, incubated overnight at 4  C with a 1:500 dilution of rabbit antibody against Ser368Cx43, a 1:250 dilution of mouse antibody against Cx43-NP, or a 1:250 dilution of goat antibody against pPKC3. They were incubated for 2 h at room temperature with a 1:7500 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibodies, as appropriate. Immunoreactive bands were detected by substrate development using NBT and BCIP in 100 mM NaCl, 100 mM Trisebase, 5 mM MgCl2, pH 9.5. In membrane stripping experiments, the blots were stripped using 25 mM glycineeHCl, pH 2.0, 1% (w/v) SDS and re-probed using a mouse antibody against b-actin (1:1000), a mouse antibody against b-tubulin (1:2000), or rabbit antibodies against PKC3

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(1:1000) and a horseradish peroxidase-conjugated secondary antibody (1:3000), Western Blotting Luminol Reagent (Santa Cruz), and Hyperfilm ECL (Amershampharmacia Biotech, Buckinghamshire, England) before X-ray film exposure and development. Bands on nitrocellulose membranes and films were scanned and quantified using Gel-Pro Analyzer 3.1 software (Media Cybernetics, MD). 2.6. Statistical analysis All data are expressed as the mean  SD for 3 independent experiments using 3 different preparations. The difference between the means was assessed by Student’s t-test and was considered as statistically significant when the P value was <0.05. 3. Results 3.1. 18b-GA causes an increase in levels of pSer368Cx43 and pPKC3 We first examined whether 18b-GA caused an increase in serine 368-phosphorylated (pSer368) Cx43 levels and whether this was prevented by a PKC inhibitor. 18b-GA induced a dose-dependent increase in pSer368Cx43 levels and a concentration of 5 mM of 18b-GA was chosen for use through this study (Fig. 1). A time-dependent increase in pSer368Cx43 levels was seen in cardiomyocytes treated with 18b-GA compared to controls (Fig. 2) and the increase

Fig. 6. Double immunofluorescence staining of cardiomyocytes showing intercellular junction distribution of pSer368Cx43 and N-cadherin. Cardiomyocytes treated with 0.1% DMSO (A, B, C, control) or with 5 mM 18b-GA for 30 min (D, E, F, GA) were double-labeled for pSer368Cx43 (pSer368, left panels) and Ncadherin (N-cad, center panels). Images were merged to show the colocalization (Merge, right panels). The arrows indicate the junctional distribution of pSer368Cx43 in the left panels and the corresponding labeling of N-cadherin in the center panels (bar ¼ 10 mm).

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induced by 30 min treatment with 18b-GA was inhibited by co-treatment with the PKC inhibitor, chelerythrine (Fig. 3). In a time-course study, levels of serine 729-phosphorylated PKC3 (pPKC3), the active form of PKC3, also showed a timedependent increase with 18b-GA treatment, although total PKC3 levels were unchanged (Fig. 4). 3.2. 18b-GA induces dispersal of pSer368Cx43immunoreactive gap junctional plaques To determine the distribution of different phosphorylated forms of Cx43 in cultured neonatal rat cardiomyocytes after treatment with 18b-GA, double immunofluorescence staining was performed using rabbit antibodies against pSer368Cx43 and mouse antibody against non-phosphorylated Ser368 of Cx43 (Cx43-NP). In control cardiomyocytes, large spots of pSer368Cx43 were observed at gap junctions (Fig. 5A), while little labeling of Cx43-NP was seen (Fig. 5B). Treatment with 18b-GA for 15 min resulted in punctate pSer368Cx43 staining of the gap junction (Fig. 5D) and prominent Cx43-NP staining (Fig. 5E). Longer incubation (30 or 60 min) induced a dispersed

