NMDA Receptor and Its Emerging Role in Cancer.

Glutamate is a key player in excitatory neurotransmission in the central nervous system (CNS). The N-methyl-D-aspartate receptor (NMDAR) is a glutamate-gated ion channel which presents several unique features and is involved in various physiological and pathological neuronal processes. Thanks to great efforts in neuroscience, its structure and the molecular mechanisms controlling its localization and functional regulation in neuronal cells are well known. The signaling mediated by NMDAR in neurons is very complex as it depends on its localization, composition, Ca2+ influx, and ion flow-independent conformational changes. Moreover, NMDA receptors are highly diffusive in the plasma membrane of neurons, where they form heterocomplexes with other membrane receptors and scaffold proteins which determine the receptor function and activation of downstream signaling. Interestingly, a recent paper demonstrates that NMDAR signaling is involved in epithelial cell competition, an evolutionary conserved cell fitness process influencing cancer initiation and progress. The idea that NMDAR signaling is limited to CNS has been challenged in the past two decades. A large body of evidence suggests that NMDAR is expressed in cancer cells outside the CNS and can respond to the autocrine/paracrine release of glutamate. In this review, we survey research on NMDAR signaling and regulation in neurons that can help illuminate its role in tumor biology. Finally, we will discuss existing data on the role of the glutamine/glutamate metabolism, the anticancer action of NMDAR antagonists in experimental models, NMDAR synaptic signaling in tumors, and clinical evidence in human cancer.

The PSI Domain of the MET Oncogene Encodes a Functional Disulfide Isomerase Essential for the Maturation of the Receptor Precursor.

The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of α and ß chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand-hepatocyte growth factor (HGF)-such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein.

Two-Chains Tissue Plasminogen Activator Unifies Met and NMDA Receptor Signalling to Control Neuronal Survival.

Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-d-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.

The Long-Lasting Protective Effect of HGF in Cardiomyoblasts Exposed to Doxorubicin Requires a Positive Feed-Forward Loop Mediated by Erk1,2-Timp1-Stat3.

Previous studies showed that the hepatocyte growth factor (HGF)-Met receptor axis plays long-lasting cardioprotection against doxorubicin anti-cancer therapy. Here, we explored the mechanism(s) underlying the HGF protective effect. DNA damage was monitored by histone H2AX phosphorylation and apoptosis by proteolytic cleavage of caspase 3. In doxorubicin-treated H9c2 cardiomyoblasts, the long-lasting cardioprotection is mediated by activation of the Ras/Raf/Mek/Erk (extracellular signal-regulated kinase 1,2) signaling pathway and requires Stat3 (signal transducer and activator of transcription 3) activation. The HGF protection was abrogated by the Erk1,2 inhibitor, PD98059. This translated into reduced Y705 phosphorylation and impaired nuclear translocation of Stat3, showing crosstalk between Erk1,2 and Stat3 signaling. An array of 29 cytokines, known to activate Stat3, was interrogated to identify the molecule(s) linking the two pathways. The analysis showed a selective increase in expression of the tissue inhibitor of metalloproteinases-1 (Timp1). Consistently, inhibition in cardiomyoblasts of Timp1 translation by siRNAs blunted both Stat3 activation and the cardioprotective effect of HGF. Thus, Timp1 is responsible for the generation of a feed-forward loop of Stat3 activation and helps cardiomyocytes to survive during the genotoxic stress induced by anthracyclines.

ERK: A Key Player in the Pathophysiology of Cardiac Hypertrophy.

Cardiac hypertrophy is an adaptive and compensatory mechanism preserving cardiac output during detrimental stimuli. Nevertheless, long-term stimuli incite chronic hypertrophy and may lead to heart failure. In this review, we analyze the recent literature regarding the role of ERK (extracellular signal-regulated kinase) activity in cardiac hypertrophy. ERK signaling produces beneficial effects during the early phase of chronic pressure overload in response to G protein-coupled receptors (GPCRs) and integrin stimulation. These functions comprise (i) adaptive concentric hypertrophy and (ii) cell death prevention. On the other hand, ERK participates in maladaptive hypertrophy during hypertension and chemotherapy-mediated cardiac side effects. Specific ERK-associated scaffold proteins are implicated in either cardioprotective or detrimental hypertrophic functions. Interestingly, ERK phosphorylated at threonine 188 and activated ERK5 (the big MAPK 1) are associated with pathological forms of hypertrophy. Finally, we examine the connection between ERK activation and hypertrophy in (i) transgenic mice overexpressing constitutively activated RTKs (receptor tyrosine kinases), (ii) animal models with mutated sarcomeric proteins characteristic of inherited hypertrophic cardiomyopathies (HCMs), and (iii) mice reproducing syndromic genetic RASopathies. Overall, the scientific literature suggests that during cardiac hypertrophy, ERK could be a "good" player to be stimulated or a "bad" actor to be mitigated, depending on the pathophysiological context.

