Heregulin-β is especially potent in activating phosphatidylinositol 3-kinase in nontransformed human mammary epithelial cells

• Published in: Journal of cellular physiology 2000-06, Vol.183 (3), p.301-313
• Main Authors: Ram, Tracy G., Hosick, Howard L., Ethier, Stephen P.
• Format: Article
• Language: English
• Subjects: Androstadienes - pharmacology; Breast - cytology; Breast Neoplasms; Cell Division - drug effects; Cell Line; Enzyme Activation; Epidermal Growth Factor - pharmacology; Epithelial Cells - cytology; Epithelial Cells - drug effects; Epithelial Cells - physiology; Female; Humans; Insulin - pharmacology; Neuregulin-1 - pharmacology; Phosphatidylinositol 3-Kinases - metabolism; Receptor, Epidermal Growth Factor - genetics; Receptor, Epidermal Growth Factor - physiology; Receptor, ErbB-2 - genetics; Receptor, ErbB-2 - physiology; Receptor, ErbB-3 - genetics; Receptor, ErbB-3 - physiology; Tumor Cells, Cultured
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/(SICI)1097-4652(200006)183:3<301::AID-JCP2>3.0.CO;2-W

The neu differentiation factors/heregulins (HRGs) comprise a family of polypeptide growth factors that activate p185erbB‐2 through direct binding to either erbB‐3 or erbB‐4 receptor tyrosine kinases. We have previously shown that HRG‐β is mitogenic for various human mammary epithelial cell lines that coexpress c‐erbB‐2 and c‐erbB‐3. Phosphatidylinositol 3‐kinase (PI3K) is activated by p185erbB‐2 /erbB‐3 heterodimers in cells stimulated by HRG, and PI3K is constitutively activated by p185erbB‐2 /erbB‐3 in breast carcinoma cells that overexpress c‐erbB‐2. To better understand the relative abilities of HRGs, epidermal growth factor (EGF), or insulin to activate PI3K under normal physiological conditions, we compared the levels of recruitment of the 85‐kDa regulatory subunit of PI3K when activated by the type I (erbB) or type II [insulin‐like growth factor (IGF)] receptor tyrosine kinases in two different nontransformed human mammary epithelial cell lines. The nontransformed H16N‐2 cells isolated from normal tissue express EGFR, p185erbB‐2 , and erbB‐3, and are highly responsive to the mitogenic effects of HRG‐β as well as to the combination of EGF and insulin in serum‐free culture. We measured the stoichiometry of p85 recruited by tyrosine‐phosphorylated proteins induced in H16N‐2 cells by either the α or the β isoform of HRG. HRG‐β was greater than 10‐fold more potent in inducing p85 recruitment than was the less biologically active HRG‐α isoform. HRG‐β was also a more potent inducer of p85 recruited by tyrosine‐phosphorylated proteins than was either EGF, insulin, or EGF and insulin combined. Furthermore, erbB‐3 principally mediated the direct recruitment of p85 in cells stimulated by HRG or EGF, indicating that, in addition to the high‐level activation of PI3K by p185erbB‐2 / erbB‐3, EGFR/erbB‐3 heterodimer interaction is essential for the weak but significant level of PI3K activated by EGF in cells that express normal EGFR levels. Studies using the PI3K inhibitor wortmannin also indicated that PI3K activation was required for the proliferation of H16N‐2 cells induced by either HRG‐β or EGF and insulin in serum‐free culture. Finally, HRG‐β was also an especially potent inducer of PI3K in the nontransformed MCF‐10A cells, which were derived spontaneously from normal reduction mammoplasty tissue. These data show, for the first time, a side‐by‐side quantitative comparison of the relative degree of PI3K activated by different growth factors in nontransformed g