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Differentiated Phenotype of Smooth Muscle Cells Depends on Signaling Pathways through Insulin-like Growth Factors and Phosphatidylinositol 3-Kinase*

  • Ken'ichiro Hayashi
    Footnotes
    Affiliations
    Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Hiroshi Saga
    Footnotes
    Affiliations
    Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Yoshihiro Chimori
    Affiliations
    Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Kazuhiro Kimura
    Affiliations
    Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Yuka Yamanaka
    Affiliations
    Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Kenji Sobue
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-6-879-3680; Fax: 81-6-879-3689;
    Affiliations
    Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Author Footnotes
    * This work was supported by grants-in-aid for COE Research (to K. S.) and in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.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.
    ‡ These authors contributed equally to this work.
Open AccessPublished:November 30, 1998DOI:https://doi.org/10.1074/jbc.273.44.28860
      Under conventional culture conditions, smooth muscle cells display their phenotypic modulation from a differentiated to a dedifferentiated state. Here, we established a primary culture system of smooth muscle cells maintaining a differentiated phenotype, as characterized by expression of smooth muscle-specific marker genes such as h-caldesmon and calponin, cell morphology, and ligand-induced contractility. Laminin retarded the progression of dedifferentiation of smooth muscle cells. Insulin-like growth factors (IGF-I and IGF-II) and insulin markedly prolonged the differentiated phenotype, with IGF-I being the more potent. In contrast, serum, epidermal growth factor, transforming growth factors, and platelet-derived growth factors potently induced dedifferentiation compared with angiotensin II, arginine-vasopressin, and basic fibroblast growth factor. Using the present culture system, we investigated signaling pathways regulating a phenotype of smooth muscle cells. In cultured cells, IGF-I specifically activated phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, protein kinase B, but not mitogen-activated protein kinases. Specific inhibitors of PI3-kinase (wortmannin and LY294002) induced dedifferentiation of smooth muscle cells even when they were cultured on laminin under IGF-I-stimulated conditions. The sole effect of laminin to retard the dedifferentiation was completely blocked by anti-IGF-I antibody, and laminin promoted the endogenous expression of IGF-I in cultured cells. The reduced promoter activity of the caldesmon gene induced by platelet-derived growth factor BB was overcome by the forced expression of the constitutive active form of PI3-kinase p110α catalytic subunit. These findings suggest that an IGF-I signaling pathway through PI3-kinase plays a critical role in maintaining a differentiated phenotype of smooth muscle cells.
      SMC
      smooth muscle cell
      ECM
      extracellular matrix
      IGF
      insulin-like growth factor
      PI3-kinase
      phosphatidylinositol 3-kinase
      MAPK
      mitogen-activated protein kinase
      ERK
      extracellular signal-regulated kinase
      JNK
      c-Jun N-terminal protein kinase
      PKB
      protein kinase B
      BSA
      bovine serum albumin
      DMEM
      Dulbecco's modified Eagle's medium
      PCR
      polymerase chain reaction
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      PI
      phosphatidylinositol
      PS
      phosphatidylserine
      CAT
      chloramphenicol acetyltransferase
      PDGF
      platelet-derived growth factor
      EGF
      epidermal growth factor, TGF, transforming growth factor
      p70S6K
      p70 ribosomal S6 kinase
      CMV
      cytomegalovirus
      pCMV
      plasmid-carrying cytomegalovirus promoter
      PIPES
      1,4-piperazinediethanesulfonic acid
      PBS
      phosphate-buffered saline
      DTT
      dithiothreitol.
      The smooth muscle cells (SMCs)1 play roles in the control of blood pressure, enteric peristalsis, and bronchial, uterus, and bladder contraction. Although the precursor cells of SMC seem to undergo phenotypic modulation into matured cells, the origin of precursor cells has not been well characterized. During embryogenesis, three different lineages (cardiac neural crest, nodosa placode, and lateral mesoderm) have been, in part, proven as precursors of SMC. In the case of vasculogenesis (
      • Folkman J.
      • D'Amore P.A.
      ,
      • Hanahan D.
      ), angioblasts originated from mesoderm differentiate into endothelial cells, which then coalesce to form a single layer of endothelial tubes. It has been proposed that these primitive vessels are involved in the recruitment of neighboring mesenchymal cells and the subsequent differentiation of SMCs. However, the precise mechanism regarding recruitment of SMC precursors has remained unknown. Phenotypic modulation of SMCs is also associated with pathological conditions, such as atherosclerosis, hypertension, and leiomyogenic tumorigenicity. In the progression of these diseases, SMCs change their phenotype from a differentiated to a dedifferentiated state (
      • Ross R.
