Regulator Of G-protein Signaling 5 Research Articles

Age-Dependent RGS5 Loss in Pericytes Induces Cardiac Dysfunction and Fibrosis.

Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown. In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts. Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFβ (transforming growth factor beta)2-dependent mechanism. Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.

RGS5 as a Biomarker of Pericytes, Involvement in Vascular Remodeling and Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by a sustained rise in mean pulmonary artery pressure. Pulmonary vascular remodeling serves an important role in PAH. Identifying a key driver gene to regulate vascular remodeling of the pulmonary microvasculature is critical for PAH management. Differentially expressed genes were identified using the Gene Expression Omnibus (GEO) GSE117261, GSE48149, GSE113439, GSE53408 and GSE16947 datasets. A co-expression network was constructed using weighted gene co-expression network analysis. Novel and key signatures of PAH were screened using four algorithms, including weighted gene co-expression network analysis, GEO2R analysis, support vector machines recursive feature elimination and robust rank aggregation rank analysis. Regulator of G-protein signaling 5 (RGS5), a pro-apoptotic/anti-proliferative protein, which regulate arterial tone and blood pressure in vascular smooth muscle cells. The expression of RGS5 was determined using reverse transcription-quantitative PCR (RT-qPCR) in PAH and normal mice. The location of RGS5 and pericytes was detected using immunofluorescence. Compared with that in the normal group, RGS5 expression was upregulated in the PAH group based on GEO and RT-qPCR analyses. RGS5 expression in single cells was enriched in pericytes in single-cell RNA sequencing analysis. RGS5 co-localization with pericytes was detected in the pulmonary microvasculature of PAH. RGS5 regulates vascular remodeling of the pulmonary microvasculature and the occurrence of PAH through pericytes, which has provided novel ideas and strategies regarding the occurrence and innovative treatment of PAH.

RGS5 augments astrocyte activation and facilitates neuroinflammation via TNF signaling

Astrocytes contribute to chronic neuroinflammation in a variety of neurodegenerative diseases, including Parkinson's disease (PD), the most common movement disorder. However, the precise role of astrocytes in neuroinflammation remains incompletely understood. Herein, we show that regulator of G-protein signaling 5 (RGS5) promotes neurodegenerative process through augmenting astrocytic tumor necrosis factor receptor (TNFR) signaling. We found that selective ablation of Rgs5 in astrocytes caused an inhibition in the production of cytokines resulting in mitigated neuroinflammatory response and neuronal survival in animal models of PD, whereas overexpression of Rgs5 had the opposite effects. Mechanistically, RGS5 switched astrocytes from neuroprotective to pro-inflammatory property via binding to the receptor TNFR2. RGS5 also augmented TNFR signaling-mediated pro-inflammatory response by interacting with the receptor TNFR1. Moreover, interrupting RGS5/TNFR interaction by either RGS5 aa 1–108 or small molecular compounds feshurin and butein, suppressed astrocytic cytokine production. We showed that the transcription of astrocytic RGS5 was controlled by transcription factor early B cell factor 1 whose expression was reciprocally influenced by RGS5-modulated TNF signaling. Thus, our study indicates that beyond its traditional role in G-protein coupled receptor signaling, astrocytic RGS5 is a key modulator of TNF signaling circuit with resultant activation of astrocytes thereby contributing to chronic neuroinflammation. Blockade of the astrocytic RGS5/TNFR interaction is a potential therapeutic strategy for neuroinflammation-associated neurodegenerative diseases.

