CREB-Regulated Transcriptional Coactivator 2 Proteome Landscape is Modulated by SREBF1.

cAMP response element-binding protein (CREB) regulated transcriptional coactivator 2 (CRTC2) is a critical transcription factor that maintains glucose homeostasis by activating CREB. Energy homeostasis is maintained through multiple pathways; therefore, CRTC2 may interact with other transcription factors, particularly under metabolic stress. CRTC2 liver-specific KO mice were created, and the global proteome, phosphoproteome, and acetylome from liver tissue under high-fat diet conditions were analyzed using liquid chromatography-tandem mass spectrometry and bioinformatics analysis. Differentially regulated proteins (DRPs) were enriched in metabolic pathways, which were subsequently corroborated through animal experiments. The consensus DRPs from these datasets were used as seed proteins to generate a protein-protein interaction network using STRING, and GeneMANIA identified fatty acid synthase as a mutually relevant protein. In an additional local-protein-protein interaction analysis of CRTC2 and fatty acid synthase with DRPs, sterol regulatory element binding transcription factor 1 (SREBF1) was the common mediator. CRTC2-CREB and SREBF1 are transcription factors, and DNA-binding motif analysis showed that multiple CRTC2-CREB-regulated genes possess SREBF1-binding motifs. This indicates the possible induction by the CRTC2-SREBF1 complex, which is validated through luciferase assay. Therefore, the CRTC2-SREBF1 complex potentially modulates the transcription of multiple proteins that fine-tune cellular metabolism under metabolic stress.

Impaired BCAA catabolism in adipose tissues promotes age-associated metabolic derangement.

Adipose tissues are central in controlling metabolic homeostasis and failure in their preservation is associated with age-related metabolic disorders. The exact role of mature adipocytes in this phenomenon remains elusive. Here we describe the role of adipose branched-chain amino acid (BCAA) catabolism in this process. We found that adipocyte-specific Crtc2 knockout protected mice from age-associated metabolic decline. Multiomics analysis revealed that BCAA catabolism was impaired in aged visceral adipose tissues, leading to the activation of mechanistic target of rapamycin complex (mTORC1) signaling and the resultant cellular senescence, which was restored by Crtc2 knockout in adipocytes. Using single-cell RNA sequencing analysis, we found that age-associated decline in adipogenic potential of visceral adipose tissues was reinstated by Crtc2 knockout, via the reduction of BCAA-mTORC1 senescence-associated secretory phenotype axis. Collectively, we propose that perturbation of BCAA catabolism by CRTC2 is critical in instigating age-associated remodeling of adipose tissue and the resultant metabolic decline in vivo.

Peli3 ablation ameliorates acetaminophen-induced liver injury through inhibition of GSK3ß phosphorylation and mitochondrial translocation.

The signaling pathways governing acetaminophen (APAP)-induced liver injury have been extensively studied. However, little is known about the ubiquitin-modifying enzymes needed for the regulation of APAP-induced liver injury. Here, we examined whether the Pellino3 protein, which has E3 ligase activity, is needed for APAP-induced liver injury and subsequently explored its molecular mechanism. Whole-body Peli3-/- knockout (KO) and adenovirus-mediated Peli3 knockdown (KD) mice showed reduced levels of centrilobular cell death, infiltration of immune cells, and biomarkers of liver injury, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), upon APAP treatment compared to wild-type (WT) mice. Peli3 deficiency in primary hepatocytes decreased mitochondrial and lysosomal damage and reduced the mitochondrial reactive oxygen species (ROS) levels. In addition, the levels of phosphorylation at serine 9 in the cytoplasm and mitochondrial translocation of GSK3ß were decreased in primary hepatocytes obtained from Peli3-/- KO mice, and these reductions were accompanied by decreases in JNK phosphorylation and mitochondrial translocation. Pellino3 bound more strongly to GSK3ß compared with JNK1 and JNK2 and induced the lysine 63 (K63)-mediated polyubiquitination of GSK3ß. In rescue experiments, the ectopic expression of wild-type Pellino3 in Peli3-/- KO hepatocytes restored the mitochondrial translocation of GSK3ß, but this restoration was not obtained with expression of a catalytically inactive mutant of Pellino3. These findings are the first to suggest a mechanistic link between Pellino3 and APAP-induced liver injury through the modulation of GSK3ß polyubiquitination.

Involvement of a novel cAMP signaling mediator for beige adipogenesis.

