losartan-potassium and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

We examined the biological functions of the gene Cbl in erythropoietin (EPO) signaling using Cbl-deficient F-36P human erythroleukemia cells by the introduction of the Cbl siRNA expression vector. Knockdown of Cbl promoted EPO-dependent proliferation and survival of F-36P cells, especially at a low concentration of EPO (0.01U/mL), similar to serum concentrations of EPO in healthy volunteers (0.005-0.04U/mL). We found that Src was degraded mainly by the proteasomal pathway because the proteasome inhibitor MG-132 but not the lysosome inhibitor NH4Cl suppressed the EPO-induced degradation of Src in F-36P cells and that knockdown of Cbl inhibited EPO-induced ubiquitination and degradation of Src in F-36P cells. The experiments using the Src inhibitor PP1 and co-expression experiments further confirmed that Cbl and the kinase activity of Src are required for the EPO-induced ubiquitination of Src. In addition, the co-expression experiments and in vitro kinase assay demonstrated that the EPO-induced tyrosine phosphorylation and ubiquitination of Cbl were dependent on the kinase activity of Src but not Jak2. Thus, Cbl negatively regulates EPO signaling mainly through the proteasome-dependent degradation of Src, and the E3 ligase activity of Cbl and its tyrosine phosphorylation are regulated by Src but not Jak2.

Topics: Ammonium Chloride; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Erythropoietin; Gene Expression Regulation; Humans; Janus Kinase 2; Leupeptins; Lymphocytes; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-cbl; Proto-Oncogene Proteins pp60(c-src); Pyrazoles; Pyrimidines; RNA, Small Interfering; Signal Transduction; Ubiquitination

Proteasome inhibitors represent a promising therapy for the treatment of relapsed and/or refractory multiple myeloma, a disease that is concomitant with osteolysis and enhanced osteoclast formation. While blockade of the proteosome pathway has been recently shown to influence osteoclast formation and function, the precise molecular cascade underlying these effects is presently unclear. Here, we provide evidence that proteasome inhibitors directly impair osteoclast formation and function via the disruption of key RANK-mediated signaling cascades. Disruption of the proteosome pathway using selective inhibitors (MG-132, MG-115, and epoxomicin) resulted in the accumulation of p62 and CYLD, and altered the subcellular targeting and distribution of p62 and TRAF6 in osteoclast-like cells. Proteosome inhibition also blocked RANKL-induced NF-kappaB activation, IkappaBalpha degradation and nuclear translocation of p65. The disruption in RANK-signaling correlated dose-dependently with an impairment in osteoclastogenesis, with relative potency epoxomicin > MG-132 > MG-115 based on equimolar concentrations. In addition, these inhibitors were found to impact osteoclastic microtubule organization and attenuate bone resorption. Based on these data we propose that deregulation of key RANK-mediated signaling cascades (p62, TRAF6, CYLD, and IkappaBalpha) underscores proteasome-mediated inhibition of osteolytic bone conditions.

Topics: Actins; Animals; Bone Resorption; Cell Line; Cysteine; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Deubiquitinating Enzyme CYLD; Erythropoietin; Humans; I-kappa B Proteins; Leupeptins; Mice; Mice, Inbred C57BL; Microtubules; NF-kappa B; NF-KappaB Inhibitor alpha; Oligopeptides; Osteoclasts; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RANK Ligand; Signal Transduction; Synaptotagmin I; TNF Receptor-Associated Factor 6; Transcription Factor TFIIH; Transcription Factors

Hypoxia-inducible factor-1alpha (HIF-1alpha) is destabilized via the ubiquitin-proteasome system. Thus HIF-1alpha expression is robustly upregulated by proteasome inhibition, but paradoxically its activity is reduced. In the present study, we investigated the mechanism underlying the paradoxical response of HIF-1alpha to proteasome inhibition. In both Hep3B and HEK293 cells, a proteasome inhibitor MG132 noticeably attenuated hypoxic induction of erythropoietin and VEGF mRNAs. MG132 inactivated HIF-1alpha C-terminal transactivation domain (CAD), independently of factor inhibiting HIF-1 (FIH) and inhibited p300 recruitment by HIF-1alpha. We next tested the possibility that CITED2 is involved in the HIF-1 inactivation. CITED2 was found to be degraded via the ubiquitin-proteasome system and thus was stabilized by proteasome inhibition. Both the activity and the p300 binding of HIF-1alpha were inhibited by CITED2 expression and recovered by CITED2 siRNA in the presence of MG132. These results suggest that CITED2 is stabilized by proteasome inhibition and inactivates HIF-1 by interfering with the HIF-1alpha-p300 interaction. This may be an important mode-of-action for proteasome inhibition-based cancer therapy.

Topics: Carcinoma, Hepatocellular; Cell Hypoxia; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; E1A-Associated p300 Protein; Erythropoietin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoprecipitation; Kidney; Leupeptins; Liver Neoplasms; Mixed Function Oxygenases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Repressor Proteins; RNA, Messenger; Trans-Activators; Transcription Factors; Ubiquitin; Vascular Endothelial Growth Factor A

The suppression of hypoxia-induced erythropoietin (EPO) expression by inflammatory cytokines like interleukin-1 (IL-1) contributes to the development of the anemia of chronic disease (ACD). However, the precise mechanism of this suppression is unclear. The 3'-EPO enhancer mediates the transcriptional response to hypoxia by binding several transcription factors, including hypoxia-inducible factor, hepatocyte nuclear factor-4alpha (HNF-4alpha) and chicken ovalbumin upstream promoter transcription factor. We investigated whether IL-1beta inhibits the activity of the 3'-EPO enhancer via HNF-4alpha. IL-1beta inhibited HNF-4alpha mRNA expression and caused proteasome-dependent degradation of HNF-4alpha protein, which resulted in a strongly reduced DNA-binding activity of HNF-4alpha. Reporter gene assays revealed that IL-1beta caused a complete suppression of the hypoxic inducibility of the 3' enhancer via inhibition of HNF-4alpha. We conclude that IL-1beta, at least partially, reduces hypoxia-induced EPO expression by down-regulation of HNF-4alpha.

Topics: Anemia; Binding Sites; Cell Hypoxia; Cell Line, Tumor; DNA; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation; Hepatocyte Nuclear Factor 4; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-1beta; Leupeptins; Luminescent Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Recombinant Fusion Proteins; RNA, Messenger