Molecular Regulation of the Response of Brain Pericytes to Hypoxia

The brain needs sufficient oxygen in order to function normally. This is achieved by a large vascular capillary network ensuring that oxygen supply meets the changing demand of the brain tissue, especially in situations of hypoxia. Brain capillaries are formed by endothelial cells and perivascular pericytes, whereby pericytes in the brain have a particularly high 1:1 ratio to endothelial cells. Pericytes not only have a key location at the blood/brain interface, they also have multiple functions, for example, they maintain blood-brain barrier integrity, play an important role in angiogenesis and have large secretory abilities. This review is specifically focused on both the cellular and the molecular responses of brain pericytes to hypoxia. We discuss the immediate early molecular responses in pericytes, highlighting four transcription factors involved in regulating the majority of transcripts that change between hypoxic and normoxic pericytes and their potential functions. Whilst many hypoxic responses are controlled by hypoxia-inducible factors (HIF), we specifically focus on the role and functional implications of the regulator of G-protein signaling 5 (RGS5) in pericytes, a hypoxia-sensing protein that is regulated independently of HIF. Finally, we describe potential molecular targets of RGS5 in pericytes. These molecular events together contribute to the pericyte response to hypoxia, regulating survival, metabolism, inflammation and induction of angiogenesis.

Implications of regulator of G-protein signaling 5 expression in the pathogenesis of primary and secondary hyperparathyroidism

ObjectiveTo study the protein and mRNA expressions of regulator of G-protein signaling 5 (RGS5) in the pathogenesis of hyperparathyroidism.MethodsThe expression of RGS5 protein in 20 primary hyperparathyroidism (PHPT), 31 secondary hyperparathyroidism (SHPT), and 20 control cases were studied by immunohistochemistry (IHC). The expression of RGS5 mRNA in 15 PHPT, 102 SHPT, and 7 normal parathyroid tissue were measured by quantitative real-time PCR (qRT-PCR) method.ResultsThe expressions of RGS5 in PHPT tissues were significantly higher than that in SHPT and normal parathyroid tissues (P < 0.05). While the differences in RGS5 protein expressions between SHPT and respective control samples were not statistically significant (P > 0.05). Likewise, the RGS5 mRNA expression in PHPT was significantly higher than that in SHPT (P < 0.05) and normal parathyroid (P < 0.05) samples. In a similar line, the differences in RGS5 gene expressions between SHPT and control tissues were not statistically significant (P > 0.05).ConclusionsThe characteristic RGS5 protein and mRNA levels in hyperparathyroidism might be helpful in discovering the pathomechanism of hyperparathyroidism and novel therapeutic targets as well.

Regulator of G-Protein Signaling 5 Protein Deficiency Differentially Influences Blood Pressure, Vascular and Behavioral Effects in Aged Male Mice.

Aging and elevated activity of the renin-angiotensin-system (RAS) are associated with hypertension, vascular and emotional behavioral abnormalities, like anxiety and depression. Many actions of the main effector hormone of the RAS, angiotensin II (Ang II), are mediated by Ang II type 1 receptor whose activity is modulated by the regulator of G-protein signaling 5 (RGS5) protein. We assessed the role of RGS5 on blood pressure, vascular and emotional behavioral outcomes in aged male mice in the presence and absence of chronically elevated Ang II levels. We used aged (∼21-month old) male RGS5-deficient (RGS5 -/- ) and wild-type (RGS5 +/+ ) mice treated with vehicle (saline) or Ang II (1 mg/kg/d for 21 days). RGS5 deficiency increased baseline and cerebral vascular superoxide levels in the presence of chronically elevated Ang II levels, suggesting that RGS5 deficiency leads to elevated blood pressure and deleterious cerebral vascular outcomes in aged mice. RGS5 deletion had no effect on Ang II-induced increases in systolic blood pressure. Chronically elevated Ang II levels increased spontaneous locomotor activity in RGS5 +/+ but not RGS5 -/- mice. RGS5 deficiency and Ang II treatment had no effect on anxiety- and depression-like behavior. This is the first study to assess the effects of deficiency of an RGS protein in the vasculature or on emotional behavioral outcomes in aged mice. We report that RGS5 has protective effects on blood pressure and the cerebral vasculature in aged mice. Clinically, these data suggest that RAS blockers may significantly reduce cerebrovascular disease risk in aged males lacking RGS5.

RGS5 Attenuates Baseline Activity of ERK1/2 and Promotes Growth Arrest of Vascular Smooth Muscle Cells.

