Mas and ET B receptors physically interact in endothelial cells (ECs) and are involved in the protective actions of angiotensin 1-7 (Ang (1-7)). We assessed whether the MAS/ET B R interaction plays a role in EC signalling and whether strategies to enhance MAS/ET B R association influence vascular responses. Human ECs were stimulated with Ang (1-7) (10 -7 M) in the presence/absence of A779 (Mas receptor antagonist, 10 -5 M) and BQ788 (ET B R antagonist, 10 -5 M). Protein expression and signalling activation were assessed by immunoblotting. NO production was evaluated by DAF-FM fluorescence and ROS production by chemiluminescence (superoxide anion) or amplex red (hydrogen peroxide (H 2 O 2 )). mRNA expression was assessed by qPCR. Endothelial function was assessed in mouse intact arteries by myography. Ang (1-7), through Mas and ET B R induced phosphorylation of eNOS (35%); followed by an increase in NO production (2.0 fold) (p<0.05 vs ctl). High throughput screening of protein:protein interaction compounds in an in-house library identified 23 potential enhancers of the MAS/ET B R interaction. Fluorescence polarization assays were used to further select the most potent enhancers and define their working concentration for testing in ECs (Enh1-4: 10 -5 M). Enh4 increased superoxide anion (55.6±26.3% vs ctl, p<0.05) and H 2 O 2 production (54.7±11.1% vs ctl, p<0.05), while Enh3 increased H 2 O 2 generation (48.1±15.4% vs ctl, p<0.05) in ECs. Moreover, Enh4 increased Nrf2 (3.0 fold), Sod1 (2.0 fold) and Nqo1 (3.1 fold) mRNA expression (p<0.05 vs ctl). Enh3 and Enh4 increased NO production (Enh3: 21.2±7.4%; Enh4: 23.6±8.2% vs veh, p<0.05) in ECs. Acetylcholine (Ach) curves were performed to assess endothelium-dependent relaxation in the absence and presence of enhancers. Enh4 increased ACh-induced relaxation (Emax%: 96.7±4.6 vs ctl: 70.4±3.3, p<0.05), while other enhancers did not improve endothelial function. Taken together, increasing MAS/ET B R interaction with specific enhancers augments protective signalling in ECs and promotes endothelial-dependent vasorelaxation, particularly with Enh4. In conclusion, enhancing interactions between MasR and ET B R may be a new vasoprotective strategy to improve vascular function in cardiovascular disease.
Vascular smooth muscle cell (SMC) proliferation and migration underlie the pathogenesis of vein graft failure and in-stent restenosis. Current interventions inhibit both SMC and endothelial cell (EC) growth, leading to thrombosis and re-occlusion. Angiotensin II (AngII) is a key regulator of VSMC proliferation and migration. A counter-regulatory axis of the renin angiotensin system (RAS) has been identified which inhibits AngII and is centred around angiotensin converting enzyme2 and Ang-(1–7) acting at Mas. We recently reported Ang-(1–9) as a novel member of this axis, acting at the angiotensin type 2 receptor (AT2R) to inhibit cardiac remodelling. Here we investigated the role of Ang-(1–9) in human SMC and EC migration and proliferation and vascular injury in vivo, and compared it to Ang-(1–7).SMC and EC were isolated from human saphenous veins. To assess migration, EC and SMC were stimulated with Ang II and Ang-(1–9) or Ang-(1–7) +/- the AT1R, AT2R or Mas antagonists losartan, PD123,319 (PD) or A779, respectively, and a wound healing assay performed. To assess proliferation, EC and SMC were stimulated with fetal calf serum (FCS) and Ang-(1–9) or Ang-(1–7) ± losartan, PD or A779. Proliferation was assessed using MTS and Edu assays. An in vivo mouse model was established via wire injury to the carotid artery. Ang-(1–7) or Ang-(1–9) ± PD or A779 were delivered subcutaneously via minipumps and neointima (NI) formation quantified 28 days post injury.Ang-(1–9) and Ang-(1–7) inhibited Ang II induced VSMC migration (Ang II 98.9 ± 1.1%, Ang-(1–9) 43.3 ± 3.3% and Ang-(1–7) 41.8 ± 4.6% wound closure; P < 0.001 vs Ang II). Furthermore, both peptides significantly inhibited FCS induced VSMC proliferation (P < 0.05). The inhibitory effects of Ang-(1–9) and Ang-(1–7) on VSMC migration and proliferation were selectively blocked by PD and A779, respectively, suggesting Ang-(1–9) acts via the AT2R and Ang-(1–7) via Mas. Neither Ang-(1–9) or Ang-(1–7) prevented EC migration or proliferation. In vivo wire injury of the mouse carotid artery induced significant NI formation at 28days (NI/media area (NI/MA) 0.80 ± 0.07 injured control vs 0.01 ± 0.01 sham; P < 0.001); this was attenuated by Ang-(1–9) (NI/MA 0.17 ± 0.1; P < 0.001 vs injured control) and Ang-(1–7) (NI/MA 0.40 ± 0.07; P < 0.05 vs injured control). The effects of Ang-(1–9) were blocked by PD (P < 0.001 vs Ang-(1–9) alone) while the effects of Ang-(1–7) were blocked by A779 (P < 0.05 vs Ang-(1–7) alone), suggesting that in vivo Ang-(1–9) acts via the AT2R and Ang-(1–7) acts via Mas.We demonstrate for a novel, direct effect of Ang-(1–9) in inhibiting VSMC proliferation and migration in vitro, and reducing neointimal formation in vivo via the AT2R. These data provide further insight into the role of the Ang-(1–9)/AT2R interaction in the vasculature and highlights the potential of Ang-(1–9) as a therapeutic agent for vascular remodelling.
