ErbB 4 Signaling During Breast and Neural Development

The erbB4 gene encodes one of the four members of the mammalian ErbB family of transmembrane tyrosine kinases. The ErbB4 protein plays a role as a receptor for the neuregulins, a large group of structurally related molecules and a few other epidermal growth factor (EGF)-related polypeptides, such as heparin-binding EGF, betacellulin and epiregulin. The importance of this receptor tyrosine kinase in development has been demonstrated by the generation of mice with a targeted inactivation of the erbB4 gene. Such mice die by embryonic day eleven due to defective trabeculation in the heart, precluding analysis of phenotypes at later stages in development and in the adult. Now, using two unique genetic approaches our laboratories succeeded in overcoming this obstacle. In the first approach, the heart defects of ErbB4 null mutant mice were rescued by transgenic expression of an ErbB4 cDNA under a cardiac-specific myosin promoter. This allowed the generation of ErbB4 mutants that develop into adulthood and are fertile. In the second approach, the role of ErbB4 during mammary gland development was specifically addressed by Cre-mediated deletion of both erbB4 alleles within the mammary epithelium. Bellow we discuss the progress made studying these genetic models in understanding the physiological roles of ErbB4 with a focus on the mammary gland and the nervous system. ERBB4 AND MAMMARY GLAND DEVELOPMENT Of the four ErbB receptors, ErbB4 appears to possess unique activities in the breast. Although all four receptors are expressed and activated in the normal mammary gland,1 ErbB4 expression appears to be selectively extinguished during the progression of breast cancer.2,3 Furthermore, while expression of EGFR(ErbB1), ErbB2/HER2/Neu (referred to here as ErbB2), and ErbB3 in primary breast tumors is associated with poor patient outcome, ErbB4 expression indicates a differentiating tumor phenotype and improved patient prognosis.4 One possible explanation for the unique activity of ErbB4 in breast cancer was revealed in experiments where endogenous ErbB4 was inactivated in the developing mammary gland by transgenic overexpression of a dominant negative mutant ErbB4 receptor. Similar to clinical observations, results from these experiments suggested that ErbB4 signaling contributed to epithelial differentiation.5 Unfortunately dominant negative ErbB receptors lack specificity, potentially attenuating signaling through all ligand bound ErbB family members. These results must therefore be considered in light of this experimental caveat. Analysis of ErbB4 function in the developing breast was further hampered because genetic deletion of both ErbB4 alleles results in embryonic lethality,6 precluding analysis of post-natal breast development. In lieu of these experimental limitations, the role of ErbB4 during mammary gland development has been recently elucidated through the analysis of two unique genetic models. Transgenically rescued ErbB4 mutants, which lack ErbB4 expression in all tissues but in the myocardium7 and mice with Cre-mediated deletions of both ErbB4 alleles within mammary epithelium,8 exhibited identical severe defects in lobuloalveolar development resulting in lactational failure. The mammary gland developmental profile culminating into a functional secretory gland can be broadly divided into three major phases; ductal morphogenesis in the virgin, alveolar proliferation during early pregnancy, and finally epithelial differentiation and lactation at the late pregnancy/parturition transition. ErbB4 appears to be dispensable for ductal branching morphogenesis in the virgin7 and early lobuloalveolar development at the onset of pregnancy.7,8 However, coincident with high levels of ErbB4 expression and activation,1 the penultimate stage of mammary gland development involving lobuloalveolar expansion and epithelial functional differentiation, absolutely requires ErbB4 expression.7,8 Molecular analysis of alveolar defects in the absence of ErbB4 revealed two divergent ErbB4 activities contributing to lactational failure at parturition. ErbB4-deficient mammary epithelium failed to express the mammary differentiation marker, sodium phosphate co-transporter type IIb (Npt2b), indicating a critical role for ErbB4 during epithelial terminal differentiation. Somewhat surprising was a concomitant defect in epithelial proliferation.8 Multiple activities for ErbB4 in the mammary gland at parturition implies that ErbB4 couples to distinct signaling pathways in this tissue. Interestingly, defects in mammary epithelial differentiation and impaired lactation are features of Stat5a-null mice.9-11 Stat5 is a member of the signal transduction and activator of transcription family and is essential for the transactivation of critical lactation products. Interestingly, Stat5 protein in ErbB4-deficient mammary glands fails to translocate to the nucleus and lacks phosphorylation at the regulatory Y694.7,8 Moreover, lactational failure in the absence of ErbB4 is associated with ablated expression of the Stat5 regulated milk-genes β-casein and whey acidic protein (WAP).8 Consistent with this observation, ErbB4 