The regulator of G-protein signaling 5 (RGS5) acts as an inhibitor of Gαq/11 and Gαi/o activity in vascular smooth muscle cells (VSMCs), which regulate arterial tone and blood pressure. While RGS5 has been described as a crucial determinant regulating the VSMC responses during various vascular remodeling processes, its regulatory features in resting VSMCs and its impact on their phenotype are still under debate and were subject of this study. While Rgs5 shows a variable expression in mouse arteries, neither global nor SMC-specific genetic ablation of Rgs5 affected the baseline blood pressure yet elevated the phosphorylation level of the MAP kinase ERK1/2. Comparable results were obtained with 3D cultured resting VSMCs. In contrast, overexpression of RGS5 in 2D-cultured proliferating VSMCs promoted their resting state as evidenced by microarray-based expression profiling and attenuated the activity of Akt- and MAP kinase-related signaling cascades. Moreover, RGS5 overexpression attenuated ERK1/2 phosphorylation, VSMC proliferation, and migration, which was mimicked by selectively inhibiting Gαi/o but not Gαq/11 activity. Collectively, the heterogeneous expression of Rgs5 suggests arterial blood vessel type-specific functions in mouse VSMCs. This comprises inhibition of acute agonist-induced Gαq/11/calcium release as well as the support of a resting VSMC phenotype with low ERK1/2 activity by suppressing the activity of Gαi/o.

RGS5-TGFβ-Smad2/3 axis switches pro- to anti-apoptotic signaling in tumor-residing pericytes, assisting tumor growth.

Regulator-of-G-protein-signaling-5 (RGS5), a pro-apoptotic/anti-proliferative protein, is a signature molecule of tumor-associated pericytes, highly expressed in several cancers, and is associated with tumor growth and poor prognosis. Surprisingly, despite the negative influence of intrinsic RGS5 expression on pericyte survival, RGS5highpericytes accumulate in progressively growing tumors. However, responsible factor(s) and altered-pathway(s) are yet to report. RGS5 binds with Gαi/q and promotes pericyte apoptosis in vitro, subsequently blocking GPCR-downstream PI3K-AKT signaling leading to Bcl2 downregulation and promotion of PUMA-p53-Bax-mediated mitochondrial damage. However, within tumor microenvironment (TME), TGFβ appeared to limit the cytocidal action of RGS5 in tumor-residing RGS5highpericytes. We observed that in the presence of high RGS5 concentrations, TGFβ-TGFβR interactions in the tumor-associated pericytes lead to the promotion of pSmad2-RGS5 binding and nuclear trafficking of RGS5, which coordinately suppressed RGS5-Gαi/q and pSmad2/3-Smad4 pairing. The RGS5-TGFβ-pSmad2 axis thus mitigates both RGS5- and TGFβ-dependent cellular apoptosis, resulting in sustained pericyte survival/expansion within the TME by rescuing PI3K-AKT signaling and preventing mitochondrial damage and caspase activation. This study reports a novel mechanism by which TGFβ fortifies and promotes survival of tumor pericytes by switching pro- to anti-apoptotic RGS5 signaling in TME. Understanding this altered RGS5 signaling might prove beneficial in designing future cancer therapy.

Abstract P096: Alterations In Vascular Rgs5 Expression In Erythropoietin-induced Hypertension Mediated By Erk1/2 Pathway Activation

