AhRR and PPP1R3C: Potential Prognostic Biomarkers for Serous Ovarian Cancer.

The lack of effective screening and successful treatment contributes to high ovarian cancer mortality, making it the second most common cause of gynecologic cancer death. Development of chemoresistance in up to 75% of patients is the cause of a poor treatment response and reduced survival. Therefore, identifying potential and effective biomarkers for its diagnosis and prognosis is a strong critical need. Copy number alterations are frequent in cancer, and relevant for molecular tumor stratification and patients' prognoses. In this study, array-CGH analysis was performed in three cell lines and derived cancer stem cells (CSCs) to identify genes potentially predictive for ovarian cancer patients' prognoses. Bioinformatic analyses of genes involved in copy number gains revealed that AhRR and PPP1R3C expression negatively correlated with ovarian cancer patients' overall and progression-free survival. These results, together with a significant association between AhRR and PPP1R3C expression and ovarian cancer stemness markers, suggested their potential role in CSCs. Furthermore, AhRR and PPP1R3C's increased expression was maintained in some CSC subpopulations, reinforcing their potential role in ovarian cancer. In conclusion, we reported for the first time, to the best of our knowledge, a prognostic role of AhRR and PPP1R3C expression in serous ovarian cancer.

MiR-4461 Inhibits Tumorigenesis of Renal Cell Carcinoma by Targeting PPP1R3C.

Background: Renal cell carcinoma (RCC) is one of the most common and malignant tumors in the urinary system. The aim of this research was to investigate the mechanism and clinical significance of miR-4461 in the RCC progression. Materials and Methods: Twenty-eight (28) paired RCC tissue samples and adjacent nontumor tissue samples, as well as RCC cell lines were used to measure the expression of miR-4461 and protein phosphatase 1 regulatory subunit 3C (PPP1R3C) transcript by real-time quantitative PCR. The target relationship between miR-4461 and PPP1R3C was predicted by TargetScan and further verified by dual-luciferase reporter gene assay and RNA pull-down assay. Cell Counting Kit-8 (CCK-8) assay and BrdU ELISA assay were performed to measure RCC cell viability and proliferation. In addition, caspase-3 activity assay and cell adhesion assay were implemented to measure RCC cell apoptosis and adhesion. Results: MiR-4461 was lowly expressed both in RCC tissues and cells, while upregulated PPP1R3C was tested in RCC tissues and cells. In addition, miR-4461 was validated to directly target PPP1R3C, thereby negatively regulating PPP1R3C. Particularly, miR-4461 exerted a clear inhibitory effect on the malignant phenotypes of RCC cells by binding and inhibiting PPP1R3C. Conclusion: MiR-4461, which served as a tumor suppressor, inhibited RCC progression by targeting and downregulating PPP1R3C.

PPP1R3C mediates metformin-inhibited hepatic gluconeogenesis.

BACKGROUND: Metformin has been widely used to alleviate hyperglycemia in patients with type 2 diabetes mainly via suppressing hepatic gluconeogenesis. However, the underlying mechanism remains incompletely clear. Here, we aimed to explore the role of PPP1R3C in metformin-mediated inhibition of hepatic gluconeogenesis. METHODS: The differentially expressed genes in primary mouse hepatocytes incubated with 8-Br-cAMP and metformin were analyzed by microarrays. Hepatic glucose production and gluconeogenic gene expressions were detected after adenovirus-mediated overexpression or silence of PPP1R3C in vitro and in vivo. The phosphorylation level and location of transducer of regulated CREB activity 2 (TORC2) were determined by Western blot and immunofluorescence. RESULTS: Metformin and adenovirus-mediated activation of AMPK suppressed 8-Br-cAMP-stimulated Ppp1r3c mRNA expression in primary mouse hepatocytes. Overexpression of PPP1R3C in primary mouse hepatocytes or the livers of wild-type mice promoted hepatic glucose production and gluconeogenic gene expressions. On the contrary, adenovirus-mediated knockdown of PPP1R3C in primary mouse hepatocytes decreased hepatic gluconeogenesis, with the suppression of cAMP-stimulated gluconeogenic gene expressions and TORC2 dephosphorylation. Notably, Ppp1r3c expression was increased in the liver of db/db mice. After PPP1R3C silence in the livers of wild-type and db/db mice, blood glucose levels and hepatic glucose production were markedly lowered, with decreased expressions of key gluconeogenic enzymes and transcript factors as well as liver glycogen content. CONCLUSION: Metformin-activated AMPK decreases hepatic PPP1R3C expression, leading to the suppression of hepatic gluconeogenesis through blocking cAMP-stimulated TORC2 dephosphorylation. Hepatic specific silence of PPP1R3C provides a promising therapeutic strategy for type 2 diabetes.

