A Preliminary Study On The Expression And Distribution Of FAM76B In Various Tissues Of Mice And The Effect Of FAM76B Gene Knockout On Liver Lipid Metabolism In Mice

FAM76B is a novel nuclear localized protein and consists of 339 amino acids which encode a 39kDa protein.Human FAM76B gene in genomic is located on 11q21,which contains 21,468 bases.The function of FAM76B is unknown.By the analysis of the FAM76B proteins of human,rat,mouse and zebrafish,a sequence alignment of these proteins was found to have high degree of conservation,which suggested the important role of FAM76B.FAM76B is the significant number of poly His-containing protein and has an ubiquitination site at Lys225.Among proteins containing His-repeats with GO(Gene Ontology terms)annotations,there was a strong over-representation of nuclear proteins related to transcriptional regulation and nervous system development.FAM76B accumulates in nuclear speckles.The nuclear speckles are subnuclear structures defined as compartments in which components of the RNA splicing machinery are stored and assembled.FAM76B is novel PGRN-interacting protein identified from two yeast twohybrid screening and confirmed by GST-pull down and coimmunoprecipitation(Co-IP)in our previous study.PGRN as a multifunctional growth factor widely expressed in a variety of tissues is involved in many important physiological events,such as early embryogenesis,wound repair and nervous system development.Furthermore,PGRN plays an important role in the tumorigenesis,neurodegenerative diseases,type 2 diabetes,nonalcoholic fatty liver disease and inmflammatory.Although FAM76B interacts with PGRN,it remains unclear so far whether FAM76B involves in the developmemt of the related disease mentioned above.In this study,murine monoclonal antibodies(MAbs)against human FAM76B were generated by using purified prokaryotic recombinant human FAM76B protein.Due to the highly conserved protein sequences between mouse and human FAM76B,MAbs against human FAM76B were shown to react with mouse FAM76B.However,little is known of the role of FAM76B gene products in vivo.To understand the physiological role of FAM76B,We examined gene expression and distribution of FAM76B in normal mouse tissues.We generated of FAM76B knockout mice and the contributed phenotyping of FAM76B knockout mice will be explored.In addition,the mechanisms involved in the diseases casused by the deficient in FAM76B gene will lay a foundation for investigating the function of FAM76B protein.The contents of the project will include the following parts.1.Production and characterization of monoclonal antibodies against a novel human nuclear protein FAM76B.Six 5-week-old female Balb/c mice were immunized with FAM76B-6His three times at 3-week intervals.The fusion of SP2/0 myeloma cells with spleen cells isolated from immunized Balb/c mouse was carried out using standard methodology.About 5 days later,the supernatants from the 96-well plates were screened by ELISA using FAM76B-His as coating antigen for specific antibody-secreting clones.The hybridoma cells of positive wells were cloned at least three times by limiting dilution in aminopterin-free selection medium and were used for producing antibody-rich fluid called ascites fluid by inoculating enlarged hybridoma cell lines into the peritoneal cavity of Balb/c mice.MAbs specific for human FAM76B were obtained and characterized by using Western blot,immunoprecipitation(IP)and immunocytochemistry(ICH)applications.2.Gene expression and distribution of FAM76B in normal mouse tissues.Analyses of the mouse FAM76B and human FAM76B pair of protein,a sequence alignment of these two proteins highlighted their high degree of conservation.MAbs specific for human FAM76B were characterized by using Western blot and immunocytochemistry tested against mouse FAM76B protein.Then we examined FAM76B gene expression and distribution of FAM76B in normal mouse tissues by Real-time PCR and immunohistochemistry.3.Generation of FAM76B knockout mice.The function of the FAM76B is unknown.Accordingly,we chose to make FAM76B knockout mice cooperated with the company.Genomic DNA of offspring was confirmed by PCR.FAM76B expression monitored by Real-time PCR compared with GAPDH as a control.Western blotting using MAbs against human FAM76B confirmed that mouse FAM76B protein in MEF cells from different genotype mice.4.Phenotype of FAM76B knockout mice.The genotype ratio and the differences by sex were determined in offspring.The weights of mice tissues and the length of mice were determined in offspring.The development of mice was monitored.5.Histopathological features of liver and fat in FAM76B-deficient mice.Liver of FAM76B-deficient mice were stained with H&E and Oil Red O at different ages.At the same time,WAT adipocytes were stained with H&E.6.Biochemical Analysis in FAM76B-deficient mice.Plasma TQ TC,HDL,LDL,aspartate aminotransferase(AST),and alanine aminotransferase(ALT)levels were measured using amodel AU480 apparatus.7.Real-time PCR Analysis gene expression of lipogenesis in FAM76B-deficient mice.Gene