BACKGROUND: Exposure to cold temperature stimulates the sympathetic nervous system that activates ß-adrenergic receptor signals in brown and beige adipocytes, leading to the induction of adaptive thermogenesis in mammals. Prominin-1 (PROM1) is a pentaspan transmembrane protein that is widely identified as a marker for stem cells, although the role of this protein as a regulator of many intracellular signaling cascades has been recently delineated. The main focus of the current study is to identify the previously unknown role of PROM1 in beige adipogenesis and adaptive thermogenesis. METHODS: Prom1 whole body knockout (Prom1 KO) mice, Prom1 adipogenic progenitor (AP) cell-specific knockout (Prom1 APKO) mice and Prom1 adipocyte-specific knockout (Prom1 AKO) mice were constructed and were subject for the induction of adaptive thermogenesis. The effect of systemic Prom1 depletion was evaluated by hematoxylin and eosin staining, immunostaining, and biochemical analysis in vivo. Flow cytometric analysis was performed to determine the identity of PROM1-expressing cell types, and the resultant cells were subject to beige adipogenesis in vitro. The potential role of PROM1 and ERM in cAMP signaling was also assessed in undifferentiated AP cells in vitro. Finally, the specific effect of Prom1 depletion on AP cell or mature adipocytes on adaptive thermogenesis was evaluated by hematoxylin and eosin staining, immunostaining, and biochemical analysis in vivo. RESULTS: Prom1 KO mice displayed an impairment in cold- or ß3-adrenergic agonist-induced adaptive thermogenesis in subcutaneous adipose tissues (SAT) but not in brown adipose tissues (BAT). By fluorescence-activated cell sorting (FACS) analysis, we identified that PROM1 positive cells are enriched in PDGFRα+Sca1+ AP cells from SAT. Interestingly, Prom1 knockout stromal vascular fractions showed reduced PDGFRα expression, suggesting a role of PROM1 in beige adipogenic potential. Indeed, we found that Prom1-deficient AP cells from SAT showed reduced potential for beige adipogenesis. Furthermore, AP cell-specific depletion of Prom1, but not adipocyte-specific depletion of Prom1, displayed defects in adaptive thermogenesis as evidenced by resistance to cold-induced browning of SAT and dampened energy expenditure in mice. CONCLUSION: We found that PROM1 positive AP cells are essential for the adaptive thermogenesis by ensuing stress-induced beige adipogenesis. Identification of PROM1 ligand might be useful in the activation of thermogenesis that could be potentially beneficial in combating obesity.

Tcf7l2 in hepatocytes regulates de novo lipogenesis in diet-induced non-alcoholic fatty liver disease in mice.

AIMS/HYPOTHESIS: Non-alcoholic fatty liver disease (NAFLD) associated with type 2 diabetes may more easily progress towards severe forms of non-alcoholic steatohepatitis (NASH) and cirrhosis. Although the Wnt effector transcription factor 7-like 2 (TCF7L2) is closely associated with type 2 diabetes risk, the role of TCF7L2 in NAFLD development remains unclear. Here, we investigated how changes in TCF7L2 expression in the liver affects hepatic lipid metabolism based on the major risk factors of NAFLD development. METHODS: Tcf7l2 was selectively ablated in the liver of C57BL/6N mice by inducing the albumin (Alb) promoter to recombine Tcf7l2 alleles floxed at exon 5 (liver-specific Tcf7l2-knockout [KO] mice: Alb-Cre;Tcf7l2f/f). Alb-Cre;Tcf7l2f/f and their wild-type (Tcf7l2f/f) littermates were fed a high-fat diet (HFD) or a high-carbohydrate diet (HCD) for 22 weeks to reproduce NAFLD/NASH. Mice were refed a standard chow diet or an HCD to stimulate de novo lipogenesis (DNL) or fed an HFD to provide exogenous fatty acids. We analysed glucose and insulin sensitivity, metabolic respiration, mRNA expression profiles, hepatic triglyceride (TG), hepatic DNL, selected hepatic metabolites, selected plasma metabolites and liver histology. RESULTS: Alb-Cre;Tcf7l2f/f essentially exhibited increased lipogenic genes, but there were no changes in hepatic lipid content in mice fed a normal chow diet. However, following 22 weeks of diet-induced NAFLD/NASH conditions, liver steatosis was exacerbated owing to preferential metabolism of carbohydrate over fat. Indeed, hepatic Tcf7l2 deficiency enhanced liver lipid content in a manner that was dependent on the duration and amount of exposure to carbohydrates, owing to cell-autonomous increases in hepatic DNL. Mechanistically, TCF7L2 regulated the transcriptional activity of Mlxipl (also known as ChREBP) by modulating O-GlcNAcylation and protein content of carbohydrate response element binding protein (ChREBP), and targeted Srebf1 (also called SREBP1) via miRNA (miR)-33-5p in hepatocytes. Eventually, restoring TCF7L2 expression at the physiological level in the liver of Alb-Cre;Tcf7l2f/f mice alleviated liver steatosis without altering body composition under both acute and chronic HCD conditions. CONCLUSIONS/INTERPRETATION: In mice, loss of hepatic Tcf7l2 contributes to liver steatosis by inducing preferential metabolism of carbohydrates via DNL activation. Therefore, TCF7L2 could be a promising regulator of the NAFLD associated with high-carbohydrate diets and diabetes since TCF7L2 deficiency may lead to development of NAFLD by promoting utilisation of excess glucose pools through activating DNL. DATA AVAILABILITY: RNA-sequencing data have been deposited into the NCBI GEO under the accession number GSE162449 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162449 ).

