Hepatocyte growth factor (HGF) and its receptor, the Met tyrosine kinase, are determinants of placenta, liver, and muscle development. Here, we show that Met function in vivo requires signaling via two carboxy-terminal tyrosines. Mutation of both residues in the mouse genome caused embryonal death, with placenta, liver, and limb muscle defects, mimicking the phenotype of met null mutants. In contrast, disrupting the consensus for Grb2 binding allowed development to proceed to term without affecting placenta and liver but caused a striking reduction in limb muscle coupled to a generalized deficit of secondary fibers. These data show that the requirements for Met signaling vary depending on the tissue and reveal a novel role for HGF/ Met in late myogenesis.

Peptide hormones are small, processed, and secreted peptides that signal via membrane receptors and play critical roles in normal and pathological physiology. The search for novel peptide hormones has been hampered by their small size, low or restricted expression, and lack of sequence similarity. To overcome these difficulties, we developed a bioinformatics search tool based on the hidden Markov model formalism that uses several peptide hormone sequence features to estimate the likelihood that a protein contains a processed and secreted peptide of this class. Application of this tool to an alignment of mammalian proteomes ranked 90% of known peptide hormones among the top 300 proteins. An analysis of the top scoring hypothetical and poorly annotated human proteins identified two novel candidate peptide hormones. Biochemical analysis of the two candidates, which we called spexin and augurin, showed that both were localized to secretory granules in a transfected pancreatic cell line and were recovered from the cell supernatant. Spexin was expressed in the submucosal layer of the mouse esophagus and stomach, and a predicted peptide from the spexin precursor induced muscle contraction in a rat stomach explant assay. Augurin was specifically expressed in mouse endocrine tissues, including pituitary and adrenal gland, choroid plexus, and the atrio-ventricular node of the heart. Our findings demonstrate the utility of a bioinformatics approach to identify novel biologically active peptides. Peptide hormones and their receptors are important diagnostic and therapeutic targets, and our results suggest that spexin and augurin are novel peptide hormones likely to be involved in physiological homeostasis.

Tumor necrosis factor-␣ (TNF-␣) modulates gene expression in endothelial cells and is angiogenic in vivo.TNF-␣ does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-␣ up-regulates in a dose-and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its coreceptor neuropilin-1 in human endothelium. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-␣ is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-␣-mediated pathological settings.

Angiogenic factors produced by tumor cells are essential for tumor growth and metastasis. In our study, the expression of Angiopoietin-1 (ANG1) and Angiopoietin-2 (ANG2) mRNA in archival human breast cancer tumor samples and in 6 breast cancer cell lines was investigated. Total RNA from biopsies of 38 breast cancer patients was extracted and ANG1 and ANG2 mRNA expression was measured by means of quantitative real-time RT-PCR (Taqmanா). Matching data with available clinicopathologic and biochemical data revealed a significant association between ANG2 expression and axillary lymph node invasion. Univariate and multivariate survival analysis, by means of Kaplan-Meier method and Cox's proportional hazards model, showed significant and independent association between ANG2 mRNA level and both disease-free (p < 0.0001) and overall survival (p < 0.0003). An important fact is that, notwithstanding the small number of cases examined, this association was confirmed also in the group of lymph node-negative patients (DFS, p < 0.003; OS, p < 0.020). Immunohistochemical analysis demonstrated that Ang2 is expressed by both tumor cells and endothelial elements. Expression in tumor cells was confirmed by studying a panel of human breast carcinoma cell lines in culture by RT-PCR. In ZR75.1 and T47D cells, expression of ANG2 mRNA was increased up to 10-fold by treatment with estrogen within 24 hr. Although preliminary, these data suggest a possible role of ANG2 as a prognostic factor for primary breast cancer.

No abstract

The biological effects of hepatocyte growth factor/scatter factor are mediated by autophosphorylation of its receptor, the Met tyrosine kinase, on two carboxyl-terminal tyrosines. These phosphotyrosines (Y1349VHVNATY1356VNV) are multifunctional docking sites for several effectors. Grb2, the adaptor for the Ras guanyl-nucleotide exchanger SOS, binds to Tyr1356 in the YVNV motif. By site-directed mutagenesis we either abrogated or duplicated the Grb2 consensus, without interfering with the other effectors. Loss of the link with Grb2 severely impaired transformation. The same mutation, however, had no effect on the "scattering" response, indicating that the level of signal which can be reached by Grb2-independent routes is permissive for motility. Duplication of the Grb2 binding site enhanced transformation and left motility unchanged. Thus, two Met-mediated biological responses, motility and growth, can be dissociated on the basis of their differential requirement for a direct link with Ras.

Numerous studies link decreased serotonin metabolites with increased impulsive and aggressive traits. However, although pharmacological depletion of serotonin is associated with increased aggression, interventions aimed at directly decreasing serotonin neuron activity have supported the opposite association. Furthermore, it is not clear if altered serotonin activity during development may contribute to some of the observed associations. Here, we used two pharmacogenetic approaches in transgenic mice to selectively and reversibly reduce the firing of serotonin neurons in behaving animals. Conditional overexpression of the serotonin 1A receptor (Htr1a) in serotonin neurons showed that a chronic reduction in serotonin neuron firing was associated with heightened aggression. Overexpression of Htr1a in adulthood, but not during development, was sufficient to increase aggression. Rapid suppression of serotonin neuron firing by agonist treatment of mice expressing Htr1a exclusively in serotonin neurons also led to increased aggression. These data confirm a role of serotonin activity in setting thresholds for aggressive behavior and support a direct association between low levels of serotonin homeostasis and increased aggression.

Sudden infant death syndrome is the leading cause of death in the postneonatal period in developed countries. Postmortem studies show alterations in serotonin neurons in the brainstem of such infants. However, the mechanism by which altered serotonin homeostasis might cause sudden death is unknown. We investigated the consequences of altering the autoinhibitory capacity of serotonin neurons with the reversible overexpression of serotonin 1A autoreceptors in transgenic mice. Overexpressing mice exhibited sporadic bradycardia and hypothermia that occurred during a limited developmental period and frequently progressed to death. Moreover, overexpressing mice failed to activate autonomic target organs in response to environmental challenges. These findings show that excessive serotonin autoinhibition is a risk factor for catastrophic autonomic dysregulation and provide a mechanism for a role of altered serotonin homeostasis in sudden infant death syndrome.