Publications by authors named "Jianqi Yang"

36 Publications

Hsa_circ_0008934 promotes the proliferation and migration of osteosarcoma cells by targeting miR-145-5p to enhance E2F3 expression.

Int J Biochem Cell Biol 2020 10 18;127:105826. Epub 2020 Aug 18.

Department of Spinal Surgery, the First People's Hospital of Foshan, Foshan 528000, Guangdong, China.

Objective: To investigate the role of hsa_circ_0008934 in osteosarcoma and the molecular mechanism involved in the regulation of the occurrence and development of osteosarcoma METHODS: Differentially expressed circRNAs in the osteosarcoma cell lines SaOS2 and MG63 and in the normal human osteoblast cell line hFOB1.19 were identified via next-generation RNA sequencing. The expression and circular morphology of hsa_circ_0008934 were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and RT-PCR analysis, respectively. Proliferation, apoptosis, cell cycle progression, migration, and invasion of SaOS2 and MG63 cells with hsa_circ_0008934 silencing or overexpression were assessed using the MTS method, colony formation assay, flow cytometry, and the transwell system, respectively. The subcellular distribution of hsa_circ_0008934 was revealed via fluorescence in situ hybridization. The binding of hsa_circ_0008934 with microRNAs was confirmed using the dual-luciferase reporter assay. The oncogenic roles of hsa_circ_0008934 in osteosarcoma were determined using an in vivo tumorigenesis assay with nude mice. qRT-PCR, western blotting, TUNEL assay, and immunohistochemistry (IHC) were used to detect the tumorigenicity of hsa_circ_0008934 in osteosarcoma cells.

Results: Many circRNAs were differentially expressed in SaOS2 and MG63 cells than in hFOB1.19 cells. Hsa_circ_0008934 expression was significantly elevated in SaOS2 and MG63 cells. Hsa_circ_0008934 silencing significantly reduced proliferation, enhanced apoptosis, blocked cell cycle progression, and impaired migration and invasion capacities of SaOS2 cells. Opposite cellular alterations were achieved by overexpressing hsa_circ_0008934 in MG63 cells. Hsa_circ_0008934 was mainly distributed in the cytosol and positively regulated E2F3 expression in osteosarcoma cells. In addition, it directly bound with miR-145-5p to repress E2F3 expression and enhanced the tumorigenesis of MG63 cells in nude mice. qRT-PCR revealed that the intracellular injection of hsa_circ_0008934 lentivirus resulted in hsa_circ_0008934 overexpression and miR-145-5p downregulation. Western blotting confirmed that E2F3 was upregulated. Moreover, the TUNEL assay showed that hsa_circ_0008934 overexpression inhibited the apoptosis of tumor cells. IHC detection revealed that the hsa_circ_0008934 overexpression could promote the expression of Ki67 and PCNA.

Conclusion: Elevated hsa_circ_0008934 expression promotes the proliferation and migration of osteosarcoma cells by sponging miR-145-5p to enhance E2F3 expression.
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http://dx.doi.org/10.1016/j.biocel.2020.105826DOI Listing
October 2020

Patterns and risk factors of peripherally inserted central venous catheter-related symptomatic thrombosis events in patients with malignant tumors receiving chemotherapy.

J Vasc Surg Venous Lymphat Disord 2020 11 21;8(6):919-929. Epub 2020 Mar 21.

Department of Medical Oncology, Clinical Medical College, Yangzhou University, Yangzhou City, Jiangsu Province, China. Electronic address:

Objective: Peripherally inserted central venous catheter (PICC)-related thrombosis (PRT) is a serious complication that can lead to interruptions in chemotherapy and other supportive care, as well as increased hospital stay and costs. We conducted a retrospective study to evaluate the patterns of symptomatic PRT in patients with cancer undergoing chemotherapy and their risk factors.

Methods: A retrospective study of 938 PICC patients from our institution between November 2014 and July 2017 was performed. Symptomatic PRT events were confirmed by color Doppler ultrasonography or computed tomography pulmonary angiography in the presence of clinical symptoms. The variables of interest were extracted from the electronic medical record system. Logistic regression analysis was used to determine the risk factors for PRT.

Results: Of the 938 patients who were followed up for more than 120,000 patient-days, 63 patients (6.7%; 0.51 per 1000 catheter-days) had symptomatic PRT. Sixty-one patients were diagnosed with upper extremity venous thrombosis (UEVT), of which 18 were isolated superficial vein thrombosis (SVT), 19 were isolated deep vein thrombosis (DVT), and 24 were extensive venous thrombosis (EVT). Two patients were diagnosed with pulmonary embolism, and two patients were diagnosed with UEVT with pulmonary embolism. The symptomatic SVT occurred in 42 of 938 patients with cancer (4.5%), which accounted for 68.9% of all UEVT events. The median time to PRT was 21 days, and the median time to catheter removal in the PRT group was 66 days as compared with 117 days in the no PRT group. Predictors associated with increased risk of PRT were age >60 years (odds ratio [OR], 2.142; 95% confidence interval [CI], 1.118-4.103) and a chemotherapy regimen containing fluorouracil (OR, 2.429; 95% CI, 1.013-5.825). Hypertension with medication was a protective factor for PRT (OR, 0.306; 95% CI, 0.113-0.828). Among the 28 patients who did not remove their PICCs immediately after PRT was diagnosed, patients with SVT, DVT, and EVT had similar success rates of retaining catheters in situ after anticoagulant therapy (SVT, 83.3%; DVT, 62.5%; EVT, 75.0%; P = .667).