Introduction: Erythropoietin (EPO)-induced hypertension is a common adverse event among patients receiving EPO therapy and is associated with risk of myocardial infarction and stroke, yet the underlying molecular mechanisms responsible for EPO related cardiovascular disease mortality are not clear. Regulator of G-protein signaling-5 ( Rgs5 ) is highly expressed in vascular smooth muscle cells (SMCs) and its action in fine tuning G-protein signaling is critical in regulating vascular tone. Whether Rgs5 plays a role in EPO-mediated hemodynamic changes is not known. We have previously shown that increased arterial pressure after EPO administration in mice is associated with significant reduction in aortic Rgs5 expression. One of the proposed mechanisms of Rgs5 mediated cellular response involves regulation of MAPK/ERK signaling pathway. Here, we investigated the role of Rgs5 and ERK1/2 pathway signaling in EPO-induced hypertension in mice. Methods: 10-12 weeks old male and female C57BL/6 mice were randomly divided into two groups: (1) Vehicle (0.9% saline-VEH), (2) EPO, (N=8). VEH and EPO were administered intraperitoneally (EPO 75U/30g, 3 times/week) for 20 days. Rgs5 silencing was performed in mouse aortic SMCs (N=3), and phophoryated-ERK1/2 (p-ERK1/2) was quantified by western blot. Results: EPO treated mice showed increased p-ERK1/2 activity in aorta and heart (fold change in aorta = 1.37, P = 0.001; fold change in heart = 1.72, P = 0.015) suggesting MAPK activation as possible mediator of EPO-induced increase in blood-pressure. In vitro , EPO increased the p-ERK activity in mouse aortic SMCs (MOVAS VEH 0.99 ± 0.05, EPO 1.31 ± 0.13, P =0.032) and interestingly Rgs5 silencing in MOVAS exacerbated EPO-induced p-ERK activation ( Rgs5 siRNA + EPO 1.70 ± 0.11, P =0.038 vs EPO), suggesting a potential role of Rgs5 in EPO mediated ERK activation and thus in regulating vascular tone. Conclusions: Our data indicated that Rgs5 is crucial in regulating EPO-induced ERK1/2 activation and that Rgs5 and its signaling pathway may serve as novel treatment targets in reducing cardiovascular complications in patients receiving EPO therapy.

Usefulness of Tumor Tissue Biopsy for Predicting the Biological Behavior of Hepatocellular Carcinoma.

Assessment of the biological behavior of tumors is important for choosing an appropriate cancer therapy. In hepatocellular carcinoma (HCC), the biological behaviour can be assessed by tumor morphology and molecular biology. This study investigated the usefulness of tumor tissue biopsy for predicting the biological behavior of HCC. We studied 43 patients who underwent hepatectomy and preoperative liver tumor biopsy for HCC. We performed clinicopathological and immunohistochemical (IHC) analyses. The expression of the following molecules was examined: regulator of G-protein signaling 5 (RGS5), glypican-3 (GPC3), keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), protein induced by vitamin K absence or antagonist-II (PIVKA-II), β-Catenin, and p53. There was an overall 83.7% agreement regarding tumor differentiation between the preoperative biopsy specimens and the resected specimens. The accuracy of IHC analysis was more than 70% for all molecules between the preoperative biopsy specimens and the resected specimens. The RGS5-positive biopsy cases had higher serum α-fetoprotein levels (p=0.04), a higher rate of moderately or poorly differentiated tumors (p=0.02) and portal vein invasion (p=0.0003) than the RGS5-negative biopsy cases. The GPC3-positive biopsy cases were younger (p=0.04), had higher serum PIVKA-II levels (p=0.01), and a higher rate of portal vein invasion (p=0.03) than the GPC3-negative biopsy cases. The PIVKA-II-positive biopsy cases had significantly higher serum PIVKA-II levels than the PIVKA-II-negative biopsy cases (p=0.02). The other molecular markers showed no significantly different clinical findings between the positive and negative cases. In HCC, there was a high agreement rate of both the histopathological and IHC findings between preoperative biopsy specimens and resected specimens. In the biopsy specimens of HCC, RGS5 and GPC3 expression were useful molecular makers for predicting portal vein invasion. Liver tumor biopsy is useful for predicting the biological behavior of HCC through histopathological and immunohistochemical findings.

RGS5 Deficiency Increases Blood Pressure and Causes Cerebral Vascular Oxidative Stress in Aged Male Mice

