Regulator Of G-protein Signaling 5 Research Articles

Abstract WMP120: Critical Role of RGS5 in Modulating Blood-Brain Barrier Integrity During Focal Cerebral Ischemia

Background: Recent studies suggest that regulator of G-protein signaling 5 (RGS5), a negative modulator of the G-protein coupled receptors (GPCR) signaling pathways, may be an important modulator of glutamate receptor activation and calcium overload in endothelial cells after ischemic stroke. While glutamate may cause neuronal damage, it is unknown how glutamate affects blood-brain barrier (BBB) permeability. Methods: To investigate the effects of RGS5 on stroke severity, wild type (WT) and RGS5 -/- mice were subjected to 30 minutes of middle cerebral artery occlusion (MCAO). Cerebral infarct volume was evaluated by TTC and Nissl staining, while neurological deficit was assessed using a 5-point motor grading scale and adhesive tape removal test. BBB permeability was analyzed by measuring brain edema with the wet/dry method and by injecting 40kDA FITC-Dextran into the femoral vein. The role of RGS5 in regulating ischemia/glutamate-induced Rho/ROCK signaling in human brain microvasculare cells (HBMEC) was investigated using lentiviral transduction of RGS5 shRNA. To mimic brain ischemic conditions in vitro , we used a model consisting of 3-hour oxygen-glucose deprivation (OGD) with L-glutamate. RhoA/ROCK activity was determined by the phosphorylation MYPT1. In vitro permeability was assessed by Transwell permeability assays. Formation of actin stress fibers was observed with Phalloidin staining. Redistribution of important tight junction (TJ) proteins was analyzed by immunostaining. Results: Following MCAO, RGS5 -/- mice exhibited larger cerebral infarct volume and greater neurological deficits compared to controls. Brain water content and FITC-Dextran leakage were higher in the ipsilateral hemisphere of RGS5 -/- mice compared to controls. Furthermore, the Transwell permeability assay demonstrated increased permeability of FITC-Dextran in RGS5-deficient HBMEC, higher Rho/ROCK activation, greater actin-stress fiber formation, and redistribution of TJ proteins. Conclusion: These findings indicate that RGS5 plays an important role in actin cytoskeleton remodeling and BBB permeability following focal cerebral ischemia, in part, through the upregulation of RhoA/ROCK pathway.

Regulator of G-protein signaling 5 enhances portal vein invasion in hepatocellular carcinoma.

Portal vein invasion (PVI) is a major prognostic factor in hepatocellular carcinoma (HCC). The aim of the present study was to identify molecules that regulate PVI. Sections of cancerous tissue, paired noncancerous tissue and the PVI area were collected from 3 frozen HCC sections, using laser microdissection. The present study focused on 3 upregulated molecules, integrin β3 (ITGB3), secreted phosphoprotein 1 (SPP1) and regulator of G-protein signaling 5 (RGS5), and 2 molecules that were downregulated in PVI tissue compared with cancer tissue, metallothionein 1G (MT1G) and metallothionein 1H (MT1H), as determined by cDNA microarray analysis. Reverse transcription-quantitative polymerase chain reaction analysis of 32 HCC cases revealed that RGS5 mRNA levels were significantly increased and MT1 G and MT1H mRNA levels were significantly decreased in cancerous tissue compared with noncancerous tissue. However, there was no significant difference in ITGB3 and SPP1 expression. There were no significant differences between the expression of these molecules and any clinicopathologic factors, including PVI. Immunohistochemical staining for RGS5 in 60 HCC cases demonstrated that RGS5 protein levels were higher in cancerous tissue compared with paired noncancerous tissue in 63.3% of HCC cases. Furthermore, high expression of RGS5 in cancerous tissue was significantly associated with PVI and tended to be associated with intrahepatic metastasis. Confluent multinodular type was significantly more frequent in cases with high expression of RGS5 in the cancerous tissue. Therefore, RGS5 may be a useful prognostic biomarker as well as a potential target of molecular therapy to treat HCC.

