Pax3 modulates expression of the c-Met receptor during limb muscle development.

Pax3 is a transcription factor whose expression has been used as a marker of myogenic precursor cells arising in the lateral somite destined to migrate to and populate the limb musculature. Accruing evidence indicates that the embryologic origins of axial and appendicular muscles are distinct, and limb muscle abnormalities in both mice and humans harboring Pax3 mutations support this distinction. The mechanisms by which Pax3 affects limb muscle development are unknown. The tyrosine kinase receptor for hepatocyte growth factor/scatter factor encoded by the c-met protooncogene is also expressed in limb muscle progenitors and, like Pax-3, is required in the mouse for limb muscle development. Here, we show that c-met expression is markedly reduced in the lateral dermomyotome of Splotch embryos lacking Pax3. We show that Pax3 can stimulate c-met expression in cultured cells, and we identify a potential Pax3 binding site in the human c-MET promoter that may contribute to direct transcriptional regulation. In addition, we have found that several cell lines derived from patients with rhabdomyosarcomas caused by a t(2;13) chromosomal translocation activating PAX3 express c-MET, whereas those rhabdomyosarcoma cell lines examined without the translocation do not. These results are consistent with a model in which Pax3 modulates c-met expression in the lateral dermomyotome, a function that is required for the appropriate migration of these myogenic precursors to the limb where the ligand for c-met (hepatocyte growth factor/scatter factor) is expressed at high levels.

Morphogenetic Effects of Neuregulin (Neu Differentiation Factor) in Cultured Epithelial Cells

Neuregulin, or neu differentiation factor, induces cell proliferation or differentiation through interaction with members of the ErbB family of receptor tyrosine kinases. We report that neuregulin can also induce profound morphogenic responses in cultured epithelial cells of different origins. These effects include scattering of small epithelial islands and rearrangement of larger cell islands into ordered ring-shaped arrays with internal lumens. The ring-forming cells are interconnected by cadherin- and β-catenin-containing adherens junctions. In confluent cultures, neuregulin treatment induces formation of circular lumenlike gaps in the monolayer. Both cell scattering and ring formation are accompanied by a marked increase in cell motility that is independent of hepatocyte growth factor/scatter factor and its receptor (c-Met). Affinity-labeling experiments implied that a combination of ErbB-2 with ErbB-3 mediates the morphogenic signal of neuregulin in gastric cells. Indeed, a similar morphogenic effect could be reconstituted in nonresponsive cells by coexpression of ErbB-2 and -3. We conclude that a heterodimer between the kinase-defective neuregulin receptor, ErbB-3, and the coreceptor, ErbB-2, mediates the morphogenetic action of neuregulin.

Androgen-repressed phenotype in human prostate cancer

An androgen-repressed human prostate cancer cell line, ARCaP, was established and characterized. This cell line was derived from the ascites fluid of a patient with advanced metastatic disease. In contrast to the behavior of androgen-dependent LNCaP and its androgen-independent C4-2 subline, androgen and estrogen suppress the growth of ARCaP cells in a dose-dependent manner in vivo and in vitro. ARCaP is tumorigenic and highly metastatic. It metastasizes to the lymph node, lung, pancreas, liver, kidney, and bone, and forms ascites fluid in athymic hosts. ARCaP cells express low levels of androgen receptor mRNA and prostate-specific antigen mRNA and protein. Immunohistochemical staining shows that ARCaP cells stain intensely for epidermal growth factor receptor, c-erb B2/neu, and c-erb B3. Staining is negative for chromogranin A and positive for bombesin, serotonin, neuron-specific enolase, and the c-met protooncogene (a hepatic growth factor/scatter factor receptor). ARCaP cells also secrete high levels of gelatinase A and B and some stromelysin, which suggests that this cell line may contain markers representing invasive adenocarcinoma with selective neuronendocrine phenotypes. Along with its repression of growth, androgen is also found to repress the expression of prostate-specific antigen in ARCaP cells as detected by a prostate-specific antigen promoter–β-galactosidase reporter assay. Our results suggest that the androgen-repressed state may be central to prostate cancer progression and that advanced prostate cancer can progress from an androgen-independent to an androgen-repressed state.

Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro.

Hepatocyte growth factor (HGF), also known as scatter factor, is a powerful motogen, mitogen, and morphogen produced by cells of mesodermal origin, acting on epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. We show that the HGF receptor is expressed by human primary osteoclasts, by osteoclast-like cell lines, and by osteoblasts. In both cell lineages, HGF stimulation triggers the receptor kinase activity and autophosphorylation. In osteoclasts, HGF receptor activation is followed by increase in intracellular Ca2+ concentration and by activation of the pp60c-Src kinase. HGF induces changes in osteoclast shape and stimulates chemotactic migration and DNA replication. Osteoblasts respond to HGF by entering the cell cycle, as indicated by stimulation of DNA synthesis. Interestingly, osteoclasts were found to synthesize and secrete biologically active HGF. These data strongly suggest the possibility of an autocrine regulation of the osteoclast by HGF and a paracrine regulation of the osteoblast by the HGF produced by the osteoclast.

Basic fibroblast growth factor can mediate the early inductive events in renal development.