stimulates Stat5 transcriptional activation of a β-casein promoter reporter plasmid when co-transfected in human breast cancer cell lines (J. Allison and F. Jones, unpublished observations). Accumulative evidence strongly implies that the loss of Stat5 activity directly contributes to impaired differentiation and lactational failure observed in ErbB4-deficient mammary glands. Molecular mechanisms regulating ErbB4 proliferation in the mammary gland remain unclear. Coordinate mammary expression of the ErbB4 ligand, HRGα, with blunted epithelial proliferation observed in ErbB4-deficient mammary glands1,12 raises the possibility that this ligand regulates ErbB4 proliferative activity. In support of this contention, HRGα-null and ErbB4-deficient mammary glands display overlapping defects in epithelial proliferation at parturition.8,13 Stat5, however, remains active in HRGα-null mammary glands (W. Long and F. Jones, unpublished observations) suggesting that HRGα regulation of ErbB4 proliferative activity would involve a Stat5 independent pathway. The essential contribution of ErbB4 to epithelial differentiation and lactation supports a novel signaling paradigm in the breast. The developmental events culminating in lactation at parturition have historically been attributed to prolactin activation of the prolactin receptor (PrlR), which is also an essential mediator of Stat5 activity in the breast. Our results however suggest that PrlR is dispensable for epithelial differentiation at late pregnancy,8 rather PrlR mediates cell specification during the pregnancy induced transition from ductal to secretory epithelium.10,14 Secretory epithelium accumulate during pregnancy in the absence of ErbB4, however, despite evidence of intact PrlR signaling, Stat5 is not activated and epithelium fail to undergo terminal differentiation in ErbB4-null mammary glands. Thus we propose that PrlR and ErbB4 cooperate to activate Stat5, supporting a “receptor switch” model of Stat5 regulation in the pregnant mammary gland (Fig. 1). The “switch” between obligate Stat5 activating receptors may be regulated by the temporal expression of receptor specific hormones. Regulation of receptor switching by ligand activity is supported by overlapping late pregnancy/parturition phenotypes of ErbB4-deficient mammary glands with mutations affecting expression or processing of the EGF-like growth factors and ErbB4 agonists HRGα,13 HB-EGF,15 and AR/EGF/TGFα.16 The mechanistic “switch” from PrlR to ErbB4 regulation of Stat5 may be driven by novel Stat5 phosphorylation events induced by ErbB4. For example, both PrlR and ErbB4 stimulate phosphorylation of Stat5 at the regulatory Y694, however, ErbB4 also induces phosphorylation of additional, unidentified, Stat5 tyrosine residues.17 Furthermore and unique to ErbB4 are phosphorylation events at multiple Stat5 serine residues (Lane R, Jones F, unpublished observations). One of these phosphorylation sites at S779 has been reported by several labs as an important event modulating Stat5 transactivation of the β-casein promoter.18-20 Interestingly, the serine/ threonine kinase p21-activated kinase (Pak) 1 mediates Stat5 phosphorylation at S779.21 Furthermore, targeted expression of a dominant negative Pak1 to the mouse mammary gland results in a lobuloalveolar phenotype that directly overlaps with phenotypes we have reported for ErbB4-null mammary glands, including lack of Stat5 phosphorylation and reduced β-casein and WAP expression.19 Although still preliminary, these results imply that coupled ErbB4 and Pak1 signaling in the developing mammary gland reveal novel ErbB4 mediated Stat5 activities essential for lactogenesis. These novel Stat5 phosphorylation events may underlie the mechanistic switch between PrlR and ErbB4 regulation of Stat5 during pregnancy. ERBB4 SIGNALING DURING BREAST AND NEURAL DEVELOPMENT e62 Cell Cycle 2003; Vol. 2 Issue 6 Figure 1. ErbB4 and PrlR cooperative to activate Stat5 during breast development. During the initial stages of pregnancy PrlR and associated tyrosine kinase, Janus-Kinase 2 (JAK2), are essential for Stat5 activation and epithelial specification as indicated by reduced expression of the ductal epithelial marker sodium potassium chloride co-transporter 1 (NKCC1). The progression of pregnancy to parturition is accompanied by an increase in EGF-like activity and this relative change in ligand activity may in part mediate the “switch” from PrlR to ErbB4 as the obligate Stat5 activating receptor. Coupled ErbB4 and Stat5 signaling is essential for epithelial terminal differentiation at late pregnancy (indicated by increased Npt2b expression) and lactation at parturition. Signaling pathways regulating ErbB4 mitogenic responses remain unclear. Interestingly, ErbB4 mediates phosphorylation of Stat5 at unique tyrosine and serine residues (P-tyr and P-ser are indicated by red and blue circles, respectively). Corroborative evidence implies that serine phosphorylation of Stat5 is mediated in part by Pak1. We propose that unique Stat5 activities associated with novel ErbB4 regulated phosphorylation events drives the late pregnancy switch from PrlR to ErbB4 as the obligate, Stat5 regulating, receptor.