Conclusions: Age >60 years and chemotherapy regimens containing fluorouracil were independent risk factors for PRT and hypertension with medication was associated with a lower risk of PRT in patients with cancer with PICCs receiving chemotherapy. PICCs-related SVT was a frequent type of PRT, which might need a better understanding and anticoagulant therapy in patients with cancer with PICCs.
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http://dx.doi.org/10.1016/j.jvsv.2020.01.010DOI Listing
November 2020

Clinical efficacy of irinotecan plus raltitrexed chemotherapy in refractory esophageal squamous cell cancer.

Anticancer Drugs 2020 04;31(4):403-410

Departments of Medical Oncology.

Our retrospective study assessed the efficacy and safety of irinotecan plus raltitrexed in esophageal squamous cell cancer (ESCC) patients who were previously treated with multiple systemic therapies. Between January 2016 and December 2018, records of 38 ESCC patients who underwent irinotecan plus raltitrexed chemotherapy after at least one line of chemotherapy were reviewed. Efficacy assessment was performed every two cycles according to the RECIST version 1.1. A total of 95 cycles of chemotherapy were administered, and the median course was 3 (range 2-6). There was no treatment-related death. Nine patients had partial response, 21 had stable disease and eight had progressive disease. The overall objective response rate was 23.68% (9/38) and the disease control rate was78.94% (30/38). After a median follow-up of 18.5 months, the median progression-free survival and overall survival were 105 and 221 days, respectively. There were five patients (13.15%) with grade 3/4 leukopenia, three patients (7.89%) with grade 3/4 neutropenia and one patient (2.63%) with grade 3/4 diarrhea. The combination of irinotecan plus raltitrexed was effective for pretreated ESCC patients. Further studies are needed to determine the optimal dose of the two drugs.
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http://dx.doi.org/10.1097/CAD.0000000000000891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077961PMC
April 2020

EDNRB Reverses Methylprednisolone-Mediated Decrease in Neural Progenitor Cell Viability via Regulating PI3K/Akt Pathway and lncRNA Expression.

J Mol Neurosci 2020 Mar 23;70(3):403-412. Epub 2019 Nov 23.

Department of Spinal Surgery, the First People's Hospital of Foshan, North Lingnan Avenue 81, Foshan, 528000, Guangdong, China.

Objective: To investigate the functions and mechanisms of methylprednisolone (MP) through endothelin receptor B (EDNRB) on the cell proliferation of neural progenitor cells (NPCs) to regulate spinal cord injury.

Methods: Primary NPCs were isolated from fetal mice and subjected to treatments with MP and IRL-1620 (EDNRB agonist). The cell viability was determined using the MTS assay. Total RNA was extracted from the cells, and RNA-seq was performed to screen for lncRNAs. The targets of the candidate lncRNAs were predicted by GO and KEGG analyses, and the expressions of lncRNAs were validated via qPCR. Furthermore, protein levels of the PI3K-AKT pathway were determined via Western blotting, and the expression of lncRNAs was detected after inhibiting the pathway with AKT inhibitor.

Results: MTS assays revealed that MP decreased the cell viability of NPCs, whereas the EDNRB agonist reversed this effect of MP. NPCs were used for RNA-seq in the following three groups: normal control (NC), MP, and MP combined with EDNRB agonist (MP + EDNRB). Our results suggested that the NONRATT030699.2, NONRATT004088.2, and NONRATT005601.2 lncRNAs might be involved in the signaling pathway that is correlated to MP and the EDNRB agonist. GO and KEGG pathway analyses revealed that this was the PI3K/AKT pathway. The relevant genes involved in the pathway were validated by Western blotting. The EDNRB agonist promoted cell proliferation mainly via the activation of the PI3K/AKT pathway; however, it suppressed the expression of p-ERK, thereby increasing the expression of cyclin D1 and attenuating the effect of MP in suppressing cell proliferation. Meanwhile, after the AKT signal pathway was inhibited, these lncRNA expressions were consistent with those in the MP + EDNRB group.

Conclusion: MP inhibits NPC proliferation, whereas EDNRB activation reverses the effect of MP via lncRNA.
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http://dx.doi.org/10.1007/s12031-019-01427-4DOI Listing
March 2020

Extramedullary leukemia behaving as solid cancer: clinical, histologic, and genetic clues to chemoresistance in organ sites.

Am J Hematol 2019 11 27;94(11):1200-1207. Epub 2019 Aug 27.

Department of Pharmacology, University of Iowa, Iowa City, Iowa.