Central role of Prominin-1 in lipid rafts during liver regeneration.

Prominin-1, a lipid raft protein, is required for maintaining cancer stem cell properties in hepatocarcinoma cell lines, but its physiological roles in the liver have not been well studied. Here, we investigate the role of Prominin-1 in lipid rafts during liver regeneration and show that expression of Prominin-1 increases after 2/3 partial hepatectomy or CCl4 injection. Hepatocyte proliferation and liver regeneration are attenuated in liver-specific Prominin-1 knockout mice compared to wild-type mice. Detailed mechanistic studies reveal that Prominin-1 interacts with the interleukin-6 signal transducer glycoprotein 130, confining it to lipid rafts so that STAT3 signaling by IL-6 is effectively activated. The overexpression of the glycosylphosphatidylinsositol-anchored first extracellular domain of Prominin-1, which is the domain that binds to GP130, rescued the proliferation of hepatocytes and liver regeneration in liver-specific Prominin-1 knockout mice. In summary, Prominin-1 is upregulated in hepatocytes during liver regeneration where it recruits GP130 into lipid rafts and activates the IL6-GP130-STAT3 axis, suggesting that Prominin-1 might be a promising target for therapeutic applications in liver transplantation.

Hepatocyte-specific Prominin-1 protects against liver injury-induced fibrosis by stabilizing SMAD7.

Prominin-1 (PROM1), also known as CD133, is expressed in hepatic progenitor cells (HPCs) and cholangiocytes of the fibrotic liver. In this study, we show that PROM1 is upregulated in the plasma membrane of fibrotic hepatocytes. Hepatocellular expression of PROM1 was also demonstrated in mice (Prom1CreER; R26TdTom) in which cells expressed TdTom under control of the Prom1 promoter. To understand the role of hepatocellular PROM1 in liver fibrosis, global and liver-specific Prom1-deficient mice were analyzed after bile duct ligation (BDL). BDL-induced liver fibrosis was aggravated with increased phosphorylation of SMAD2/3 and decreased levels of SMAD7 by global or liver-specific Prom1 deficiency but not by cholangiocyte-specific Prom1 deficiency. Indeed, PROM1 prevented SMURF2-induced SMAD7 ubiquitination and degradation by interfering with the molecular association of SMAD7 with SMURF2. We also demonstrated that hepatocyte-specific overexpression of SMAD7 ameliorated BDL-induced liver fibrosis in liver-specific Prom1-deficient mice. Thus, we conclude that PROM1 is necessary for the negative regulation of TGFß signaling during liver fibrosis.

A novel role of CRTC2 in promoting nonalcoholic fatty liver disease.

