Beta-arrestin 1 Mediates Liver Thyrotropin Regulation Of Cholesterol Metabolism Via The AKT-dependent Pathway

Back groundCholesterol metabolism is one of the most important functions of liver.Therefore,hepatocytes play a key role to maintain and regulate cholesterol homeostasis.Hypercholesterolemia,caused by an imbalance of cholesterol metabolism,had already become an independent risk factor of atherosclerotic disease.Further study of the molecular mechanism of liver cholesterol metabolism will provide a strong biological basis for understanding the imbalance of cholesterol metabolism,the pathology of atherosclerotic diseases,and help us to develop novel drugs and more effective treatments.Thyroid-stimulating hormone(TSH),positively correlating with serum total cholesterol levels,has caught the scientists' eyeballs in recent years.In our previous study,thyroid stimulating hormone receptor(TSHR),expressing on the surface of hepatocytes,is direct sponsor to peripheric TSH and mediates its function through highly specific interaction.TSHR is one of the members of the G Protein-Coupled Receptor(GPCR)family.A major role of TSHR in cholesterol metabolism was illustrated,as TSH could regulate hepatic cholesterol metabolism via cAMP/PKA/CREB/HMGCR and SREBP2/HNF4a/CYP7A1 pathways to regulate synthesis and degradation of hepatic cholesterol.Our further study raised up another TSHR regulator in liver tissues,?-arrestins(ARRBs).It's interesting that ARRBs have dual signal transduction activity.On the one hand,they can mediate desensitization of activated GPCR.And on the other hand,they can initiate G protein-independent signal transduction processes.ARRB1 and ARRB2,principle members of ARRBs family,are widely distributed and play different roles in the molecular mechanisms involved in TSHR.However,the detailed mechanisms of ARRBs in TSH-initiated cholesterol metabolism has not been reported.In our study,the effects of ARRBs on TSH-regulated cholesterol metabolism were investigated.Cholesterol levels in ARRB1/2-knockout(ARRB1-KO and ARRB2-KO)mice and ARRB1/2-knockdown(ARRB1-KD and ARRB2-KD)cells were measured.Molecules participated in cholesterol metabolism were analyzed.It turned out that deficiencies in ARRB1 led to decreased cholesterol levels and decreased TSH-stimulated AKT phosphorylation.Subsequently,the inhibitory effect to CYP7A1 by SREBP2 was reduced due to lowered mature SREBP2 level.Other than the failures of TSH in ARRB1/2-KD cells,AKT activator could enhance AKT phosphorylation and mature SREBP2 level.Our results demonstrate that ARRBs,especially ARRB1,are involved in TSH-regulated cholesterol metabolism through AKT pathway.TSH increased ARRB1 expression,enhanced AKT phosphorylation,up-regulated SREBP2 levels,inhibited CYP7A1 expression,thereby reducing the conversion of cholesterol to bile acids.Objectives1.To confirm the effect of cholesterol levels caused by deficiency of ARRBs in the liver.2.To clarify the moleculars of ARRBs-mediated signaling pathways related to cholesterol metabolism during TSH stimulation in the liver.Materials and methods1.Animal modles(1)Genetically arrb1-KO and arrb2-KOmice(C57BL/6J)were kindly provided by Prof.Jinpeng Sun,Medical College of Shandong University.The wild-type mice and genetically manipulated mice were housed at 23 ? under a 12-hour light-dark cycle and in a humidity-controlled(60%)environment.The mice were allowed to drink and eat freely.(2)Subclinical hypothyroidism(SCH)mouse model:male C57BL/6 mice(8 weeks old)were given methimazole(MMI,0.04 mg/kg.d.)in drinking water to inhibit thyroid hormone(TH)synthesis.Mice were weighed each week,and the MMI doses were adjusted according to the body weight.After MMI was administered for 12 weeks,the mice were harvested.(3)Biochemical and Molecular Analysis of Model Animals:mice were fasted for 6 hour and then euthanized using sodium pentobarbital.Serum samples were collected immediately prior to sacrificing the mice and were tested for free thyroxine(FT4),free triiodothyonie(FT3)and TSH levels.Livers were excised,washed in PBS and stored in liquid nitrogen for further analysis.Total mRNA was extracted using Trizol standard method.Total protein was extracted according to standard protocols.The protein concentration was measured using the BCA method.2.Cell modles(1)HepG2 cells were routinely cultured and passaged according to standard culture method recommended by ATCC.(2)ARRB1/2-KD cell:CRISPR/Cas9 system was used to generate stable ARRB1/2-KD cultures,when HepG2 cells were achieved to 70-80%confluence.The knockdown efficiencies of tree different candidate oligos were optimized according to target protein level semi-quantified by westernblot and gray scale assay.(3)TSH stimulating cell:The cells were starved overnight when reached 70-80%confluence,4 ?M bovine TSH was added,and stimulation was conducted for a time-course of 24 hours in serum-free DMEM.Cells were harvested according to the experiment arrangement.