expression of lipogenesis monitored by Real-time PCR compared with GAPDH as a control at 1,3,6,9,12 and 15 month,such as FASN,SCD,ACC,Dgat land Dgat 2 in de novo lipogenesis;Cptl,Acox,Acadv and Mcad in fatty acid beta oxidation;FABP and Apoa4 in uptake of free fatty acids into the liver;and Mtp,Vldlr in TG secretion.Lipin 1,SREBP-1,PPARa and PGC-la expression monitored by Real-time PCR compared with GAPDH as a control at 1,3,6,9,12 and 15 month.8.The expression of F4/80,TNFa and PGEN in FAM76B+/-mice livers.The expression of F4/80,TNFa and PGRN were monitored by Real-time PCR compared with GAPDH as a control at 1,3,6,9,12 and 15 month.Some results were obtained from the the project as follows:1.Production and characterization of monoclonal antibodies against a novel human nuclear protein FAM76BThe characterization of the six monoclonal antibodies investigated,mAbs No.1,No.2 and No.5 seem to have the highest affinities for human FAM76B and were effective in all assays tested.The study should provide a useful tool for investigating the biological function of FAM76B.In order to identify the epitope mapping,the regions of the FAM76B were determined by each mAb.Plasmids encoding FAM76B truncation mutants representing different regions of the protein were expressed in Escherichia coli BL21(DE3).They were separated by SDS-PAGE and analyzed by Western blot to test the FAM76B MAbs.Antibodies No.3 and No.6 reacted with epitopes in aa97-163.No.2 epitope in aa187-262 and No.5 might recognize aa263-265.No.1 reacted with aa266-339.HEK 293 cells were transfected with the vectors expressed the FAM76B truncation mutants were tested by different FAM76B MAbs immunohistochemically,which showed that nuclear localization signal was in aa187-265,C-terminal polyhistidine repeat domain.2.Gene expression and distribution of FAM76B in normal mouse tissues.MAbs No.1,No.2 and No.5 seemed to detect mouse FAM76B usefully and were effective in Western blot and immunocytochemistry tested.We examined FAM76B gene expression in mice by Real-time PCR and immunohistochemistry.Brain,liver and spleen expressed FAM76B at high levels,especially in brain,3.Generation of FAM76B knockout miceGenomic DNA of heterozygous,homozygous mice and wild type mice were confirmed by PCR.FAM76B expression was monitored by Real-time PCR.FAM76B mRNA levels in the MEF were undetectable from homozygous mice.Real-time PCR likewise detected MEF from heterozygous mice.About half of messenger for FAM76B compared to WT.Western blotting using MAbs against human FAM76B confirmed that FAM76B protein was markedly diminished in the MEF from homozygous mice.Protein contents of MEF from heterozygous mice were shows?50%reduction.These experiments demonstrate that FAM76B knockout mice had been successful.4.Phenotype of FAM76B knockout miceHowever,no significant differences by physical exanination could be detected.Homozygotes generated from offspring survived at a significantly lower frequency than the expected 1:2:1 Mendelian ratio(39.5%+/+,51.5%+/-,9%-/-).This data indicates that FAM76B-deficient mice have unkown lethal problems between the embryonic stage and weaning,caused the prenatal or postnatal death.Body weight was not significantly different between FAM76B+/-mice and WT animals when the feeding trials started.When maintained on for 5 months,body weight changes did not differ significantly between FAM76B+/-and WT mice.Until 8 months,FAM76B+/-mice showed higher body weight,but showed no significantly difference compared with WT mice.After 9 months,FAM76B+/-mice showed a significantly higher body weight compared with WT mice.This effect reached statistical significance from month 9 to month 15.Liver weight and fat mass had a similar trend during the feeding trials.In this study,we demonstrated that ablation of FAM76B induced obesity in mice.5.Histopathological features of liver and fat in FAM76B knockout miceHistological examination of livers from FAM76B knockout mice and WT mice demonstrated the progressive development of substantial steatosis and inflammatory reaction.Until 3 months,livers from FAM76B+/-mice demonstrated no evidence of steatosis at any time.At 4 months and 5 months,FAM76B+/-mice had substantially less steatosis.Small fat droplets were presented in hepatocytes at 6 months and 8 months,but these did not fill the cytoplasm.The amount of fat and size of the droplets increased in hepatocytes over time such that by 9 and 15months.Also by 9 months,small-droplet fat accrued in perivenous hepatocytes,and this was more pronounced at 15 months.Inflammatory foci were scattered across the lobule was seen at 15 months.The NAFLD activity score(NAS)was used to assess in FAM76B+/-mice and WT mice.FAM76B+/-mice evaluated nonalcoholic fatty liver disease activity score 1 at 8 months.and 9 months.FAM76B+/-mice classified nonalcoholic fatty liver disease activity score 2 at 15 months.Histological examination of livers from FAM76B mice demonstrated the progressive development of substantial steatosis.FAM76B deficient induces NAFLD in miceOil Red O staining of liver sections