Study On The Roles Of Lipinl In The Regulation Of Cell Metabolism

As an important member of lipins family member,lipinl exerts dual functions as a phosphatidate phosphatase enzyme and a co-transcriptional regulator in lipid metabolism.In fact,it is also involved in many other cell processes.However its regulation mechanism is unknown.In this study,a lipinl overexpression hepatoma BEL7402 stable cell line was constructed.We found that lipinl overexpression promoted the expression of PPARa and SREBP1，leading to the reduction in triglycerides synthesis.Meanwhile,lipinl overexpression could induce cell cycle G0/G1 phase arrest and slow down hepatoma cancer cell proliferation.To demonstrate the possible mechanism by which lipinl regulated cellular processes involved in cell cycle,a proteomic technique,two-dimensional polyacrylamide gel electrophoresis(2D-PAGE)and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF/TOF-MS),was used to analyze the effect of lipinl overexpression on cell proteome expression.Seven proteins were found to be differential in lipinl overexpression stable cells compared to the control(cells stablely transfected with empty vector pcDNA3.1<+>).Among the identified proteins,lamin A/C was the most significantly upregulated protein in lipinl overexpression cells.Since previous studies show that the p53-p21 axis is found to be involved in the cell proliferation caused by the inhibition of lamin A/C expression.Here,we detected whether the upregulation of lamin A/C expression caused by lipinl overexpression was also related to the p53-p21 axis.The result of Western blotting substantiated that lipinl induced the upregulation of p53 tumor suppressor and its downstream target protein,cyclin inhibitors p21 and p27.In contrary,after lipinl was knocked down with siRNA,p53 and p21 was down regulated and the cell proliferation was increased accompanied with the decrease in the expression of lamin A/C.Thus,these data demonstrate that lipinl induced cell cycle arrest and inhibited proliferation of hepatoma cells through lamin A/C-p53-p21 signaling pathway without the involvement of Erk1/2 in the hepatoma cells.Secondly,metal ions play essential roles in various critical biological processes,including proliferation and cell cycle.Previous studies indicate that the levels of cellular iron regulate cell cycle through p53-p21-p27-mediated signaling pathways.In this study,we detected the contents of eight metal ions(potassium,calcium,sodium,magnesium,manganese,zinc,iron and copper).Our results showed that only content of intracellular iron was significantly decreased by lipin1 overexpression.The levels of mRNAs of iron related proteins suggested that the intracellular iron reduction was positively related to the increase in the expression of ferroportin,iron export protein,in the stable cells overexpressed lipinl.And Western blotting furtherly substantiated that lipinl overexpression up-regulated ferroportin.In addition,after inhibiton of lipinl with lipinl siRNA,we found that intracellular iron content was increased by the decrease in the expression of ferroportin.Meanwhile,the expression of p53-p21 had also changed during the regulation of iron homeostasis by lipinl.Therefore,our data indicate that lipinl may modulate the cell cycle through p53-p21-p27-mediated signaling pathways by interfering with the expression of ferroportin,reducing iron levels in cells.Our findings further demonstrate that iron reduction could be a potential strategy of cancer prevention and treatment.Finally,for further researching the regulation mechanism of lipin1 in proliferation,protein-protein interaction networks of lipinl were unreaveled by utilizing pull down assay coupled with mass spectrometry(MS)in 293T human emborynic kidney cells with transient transfection of plasmid pcDNA3.1/myc-HisA-lipin1.Pull-down assay on the Ni2+-chelating column was used to isolate lipinl complexes from 293T cells with 6-His tagged lipinl.The lipinl complexes were analyzed on Q Exactive mass spectrometer.A total of 30 proteins were identified from label free quantitation of the MS data by Proteome Discoverer Platform,which could not be found from MS data of control(cells transfected with empty vector).Among these interacting proteins,the physical interaction between lipinl and eEFlAl was further affirmed in 293T cells transfected with 6-His tagged lipinl and hepatocyte SMMC7721 cells by protein immunoprecipitation and immunofluorescence microscopy.Lipinl also interacted with HIST1H2BK,which was confirmed in SMMC7721 cells by protein immunoprecipitation.Meanwhile,proteomic change was investigated in 293T cells overexpressed lipinl by 2D-PAGE with MALDI-TOF/TOF MS,and expressions of eleven proteins were found to be differential.Among them,three proteins(eEF-1 Bγ,CCT1 and CCT3)were up-regulated and other eight proteins(NDKA,Stathmin,HNRNP A1,TK,KRT1,PKM,RanBP1 and LDHB)were down-regulated.Western blotting was used to affirm the expression of RanBPl in 293T cells transiently overexpressed lipinl.The bioinformatic analysis showed that these proteins were relevant to cell part,metabolism process,catalytic activity and chaperone.The results highlight the multi-potential roles of lipinl involved in many cellular metabolism processes。Additionally,in order to explore the possibility of lipinl as a potential drug target for the treatment of nonalcoholic fatty liver disease(NAFLD),we investigated disturbance effects of some drugs on NAFLD and biological role of lipinl.The human hepatocarcinoma SMMC-7721 cells were treated with oleic acid to sucessfully establish an in vitro model of NAFLD.Interestingly,artemisinin,a kind of sesquiterpene lactone endoperoxide isolated from Artemisia annua.,which has been used to effectively treat different forms of malaria,could reduce intracellular lipid accumulation and triacylglycerol concentration in our cellular model of NAFLD.Effects of artemisinin on expression of lipogenic and lipolytic genes in SMMC-7721 cells were analyzed by RT-PCR,real time RT-PCR and Western blotting.The addition of artemisnin induced a decrease in the mRNA and protein level of CD36,while it had no effect on both mRNA and protein level of lipinl,PPARa and SREBP1.Artemisinin can reduce hepatocellular lipid accumulation and triacylglycerol content via the inhibition of CD36 expression in the in vitro model of NAFLD.Unfortunately,no lipinl targeted compounds have been screened yet.In summary,our data substantiate that besides its regulation of lipid metabolism,lipinl could also regulate cell proliferation through lamin A/C-p53-p21-mediated signaling patyway in the hepatic carcinoma cells.Meanwhile,our findings for the first time show that lipinl participated in the regulation of iron homeostasis in human hepatic carcinoma cells.Nevertheless a few tumor types are deficient in lipinl and some of which do express lipinl,further studies need to be done to explore the direct relationship between lipinl and intracellular iron level in different types of cancer cells.It is critical for the significant therapeutic implications of lipinl overexpression to inhibit the development of cancer.Furthemore we reported 30 lipinl interacting partners by using His pull down assay with nano-LC/ESI/MS/MS methodology,which will lay a good foundation for further study of cell function of lipin1.Our studies will help to improve the understanding of the cellular metabolic regulation of lipinl and be expected to broaden new insights into the novel functions of lipinl.