Regulator of G-protein signaling 5 protects cardiomyocytes against apoptosis during in vitro cardiac ischemia-reperfusion in mice by inhibiting both JNK1/2 and P38 signaling pathways

Ischemic heart disease is one of the most common diseases in modern society. Ischemic myocardium can be salvaged by vascular recanalization therapy, but its benefit is attenuated by injury that can occur during reperfusion. And apoptotic cell death plays an important part in myocardial ischemia-reperfusion (IR) injury. Regulator of G-protein signaling 5 (RGS5), highly expressed in different cell types of the human adult heart, is a guanosine triphosphatase-activating protein to inhibit many signaling pathways such as c-Jun NH2-terminal kinase 1/2 (JNK1/2) and p38 which promote cardiac IR-induced apoptosis. However the role of RGS5 in cardiac IR-induced apoptosis remains unclear. An in vitro IR model was applied to the isolated hearts of wild type mice (WT), RGS5-transgenic mice (TG), and RGS5-knockout mice (KO). Our results revealed that compared with either WT or KO mice, TG mice showed inhibition of cardiomyocyte apoptosis as indicated by a greater increase of B cell lymphoma/lewkmia-2 (Bcl-2), and an obvious reduction in the positive expression of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), Bcl-2 Associated X protein (Bax), and active caspase-3. Moreover, the inhibition of both JNK1/2 and p38 signaling markedly reversed IR-induced cardiomyocyte apoptosis in RGS5-KO mice. These studies show that RGS5 protects cardiomyocytes against apoptosis during IR through inhibiting both JNK1/2 and p38 signaling pathways.

Regulator of G-Protein Signaling 5 Prevents Smooth Muscle Cell Proliferation and Attenuates Neointima Formation.

Regulator of G-protein signaling 5 (RGS5) is abundantly expressed in vascular smooth muscle cells (SMCs) and inhibits G-protein signaling by enhancing the guanosine triphosphate-hydrolyzing activity of Gα-subunits. In the present study, we investigated the effects of RGS5 on vascular SMC function in vitro and neointima formation after wire-induced injury in mice and determined the underlying mechanisms. We found a robust expression of RGS5 in native arteries of C57BL/6 mice and a highly significant downregulation within neointimal lesions 10 and 21 days after vascular injury as assessed by quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. In vitro, RGS5 was found significantly downregulated after mitogenic stimulation of human coronary artery SMCs. To restore RGS5 levels, SMCs were transduced with adenoviral vectors encoding wild-type RGS5 or a nondegradable mutant. RGS5-WT and, even more prominently, the C2A-RGS5 mutant prevented SMC proliferation and migration. In contrast, the siRNA-mediated knockdown of RGS5 significantly augmented SMC proliferation. Following overexpression of RGS5, fluorescence-activated cell sorting analysis of propidium iodide-stained cells indicated cell cycle arrest in G0/G1 phase. Mechanistically, inhibition of the phosphorylation of the extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase downstream signaling was shown to be responsible for the anti-proliferative effect of RGS5. Following wire-induced injury of the femoral artery in C57BL/6 mice, adenoviral-mediated overexpression of RGS5-WT or C2A-RGS5 significantly reduced SMC proliferation and neointima formation in vivo. Downregulation of RGS5 is an important prerequisite for SMC proliferation in vitro and in vivo. Therefore, reconstitution of RGS5 levels represents a promising therapeutic option to prevent vascular remodeling processes.

Abstract 17947: Regulator of G-Protein Signaling 5 Prevents Smooth Muscle Cell Proliferation and Attenuates Neointima Formation

Introduction: Regulator of G-protein signaling 5 (RGS5) is abundantly and specifically expressed in vascular smooth muscle cells (SMCs) and inhibits G-protein signaling by enhancing the GTP-hydrolyzing activity of Gα-subunits. Hypothesis: In the present study, we aimed to investigate the effects of RGS5 on vascular SMC function in vitro and neointima formation in vivo and to determine the underlying molecular mechanisms. Methods: Expression of RGS5 was assessed in vitro and in vivo by qPCR, immunoblotting and immunohistochemistry. Proliferation and migration of human coronary artery SMC were assessed in vitro following the adenoviral-mediated over expression of wild type (WT) RGS5, of a non-degradable mutant (C2A-RGS5) or siRNA-mediated knock down. In vivo, the proliferation and neointima formation were assessed in a wire-mediated injury model of the femoral artery in C57BL/6J mice. Results: We found a robust expression of RGS5 in native arteries of C57BL/6 mice and a significant down-regulation within neointimal lesions 10 and 21 days after vascular injury as assessed by qPCR, immunohistochemistry and immunoblotting. In vitro, RGS5 was found significantly down-regulated after mitogenic stimulation of SMCs. Overexpression of RGS5 or, even more prominently, the C2A-RGS5 mutant prevented SMC proliferation and migration. In contrast, the siRNA-mediated knockdown of RGS5 significantly augmented SMC proliferation. Following overexpression of RGS5, FACS analysis of propidium iodide-stained cells indicated cell cycle arrest in G0/G1 phase. Mechanistically, inhibition of ERK1/2 phosphorylation and MAPK downstream signaling was found to be responsible for the anti-proliferative effect since over expression of a constitutive active form of MEK reversed the growth-inhibitory effect of of RGS5. Following wire-induced injury, adenoviral-mediated overexpression of RGS5 or C2A-RGS5 significantly decreased ERK phosphorylation, SMC proliferation and neointima formation in vivo (n=8; p<0.001). Conclusions: Down-regulation of RGS5 is an important prerequisite for SMC proliferation and neointima Formation. Therefore, reconstitution of RGS5 levels might represent a promising therapeutic option to prevent vascular remodeling processes.