The traditional Chinese formulae Ling-gui-zhu-gan decoction alleviated non-alcoholic fatty liver disease via inhibiting PPP1R3C mediated molecules.

BACKGROUND: Ling-gui-zhu-gan decoction (LGZG), a classic traditional Chinese medicine formula, has been confirmed to be effective in improving steatosis in non-alcoholic fatty liver disease (NAFLD). However, the mechanism under the efficacy remains unclear. Hence, this study was designed to investigate the mechanisms of LGZG on alleviating steatosis. METHODS: Twenty four rats were randomly divided into three groups: normal group, NAFLD group, fed with high fat diet (HFD) and LGZG group (fed with HFD and supplemented with LGZG). After 4 weeks intervention, blood and liver were collected. Liver steatosis was detected by Oil Red O staining, and blood lipids were biochemically determined. Whole genome genes were detected by RNA-Seq and the significant different genes were verified by RT-qPCR. The protein expression of Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) and key molecules of glycogen and lipid metabolism were measured by western blot. Chromophore substrate methods measured glycogen phosphorylase (GPa) activity and glycogen content. RESULTS: HFD can markedly induce hepatic steatosis and promote liver triglyceride (TG) and serum cholesterol (CHOL) contents, while liver TG and serum CHOL were both markedly decreased by LGZG treatment for 4 weeks. By RNA sequencing, we found that NAFLD rats showed significantly increase of PPP1R3C expression and LGZG reduced its expression. RT-qPCR and Western blot both verified the alteration of PPP1R3C upon LGZG intervention. LGZG also promoted the activity of glycogen phosphorylase liver type (PYGL) and inhibited the activity of glycogen synthase (GS) in NAFLD rats, resulting in glycogenolysis increase and glycogen synthesis decrease in the liver. By detecting glycogen content, we also found that LGZG reduced hepatic glycogen in NAFLD rats. In addition, we analyzed the key molecules in hepatic de novo lipogenesis and cholesterol synthesis, and indicated that LGZG markedly inhibited the activity of acetyl-CoA carboxylase (ACC), sterol receptor element-binding protein-1c (SREBP-1c) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), resulting in lipid synthesis decrease in the liver. CONCLUSION: Our data highlighted the role of PPP1R3C targeting pathways, and found that hepatic glycogen metabolism might be the potential target of LGZG in preventing NAFLD.

Role of glycogen phosphorylase in liver glycogen metabolism.

