ABSTRACT
Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.
Subject(s)
Biosynthetic Pathways , Cyclin D1 , Hepatocytes , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/metabolism , Hepatocytes/metabolism , Proteomics , Pyrimidines/biosynthesis , Humans , Animals , Mice , Cell LineABSTRACT
Black adolescent males use available mental health services at a disproportionately lower rate compared to males of other racial groups. This study examines barriers to school-based mental health resource (SBMHR) use among Black adolescent males, as a means of addressing reduced usage of available mental health resources and to improve these resources to better support their mental health needs. Secondary data for 165 Black adolescent males were used from a mental health needs assessment of two high schools in southeast Michigan. Logistic regression was employed to examine the predictive power of psychosocial (self-reliance, stigma, trust, and negative previous experience) and access barriers (no transportation, lack of time, lack of insurance, and parental restrictions) on SBMHR use, as well as the relationship between depression and SBMHR use. No access barriers were found to be significantly associated with SBMHR use. However, self-reliance and stigma were statistically significant predictors of SBMHR use. Participants who identified self-reliance in addressing their mental health symptoms were 77% less likely to use available mental health resources in their school. However, participants who reported stigma as a barrier to using SBMHR were nearly four times more likely to use available mental health resources; this suggests potential protective factors in schools that can be built into mental health resources to support Black adolescent males' use of SBMHRs. This study serves as an early step in exploring how SBMHRs can better serve the needs of Black adolescent males. It also speaks to potential protective factors that schools provide for Black adolescent males who have stigmatized views of mental health and mental health services. Future studies would benefit from a nationally representative sample allowing for more generalizable results regarding barriers and facilitators to Black adolescent males' use of school-based mental health resources.
Subject(s)
Hepatoblastoma , Liver Neoplasms , Cell Differentiation , Cell Proliferation , Epigenesis, Genetic , HumansABSTRACT
BACKGROUND/AIMS: The development of decompensation in cirrhosis demarcates a marked change in the natural history of chronic liver disease. HMG-CoA reductase inhibitors (statins) exert pleiotropic effects that reduce inflammation and fibrosis as well as improve vascular reactivity. Retrospective studies uniformly have associated statin utilization with improved outcomes for patients with cirrhosis. Prospective human studies have shown that statins reduce portal hypertension and reduce death in patients with decompensated cirrhosis after variceal hemorrhage when added to standard therapy with an acceptable safety profile. This proposal aims to extend these findings to demonstrate that simvastatin reduces incident hepatic decompensation events among cirrhotic patients at high risk for hepatic decompensation. METHODS: We will perform the SACRED Trial (NCT03654053), a phase III, prospective, multi-center, double-blind, randomized clinical trial at 11 VA Medical Centers. Patients with compensated cirrhosis with clinically significant portal hypertension will be stratified based upon the concomitant use of nonselective beta-blockers and randomized to simvastatin 40 mg/day versus placebo for up to 24 months. Patients will be observed for the development of hepatic decompensation (variceal hemorrhage, ascites, encephalopathy), hepatocellular carcinoma, liver-related death, death from any cause, and/or complications of statin therapy. Ancillary studies will evaluate patient-reported outcomes and pharmacogenetic corollaries of safety and/or efficacy. CONCLUSION: Statins have a long track-record of safety and tolerability. This class of medications is generic and inexpensive, and thus, if the hypothesis is proven, there will be few barriers to widespread acceptance of the role of statins to prevent decompensation in patients with compensated cirrhosis. ClinicalTrials.gov Identifier: NCT03654053.