punctate staining for pSer368Cx43 (Fig. 5G, J) and some cytoplasmic staining for Cx43-NP (Fig. 5H, K, arrowheads). In both controls and treated cells, Cx43-NP was partially colocalized with pSer368Cx43 at gap junction plaques (Fig. 5, arrows). The junctional localization of pSer368Cx43 was confirmed by double-staining of pSer368Cx43 and N-cadherin at cellecell contact areas (Fig. 6). 3.3. Chelerythrine prevents 18b-GA-induced change in pSer368Cx43 staining pattern We examined the effects of chelerythrine on the 18b-GAinduced change in pSer368Cx43 and Cx43-NP immunoreactivity in cardiomyocytes. Although treatment with chelerythrine alone also resulted in punctate pSer368Cx43 staining at cellecell contact areas (Fig. 7G), Cx43-NP staining was weak (Fig. 7H). Co-treatment with chelerythrine attenuated 18b-GA-induced change in pSer368Cx43 staining pattern by preserving pSer368Cx43 aggregates at cellecell contacts (Fig. 7J), whereas 18b-GA-induced Cx43-NP staining was unaffected and remained prominent at gap junctions (Fig. 7K).

Fig. 7. Effects of chelerythrine on pSer368Cx43 and Cx43-NP staining in cardiomyocytes. Cardiomyocytes treated for 30 min with 0.1% DMSO (control) (A, B, C), 5 mM 18b-GA (GA) (D, E, F), or 0.5 mM chelerythrine (Chel) (G, H, I) or co-treated with 5 mM 18b-GA and 0.5 mM chelerythrine (GA þ Chel) (J, K, L) were double-labeled with antibody against pSer368Cx43 (pSer368, top panels) and antibody against Cx43-NP (NP, center panels). Images were merged to show the colocalization (Merge, bottom panels). The arrows indicate the junctional distribution of pSer368Cx43 in the upper panels and the corresponding Cx43-NP labeling in the center panels (Bar ¼ 10 mm).

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3.4. Calyculin A blocks the 18b-GA-induced increase in pSer368Cx43 levels In our previous study in cardiomyocytes (Chung et al., 2007), 18b-GA treatment induced a global serine/threonine dephosphorylation of Cx43 and a concurrent increase in Cx43NP levels, which was blocked by co-treatment with the protein phosphatase inhibitor, calyculin A (Table 1). We therefore examined the effects of calyculin A on the 18b-GA-induced change in pSer368Cx43 and Cx43-NP levels in cardiomyocytes. 18b-GA treatment induced Cx43 dephosphorylation (Fig. 8) and increased Cx43-NP levels (Fig. 9A, C) and pSer368Cx43 levels (Fig. 9B, C), and both effects were inhibited by calyculin A co-treatment. 3.5. 18b-GA increases the association of total Cx43 with PKC3 in cardiomyocytes Double immunofluorescence staining and immunoprecipitation were performed to determine whether the 18bGA-induced activation of PKC3 promoted its association with Cx43. 18b-GA treatment induced an increase of PKC3 immunoreactivity at cellecell junctions, which co-localized with Cx43 (Fig. 10D-F, arrows). Cardiomyocyte extracts were immunoprecipitated with a rabbit polyclonal antibody against total Cx43 and the immune complex was probed for PKC3 using a mouse monoclonal antibody. The results showed that 18b-GA increased the co-immunoprecipitation of total Cx43 and PKC3 and that this effect was prevented by calyculin A co-treatment (Fig. 10G).

Fig. 8. Immunoblots of cardiomyocyte lysates showing an increase in Cx43NP with 18b-GA treatment. Cardiomyocytes treated for 15 min with 0.1% DMSO in the absence (Cont) and presence of alkaline phosphatase (AP) or 5 mM 18b-GA (GA) were harvested and the cell lysates analyzed by 10% polyacrylamide SDS gel electrophoresis and immunoblotting. The blots were probed with antibody against Cx43-NP (NP), then stripped and re-probed with anti-b-tubulin antibody (b-tub) as a loading control.

Phosphorylation of Cx43 by PKC regulates the function and assembly/disassembly of gap junctions in cardiomyocytes. Activation of PKC by TPA causes a decrease in dye coupling and an increase in electrical coupling between neonatal rat cardiomyocytes (Kwak et al., 1995). However, inhibition of basal PKC activity by the protein kinase inhibitor, staurosporine, reduces electrical coupling in cardiomyocytes and that this effect is reversed by TPA (Sa´ez et al., 1997). Huang et al. (2004) showed that oleic acid increases pSer368Cx43 levels and induces gap junction disassembly by activation of PKC3 in cardiomyocytes. In the present study, treatment of cardiomyocytes with chelerythrine prevented 18b-GA-induced

