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Akt signaling is involved in the disruption of gap junctional communication caused by v-Src and TNF-α
Biochemical and Biophysical Research Communications 400 (2010) 230–235
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
PI3K/Akt signaling is involved in the disruption of gap junctional communication caused by v-Src and TNF-a Satoko Ito a, Toshinori Hyodo a, Hitoki Hasegawa a, Hong Yuan b, Michinari Hamaguchi a, Takeshi Senga a,⇑ a b
Division of Cancer Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan Depertment of Obsterics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
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Article history: Received 12 August 2010 Available online 19 August 2010 Keywords: Gap junction Connexin43 v-Src Akt TNF-a
a b s t r a c t Gap junctional communication, which is mediated by the connexin protein family, is essential for the maintenance of normal tissue function and homeostasis. Loss of intercellular communication results in a failure to coordinately regulate cellular functions, and it can facilitate tumorigenesis. Expression of oncogenes and stimulation with cytokines has been shown to suppress intercellular communication; however, the exact mechanism by which intercellular communication is disrupted by these factors remains uncertain. In this report, we show that Akt is essential for the disruption of gap junctional communication in v-Src-transformed cells. In addition, inhibition of Akt restores gap junctional communication after it is suppressed by TNF-a signaling. Furthermore, we demonstrate that the expression of a constitutively active form of Akt1, but not of Akt2 or Akt3, is sufficient to suppress gap junctional communication. Our results clearly define Akt1 as one of the critical regulators of gap junctional communication. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Gap junctions are transmembrane channels that mediate communication between adjacent cells and allow the direct intercellular exchange of small molecules of less than 1 kDa [1,2]. Gap junctional communication is thought to play an important role in embryonic development, regulation of normal cell growth and differentiation, and electrical coupling. Gap junctions are composed of integral transmembrane proteins termed ‘‘connexins”. At least 20 distinct members of the connexin gene family, including connexin26, 32, 43 and 46, have been identified in mammals. Among these, connexin43 (Cx43) is one of the most ubiquitously expressed connexins in tissues and cell lines, and its regulation has been well characterized [3,4]. Gap junctional communication is often disrupted in transformed cells. v-Src, a transforming product of the Rous sarcoma virus, phosphorylates tyrosine residues of various cellular proteins to cause cellular transformation. v-Src-transformed cells are characterized by disrupted intercellular communication [5], along with reduced organization of the actin cytoskeleton, cell–cell adhesions and integrin-associated focal adhesions [6]. The mechanism by which intercellular communication is disrupted in v-Src-transformed cells has been extensively studied [7,8]. Tyr247 and Tyr265 of Cx43 are directly phosphorylated by v-Src, and expression of a Cx43 whose
⇑ Corresponding author. Fax: +81 52 744 2464. E-mail address: [email protected] (T. Senga). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.08.045
Tyr247 and Tyr265 were substituted to alanine restored gap junctional communication after disruption by v-Src expression [9]. Other studies, however, indicated that phosphorylation of Cx43 by kinases such as MAPK and PKC are involved in the disruption of intercellular communication by v-Src [8,10,11]. Recent studies using a phospho-specific antibody demonstrated that serine residues of Cx43 were phosphorylated in v-Src-transformed cells [12]. In addition, we previously showed that Ras signaling was critical for the inhibition of gap junctional communication in v-Srctransformed cells, independent of tyrosine phosphorylation of Cx43 [13]. It still remains controversial whether Src phosphorylation of Cx43 inhibits intercellular communication or whether other signaling pathways play a more essential role in the regulation of gap junctional communication. Disruption of gap junctional communication is also observed after treatment with proinflammatory mediators such as TNF-a, IL-1b and LPS [14]. It has been reported that intercellular communication was transiently disrupted by TNF-a stimulation, and this disruption was mediated by tyrosine phosphorylation of Cx43 by activated Src [15]. In addition, TNF-a reduced Cx43 expression by inhibiting the promoter activity of the Cx43 gene [16]. Because multiple pathways are involved in the regulation of Cx43 function, kinases other than Src may also regulate TNF-a-mediated suppression of gap junctional communication. In this report, we studied the role of Akt signaling in the regulation of intercellular communication. The serine/threonine kinase Akt acts downstream of phosphatidylinositol-3-kinase (PI3K), and it plays a pivotal role in cell proliferation, survival, metabolism
S. Ito et al. / Biochemical and Biophysical Research Communications 400 (2010) 230–235
and cancer progression [17,18]. The Akt kinase family has three highly homologous isoforms (Akt1, Akt2 and Akt3), and several recent findings indicated that functional differences exist between the isoforms [19]; however, the specific roles of each isoform are still uncertain. Here we show that Akt activation plays a pivotal role in the disruption of gap junctional communication in v-Src-transformed cells and TNF-a-stimulated cells. In addition, we demonstrate that expression of active Akt1, but not Akt2 or Akt3, is sufficient to disrupt gap junctional communication. 2. Materials and methods 2.1. Cell culture The immortalized rat fibroblast cell line 3Y1, v-Src-transformed 3Y1 (SR3Y1) cells and the immortalized mouse fibroblast cell line Balb3T3 were purchased from RIKEN BioResource Center (Ibaragi, Japan). 3Y1, SR3Y1 and Balb3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin.
