Phenotypic modulation of smooth muscle cells through EGF family ligand- and EGFR-triggered signaling pathways

Yuka Yamanaka1, KenÕichiro Hayashi1, Toshi Komurasaki2, Shigeto Morimoto1, Toshio Ogihara1 and Kenji Sobue2

1Department of Neuroscience, Osaka University Graduate School of Medicine D13, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan, 2Research Center, Taisho Pharmaceutical Co. Ltd., 403-1 Yoshino-cho, Omiya, Saitama 330-8530, Japan,

AIM: The phenotypic modulation of smooth muscle cells (SMCs) is closely associated with the development and progression of various SMC diseases. We investigated the molecular mechanism of phenotypic modulation triggered by EGF family ligands using a primary culture system of differentiated SMCs, among four EGF-receptor (EGFR) family members, the EGFR was solely activated by EGF, heparin-binding EGF (HB-EGF), transforming growth factor alpha (TGFa), epiregulin (ER), and betacellulin (BTC) .METHODS: Antibodies. Antibodies against ERK, p38MAPK, phosphorylated ERK, phosphorylated p38MAPK, the EGFR family members (EGFR, ErbB-2, ErbB-3, and ErbB-4), and phosphotyrosine were purchased from Santa Cruz Biotechnology (USA). Cell culture. Visceral SMCs were prepared from 15-day-old chicken embryo gizzards and vascular SMCs of 5-week-old SD male rats were cultured on laminin-coated dishes under the culture conditions . The ligand-induced contractility of cultured SMCs was monitored for at least 5days.Northern blotting. Total RNAs from cultured SMCs under the indicated conditions was separated on 1.0% agarose-formaldehyde denaturing gels and transferred to nylon membranes (GeneScreen, NEN Life Science Products). The blots were hybridized with 32P-labelled antisense strands corresponding to the common part of h- and l-caldesmon (CaD) cDNA and calponin (CN) cDNA . To quantify the amount of RNAs in the northern blots, ribosomal RNAs were stained with 0.02% methylene blue. Immunoblotting. After stimulation with EGF family ligands, cultured SMCs were lysed with 2% SDS sample buffer. The proteins were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Target proteins on the membrane were visualized with an ECL Western blotting kit (Amersham) using the appropriate antibodies. Immunoprecipitation. The cell lysates were prepared from cultured SMCs maintaining a differentiated phenotype and were immunoprecipitated with antibodies against EGFR, ErbB-2, ErbB-3, or ErbB-4. The immunoprecipitates thus obtained were analyzed by immunoblotting with the respective EGFR family or phosphotyrosine antibodies. Protein kinase assays. We assayed the incorporations of [32P]ATP into their respective substrates, myelin basic protein (MBP) for ERK and GST-ATF2 for p38MAPK. RESULTS:EGFR, ErbB-2, ErbB-3, and ErbB-4 were detected in differentiated SMCs. EGF, HB-EGF, TGFa, ER, and BTC caused a decrease in total CaD and CN(SMC-differentiation marker genes ) expression and an increase in the isoform conversion of CaD from the h- to l-forms in a dose-dependent manner. In contrast, HRG showed no significant effect on the differentiated SMC phenotype. We then used specific kinase inhibitors for ERGR and ErbB-2, AG1478 and AG825, respectively, to address the involvement of these EGFR family members in phenotypic modulation. Pre-treatment with AG1478 completely blocked the change in the expression of SMC-differentiation marker genes, but pre-treatment with AG825 did not. EGF, HB-EGF, TGFa, ER, and BTC significantly phosphorylated the EGFR, and its phosphorylation was suppressed by pre-treatment with AG1478.We next characterized the downstream signaling pathways triggered by EGF family ligands. EGF, HB-EGF, TGFa, ER, and BTC significantly phosphorylated ERK and p38MAPK, but HRG did not. The phosphorylation of ERK and p38MAPK were completely blocked by pre-treatment with AG1478, indicating that the EGF family ligands mediated the ERK and p38MAPK signaling pathways through EGFR. In contrast, all of the EGF family ligands examined here potently activated the JNK signaling pathway . Pre-treatment with the ERK kinase inhibitor, PD98059, and the p38MAPK inhibitor, SB203580, suppressed the activities of ERK and p38MAPK in EGF-stimulated SMCs. Northern blot analysis revealed that the EGF-induced change in the expression of SMC-differentiation marker genes was partially inhibited by pre-treatment with either PD98059 or SB203580, and was completely inhibited by pre-treatment with both drugs. CONCLUSION: We found that EGF family ligands potently induced the transition of SMCs from a differentiated phenotype to dedifferentiated one through the activation of the EGFR, and that this phenotypic modulation depended on the coordinated activation of the downstream signaling pathways involving ERK and p38MAPK.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Molecular Pathology.