Recent studies of leukemic tumors in individual extramedullary sites showed they adopt the clinical and metastatic behavior of solid cancers originating in those sites. To elucidate features of leukemic tumors that render them resistant to agents effective against marrow leukemia, we analyzed a series of AML breast tumors by histology, immunohistochemistry, and RNA sequencing. Striking histologic similarities to solid cancers were found: a single-filing architectural pattern virtually identical to that of invasive lobular breast carcinoma and dense desmoplastic keloid-like fibrosis similar to colon, gallbladder, and pancreas carcinomas. Sequencing found 2157 genes significantly downregulated in AML breast tumors compared to normal breast. Comparison to triple-negative breast cancer found 859 genes similarly downregulated. At least 30 of these genes have been associated with poor prognosis in breast cancers. Five were reported in AML marrow studies to correlate with poor prognosis. The findings of this pilot study suggest the seed-and-soil interaction recognized in solid cancer growth may help explain how leukemic cells, in some patients, adopt solid tumor behavior in non-marrow sites. Transformed cells that metastasize from tumor to marrow can impart chemoresistance and be an unrecognized cause of treatment failure and death. Further studies comparing leukemic tumor to simultaneous marrow could potentially identify biomarkers that predict extramedullary resistance and lead to new therapeutic targets. Recognizing the potential for leukemia to adopt solid tumor phenotype, and implementation of body scanning and ablative tumor treatment, could decrease the persistently high rates of marrow resistance and treatment failure.
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http://dx.doi.org/10.1002/ajh.25594DOI Listing
November 2019

Age-dependent nigral dopaminergic neurodegeneration and α-synuclein accumulation in RGS6-deficient mice.

JCI Insight 2019 05 23;5. Epub 2019 May 23.

Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA.

Parkinson's is primarily a non-familial, age-related disorder caused by α-synuclein accumulation and the progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). G protein-coupled receptor (GPCR)-cAMP signaling has been linked to a reduction in human Parkinson's incidence and α-synuclein expression. Neuronal cAMP levels are controlled by GPCRs coupled to Gs or Gi/o, which increase or decrease cAMP, respectively. Regulator of G protein signaling 6 (RGS6) powerfully inhibits Gi/o signaling. Therefore, we hypothesized that RGS6 suppresses D2 autoreceptor- Gi/o signaling in SNc dopamine neurons promoting neuronal survival and reducing α-synuclein expression. Here we provide novel evidence that RGS6 critically suppresses late-age-onset SNc dopamine neuron loss and α-synuclein accumulation. RGS6 is restrictively expressed in human SNc dopamine neurons and, despite their loss in Parkinson's, all surviving neurons express RGS6. RGS6-/- mice exhibit hyperactive D2 autoreceptors with reduced cAMP signaling in SNc dopamine neurons. Importantly, RGS6-/- mice recapitulate key sporadic Parkinson's hallmarks, including: SNc dopamine neuron loss, reduced nigrostriatal dopamine, motor deficits, and α-synuclein accumulation. To our knowledge, Rgs6 is the only gene whose loss phenocopies these features of human Parkinson's. Therefore, RGS6 is a key regulator of D2R-Gi/o signaling in SNc dopamine neurons, protecting against Parkinson's neurodegeneration and α-synuclein accumulation.
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http://dx.doi.org/10.1172/jci.insight.126769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629243PMC
May 2019

MicroRNA-365 suppresses cell growth and invasion in esophageal squamous cell carcinoma by modulating phosphoserine aminotransferase 1.

Cancer Manag Res 2018 15;10:4581-4590. Epub 2018 Oct 15.

Department of Thoracic and Cardiovascular Surgery, Subei People's Hospital of Jiangsu Province, Yangzhou, Jiangsu, China,

Background: A number of studies have indicated that expression of miRNA-365 (miR-365) is suppressed in various cancers, suggesting its cancer-suppressive role. In the present investigation, we evaluated the regulation and character of miR-365 in human esophageal squamous cell carcinoma (ESCC).

Patients And Methods: The tumor tissues and adjacent nontumor tissue samples were collected from 30 patients having ESCC, and the expression levels of miR-365 were studied by quantitative real-time polymerase chain reaction (PCR). MTT and cell invasion by Matrigel assay were done to study the effect of miR-365 on proliferation and metastasis of ESCC cells. An in vivo tumor model was generated by inoculating ESCC cells subcutaneously into BALB nude mice. A study of various biomarkers such as quantitative polymerase chain reaction (qPCR), luciferase activity assay, and Western blot was done to confirm the targets of miR-365.

Results: In tumor tissues, a significant downregulation of miR-365 was observed versus the nontumor adjacent tissues and ESCC cells versus the selected esophageal endothelial cells. It was observed that higher expression levels of miR-365 inhibited the cell invasion, colony formation, growth in esophageal cancer cell lines in vitro, and tumor development in vivo. The study of biomarkers suggests involvement of phosphoserine aminotransferase 1 (PSAT1) as a favorable target for miR-365, and its abnormal expression inverted the miR-365-arbitrated suppression of invasion, viability, and epithelial-mesenchymal transition in esophageal cancer cells. A negative correlation existed with expression of miR-365 and PSAT1 in human esophageal cancer tissue samples.

Conclusion: The study established that miR-365 exhibits tumor-suppressive action via regulating the levels of PSAT1 and leads to invasion and progressiveness of esophageal cancer.
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http://dx.doi.org/10.2147/CMAR.S157858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197828PMC
October 2018

Essentiality of Regulator of G Protein Signaling 6 and Oxidized Ca/Calmodulin-Dependent Protein Kinase II in Notch Signaling and Cardiovascular Development.

J Am Heart Assoc 2017 Oct 27;6(11). Epub 2017 Oct 27.

Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA

Background: Congenital heart defects are the most common birth defects worldwide. Although defective Notch signaling is the major cause of mouse embryonic death from cardiovascular defects, how Notch signaling is regulated during embryonic vasculogenesis and heart development is poorly understood.

