Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis

Hepatocyte growth factor (HGF) has been proposed as an endogenous cardioprotective agent against oxidative stress. The mechanism of HGF action in the heart, however, has not yet been elucidated. The present study demonstrates that HGF protects adult cardiac myocytes against oxidative stress-induced apoptosis. HGF, at the concentrations which can be detected in the plasma of humans subsequent to myocardial infarction, effectively attenuated death of isolated adult rat cardiac myocytes and cultured HL-1 cardiac muscle cells induced by apoptosis-inducing oxidative stress stimuli such as daunorubicin, serum deprivation, and hydrogen peroxide. We identified expression of c-Met HGF receptor in adult cardiac myocytes, which can be rapidly tyrosine phosphorylated in response to HGF treatment. HGF also activated MEK, p44/42 MAPK, and p90RSK. To determine if MEK-MAPK pathway may be involved in the mechanism of HGF-mediated cardiac myocyte protection, effects of a specific MEK inhibitor, PD98059, were studied. Pretreatment of cells with PD98059 partially blocked HGF signaling for protection against hydrogen peroxide-induced cell death. Thus, HGF protects cardiac myocytes against oxidative stress, in part, via activating MEK-MAPK pathway.     

Treatment with an adenoviral vector encoding hepatocyte growth factor mitigates established cardiac dysfunction in doxorubicin-induced cardiomyopathy

Hepatocyte growth factor (HGF) reportedly exerts beneficial effects on the heart following myocardial infarction and during nonischemic cardiomyopathy, but the precise mechanisms underlying the latter have not been well elucidated. We generated nonischemic cardiomyopathy in mice by injecting them with doxorubicin (15 mg/kg ip). Two weeks later, when cardiac dysfunction was apparent, an adenoviral vector encoding human HGF gene (Ad.CAG-HGF, 1x10(11) particles/mouse) was injected into the hindlimb muscles; LacZ gene served as the control. Left ventricular dilatation and dysfunction normally seen 4 wk after doxorubicin administration were significantly mitigated in HGF-treated mice, as were the associated cardiomyocyte atrophy/degeneration and myocardial fibrosis. Myocardial expression of GATA-4 and a sarcomeric protein, myosin heavy chain, was downregulated by doxorubicin, but the expression of both was restored by HGF treatment. The protective effect of HGF against doxorubicin-induced cardiomyocyte atrophy was confirmed in an in vitro experiment, which also showed that neither cardiomyocyte apoptosis nor proliferation plays significant roles in the present model. Upregulation of c-Met/HGF receptor was noted in HGF-treated hearts. Among the mediators downstream of c-Met, the activation of extracellular signal-regulated kinase (ERK) was reduced by doxorubicin, but the activity was restored by HGF. Levels of transforming growth factor-beta1 and cyclooxygenase-2 did not differ between the groups. Our findings suggest the HGF gene delivery exerts therapeutic antiatrophic/degenerative and antifibrotic effects on myocardium in cases of established cardiac dysfunction caused by doxorubicin. These beneficial effects appear to be related to HGF-induced ERK activation and upregulation of c-Met, GATA-4, and sarcomeric proteins.     

S100B: a multifunctional role in cardiovascular pathophysiology

S100B, a calcium-binding protein of the EF-hand type exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures, and high level expression of S100B in transgenic mice hearts and aortic smooth muscle cells inhibit cardiac hypertrophy and the associated phenotype, arterial smooth muscle proliferation, respectively, but demonstrate increased apoptosis following α₁-adrenergic stimulation or myocardial infarction. Knocking out S100B, augmented hypertrophy, decreased apoptosis and preserved cardiac function following myocardial infarction. S100B induces apoptosis by an extracellular mechanism by interacting with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular, and extracellular, roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular disease.     

Bcl-x(S) induces an NGF-inhibitable cytochrome c release

Bcl-x(S), a pro-apoptotic member of the Bcl-2 protein family, is localized in the mitochondrial outer membrane and induces caspase-dependent and nerve growth factor (NGF)-inhibitable apoptosis in PC12 cells. The mechanism of action of Bcl-x(S) and how NGF inhibits this death are not fully understood. It is still unknown whether Bcl-x(S) induces mitochondrial cytochrome c release, and which apoptotic step NGF inhibits. We show that Bcl-x(S) induces cytochrome c release and caspase-3 activation in several cell types, and that in PC12 cells, these events are inhibited by NGF treatment. The survival effect of NGF was inhibited by inhibitors of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI 3-kinase), and the mitogen-activated protein kinase kinase (MEK) inhibitors GF109203X, LY294002, and U0126. These findings show that cytochrome c release and caspase-3 activation participate in Bcl-x(S)-induced apoptosis, and that NGF inhibits Bcl-x(S)-induced apoptosis at the mitochondrial level via the PKC, PI 3-kinase, and MEK signaling pathways.     

Bcl-x(S) can form homodimers and heterodimers and its apoptotic activity requires localization of Bcl-x(S) to the mitochondria and its BH3 and loop domains

Proteins of the Bcl-2 family regulate apoptosis, some antagonizing cell death and others, such as Bcl-x(S), promoting it. We previously showed that expression of Bcl-x(S) in PC12 cells is a useful system for studying the mechanism of Bcl-x(S)-induced apoptosis. To further investigate this apoptotic effect and its prevention by anti-apoptotic agents, we assessed the role of distinct Bcl-x(S) domains, via the study of their mutations, on the ability of Bcl-x(S) to induce apoptosis and to localize to the mitochondria, as well as the ability of these domains to counteract the effects of anti-apoptotic agents on Bcl-x(S). Deletion of the transmembrane domain (DeltaTM) prevented the localization of Bcl-x(S) DeltaTM to the mitochondria and the ability of this mutant to induce apoptosis. Deletion of the amino acids GD 94-95 from the BH3 domain, or deletion of the loop region, impaired the ability of these mutants to induce apoptosis but not their localization to the mitochondria. Deletion of the BH4 domain or destruction of the caspase cleavage site in the loop region (by replacing amino acid D61 with A61) did not affect either the localization of these mutants to the mitochondria or their ability to induce cell death. It thus appears that Bcl-x(S)-induced apoptosis in PC12 cells is mediated by localization of Bcl-x(S) to the mitochondria by a process that requires the transmembrane domain. Furthermore, once localized to the mitochondria Bcl-x(S) requires the BH3 domain, and to a lesser extent the loop domain, for its subsequent activity. The anti-apoptotic agents Bcl-2 and Bcl-x(L), the caspase inhibitor Z-VAD-FMK, and nerve growth factor (NGF) did not prevent Bcl-x(S) localization to the mitochondria, and did not require the BH4 or the loop domains of Bcl-x(S) for their survival effect. Bcl-x(S) is capable of forming homodimers with itself and heterodimers with Bcl-x(L) or Bcl-2. Accordingly co-expression of Bcl-x(S) DeltaTM with Bcl-x(S), Bcl-2, or Bcl-x(L) leads to a change in the subcellular distribution of Bcl-x(S) DeltaTM, from a diffuse distribution throughout the cell to a more defined distribution. Moreover co-immunoprecipitation experiments directly demonstrated that Bcl-x(S) can associate with GFP-Bcl-x(S), Bcl-x(L), or Bcl-2. These results suggest that such Bcl-x(S) interactions may be important for the mechanism of action of this protein.     

Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants

Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner.     

Impact of combined mTOR and MEK inhibition in uveal melanoma is driven by tumor genotype

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.