4. Discussion Treatment of cardiomyocytes with 18b-GA induced an increase in pSer368Cx43 levels, concurrent with changes of pSer368Cx43-containing gap junction plaques from large aggregates to dispersed punctates. The 18b-GA-induced increase in pSer368Cx43 levels and change in pSer368Cx43 staining pattern were both prevented by co-treatment with the PKC inhibitor, chelerythrine, suggesting a signaling mechanism involving PKC. These results are in good agreement with those of a previous in vitro study of mouse four-cell embryos treated with 12-O-tetradecanoylphorbol-13-acetate, TPA (PMA), showing an increase in the levels of phosphorylated Cx43 and a concurrent decrease in Cx43 spot intensity at cellecell contact sites (Ogawa et al., 2000). Table 1 Effects of 18b-glycyrrhetinic acid on Cx43 gap junctions in cardiomyocytes Effects

Blockers

Dose-dependent inhibition of dye coupling Increase association of c-Src with Cx43 Selective Cx43 dephosphorylation Increase association of PKC3 with Cx43 Increase Cx43 phosphorylation at Ser368

PP2a PP2a Calyculin Aa Calyculin A Chelerythrine and calyculin A

a

Chung et al., 2007.

Fig. 9. Calyculin A prevents the 18b-GA-induced increase in Ser368Cx43 levels. Cardiomyocytes treated for 30 min with 0.1% DMSO (control), 5 mM 18b-GA (GA), or 10 nM calyculin A (CA) or co-treated with 5 mM 18b-GA and 10 nM calyculin A (GA þ CA) were harvested and the cell lysates analyzed by SDSepolyacrylamide gel electrophoresis and immunoblotting. (A) Blots probed with antibody against Cx43-NP (NP) and (B) duplicate blots probed with antibody against pSer368Cx43 (pSer368); both blots were stripped and re-probed with anti-b-tubulin antibody (b-tub). (C) Densitometric analysis of the pSer368Cx43 and Cx43-NP blots from 3 experiments. *P < 0.01, compared to the control lane, yP < 0.05, compared to the GA lane by the unpaired Student’s t-test.

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Fig. 10. 18b-GA increases the association of total Cx43 with PKC3 in cardiomyocytes. Cardiomyocytes treated for 30 min with 0.1% DMSO (control) (A, B, C) or 5 mM 18b-GA (GA) (D, E, F) were double-labeled with antibody against PKC3 (PKC3, left panels) and antibody against total Cx43 (Cx43, center panels). Images were merged to show the colocalization (Merge, right panels). The arrows indicate the junctional distribution of PKC3 in the left panels and the corresponding Cx43 labeling in the center panels (bar ¼ 10 mm). (G) Cardiomyocytes treated for 60 min with 0.1% DMSO (control), 5 mM 18b-GA (GA), or co-treated with 5 mM 18b-GA and 10 nM calyculin A (GA þ CA) were harvested, and the cell lysates immunoprecipitated using anti-Cx43 antibody (IP: Total Cx43), then the coprecipitated complexes were probed for pPKC3 and total Cx43 by immunoblotting (IB). The lower blots were stripped and re-probed with antibodies against rabbit immunoglobulin G (IgG), which was used as a loading control.

dispersal of pSer368Cx43-immunoreactive gap junctional plaques, indicating an effect of 18b-GA on gap junctions by PKC activation. Moreover, PKC activation by 18b-GA or PKC inhibition by chelerythrine both induced gap junction dispersal. Thus, the maintenance of Cx43 phosphorylation in a state of equilibrium by basal PKC activity seems to be required for cardiomyocytes to maintain the integrity and function of gap junctions. Low concentrations of 18b-GA enhance the activity of purified rat brain PKC in vitro (O’Brian et al., 1990). Our results showed that 18b-GA induced an increase in levels of pPKC3 (the active form of PKC3), but not of total PKC3, indicating that activation of PKC3 occurs following 18b-GA stimulation. In cardiomyocytes stimulated by PMA, PKC3 is found at cellecell interface membranes (Disatnik et al., 1994). Fibroblast growth factor-2 stimulates Cx43 phosphorylation and increases the colocalization of Cx43 with PKC3 at celle cell interfaces in cardiomyocytes (Doble et al., 2000). Furthermore, PKC activated by PMA has been shown to directly phosphorylate Cx43 at serine 368 (Lampe et al.,