OBJECTIVE: Diet-induced obesity is often associated with nonalcoholic fatty liver disease (NAFLD), which instigates severe metabolic disorders, including cirrhosis, hepatocellular carcinoma, and type 2 diabetes. We have shown that hepatic depletion of CREB regulated transcription co-activator (CRTC) 2 protects mice from the progression of diet-induced fatty liver phenotype, although the exact mechanism by which CRTC2 modulates this process is elusive to date. Here, we investigated the role of hepatic CRTC2 in the instigation of NAFLD in mammals. METHODS: Crtc2 liver-specific knockout (Crtc2 LKO) mice and Crtc2 flox/flox (Crtc2 f/f) mice were fed a high fat diet (HFD) for 7-8 weeks. Body weight, liver weight, hepatic lipid contents, and plasma triacylglycerol (TG) levels were determined. Western blot analysis was performed to determine Sirtuin (SIRT) 1, tuberous sclerosis complex (TSC) 2, and mammalian target of rapamycin complex (mTORC) 1 activity in the liver. Effects of Crtc2 depletion on lipogenesis was determined by measuring lipogenic gene expression (western blot analysis and qRT-PCR) in the liver as well as Oil red O staining in hepatocytes. Effects of miR-34a on mTORC1 activity and hepatic lipid accumulation was assessed by AAV-miR-34a virus in mice and Ad-miR-34a virus and Ad-anti-miR-34a virus in hepatocytes. Autophagic flux was assessed by western blot analysis after leupeptin injection in mice and bafilomycin treatment in hepatocytes. Lipophagy was assessed by transmission electron microscopy and confocal microscopy. Expression of CRTC2 and p-S6K1 in livers of human NAFLD patients was assessed by immunohistochemistry. RESULTS: We found that expression of CRTC2 in the liver is highly induced upon HFD-feeding in mice. Hepatic depletion of Crtc2 ameliorated HFD-induced fatty liver disease phenotypes, with a pronounced inhibition of the mTORC1 pathway in the liver. Mechanistically, we found that expression of TSC2, a potent mTORC1 inhibitor, was enhanced in Crtc2 LKO mice due to the decreased expression of miR-34a and the subsequent increase in SIRT1-mediated deacetylation processes. We showed that ectopic expression of miR-34a led to the induction of mTORC1 pathway, leading to the hepatic lipid accumulation in part by limiting lipophagy and enhanced lipogenesis. Finally, we found a strong association of CRTC2, miR-34a and mTORC1 activity in the NAFLD patients in humans, demonstrating a conservation of signaling pathways among species. CONCLUSIONS: These data collectively suggest that diet-induced activation of CRTC2 instigates the progression of NAFLD by activating miR-34a-mediated lipid accumulation in the liver via the simultaneous induction of lipogenesis and inhibition of lipid catabolism. Therapeutic approach to specifically inhibit CRTC2 activity in the liver could be beneficial in combating NAFLD in the future.

Activation of ectopic olfactory receptor 544 induces GLP-1 secretion and regulates gut inflammation.

Olfactory receptors are ectopically expressed in extra-nasal tissues. The gut is constantly exposed to high levels of odorants where ectopic olfactory receptors may play critical roles. Activation of ectopic olfactory receptor 544 (Olfr544) by azelaic acid (AzA), an Olfr544 ligand, reduces adiposity in mice fed a high-fat diet (HFD) by regulating fuel preference to fats. Herein, we investigated the novel function of Olfr544 in the gut. In GLUTag cells, AzA induces the cAMP-PKA-CREB signaling axis and increases the secretion of GLP-1, an enteroendocrine hormone with anti-obesity effects. In mice fed a HFD and orally administered AzA, GLP-1 plasma levels were elevated in mice. The induction of GLP-1 secretion was negated in cells with Olfr544 gene knockdown and in Olfr544-deficient mice. Gut microbiome analysis revealed that AzA increased the levels of Bacteroides acidifaciens and microbiota associated with antioxidant pathways. In fecal metabolomics analysis, the levels of succinate and trehalose, metabolites correlated with a lean phenotype, were elevated by AzA. The function of Olfr544 in gut inflammation, a key feature in obesity, was further investigated. In RNA sequencing analysis, AzA suppressed LPS-induced activation of inflammatory pathways and reduced TNF-α and IL-6 expression, thereby improving intestinal permeability. The effects of AzA on the gut metabolome, microbiome, and colon inflammation were abrogated in Olfr544-KO mice. These results collectively demonstrated that activation of Olfr544 by AzA in the gut exerts multiple effects by regulating GLP-1 secretion, gut microbiome and metabolites, and colonic inflammation in anti-obesogenic phenotypes and, thus, may be applied for obesity therapeutics.

Liver-Specific Deletion of Mouse CTCF Leads to Hepatic Steatosis via Augmented PPARγ Signaling.

