Identification Of The Functional Domain Of ZHX2 That Inhibits LPL Expression

ZHX2(zinc fingers and homoboxes2),along with ZHX1 and ZHX3,belongs to zinc finger and homoboxes family.ZHX2 regulates diversity of life processes as a transcription repressor. Recently, ZHX2 is identified as a new tumor suppressor gene for its down regulation in many malignant tumors, such as hepatocellular carcinoma, lung cancer and Hodgkin lymphoma and for its role in arresting cell cycle and inhibiting cell proliferation. In addition,ZHX2 inhibits the expression of tumor markers of hepatocellular carcinoma (AFP, GPC3) in transcriptional level. These data proved that ZHX2 plays an important role in the development of tumor development and progression and provided a new candidate for diagnosis and prognosisof liver cancer.Balb/cJ mice express a lower level of ZHX2 in liver than other mice with similar genetic background. More importantly, Balb/cJ mice are resistent to hyperlipidemia when fed with high fat diet. According to the quantitative trait loci mapping identification,ZHX2 gene is found to be associated with this phenotype. Interestingly, LPL,a well-known lipid metabolism related gene,had a relatively high expression level in Balb/cJ mice.this may explains the phenotype descriped above owing to Balb/cJ mice’s higher expression level of LPL in liver which take up more serum TGthan other mice strain. LPL isa rate-limiting enzyme to hydrolysis triglycerides in vivo. LPL (lipoprotein lipase), releases FFAs from CM,VLDL(which are riched in TG) circulating in blood to supply energy needed organs like brain, cardiac muscle or simply transports FFAs to adipose tissue. LPL forms homodimer to anchor on the surface of endothelial cells through the heparin binding site. Similar to AFP, LPL highly expresses in fetal liver, while extinguishes in the adult liver. According to the aforementioned data, We hypothesized that there might be a regulatory relationship between ZHX2 and LPL. Our previous work showed that, the full-length ZHX2 bound to LPL promoter and inhibited LPL expression at the transcriptional level. Since ZHX2 is a protein of multi domains with total length of 837 amino acids,containing two zinc finger domains and five homodomains, a proline-rich region lies between Homology domain 1 and homology domain 2,we aims to further clarify the functional domain of ZHX2 that inhibits LPL expression. Hence, we constructed the different truncated ZHX2 expression plasmids and studiedtheir inhibitory effects on the expression of LPL.Objectives1. To construct over-expression plasmids which contains different functional domains of ZHX2, then to transfect them in cultured cells to screen the functional domain that inhibits the expression of LPL.2.To detect the effect of full-length or truncated ZHX2 protein identified above on LPL core promoter activity to further verify the transcription inhibitory domain of ZHX2 that regulates LPL expressionMethods:1. Construction of truncated ZHX2 expression vectorAccording to the existing reports, ZHX2 homeodomain 4,5 did not show obvious transcription inhibitory function. Our laboratory already established three truncated ZHX2 plasmids:ZHX2(1-501aa) containing zinc finger domain 1 to homology domain 2, ZHX2(242-501aa) containing homology domain 1 to homology domain 2, ZHX2(242-338aa) containing homology domain 1 alone. Here, another two truncated ZHX2fragments, ZHX2(242-446aa) containing homology domain 1 to proline-rich region with nuclear localization sequence and ZHX2(242-439aa) containing homology domain 1 to partial proline-rich region without nuclear localization sequence plasmids, were amplified with full-length ZHX2 plamids as PCR template. Then, PCR products were inserted into pcDNA3. Double restriction enzyme digestion and DNA sequencing were performed for verification of the constructs.2. Screening of the function domain of ZHX2 that inhibits the expression of LPLHepatocellular carcinoma cell line SMMC7721 and HepG2 were transfected with full length or various truncated ZHX2plasmids. RNA were harvested after 48 hours. RT-PCR were performed to detect the expression level of LPL.3. Effects of function domain