Experimental Study On Syndecan-1 Shedding Mediated VLDL Binding And Uptake To Human Hepatocytes

Thrombotic Triglyceride-rich lipoproteins (TRLs) include chylomicrons, very low density lipoproteins (VLDL) and their remnants. Their abnormal accumulation in the circulation results in hypertriglyceridemia. Hypertriglyceridemia is a common disorder that increases the risk of atherosclerosis and coronary artery disease. The body clears these remnant lipoproteins from the circulation via endocytic receptors in the liver, including the low density lipoprotein receptors (LDLR), the LDLR-related proteins (LRPs), lipolysis stimulated receptors (LSR), and one or more heparan sulfate proteoglycans (HSPGs). Syndecan-1 is type I transmembrane proteoglycans bearing three heparan sulfate chains on its core protein. Our previous work on lipoprotein clearance shows that syndecan-1 is the primary heparan sulfate proteoglycans mediating hepatic clearance of triglyceride-rich lipoproteins in mice. However, the function of syndecan-1 on lipoprotein clearance in human hepatocytes is less clear. In addition, syndecan-1 can be shed from many culture cells spontaneously. Many inducers including growth factors, bacterial virulence factors can accelerate syndcan-1 shedding from cell surface. Metalloproteinases mediate the cleavage of syndecan-1 on cell surface. Although the exact biological significance of syndecan-1 shedding is unclear in many conditions, it regulates the chemokine activity during wound healing and bacterial infection. The biological function of syndecan-1 shedding lipoprotein clearance is unknown.Here, we extended our syndecan-1 and lipoprotein research to human hepatocytes, including Hep3B cells, HepG2 cells, and primary normal human hepatocytes. We examined the activity of syndcan-1 in VLDL binding and uptake to human cell, the mechanism of syndecan-1 shedding from human hepatocytes and its role on VLDL clearance base on RNA interference, VLDL binding and uptake assay, syndecan-1 shedding assay, and mouse model in vivo assay.1. Expression of Heparan sulfate proteoglycans in human hepatocytes.In order to detect HSPGs expression in Hep3B cells, HepG2 cells, and primary normal human hepatocytes systematically, we examined the mRNA levels of HSPGs through qPCR assay, and protein levels of HSPGs through western blotting. We show those human hepatocytes express multiple HSPGs both in mRNA level and protein level, mainly including syndecan-1, syndecan-2, and syndecan-4.2. Biological function of HSPGs and syndecan-1 in VLDL binding and uptake to human hepatocytes.(1) Human VLDL (δ<1.006 g/ml) was isolated from plasma by buoyant density ultracentrifugation. Detection of apolipoproteins by silver staining shows the isolated VLDL contains apoB100, apoB48, and apoE et al. VLDL was fluorescently labeled by DiD through co-incubation. Labeled DiD-VLDL shows increased fluorescence activity along with increased amount of VLDL particles.(2) Binding and uptake curves of DiD-VLDL to Hep3B cells were measured by incubating cells with indicated concentration of DiD-VLDL for indicated time at 4℃and 37℃, respectively. Uptake of VLDL to Hep3B cells at 37℃.increased with the concentration of VLDL and the incubation time. Binding of VLDL to Hep3B cell at 4℃occurred in a saturable manner both respect to time and concentration. According to those results, we determined to apply 1h incubation with 100μg/ml DiD-VLDL to be the subsequent experimental protocol. Treatment of the cells with a mixture of heparin lyases significantly reduced binding and uptake to Hep3B cells. In the same way, heparin lyases treatment to primary human hepatocytes also reduced VLDL binding and uptake significantly.(3) The role of syndecan-1 in VLDL binding and uptake was examined by knocking down synden-1 expression in Hep3B cells using syndecan-1 specific siRNAs. SiRNAs reduced syndecan-1 expression by 80% in general based on flow cytometry and western blotting. Syndecan-1 knock down reduced VLDL binding to Hep3B cells by 40% and uptake by25%.3. Hepatocytes syndecan-1 shedding and its mechanism(1) Spontaneous syndecan-1 shedding was measured by detecting syndecan-1 ectodomains in 3h, 6h, and 12h conditioned media from Hep3B cells, HepG2 cells, and primary human hepatocytes using dot blotting. Syndecan-1 ectodomains accumulated progressively in the condition media over time in all three cell types. Inclusion of broad spectrum metalloproteinase inhibited spontaneous syndecan-1 shedding. ADAM-17 inhibitor GW280264 blocked syndcan-1 shedding suggests that ADAM-17 may be the key enzyme that mediates spontaneous syndecan-1 shedding. Other metalloproteinases may also contribute to this process due to partly inhibition by GI254023.