BACKGROUND & AIMS: The liver is the major organ for metabolizing lipids, and malfunction of the liver leads to various diseases. Nonalcoholic fatty liver disease is rapidly becoming a major health concern worldwide and is characterized by abnormal retention of excess lipids in the liver. CCCTC-binding factor (CTCF) is a highly conserved zinc finger protein that regulates higher-order chromatin organization and is involved in various gene regulation processes. Here, we sought to determine the physiological role of CTCF in hepatic lipid metabolism. METHODS: We generated liver-specific, CTCF-ablated and/or CD36 whole-body knockout mice. Overexpression or knockdown of peroxisome proliferator-activated receptor (PPAR)γ in the liver was achieved using adenovirus. Mice were examined for development of hepatic steatosis and inflammation. RNA sequencing was performed to identify genes affected by CTCF depletion. Genome-wide occupancy of H3K27 acetylation, PPARγ, and CTCF were analyzed by chromatin immunoprecipitation sequencing. Genome-wide chromatin interactions were analyzed by in situ Hi-C. RESULTS: Liver-specific, CTCF-deficient mice developed hepatic steatosis and inflammation when fed a standard chow diet. Global analysis of the transcriptome and enhancer landscape revealed that CTCF-depleted liver showed enhanced accumulation of PPARγ in the nucleus, which leads to increased expression of its downstream target genes, including fat storage-related gene CD36, which is involved in the lipid metabolic process. Hepatic steatosis developed in liver-specific, CTCF-deficient mice was ameliorated by repression of PPARγ via pharmacologic blockade or adenovirus-mediated knockdown, but hardly rescued by additional knockout of CD36. CONCLUSIONS: Our data indicate that liver-specific deletion of CTCF leads to hepatosteatosis through augmented PPARγ DNA-binding activity, which up-regulates its downstream target genes associated with the lipid metabolic process.

Depletion of Prmt1 in Adipocytes Impairs Glucose Homeostasis in Diet-Induced Obesity.

Protein arginine methyltransferase (PRMT) 1 is involved in the regulation of various metabolic pathways such as glucose metabolism in liver and atrophy in the skeletal muscle. However, the role of PRMT1 in the fat tissues under the disease state has not been elucidated to date. In this study, we delineate the function of this protein in adipocytes in vivo. PRMT1 expression was abundant in the white adipose tissues (WAT), which was induced upon a high-fat diet in mice and by obesity in humans. We found that adipocyte-specific depletion of Prmt1 resulted in decreased fat mass without overall changes in body weight in mice. Mechanistically, the depletion of Prmt1 in WAT led to the activation of the AMPK pathway, which was causal to the increased lipophagy, mitochondrial lipid catabolism, and the resultant reduction in lipid droplet size in WAT in vivo. Interestingly, despite the increased energy expenditure, we observed a promotion of adipose tissue inflammation and an ectopic accumulation of triglycerides in the peripheral tissues in Prmt1 adipocyte-specific knockout mice, which promoted the impaired insulin tolerance that is reminiscent of mouse models of lipodystrophy. These data collectively suggest that PRMT1 prevents WAT from excessive degradation of triglycerides by limiting AMPK-mediated lipid catabolism to control whole-body metabolic homeostasis in diet-induced obesity conditions.

Prominin-1-Radixin axis controls hepatic gluconeogenesis by regulating PKA activity.

Prominin-1 (Prom1) is a major cell surface marker of cancer stem cells, but its physiological functions in the liver have not been elucidated. We analyzed the levels of mRNA transcripts in serum-starved primary WT (Prom1+/+ ) and KO (Prom1-/- ) mouse hepatocytes using RNA sequencing (RNA-seq) data, and found that CREB target genes were downregulated. This initial observation led us to determine that Prom1 deficiency inhibited cAMP response element-binding protein (CREB) activation and gluconeogenesis, but not cyclic AMP (cAMP) accumulation, in glucagon-, epinephrine-, or forskolin-treated liver tissues and primary hepatocytes, and mitigated glucagon-induced hyperglycemia. Because Prom1 interacted with radixin, Prom1 deficiency prevented radixin from localizing to the plasma membrane. Moreover, systemic adenoviral knockdown of radixin inhibited CREB activation and gluconeogenesis in glucagon-treated liver tissues and primary hepatocytes, and mitigated glucagon-elicited hyperglycemia. Based on these results, we conclude that Prom1 regulates hepatic PKA signaling via radixin functioning as an A kinase-anchored protein (AKAP).