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equal amounts of protein were incubated with anti-Cx43 polyclonal antibody (Santa Cruz Biotechnology) coupled to protein Aagarose beads (Thermo Scientific, Waltham, MA) at 4 °C for 1 h. After extensive washing with lysis buffer, proteins were eluted with Laemmli sample buffer followed by boiling and then subjected to immunoblotting. 2.5. Measurement of gap junctional communication To measure gap junctional communication, confluent cells were microinjected with Lucifer Yellow (Sigma, Louis, MO) using the Eppendorf microinjection system, FemtoJet and InjectMan NI2 (Eppendorf, Tokyo, Japan), and Olympus IX71 (Olympus, Tokyo, Japan). Two minutes after microinjection, the cells were washed and fixed with 4% paraformaldehyde. Dye transfer was monitored by fluorescent microscopy and the number of fluorescent cells around injected cells was counted. The statistical analysis of the results was performed using Student’s t-test. Values of P < 0.05 were considered statistically significant. 2.6. Soft agar colony formation assay
2.2. Antibodies and reagents Mouse TNF-a was obtained from Pepro Tech (Rocky Hill, NJ) and LY294002 and wortmannin from Wako (Tokyo, Japan). Anti-Connexin43 monoclonal antibody was purchased from BD Biosciences (San Jose, CA), anti-b-actin antibody was from Sigma, anti-Akt and anti-phospho-Akt antibodies were from Cell Signaling Technology (Danvers, MA), and anti-ERK2 and anti-Connexin43 polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). 2.3. Plasmids and generation of stable expression cell lines Wild-type, dominant negative and constitutively active mouse Akt1 expression plasmids were purchased from Millipore (Billerica, MA). Akt2 and Akt3 were amplified by PCR from a mouse fetal cDNA library. Active Akt2 and Akt3 were made by fusing a myristoylation signal to the N-terminus. To establish cell lines that constitutively express dominant negative Akt1, cells were transfected with the expression plasmid and cultured in the presence of 400 lg/ml neomycin. Colonies that expressed dominant negative Akt1 were selected and expanded for further studies. To establish Balb3T3 cells that express the active form of the Akt isoforms, cDNAs were cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) with an N-terminal myc-tag. BalB3T3 cells were transfected with the plasmid and cell lines that constitutively expressed each active Akt isoform were established. 2.4. Immunoblotting and immunoprecipitation Cells were lysed with Laemmli sample buffer and boiled for 5 min. Protein concentrations of lysates were measured using RCDC Protein Assay (BIO-RAD, Hercules, CA). Equal protein quantities were loaded onto SDS–polyacrylamide electrophoresis (SDS– PAGE) gels, and transferred to PVDF membrane (Millipore). The membrane was blocked with 5% nonfat skim milk, incubated with each primary antibody for 1 h, washed with TBS-T buffer (100 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.05% Tween20) and then incubated with secondary antibodies. Proteins were visualized by enhanced chemiluminescence (GE Healthcare, Uppsala, Sweden). To detect the tyrosine phosphorylation of Cx43, cells were lysed with RIPA buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% TritonX100, 1% sodium deoxycholate, 0.1% SDS) and centrifuged at 15,000 rpm for 20 min to clear cell debris. Cell lysates containing
Cells (5 104) were mixed with 0.36% agar in DMEM supplemented with 10% FBS and overlaid onto 0.72% agar layers in 6-well plates. After three weeks of incubation, colonies in randomly selected fields (40 magnification) were counted.
