Heparin‐binding epidermal growth factor (HB‐EGF) and betacellulin (BTC) are activating ligands for EGF receptor (EGFR/ErbB1) and ErbB4. To identify their physiological functions, we disrupted mouse HB‐EGF and BTC alleles by homologous recombination. Most HB‐EGF−/− mice died before weaning, and survivors had enlarged, dysfunctional hearts and reduced lifespans. Although BTC−/− mice were viable and fertile and displayed no overt defects, the lifespan of double null HB‐EGF−/−/BTC−/− mice was further reduced, apparently due to accelerated heart failure. HB‐EGF−/− newborns had enlarged and malformed semilunar and atrioventricular heart valves, and hypoplastic, poorly differentiated lungs. Defective cardiac valvulogenesis was the result of abnormal mesenchymal cell proliferation during remodeling, and was associated with dramatic increases in activated Smad1/5/8. Consistent with the phenotype, HB‐EGF transcripts were localized to endocardial cells lining the margins of wild‐type valves. Similarly defective valvulogenesis was observed in newborn mice lacking EGFR and tumor necrosis factor‐α converting enzyme (TACE). These results suggest that cardiac valvulogenesis is dependent on EGFR activation by TACE‐derived soluble HB‐EGF, and that EGFR signaling is required to regulate bone morphogenetic protein signaling in this context.

It is becoming clear that converging pathways coordinate early heart valve development and remodeling into functional valve leaflets. The integration of these pathways begins with macro and molecular interactions outside the cell in the extracellular matrix separating the myocardial and endocardial tissue components of the rudimentary heart. Such interactions regulate events at the cell surface through receptors, proteases, and other membrane molecules which in turn transduce signals into the cell. These signals trigger intracellular cascades that transduce cellular responses through both transcription factor and cofactor activation mediating gene induction or suppression. Chamber septation and valve formation occur from these coordinated molecular events within the endocardial cushions to sustain unidirectional blood flow and embryo viability. This review discusses the emerging connection between extracellular matrix and growth factor receptor signaling during endocardial cushion morphogenesis by highlighting the extracellular component, hyaluronan, and erbB receptor functions during early valve development.

EGF family growth factors, including transforming growth factor-alpha (TGFalpha), amphiregulin (AR), and heparin-binding EGF (HB-EGF), are invariably expressed as transmembrane precursors that are cleaved at one or more sites in the extracellular domain to release soluble growth factor. Considerable attention has focused on the identification of proteases responsible for these processing events. We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the generation of soluble TGFalpha from its transmembrane precursor, proTGFalpha. Here, we review our findings that primary keratinocytes from Tace(deltaZn/deltaZn) mice, which express a nonfunctional TACE, released dramatically lower levels of soluble TGFalpha compared to their normal counterparts, even though TGFalpha mRNA and cell-associated protein levels were similar in the two cell populations. Restoration of TACE activity in Tace(deltaZn/deltaZn) cells increased shedding of TGFalpha species, including the mature, 6-kDa protein. Further, exogenous TACE enzyme accurately cleaved the N-terminal processing site of proTGFalpha in cell lysates, as well as both physiologic sites of a soluble proTGFalpha ectodomain. TACE also accurately cleaved peptide substrates corresponding to the processing sites of several additional EGF family members, and restoration of TACE activity enhanced the shedding of soluble AR and HB-EGF proteins from Tace(deltaZn/deltaZn) cells. Finally, reduction of functional TACE gene dosage greatly exacerbated the open-eye defect of Egfr(wa-2/wa-2) newborns, which is regulated by redundant actions of several EGF family ligands. The implications of these results for the biology of the EGF family and TACE are discussed.