Role of CRTC2 in Metabolic Homeostasis: Key Regulator of Whole-Body Energy Metabolism?

Cyclic adenosine monophosphate (cAMP) signaling is critical for regulating metabolic homeostasis in mammals. In particular, transcriptional regulation by cAMP response element-binding protein (CREB) and its coactivator, CREB-regulated transcription coactivator (CRTC), is essential for controlling the expression of critical enzymes in the metabolic process, leading to more chronic changes in metabolic flux. Among the CRTC isoforms, CRTC2 is predominantly expressed in peripheral tissues and has been shown to be associated with various metabolic pathways in tissue-specific manners. While initial reports showed the physiological role of CRTC2 in regulating gluconeogenesis in the liver, recent studies have further delineated the role of this transcriptional coactivator in the regulation of glucose and lipid metabolism in various tissues, including the liver, pancreatic islets, endocrine tissues of the small intestines, and adipose tissues. In this review, we discuss recent studies that have utilized knockout mouse models to delineate the role of CRTC2 in the regulation of metabolic homeostasis.

PRMT1 Is Required for the Maintenance of Mature ß-Cell Identity.

Loss of functional ß-cell mass is an essential feature of type 2 diabetes, and maintaining mature ß-cell identity is important for preserving a functional ß-cell mass. However, it is unclear how ß-cells achieve and maintain their mature identity. Here we demonstrate a novel function of protein arginine methyltransferase 1 (PRMT1) in maintaining mature ß-cell identity. Prmt1 knockout in fetal and adult ß-cells induced diabetes, which was aggravated by high-fat diet-induced metabolic stress. Deletion of Prmt1 in adult ß-cells resulted in the immediate loss of histone H4 arginine 3 asymmetric dimethylation (H4R3me2a) and the subsequent loss of ß-cell identity. The expression levels of genes involved in mature ß-cell function and identity were robustly downregulated as soon as Prmt1 deletion was induced in adult ß-cells. Chromatin immunoprecipitation sequencing and assay for transposase-accessible chromatin sequencing analyses revealed that PRMT1-dependent H4R3me2a increases chromatin accessibility at the binding sites for CCCTC-binding factor (CTCF) and ß-cell transcription factors. In addition, PRMT1-dependent open chromatin regions may show an association with the risk of diabetes in humans. Together, our results indicate that PRMT1 plays an essential role in maintaining ß-cell identity by regulating chromatin accessibility.

Salt-inducible kinase 1 regulates bone anabolism via the CRTC1-CREB-Id1 axis.

New bone anabolic agents for the effective treatment of bone metabolic diseases like osteoporosis are of high clinical demand. In the present study, we reveal the function of salt-inducible kinase 1 (SIK1) in regulating osteoblast differentiation. Gene knockdown of SIK1 but not of SIK2 or SIK3 expression in primary preosteoblasts increased osteoblast differentiation and bone matrix mineralization. SIK1 also regulated the proliferation of osteoblastic precursor cells in osteogenesis. This negative control of osteoblasts required the catalytic activity of SIK1. SIK1 phosphorylated CREB regulated transcription coactivator 1 (CRTC1), preventing CRTC1 from enhancing CREB transcriptional activity for the expression of osteogenic genes like Id1. Furthermore, SIK1 knockout (KO) mice had higher bone mass, osteoblast number, and bone formation rate versus littermate wild-type (WT) mice. Preosteoblasts from SIK1 KO mice showed more osteoblastogenic potential than did WT cells, whereas osteoclast generation among KO and WT precursors was indifferent. In addition, bone morphogenic protein 2 (BMP2) suppressed both SIK1 expression as well as SIK1 activity by protein kinase A (PKA)-dependent mechanisms to stimulate osteogenesis. Taken together, our results indicate that SIK1 is a key negative regulator of preosteoblast proliferation and osteoblast differentiation and that the repression of SIK1 is crucial for BMP2 signaling for osteogenesis. Therefore, we propose SIK1 to be a useful therapeutic target for the development of bone anabolic strategies.

Essential Role of Protein Arginine Methyltransferase 1 in Pancreas Development by Regulating Protein Stability of Neurogenin 3.