Abstract 441: Rgs5 Controls Myogenic Responses of Vascular Smooth Muscle Cells

Contraction of vascular smooth muscle cells (VSMCs) in response to an increase in blood pressure is pivotal for maintaining blood flow and constitutes both consequence and cause of chronic hypertension. While a plethora of contraction-mediating mechanisms has been delineated, not much is known about molecular determinants regulating the responses of VSMCs to an increase in wall stress or biomechanical stretch. In this context, we reported that conditional ablation of Junb - a subunit of the transcription factor AP-1 (activator protein 1) - interferes with the expression of myosin light chain 9 and consequently impaired myogenic responses of arteries. We hypothesized that the arterial circumferential wall stress is increased under these conditions which may influence the VSMC phenotype and vessel wall architecture. In fact, Junb-deficient arteries showed structural remodeling as evidenced by a ~2-3-fold decrease in collagen type I and IV content (n=5, p<0.05). cDNA microarray analyses revealed that expression of regulator of G-protein signaling 5 (RGS5) was up-regulated in VSMCs (~6-fold, n=4, p<0.001) isolated from Junb-deficient arteries which was confirmed in vivo but not mediated by AP-1. However, elevation of wall stress by volume-induced hypertension in vivo or exposure of VSMCs to biomechanical stretch in vitro was sufficient to trigger RGS5 expression (3-fold, n=3, p<0.05). Overexpression of this regulator activates RhoA and enhanced stretch-induced responses of VSMCs such as stress fiber formation while its knockdown showed opposite effects. In line with this, RGS5-deficient arteries show an impaired pressure-mediated constriction (n=8, p<0.05) and both the hypertension-induced increase in diastolic blood pressure as well as structural remodeling of the arterial vessel wall were significantly attenuated in RGS5-deficient mice. Collectively, these findings indicate that biomechanical stretch mediates the expression of RGS5 in VSMCs to enhance stretch-dependent responses as a prerequisite for adequate adaptive vascular remodeling processes. This mechanism may establish RGS5 as a promising target for future strategies to combat, e.g. the deleterious consequences of arterial hypertension such as arterial stiffening.

Regulator of G-protein signaling-5 is a marker of hepatic stellate cells and expression mediates response to liver injury.

Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4)-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.

THE ROLE OF RGS5 IN MEDIATING THE PLEIOTROPIC EFFECTS OF STATINS IN VASCULAR SMOOTH MUSCLE CELLS

Statins have been demonstrated to exert pleiotropic anti-atherogenic effects in vascular smooth muscle cells (VSMCs), including inhibition of VSMC migration and proliferation. However, the mechanism underlying this inhibition is not fully understood. A previous microarray study on aortas of statin-treated mice have demonstrated regulator of G-protein signaling 5 (RGS5) to show the greatest increase in expression among non-cytoskeletal proteins. We hypothesized that by inhibiting G-protein signaling involved in VSMC recruitment, RGS5 may contribute to the pleiotropic effects of statins in VSMCs. VSMCs were isolated from mouse aortas and incubated at different concentrations of fluvastatin for 24 hours. Quantitative real-time polymerase chain reaction analysis demonstrated that RGS5 expression significantly increased with 10 μM and 100 μM fluvastatin as compared to vehicle-treated controls (P<0.05), but not with 1 μM fluvastatin. Preliminary time profile studies with 10 μM fluvastatin suggest that the upregulation of RGS5 occurs after 18 hours, which remains stable for up to 48 hours. In order to assess the role of RGS5 during atherosclerosis progression, RGS5/apoE dKO mice, along with RGS5 WT/apoE KO littermate mice were generated and placed on a high cholesterol diet for 12 weeks. During this period, RGS5/apoE dKO mice displayed markedly reduced survival as compared to RGS5 WT/apoE KO mice (P<0.05). Subsequent Oil-Red O stained plaque size analysis in the thoracic aorta demonstrated a larger normalized lesion size in RGS5/apoE dKO mice as compared to littermate RGS5 WT/apoE KO mice (P<0.05). These results suggest an inhibitory role of RGS5 in the progression of atherosclerosis, and that its upregulation in VSMCs following statin treatment may contribute to the therapeutic pleiotropic effects of statins. Future experiments will assess the role of the statin-induced RGS5 upregulation in modulating signaling pathways involved in VSMC recruitment.

