endothelin-1 and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Endothelin-1 (ET-1) dose-dependently increased HIF1α expression in pulmonary artery smooth muscle cells (PASMCs). Inhibition of protein synthesis did not affect ET-1-induced HIF1α expression. The maximum effect of ET-1 was similar to that caused by proteasome inhibitor MG132. Further study indicates that ET-1 also dose-dependently stimulated calcineurin activation, specific calcineurin inhibitor cyclosporine A (CsA), abolished ET-1-induced HIF1α elevation, and reversed ET-1-induced RACK1 (receptor of activated protein kinase C 1) de-phosphorylation. Endothelin receptor A was found to specifically mediate the effects of ET-1. To examine whether RACK1 is particularly involved in proteasome-dependent HIF1α degradation, RACK1 was silenced by siRNA transfection. Cells lacking RACK1 exhibited significant elevation of HIF1α protein level. Taken together, our study suggests that ET-1 suppressed proteasome-dependent HIF1α degradation by calcineurin-dependent RACK1 de-phosphorylation.

Topics: Animals; Calcineurin; Cyclosporine; Endothelin-1; Gene Expression Regulation; Gene Silencing; GTP-Binding Proteins; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Myocytes, Smooth Muscle; Phosphorylation; Primary Cell Culture; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Biosynthesis; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptors for Activated C Kinase; Receptors, Endothelin; Signal Transduction

The zebrafish pharyngeal cartilage is derived from the pharyngeal apparatus, a vertebrate-specific structure derived from all three germ layers. Developmental aberrations of the pharyngeal apparatus lead to birth defects such as Treacher-Collins and DiGeorge syndromes. While interactions between endoderm and neural crest (NC) are known to be important for cartilage formation, the full complement of molecular players involved and their roles remain to be elucidated. Activated leukocyte cell adhesion molecule a (alcama), a member of the immunoglobulin (Ig) superfamily, is among the prominent markers of pharyngeal pouch endoderm, but to date no role has been assigned to this adhesion molecule in the development of the pharyngeal apparatus. Here we show that alcama plays a crucial, non-autonomous role in pharyngeal endoderm during zebrafish cartilage morphogenesis. alcama knockdown leads to defects in NC differentiation, without affecting NC specification or migration. These defects are reminiscent of the phenotypes observed when Endothelin 1 (Edn1) signaling, a key regulator of cartilage development is disrupted. Using gene expression analysis and rescue experiments we show that Alcama functions downstream of Edn1 signaling to regulate NC differentiation and cartilage morphogenesis. In addition, we also identify a role for neural adhesion molecule 1.1 (nadl1.1), a known interacting partner of Alcama expressed in neural crest, in NC differentiation. Our data shows that nadl1.1 is required for alcama rescue of NC differentiation in edn1(-/-) mutants and that Alcama interacts with Nadl1.1 during chondrogenesis. Collectively our results support a model by which Alcama on the endoderm interacts with Nadl1.1 on NC to mediate Edn1 signaling and NC differentiation during chondrogenesis.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Animals; Branchial Region; Cell Differentiation; Chondrogenesis; Cloning, Molecular; DNA Primers; Endothelin-1; Gene Expression Profiling; Gene Knockout Techniques; Immunohistochemistry; In Situ Hybridization, Fluorescence; Leupeptins; Models, Biological; Neural Cell Adhesion Molecules; Neural Crest; Signal Transduction; Zebrafish; Zebrafish Proteins

Acute kidney dysfunction after ischemia-reperfusion injury (IRI) may be a consequence of persistent intrarenal vasoconstriction. Regulators of G-protein signaling (RGSs) are GTPase activators of heterotrimeric G proteins that can regulate vascular tone. RGS4 is expressed in vascular smooth muscle cells in the kidney; however, its protein levels are low in many tissues due to N-end rule-mediated polyubiquitination and proteasomal degradation. Here, we define the role of RGS4 using a mouse model of IRI comparing wild-type (WT) with RGS4-knockout mice. These knockout mice were highly sensitized to the development of renal dysfunction following injury exhibiting reduced renal blood flow as measured by laser-Doppler flowmetry. The kidneys from knockout mice had increased renal vasoconstriction in response to endothelin-1 infusion ex vivo. The intrinsic renal activity of RGS4 was measured following syngeneic kidney transplantation, a model of cold renal IRI. The kidneys transplanted between knockout and WT mice had significantly reduced reperfusion blood flow and increased renal cell death. WT mice administered MG-132 (a proteasomal inhibitor of the N-end rule pathway) resulted in increased renal RGS4 protein and in an inhibition of renal dysfunction after IRI in WT but not in knockout mice. Thus, RGS4 antagonizes the development of renal dysfunction in response to IRI.