Cardiac concentric hypertrophy promoted by activated Met receptor is mitigated in vivo by inhibition of Erk1,2 signalling with Pimasertib.

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies.

Cellular and molecular mechanisms of HGF/Met in the cardiovascular system.

Met tyrosine kinase receptor, also known as c-Met, is the HGF (hepatocyte growth factor) receptor. The HGF/Met pathway has a prominent role in cardiovascular remodelling after tissue injury. The present review provides a synopsis of the cellular and molecular mechanisms underlying the effects of HGF/Met in the heart and blood vessels. In vivo, HGF/Met function is particularly important for the protection of the heart in response to both acute and chronic insults, including ischaemic injury and doxorubicin-induced cardiotoxicity. Accordingly, conditional deletion of Met in cardiomyocytes results in impaired organ defence against oxidative stress. After ischaemic injury, activation of Met provides strong anti-apoptotic stimuli for cardiomyocytes through PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase) cascades. Recently, we found that HGF/Met is also important for autophagy regulation in cardiomyocytes via the mTOR (mammalian target of rapamycin) pathway. HGF/Met induces proliferation and migration of endothelial cells through Rac1 (Ras-related C3 botulinum toxin substrate 1) activation. In fibroblasts, HGF/Met antagonizes the actions of TGFß1 (transforming growth factor ß1) and AngII (angiotensin II), thus preventing fibrosis. Moreover, HGF/Met influences the inflammatory response of macrophages and the immune response of dendritic cells, indicating its protective function against atherosclerotic and autoimmune diseases. The HGF/Met axis also plays an important role in regulating self-renewal and myocardial regeneration through the enhancement of cardiac progenitor cells. HGF/Met has beneficial effects against myocardial infarction and endothelial dysfunction: the cellular and molecular mechanisms underlying repair function in the heart and blood vessels are common and include pro-angiogenic, anti-inflammatory and anti-fibrotic actions. Thus administration of HGF or HGF mimetics may represent a promising therapeutic agent for the treatment of both coronary and peripheral artery disease.

The MET Oncogene: Thirty Years of Insights into Molecular Mechanisms Driving Malignancy.

The discovery and subsequent research on the MET oncogene's role in cancer onset and progression have illuminated crucial insights into the molecular mechanisms driving malignancy. The identification of MET as the hepatocyte growth factor (HGF) receptor has paved the path for characterizing the MET tyrosine kinase activation mechanism and its downstream signaling cascade. Over the past thirty years, research has established the importance of HGF/MET signaling in normal cellular processes, such as cell dissociation, migration, proliferation, and cell survival. Notably, genetic alterations that lead to the continuous activation of MET, known as constitutive activation, have been identified as oncogenic drivers in various cancers. The genetic lesions affecting MET, such as exon skipping, gene amplification, and gene rearrangements, provide valuable targets for therapeutic intervention. Moreover, the implications of MET as a resistance mechanism to targeted therapies emphasize the need for combination treatments that include MET inhibitors. The intriguing "flare effect" phenomenon, wherein MET inhibition can lead to post-treatment increases in cancer cell proliferation, underscores the dynamic nature of cancer therapeutics. In human tumors, increased protein expression often occurs without gene amplification. Various mechanisms may cause an overexpression: transcriptional upregulation induced by other oncogenes; environmental factors (such as hypoxia or radiation); or substances produced by the reactive stroma, such as inflammatory cytokines, pro-angiogenic factors, and even HGF itself. In conclusion, the journey to understanding MET's involvement in cancer onset and progression over the past three decades has not only deepened our knowledge, but has also paved the way for innovative therapeutic strategies. Selective pharmacological inactivation of MET stands as a promising avenue for achieving cancer remission, particularly in cases where MET alterations are the primary drivers of malignancy.

Gene expression profiling of HGF/Met activation in neonatal mouse heart.