BACKGROUND: Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells. Recent studies have revealed that PRMT1 plays important roles in the development of various tissues. However, its role in pancreas development has not yet been elucidated. METHODS: Pancreatic progenitor cell-specific Prmt1 knock-out (Prmt1 PKO) mice were generated and characterized for their metabolic and histological phenotypes and their levels of Neurog3 gene expression and neurogenin 3 (NGN3) protein expression. Protein degradation assays were performed in mPAC cells. RESULTS: Prmt1 PKO mice showed growth retardation and a severely diabetic phenotype. The pancreatic size and ß-cell mass were significantly reduced in Prmt1 PKO mice. Proliferation of progenitor cells during the secondary transition was decreased and endocrine cell differentiation was impaired. These defects in pancreas development could be attributed to the sustained expression of NGN3 in progenitor cells. Protein degradation assays in mPAC cells revealed that PRMT1 was required for the rapid degradation of NGN3. CONCLUSION: PRMT1 critically contributes to pancreas development by destabilizing the NGN3 protein.

Skeletal muscle-specific Prmt1 deletion causes muscle atrophy via deregulation of the PRMT6-FOXO3 axis.

Protein arginine methyltransferases (PRMTs) have emerged as important regulators of skeletal muscle metabolism and regeneration. However, the direct roles of the various PRMTs during skeletal muscle remodeling remain unclear. Using skeletal muscle-specific prmt1 knockout mice, we examined the function and downstream targets of PRMT1 in muscle homeostasis. We found that muscle-specific PRMT1 deficiency led to muscle atrophy. PRMT1-deficient muscles exhibited enhanced expression of a macroautophagic/autophagic marker LC3-II, FOXO3 and muscle-specific ubiquitin ligases, TRIM63/MURF-1 and FBXO32, likely contributing to muscle atrophy. The mechanistic study reveals that PRMT1 regulates FOXO3 through PRMT6 modulation. In the absence of PRMT1, increased PRMT6 specifically methylates FOXO3 at arginine 188 and 249, leading to its activation. Finally, we demonstrate that PRMT1 deficiency triggers FOXO3 hyperactivation, which is abrogated by PRMT6 depletion. Taken together, PRMT1 is a key regulator for the PRMT6-FOXO3 axis in the control of autophagy and protein degradation underlying muscle maintenance. Abbreviations: Ad-RNAi: adenovirus-delivered small interfering RNA; AKT: thymoma viral proto-oncogene; AMPK: AMP-activated protein kinase; Baf A1: bafilomycin A1; CSA: cross-sectional area; EDL: extensor digitorum longus; FBXO32: F-box protein 32; FOXO: forkhead box O; GAS: gatrocnemieus; HDAC: histone deacetylase; IGF: insulin-like growth factor; LAMP: lysosomal-associated membrane protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mKO: Mice with skeletal muscle-specific deletion of Prmt1; MTOR: mechanistic target of rapamycin kinase; MYH: myosin heavy chain; MYL1/MLC1f: myosin, light polypeptide 1; PRMT: protein arginine N-methyltransferase; sgRNA: single guide RNA; SQSTM1: sequestosome 1; SOL: soleus; TA: tibialis anterior; TRIM63/MURF-1: tripartite motif-containing 63; YY1: YY1 transcription factor.

Cardiac specific PRMT1 ablation causes heart failure through CaMKII dysregulation.

Dysregulation of Ca2+/calmodulin-dependent protein kinase (CaMK)II is closely linked with myocardial hypertrophy and heart failure. However, the mechanisms that regulate CaMKII activity are incompletely understood. Here we show that protein arginine methyltransferase 1 (PRMT1) is essential for preventing cardiac CaMKII hyperactivation. Mice null for cardiac PRMT1 exhibit a rapid progression to dilated cardiomyopathy and heart failure within 2 months, accompanied by cardiomyocyte hypertrophy and fibrosis. Consistently, PRMT1 is downregulated in heart failure patients. PRMT1 depletion in isolated cardiomyocytes evokes hypertrophic responses with elevated remodeling gene expression, while PRMT1 overexpression protects against pathological responses to neurohormones. The level of active CaMKII is significantly elevated in PRMT1-deficient hearts or cardiomyocytes. PRMT1 interacts with and methylates CaMKII at arginine residues 9 and 275, leading to its inhibition. Accordingly, pharmacological inhibition of CaMKII restores contractile function in PRMT1-deficient mice. Thus, our data suggest that PRMT1 is a critical regulator of CaMKII to maintain cardiac function.