3. Results To examine whether Akt signaling has a functional role in the regulation of intercellular communication, we used PI3K inhibitors, LY294002 and wortmannin, to suppress Akt activation. A rat fibroblast cell line, 3Y1, and 3Y1 cells transformed by v-Src (SR3Y1), were used in this study. Treatment of SR3Y1 cells with either inhibitor suppressed phosphorylation of Akt, but did not suppress Cx43 expression and tyrosine phosphorylation of cellular proteins (Fig. 1A). Tyrosine phosphorylation of Cx43 by v-Src has been reported to regulate gap junctional communication; therefore, we checked tyrosine phosphorylation of Cx43 in the presence of the inhibitors. As shown in Fig. 1B, addition of the inhibitors to the SR3Y1 cells did not affect tyrosine phosphorylation of Cx43. Although gap junctional communication is regulated by other types of connexins, previous reports indicated that Cx43 is the major connexin expressed in 3Y1 and SR3Y1 cells [13]. We next examined the effect of the inhibitors on the ability of the cells to communicate through gap junctional channels. To measure intercellular communication, single-cell microinjection of Lucifer Yellow dye was performed using a computer-assisted microinjection system. Cells were grown to confluency, treated with or without the inhibitors for 30 min, and then the dye was microinjected to the parental cells. Two minutes after injection, the cells were fixed and the number of adjacent cells that became fluorescent was counted. Although the 3Y1 cells showed a clear spread of the dye to the adjacent cells, the transfer of the dye was significantly suppressed in SR3Y1 cells. In contrast, dye transfer to the surrounding cells was clearly observed in SR3Y1 cells treated with the inhibitors, indicating that inhibition of PI3K/Akt signaling in SR3Y1 cells restored gap junctional communication (Fig. 1C). To further confirm the role of Akt signaling in the regulation of intercellular communication, we established SR3Y1 cell lines that constitutively expressed a dominant negative form of Akt1. We used two cell lines, dnAkt-SR6 and dnAkt-SR20, for further investigation. Both dnAkt-SR6 and dnAkt-SR20 expressed dominant negative Akt at similar levels, and overall tyrosine phosphorylation of cellular proteins was not affected (Fig. 2A). In addition, tyrosine
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Fig. 1. Suppression of Akt activation restores gap junctional communication in v-Src-transformed cells. (A) 3Y1 and SR3Y1 cells were treated with or without 20 lM LY294002 or 100 nM wortmannin for 30 min, and then phosphorylation and expression levels of the indicated proteins were examined by immunoblotting. (B) Cells were treated with or without the indicated inhibitors for 30 min and immunoprecipitated with anti-Cx43 antibody. The immunoprecipitates were blotted with anti-pTyr antibody to detect tyrosine phosphorylation of Cx43. (C) Cells were treated with or without the inhibitors for 30 min, and then Lucifer Yellow was microinjected into cells. Two minutes after the dye injection, the cells were fixed and the number of cells that incorporated the dye was counted. Images that are representative of these experiments are shown. Results from three independent experiments are indicated on the graph (mean ± SD). Asterisks indicate P values in comparison with SR3Y1 (*P < 0.01).
phosphorylation of Cx43 was not affected by the expression of dominant negative Akt (Fig. 2B). We then examined gap junctional communication by dye injection. As shown in Fig. 2C, the number of adjacent cells that became fluorescent after the dye injection was increased by the expression of dominant negative Akt. Gap junctional communication has been reported to be suppressed by stimulation with cytokines such as TNF-a and IL-1b [14]; therefore, we studied the role of Akt signaling in the disruption of intercellular communication induced by TNF-a. Consistent with the previous report [15], we observed transient suppression of gap junctional-mediated dye transfer in Balb3T3 cells stimulated by TNF-a (Fig. 3A). To examine whether Akt activation was involved in TNF-a-mediated disruption of intercellular communication, we investigated Akt activation after TNF-a stimulation. As shown in Fig. 3B, Akt was clearly activated by TNF-a stimulation. Time course analysis of Akt activation demonstrated that suppression of dye transfer correlated with activation of Akt. Next, we tested whether inhibition of gap junctional communication was mediated by activation of Akt. Balb3T3 cells were pretreated with or without LY294002 for 30 min, stimulated with TNF-a and then microinjected with Lucifer Yellow. As shown in Fig. 3C, transient suppression of dye transfer after TNF-a stimulation was restored in the presence of LY294002. To further confirm whether Akt activation is required for the suppression of intercellular communication after TNF-a treatment, we established Balb3T3 cells that constitutively expressed dominant negative Akt (dnAkt-Balb3T3). As shown in Fig. 3D, dnAkt-Balb3T3 cells were resistant to TNFa-mediated suppression of intercellular communication. These results indicate that activation of Akt is essential for TNF-a-mediated disruption of gap junctional communication.
Lastly, we tested whether activation of Akt was sufficient to disrupt gap junctional communication. We created constitutively active forms of the three Akt isoforms (Akt1, Akt2 and Akt3) by fusing a myristoylation signal to the N-terminus of the proteins. Balb3T3 cells were transfected with a plasmid that encoded each active isoform of Akt, and cell lines were established. The expression levels of each Akt isoform were similar in each cell line, and there was no difference in the levels of Cx43 expression between cell lines (Fig. 4A). We examined whether expression of active Akt could promote anchorage-independent growth of Balb3T3 cells. Each cell line was cultured in soft agar for 3 weeks, and the formation of colonies was examined. As shown in Fig. 4B, the different cell lines formed colonies that were similar in size and number. We then performed dye microinjection to measure gap junctional communication of these cells. As shown in Fig. 4C, dye transfer to the surrounding cells was reduced in active Akt1-expressing cells, but not in Akt2- or Akt3-expressing cells, as compared to parental Balb3T3 cells. This indicates that gap junctional communication is specifically regulated by Akt1. 4. Discussion Accumulating evidence has suggested that human cancers are deficient in gap junctional communication due to downregulation of connexin expression or the inability to form functional channels [20]. Although several studies have indicated that sustained connexin expression is related to the invasiveness of transformed cells [21,22], genetically engineered mice lacking connexins have been shown to be susceptible to radiation or chemically-induced tumorigenesis [23,24]. In addition, restoration of gap junctional commu-
S. Ito et al. / Biochemical and Biophysical Research Communications 400 (2010) 230–235
Fig. 2. Expression of dominant negative Akt in SR3Y1 restored gap junctional communication. (A) Tyrosine phosphorylation of cellular proteins and expression of indicated proteins were examined by immunoblotting. (B) SR3Y1 cells were lysed and immunoprecipitated with anti-Cx43 antibody and blotted with anti-pTyr antibody to detect tyrosine phosphorylation of Cx43. (C) SR3Y1 cells were microinjected with Lucifer Yellow, and 2 min later, the cells were fixed and the fluorescent cells were counted. Images that are representative of these experiments are shown. Results from three independent experiments are indicated on the graph (mean ± SD). Asterisks indicate P values in comparison with SR3Y1 (*P < 0.01).