Subject(s)
Esophageal and Gastric Varices , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Liver Neoplasms , Fibrosis , Gastrointestinal Hemorrhage , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Liver Cirrhosis/complications , Liver Cirrhosis/drug therapy , Liver Neoplasms/drug therapy , Prospective Studies , Retrospective Studies , Simvastatin/therapeutic useABSTRACT
The liver is organized into zones in which hepatocytes express different metabolic enzymes. The cells most responsible for liver repopulation and regeneration remain undefined, because fate mapping has only been performed on a few hepatocyte subsets. Here, 14 murine fate-mapping strains were used to systematically compare distinct subsets of hepatocytes. During homeostasis, cells from both periportal zone 1 and pericentral zone 3 contracted in number, whereas cells from midlobular zone 2 expanded in number. Cells within zone 2, which are sheltered from common injuries, also contributed to regeneration after pericentral and periportal injuries. Repopulation from zone 2 was driven by the insulin-like growth factor binding protein 2-mechanistic target of rapamycin-cyclin D1 (IGFBP2-mTOR-CCND1) axis. Therefore, different regions of the lobule exhibit differences in their contribution to hepatocyte turnover, and zone 2 is an important source of new hepatocytes during homeostasis and regeneration.
Subject(s)
Hepatocytes/physiology , Liver Regeneration , Liver/physiology , Animals , Biliary Tract/cytology , Biliary Tract Diseases/physiopathology , Cell Proliferation , Cyclin D1/metabolism , Gene Knock-In Techniques , Homeostasis , Insulin-Like Growth Factor Binding Protein 2/metabolism , Liver/cytology , Mice , TOR Serine-Threonine Kinases/metabolismABSTRACT
Background: Nearly one-third of youth are affected by a mental health disorder, and the majority do not receive adequate care. To improve clinical outcomes among youth, efforts have been made to train providers in evidence-based mental health practices, such as cognitive behavioral therapy (CBT). Such efforts call for valid assessment measures that can inform and evaluate training activities. Aims: This study presents the development and validation of the CBT Competence Scale (CCS), a brief self-report measure to assess provider competence for CBT delivery. Method: Participants were 387 school mental health professionals (SMHPs) working with students in Michigan, USA. Initial items (n=59) were developed to evaluate competence in delivering common elements of CBT, with competence conceptualized as covering domains of knowledge, perception, and use of CBT techniques. CCS validation proceeded in three steps: using item response theory to select the most important items for assessing knowledge, evaluating the factor structure using exploratory and then confirmatory factor analyses, and examining reliability and validity of the resultant measure. Results: The validated CCS measure consists of four dimensions of CBT competence across 33 items: Non-behavioral skills, Behavioral skills, Perceptions, and Knowledge. The CCS demonstrated excellent internal consistency and good construct-based validity. Conclusions: The CCS holds promise as a valid, informative measure of CBT competence appropriate for the school setting, with potential for application in other environments such as mental health clinics.
ABSTRACT
BACKGROUND & AIMS: Type II diabetes mellitus worsens the prognosis of cirrhosis. Multiple medications including metformin and statins often are co-administered to manage patients with diabetes. The aim of this study was to assess the impact of metformin exposure on mortality, hepatic decompensation, and hepatocellular carcinoma in individuals with diabetes and cirrhosis, controlling for multiple concomitant exposures. METHODS: We performed a retrospective cohort study of patients with cirrhosis diagnosed between January 1, 2008, through June 30, 2016, in the Veterans Health administration. Marginal structural models and propensity-matching approaches were implemented to quantify the treatment effect of metformin in patients with pre-existing diabetes with or without prior metformin exposure. RESULTS: Among 74,984 patients with cirrhosis, diabetes mellitus was present before the diagnosis of cirrhosis in 53.8%, and was diagnosed during follow-up evaluation in 4.8%. Before the diagnosis of cirrhosis, 11,114 patients had active utilization of metformin. In these patients, metformin, statin, and angiotensinogen-converting enzyme inhibitor/angiotensin-2-receptor blocker exposure were associated independently with reduced mortality (metformin hazard ratio, 0.68; 95% CI, 0.61-0.75); metformin was not associated with reduced hepatocellular carcinoma or hepatic decompensation after adjustment for concomitant statin exposure. For patients with diabetes before a diagnosis of cirrhosis but no prior metformin exposure, metformin similarly was associated with reduced mortality (hazard ratio, 0.72; 95% CI, 0.35-0.97), but not with reduced hepatocellular carcinoma or hepatic decompensation. CONCLUSIONS: Metformin use in patients with cirrhosis and diabetes appears safe and is associated independently with reduced overall, but not liver-related, mortality, hepatocellular carcinoma, or decompensation after adjusting for concomitant statin and angiotensinogen-converting enzyme inhibitor/angiotensin-2-receptor blocker exposure.