Antiatherosclerotic Effect of Korean Red Ginseng Extract Involves Regulator of G‐Protein Signaling 5

Regulator of G-protein signaling 5 (RGS5), an inhibitor of Gα(q) and Gα(i) activation, has been reported to have antiatherosclerosis. Previous studies showed antiatherosclerotic effect of Korean red ginseng water extract (KRGE) via multiple signaling pathways. However, potential protective effect of KRGE through RGS5 expression has not been elucidated. Here, we investigated the antiatherosclerotic effect of KRGE in vivo and in vitro and its role on RGS5 mRNA expression. Elevated levels of total cholesterol, lactate dehydrogenase (LDH), and triglyceride (TG) in western diet groups of low-density lipoprotein receptor deficient LDLr−/− mice were reversed by oral administration of KRGE. KRGE suppressed transcriptional activity of tumor necrotic factor alpha (TNF-α), interleukin-6 (IL-6), and leptin in adipose tissue. It also potently repressed western diet-induced atheroma formation in aortic sinus. While KRGE showed reduced mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells, it enhanced mRNA expression of RGS5. Moreover, RGS5 siRNA transfection of microglia cells pretreated with KRGE reversed its inhibitory effect on the expression of iNOS, COX-2, and IL-1β mRNA. In conclusion, KRGE showed antiatherosclerotic and anti-inflammatory effects in western diet fed LDLr−/− mice and this effect could partly be mediated by RGS5 expression.

Roles of regulator of G-protein signaling 5 in tumors

Regulator of G-protein signaling 5 (RGS5) belongs to RGS family,which can negatively regulate the conduction of this signaling pathway.RGS5 mainly expresses in vascular pericyte,and is closely related to the occurrence,development and maturation of the blood vessels.Loss of RGS5 results in pericyte maturation,tumor vascular normalization,and these changes can improve the curative effect combined with chemotherapy and immunotherapy,indicating that RGS5 may become a new target of anti-tumor treatment.In addition,RGS5 involves in tumor metastasis and apoptosis,which can improve antineoplastic effect by inducing tumor cells apoptosis. Key words: Neoplasms; Regulator of G-protein signaling 5

Over-expression of regulator of G protein signaling 5 promotes tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. The regulator of G-protein signaling 5 (RGS5) has been reported to be highly expressed in some malignant tumors. However, its expression and role in HCC has not been reported. The expression of RGS5 was examined in liver cancer tissues and cell lines by real-time quantitative PCR. The cell migration and invasion was investigated by wound healing and transwell invasion assay. The epithelial-mesenchymal transition markers were detected by Western blotting or immunofluorescence. We observed that RGS5 is over-expressed in most of liver cancer tissue samples and cell lines compared with matched normal samples. Further analysis showed that the over-expression of RGS5 is associated with liver cancer recurrence, venous infiltration, and patients' poor survival. Next, we found that knockdown of RGS5 significantly inhibits liver cancer cell migration and invasion in highly invasive liver cancer cells. Furthermore, we found that over-expression of RGS5 induces epithelial-mesenchymal transition in epithelial liver cancer cells. These results indicate that over-expression of RGS5 promotes tumor metastasis by inducing epithelial-mesenchymal transition in HCC.