(2) Syndecan-1 shedding can be accelerated by inducers. PMA, ceramide, and ionomycin et al can induce syndencan-1 shedding from cell surface. We used 0.25μM PMA to stimulate the cells to induce syndecan-1 shedding according to the PMA stimulation time and dose curves. Marimastat and GW280264 blocked PMA induced syndecan-1 shedding, whereas, GI254023 was without effect. PMA also failed to induced syndecan-1 shedding from ADAM-17 silenced Hep3B cell and HepG2 cells. These results indicate that ADAM-17 is the key enzyme that mediates PMA induced syndecan-1 shedding from human hepatocytes.(3) The signaling pathway involved in PMA induced syndecan-1 shedding was determined by different signaling pathway inhibitory assay. PKC signaling pathway inhibitor BIM I profoundly blocked PMA induced syndecan-1 shedding. In contrast, the MEK inhibitor U0216 and the p28 MAPK inhibitor failed to inhibit PMA induced syndecan-1 shedding.4. Effect of syndecan-1 shedding on VLDL binding and uptake to hepatocytes(1) DiD-VLDL was incubated with PMA induced Hep3B cells and primary human hepatocytes to measure the binding and uptake. PMA induced cells showed significant reduced VLDL binding and uptake ability comparing to the DMSO control cells.(2)Theoretically, syndecan-1 ectodomains have the ability to bind VLDL particles due to their covalently attached heparan sulfate chains. In order to test this, we labeled syndecan-1 with 35S and collected the ectodmains after PMA stimulation followed by DEAE purification and concentration. 35S labeled ectodomains were incubated with VLDL at room temperature in iodixanol (δ=1.019) followed by buoyant density ultracentrifugation. Fractions taken sequentially from the top of the tubes were assay for radioactivity and syndecan-1 ectodomains. Around 65% of counts appeared in the top four fractions, whereas in the absence of VLDL, less than 5% of the counts were found in the top 4 fractions. Dot blotting analysis of the pooled top four fractions showed syndecan-1 ectodomains were present in samples containing VLDL. Inclusion of heparin reduced the radioactivity in the top 4 fractions. Removal of syndecan-1 ectodomains from the solution by immunop- recipitation also reduced the counts in the top 4 fractions by 50%.5. LPS induced syndecan-1 shedding in mice and its relationship with plasma triglyceride-rich lipoproteins level(1) Syndecan-1 ectodomains shed from primary mouse hepatocytes spontaneously. However, metalloproteinase inhibitor Marimastat failed to inhibite the shedding. PMA and LPS couldn't induce the syndecan-1 shedding from the primary mouse hepatocytes. (2) Syndecan-1 shedding in vivo could be induced by intraperitoneal injection of LPS (4.5 mg/ kg body weight). Syndecan-1 ectodomains accumulated significantly in the plasma 24 hours after LPS injection based on the dot blotting analysis. Immuno- histochemistry staining also showed the significant syndecan-1 loss in the liver after LPS injection. In the meantime, plasma triglyceride level increased two-fold in the LPS injected mice, whereras total plasma cholesterol lever did not change. Triglyceride level increased further in LDLR-/- mice after LPS injection.(3) Human VLDL clearance in LPS or PBS injected mouse was determined by ELISA assay. Human VLDL was injected to mice via tail vein. Blood samples were collected at different time points. The amount of remaining human apolipoprotein B was determined by ELISA. The LPS injected mice cleared human VLDL particles less extensively comparing to the PBS injected control mice.Taken together, human hepatocytes expressin multiple HSPGs. Syndecan-1 is one of them and can mediate binding and uptake of VLDL to the hepatocytes. Syndecan-1 undergoes spontaneous shedding from hepatocytes through the metalloproteinase proteolysis. PMA induces syndecan-1 shedding from hepatocytes through a PKC dependent activation of ADAM-17. PMA stimulation significantly reduces VLDL binding and uptake to hepatocytes. Shed syndecan-1 ectodomains can bind to VLDL in vitro. LPS injection causes syndecan-1 shedding in the mouse liver and plasma increased triglyceride level. Here, we demonstrate the role of syndecan-1 in VLDL binding and uptake, the mechanism of syndecan-1 shedding inhuman hepatocytes and its effect on VLDL binding and uptake. We analyzed the in vivo syndecan-1 shedding caused by LPS injection and its relationship with increased plasma triglyceride level. We propose that syndecan-1 shedding maybe contribute in part to the Hypertriglyceridemia in LPS injected mice and even patients with sepsis.