Subject(s)
Carcinoma, Hepatocellular , Diabetes Mellitus, Type 2 , Liver Neoplasms , Metformin , Carcinoma, Hepatocellular/epidemiology , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/drug therapy , Humans , Liver Cirrhosis/complications , Liver Cirrhosis/epidemiology , Liver Neoplasms/epidemiology , Metformin/therapeutic use , Retrospective StudiesABSTRACT
Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.
Subject(s)
Cell Cycle/physiology , Cyclin D1/genetics , Cyclin D1/metabolism , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Liver/metabolism , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Female , Gene Knockdown Techniques , Hepatocytes/metabolism , Hepatocytes/pathology , Liver Regeneration/genetics , Liver Regeneration/physiology , Male , Mice, Inbred BALB C , Mice, KnockoutABSTRACT
During normal proliferation, hepatocytes accumulate triglycerides (TGs) in lipid droplets (LDs), but the underlying mechanisms and functional significance of this steatosis are unknown. In the current study, we examined the coordinated regulation of cell cycle progression and LD accumulation. As previously shown, hepatocytes develop increased LD content after mitogen stimulation. Cyclin D1, in addition to regulating proliferation, was both necessary and sufficient to promote LD accumulation in response to mitogens. Interestingly, cyclin D1 promotes LD accumulation by inhibiting the breakdown of TGs by lipolysis through a mechanism involving decreased lipophagy, the autophagic degradation of LDs. To examine whether inhibition of lipolysis is important for cell cycle progression, we overexpressed adipose TG lipase (ATGL), a key enzyme involved in TG breakdown. As expected, ATGL reduced LD content but also markedly inhibited hepatocyte proliferation, suggesting that lipolysis regulates a previously uncharacterized cell cycle checkpoint. Consistent with this, in mitogen-stimulated cells with small interfering RNA-mediated depletion of cyclin D1 (which inhibits proliferation and stimulates lipolysis), concurrent ATGL knockdown restored progression into S phase. Following partial hepatectomy, a model of robust hepatocyte proliferation in vivo, ATGL overexpression led to decreased LD content, cell cycle inhibition, and marked liver injury, further indicating that down-regulation of lipolysis is important for normal hepatocyte proliferation. Conclusion: We suggest a new relationship between steatosis and proliferation in hepatocytes: cyclin D1 inhibits lipolysis, resulting in LD accumulation, and suppression of lipolysis is necessary for cell cycle progression.
ABSTRACT
BACKGROUND & AIMS: Concerns related to hepatotoxicity frequently lead to discontinuation or non-initiation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase therapy in patients with cirrhosis despite data supporting statin use. We investigated the independent effects of hyperlipidemia and statin exposure on mortality, hepatic decompensation, and hepatocellular carcinoma development in a large national cohort of patients with cirrhosis. METHODS: We performed a retrospective cohort study of patients with newly diagnosed cirrhosis from January 1, 2008 through June 30, 2016 in the Veterans Health Administration. Subjects were divided into 2 cohorts: 21,921 patients with prior statin exposure (existing users) and 51,023 statin-naïve individuals, of whom 8794 subsequently initiated statin therapy (new initiators) and 44,269 did not (non-initiators). Multivariable Cox proportional hazard models with inverse probability weighting were constructed to assess the effects of time-updating lipid profiles and cumulative exposure to statins on survival and hepatic decompensation. Statin-naïve new initiators were propensity matched with non-initiators to simulate a randomized controlled trial of statin use in cirrhosis. RESULTS: In statin-naïve subjects, every 10-mg/dL increase in baseline total cholesterol was associated with a 3.6% decrease in mortality. In existing users, each year of continued statin exposure was associated with a hazard ratio of 0.920 (95% confidence interval 0.0.897-0.943) for mortality. After risk-set matching, each year of statin exposure among new initiators was associated with a hazard ratio of 0.913 (95% confidence interval 0.890-0.937) for mortality. CONCLUSIONS: In a retrospective cohort study of veterans with a new diagnosis of cirrhosis, we associated hypercholesterolemia with well-preserved hepatic function and decreased mortality. Nonetheless, each cumulative year of statin exposure was associated with an independent 8.0%-8.7% decrease of mortality of patients with cirrhosis of Child-Turcotte-Pugh classes A and B.