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nication by forced expression of Cx43 in cancer cells decreased neoplastic potential [25,26]. Thus, it is widely accepted that disruption of intercellular communication plays a role in carcinogenesis. Akt is a critical regulator of signal transduction induced by cytokines, growth factors and other cellular stimuli that control multiple cellular functions [17]. Aberrant activation of Akt has been demonstrated in a wide range of cancer types [19]. Activated Akt phosphorylates various target proteins to regulate cancer cell invasion and metastasis; however, it remained uncertain whether activation of Akt was related to the disruption of gap junctional communication. In this report, we showed that disruption of intercellular communication in v-Src-transformed cells is dependent on Akt signaling. In addition, activation of Akt1 was sufficient for the disruption of gap junctional communication. Our results suggest a possible role for Akt1 in the suppression of gap junctional communication, which in turn promotes cancer progression. The Akt kinase family is comprised of three highly homologous isoforms. Analysis of Akt isoform knockout mice demonstrated isoform-specific functions in the regulation of cell growth, neuronal development and metabolism [19]. Although our knowledge about the distinct functions of the different Akt isoforms in tumor development and invasion is limited, several studies have pointed toward different roles for Akt1 and Akt2 in cancer cell migration and invasion. Active Akt1 inhibits cell migration and invasion by inhibiting Rho activity [27]. In contrast, overexpression of Akt2 promotes invasiveness of breast cancer cells [28]. We have demonstrated that although expression of each active Akt isoform caused similar levels of anchorage-independent growth, gap junctional communication was specifically disrupted by active Akt1 in Balb3T3 cells. Because the disruption of intercellular communication is related to cancer progression, our results together with pre-
Fig. 3. Activation of Akt is required for the suppression of TNF-a-mediated gap junctional communication. (A) Cells were treated with 3 nM TNF-a, and at the indicated time points Lucifer Yellow was injected to evaluate gap junctional communication. Images that are representative of the dye spreading experiments are shown. Results from three independent experiments are indicated on the graph (mean ± SD). (B) Balb3T3 cells were treated with 3 nM TNF-a and activation of Akt, at the indicated time points, was examined by immunoblotting. (C) Balb3T3 cells were treated or untreated with LY294002 for 30 min and then stimulated with TNF-a. Thirty minutes after the stimulation, dye injection was performed to evaluate gap junctional communication. The graph shows results from three independent experiments (mean ± SD). (D) Cells were stimulated with TNF-a and 30 min later, gap junctional communication was evaluated by dye injection. Results from three independent experiments are shown on the graph (mean ± SD).
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Fig. 4. Expression of active Akt1, but not Akt2 or Akt3, suppresses gap junctional communication. (A) Expression of the indicated proteins in each cell line was examined by immunoblotting. (B) Each cell line was cultured in soft agar for three weeks to evaluate the ability of the cells to grow anchorage independently. The representative images of colonies of each cell line are shown. The number of colonies were counted, as seen on the graph. Data represent mean ± SD from three independent experiments. (C) Cells were microinjected with Lucifer Yellow to evaluate gap junctional communication. Representative images of the dye spreading experiments from each cell line are shown. The graph shows results from three independent experiments (mean ± SD). Asterisks indicate P values in comparison with vector (*P < 0.05, **P > 0.05).