Aging is associated with elevated blood pressure, vascular oxidative stress and emotional behavioral abnormalities, such as anxiety and depression. Angiotensin II (Ang II) is reportedly involved in both vascular and emotional behavioral abnormalities. The actions of Ang II are mediated by the Ang II type 1 receptor whose activity is modulated by regulator of G‐protein signaling 5 (RGS5) protein. We have previously reported that RGS5 deficiency exacerbates Ang II‐induced elevations in blood pressure, and emotional behaviors in the absence and presence of elevated Ang II levels in adult (~26 week old) male mice. In this study we assessed the role of RGS5 on blood pressure, vascular and emotional behavioral outcomes in aged male mice in the absence and presence of elevated Ang II levels (1 mg/kg/d for 21 days). Specifically, we used aged (16–20 month old) male mice lacking RGS5 and their wild type counterparts to assess effects of saline or Ang II on systolic blood pressure (SBP), vascular function and oxidative stress, locomotor activity, and anxiety‐ and depression‐like behavior. RGS5 deficiency increased SBP under normal conditions, and increased cerebral vascular superoxide levels in the absence and presence of elevated Ang II levels, suggesting that RGS5 deficiency leads to elevated blood pressure and cerebral vascular oxidative stress in aged mice. RGS5 deficiency did not impair relaxation responses to acetylcholine, or contraction to the nitric oxide synthase inhibitor l‐NAME in aorta, suggesting that RGS5 deficiency had no effect on endothelial function in aorta. Interestingly, elevated Ang II levels increased spontaneous locomotor activity in wild‐type mice, but not in mice lacking RGS5. These data suggest that RGS5 deficiency attenuated responsiveness to Ang II‐induced increases in spontaneous locomotor activity. RGS5 deficiency and Ang II treatment had no effect on anxiety‐ and depression‐like behavior. In summary, these data suggest that RGS5 protects against elevated blood pressure, and has protective effects in the cerebral circulation in the absence and presence of elevated Ang II levels in aged male mice. Clinically, these data suggest that inhibitors of the renin angiotensin system may reduce the risk of cerebrovascular disease in aged males lacking RGS5.Support or Funding InformationThis work was supported by funds from the College of Pharmacy, Ohio Northern University.

Human cytomegalovirus promoting endothelial cell proliferation by targeting regulator of G-protein signaling 5 hypermethylation and downregulation

Interactions between human cytomegalovirus (HCMV) infection and environmental factors can increase susceptibility to essential hypertension (EH). Although endothelial dysfunction is the initial factor of EH, the epigenetic mechanisms through which HCMV infection induces endothelial cell dysfunction are poorly understood. Here, we evaluated whether HCMV regulated endothelial cell function and assessed the underlying mechanisms. Microarray analysis in human umbilical vein endothelial cells (HUVECs) treated with HCMV AD169 strain in the presence of hyperglycemia and hyperlipidemia revealed differential expression of genes involved in hypertension. Further analyses validated that the regulator of G-protein signaling 5 (RGS5) gene was downregulated in infected HUVECs and showed that HCMV infection promoted HUVEC proliferation, whereas hyperglycemia and hyperlipidemia inhibited HUVEC proliferation. Additionally, treatment with decitabine (DAC) and RGS5 reversed the effects of HCMV infection on HUVEC proliferation, but not triggered by hyperglycemia and hyperlipidemia. In summary, upregulation of RGS5 may be a promising treatment for preventing HCMV-induced hypertension.

Regulator of G-protein signaling 5 protein protects against anxiety- and depression-like behavior.

Anxiety and depression are a major health burden. Angiotensin II, via activation of angiotensin II type 1 receptor (AT1R)-mediated brain oxidative stress and inflammation may contribute to these emotional abnormalities. In this study, we investigated the role of a regulator of G-protein signaling 5 (RGS5) protein, which regulates AT1R activity, in angiotensin II-induced brain oxidative stress, inflammation and anxiety-, and depression-like behavior. We hypothesized that deletion of the RGS5 protein would worsen angiotensin II-induced anxiety- and depression-like behavior, cerebral vascular oxidative stress, and brain inflammation. Adult male wild-type and RGS5-deficient mice were implanted with osmotic minipumps delivering either vehicle (saline) or angiotensin II (1 mg/kg/d) for three weeks. Subsequently, mice were tested for locomotor activity, anxiety-like behavior (using the elevated plus maze), and depression-like behavior (using the tail suspension test). After behavioral testing, brain tissue was collected to assess oxidative stress and inflammatory proteins. RGS5 deletion resulted in anxiety-like but not depression-like behavior when compared to wild-type mice. Combined deletion of RGS5 and angiotensin II treatment did not further worsen anxiety-like behavior observed in RGS5-deficient mice. In contrast, depression-like behavior was worsened in RGS5-deficient mice treated with angiotensin II. Interestingly, RGS5 deficiency and angiotensin II treatment had no effect on cerebral vascular oxidative stress, or on expression of the inflammatory marker vascular cell adhesion molecule-1 in the brain. RGS5 deficiency was also associated with decreased blood pressure and an enhanced pressor response to angiotensin II. These data suggest that RGS5 protects against anxiety-like behavior and against angiotensin II-induced depression-like behavior.

