Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein–coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal–regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.

Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin promoting migration, proliferation, and survival in a wide spectrum of cells, can also modulate different biological responses in stem cells, but the mechanisms involved are not completely understood so far. In this context, we show that short-term exposure of mesenchymal stem cells (MSCs) to HGF can induce the activation of its cognate Met receptor and the downstream effectors ERK1/2, p38MAPK, and PI3K/Akt, while long-term exposure to HGF resulted in cytoskeletal rearrangement, cell migration, and marked inhibition of proliferation through the arrest in the G 1 -S checkpoint.When added to MSCs, the K252A tyrosine kinase inhibitor prevented HGF-induced responses. HGF's effect on MSC proliferation was reversed by p38 inhibitor SB203580, while the effects on cell migration were abrogated by PI3K inhibitor Wortmannin, suggesting that HGF acts through different pathways to determine its complex effects on MSCs. Prolonged treatment with HGF induced the expression of cardiac-specific markers (GATA-4, MEF2C, TEF1, desmin, ␣-MHC, ␤-MHC, and nestin) with the concomitant loss of the stem cell markers nucleostemin, c-kit, and CD105. STEM CELLS 2006;24: 23-33

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The human c-MET oncogene encodes a transmembrane tyrosine kinase (p190c-met) with structural and functional features of a growth-factor receptor. Monoclonal antibodies (MAbs) have been used to investigate the distribution of the c-Met protein in human normal and neoplastic tissues. By immunofluorescence microscopy homogeneous expression was detected in normal hepatocytes as well as in epithelial cells lining the stomach, the small and the large intestine. Positive staining was also found in epithelial cells of the endometrium and ovary, and in basal keratinocytes of esophagus and skin. By Northern blot analysis, high levels of c-met messenger RNA were detected in specimens of liver, gastro-intestinal tract and kidney. c-met-specific mRNA was also found in thyroid, pancreas and placenta, in which organs c-Met protein was barely detectable by immunofluorescence. The antibodies revealed expression of c-MET protein in hepatomas (11/14), carcinomas of colon and rectum (19/21), stomach (11/22), kidney (16/19), ovary (9/17) and skin (7/17). Carcinomas of the lung (13/20), thyroid (11/13) and pancreas (5/7) were also positive. In these last cases (lung, thyroid and pancreas) tumor cells were homogeneously stained by the antibodies, whereas in their normal counterparts staining was barely detectable. These data suggest that the receptor encoded by c-MET plays a physiological role in epithelial cell growth and that its expression is altered in human carcinomas.

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Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vm. of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.Tyrosine kinases are crucially involved in the transduction of growth-promoting stimuli to the cell interior. Transmembrane growth factor receptors such as the epidermal and platelet-derived growth factor, insulin, and colony-stimulating factor 1 receptors, are endowed with ligand-induced tyrosine kinase activity. Upon ligand binding, they display a short-lived pulse of activity leading to their autophosphorylation and to the phosphorylation of cellular substrates on tyrosine residues (for a review, see references 52 and 56). Membrane-associated tyrosine kinases of the src family are thought to be similarly involved in signal transduction operated by a distinct set of receptors. The most abundant class of oncogenes comprises the genes coding for both types of tyrosine kinases. Whatever the mechanism, activation of their transforming potential leads to the expression of a protein with nonregulated, often enhanced enzymatic activity (reviewed in references 28, 32, and 33).The enzymatic activity of tyrosine kinases can be modulated in several ways (reviewed in references 33, 52, and 56). Ligands are well-known activators of the receptors. Structural alterations such as N-and C-terminal truncation and point mutations are critical for the transforming proteins. In addition, phosphorylation is a ubiquitous way of transiently modulating tyrosine kinase activity. Tyrosine autophosphorylation has often been associated with activation. Protein kinase C-mediated serine or threonine phosphorylation has been shown to be inhibitory for some gr...

Summary Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The MetlHGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P=0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P<0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer.

New biomimetic magnetite nanoparticles (hereafter BMNPs) with sizes larger than most common superparamagnetic nanoparticles were produced in the presence of the recombinant MamC protein from Magnetococcus marinus MC-1 and functionalized with doxorubicin (DOXO) intended as potential drug nanocarriers. Unlike inorganic magnetite nanoparticles, in BMNPs the MamC protein controls their size and morphology, providing them with magnetic properties consistent with a large magnetic moment per particle; moreover, it provides the nanoparticles with novel surface properties. BMNPs display the isoelectric point at pH 4.4, being strongly negatively charged at physiological pH (pH 7.4). This allows both (i) their functionalization with DOXO, which is positively charged at pH 7.4, and (ii) the stability of the DOXO–surface bond and DOXO release to be pH dependent and governed by electrostatic interactions. DOXO adsorption follows a Langmuir–Freundlich model, and the coupling of DOXO to BMNPs (binary biomimetic nanoparticles) is very stable at physiological pH (maximum release of 5% of the drug adsorbed). Conversely, when pH decreases, these electrostatic interactions weaken, and at pH 5, DOXO is released up to ∼35% of the amount initially adsorbed. The DOXO–BMNPs display cytotoxicity on the GTL-16 human gastric carcinoma cell line in a dose-dependent manner, reaching about ∼70% of mortality at the maximum amount tested, while the nonloaded BMNPs are fully cytocompatible. The present data suggest that BMNPs could be useful as potential drug nanocarriers with a drug adsorption-release governed by changes in local pH values.