Suppressor of cytokine signalling 3 (SOCS3) is an inducible specificity factor for a multi‐subunit E3 ubiquitin ligase complex. However the full spectrum of ubiquitylated SOCS3 substrates is unknown. By performing an unbiased SILAC‐facilitated comparison of tandem affinity purified ubiquitinomes from WT and SOCS3‐null fibroblasts, we have identified cavin‐1/PTRF as a protein whose ubiquitylation status is significantly enhanced in SOCS3‐expressing versus SOCS3‐null cells. Importantly, studies of endogenous cavin‐1 expressed in adipocytes and fibroblasts have shown it to be ubiquitylated. Co‐immunoprecipitations and overlapping peptide array overlays confirmed that SOCS3 interacted directly with two distinct regions within cavin‐1 in an interaction that did not require tyrosine phosphorylation. Cavin‐1 ubiquitylation was also found to be enhanced in the presence of SOCS3 and its cognate Cul‐Rbx‐elongin B/C E3 ligase scaffold. In summary, our data suggest a potentially novel role for SOCS3 in regulating caveolae assembly via cavin‐1 ubiquitylation. This provides a novel mechanism by which SOCS3‐inducing stimuli may influence caveolar architecture and function.
The microphthalmia‐associated transcription factor (MITF) is a master regulator of development and differentiation within the melanocyte lineage. However, aberrant MITF activity can lead to multiple malignancies such as skin cancer, where it plays a key role in modulating the proliferation and invasiveness of melanoma. MITF is a basic helix‐loop‐helix leucine zipper transcription factor that binds to specific gene promoters via a central DNA‐binding domain. MITF also recruits transcriptional co‐activators, such as the histone acetyltransferase CREB‐binding protein and its homologue p300 (CBP/p300) through an N‐terminal acidic transactivation domain (TAD), however the details of these interactions are not yet fully understood. In order to gain insight into the mechanisms of gene regulation by MITF, we investigated the structure and functional interaction between MITF‐TAD and the transcription adapter putative zinc finger (TAZ2) domain of CBP/p300. In mammalian‐one‐hybrid assays MITF transcriptional activity was enhanced in the presence of co‐transfected CBP/p300 and abolished upon deletion of residues within the MITF‐TAD. Peptide microarrays indicated that no one residue of MITF is essential for TAZ2 binding, however, deletion of multiple residues in MITF‐TAD ablated its ability to bind TAZ2. NMR‐based chemical shift mapping experiments determined that MITF‐TAD interacts with the same surface of TAZ2 as the adenoviral protein E1A, which has been shown to inhibit MITF function. We determined that E1A and MITF‐TAD directly compete for CBP/p300 through the TAZ2 domain using NMR‐based titrations, pulldown, and mammalian‐hybrid assays. Furthermore, we used qPCR to measure the effect of disrupting these interactions on the transcription of MITF‐specific target genes in melanoma‐derived cell lines. Understanding mechanistic details regarding the interaction between MITF and its co‐activators is fundamental to our understanding of gene regulation by MITF and may outline a potential new strategy to inhibit MITF function. Support or Funding Information This research was funded by the Beatrice Hunter Cancer Research Institute, Nova Scotia Heath Research Foundation Scotia Scholars Award, a Canadian Institutes of Health Research studentship, Dalhousie Nova Scotia Graduate Scholarship, and Izaak Walton Killam Predoctoral Scholarship.