The mechanisms that regulate the transition between the initial priming phase and DNA replication in liver regeneration are poorly understood. To study this transition, we compared events occurring after standard two-thirds partial hepatectomy, which elicits full regeneration, with response to a reduced hepatectomy, onethird partial hepatectomy (1/3PH), which leads to little DNA replication. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between the two procedures, cell cycle progression was significantly blunted in 1/3PH mice. Among the main defects observed in 1/3PH mice were an almost complete deficiency in retinoblastoma phosphorylation and the lack of increase in kinase activity associated with cyclin E. We report that, in two-thirds partial hepatectomy mice, the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) preceded the start of DNA replication and was not detectable in 1/3PH animals. Injection of HB-EGF into 1/3PH mice resulted in a >15-fold increase in DNA replication. Moreover, we show that hepatocyte DNA replication was delayed in HB-EGF knock-out mice. In summary, we show that HB-EGF is a key factor for hepatocyte progression through G 1 /S transition during liver regeneration.

Abstract-Stimulation of ␣ 1 -adrenoceptors induces proliferation of vascular smooth muscle cells (SMCs) and contributes to arterial remodeling. Although activation of NAD(P)H oxidase and generation of reactive oxygen species (ROS) are required, little is known about this pathway. In this study, we examined the hypothesis that epidermal growth factor receptor (EGFR) transactivation and extracellular regulated kinases (ERK) are involved in ␣ 1 -adrenoceptor-mediated SMC growth. Phenylephrine increased protein synthesis in association with a rapid (Յ5 minutes) and sustained (Ն60 minutes) doubling of phosphorylation of EGFR and ERK1/2, but not p38 or JNK in the media of rat aorta maintained in organ culture. Antagonists of EGFR phosphotyrosine activity (AG-1478) and ERK phosphorylation (PD-98059, U-0126) abolished phenylephrine-induced protein synthesis, whereas antagonists of p38 or JNK phosphorylation had no specific effect. A competitive antagonist (P22) for heparin binding EGF-like growth factor (HB-EGF) blocked phenylephrine-induced protein synthesis, as did downregulation of pro-HB-EGF (CRM197). Phenylephrine-induced protein synthesis was inhibited by neutralizing antibody to HB-EGF and absent in HB-EGF Ϫ/Ϫ SMCs. Inhibitors of metalloproteinases (BiPS, KB-R7785) also blocked adrenergic growth. The neutralizing antibody against HB-EGF had no effect on the two-fold increase in ROS generation induced by phenylephrine (DCF fluorescence), suggesting that stimulation of NAD(P)H oxidase by ␣ 1 -adrenoceptor occupation precedes HB-EGF release. Cell culture studies confirmed and extended these findings. These data suggest that ␣ 1 -adrenoceptor-mediated SMC growth requires ROS-dependent shedding of HB-EGF, transactivation of EGFR, and activation of the MEK1/2-dependent MAP kinase pathway. This trophic pathway may link sympathetic activity to arterial wall growth in adaptive remodeling and hypertrophic disease. Key Words: ␣-adrenergic receptor Ⅲ vascular smooth muscle cell proliferation Ⅲ signal transduction Ⅲ reactive oxygen species Ⅲ metalloproteinase V ascular smooth muscle cell (SMC) proliferation, hypertrophy, and migration are central to development of vascular disease such as restenosis after vessel injury, atherosclerosis, and wall hypertrophy. In addition to evidence that prolonged elevation of plasma catecholamines is a risk factor for vascular diseases, 1,2 recent reports have shown that catecholamines directly induce hypertrophy of the arterial wall by stimulation of ␣ 1 -adrenoceptors (␣ 1 -ARs), which are G-protein-coupled receptors (GPCRs). Catecholamine stimulation in cell and organ culture induces dose-dependent proliferation, protein synthesis, and migration of SMCs and adventitial fibroblasts and promotes dedifferentiation of the SMC phenotype. [3][4][5][6][7][8] Furthermore, the potency of these effects is strongly augmented in injured arteries. 7 Similar effects are seen in vivo, where endogenous vascular wall catecholamines contribute to hypertrophy, fibrosis, and lumen loss after balloon injury of...