vious findings suggest that the Akt kinases have isoform-specific functions in cancer development. It still remains uncertain how gap junctional communication is disrupted by Akt1 activation. A recent study has shown that Akt1 can directly phosphorylate Ser373 and Ser369 on Cx43 to induce association with 14-3-3 [29]; however, the physiological role of the phosphorylation still remains unknown. Lysophosphatidic acid (LPA) can activate Akt and disrupt gap junctional communication in rat ovarian surface epithelial (ROSE199) cells; nevertheless, inhibition of Akt did not restore gap junctional communication [30]. In our study, recovery of intercellular communication in v-Src-transformed cells by inhibition of Akt was around 60% of the level of normal cells, indicating that additional pathways are also important for the disruption of gap junctional communication; therefore, pathways not involving Akt may play essential roles in the disruption of gap junctional communication caused by LPA. Further studies are needed to elucidate the molecular mechanism by which Akt1 activation disrupts gap junctional communication and the functional role of phosphorylation of Ser373 and Ser369 on Cx43 by Akt. In conclusion, we have demonstrated that Akt activation is crucial for the disruption of intercellular communication in v-Src-transformed cells and TNF-a-stimulated cells. Furthermore, active Akt1, but not active Akt2 or Akt3, was sufficient for the disruption of gap junctional communication in Balb3T3 cells. Gap junctional communication has been reported to be regulated by various protein kinases such as PKC and MAPK, but our results clearly define Akt as one of the critical regulators of gap junctional communication. Acknowledgments We thank the members of the Division of Cancer Biology for helpful discussions and technical assistance. This research was funded in part by a Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
PI3K/Akt signaling is involved in the disruption of gap junctional communication caused by v-Src and TNF-a Satoko Ito a, Toshinori Hyodo a, Hitoki Hasegawa a, Hong Yuan b, Michinari Hamaguchi a, Takeshi Senga a,⇑ a b
Division of Cancer Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan Depertment of Obsterics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
a r t i c l e
i n f o
Article history: Received 12 August 2010 Available online 19 August 2010 Keywords: Gap junction Connexin43 v-Src Akt TNF-a
a b s t r a c t Gap junctional communication, which is mediated by the connexin protein family, is essential for the maintenance of normal tissue function and homeostasis. Loss of intercellular communication results in a failure to coordinately regulate cellular functions, and it can facilitate tumorigenesis. Expression of oncogenes and stimulation with cytokines has been shown to suppress intercellular communication; however, the exact mechanism by which intercellular communication is disrupted by these factors remains uncertain. In this report, we show that Akt is essential for the disruption of gap junctional communication in v-Src-transformed cells. In addition, inhibition of Akt restores gap junctional communication after it is suppressed by TNF-a signaling. Furthermore, we demonstrate that the expression of a constitutively active form of Akt1, but not of Akt2 or Akt3, is sufficient to suppress gap junctional communication. Our results clearly define Akt1 as one of the critical regulators of gap junctional communication. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Gap junctions are transmembrane channels that mediate communication between adjacent cells and allow the direct intercellular exchange of small molecules of less than 1 kDa [1,2]. Gap junctional communication is thought to play an important role in embryonic development, regulation of normal cell growth and differentiation, and electrical coupling. Gap junctions are composed of integral transmembrane proteins termed ‘‘connexins”. At least 20 distinct members of the connexin gene family, including connexin26, 32, 43 and 46, have been identified in mammals. Among these, connexin43 (Cx43) is one of the most ubiquitously expressed connexins in tissues and cell lines, and its regulation has been well characterized [3,4]. Gap junctional communication is often disrupted in transformed cells. v-Src, a transforming product of the Rous sarcoma virus, phosphorylates tyrosine residues of various cellular proteins to cause cellular transformation. v-Src-transformed cells are characterized by disrupted intercellular communication [5], along with reduced organization of the actin cytoskeleton, cell–cell adhesions and integrin-associated focal adhesions [6]. The mechanism by which intercellular communication is disrupted in v-Src-transformed cells has been extensively studied [7,8]. Tyr247 and Tyr265 of Cx43 are directly phosphorylated by v-Src, and expression of a Cx43 whose
⇑ Corresponding author. Fax: +81 52 744 2464. E-mail address: [email protected] (T. Senga). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.08.045
Tyr247 and Tyr265 were substituted to alanine restored gap junctional communication after disruption by v-Src expression [9]. Other studies, however, indicated that phosphorylation of Cx43 by kinases such as MAPK and PKC are involved in the disruption of intercellular communication by v-Src [8,10,11]. Recent studies using a phospho-specific antibody demonstrated that serine residues of Cx43 were phosphorylated in v-Src-transformed cells [12]. In addition, we previously showed that Ras signaling was critical for the inhibition of gap junctional communication in v-Srctransformed cells, independent of tyrosine phosphorylation of Cx43 [13]. It still remains controversial whether Src phosphorylation of Cx43 inhibits intercellular communication or whether other signaling pathways play a more essential role in the regulation of gap junctional communication. Disruption of gap junctional communication is also observed after treatment with proinflammatory mediators such as TNF-a, IL-1b and LPS [14]. It has been reported that intercellular communication was transiently disrupted by TNF-a stimulation, and this disruption was mediated by tyrosine phosphorylation of Cx43 by activated Src [15]. In addition, TNF-a reduced Cx43 expression by inhibiting the promoter activity of the Cx43 gene [16]. Because multiple pathways are involved in the regulation of Cx43 function, kinases other than Src may also regulate TNF-a-mediated suppression of gap junctional communication. In this report, we studied the role of Akt signaling in the regulation of intercellular communication. The serine/threonine kinase Akt acts downstream of phosphatidylinositol-3-kinase (PI3K), and it plays a pivotal role in cell proliferation, survival, metabolism
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and cancer progression [17,18]. The Akt kinase family has three highly homologous isoforms (Akt1, Akt2 and Akt3), and several recent findings indicated that functional differences exist between the isoforms [19]; however, the specific roles of each isoform are still uncertain. Here we show that Akt activation plays a pivotal role in the disruption of gap junctional communication in v-Src-transformed cells and TNF-a-stimulated cells. In addition, we demonstrate that expression of active Akt1, but not Akt2 or Akt3, is sufficient to disrupt gap junctional communication. 2. Materials and methods 2.1. Cell culture The immortalized rat fibroblast cell line 3Y1, v-Src-transformed 3Y1 (SR3Y1) cells and the immortalized mouse fibroblast cell line Balb3T3 were purchased from RIKEN BioResource Center (Ibaragi, Japan). 3Y1, SR3Y1 and Balb3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin.