Programmed death-ligand 1 expression is an unfavorable prognostic factor of hepatocellular carcinoma after archiving sustained virologic response for hepatitis C virus infection.

The aim of the present study was to study the pathological prognostic factor of initial hepatocellular carcinoma (HCC) after archiving sustained virologic response (SVR) for hepatitis C virus (HCV) infection. A single-center retrospective analysis was performed for patients who underwent hepatectomy between 2003 and 2017. We studied clinico-pathological findings of resected liver tissues in 35 patients with HCC after SVR treated by interferon (IFN group) and 13 patients with HCC after SVR treated by direct acting antivirals (DAA group). We also performed immunohistochemical staining using antibodies against programmed death-ligand 1 (PD-L1), cytokeratin 19, epithelial cell adhesion molecule (EpCAM) and regulator of G-protein signaling 5 (RGS5). PD-L1 positive HCC was observed in 6 cases of the IFN group and 4 cases of the DAA group. In the IFN group, in univariate analysis of recurrence free survival after surgery (RFS), the PD-L1 expression had a statistically significant impact (HR=6.01; P=0.02). In the multivariate analysis of RFS, PD-L1 expression significantly remained (HR=5.01; P=0.03). For both RFS and overall survival, Kaplan-Meier curves confirmed that patients with PD-L1 expression showed significantly worse prognosis (log-rank test P<0.01). Nuclear grade, RGS5 expression, and EpCAM expression were significantly higher in the PD-L1-positive HCC group compared with the PD-L1-negative HCC group (P<0.05). Therefore, PD-L1 expression may be an independent prognostic factor of surgically resected HCC after achieving SVR.

Regulator of G-protein signaling 5 regulates the shift from perivascular to parenchymal pericytes in the chronic phase after stroke.

Poststroke recovery requires multiple repair mechanisms, including vascular remodeling and blood-brain barrier (BBB) restoration. Brain pericytes are essential for BBB repair and angiogenesis after stroke, but they also give rise to scar-forming platelet-derived growth factor receptor β (PDGFR-β)–expressing cells. However, many of the molecular mechanisms underlying this pericyte response after stroke still remain unknown. Regulator of G-protein signaling 5 (RGS5) has been associated with pericyte detachment from the vascular wall, but whether it regulates pericyte function and vascular stabilization in the chronic phase of stroke is not known. Using RGS5–knockout (KO) mice, we study how loss of RGS5 affects the pericyte response and vascular remodeling in a stroke model at 7 d after ischemia. Loss of RGS5 leads to a shift toward an increase in the number of perivascular pericytes and reduction in the density of parenchymal PDGFR-β–expressing cells associated with normalized PDGFR-β activation after stroke. The redistribution of pericytes resulted in higher pericyte coverage, increased vascular density, preservation of vessel lengths, and a significant reduction in vascular leakage in RGS5-KO mice compared with controls. Our study demonstrates RGS5 in pericytes as an important target to enhance vascular remodeling.—Roth, M., Gaceb, A., Enström, A., Padel, T., Genové, G., Özen, I., Paul, G. Regulator of G-protein signaling 5 regulates the shift from perivascular to parenchymal pericytes in the chronic phase after stroke.