Regulator of G‐protein Signaling 5 reduces HeyA8 ovarian cancer cell proliferation and extends survival in a murine tumor model

The regulator of G‐protein signaling 5 (RGS5) belongs to a family of GTPase activators that terminate signaling cascades initiated by extracellular mediators and GPCRs. One RGS5 role is as a pericyte biomarker influencing the switch to angiogenesis during malignant progression. Its other role is to promote apoptosis in hypoxic environments. We set out to clarify the extent to which RGS5 expression regulates tumor progression – whether it plays a pathogenic or protective role. We thus constructed an inducible gene expression system to achieve RGS5 expression in HeyA8‐ MDR ovarian cancer cells. Through this we observed that inducible RGS5 expression significantly reduces in vitro BrdU‐positive HeyA8‐MDR cells. Interestingly, mice bearing RGS5‐ expressing tumors demonstrated an increase in survival compared with controls, which might be attributed to the vast regions of necrosis observed by pathological examination. Additionally, mice bearing RGS5‐expressing tumors were less likely to have ulcerated tumors. Taken together, this data supports the idea that temporal expression and stabilization of RGS5 could be a valuable tactic within the context of a multi‐component approach for modulating tumor progression.

Nanogold and 5-fluorouorouracil conjugation affecting hepatic tumor vascular and inhibiting tumor growth

Objective To observe the effect of hepatic tumor vascular and the carcinoma growth with nangold and 5-fluorouracil (5-Fu) conjugation.Methods Hepatocellular cancer cells of the line H22 were injected subcutaneously into the right armpits of 21 BALB/C nude mice.When the size of transplanted tumor reached about 7-8 mm,they were divided into 3 groups,7 mice in each group,5-Fu and nanogold 0.2 ml was injected respectively in conjugation group,only 5-Fu was injected in 5-Fu group,and the same volume normal saline was used in control group.After continuous drug use for 7 days,the mice were sacrifled,the tumor size and weight were measured,the microvacular density (MVD) and mature pericyte coverage index (MPI) of tumor were detected by immunohistechemical staining,concentration of 5-Fu in tumor was detected by high performance liquid chromatography (HPLC) ; regulator of G-protein signaling-5(RGS-5) was detected by western blotting.Results (1) The tumor volume and the MVD in the conjugation group were (1.487 ± 0.859) cm3 and (23 ± 7) vessels/mm2,less than the 5-Fu group [(2.137 ±1.047) cm3 and (43 ± 12) vessels/mm2] ; Also the MPI and the percentage of 5-Fu in tumor were (37.5 ± 11.3)％ and (5.37 ±0.41) mg/L respectively in the conjugation group,more than the other two groups (P ＜0.05).(2)Western blotting showed the expression of RGS-5 in conjugation group were less than the other groups.Conclusion (1)Nangold enhanced the effect of 5-Fu anti-hepatic tumor,inhibiting the growth of carcinoma,it could be benefit for the 5-Fu penetrating the tumor inside.(2) Nangold could inhibit the expression of RGS-5 and upward MPI,make tumor microvessel normal. Key words: Nanogold; Fluorouacil; Tumor vascular; G-protein signaling-5

Regulator of G-Protein Signaling 5 Controls Blood Pressure Homeostasis and Vessel Wall Remodeling

Regulator of G-protein signaling 5 (RGS5) modulates G-protein-coupled receptor signaling and is prominently expressed in arterial smooth muscle cells. Our group first reported that RGS5 is important in vascular remodeling during tumor angiogenesis. We hypothesized that RGS5 may play an important role in vessel wall remodeling and blood pressure regulation. To demonstrate that RGS5 has a unique and nonredundant role in the pathogenesis of hypertension and to identify crucial RGS5-regulated signaling pathways. We observed that arterial RGS5 expression is downregulated with chronically elevated blood pressure after angiotensin II infusion. Using a knockout mouse model, radiotelemetry, and pharmacological inhibition, we subsequently showed that loss of RGS5 results in profound hypertension. RGS5 signaling is linked to the renin-angiotensin system and directly controls vascular resistance, vessel contractility, and remodeling. RGS5 deficiency aggravates pathophysiological features of hypertension, such as medial hypertrophy and fibrosis. Moreover, we demonstrate that protein kinase C, mitogen-activated protein kinase/extracellular signal-regulated kinase, and Rho kinase signaling pathways are major effectors of RGS5-mediated hypertension. Loss of RGS5 results in hypertension. Loss of RGS5 signaling also correlates with hyper-responsiveness to vasoconstrictors and vascular stiffening. This establishes a significant, distinct, and causal role of RGS5 in vascular homeostasis. RGS5 modulates signaling through the angiotensin II receptor 1 and major Gαq-coupled downstream pathways, including Rho kinase. So far, activation of RhoA/Rho kinase has not been associated with RGS molecules. Thus, RGS5 is a crucial regulator of blood pressure homeostasis with significant clinical implications for vascular pathologies, such as hypertension.