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equal amounts of protein were incubated with anti-Cx43 polyclonal antibody (Santa Cruz Biotechnology) coupled to protein Aagarose beads (Thermo Scientific, Waltham, MA) at 4 °C for 1 h. After extensive washing with lysis buffer, proteins were eluted with Laemmli sample buffer followed by boiling and then subjected to immunoblotting. 2.5. Measurement of gap junctional communication To measure gap junctional communication, confluent cells were microinjected with Lucifer Yellow (Sigma, Louis, MO) using the Eppendorf microinjection system, FemtoJet and InjectMan NI2 (Eppendorf, Tokyo, Japan), and Olympus IX71 (Olympus, Tokyo, Japan). Two minutes after microinjection, the cells were washed and fixed with 4% paraformaldehyde. Dye transfer was monitored by fluorescent microscopy and the number of fluorescent cells around injected cells was counted. The statistical analysis of the results was performed using Student’s t-test. Values of P < 0.05 were considered statistically significant. 2.6. Soft agar colony formation assay
2.2. Antibodies and reagents Mouse TNF-a was obtained from Pepro Tech (Rocky Hill, NJ) and LY294002 and wortmannin from Wako (Tokyo, Japan). Anti-Connexin43 monoclonal antibody was purchased from BD Biosciences (San Jose, CA), anti-b-actin antibody was from Sigma, anti-Akt and anti-phospho-Akt antibodies were from Cell Signaling Technology (Danvers, MA), and anti-ERK2 and anti-Connexin43 polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). 2.3. Plasmids and generation of stable expression cell lines Wild-type, dominant negative and constitutively active mouse Akt1 expression plasmids were purchased from Millipore (Billerica, MA). Akt2 and Akt3 were amplified by PCR from a mouse fetal cDNA library. Active Akt2 and Akt3 were made by fusing a myristoylation signal to the N-terminus. To establish cell lines that constitutively express dominant negative Akt1, cells were transfected with the expression plasmid and cultured in the presence of 400 lg/ml neomycin. Colonies that expressed dominant negative Akt1 were selected and expanded for further studies. To establish Balb3T3 cells that express the active form of the Akt isoforms, cDNAs were cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) with an N-terminal myc-tag. BalB3T3 cells were transfected with the plasmid and cell lines that constitutively expressed each active Akt isoform were established. 2.4. Immunoblotting and immunoprecipitation Cells were lysed with Laemmli sample buffer and boiled for 5 min. Protein concentrations of lysates were measured using RCDC Protein Assay (BIO-RAD, Hercules, CA). Equal protein quantities were loaded onto SDS–polyacrylamide electrophoresis (SDS– PAGE) gels, and transferred to PVDF membrane (Millipore). The membrane was blocked with 5% nonfat skim milk, incubated with each primary antibody for 1 h, washed with TBS-T buffer (100 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.05% Tween20) and then incubated with secondary antibodies. Proteins were visualized by enhanced chemiluminescence (GE Healthcare, Uppsala, Sweden). To detect the tyrosine phosphorylation of Cx43, cells were lysed with RIPA buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% TritonX100, 1% sodium deoxycholate, 0.1% SDS) and centrifuged at 15,000 rpm for 20 min to clear cell debris. Cell lysates containing
Cells (5 104) were mixed with 0.36% agar in DMEM supplemented with 10% FBS and overlaid onto 0.72% agar layers in 6-well plates. After three weeks of incubation, colonies in randomly selected fields (40 magnification) were counted.
3. Results To examine whether Akt signaling has a functional role in the regulation of intercellular communication, we used PI3K inhibitors, LY294002 and wortmannin, to suppress Akt activation. A rat fibroblast cell line, 3Y1, and 3Y1 cells transformed by v-Src (SR3Y1), were used in this study. Treatment of SR3Y1 cells with either inhibitor suppressed phosphorylation of Akt, but did not suppress Cx43 expression and tyrosine phosphorylation of cellular proteins (Fig. 1A). Tyrosine phosphorylation of Cx43 by v-Src has been reported to regulate gap junctional communication; therefore, we checked tyrosine phosphorylation of Cx43 in the presence of the inhibitors. As shown in Fig. 1B, addition of the inhibitors to the SR3Y1 cells did not affect tyrosine phosphorylation of Cx43. Although gap junctional communication is regulated by other types of connexins, previous reports indicated that Cx43 is the major connexin expressed in 3Y1 and SR3Y1 cells [13]. We next examined the effect of the inhibitors on the ability of the cells to communicate through gap junctional channels. To measure intercellular communication, single-cell microinjection of Lucifer Yellow dye was performed using a computer-assisted microinjection system. Cells were grown to confluency, treated with or without the inhibitors for 30 min, and then the dye was microinjected to the parental cells. Two minutes after injection, the cells were fixed and the number of adjacent cells that became fluorescent was counted. Although the 3Y1 cells showed a clear spread of the dye to the adjacent cells, the transfer of the dye was significantly suppressed in SR3Y1 cells. In contrast, dye transfer to the surrounding cells was clearly observed in SR3Y1 cells treated with the inhibitors, indicating that inhibition of PI3K/Akt signaling in SR3Y1 cells restored gap junctional communication (Fig. 1C). To further confirm the role of Akt signaling in the regulation of intercellular communication, we established SR3Y1 cell lines that constitutively expressed a dominant negative form of Akt1. We used two cell lines, dnAkt-SR6 and dnAkt-SR20, for further investigation. Both dnAkt-SR6 and dnAkt-SR20 expressed dominant negative Akt at similar levels, and overall tyrosine phosphorylation of cellular proteins was not affected (Fig. 2A). In addition, tyrosine