Subject(s)
Carcinoma, Hepatocellular/epidemiology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hypercholesterolemia/complications , Hypercholesterolemia/drug therapy , Liver Cirrhosis/mortality , Liver Neoplasms/epidemiology , Aged , Cholesterol/blood , Female , Heart Failure/epidemiology , Humans , Hypercholesterolemia/blood , Liver Cirrhosis/physiopathology , Male , Middle Aged , Myocardial Infarction/epidemiology , Propensity Score , Proportional Hazards Models , Randomized Controlled Trials as Topic , Retrospective Studies , Stroke/epidemiology , Survival Rate , United States/epidemiologySubject(s)
Blood Coagulation Disorders/etiology , Craniopharyngioma/complications , Hypopituitarism/etiology , Liver Cirrhosis/etiology , Non-alcoholic Fatty Liver Disease/etiology , Obesity/etiology , Pituitary Neoplasms/complications , Adolescent , Craniopharyngioma/diagnostic imaging , Craniopharyngioma/pathology , Craniopharyngioma/surgery , Humans , Hypopituitarism/metabolism , Leptin/metabolism , Male , Pituitary Neoplasms/diagnostic imaging , Pituitary Neoplasms/pathology , Pituitary Neoplasms/surgeryABSTRACT
BACKGROUND: REP 2139 clears circulating hepatitis B virus (HBV) surface antigen (HBsAg), enhancing the restoration of functional control of HBV infection by immunotherapy. We assessed the safety and efficacy of REP 2139 and pegylated interferon alfa-2a in patients with chronic HBV and hepatitis D virus (HDV) co-infection. METHODS: In this open-label, non-randomised, phase 2 trial, patients aged 18-55 years, who were treatment naive, hepatitis B e antigen [HBeAg] negative, anti-hepatitis D antigen [HDAg] positive, and HDV RNA positive, with serum HBsAg concentrations of more than 1000 IU/mL, and a history of HDV infection for 6 months or more before treatment, were recruited at Toma Ciorba Hospital of Infectious Diseases in ChiÈinau, Moldova. Patients were excluded if they had HDV superinfection, liver infections other than HBV and HDV, or liver cirrhosis. Patients received 500 mg intravenous REP 2139 once per week for 15 weeks, followed by combined therapy with 250 mg intravenous REP 2139 and 180 µg subcutaneous pegylated interferon alfa-2a once per week for 15 weeks, then monotherapy with 180 µg pegylated interferon alfa-2a once per week for 33 weeks. The primary endpoints assessed at the end of treatment were the safety and tolerability of the treatment regimen, analysed in the intention-to-treat population. Secondary outcomes included the proportion of patients with serum HBsAg less than 50 IU/mL, the proportion of patients with suppressed HBV DNA, and the proportion of patients who maintained these responses through follow-up. The REP 301 trial is registered with ClinicalTrials.gov, number NCT02233075. We also did an additional follow-up at 1 year after the end of treatment, as an interim analysis of the REP 301-LTF trial (planned duration 3 years), registered with ClinicalTrials.gov, number NCT02876419, which is ongoing but not recruiting patients. FINDINGS: Between Sept 8, 2014, and Jan 27, 2015, we enrolled 12 patients into the REP 301 study. All 12 patients experienced at least one adverse event during treatment: two (17%) patients experienced anaemia, eight (67%) neutropenia, and ten (83%) thrombocytopenia. Five (42%) patients had raised alanine aminotransferase levels, four (33%) had raised aspartate aminotransferase levels, and two (17%) had increased bilirubin concentrations. Four (33%) patients had a serious adverse event, and 12 (100%) patients had treatment-emergent lab abnormalities. Six patients had HBsAg levels less than 50 IU/mL by the end of treatment (all <0·05 IU/mL); five maintained this level of suppression at the end of 1 year follow-up. Six patients had hepatitis B surface antibody (anti-HBs) titres above 10 mIU/mL at the end of treatment (five had maximum anti-HBs concentrations of 7681-86â532 mIU/mL during treatment), which were maintained at the end of 1 year follow-up in these five patients. Elevated alanine and aspartate aminotransferase concentrations and profound elevations of anti-HBs titres were restricted to patients who had HBsAg