Effects of nangold on proliferation and apoptosis of human microvascular pericyte cells in vitro

Objective To observe the effects of nangold(GNP) on proliferation and the expression of regulator of G-protein signaling5 (RGS5) of human microvascular pericytes.Methods The GNP solutions of 60 nmol/L with diameters of 25,50 and 100 nm were prepared.HBVP-1200 cells were cultured in vitro.The methyl thiazolium tetrazolium (MTT) was used to detect the effects of ditferent diameters of GNP on proliferation of pericytes.Atomic force microscope (AFM) was used to observe morphological changes of pericytes.The expression of RGS5 protein was detected by using Western blotting.Results GNP could euhance the proliferation of human microvascular pericytes.After treatment with different diameters of GNP (25,50,and 100 nm),the proliferation rate of pericytes was ( 147.9 ± 5.9) ％,( 121.7 ± 3.4) ％ and ( 107.6 ± 2.1 ) ％ respectively.When the diameter of GNP was smaller,the proliferation rate was increased significantly (P ＜0.05).Under AFM,the morphological changes of pericytes treated with GNP were observed,including invagination,obvious shrinked cell membrane and much rougher surface.The protein expression of RGS5 was reduced when the GNP was given 24 h later,especially when the diameter of GNP was 25 nm.Conclusion GNP could enhance the proliferation of human microvascular pericytes,and restrain the RGS5 protein expression.The smaller the diameter of GNP,the stronger effects of GNP on the pericytes. Key words: Gold nanoparticles; Pericyte; Regulator of G-protein signaling 5; Atomic force

PO-86 Cancer cells modify the microenvironment: RGS5, a novel endothelial protein of the abnormal tumor vasculature

PO-86 Cancer cells modify the microenvironment: RGS5, a novel endothelial protein of the abnormal tumor vasculature

Regulator of G-Protein Signaling–5 Inhibits Bronchial Smooth Muscle Contraction in Severe Asthma

Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein-coupled receptor (GPCR)-induced Ca(2+) mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca(2+) mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling-5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca(2+) to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.

Regulator of G-Protein Signaling 5 Reduces HeyA8 Ovarian Cancer Cell Proliferation and Extends Survival in a Murine Tumor Model.

The regulator of G-protein signaling 5 (RGS5) belongs to a family of GTPase activators that terminate signaling cascades initiated by extracellular mediators and G-protein-coupled receptors. RGS5 has an interesting dual biological role. One functional RGS5 role is as a pericyte biomarker influencing the switch to angiogenesis during malignant progression. Its other functional role is to promote apoptosis in hypoxic environments. We set out to clarify the extent to which RGS5 expression regulates tumor progression—whether it plays a pathogenic or protective role in ovarian tumor biology. We thus constructed an inducible gene expression system to achieve RGS5 expression in HeyA8-MDR ovarian cancer cells. Through this we observed that inducible RGS5 expression significantly reduces in vitro BrdU-positive HeyA8-MDR cells, although this did not correlate with a reduction in tumor volume observed using an in vivo mouse model of ovarian cancer. Interestingly, mice bearing RGS5-expressing tumors demonstrated an increase in survival compared with controls, which might be attributed to the vast regions of necrosis observed by pathological examination. Additionally, mice bearing RGS5-expressing tumors were less likely to have ulcerated tumors. Taken together, this data supports the idea that temporal expression and stabilization of RGS5 could be a valuable tactic within the context of a multicomponent approach for modulating tumor progression.

Biochemical pathways in cancer.