confirms raise of steatosis in FAM76B+/-mice.Until 3 months,livers from FAM76B+/-mice demonstrated no evidence of lipid droplets(LDs)at any time.At 4 months and 5 months,there were some FAM76B+/-mice had substantially less LDs.The diameter and density of the LDs were smaller.The amount of large LDs increased in hepatocytes at 6 months and 8 months.Large LDs were more marked at 9 and 15months.WAT adipocytes were stained with H&E.The areas of white adipocytes were in accordance with the morphological observations.Compared with WT mice,the areas of the white adipocytes in FAM76B+/-mice showed no significantly difference until 3 months.The areas of the white adipocytes in FAM76B+/-mice were increased from 4 months to 7 months,but had no significantly difference.After 8 months,the areas of the white adipocytes in FAM76B+/-mice were significantly increased6.Biochemical Analysis in FAM76B-deficient miceThe serum TG levels were significantly increased in FAM76B-deficient mice at 1 and 3months compared with WT mice.FAM76B-deficient mice display elevated levels of plasma CHOL at 1,3,9 and 12 months.HDL levels were not significantly increased in FAM76B-deficient mice except for 12 months.However,the FAM76B-deficient mice did not display significantly increased LDL levels.AST and ALT levels in FAM76B+/-mice showed no significantly difference compared with WT mice,except for ALT levels were significantly increased at 15 months.The above results suggested FAM76B deletion showed no noteworthy impacts on the lipid plasma level and induced liver injury in mice at 15 months.7.Real-time PCR Analysis gene expression of lipogenesis in FAM76B-deficient mice.Indeed,we found that the expression of Fasn,Scdl and vldlr were significantly induced in the livers of FAM76B+/-mice.Accumulation of lipid in the liver of FAM76B+/-mice can be traced by the increased incidence of de novo lipogenesis and the decreased of TG secretion.Accumulation of lipid in the liver resulted in the progression of non-alcoholic fatty liver disease(NAFLD).The expression of Srebpl was significantly induced in the livers of FAM76B+/-mice,consistenting with its lipogenic targets,Fasn and Scd1.We considered the possibility that FAM76B+/-mice livers might have an elavation in SREBPlc induction that could account for their increased TG levels by induced the fatty acid biosynthesis.On the other hand,the expression of lipinl and PGC-la were significantly reduced in the livers of FAM76B+/-mice.The studies have demonstrated that enhanced expression of lipinl or PGC-1? stimulated VLDL secretion.We considered the possibility that FAM76B+/-mice livers might have a defect in lipinl and PGC-la induction that could account for their increased TG levels by reduced TG secretion.The expression of PPARa did not display significantly decreased in the FAM76B+/-mice.FAM76B+/-mice livers had no effect on the PGC-la/PPAR?/lipinl Pathway leading to decreased Fatty Acid Oxidation,consistenting with the expression of Cpt1,Acox,Acadv and Mead in Fatty acid oxidative pathways.8.The expression of F4/80,TNFaand PGRN in FAM76B+/-mice liversLiver F4/80 and TNFa mRNA levels did not display significantly decreased in the FAM76B+/-mice until 3 months.After 6 months,the expression of F4/80 and TNFa were significantly induced in the livers of FAM76B+/-mice,especially fold increased over WT mice at 15 months,consistenting with slight lobular inflammation.F4/80 is the phenotypic characterization of mouse tissue macrophages.TNFa is proinflammatory cytokines.At this time point,NASH in FAM76B+/-mice was characterized and by marked upregulation in liver levels of TNFa,consistenting with progressive non-alcoholic fatty liver disease.However,it suggests that FAM76B play a suppressor in liver.The expression of PGRN was significantly induced in the livers of FAM76B+/-mice at 15 months,consistenting with levels of TNFa and the progressive non-alcoholic fatty liver disease.It suggested FAM76B interacted with PGRN in inflammatory of NAFLD.In summary,brain,liver and spleen expressed FAM76B at high levels,especially brain.We examined abnormal hepatic lipid metabolism and inflammatory in FAM76B+/-mice.We delineated the molecular mechanisms for lipid accumulation in the liver of FAM76B+/-mice as increased the fatty acid biosynthesis and reduced VLDL secretion.The FAM76B+/-mice livers might have elavations in SREBP1,FASN and SCD induction that could account for their increased TG levels by induced the fatty acid biosynthesis.FAM76B+/-mice livers might have a defect in lipinl and PGC-la induction that could account for their increased TG levels by reduced TG secretion.On the other hand,the expression of TNFa and PGRN were significantly induced in the livers of FAM76B+/-mice.Inflammatory was upregulation in liver levels of TNFa.NAFLD was stemmed from induced the fatty acid biosynthesis,reduced TG secretion and inflammatory.All the findings above will lay a basis for the elucidation of FAM76B biological functions,its molecular mechanisms in nonalcoholic fatty liver disease and FAM76B interacted with PGRN in NAFLD.