of ZHX2 on the activity of LPL core promoterHEK293 cells were co-transfected with the LPL core promoter reporter plasmid and full-length or truncated ZHX2 plasmids, together with pRL-TK plasmid as the transfection efficiency control.48 hours after transfection, celllysis was collected and Dual luciferase report assay wasperformed to measure LPL core promoter activity.Results1. The construction and validation of truncated ZHX2 expression plasmidsAfter PCR amplification, DNA fragment for ZHX2(242-446aa) or ZHX2(242-439aa) was digested with EcoR I and Kpn I and then inserted into pcDNA3 plasmids. Double restriction enzyme digestion and DNA sequencing showed the constructs containing the exact truncated ZHX2 fragments.2. ZHX2 (242-446aa) is the function domain that inhibits LPL expressionHepatocellular carcinoma cell line SMMC7721 and HepG2 were transfected with full-length and truncated ZHX2 plasmids encoding different function domain. RT-PCR results showed that, compared with pcDNA3 control group, full length ZHX2, ZHX2(1-501aa), ZHX2(242-501aa), and ZHX2(242-446aa) had inhibitory effect on LPL expression, while ZHX2 (242-439aa) and ZHX2(242-338aa) lost the suppression ability. Therefore, ZHX2(242-446aa) is the functional domain transcriptionally inhibiting LPL expression.3. ZHX2(242-446aa) inhibits the LPL core promoter activityHEK293 cells were co-transfected with ZHX2 full-length or ZHX2(242-446aa) expression plasmid with LPL core promoter(-197-+96) reporter plasmid.Dual luciferasereport assay were performed to measure the promoter activity. Results showed that, compared with the control group, ZHX2(242-446aa) showed the repression effect of the LPL core promoter activity, further confirming the transcriptional inhibition of ZHX2 on LPL expression.Conclusions and significance:1. ZHX2 inhibits the expression of LPL at the transcriptional level. The function domain of ZHX2 inhibiting LPL expression is 242-446aa.2. LPL is the rate-limiting enzyme that hydrolyzes triglyceride and is related with lipid metabolism diseases such as diabetes and atherosclerosis. This study aims to give a further insight into regulation relationship between ZHX2 and LPL, providing new understanding of the pathogenesis of lipid metabolism related disease and new therapeutic targets for clinical treatment of obesity, atherosclerosis and diabetes.ZHX2 is widely expressed in various organs and tissues. Northern blot results showed that, ZHX2 mRNA existd in a majority of body systems, including the immune system.ZHX2 mRNA was detected in peripheral blood lymphocytes, liver, spleen and thymus. Furthermore, it has been showed that ZHX2 is involved in many biological processes such as cell cycle progression, organ and tissue development. During cerebral cortex development, ZHX2 is expressed in neural progenitor cells and interacts with Ephrin B1 to maintain the phenotype of neural progenitor cell. ZHX2 expression is down regulated along with erythroid differentiation process and increases gradually in the development of B cells. In the kidney, the ZHX protein family regulates gene expression profile that controls podocyte function. On the other way, ZHX2 and ZHX1, acting as transcription factors, can change the expression levels of primary glomerular disease related genes. However, the effects of ZHX2 on the immune system and inflammatory related diseases havenot been reported yet.Liver is the largest solid organ in body adjacent to spleen and small intestine. Blood flow through the portal vein and hepatic artery brings a variety of antigens derived from self-apoptotic cells or intestinal bacteria into the liver. In addition to the parenchyma cell, liver contains a large number of non parenchymal cells, most of which are immune cells, such as T cells, NK cells, DC cells and Kupffer cells. With such a complex immune microenvironment, liver is also defined as the largest immune organ. Kupffer cells are resident macrophages in the liver, accounts for 80%～90%of the total body macrophage pool. Macrophages secrete large number of pro-inflammatory cytokines and chemokines during inflammation and recruit other immune cells. Kupffer cells also express co-stimulatory