levels of less than <1 IU/mL before the introduction of pegylated interferon alfa-2a. Nine patients had suppressed HBV DNA (<10 IU/mL]) at the end of treatment, which was maintained by seven patients and newly established in an eighth patient at the end of 1 year follow-up. 11 patients became HDV RNA negative during treatment, with nine remaining HDV RNA negative at the end of treatment; seven of these patients remained HDV RNA negative by the end of 1 year follow-up. By the end of 1 year follow-up, normalisation of serum aminotransferases occurred in nine of 12 patients. INTERPRETATION: Combined REP 2139 and pegylated interferon alfa-2a therapy is safe, well tolerated, and establishes functional control of HBV and HDV co-infection and normalisation of serum aminotransferases in a high proportion of patients 1 year after therapy. This combination therapy approach might provide a new treatment option for patients with HBV and HDV co-infection. FUNDING: Replicor.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis B/drug therapy , Hepatitis D/drug therapy , Interferon-alpha/therapeutic use , Nucleic Acids/therapeutic use , Polyethylene Glycols/therapeutic use , Polymers/therapeutic use , Adolescent , Adult , Alanine Transaminase/blood , Anemia/chemically induced , Antiviral Agents/adverse effects , Aspartate Aminotransferases/blood , Coinfection/drug therapy , Drug Therapy, Combination , Female , Humans , Interferon-alpha/adverse effects , Liver/enzymology , Male , Middle Aged , Neutropenia/chemically induced , Nucleic Acids/adverse effects , Polyethylene Glycols/adverse effects , Polymers/adverse effects , Recombinant Proteins/adverse effects , Recombinant Proteins/therapeutic use , Thrombocytopenia/chemically induced , Young AdultSubject(s)
Bile Duct Neoplasms/epidemiology , Carcinoma, Hepatocellular/epidemiology , Cholangiocarcinoma/epidemiology , Liver Neoplasms/epidemiology , Veterans/statistics & numerical data , Adenocarcinoma/epidemiology , Adenocarcinoma/secondary , Aged , Autopsy , Carcinoma, Squamous Cell/epidemiology , Carcinoma, Squamous Cell/secondary , Colonic Neoplasms/epidemiology , Colonic Neoplasms/pathology , Hepatitis C, Chronic/epidemiology , Humans , Liver Cirrhosis/epidemiology , Liver Neoplasms/secondary , Lung Neoplasms/epidemiology , Lung Neoplasms/pathology , Male , Pancreatic Neoplasms/epidemiology , Pancreatic Neoplasms/pathology , Prevalence , Small Cell Lung Carcinoma/epidemiology , Small Cell Lung Carcinoma/secondary , United States/epidemiologyABSTRACT
Cyclin D1 is a cell cycle protein that promotes proliferation by mediating progression through key checkpoints in G1 phase. It is also a proto-oncogene that is commonly overexpressed in human cancers. In addition to its canonical role in controlling cell cycle progression, cyclin D1 affects other aspects of cell physiology, in part through transcriptional regulation. In this study, we find that cyclin D1 inhibits the activity of a key metabolic transcription factor, peroxisome proliferator-activated receptor α (PPARα), a member of nuclear receptor family that induces fatty acid oxidation and may play an anti-neoplastic role. In primary hepatocytes, cyclin D1 inhibits PPARα transcriptional activity and target gene expression in a cdk4-independent manner. In liver and breast cancer cells, knockdown of cyclin D1 leads to increased PPARα transcriptional activity, expression of PPARα target genes, and fatty acid oxidation. Similarly, cyclin D1 depletion enhances binding of PPARα to target sequences by chromatin immunoprecipitation. In proliferating hepatocytes and regenerating liver in vivo, induction of endogenous cyclin D1 is associated with diminished PPARα activity. Cyclin D1 expression is both necessary and sufficient for growth factor-mediated repression of fatty acid oxidation in proliferating hepatocytes. These studies indicate that in addition to playing a pivotal role in cell cycle progression, cyclin D1 represses PPARα activity and inhibits fatty acid oxidation. Our findings establish a new link between cyclin D1 and metabolism in both tumor cells and physiologic hepatocyte proliferation.