The earliest characterized events during induction of tubulogenesis in renal anlage include the condensation or compaction of metanephrogenic mesenchyme with the concurrent upregulation of WT1, the gene encoding the Wilms tumor transcriptional activator/suppressor. We report that basic fibroblast growth factor (FGF2) can mimic the early effects of an inductor tissue by promoting the condensation of mesenchyme and inhibiting the tissue degeneration associated with the absence of an inductor tissue. By in situ hybridization, FGF2 was also found to mediate the transcriptional activation of WT1 and of the hepatocyte growth factor receptor gene, c-met. Although FGF2 can induce these early events of renal tubulogenesis, it cannot promote the epithelial conversion associated with tubule formation in metanephrogenic mesenchyme. For this, an undefined factor(s) from pituitary extract in combination with FGF2 can cause tubule formation in uninduced mesenchyme. These findings support the concept that induction in kidney is a multiphasic process that is mediated by more than a single comprehensive inductive factor and that soluble molecules can mimic these inductive activities in isolated uninduced metanephrogenic mesenchyme.

Unusual phenotypic alteration of β amyloid precursor protein (βAPP) maturation by a new Val-715 → Met βAPP-770 mutation responsible for probable early-onset Alzheimer’s disease

We have identified a novel β amyloid precursor protein (βAPP) mutation (V715M-βAPP770) that cosegregates with early-onset Alzheimer’s disease (AD) in a pedigree. Unlike other familial AD-linked βAPP mutations reported to date, overexpression of V715M-βAPP in human HEK293 cells and murine neurons reduces total Aβ production and increases the recovery of the physiologically secreted product, APPα. V715M-βAPP significantly reduces Aβ40 secretion without affecting Aβ42 production in HEK293 cells. However, a marked increase in N-terminally truncated Aβ ending at position 42 (x-42Aβ) is observed, whereas its counterpart x-40Aβ is not affected. These results suggest that, in some cases, familial AD may be associated with a reduction in the overall production of Aβ but may be caused by increased production of truncated forms of Aβ ending at the 42 position.

The Multisubstrate Docking Site of the MET Receptor Is Dispensable for MET-mediated RAS Signaling and Cell Scattering

The scatter factor/hepatocyte growth factor regulates scattering and morphogenesis of epithelial cells through activation of the MET tyrosine kinase receptor. In particular, the noncatalytic C-terminal tail of MET contains two autophosphorylation tyrosine residues, which form a multisubstrate-binding site for several cytoplasmic effectors and are thought to be essential for signal transduction. We show here that a MET receptor mutated on the four C-terminal tyrosine residues, Y1311F, Y1347F, Y1354F, and Y1363F, can induce efficiently a transcriptional response and cell scattering, whereas it cannot induce cell morphogenesis. Although the mutated receptor had lost its ability to recruit and/or activate known signaling molecules, such as GRB2, SHC, GAB1, and PI3K, by using a sensitive association–kinase assay we found that the mutated receptor can still associate and phosphorylate a ∼250-kDa protein. By further examining signal transduction mediated by the mutated MET receptor, we established that it can transmit efficient RAS signaling and that cell scattering by the mutated MET receptor could be inhibited by a pharmacological inhibitor of the MEK-ERK (MAP kinase kinase–extracellular signal-regulated kinase) pathway. We propose that signal transduction by autophosphorylation of the C-terminal tyrosine residues is not the sole mechanism by which the activated MET receptor can transmit RAS signaling and cell scattering.

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used 1H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small ΔG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger ΔG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.

Cytochrome c′ folding triggered by electron transfer: Fast and slow formation of four-helix bundles

Reduced (FeII) Rhodopseudomonas palustris cytochrome c′ (Cyt c′) is more stable toward unfolding ([GuHCl]1/2 = 2.9(1) M) than the oxidized (FeIII) protein ([GuHCl]1/2 = 1.9(1) M). The difference in folding free energies (ΔΔGf° = 70 meV) is less than half of the difference in reduction potentials of the folded protein (100 mV vs. NHE) and a free heme in aqueous solution (≈−150 mV). The spectroscopic features of unfolded FeII–Cyt c′ indicate a low-spin heme that is axially coordinated to methionine sulfur (Met-15 or Met-25). Time-resolved absorption measurements after CO photodissociation from unfolded FeII(CO)–Cyt c′ confirm that methionine can bind to the ferroheme on the microsecond time scale [kobs = 5(2) × 104 s−1]. Protein folding was initiated by photoreduction (two-photon laser excitation of NADH) of unfolded FeIII–Cyt c′ ([GuHCl] = 2.02–2.54 M). Folding kinetics monitored by heme absorption span a wide time range and are highly heterogeneous; there are fast-folding (≈103 s−1), intermediate-folding (102–101 s−1), and slow-folding (10−1 s−1) populations, with the last two likely containing methionine-ligated (Met-15 or Met-25) ferrohemes. Kinetics after photoreduction of unfolded FeIII–Cyt c′ in the presence of CO are attributable to CO binding [1.4(6) × 103 s−1] and FeII(CO)–Cyt c′ folding [2.8(9) s−1] processes; stopped-flow triggered folding of FeIII–Cyt c′ (which does not contain a protein-derived sixth ligand) is adequately described by a single kinetics phase with an estimated folding time constant of ≈4 ms [ΔGf° = −33(3) kJ mol−1] at zero denaturant.

Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells.

Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligand-induced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.