The revolutionary advances in cellular and molecular biochemistry in the last quarter century have provided an unprecedented opportunity for systematic approaches to understand the cancer process. With these new advances and the knowledge acquired as a direct result, an enormous impact has positively affected the clinical areas of cancer prevention, disease management, and treatment of malignancy. Despite these scientific achievements, we are still faced with the challenge of fully understanding the complex nature of cancer, including issues of disease recurrence and drug resistance. Therefore, dissecting biochemical pathways that underlie the development of cancer should be given high priority to improve knowledge of human health. In this special issue, we will explore the various aspects of cancer-related biochemical pathways. The papers in this issue discuss multiple signaling pathways involved in the regulation of proliferation, senescence, and death of cancer cells. M. K. Altman et al. demonstrated that the regulator of G-protein signaling 5 (RGS5) is able to reduce the proliferation of ovarian cancer cells and extend the survival of mice. The tumor suppressor p53 plays an essential role in cell proliferation, cell cycle progression, cell death, and senescence [1, 2]. Reisman et al. reviewed the evidence that DNA-binding factors RBP-Jκ and C/EBPβ-2 transcriptionally activate p53 to ensure a rapid cellular response to DNA damage. p53 is also involved in complex networks of mitotic kinase signaling in response to mitotic spindle damage to ensure the proper cell cycle progression. G. H. Ha and E. K. Breuer discussed a mutual regulation network between mitotic kinase and p53 signaling. The tumor suppressor p16INK4A has shown to be implicated in replicative senescence. Here, the role of p16INK4A in replicative senescence and DNA damage-induced premature senescence in the absence of p53 was reviewed by R. Mirzayans et al. The authors also discussed a possible existence of a negative regulatory relationship between p53 and p16INK4A. R. Jager and H. O. Fearnhead reviewed cancer cell proliferation and death in a different point of view. These authors suggested that cancer cell proliferation might be a consequence of inappropriate or corrupted tissue-repair programs, initiated by a signal from dying cells. Mounting evidence suggested that dissecting the biochemical process of genetic alterations will help us understand mechanisms underlying the genesis and spread of cancer and develop novel strategies for targeted therapy and tailored cancer management [3, 4]. J. L. Johnson et al. discussed the genetic and biochemical alternations in heterogeneous cell signaling pathways in non-small-cell lung cancer and its implications in targeted cancer therapy. The papers also provide new insights into the pathogenesis of cancer and therapeutic intervention strategies. As a new therapeutic target, S. Mohanan et al. suggested that peptidylarginine deiminases (PADs) play an important role in cancer pathogenesis and its inhibition may have a profound impact on the regulation of the inflammatory tumor microenvironment and tumor growth. To improve the efficacy of chemotherapy in hormone-refractory prostate cancer, E. Tsakalozou et al. investigated the combination effects of docetaxel and doxorubicin and proposed the synergistic ratios of drug combinations. We believe that a better understanding of cellular biochemical pathways and activities will ultimately have important implications in the management of cancer patients. Previous work in this area has demonstrated the power of translating molecular and biochemical findings to therapeutics and the clinic. It is our hope that the papers in this issue will be of great help in understanding the complex nature of cancer and in designing and conducting preventive and therapeutic interventions in the near future. Eun-Kyoung Yim Breuer Mandi M. Murph Rolf J. Craven

Regulator of G-protein signaling 5 (RGS5) protein: a novel marker of cancer vasculature elicited and sustained by the tumor’s proangiogenic microenvironment

We previously identified regulator of G-protein signaling 5 (RGS5) among several genes expressed by tumor-derived endothelial cells (EC). In this study, we provide the first in vivo/ex vivo evidence of RGS5 protein in the vasculature of ovarian carcinoma clinical specimens and its absence in human ovaries. Consistent with this, we show higher amounts of Rgs5 transcript in EC isolated from human cancers (as opposed to normal tissues) and demonstrate that expression is sustained by a milieu of factors typical of the proangiogenic tumor environment, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2). Supporting these findings, we show elevated levels of Rgs5 mRNA in the stroma from strongly (as opposed to weakly) angiogenic ovarian carcinoma xenografts and accordingly, we also show more of the protein associated to the abnormal vasculature. RGS5 protein predominantly colocalizes with the endothelium expressing platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) and to a much lesser extent with perivascular/mural cells expressing platelet-derived growth factor receptor-beta (PDGFR-β) or alpha smooth muscle actin (αSMA). To toughen the relevance of the findings, we demonstrate RGS5 in the blood vessels of other cancer models endowed with a proangiogenic environment, such as human melanoma and renal carcinoma xenografts; to the contrary, it was undetectable in the vasculature of normal mouse tissues. RGS5 expression by the cancer vasculature triggered and retained by the proangiogenic microenvironment supports its exploitation as a novel biomarker and opens the path to explore new possibilities of therapeutic intervention aimed at targeting tumor blood vessels.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-011-0862-8) contains supplementary material, which is available to authorized users.