Parathyroid‐targeted overexpression of Regulator of G‐Protein Signaling 5 (RGS5) causes hyperparathyroidism in transgenic mice

The relationship between impaired calcium sensing, dysregulated parathyroid hormone (PTH) secretion, and parathyroid cell proliferation in parathyroid neoplasia is not understood. We previously reported that a GTPase activating protein, regulator of G‐protein signaling 5 (RGS5) is overexpressed in a subset of parathyroid tumors associated with primary hyperparathyroidism (PHPT), and that RGS5 can inhibit signaling from the calcium‐sensing receptor (CASR). In vivo, we found that RGS5‐null mice have abnormal PTH secretion. To gain a better understanding of the potential role of RGS5 overexpression in parathyroid neoplasia and PHPT and to investigate whether inhibition of CASR signaling can lead to parathyroid neoplasia, we created and characterized a transgenic mouse strain over‐expressing RGS5 specifically in the parathyroid gland. These mice develop hyperparathyroidism, bone changes reflective of elevated PTH, and parathyroid neoplasia. Further, expression of exogenous RGS5 in normal human parathyroid cells results in impaired signaling from CASR and negative feedback on PTH secretion. These results provide evidence supporting the setpoint hypothesis of parathyroid neoplasia and define a role for RGS5 in the pathogenesis of PHPT through inhibition of CASR signaling.Support or Funding InformationPhRMA Foundation and American Cancer Society research grants.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Parathyroid-Targeted Overexpression of Regulator of G-Protein Signaling 5 (RGS5) Causes Hyperparathyroidism in Transgenic Mice.

The relationship between impaired calcium sensing, dysregulated parathyroid hormone (PTH) secretion, and parathyroid cell proliferation in parathyroid neoplasia is not understood. We previously reported that a GTPase activating protein, regulator of G-protein signaling 5 (RGS5) is overexpressed in a subset of parathyroid tumors associated with primary hyperparathyroidism (PHPT) and that RGS5 can inhibit signaling from the calcium-sensing receptor (CASR). In vivo, we found that RGS5-null mice have abnormally low PTH levels. To gain a better understanding of the potential role of RGS5 overexpression in parathyroid neoplasia and PHPT and to investigate whether inhibition of CASR signaling can lead to parathyroid neoplasia, we created and characterized a transgenic mouse strain overexpressing RGS5 specifically in the parathyroid gland. These mice develop hyperparathyroidism, bone changes reflective of elevated PTH, and parathyroid neoplasia. Further, expression of exogenous RGS5 in normal human parathyroid cells results in impaired signaling from CASR and negative feedback on PTH secretion. These results provide evidence that RGS5 can modulate signaling from CASR and support a role for RGS5 in the pathogenesis of PHPT through inhibition of CASR signaling. © 2019 American Society for Bone and Mineral Research.

RGS5 decreases the proliferation of human ovarian carcinoma‑derived primary endothelial cells through the MAPK/ERK signaling pathway in hypoxia.

Regulator of G-protein signaling 5 (RGS5), a tissue-specific signal-regulating molecule, plays a key role in the development of the vasculature. It was recently found that RGS5 is abundantly expressed in epithelial ovarian cancer (EOC) compared with the normal ovaries. However, the distribution of RGS5 in EOC and its significance require further investigation. The aim of the present study was to investigate the expression of RGS5 in EOC, as well as its association with cancer differentiation, metastasis and clinicopathological parameters. Immunohistochemistry (IHC), western blotting, RT-PCR, wound-healing, cell proliferation and flow cytometric assays were the methods used in the present study. RGS5 was highly expressed in the cytoplasm of ovarian carcinoma cells and in microvascular structures. The expression of RGS5 in EOC was negatively associated with peritoneal metastasis (P=0.004), but it was not found to be associated with age, tumor size, clinical stage or lymph node metastasis (P>0.05). EOC patients with high RGS5 expression had a prolonged progression-free survival (72.34±8.41 vs. 43.56±5.41 months, P<0.001). High expression of RGS5 was correlated with significantly lower microvascular density (MVD) as indicated by the expression of CD34, whereas the opposite was observed in tissues with low RGS5 expression (P<0.05). Hypoxia increased RGS5 expression in ovarian carcinoma-derived endothelial cells (ODMECs), whereas the proliferative capacity of ODMECs exhibited a significant increase following RNAi-mediated reduction of RGS5 expression. These data indicated that RGS5 plays a key role in angiogenesis in ovarian carcinoma. In addition, RGS5 downregulated the expression of the downstream proteins CDC25A, CDK2 and cyclin E, which are mediated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, causing ODMEC arrest in the G1 phase of the cell cycle under hypoxic conditions. Collectively, our data indicated that RGS5 is crucial for the occurrence and development of ovarian cancer, and that RGS5 and its signaling pathway may serve as anti-angiogenesis targets for the treatment of ovarian cancer.