Methods And Results: Regulator of G protein signaling 6 (RGS6)/Ca/calmodulin-dependent protein kinase II (CaMKII) double mutant mice were developed by crossing RGS6 mice with mice expressing an oxidation-resistant CaMKIIδ (CaMKII), and the resulting embryonic defects/lethality were investigated using E7.5 to E15.5 embryos. While loss of either RGS6 or oxidized CaMKIIδ does not alter embryogenesis, their combined loss causes defective Notch signaling, severe cardiovascular defects, and embryonic lethality (≈E10.5-11.5). Embryos lacking RGS6 and expressing oxidation-resistant CaMKIIδ exhibit reduced myocardial wall thickness, abnormal trabeculation, and arterial specification defects. Double mutants show vascular remodeling defects, including reduced neurovascularization, delayed neural tube maturation, and small dorsal aortae. These striking cardiovascular defects were accompanied by placental and yolk sac defects in angiogenesis, hematopoiesis, and vascular remodeling similar to what is seen with defective Notch1 signaling. Double mutant hearts, embryos, and yolk sacs exhibit profound downregulation of Notch1, Jagged 1, and Notch downstream target genes Hey1, Hey2, and Hey1L as well as impaired Notch1 signaling in embryos/hearts.

Conclusions: RGS6 and oxidized CaMKIIδ together function as novel critical upstream modulators of Notch signaling required for normal cardiovascular development and embryo survival. Their combined need indicates that they function in parallel pathways needed for Notch1 signaling in yolk sac, placenta and embryos. Thus, dysregulated embryonic RGS6 expression and oxidative activation of CaMKII may potentially contribute to congenital heart defects.
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http://dx.doi.org/10.1161/JAHA.117.007038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721783PMC
October 2017

Snail Enhances Glycolysis in the Epithelial-Mesenchymal Transition Process by Targeting FBP1 in Gastric Cancer.

Cell Physiol Biochem 2017 24;43(1):31-38. Epub 2017 Aug 24.

Department of Oncology, Clinical Medical College of Yangzhou University, Subei People's Hospital of Jiangsu Province, Yangzhou, China.

Background: Snail is a key regulator of epithelial-mesenchymal transition (EMT) in cancer. However, the regulatory role and underlying mechanisms of Snail in gastric cancer metabolism are unknown. In this study, we characterized the regulation of aerobic glycolysis by Snail in gastric cancer.

Methods: The impact of Snail on glucose metabolism was studied in vitro. Combining maximum standardized uptake value (SUVmax), which was obtained preoperatively via a PET/CT scan, with immunohistochemistry staining, we further analyzed the correlation between SUVmax and Snail expression in gastric cancer tissues.

Results: Increased expression of Snail promoted lactate production, glucose utilization, and decreased FBP1 expression at both mRNA and protein level. The expression level of Snail was positively associated with SUVmax in gastric cancer patients (P=0.022). Snail and FBP1 expression were inversely correlated at both mRNA and protein level (P=0.002 and P=0.015 respectively) in gastric cancer tissues. Further studies demonstrated that Snail inhibited the FBP1 gene expression at the transcriptional level. Restoring FBP1 expression reversed the effects of glycolysis and EMT induced by Snail in gastric cancer cells.

Conclusions: Our results thus reveal that Snail serves as a positive regulator of glucose metabolism through regulation of the FBP1 in gastric cancer. Disrupting the Snail-FBP1 signaling axis may be effective to prevent primary tumor EMT and glycolysis process.
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http://dx.doi.org/10.1159/000480314DOI Listing
November 2017

miRNA-148a serves as a prognostic factor and suppresses migration and invasion through Wnt1 in non-small cell lung cancer.

PLoS One 2017 15;12(2):e0171751. Epub 2017 Feb 15.

Department of Medical Oncology, Northern Jiangsu People's Hospital, Clinical Medical College of Yangzhou University, Yangzhou, Jiangsu, China.

Lung cancer is the leading cause of cancer death in the world, and aberrant expression of miRNA is a common feature during the cancer initiation and development. Our previous study showed that levels of miRNA-148a assessed by quantitative real-time polymerase chain reaction (qRT-PCR) were a good prognosis factor for non-small cell lung cancer (NSCLC) patients. In this study, we used high-throughput formalin-fixed and paraffin-embedded (FFPE) lung cancer tissue arrays and in situ hybridization (ISH) to determine the clinical significances of miRNA-148a and aimed to find novel target of miRNA-148a in lung cancer. Our results showed that there were 86 of 159 patients with low miRNA-148a expression and miRNA-148a was significantly down-regulated in primary cancer tissues when compared with their adjacent normal lung tissues. Low expression of miRNA-148a was strongly associated with high tumor grade, lymph node (LN) metastasis and a higher risk of tumor-related death in NSCLC. Lentivirus mediated overexpression of miRNA-148a inhibited migration and invasion of A549 and H1299 lung cancer cells. Furthermore, we validated Wnt1 as a direct target of miRNA-148a. Our data showed that the Wnt1 expression was negatively correlated with the expression of miRNA-148a in both primary cancer tissues and their corresponding adjacent normal lung tissues. In addition, overexpression of miRNA-148a inhibited Wnt1 protein expression in cancer cells. And knocking down of Wnt-1 by siRNA had the similar effect of miRNA-148a overexpression on cell migration and invasion in lung cancer cells. In conclusion, our results suggest that miRNA-148a inhibited cell migration and invasion through targeting Wnt1 and this might provide a new insight into the molecular mechanisms of lung cancer metastasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171751PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310808PMC
August 2017

Regulator of G Protein Signaling 6 Protects the Heart from Ischemic Injury.