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Fig. 1. Suppression of Akt activation restores gap junctional communication in v-Src-transformed cells. (A) 3Y1 and SR3Y1 cells were treated with or without 20 lM LY294002 or 100 nM wortmannin for 30 min, and then phosphorylation and expression levels of the indicated proteins were examined by immunoblotting. (B) Cells were treated with or without the indicated inhibitors for 30 min and immunoprecipitated with anti-Cx43 antibody. The immunoprecipitates were blotted with anti-pTyr antibody to detect tyrosine phosphorylation of Cx43. (C) Cells were treated with or without the inhibitors for 30 min, and then Lucifer Yellow was microinjected into cells. Two minutes after the dye injection, the cells were fixed and the number of cells that incorporated the dye was counted. Images that are representative of these experiments are shown. Results from three independent experiments are indicated on the graph (mean ± SD). Asterisks indicate P values in comparison with SR3Y1 (*P < 0.01).
phosphorylation of Cx43 was not affected by the expression of dominant negative Akt (Fig. 2B). We then examined gap junctional communication by dye injection. As shown in Fig. 2C, the number of adjacent cells that became fluorescent after the dye injection was increased by the expression of dominant negative Akt. Gap junctional communication has been reported to be suppressed by stimulation with cytokines such as TNF-a and IL-1b [14]; therefore, we studied the role of Akt signaling in the disruption of intercellular communication induced by TNF-a. Consistent with the previous report [15], we observed transient suppression of gap junctional-mediated dye transfer in Balb3T3 cells stimulated by TNF-a (Fig. 3A). To examine whether Akt activation was involved in TNF-a-mediated disruption of intercellular communication, we investigated Akt activation after TNF-a stimulation. As shown in Fig. 3B, Akt was clearly activated by TNF-a stimulation. Time course analysis of Akt activation demonstrated that suppression of dye transfer correlated with activation of Akt. Next, we tested whether inhibition of gap junctional communication was mediated by activation of Akt. Balb3T3 cells were pretreated with or without LY294002 for 30 min, stimulated with TNF-a and then microinjected with Lucifer Yellow. As shown in Fig. 3C, transient suppression of dye transfer after TNF-a stimulation was restored in the presence of LY294002. To further confirm whether Akt activation is required for the suppression of intercellular communication after TNF-a treatment, we established Balb3T3 cells that constitutively expressed dominant negative Akt (dnAkt-Balb3T3). As shown in Fig. 3D, dnAkt-Balb3T3 cells were resistant to TNFa-mediated suppression of intercellular communication. These results indicate that activation of Akt is essential for TNF-a-mediated disruption of gap junctional communication.
Lastly, we tested whether activation of Akt was sufficient to disrupt gap junctional communication. We created constitutively active forms of the three Akt isoforms (Akt1, Akt2 and Akt3) by fusing a myristoylation signal to the N-terminus of the proteins. Balb3T3 cells were transfected with a plasmid that encoded each active isoform of Akt, and cell lines were established. The expression levels of each Akt isoform were similar in each cell line, and there was no difference in the levels of Cx43 expression between cell lines (Fig. 4A). We examined whether expression of active Akt could promote anchorage-independent growth of Balb3T3 cells. Each cell line was cultured in soft agar for 3 weeks, and the formation of colonies was examined. As shown in Fig. 4B, the different cell lines formed colonies that were similar in size and number. We then performed dye microinjection to measure gap junctional communication of these cells. As shown in Fig. 4C, dye transfer to the surrounding cells was reduced in active Akt1-expressing cells, but not in Akt2- or Akt3-expressing cells, as compared to parental Balb3T3 cells. This indicates that gap junctional communication is specifically regulated by Akt1. 4. Discussion Accumulating evidence has suggested that human cancers are deficient in gap junctional communication due to downregulation of connexin expression or the inability to form functional channels [20]. Although several studies have indicated that sustained connexin expression is related to the invasiveness of transformed cells [21,22], genetically engineered mice lacking connexins have been shown to be susceptible to radiation or chemically-induced tumorigenesis [23,24]. In addition, restoration of gap junctional commu-
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Fig. 2. Expression of dominant negative Akt in SR3Y1 restored gap junctional communication. (A) Tyrosine phosphorylation of cellular proteins and expression of indicated proteins were examined by immunoblotting. (B) SR3Y1 cells were lysed and immunoprecipitated with anti-Cx43 antibody and blotted with anti-pTyr antibody to detect tyrosine phosphorylation of Cx43. (C) SR3Y1 cells were microinjected with Lucifer Yellow, and 2 min later, the cells were fixed and the fluorescent cells were counted. Images that are representative of these experiments are shown. Results from three independent experiments are indicated on the graph (mean ± SD). Asterisks indicate P values in comparison with SR3Y1 (*P < 0.01).