Topics: Acute Kidney Injury; Animals; Cysteine Proteinase Inhibitors; Disease Models, Animal; Endothelin-1; Enzyme Activation; GTP-Binding Protein alpha Subunits, Gq-G11; Kidney; Kidney Transplantation; Laser-Doppler Flowmetry; Leupeptins; Ligation; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Nephrectomy; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Renal Circulation; Reperfusion Injury; RGS Proteins; Time Factors; Vasoconstriction; Vasoconstrictor Agents

Protein kinase A (PKA) is an important effector enzyme commonly activated by cAMP. The present study focuses on our finding that the vasoactive peptide endothelin-1 (ET1), whose signaling is not coupled to cAMP production, stimulates PKA in two independent cellular models. Using an in vivo assay for PKA activity, we found that ET1 stimulated PKA in HeLa cells overexpressing ET1 receptors and in aortic smooth muscle cells expressing endogenous levels of ET1 receptors. In these cell models, ET1 did not stimulate cAMP production, indicating a novel mechanism for PKA activation. The ET1-induced activation of PKA was found to be dependent on the degradation of inhibitor of kappaB, which was previously reported to bind and inhibit PKA. ET1 potently stimulated the nuclear factor-kappaB pathway, and this effect was inhibited by overexpression of the inhibitor of kappaB dominant negative mutant (IkappaBalpham) and by treatment with the proteasome inhibitor MG-132. Importantly, IkappaBalpham and MG-132 had similar inhibitory effects on ET1-induced activation of PKA without affecting G(s)-mediated activation of PKA or ET1-induced phosphorylation of mitogen-activated protein kinase. Finally, another vasoactive peptide, angiotensin II, also stimulated PKA in a cAMP-independent manner in aortic smooth muscle cells. These findings suggest that cAMP-independent activation of PKA might be a general response to vasoactive peptides.

Topics: Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Endothelin-1; Enzyme Activation; HeLa Cells; Humans; I-kappa B Proteins; Isoproterenol; Leupeptins; Mice; Multienzyme Complexes; NF-kappa B; NF-KappaB Inhibitor alpha; Proteasome Endopeptidase Complex; Receptor, Endothelin A; Receptors, Endothelin; Recombinant Proteins; Transfection

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype and proliferate and synthesize fibrosis components. Endothelin-1 (ET-1), which inhibited the growth of human myofibroblastic HSC, increased the formation of two NF-kappaB DNA binding complexes; this effect was also observed with tumor necrosis factor-alpha (TNF-alpha). The complexes were identified as the p50/p50 and p50/p65 NF-kappaB dimers. Activation of NF-kappaB was associated with the degradation of the inhibitory protein IkappaB-alpha; no IkappaB-beta was detected. Activation of NF-kappaB and degradation of IkappaB-alpha were prevented by the NF-kappaB inhibitors sodium salicylate and MG-132. In addition to cyclooxygenase-1 (COX-1), COX-2 is also constitutively expressed in human HSC, and the use of dexamethasone and of SC-58125, a selective COX-2 inhibitor, revealed that COX-2 accounts for basal COX activity. Moreover, COX-2 mRNA and protein were up-regulated by ET-1 and TNF-alpha, whereas COX-1 was unaffected. Induction of COX-2 and stimulation of COX activity by ET-1 and TNF-alpha were prevented by sodium salicylate and MG-132, suggesting that activation of NF-kappaB by either factor is needed for stimulation of COX-2. Finally, SC-58125 and dexamethasone reduced the growth inhibitory effect of ET-1 and TNF-alpha, indicating that activation of COX-2 is required for inhibition of HSC proliferation. Taken together, our results suggest that NF-kappaB, by inducing COX-2 expression, may play an important role in the negative regulation of human myofibroblastic HSC proliferation.

Topics: Adipocytes; Antineoplastic Agents; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dexamethasone; Dimerization; DNA-Binding Proteins; Endothelin-1; Fibroblasts; Humans; I-kappa B Proteins; Isoenzymes; Leupeptins; Liver; Membrane Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Receptor, Endothelin B; Receptors, Endothelin; Sodium Salicylate; Tumor Necrosis Factor-alpha; Up-Regulation; Viper Venoms