Hepatocyte Growth Factor (HGF) controls growth and differentiation in different cell types, including cardiac cells. However, its downstream effectors are poorly understood. To investigate the transcriptional targets of HGF, we analyzed the hearts of neonatal mice with cardiomyocyte-specific HGF overexpression with whole genome DNA microarrays. When comparing HGF expressing versus control hearts, we found a total of 249 transcripts with significant gene expression changes (210 upregulated and 39 downregulated). Gene Ontology (GO) annotation analysis revealed that the transcripts modulated by HGF were enriched for metabolic functions including: protein translation, vesicle-mediated transport, regulation of transcription, regulation of muscle development. Using an automated literature meta-analysis approach, we obtained a co-occurrence network oriented to the positive regulatory role of Myc and Notch1 in controlling some of the genes which are downstream to HGF. GO analysis of this network returned genes involved in the regulation of heart development. HGF positively controls MyocD, an activator of cardiac gene expression, and Hdac5, an inhibitor of cardiac growth. These results may unveil a new role of HGF in the modulation of signaling pathways implicated in the activation or repression of cardiomyogenesis.

Digoxin and ouabain induce the efflux of cholesterol via liver X receptor signalling and the synthesis of ATP in cardiomyocytes.

Cardioactive glycosides exert positive inotropic effects on cardiomyocytes through the inhibition of Na(+)/K(+)-ATPase. We showed previously that in human hepatoma cells, digoxin and ouabain increase the rate of the mevalonate cascade and therefore have Na(+)/K(+)-ATPase-independent effects. In the present study we found that they increase the expression and activity of 3-hydroxy-3 methylglutaryl-CoA reductase and the synthesis of cholesterol in cardiomyocytes, their main target cells. Surprisingly this did not promote intracellular cholesterol accumulation. The glycosides activated the liver X receptor transcription factor and increased the expression of ABCA1 (ATP-binding cassette protein A1) transporter, which mediates the efflux of cholesterol and its delivery to apolipoprotein A-I. By increasing the synthesis of ubiquinone, another derivative of the mevalonate cascade, digoxin and ouabain simultaneously enhanced the rate of electron transport in the mitochondrial respiratory chain and the synthesis of ATP. Mice treated with digoxin showed lower cholesterol and higher ubiquinone content in their hearts, and a small increase in their serum HDL (high-density lipoprotein) cholesterol. The results of the present study suggest that cardioactive glycosides may have a role in the reverse transport of cholesterol and in the energy metabolism of cardiomyocytes.

MET Oncogene Enhances Pro-Migratory Functions by Counteracting NMDAR2B Cleavage.

The involvement of the N-methyl-D-aspartate receptor (NMDAR), a glutamate-gated ion channel, in promoting the invasive growth of cancer cells is an area of ongoing investigation. Our previous findings revealed a physical interaction between NMDAR and MET, the hepatocyte growth factor (HGF) receptor. However, the molecular mechanisms underlying this NMDAR/MET interaction remain unclear. In this study, we demonstrate that the NMDAR2B subunit undergoes proteolytic processing, resulting in a low-molecular-weight form of 100 kDa. Interestingly, when the NMDAR2B and MET constructs were co-transfected, the full-size high-molecular-weight NMDAR2B form of 160 kDa was predominantly observed. The protection of NMDAR2B from cleavage was dependent on the kinase activity of MET. We provide the following evidence that MET opposes the autophagic lysosomal proteolysis of NMDAR2B: (i) MET decreased the protein levels of lysosomal cathepsins; (ii) treatment with either an inhibitor of autophagosome formation or the fusion of the autophagosome and lysosome elevated the proportion of the NMDAR2B protein's uncleaved form; (iii) a specific mTOR inhibitor hindered the anti-autophagic effect of MET. Finally, we demonstrate that MET coopts NMDAR2B to augment cell migration. This implies that MET harnesses the functionality of NMDAR2B to enhance the ability of cancer cells to migrate.

Signaling to cardiac hypertrophy: insights from human and mouse RASopathies.

Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli, some of which might finally lead up to a maladaptive state. An integral part of the pathogenesis of the hypertrophic cardiomyopathy disease (HCM) is the activation of the rat sarcoma (RAS)/RAF/MEK (mitogen-activated protein kinase kinase)/MAPK (mitogen-activated protein kinase) cascade. Therefore, the molecular signaling involving RAS has been the subject of intense research efforts, particularly after the identification of the RASopathies. These constitute a class of developmental disorders caused by germline mutations affecting proteins contributing to the RAS pathway. Among other phenotypic features, a subset of these syndromes is characterized by HCM, prompting researchers and clinicians to delve into the chief signaling constituents of cardiac hypertrophy. In this review, we summarize current advances in the knowledge of the molecular signaling events involved in the pathogenesis of cardiac hypertrophy through work completed on patients and on genetically manipulated animals with HCM and RASopathies. Important insights are drawn from the recognition of parallels between cardiac hypertrophy and cancer. Future research promises to further elucidate the complex molecular interactions responsible for cardiac hypertrophy, possibly pointing the way for the identification of new specific targets for the treatment of HCM.

MET Oncogene Controls Invasive Growth by Coupling with NMDA Receptor.

The N-methyl-D-aspartate receptor (NMDAR) is a glutamate-gated ion channel involved in excitatory synaptic transmission. Outside the nervous system, the NMDAR is expressed in a variety of tissues and in cancers, notably in the highly invasive and metastatic triple-negative breast carcinoma. MET encodes the tyrosine kinase receptor for HGF and is a master regulator gene for "invasive growth". In silico analysis shows that high expression of the NMDAR2B subunit is a negative prognostic factor in human invasive breast carcinoma. Here, we show that in triple-negative breast cancer cell lines NMDAR2B and MET proteins are coexpressed. HGF stimulation of these cells is followed by autophosphorylation of the MET kinase and phosphorylation of the NMDAR2B subunit at tyrosines 1252 and 1474. MET and phosphorylated NMDAR2B are physically associated, as demonstrated by co-immunoprecipitation, confocal immunofluorescence, and proximity ligation assays. Notably, pharmacological inhibition of NMDAR by MK801 and ifenprodil blunts the biological response to HGF. These results demonstrate the existence of a MET-NMDAR crosstalk driving the invasive program, paving the way for a new combinatorial therapy.

A mouse model for spatial and temporal expression of HGF in the heart.

In order to study the effects of Hepatocyte Growth Factor (HGF) in the heart, two transgenic mice were developed, one carrying a bidirectional HGF-TetO-GFP responder construct and the other carrying a α-MHC-tTA transactivator construct. Crosses were carried out between heterozygotes, so that litters contained bitransgenic α-MHC-tTA/HGF-TetO-GFP+, thus expressing HGF and GFP exclusively in the heart and only in the absence of Doxycycline. Our data show that the expression of HGF was indeed restricted to the heart and that the expression was limited to the timeframe of the absence of Doxycycline. Surprisingly the expression was variable even between bitransgenic littermates. In the setting of a model of ischemia-reperfusion, the expression of HGF ameliorates cardiac functionality, enhances proliferation and diminishes the scarred area, proving that this is a good model to study the beneficial influences and functional roles of HGF in the heart.

HGF and MET: From Brain Development to Neurological Disorders.

Hepatocyte growth factor (HGF) and its tyrosine kinase receptor, encoded by the MET cellular proto-oncogene, are expressed in the nervous system from pre-natal development to adult life, where they are involved in neuronal growth and survival. In this review, we highlight, beyond the neurotrophic action, novel roles of HGF-MET in synaptogenesis during post-natal brain development and the connection between deregulation of MET expression and developmental disorders such as autism spectrum disorder (ASD). On the pharmacology side, HGF-induced MET activation exerts beneficial neuroprotective effects also in adulthood, specifically in neurodegenerative disease, and in preclinical models of cerebral ischemia, spinal cord injuries, and neurological pathologies, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). HGF is a key factor preventing neuronal death and promoting survival through pro-angiogenic, anti-inflammatory, and immune-modulatory mechanisms. Recent evidence suggests that HGF acts on neural stem cells to enhance neuroregeneration. The possible therapeutic application of HGF and HGF mimetics for the treatment of neurological disorders is discussed.

Factor XII protects neurons from apoptosis by epidermal and hepatocyte growth factor receptor-dependent mechanisms.