J Pharmacol Exp Ther 2017 03 29;360(3):409-416. Epub 2016 Dec 29.

Department of Pharmaceutical and Biomedical Sciences, Raabe College of Pharmacy, Ohio Northern University, Ada, Ohio (B.R.R., N.W.M., S.L.S., A.D.B.); Department of Pharmacology, Carver College of Medicine, University of Iowa, Iowa City, Iowa (B.C., J.Y., R.A.F.); and Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan (S.W.W., R.R.N.).

Gαi-coupled receptors play important roles in protecting the heart from ischemic injury. Regulator of G protein signaling (RGS) proteins suppress Gαi signaling by accelerating the GTPase activity of Gαi subunits. However, the roles of individual RGS proteins in modulating ischemic injury are unknown. In this study, we investigated the effect of RGS6 deletion on myocardial sensitivity to ischemic injury. Hearts from RGS6 knockout (RGS6) and RGS6 wild-type (RGS6) mice were subjected to 30 minutes of ischemia and 2 hours of reperfusion on a Langendorff heart apparatus. Infarcts in RGS6 hearts were significantly larger than infarcts in RGS6 hearts. RGS6 hearts also exhibited increased phosphorylation of β-adrenergic receptors and G protein-coupled receptor kinase 2 (GRK2). Mitochondrial GRK2 as well as caspase-3 cleavage were increased significantly in RGS6 hearts compared with RGS6 hearts after ischemia. Chronic propranolol treatment of mice prevented the observed increases in ischemic injury and the GRK2 phosphorylation observed in RGS6 hearts. Our findings suggest that loss of RGS6 predisposes the ventricle to prodeath signaling through a βAR-GRK2-dependent signaling mechanism, and they provide evidence for a protective role of RGS6 in the ischemic heart. Individuals expressing genetic polymorphisms that suppress the activity of RGS6 may be at increased risk of cardiac ischemic injury. Furthermore, the development of agents that increase RGS6 expression or activity might provide a novel strategy for the treatment of ischemic heart disease.
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http://dx.doi.org/10.1124/jpet.116.238345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5325075PMC
March 2017

RGS6 is an essential tumor suppressor that prevents bladder carcinogenesis by promoting p53 activation and DNMT1 downregulation.

Oncotarget 2016 Oct;7(43):69159-69172

Department of Pharmacology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.

Urinary bladder cancer (UBC) is largely caused by exposure to toxic chemicals including those in cigarette smoke (i.e. BBN). An activating SNP in RGS6 is associated with a pronounced reduction in UBC risk, especially among smokers. However, the mechanism underlying this reduction remains unknown. Here we demonstrate that RGS6 is robustly expressed in human urothelium, where urothelial cell carcinoma originates, and is downregulated in human UBC. Utilizing RGS6-/- mice we interrogated a possible role for RGS6 as a tumor suppressor using the BBN-induced bladder carcinogenesis model that closely recapitulates human disease. As in humans, RGS6 is robustly expressed in mouse urothelium. RGS6 loss dramatically accelerates BBN-induced bladder carcinogenesis, with RGS6-/- mice consistently displaying more advanced pathological lesions than RGS6+/+ mice. Furthermore, BBN treatment promotes urothelial RGS6 mRNA and protein downregulation. RGS6 loss impairs p53 activation and promotes aberrant accumulation of oncogenic protein DNMT1 in urothelium. Tumor suppressor RASSF1A, a DNMT1-regulated gene, is also silenced, likely via methylation of its promoter during BBN exposure. We hypothesize that this BBN-induced RGS6 loss represents a critical hit in UBC as it irrevocably impairs the anti-proliferative actions of the ATM/p53 and RASSF1A pathways. Consistent with these findings, RGS6-/- mice treated with CP-31398, a p53-stablizing agent, and/or 5-Aza, a DNMT1 inhibitor, are protected from BBN-induced tumorigenesis. Together, our data identify RGS6 as a master tumor suppressor modulating two critical signaling pathways that are often dysregulated in UBC; therefore, RGS6 represents a potential novel biomarker for UBC diagnosis/prognosis and an appealing new target in its treatment.
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http://dx.doi.org/10.18632/oncotarget.12473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342467PMC
October 2016

Regulator of G protein signaling 6 is a critical mediator of both reward-related behavioral and pathological responses to alcohol.

Proc Natl Acad Sci U S A 2015 Feb 2;112(7):E786-95. Epub 2015 Feb 2.

Department of Pharmacology and

Alcohol is the most commonly abused drug worldwide, and chronic alcohol consumption is a major etiological factor in the development of multiple pathological sequelae, including alcoholic cardiomyopathy and hepatic cirrhosis. Here, we identify regulator of G protein signaling 6 (RGS6) as a critical regulator of both alcohol-seeking behaviors and the associated cardiac and hepatic morbidities through two mechanistically divergent signaling actions. RGS6(-/-) mice consume less alcohol when given free access and are less susceptible to alcohol-induced reward and withdrawal. Antagonism of GABA(B) receptors or dopamine D2 receptors partially reversed the reduction in alcohol consumption in RGS6(-/-) animals. Strikingly, dopamine transporter inhibition completely restored alcohol seeking in mice lacking RGS6. RGS6 deficiency was associated with alterations in the expression of genes controlling dopamine (DA) homeostasis and a reduction in DA levels in the striatum. Taken together, these data implicate RGS6 as an essential regulator of DA bioavailability. RGS6 deficiency also provided dramatic protection against cardiac hypertrophy and fibrosis, hepatic steatosis, and gastrointestinal barrier dysfunction and endotoxemia when mice were forced to consume alcohol. Although RGS proteins canonically function as G-protein regulators, RGS6-dependent, alcohol-mediated toxicity in the heart, liver, and gastrointestinal tract involves the ability of RGS6 to promote reactive oxygen species-dependent apoptosis, an action independent of its G-protein regulatory capacity. We propose that inhibition of RGS6 might represent a viable means to reduce alcohol cravings and withdrawal in human patients, while simultaneously protecting the heart and liver from further damage upon relapse.
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http://dx.doi.org/10.1073/pnas.1418795112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343156PMC
February 2015