Liver glycogen is synthesized after a meal in response to an increase in blood glucose concentration in the portal vein and endocrine and neuroendocrine signals, and is degraded to glucose between meals to maintain blood glucose homeostasis. Glycogen degradation and synthesis during the diurnal cycle are mediated by changes in the activities of phosphorylase and glycogen synthase. Phosphorylase is regulated by phosphorylation of serine-14. Only the phosphorylated form of liver phosphorylase (GPa) is catalytically active. Interconversion between GPa and GPb (unphosphorylated) is dependent on the activities of phosphorylase kinase and of phosphorylase phosphatase. The latter comprises protein phosphatase-1 in conjunction with a glycogen-targeting protein (G-subunit) of the PPP1R3 family. At least two of six G-subunits (GL and PTG) expressed in liver are involved in GPa dephosphorylation. GPa to GPb interconversion is dependent on the conformational state of phosphorylase which can be relaxed (R) or tense (T) depending on the concentrations of allosteric effectors such as glucose, glucose 6-phosphate and adenine nucleotides and on the acetylation state of lysine residues. The G-subunit, GL, encoded by PPP1R3B gene is expressed at high levels in liver and can function as a phosphorylase phosphatase and a synthase phosphatase and has an allosteric binding site for GPa at the C-terminus which inhibits synthase phosphatase activity. GPa to GPb conversion is a major upstream event in the regulation of glycogen synthesis by glucose, its downstream metabolites and extracellular signals such as insulin and neurotransmitters.

Molecular characterization and expression analysis of PPP1R3C in hypoxia-tolerant Indian catfish, Clarias batrachus (Linnaeus, 1758) under hypoxia.

Hypoxia is an important environmental stressor that leads to rapid adaptive changes in metabolic organization. However, the molecular mechanisms of hypoxia tolerance in fish remain largely unknown. The present work was focused on understanding the molecular mechanisms and signaling pathways that may lead to tolerance of Clarias batrachus to hypoxic stress. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) is a new hypoxia-inducible factor (HIF) targeted gene and is regulated by HIF-1 under hypoxic conditions. Overexpression of PPP1R3C increases glycogen accumulation through activation of several enzymes and processes. In this study, for the first time, full length cDNA of PPP1R3C from C. batrachus was characterized and its expression pattern in the brain, liver, muscle and spleen under short (progressive hypoxia; PH, 1h, 6h and 12h) and long-term (natural) hypoxic conditions was investigated. The complete cDNA of PPP1R3C was of 1499 bp, encoding 285 amino acid residues. The identified protein had a protein phosphatase 1 binding motif and a carbohydrate binding domain, thought to be involved in the regulation of glycogen metabolism. Short-term hypoxia exposure caused significant increase in PPP1R3C transcripts in the liver (6h; 6.96 fold and 12h; 3.91 fold) and muscle (progressive hypoxia; 3.46 fold), while, after long-term hypoxia exposure, significant up-regulation in the liver (7.77 fold) and spleen (6.59 fold) tissues was observed. No significant differences were observed in the brain for any time periods. Thus PPP1R3C may play an important role in the tolerance of C. batrachus to hypoxia.

High NaCl-induced inhibition of PTG contributes to activation of NFAT5 through attenuation of the negative effect of SHP-1.

Activation of the transcription factor NFAT5 by high NaCl involves changes in phosphorylation. By siRNA screening, we previously found that protein targeting to glycogen (PTG), a regulatory subunit of protein phosphatase1 (PP1), contributes to regulation of high NaCl-induced NFAT5 transcriptional activity. The present study addresses the mechanism involved. We find that high NaCl-induced inhibition of PTG elevates NFAT5 activity by increasing NFAT5 transactivating activity, protein abundance, and nuclear localization. PTG acts via a catalytic subunit PP1γ. PTG associates physically with PP1γ, and NaCl reduces both this association and remaining PTG-associated PP1γ activity. High NaCl-induced phosphorylation of p38, ERK, and SHP-1 contributes to activation of NFAT5. Knockdown of PTG does not affect phosphorylation of p38 or ERK. However, PTG and PP1γ bind to SHP-1, and knockdown of either PTG or PP1γ increases high NaCl-induced phosphorylation of SHP-1-S591, which inhibits SHP-1. Mutation of SHP-1-S591 to alanine, which cannot be phosphorylated, increases inhibition of NFAT5 by SHP-1. Thus high NaCl reduces the stimulatory effect of PTG and PP1γ on SHP-1, which in turn reduces the inhibitory effect of SHP-1 on NFAT5. Our findings add to the known functions of PTG, which was previously recognized only for its glycogenic activity.