Subject(s)
Cyclin D1/metabolism , Fatty Acids/metabolism , Liver Neoplasms, Experimental/metabolism , PPAR alpha/metabolism , Animals , Cell Line, Tumor , Hep G2 Cells , Humans , Lipid Metabolism , Liver/metabolism , Liver/pathology , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred BALB C , Oxidation-Reduction , Proto-Oncogene Mas , Rats , Rats, Sprague-Dawley , TransfectionABSTRACT
Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced ß-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and ß-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.
Subject(s)
Kruppel-Like Transcription Factors/metabolism , Liver Regeneration , Sulfatases/metabolism , Wnt Signaling Pathway , Animals , Cell Proliferation/drug effects , Down-Regulation/drug effects , Hedgehog Proteins/metabolism , Hepatectomy , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Kruppel-Like Transcription Factors/genetics , Liver Regeneration/drug effects , Mice , Mice, Inbred C57BL , Models, Biological , Sulfatases/deficiency , Wnt Signaling Pathway/drug effects , Wnt3A Protein/pharmacology , Zinc Finger Protein GLI1 , beta Catenin/metabolismABSTRACT
Force-based atomic force microscopy (AFM) was used to detect HCV (hepatitis C virus) RNA directly and to quantitatively analyse it without the need for reverse transcription or amplification. Capture and detection DNA probes were designed. The former was spotted onto a substrate with a conventional microarrayer, and the latter was immobilized on an AFM probe. To control the spacing between the immobilized DNAs on the surface, dendron self-assembly was employed. Force-distance curves showed that the mean force of the specific unbinding events was 32 ± 5 pN, and the hydrodynamic distance of the captured RNA was 30-60 nm. Adhesion force maps were generated with criteria including the mean force value, probability of obtaining the specific curves and hydrodynamic distance. The maps for the samples whose concentrations ranged from 0.76 fM to 6.0 fM showed that cluster number has a linear relationship with RNA concentration, while the difference between the observed number and the calculated one increased at low concentrations. Because the detection limit is expected to be enhanced by a factor of 10 000 when a spot of 1 micron diameter is employed, it is believed that HCV RNA of a few copy numbers can be detected by the use of AFM.
Subject(s)
Hepacivirus/genetics , Microscopy, Atomic Force/methods , RNA, Viral/analysis , Base Sequence , DNA Probes , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Viral/chemistryABSTRACT
Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.
Subject(s)
Cyclin D1/metabolism , Glucose/pharmacology , Hepatocyte Nuclear Factor 4/metabolism , Lipogenesis/drug effects , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Cells, Cultured , Cyclin D1/antagonists & inhibitors , Cyclin D1/genetics , Cyclin-Dependent Kinase 4/metabolism , Gene Expression Regulation/drug effects , Hepatocytes/cytology , Hepatocytes/metabolism , Lipid Metabolism/drug effects , Mice , Nuclear Proteins/genetics , Protein Binding , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering/metabolism , Transcription Factors/geneticsABSTRACT
Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.
Subject(s)
Cell Cycle , Cyclin D1/metabolism , Vestibule, Labyrinth/cytology , Adult , Apoptosis , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , DNA Damage , HumansABSTRACT
Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.