We demonstrated that Mas1 and ET B R physically interact in endothelial cells inducing Ang-(1-7) vascular protection. Using high throughput screening of >20,000 druggable compounds, we identified a number of molecules that enhance Mas1:ET B R interactions. Of these, 2 potently enhanced interaction between the receptors. These enhancers (Enh), termed Enh3 and Enh4 were used to assess Mas1:ET B R interaction and cellular functional responses in vascular smooth muscle cells (VSMCs) from normotensive WKY and hypertensive SHRSP rats. Cells were exposed to Enh 3 and Enh4 (10 -5 M) for short (5,15 and 30min) and long timepoints (5hours). Expression of signaling molecules was assessed by immunoblotting and Ca 2+ influx was evaluated using fluorescence microscopy. In WKY VSMCs, Enh 3 short term stimulation reduced basal ERK1/2 phosphorylation (26.8±5.9% vs veh, p<0.01), an effect absent in SHRSP VSMCs, while Enh 4 had no effect. Short term exposure to Enh 4, but not Enh3, reduced basal MLC20 phosphorylation (54.9±7.5% vs veh, p<0.001) in WKY but not in SHRSP VSMCs. In SHRSP VSMCs, Enh 3, but not Enh4, reduced basal AKT phosphorylation (63.5±8.9% vs veh, p<0.001). Long term stimulation with Enh 3 reduced expression of AKT (38.0±2.0% vs veh, p<0.001), PCNA (60.0±7.0% vs veh, p<0.001), and VCAM-1 (35.0±8.0% vs veh, p<0.001) in WKY VSMCs. Similarly, AKT (35.0±12.0% vs veh, p<0.05) expression was reduced by Enh 3, with no effect on other markers in SHRSP VSMCs. Enh 4 long-term stimulation reduced AKT expression (30.0±10.0% vs veh, p<0.05) in WKY VSMCs, without effect on SHRSP VSMCs. ET-1 induced Ca 2+ influx in WKY and SHRSP VSMCs was unaffected by Enh3 and Enh4. In conclusion, enhancing Mas1:ET B R interaction attenuates mitogenic and pro-inflammatory signaling pathways in WKY and SHRSP VSMCs. Enhancing interaction between these receptors does not increase Ca 2+ signaling, important in VSMC contraction. Our data suggest that Enh3 and Enh4 may have VSMC protective effects and that they do not amplify injurious signaling induced by ET-1/ETBR. These findings identify a potential new strategy in vasoprotection.
Epidermal growth factor (EGF), signals throught the EGF receptor (EGFR) and plays an important role in the pathogenesis of vascular remodeling. Transient receptor potential melastatin 7 (TRPM7) is a channel bound to a kinase domain important for Mg 2+ , Zn 2+ and Ca 2+ homeostasis. Cancer patients treated with EGFR inhibitors develop hypomagnesemia, suggesting a relationship between EGFR and TRPM7. Here we investigated the role of TRPM7 in EGF signaling in vascular smooth muscle cell (VSMC) from humans (hVSMC) and rats (rVSMC). VSMCs were stimulated with EGF (50ng/ml) for 5min and 24h with/without pretreatment of gefitinib (1μM), PP2 (10μM), 2APB (30μM) and NS8593 (40μM), inhibitors of EGFR, c-Src kinase and TRPM7 respectively. Aortas were isolated from wild type (WT), TRPM7-deficient (TRPM7 +/Δkinase ) and kinase-dead (TRPM7 R/R ) mice. Protein expression was assessed by immunoblotting. Ca 2+ and Mg 2+ were assessed using Cal-520 and Mg-green probes respectively. EGFR/TRPM7 interaction was investigated by proximity ligation assay (PLA), immunoprecipitation and confocal microscopy. VSMC migration and proliferation were examined by wound healing and CFSE proliferation assays. In hVSMC and rVSMC, EGF increased TRPM7 expression (47%) and phosphorylation (21%), (p<0.05); effects abolished by gefitinib and PP2. EGF-induced Mg 2+ and Ca 2+ influx was attenuated by gefitinib (4% and 8% respectively), NS8593 (5% for Mg 2+ ) and 2-APB (6% and 13% respectively). EGF enhanced ERK1/2 phosphorylation (3-fold) through c-Src, EGFR and TRPM7, p<0.05. Cell migration (26%) and proliferation (17%) were enhanced by EGF, and reduced by inhibitors of EGFR, TRPM7 and ERK1/2, p<0.05. EGF induced TRPM7-EGFR interaction (51%), which was reduced by gefitinib (34%) and PP2 (25%). VSMC from TRPM7 +/Δkinase showed reduced EGFR expression (73%), phospho-c-Src (22%), and phospho-ERK1/2 (90%). Aortas from TRPM7 R/R exhibited reduced phospho-EGFR (63%) and phospho-ERK1/2 (36%). Vessels from TRPM7 +/Δkinase showed reduced wall thickness (35%). Our findings demonstrate that interaction between EGFR/TRPM7 is a key process underlying EGF-induced VSMC migration and growth. This novel EGF-c-Src-EGFR-TRPM7 pathway may play an important role in vascular remodeling.
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