      ). Recent studies have focused on the molecular mechanism of SMC phenotype-dependent expression and/or isoform conversion of α-SM actin (
      • Campbell J.H.
      • Kocher O.
      • Skalli O.
      • Gabbiani G.
      • Campbell G.R.
      ,
      • Blank R.S.
      • McQuinn T.C.
      • Yin K.C.
      • Thompson M.M.
      • Takeyasu K.
      • Schwartz R.J.
      • Owens G.K.
      ), smooth muscle myosin heavy chain (
      • Nagai R.
      • Kuro-o M.
      • Babij P.
      • Periasamy M.
      ,
      • Madsen C.S.
      • Hershey J.C.
      • Hautmann M.B.
      • White S.L.
      • Owens G.K.
      ), α- and β-tropomyosins (
      • Lees-Miller J.P.
      • Helfman D.M.
      ,
      • Kashiwada K.
      • Nishida W.
      • Hayashi K.
      • Ozawa K.
      • Yamanaka Y.
      • Saga H.
      • Yamashita T.
      • Tohyama M.
      • Shimada S.
      • Sato K.
      • Sobue K.
      ), calponin (
      • Gimona M.
      • Sparrow M.P.
      • Strasser P.
      • Herzog M.
      • Small J.V.
      ,
      • Shanahan C.M.
      • Weissberg P.L.
      • Necalfe J.C.
      ), SM22α (
      • Gimona M.
      • Sparrow M.P.
      • Strasser P.
      • Herzog M.
      • Small J.V.
      ,
      • Shanahan C.M.
      • Weissberg P.L.
      • Necalfe J.C.
      ), h-caldesmon (
      • Kashiwada K.
      • Nishida W.
      • Hayashi K.
      • Ozawa K.
      • Yamanaka Y.
      • Saga H.
      • Yamashita T.
      • Tohyama M.
      • Shimada S.
      • Sato K.
      • Sobue K.
      ,
      • Yano H.
      • Hayashi K.
      • Momiyama T.
      • Saga H.
      • Haruna M.
      • Sobue K.
      ), and α1 integrin (
      • Glukhova M.
      • Koteliansky V.
      • Fondacci C.
      • Marotte F.
      • Rappaport L.
      ,
      • Obata H.
      • Hayashi K.
      • Nishida W.
      • Momiyama T.
      • Uchida A.
      • Ochi T.
      • Sobue K.
      ). These proteins, which are specifically expressed in SMCs, are collectively termed as SMC-specific molecular markers. The expressions of α-SM actin, caldesmon, calponin, SM22α, β-tropomyosin, smooth muscle myosin heavy chain, and α1 integrin are increased in differentiated SMCs but are decreased in dedifferentiated SMCs. These changes are regulated at transcription levels. Isoform changes of caldesmon, α-tropomyosin, and smooth muscle myosin heavy chain are controlled by alternative splicing in an SMC phenotype-dependent manner. Changes to these molecular markers are, therefore, favorable for quantification to SMC phenotypes (
      • Sobue K.
      • Hayashi K.
      • Nishida W.
      ).
      In order to elucidate the molecular mechanism of such phenotypic modulation, it is necessary to use cultured SMCs. However, the SMCs in conventional culture display their phenotype from a differentiated to a dedifferentiated state (
      • Chamley-Campbell J.
      • Campbell G.R.
      • Ross R.
      ). In addition, passaged SMCs do not show a differentiated phenotype even if they are cultured under quiescent conditions. Serum growth factors and extracellular matrices (ECMs) have been reported to be involved in such phenotypic modulation. Using morphological criteria, Hedin et al. (
      • Hedin U.
      • Bottger B.A.
      • Forsberg E.
      • Johansson S.
      • Thyberg J.