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nication by forced expression of Cx43 in cancer cells decreased neoplastic potential [25,26]. Thus, it is widely accepted that disruption of intercellular communication plays a role in carcinogenesis. Akt is a critical regulator of signal transduction induced by cytokines, growth factors and other cellular stimuli that control multiple cellular functions [17]. Aberrant activation of Akt has been demonstrated in a wide range of cancer types [19]. Activated Akt phosphorylates various target proteins to regulate cancer cell invasion and metastasis; however, it remained uncertain whether activation of Akt was related to the disruption of gap junctional communication. In this report, we showed that disruption of intercellular communication in v-Src-transformed cells is dependent on Akt signaling. In addition, activation of Akt1 was sufficient for the disruption of gap junctional communication. Our results suggest a possible role for Akt1 in the suppression of gap junctional communication, which in turn promotes cancer progression. The Akt kinase family is comprised of three highly homologous isoforms. Analysis of Akt isoform knockout mice demonstrated isoform-specific functions in the regulation of cell growth, neuronal development and metabolism [19]. Although our knowledge about the distinct functions of the different Akt isoforms in tumor development and invasion is limited, several studies have pointed toward different roles for Akt1 and Akt2 in cancer cell migration and invasion. Active Akt1 inhibits cell migration and invasion by inhibiting Rho activity [27]. In contrast, overexpression of Akt2 promotes invasiveness of breast cancer cells [28]. We have demonstrated that although expression of each active Akt isoform caused similar levels of anchorage-independent growth, gap junctional communication was specifically disrupted by active Akt1 in Balb3T3 cells. Because the disruption of intercellular communication is related to cancer progression, our results together with pre-
Fig. 3. Activation of Akt is required for the suppression of TNF-a-mediated gap junctional communication. (A) Cells were treated with 3 nM TNF-a, and at the indicated time points Lucifer Yellow was injected to evaluate gap junctional communication. Images that are representative of the dye spreading experiments are shown. Results from three independent experiments are indicated on the graph (mean ± SD). (B) Balb3T3 cells were treated with 3 nM TNF-a and activation of Akt, at the indicated time points, was examined by immunoblotting. (C) Balb3T3 cells were treated or untreated with LY294002 for 30 min and then stimulated with TNF-a. Thirty minutes after the stimulation, dye injection was performed to evaluate gap junctional communication. The graph shows results from three independent experiments (mean ± SD). (D) Cells were stimulated with TNF-a and 30 min later, gap junctional communication was evaluated by dye injection. Results from three independent experiments are shown on the graph (mean ± SD).
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Fig. 4. Expression of active Akt1, but not Akt2 or Akt3, suppresses gap junctional communication. (A) Expression of the indicated proteins in each cell line was examined by immunoblotting. (B) Each cell line was cultured in soft agar for three weeks to evaluate the ability of the cells to grow anchorage independently. The representative images of colonies of each cell line are shown. The number of colonies were counted, as seen on the graph. Data represent mean ± SD from three independent experiments. (C) Cells were microinjected with Lucifer Yellow to evaluate gap junctional communication. Representative images of the dye spreading experiments from each cell line are shown. The graph shows results from three independent experiments (mean ± SD). Asterisks indicate P values in comparison with vector (*P < 0.05, **P > 0.05).
vious findings suggest that the Akt kinases have isoform-specific functions in cancer development. It still remains uncertain how gap junctional communication is disrupted by Akt1 activation. A recent study has shown that Akt1 can directly phosphorylate Ser373 and Ser369 on Cx43 to induce association with 14-3-3 [29]; however, the physiological role of the phosphorylation still remains unknown. Lysophosphatidic acid (LPA) can activate Akt and disrupt gap junctional communication in rat ovarian surface epithelial (ROSE199) cells; nevertheless, inhibition of Akt did not restore gap junctional communication [30]. In our study, recovery of intercellular communication in v-Src-transformed cells by inhibition of Akt was around 60% of the level of normal cells, indicating that additional pathways are also important for the disruption of gap junctional communication; therefore, pathways not involving Akt may play essential roles in the disruption of gap junctional communication caused by LPA. Further studies are needed to elucidate the molecular mechanism by which Akt1 activation disrupts gap junctional communication and the functional role of phosphorylation of Ser373 and Ser369 on Cx43 by Akt. In conclusion, we have demonstrated that Akt activation is crucial for the disruption of intercellular communication in v-Src-transformed cells and TNF-a-stimulated cells. Furthermore, active Akt1, but not active Akt2 or Akt3, was sufficient for the disruption of gap junctional communication in Balb3T3 cells. Gap junctional communication has been reported to be regulated by various protein kinases such as PKC and MAPK, but our results clearly define Akt as one of the critical regulators of gap junctional communication. Acknowledgments We thank the members of the Division of Cancer Biology for helpful discussions and technical assistance. This research was funded in part by a Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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