Rgs6 is required for adult maintenance of dopaminergic neurons in the ventral substantia nigra.

PLoS Genet 2014 Dec 11;10(12):e1004863. Epub 2014 Dec 11.

Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal (IRCM) Montréal, Quebec, Canada; Division of Experimental Medicine, McGill University, Montréal, Quebec, Canada.

Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to Pitx3-/- mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of Rgs6-/- mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.
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http://dx.doi.org/10.1371/journal.pgen.1004863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263397PMC
December 2014

G-protein inactivator RGS6 mediates myocardial cell apoptosis and cardiomyopathy caused by doxorubicin.

Cancer Res 2013 Mar 21;73(6):1662-7. Epub 2013 Jan 21.

Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242, USA.

Clinical use of the widely used chemotherapeutic agent doxorubicin is limited by life-threatening cardiotoxicity. The mechanisms underlying doxorubicin-induced cardiomyopathy and heart failure remain unclear but are thought to involve p53-mediated myocardial cell apoptosis. The tripartite G-protein inactivating protein RGS6 has been implicated in reactive oxygen species (ROS) generation, ATM/p53 activation, and apoptosis in doxorubicin-treated cells. Thus, we hypothesized that RGS6, the expression of which is enriched in cardiac tissue, might also be responsible for the pathologic effects of doxorubicin treatment in heart. In this study, we show that RGS6 expression is induced strongly by doxorubicin in the ventricles of mice and isolated ventricular myocytes via a posttranscriptional mechanism. While doxorubicin-treated wild-type (WT) mice manifested severe left ventricular dysfunction, loss of heart and body mass, along with decreased survival 5 days after doxorubicin administration, mice lacking RGS6 were completely protected against these pathogenic responses. Activation of ATM/p53 apoptosis signaling by doxorubicin in ventricles of WT mice was also absent in their RGS6(-/-) counterparts. Doxorubicin-induced ROS generation was dramatically impaired in both the ventricles and ventricular myocytes isolated from RGS6(-/-) mice, and the apoptotic response to doxorubicin in ventricular myocytes required RGS6-dependent ROS production. These results identify RGS6 as an essential mediator of the pathogenic responses to doxorubicin in heart, and they argue that RGS6 inhibition offers a rational means to circumvent doxorubicin cardiotoxicity in human patients with cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-3453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602152PMC
March 2013

Defective retinal depolarizing bipolar cells in regulators of G protein signaling (RGS) 7 and 11 double null mice.

J Biol Chem 2012 Apr 27;287(18):14873-9. Epub 2012 Feb 27.

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, USA.

Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gα(o)/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6-8 deleted. The RGS7(-/-) mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7(-/-) mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7(-/-) retina, but the Gβ5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gβ5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gβ5(-/-) mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gβ5S is undetectable, whereas levels of the photoreceptor-specific Gβ5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gβ5S expression in retina, the DBC-related defects in Gβ5(-/-) mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gβ5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.
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http://dx.doi.org/10.1074/jbc.M112.345751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340290PMC
April 2012

Regulator of G protein signaling 6 (RGS6) protein ensures coordination of motor movement by modulating GABAB receptor signaling.

J Biol Chem 2012 Feb 16;287(7):4972-81. Epub 2011 Dec 16.

Department of Pharmacology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.

γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.
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http://dx.doi.org/10.1074/jbc.M111.297218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281673PMC
February 2012

Regulator of G protein signaling 6 mediates doxorubicin-induced ATM and p53 activation by a reactive oxygen species-dependent mechanism.

Cancer Res 2011 Oct 22;71(20):6310-9. Epub 2011 Aug 22.

Department of Pharmacology, University of Iowa, Carver College of Medicine, Iowa City, Iowa 52242, USA.

Doxorubicin (DXR), among the most widely used cancer chemotherapy agents, promotes cancer cell death via activation of ataxia telangiectasia mutated (ATM) and the resultant upregulation of tumor suppressor p53. The exact mechanism by which DXR activates ATM is not fully understood. Here, we discovered a novel role for regulator of G protein signaling 6 (RGS6) in mediating activation of ATM and p53 by DXR. RGS6 was robustly induced by DXR, and genetic loss of RGS6 dramatically impaired DXR-induced activation of ATM and p53, as well as its in vivo apoptotic actions in heart. The ability of RGS6 to promote p53 activation in response to DXR was independent of RGS6 interaction with G proteins but required ATM. RGS6 mediated activation of ATM and p53 by DXR via a reactive oxygen species (ROS)-dependent and DNA damage-independent mechanism. This mechanism represents the primary means by which DXR promotes activation of the ATM-p53 apoptosis pathway that underlies its cytotoxic activity. Our findings contradict the canonical theories that DXR activates ATM primarily by promoting DNA damage either directly or indirectly (via ROS) and that RGS6 function is mediated by its interactions with G proteins. These findings reveal a new mechanism for the chemotherapeutic actions of DXR and identify RGS6 as a novel target for cancer chemotherapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-3397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196377PMC
October 2011

Function of phosphoenolpyruvate carboxykinase in mammary gland epithelial cells.