molecules on the surfaces as APC to activate other immune cells, taking an important part in immune system to defend against foreign invasion.Viral infections, autoimmune diseases, alcohol and some drugs can cause acute or chronic hepatitis. Persistent inflammation may lead to liver cirrhosis and hepatocellular carcinoma. ConA or LPS/GalN induced acute hepatitis in mice are models used to simulate the progression of human liver injury. In the present work, we focus on the ConA induced hepatitis mouse model to study the possible role of ZHX2 in liver injury.Objectives1. To detect the changes of ZHX2 expression in the liver of acute hepatitis mice and to study the possible role of ZHX2 in acute hepatitis.2. To detect the ZHX2 expression in macrophages and to explore its role in regulating macrophage activation.Methods1. Changes of ZHX2 expression in the liver of acute hepatitis mice1.1 The establishment of ConA induced acute hepatitis mouse model6-8 week male C57BL/6 mice were divided into two groups,4 mice per group.The experimental group was induced for acute hepatitis by a single dose of ConA (20mg/kg) via intravenous injection. The control group was injected with PBS instead.8h later, the mice were sacrificed and serum was collected to detect ALT level. Liver tissue was freezing in -80℃ for RNA extraction to detect ZHX2 expression.1.2 The establishment of LPS/GalN induced acute hepatitis mouse model6-8 week male C57BL/6 mice were intraperitoneally injected with LPS/GalN (LPS 50 ug/kg, D-GalN 800mg/kg) to induce acute hepatitis in mice. The control group of mice was intraperitoneally injected with an equal volume of PBS.5h later, the mice were sacrificed and serum was collected to detect ALT level. Liver tissue was freezing in -80℃ for RNA extraction to detect ZHX2 expression.2. The effect of ZHX2 on ConA induced hepatitis in vivoIn order to avoid hydro-dynamic tail vein injection caused liver injury, we used in vivo transfection reagent PEI to achieve ZHX2 over-expression in liver.6-8 week male C57BL/6 mice were injected with PEI-plasmids mixture (350ul/mice) intravenously. Control group was using pcDNA3 plasmids instead.48h after transfection, 10mg/kg ConA was injected to mice via tail vein to induce acute hepatitis.6h after injection, mice were sacrificed and serum was collected to detect ALT level. Part of liver tissue was fixed in 4% paraformaldehyde to prepare paraffin sections. For further observation of hepatocyte cell death and infiltration of inflammatory cells, HE staining and TUNEL staining were performed.Liver samples were saved and frozen at -80℃ for RNA and protein extraction. RT-PCR and Western blot were performed to detect ZHX2 over-expression efficiency and the expression of cytokines and inflammatory-related molecules. The remaining parts of the livers were used for isolating liver mononuclear cells.The isolated cells were dissolved in TRIzol reagent for further RNA extraction.3. The expression of ZHX2 in macrophages and its role in regulating macrophage activation3.1 Effects of ConA and LPS on ZHX2 expression in macrophagesStarch induced peritoneal macrophages were stimulated with a series doses of ConA or LPS or a single dose of stimulation at different time points.Total RNA was extracted to perform RT-PCR. Expressions of ZHX2 and pro-inflammatory cytokines were detected.3.2 The effect of ZHX2 on the expression of inflammatory cytokines in macrophagesIn order to further clarify the regulatory role of ZHX2 in macrophage activation, we utilized the starch induced peritoneal macrophages or bone marrow-derived macrophages and then siRNA specifically against ZHX2 were transfected for 48 hours before LPS stimulation at different dosages, NC siRNA was used as negative control. Total RNA was extracted to perform RT-PCR. Expressions of ZHX2 knockdown efficiency and pro-inflammatory cytokines IL-6, TNF-a and iNOS were detected.Results1. ZHX2 aggravated the liver injury in ConA induced hepatitis1.1 ZHX2 was up-regulated in the liver of ConA induced hepatitisC57BL/6 mice were treated with ConA to induce acute hepatitis.8h after the injection, mice were sacrificed.Results of serum ALT level showed that, compared