Loss of Regulator of G-Protein Signaling 5 Leads to Neurovascular Protection in Stroke.

Background and Purpose- In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Pericyte detachment contributes to BBB disruption and neurovascular dysfunction, but little is known about its regulation in stroke. Here, we investigated how loss of RGS5 (regulator of G protein signaling 5) in pericytes affects BBB breakdown in stroke and its consequences. Method- We used RGS5 knockout and control mice and applied a permanent middle cerebral occlusion model. We analyzed pericyte numbers, phenotype, and vessel morphology using immunohistochemistry and confocal microscopy. We investigated BBB breakdown by measuring endothelial coverage, tight junctions, and AQP4 (aquaporin 4) in addition to BBB permeability (fluorescent-conjugated dextran extravasation). Tissue hypoxia was assessed with pimonidazole hydrochloride and neuronal death quantified with the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results- We demonstrate that loss of RGS5 increases pericyte numbers and their endothelial coverage, which is associated with higher capillary density and length, and significantly less BBB damage after stroke. Loss of RGS5 in pericytes results in reduced vascular leakage and preserved tight junctions and AQP4, decreased cerebral hypoxia, and partial neuronal protection in the infarct area. Conclusions- Our findings show that loss of RGS5 affects pericyte-related BBB preservation in stroke and identifies RGS5 as an important target for neurovascular protection.

Abstract 416: Are Good and Evil Polarized? - How Murine Rgs5 knock-out Influences Macrophage Polarization and Aortic Atherosclerosis and Dissection

Background: Aortic atherosclerosis and dissection cause significant morbidity and mortality. Although atherosclerosis is associated with aortic dissection, points of interaction are unclear. Regulator-of-G-protein signaling 5 (RGS5) is still insufficiently characterized regarding its role in aortic atherosclerosis and dissection. Objectives: We investigated Rgs5-deficiencie’s effects on aortic atherosclerosis and dissection. Methods: ApoE -/- Rgs5 GFP/GFP double knockout mice (rraa, n=15) and WT littermates (RRaa, n=17) were fed a cholesterol-rich diet for 14weeks. Plaque size, plaque morphology (composition and a validated vulnerability score based on Stary criteria), macrophage phenotype and aortic dissection were evaluated. Results: rraa mice displayed more aortic dissections (5/15) compared to WT (0/17; p&lt;0.05 in Chi 2 -test). Surprisingly, rraa showed significantly smaller (rraa plaque size 190 657 μm 2 ± 93184 vs. control 398 279 μm 2 ± 153339) and less complicated plaques (5 of 15 mice with &gt;2 vulnerability features) compared to RRaa (14 of 15 with &gt;2 vulnerability features, p&lt;0.01). While relative areas of aSMA and lipids did not differ between genotypes, relative plaque areas positive for collagen and CD45 were increased in rraa (n=15/17, p&lt;0.01) compared to RRaa. Due to smaller plaque sizes were anti-inflammatory macrophages reduced and pro-inflammatory macrophages increased in absolute numbers (p&lt;0.05). Phenotyping of CD45 + -myeloid cells revealed increased proportions of macrophages staining positive for pro-inflammatory markers (Cd86/Cd45: 24.71 area% ± 3.75 for RRaa; 60.95 area% ± 5.909, for rraa and iNOS/Cd45: 37.22 area% ± 6.37, for RRaa; 73.99 area% ± 3.997 for rraa, p&lt;0.01), while anti-inflammatory markers (Cd163/Cd45 and Dectin/Cd45 double stainings) remained unchanged. Conclusions: rraa mice are more prone to aortic dissection despite smaller and less complex plaques with reduced anti-inflammatory but increased numbers of pro-inflammatory macrophages. We hypothesize that pro-inflammatory macrophage polarization along with evasion of anti-inflammatory macrophages in rraa mice could be causative for higher dissection numbers. Our data warrant further studies into the diverging pathomechanistic roles of Rgs5 in aortic dissection and atherosclerosis.