J Lipid Res 2011 Jul 19;52(7):1352-62. Epub 2011 Apr 19.

Department of Nutrition, Case Western Reserve University, Cleveland, OH 44106, USA.

Previously, we have shown that Pck1 expression in mammary gland adipocytes and white adipose tissue maintains triglyceride stores through glyceroneogenesis, and these lipids were used for synthesis of milk triglycerides during lactation. Reduced milk triglycerides during lactation resulted in patterning of the newborn for insulin resistance. In this study, the role of Pck1 in mammary gland epithelial cells was analyzed. The developmental expression of Pck1 decreased in isolated mouse mammary gland epithelial cells through development and during lactation. Using HC11, a clonal mammary epithelial cell line, we found that both Janus kinase 2 signal transducers and activators of transcription 5 and the AKT pathways contributed to the repression of Pck1 mRNA by prolactin. These pathways necessitate three accessory factor regions of the Pck1 promoter for repression by prolactin. Using [U-(13)C(6)]glucose, [U-(13)C(3)]pyruvate, and [U-(13)C(3)]glycerol in HC11 cells, we determined that Pck1 functions in the pathway for the conversion of gluconeogenic precursors to glucose and contributes to glycerol-3-phosphate synthesis through glyceroneogenesis. Therefore, Pck1 plays an important role in both the mammary gland adipocytes and epithelial cells during lactation.
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http://dx.doi.org/10.1194/jlr.M012666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122918PMC
July 2011

Acetylation-deacetylation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) regulates its transcriptional activity and nucleocytoplasmic localization.

J Biol Chem 2011 Mar 31;286(9):7629-40. Epub 2010 Dec 31.

Department of Physiology, Meharry Medical College, Nashville, Tennessee 37208-3599, USA.

Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys(588) and Lys(591) of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export.
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http://dx.doi.org/10.1074/jbc.M110.208173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045017PMC
March 2011

Regulator of G protein signaling 6 (RGS6) induces apoptosis via a mitochondrial-dependent pathway not involving its GTPase-activating protein activity.

J Biol Chem 2011 Jan 1;286(2):1409-19. Epub 2010 Nov 1.

Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.

Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨ(m)) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨ(m) was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer.
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http://dx.doi.org/10.1074/jbc.M110.186700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020749PMC
January 2011

RGS6, a modulator of parasympathetic activation in heart.

Circ Res 2010 Nov 23;107(11):1345-9. Epub 2010 Sep 23.

Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.

Rationale: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein-coupled muscarinic M2 receptors, which activate heterotrimeric G(i/o) proteins to promote G protein-coupled inwardly rectifying K(+) (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart.

Objective: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart.

Methods And Results: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (I(K)(ACh)) activation and deactivation, as well as the extent of I(K)(ACh) desensitization.

Conclusions: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels.
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http://dx.doi.org/10.1161/CIRCRESAHA.110.224220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2997524PMC
November 2010

A novel mechanism involving coordinated regulation of nuclear levels and acetylation of NF-YA and Bcl6 activates RGS4 transcription.

J Biol Chem 2010 Sep 14;285(39):29760-9. Epub 2010 Jul 14.

Departments of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.

Neuronally enriched RGS4 plays a critical role attenuating G protein signaling in brain, although the mechanisms regulating RGS4 expression are unknown. Here we describe a novel mechanism for transcriptional activation of RGS4 in neuron-like PC6 cells, where RGS4 is markedly induced during confluence-induced growth arrest. Transcriptional activation of RGS4 in confluent PC6 cells was accompanied by impaired G(i/o)-dependent MAPK activation. In the human RGS4 gene promoter, we identified three phylogenetically conserved cis-elements: an inverted CCAAT box element (ICE), a cAMP response element, and a B-cell lymphoma 6 (Bcl6)-binding site. The ICE and the cAMP response element mediate activation, and the Bcl6 site mediates repression of RGS4 transcription. Activation of RGS4 transcription in confluent PC6 cells is accompanied by increases in NF-YA and C/EBPβ and decreases in Bcl6 levels in the nucleus. Increases in NF-YA and C/EBPβ lead to their increased binding to the RGS4 promoter in vivo, and dominant negative forms of these proteins repressed RGS4 promoter activity. Acetylation of NF-YA and Bcl6 were increased in postconfluent cells. Trichostatin A stimulation of RGS4 promoter activity, accompanied by increased binding of NF-YA and decreased binding of Bcl6 to the promoter, was abolished by mutation of the ICE and enhanced by mutation of the Bcl6 site. These findings demonstrate a dynamic and coordinated regulation of nuclear levels and acetylation status of trans-acting factors critical in determining the off/on state of the RGS4 promoter.
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http://dx.doi.org/10.1074/jbc.M110.121459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943308PMC
September 2010

Regulation of hepatic gluconeogenesis by an ER-bound transcription factor, CREBH.

Cell Metab 2010 Apr;11(4):331-9

Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, 300 Chunchun-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Korea.