      ) demonstrated that in primary cultured vascular SMCs, laminin induced a delay in the progression of dedifferentiation, whereas fibronectin stimulated it. There are some reports regarding the establishment of cell lines that display partial characteristics of differentiated SMCs as monitored by morphological and biochemical indicators. Despite these efforts, primary culture systems or cell lines of SMC maintaining a differentiated phenotype have not been presented. Here, we achieved the first establishment of a primary culture system of SMCs to maintain a differentiated phenotype for a long culture using SMC-specific molecular markers, cell morphology, and ligand-induced contractility as indicators. We confirmed that laminin retarded the progression of SMC dedifferentiation and found that insulin-like growth factor-I (IGF-I) had a highly potent activity to maintain a differentiated phenotype. Using these culture conditions, we investigated the IGF-I-generated signaling pathway in SMCs and demonstrated the critical role of phosphatidylinositol 3-kinase (PI3-kinase) in maintaining a differentiated phenotype. This culture system seems to be useful for future analyses of SMC-specific signal transduction and transcription and for identification of factors affecting the SMC phenotype.

      DISCUSSION

      Under conventional culture conditions, SMCs convert their phenotype from a differentiated to a dedifferentiated state (
      • Chamley-Campbell J.
      • Campbell G.R.
      • Ross R.
      ). During dedifferentiation, the SMC irreversibly changes its morphology from a long spindle-like to a proliferative fibroblast-like shape and loses the expression of SMC-specific marker genes and its ligand-induced contractility. To our knowledge, there have been no reports regarding the establishment of primary culture systems or clonal cell lines of SMC that can control its own phenotype. As monitored by SMC-specific molecular markers, cell morphology, and function, we achieved the first establishment of a primary culture system of SMCs maintaining a differentiated phenotype by a combination of IGF-I and laminin. Using this culture system, we demonstrated that PI3-kinase activation by IGF-I is a key signaling event involved in maintenance of a differentiated phenotype of SMCs.
      As shown in Fig. 2, IGF-I, IGF-II, and insulin prolonged the differentiated phenotype of SMCs. Among them, IGF-1 was the most potent factor (Fig. 3). Because the order of concentrations of IGFs and insulin required for maintenance of half-maximum expression levels of caldesmon and calponin genes was IGF-I < IGF-II ≪ insulin, the effects of these growth factors would be mediated through IGF-I receptor. It has been reported that in cultured vascular SMCs, IGF-I stimulates SMC proliferation and migration (
      • Clemmons D.R.
      ,
      • Bornfeldt K.E.
      • Arnqvist H.L.
      • Norstedt G.
      ,
      • Delafontaine P.
      • Lou H.
      • Alexander R.W.
      ,
      • Bronfeldt K.E.
      • Rainco E.W.
      • Nateano T.
      • Groves L.M.
      • Krebs E.
      • Ross R.
      ). Balloon denaturation of the rat aorta results in a 7-fold increase in IGF-I expression, and this increase is closely associated with cell growth (
      • Creck B.
      • Fishbein M.C.
      • Forrester J.S.
      • Helfant R.H.
      • Fagin J.A.
      ). Antisense transcripts of IGF-I receptor cDNA inhibit vascular SMC growth (
      • Du J.
      • Delafontaine P.
      ). This accumulated evidence suggests that IGF-I is a factor inducing cell growth and/or migration in vascular SMCs. On the other hand, in our culture system of visceral SMCs, IGF-I was the most potent factor maintaining a differentiated phenotype, but it is not involved in dedifferentiation characterized by cell growth and migration. This discrepancy would be due to a difference in the origin of the SMCs or experimental conditions. In fact, vascular SMCs reported by previous investigators have been passaged under serum-stimulated conditions. The response to IGF-I in these cells would be changed by serum-induced phenotypic modulation. Additionally, our culture conditions are in part applicable for vascular SMCs. Therefore, a difference in experimental conditions is a likely cause of the discrepancy.
      Using a variety of cell lines, it has been well studied that binding of IGF-I to its receptor activates the intrinsic tyrosine kinase activity of the IGF-I receptor, resulting in autophosphorylation of its own receptor and phosphorylation of target molecules such as insulin receptor substrate-1 and Shc proteins (
      • Myers Jr., M.G.
      • Sun X.J.
      • Cheatham B.
      • Jachna B.R.
      • Glasheen E.M.
      • Backer J.M.
      • White M.F.
      ,
      • Sasaoka T.
      • Draznin B.
      • Leitner J.W.
      • Langlois W.J.
      • Olefsky J.M.
      ). Multiple phosphotyrosine sites in insulin receptor substrate-1 serve as binding sites of various signaling molecules, including PI3-kinase (
      • Giorgetti S.
      • Pelicci P.G.
      • Pelicci G.
      • van Obberghen E.
      ). Stimulation of PI3-kinase leads to the activation of downstream signaling molecules, including Akt/PKB and p70S6K (
      • Dardevet D.