2000). In our present study, activation of PKC3 by 18b-GA in cardiomyocytes was coincident with the increase in pSer368Cx43 levels, which was also prevented by co-treatment with chelerythrine, suggesting that phosphorylation of Ser368 in Cx43 is mediated by PKC3. This idea was supported by our immunofluorescence staining showing increased colocalization of PKC3 with Cx43 at gap junction plaques and immunoprecipitation analysis showing increased association of pPKC3 with Cx43 after 18b-GA treatment. In WB-F344 liver epithelial cells, 18b-GA reversibly induces Cx43 dephosphorylation and gap junction disassembly, as shown by decreased junctional staining and increased cytoplasmic staining for Cx43 (Guan et al., 1996). In cardiomyocytes, 18b-GA induces Cx43 dephosphorylation in a protein phosphatase-dependent manner, as the 18b-GAinduced Cx43 dephosphorylation is prevented by the protein phosphatase type 1 and type 2A inhibitor, calyculin A (Chung et al., 2007; Table 1). The mouse monoclonal antibody (138300, Cx43-NP) is specific for non-phosphorylated Ser368 of Cx43, whereas the target of the rabbit polyclonal antibody

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(AB3841, pSer368Cx43) is phosphorylated Ser368 of Cx43 (Solan et al., 2003). Thus, both pSer368Cx43 and Cx43-NP antibodies could not recognize the same molecule. However, our results showed that Cx43-NP was partially co-localized with pSer368Cx43 at gap junction plaques in both controls and treated cells, implying co-existence of pSer368Cx43 and Cx43-NP in the same plaques. In the present study, despite an increase of Cx43-NP levels seen after 18b-GA treatment, an increase in pSer368Cx43 levels was also seen after 18b-GA stimulation. Furthermore, co-treatment with calyculin A not only prevented 18b-GA-induced Cx43 dephosphorylation, but also inhibited the increase in pSer368Cx43 levels (Table 1). Phosphorylation of serine 368 in Cx43 occurs in both the plasma membrane and cytoplasm, and Cx43-NP can be phosphorylated at Ser368 by TPA stimulation (Solan et al., 2003). Since 18b-GA induces a rapid accumulation of Cx43NP in cardiomyocytes (Chung et al., 2007), the accumulated Cx43-NP may serve as a substrate pool for PKC3. Once formation of Cx43-NP is blocked by calyculin A and less Cx43-NP is available for PKC3, pSer368Cx43 levels would no longer be increased by 18b-GA stimulation. This hypothesis would imply that phosphorylation at other sites are increased by the phosphatase inhibitor, to account for the decreased nonphosphorylated Cx43. This idea was further supported by our immunoprecipitation data showing that calyculin A co-treatment prevented 18b-GA-induced Cx43 dephosphorylation by shifting Cx43 to a slower mobility and decreased the association of pPKC3 with total Cx43 (Fig. 10G). However, the possibility of a direct inhibitory effect of calyculin A on 18bGA cannot be ruled out. In an in vitro study, Vondriska et al. (2001) demonstrated that recombinant PKC3 and Src proteins are binding partners to each other and association of PKC3 with Src enhances the PKC3-associated Src kinase activity. In our previous study, 18b-GA was shown to induce Src phosphorylation and enhance the binding of p-Src to Cx43 (Chung et al., 2007). Whether 18b-GA induces association of PKC3 with Src and whether the 18b-GA-induced binding of p-Src to Cx43 is regulated by PKC3 require further investigation. In conclusion, we have demonstrated that, in cardiomyocytes, 18b-GA, via PKC3 activation, causes an increase in pSer368Cx43 levels concomitant with disassembly of gap junctional plaques. The 18b-GA-induced increase in pSer368Cx43 levels is prevented by chelerythrine and calyculin A. Further studies on the interaction between Cx43 and its binding proteins and on cross-talk between PKC3 and Src kinase will help to elucidate the mechanism by which 18b-GA inhibits gap junction intercellular communication. Acknowledgements This work was supported by Taiwan National Science Council grant NSC-93-2320-B002-098.

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