Amphiregulin is a potent mitogen for the vascular smooth muscle cell line, A7r5

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Abstract

The regulation of amphiregulin, an epidermal growth factor (EGF) family member, and its effect on vascular smooth muscle cells (VSMC) were examined. Amphiregulin mRNA was upregulated by amphiregulin itself as well as α-thrombin. Amphiregulin caused an approximate 3-fold increase in DNA synthesis. Its effect on growth was compared with those of other mitogens, and was found to be approximately 3.5-, 2.4-, and 1.0-fold greater than those of endothelin-I (ET-I), α-thrombin, and platelet-derived growth factor-AB (PDGF-AB), respectively. As evidenced by Western blot analysis, amphiregulin stimulated the phosphorylation of p42/p44-mitogen-activated protein kinase (MAPK), p38-MAPK, c-Jun NH2-terminal protein kinase (JNK), and Akt/protein kinase B (PKB), respectively. By statistical analysis, the amphiregulin-induced growth effect was significantly decreased by the MAP kinase/ extracellular regulated kinase kinase-1 (MEK-1) inhibitor PD98059, p38-MAPK inhibitor SB203580, and phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, respectively, but was not decreased by JNK inhibitor SP600125. These results suggest that amphiregulin is the most potent mitogen of the mitogens tested, and its growth effect is mediated at least in part through the p42/p44-MAPK, p38-MAPK, and PI-3 kinase-Akt/PKB pathways in VSMC.

Section snippets

Methods

Cell line and cell culture. A7r5, immortal embryonic rat thoracic aorta smooth muscle cell line, was obtained from the American Type Culture Collection (ATCC No. CRL1444). The A7r5 cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS) under 5% CO2 in air at 37 °C in a humidified incubator.

Reagents. p42/p44-MAPK, p38-MAPK, JNK, phospho-p42/p44-MAPK, phospho-p38-MAPK antibodies, and phospho and nonphospho-Akt/PKB antibodies were from Cell Signaling

Expression of the EGF family and the EGF receptor family in A7r5 cells

It is generally believed that the EGF family plays an important role in cell proliferation, survival, migration, and differentiation. These factors exert their cellular effects through binding to cell surface receptors. The EGF family of receptors include epidermal growth factor receptor (EGFR also called ErbB1/HER1) [15], ErbB2 (Neu/HER2) [16], ErbB3 (HER3) [17], and ErbB4 (HER4) [18]. We initially addressed the issue of whether the EGF family and its receptor family were expressed in A7r5

Discussion

In the present study, we demonstrated that amphiregulin, epiregulin, heregulin, and HB-EGF are expressed as evidenced by RT-PCR analysis. The EGF family can be divided into two subgroups on the basis of their direct binding to specific receptors [20], [21]. Members of the first group, which includes amphiregulin, epiregulin, HB-EGF, EGF, TGF-α, and betacellulin, bind directly to the classic EGF receptor. Members of the other group, which includes heregulin, bind directly to the ErbB3 and ErbB4.

Acknowledgements

We are grateful to Dr. Takio Hayashi for encouragement and Mrs. Mari Kurata for excellent technical assistance.

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