BACKGROUND: Factor XII (FXII) is a serine protease that participates in the intrinsic coagulation pathway. Several studies have shown that plasma FXII exerts a deleterious role in cerebral ischemia and traumatic brain injury by promoting thrombo-inflammation. Nevertheless, the impact of FXII on neuronal cell fate remains unknown. OBJECTIVES: We investigated the role of FXII and FXIIa in neuronal injury and apoptotic cell death. METHODS: We tested the neuroprotective roles of FXII and FXIIa in an experimental model of neuronal injury induced by stereotaxic intracerebral injection of N-methyl-D-aspartic acid (NMDA) in vivo and in a model of apoptotic death of murine primary neuronal cultures through serum deprivation in vitro. RESULTS: Here, we found that exogenous FXII and FXIIa reduce brain lesions induced by NMDA injection in vivo. Furthermore, FXII protects cultured neurons from apoptosis through a growth factor--like effect. This mechanism was triggered by direct interaction with epidermal growth factor (EGF) receptor and subsequent activation of this receptor. Interestingly, the "proteolytically" active and two-chain form of FXII, FXIIa, exerts its protective effects by an alternative signaling pathway. FXIIa activates the pro-form of hepatocyte growth factor (HGF) into HGF, which in turn activated the HGF receptor (HGFR) pathway. CONCLUSION: This study describes two novel mechanisms of action of FXII and identifies neurons as target cells for the protective effects of single and two-chain forms of FXII. Therefore, inhibition of FXII in neurological disorders may have deleterious effects by preventing its beneficial effects on neuronal survival.

Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor.

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.

Identification of novel circulating microRNAs in advanced heart failure by next-generation sequencing.

AIMS: Risk stratification in patients with advanced chronic heart failure (HF) is an unmet need. Circulating microRNA (miRNA) levels have been proposed as diagnostic and prognostic biomarkers in several diseases including HF. The aims of the present study were to characterize HF-specific miRNA expression profiles and to identify miRNAs with prognostic value in HF patients. METHODS AND RESULTS: We performed a global miRNome analysis using next-generation sequencing in the plasma of 30 advanced chronic HF patients and of matched healthy controls. A small subset of miRNAs was validated by real-time PCR (P < 0.0008). Pearson's correlation analysis was computed between miRNA expression levels and common HF markers. Multivariate prediction models were exploited to evaluate miRNA profiles' prognostic role. Thirty-two miRNAs were found to be dysregulated between the two groups. Six miRNAs (miR-210-3p, miR-22-5p, miR-22-3p, miR-21-3p, miR-339-3p, and miR-125a-5p) significantly correlated with HF biomarkers, among which N-terminal prohormone of brain natriuretic peptide. Inside the cohort of advanced HF population, we identified three miRNAs (miR-125a-5p, miR-10b-5p, and miR-9-5p) altered in HF patients experiencing the primary endpoint of cardiac death, heart transplantation, or mechanical circulatory support implantation when compared with those without clinical events. The three miRNAs added substantial prognostic power to Barcelona Bio-HF score, a multiparametric and validated risk stratification tool for HF (from area under the curve = 0.72 to area under the curve = 0.82). CONCLUSIONS: This discovery study has characterized, for the first time, the advanced chronic HF-specific miRNA expression pattern. We identified a few miRNAs able to improve the prognostic stratification of HF patients based on common clinical and laboratory values. Further studies are needed to validate our results in larger populations.

Ghrelin and des-acyl ghrelin promote differentiation and fusion of C2C12 skeletal muscle cells.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

Molecular Engineering Strategies Tailoring the Apoptotic Response to a MET Therapeutic Antibody.

The MET oncogene encodes a tyrosine kinase receptor involved in the control of a complex network of biological responses that include protection from apoptosis and stimulation of cell growth during embryogenesis, tissue regeneration, and cancer progression. We previously developed an antagonist antibody (DN30) inducing the physical removal of the receptor from the cell surface and resulting in suppression of the biological responses to MET. In its bivalent form, the antibody displayed a residual agonist activity, due to dimerization of the lingering receptors, and partial activation of the downstream signaling cascade. The balance between the two opposing activities is variable in different biological systems and is hardly predictable. In this study, we generated and characterized two single-chain antibody fragments derived from DN30, sharing the same variable regions but including linkers different in length and composition. The two engineered molecules bind MET with high affinity but induce different biological responses. One behaves as a MET-antagonist, promoting programmed cell death in MET "addicted" cancer cells. The other acts as a hepatocyte growth factor (HGF)-mimetic, protecting normal cells from doxorubicin-induced apoptosis. Thus, by engineering the same receptor antibody, it is possible to generate molecules enhancing or inhibiting apoptosis either to kill cancer cells or to protect healthy tissues from the injuries of chemotherapy.