(4)AKT activator SC79 stimulation cell model:The corresponding cell model was treated with SC79 and maintained for 24 hours.Cells were harvested according to the experiment arrangement.3.GloSensorTM cAMP assayIntracellular concentration of cAMP,a second messenger of TSH-TSHR signal transduction,commonly used to indicate the intracellular activity of TSHR pathway on real-time.The GloSensor assay is a method for detecting cAMP in a cell using a bioluminescence method,and detects cAMP produced in response to a test compound on a GPCR in vivo.The test was used to monitor the baseline and curve of cAMP before and after TSH stimulating on the cells.4.Cholesterol measurementsFree and total cholesterol in tissues and cells were measured using cholesterol assay kits.Experiment operations were performed according to the kit manu.The level of cholesterol were corrected by protein concentration.5.Molecular analysis of cholesterol metabolism-related proteinsTotal mRNA and protein were prepared for qRT-PCR and westemblot analysis.6.Graphics and statistical analysisThe data were analyzed using SPSS 17.0 and expressed as the mean±SE.Error bars represent standard error(s.e.m.).The density of immunoblotting bands were compared on densitometric scan by Image J,antified by Origin 9.0 and set as grayscale analysis.Statistical significance was assessed by t tests for differences between two experimental groups and by one-way ANOVA for multiple groups.P<0.05 was considered statistically significant.Graphics were generated using Origin 9.0 and assembled in Adobe Illustrator CS5.Results1.The expression of ARRBs were upregulated under pathological increase of peripheral TSH level.The serum TSH in SCH mice were significantly higher than those in their wild-type counterparts,but FT4 were similar between them.At the same time,hepatocellular ARRBs mRNA significantly increased(ARRB1 p<0.05,ARRB2 p<0.01)of SCH mice compared with wild-type mice.Similarly,ARRBs accumulation also detected by westernblot after 4 ?M TSH stimulation on HepG2 cells for 24h.Interestingly,a dramatic increase in ARRB2 was observed quickly within 1-2 hours(p<0.01),while ARRB1 chronically increased on 12-24 hours with statistical significance(p<0.05).2.Knockout or konckdown of ARRBs led to decreased cholesterol levelsFree and total cholesterol levels in the liver tissues of arrbl-KO mice were lower than wild-type counterparts(p<0.05),whereas arrb2-KO mice did not show obvious differences.We also found the same phenomenon in the ARRB1/2-KD cell model.Artificial reduction of ARRB1,but not of ARRB2,is associated with a significant decrease in total cholesterol levels(p<0.05).3.The role of ARRBs in TSH-mediated hepatic cholesterol metabolism is relatedto inhibition of cholesterol synthesis and increase of cholesterol conversion.(1)Inhibition of ARRBs increases hepatic cellular cholesterol synthesisHMGCR and CYP7A1,rate-limiting enzymes for cholesterol metabolism,were tested in both "ARRBs-konckout or knockdown" models.In the livers of arrb1/2-KO mice,the level of HMGCR was increased on account of ARRBs deficiency(p<0.05).For the cholesterol conversion process,CYP7A1 showed increased expression in response to ARRB1 deficiency.In the ARRB1/2-KD cell,HMGCR increased(p<0.05),while CYP7A1 increased only in ARRB1-KD cell,showing a similar tendency to that in arrbl/2-KO animals.These results suggest that Inhibition of ARRBs increases hepatic cellular cholesterol synthesis.Furthermore,the increase of CYP7A1 led by deficiency of ARRB1 suggest that ARRB1 may play a more important role than ARRB2 in liver cholesterol metabolism.(2)ARRBs were involved in the regulation of downstream G protein-mediated signaling pathway activity of TSHR.To measure the activated efficiency of TSHR signaling pathway under the condition of ARRBs down-regulation,the change of intracellular cAMP level stimulated with 4?M TSH,forskolin(Adenylate cyclase activator)and PBS were quantitatively analyzed with GloSensor in the control and ARRB1/2-KD cells.Forskolin and PBS were used as positive and negative controls,respectively.After 2 hours' treatment,the change of cAMP levels of ARRB2-KD cells were obviously higher than that of ARRB1-KD cells and the control cells.In consequences,it's supposed that ARRB2 deficiency increased cAMP level,which was regarded as a convictive "readout" of TSH-TSHR-PKA-cAMP effect.(3)The regulation of the expression of cholesterol metabolism enzymes by ARRBs.In normal HepG2 cells,TSH stimulation could up-regulate the protein level of HMGCR,while the level of CYP7A1 was reduced.In ARRB1/2-KD cells,the effect of TSH to HMGCR was not obviously influenced by ARRBs-deficiencies.However,the protein level of CYP7A1 tended to be higher in ARRB1-KD cells than in ARRB2-KD and wild-type counterparts after 24 h TSH treatment.This result means the inhibitory effects of TSH on CYP7A1 were weakened in ARRB1-KD cells,but remained unchanged in ARRB2-KD cells,which indicated that ARRB1 may play more important roles in TSH regulation of cholesterol conversion.4.ARRBs-mediated signaling pathways related to cholesterol metabolism during TSH stimulation was regulated by AKTser308 phosphorylation.Based on all above phenomenon,we further studied the regulation of ARRBs-mediated TSH-TSHR signaling pathway.In the control cells,the phosphorylation levels of Thr308 and Ser473 of AKT were dramatically increased within 60 minutes of stimulation with 4 ?M TSH.In ARRB1/2-KD cells,AKT Ser473 phosphorylation levels were not dramatically different from those in the control cells,but the level of AKT Thr 308 phosphorylation was reduced,especially in ARRB1-KD cells.ARRB1/2-KD cells along with the control cells were treated with the AKT activator SC79.The result turned out that compared to TSH,ARRB1-KD cells treated with SC79 showed a decreased level of CYP7A1.It is suggested that the application of AKT agonists can reverse the changes in the signaling pathways caused by ARRB1 deletion.ConclusionsARRB1 was involved in TSH-regulated cholesterol metabolism through AKT pathway.TSH increased ARRB1 expression,enhanced AKT phosphorylation,up-regulated SREBP2 levels,inhibited CYP7A1 expression,thereby reducing the conversion of cholesterol to bile acids.