with PBS control group, serum ALT level from ConA treated mice increased significantly, indicating the hepatitis model was successfully induced. Expression of ZHX2 in liver tissues was detected using RT-PCR. The results showed that, compared with PBS control group, ZHX2 expression level increased significantly in the liver of ConA treated group.1.2 ZHX2 was up-regulated in liver of LPS/GalN induced hepatitisC57BL/6 mice were intraperitoneally injected with LPS/GalN to induce acute hepatitis,5h after treatment, mice were sacrificed and the detection.Results of serum ALT level showed that, compared with PBS control group, serum ALT level from LPS/GalN treated mice increased significantly, indicating the hepatitis model was successfully induced. Expression of ZHX2 in liver tissues was detected using RT-PCR. The results showed that, compared with PBS control group, ZHX2 expression level increased significantly in LPS/GalN treated group.2. Over-expression of ZHX2 in vivo exacerbated ConA induced liver injuryPEI carriedwas used for transfecting ZHX2 over-expression plasmids to over-express ZHX2 into C57BL/6 mice via tail vein injection.48h later,lOmg/kg ConA was given to induce acute hepatitis. The mice were sacrificed 6 h after injectionand blood was collected to measure the level of serum ALT. The results showed that serum ALT level of mice over-expressing ZHX2 was higher than that of pcDNA3 group, indicating that over-expression of ZHX2 in vivo might exacerbate liver injury. HE staining of liver tissue sections showed that, compared with pcDNA3 control group, liver tissue from ZHX2 over-expression groups presented a larger number of degeneration and necrosis of hepatocytes, more severe tissue hyperemia and infiltration of inflammatory cells. Results of TUNEL staining manifested that ZHX2 over-expression mice group had moreTUNEL positive staining cells than pcDNA3 group.RNA was extracted from hepatocytes and liver mononuclear cells. RT-PCR results showed that over-expression of ZHX2 in mice was successful and the expressions of the pro-inflammatory cytokines like IFN-γ, TNF-α, IL-1β in ZHX2-overexpressed mice were higher than that in pcDNA3 group. Taken together, these results suggested that, over expression of ZHX2 in vivo could aggravate the liver injury and the secretion of inflammatory cytokines induced by ConA.3.ZHX2 expression in macrophages and its role in regulating macrophage activation3.1 LPS and ConA stimulation up-regulated ZHX2 expression in peritoneal macrophagesStarch induced peritoneal macrophages were stimulated with a series doses of ConA or LPS or a single dose of stimulation at different time points.Total RNA was extracted to perform RT-PCR. The results showed that, ConA up-regulated the expression of ZHX2 in starch induced peritoneal macrophages,while the mRNA level of inflammatory factors IL-1β, IL-6 were not significantly altered. However, after LPS stimulation, ZHX2 expression was up-regulated accompanied by increased mRNA level of inflammatory factors IL-1β and IL-6.3.2 ZHX2 knockdown inhibitedthe secretion of inflammatory cytokines in macrophageStarch induced peritoneal macrophages or bone marrow-derived macrophages were transfected with ZHX2 specific siRNA and 48 hours later, different dosage of LPS was administrated. NC siRNA were used as negative control. The RT-PCR results showed that after knockdown of ZHX2, the mRNA level of inflammatory cytokines TNF-a, IL-6 and iNOS were lower than that of NC group cells.These results preliminarily suggested that ZHX2 could promote the secretion of inflammatory cytokines after macrophage activation.Conclusion and significance1. The role of ZHX2 in inflammatory diseases has not been reported, this is the first study to demonstrate the over-expression of ZHX2 in vivo can aggravate the liver injury induced by ConA, providing new evidence for functional study of ZHX2.2 It is the first study of ZHX2 in modulating macrophage activation which indicates that knock down of ZHX2 could affect the secretion of inflammatory cytokines by macrophages,providing novel datafor the elucidation of mechanisms involved in macrophage activation and the related inflammatory disease.