Endoplasmic reticulum (ER)-bound transcription factor families are shown to be involved in the control of various metabolic pathways. Here, we report a critical function of ER-bound transcription factor, CREBH, in the regulation of hepatic gluconeogenesis. Expression of CREBH is markedly induced by fasting or in the insulin-resistant state in rodents in a dexamethasone- and PGC-1alpha-dependent manner, which results in the accumulation of active nuclear form of CREBH (CREBH-N). Overexpression of constitutively active CREBH activates transcription of PEPCK-C or G6Pase by binding to its enhancer site that is distinct from the well-characterized CREB/CRTC2 regulatory sequences in vivo. Of interest, knockdown of CREBH in liver significantly reduces blood glucose levels without altering expression of genes involved in the ER stress signaling cascades in mice. These data suggest a crucial role for CREBH in the regulation of hepatic glucose metabolism in mammals.
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http://dx.doi.org/10.1016/j.cmet.2010.02.016DOI Listing
April 2010

Activation of SIRT1 by resveratrol represses transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) by deacetylating hepatic nuclear factor 4alpha.

J Biol Chem 2009 Oct 3;284(40):27042-53. Epub 2009 Aug 3.

Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935, USA.

The SIRT1 activators isonicotinamide (IsoNAM), resveratrol, fisetin, and butein repressed transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C). An evolutionarily conserved binding site for hepatic nuclear factor (HNF) 4alpha (-272/-252) was identified, which was required for transcriptional repression of the PEPCK-C gene promoter caused by these compounds. This site contains an overlapping AP-1 binding site and is adjacent to the C/EBP binding element (-248/-234); the latter is necessary for hepatic transcription of PEPCK-C. AP-1 competed with HNF4alpha for binding to this site and also decreased HNF4alpha stimulation of transcription from the PEPCK-C gene promoter. Chromatin immunoprecipitation experiments demonstrated that HNF4alpha and AP-1, but not C/EBPbeta, reciprocally bound to this site prior to and after treating HepG2 cells with IsoNAM. IsoNAM treatment resulted in deacetylation of HNF4alpha, which decreased its binding affinity to the PEPCK-C gene promoter. In HNF4alpha-null Chinese hamster ovary cells, IsoNAM and resveratrol failed to repress transcription from the PEPCK-C gene promoter; overexpression of HNF4alpha in Chinese hamster ovary cells re-established transcriptional inhibition. Exogenous SIRT1 expression repressed transcription, whereas knockdown of SIRT1 by RNA interference reversed this effect. IsoNAM decreased the level of mRNA for PEPCK-C but had no effect on mRNA for glucose-6-phosphatase in AML12 mouse hepatocytes. We conclude that SIRT1 activation inhibited transcription of the gene for PEPCK-C in part by deacetylation of HNF4alpha. However, SIRT1 deacetylation of other key regulatory proteins that control PEPCK-C gene transcription also likely contributed to the inhibitory effect.
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http://dx.doi.org/10.1074/jbc.M109.047340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785634PMC
October 2009

Aspects of the control of phosphoenolpyruvate carboxykinase gene transcription.

J Biol Chem 2009 Oct 27;284(40):27031-5. Epub 2009 Jul 27.

Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4936, USA.

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http://dx.doi.org/10.1074/jbc.R109.040535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785632PMC
October 2009

What is the metabolic role of phosphoenolpyruvate carboxykinase?

J Biol Chem 2009 Oct 27;284(40):27025-9. Epub 2009 Jul 27.

Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4936, USA.

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http://dx.doi.org/10.1074/jbc.R109.040543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785631PMC
October 2009

The genome sequence of taurine cattle: a window to ruminant biology and evolution.

Science 2009 Apr;324(5926):522-8

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.
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http://dx.doi.org/10.1126/science.1169588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943200PMC
April 2009

Identification of conserved regulatory elements in mammalian promoter regions: a case study using the PCK1 promoter.

Genomics Proteomics Bioinformatics 2008 Dec;6(3-4):129-43

Bovine Functional Genomics Laboratory, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA.

A systematic phylogenetic footprinting approach was performed to identify conserved transcription factor binding sites (TFBSs) in mammalian promoter regions using human, mouse and rat sequence alignments. We found that the score distributions of most binding site models did not follow the Gaussian distribution required by many statistical methods. Therefore, we performed an empirical test to establish the optimal threshold for each model. We gauged our computational predictions by comparing with previously known TFBSs in the PCK1 gene promoter of the cytosolic isoform of phosphoenolpyruvate carboxykinase, and achieved a sensitivity of 75% and a specificity of approximately 32%. Almost all known sites overlapped with predicted sites, and several new putative TFBSs were also identified. We validated a predicted SP1 binding site in the control of PCK1 transcription using gel shift and reporter assays. Finally, we applied our computational approach to the prediction of putative TFBSs within the promoter regions of all available RefSeq genes. Our full set of TFBS predictions is freely available at http://bfgl.anri.barc.usda.gov/tfbsConsSites.
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http://dx.doi.org/10.1016/S1672-0229(09)60001-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054123PMC
December 2008

Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2.

J Biol Chem 2008 Apr 31;283(14):8984-94. Epub 2008 Jan 31.

School of Medicine, Meharry Medical College, Nashville, TN 37208-3599, USA.

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.
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http://dx.doi.org/10.1074/jbc.M709040200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276363PMC
April 2008
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