      • Sornet C.
      • Vary T.
      • Grizard J.
      ). PI3-kinase also directly interacts with the PDGF receptor. The Shc proteins have specific tyrosine phosphorylation sites to associate with Grb2 and the GTP-binding protein Ras (
      • Pelicci G.
      • Lanfrancone L.
      • Grignani F.
      • McGlade J.
      • Cavallo F.
      • Forni G.
      • Nicoletti I.
      • Pawson T.
      • Pelicci P.G
      ,
      • Sasaoka T.
      • Rose D.W.
      • Jhun B.H.
      • Saltiel A.R.
      • Draznin B.
      • Olefsky J.M.
      ). However, these are no reports regarding the IGF-I generated signaling in SMCs. In this study, we found in our culture system that IGF-I enhanced PI3-kinase and Akt/PKB activities and that these activations were completely blocked by wortmannin and LY294002 (Fig. 7, A and B). Consistent with this, the PI3-kinase inhibitors induced the dedifferentiation of SMCs cultured on laminin under IGF-I stimulated conditions (Fig. 8). Forced activation of PI3-kinase further resulted in an increase in the promoter activity of caldesmon gene in dedifferentiated SMCs induced by PDGF-BB (Fig. 9). In contrast, IGF-I did not activate the signaling pathways through MAPKs (Fig. 7,C–E). These results suggest that a signaling pathway of PI3-kinase plays a crucial role in maintaining a differentiated phenotype of SMC. At present, we have not yet characterized the downstream signaling molecules of PI3-kinase, except for Akt/PKB. Because the activity of Akt/PKB was potentiated by IGF-I stimulation in a PI3-kinase-dependent manner and activation of this kinase lasted for more than 3 h (Fig. 7 B), this kinase would be one of the downstream targets of PI3-kinase. It is now necessary to reveal whether Akt/PKB is functionally involved in phenotypic modulation of SMC and to identify the further downstream targets. Rapamycin did not affect a differentiated phenotype of SMCs cultured on laminin under IGF-I stimulated conditions (Fig. 8), indicating that p70S6K would not be involved in this signaling. As demonstrated here, the signaling pathway from IGF-I to PI3-kinase plays a key role in maintaining a differentiated phenotype of SMCs. IGF-I-stimulation was, however, not able to convert a dedifferentiated state of SMCs to a differentiated state even when IGF-I receptor was fully expressed (data not shown). Therefore, the signaling pathway inducing differentiation of SMCs would be different from that maintaining a differentiated phenotype.
      Regarding the effect of laminin on SMCs, laminin enhanced endogenous expression and secretion of IGF-I. This finding is based on the inhibitory effect of neutralizing anti-IGF-I antibody, endogenous expression of IGF-I mRNAs (Figs. 5 and 6), and semi-estimation of IGF-I in cultured medium for maintaining the expression of SMC-specific marker genes. The effect of laminin mediated through endogenous expression and secretion of IGF-I on differentiated SMCs was abolished by the specific PI3-kinase inhibitors wortmannin and LY294002 (data not shown). These observations suggest that a low level of endogenously produced IGF-I might contribute to delay the dedifferentiation. It has been previously reported that laminin delayed the progression of dedifferentiation of primary cultured vascular SMCs, whereas fibronectin stimulates it (
      • Hedin U.
      • Bottger B.A.
      • Forsberg E.
      • Johansson S.
      • Thyberg J.
      ). Such an effect of laminin on cultured vascular SMCs might be due to the enhanced production and secretion of IGF-I by laminin.
      In this paper, we established for the first time a culture system of visceral SMCs maintaining a differentiated state for a long culture. This culture system may be applicable for vascular SMCs. Furthermore, the SMC culture system presented here is useful for analyzing SMC-specific molecular events, such as signal transduction and gene regulation, including transcription and splicing. In this paper, we showed a partial characterization of caldesmon gene expression in association with signaling pathways (Fig. 9). Using the present culture system, we are currently conducting detailed analyses of the gene regulation of SMC-specific markers and screening of novel factors involved in phenotypic modulation of SMCs. Information obtained by this culture system should be effective in clarifying the molecular mechanism of SMC phenotypic determination, including vascular and visceral SMCs.

      ACKNOWLEDGEMENTS

      We thank Drs. H. Kurosu and T. Katada (Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo) for providing a constitutive active form of PI3-kinase p110α subunit cDNA.

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