Molecular Mechanisms Of Sulfated Lactosyl Ceramide Regulation Of Integrin αV In Humam Hepatocelluar Carcinoma Cells

Cerebroside,which is a class of membrane lipid molecules, has an important biological role.CST, firstly purified from human renal cancer by K. Honke, is a kind of cerebroside sulfotransferase. CST can catalyze the transferring reaction of active sulfate group from the sulfate group of3’-phospho-adenosine-5’-phosphosullfate (PAPS), to the3’hydroxyl group of terminal galactose on cerebroside, to become sulfated cerebroside. These enzyme products include sulfated lactosyl ceramide (sulfo-lacto-cer, SM3) or galactosylcermide sulfate (SM4), while SM3is the main form in the hepatoma cells. CST gene is22kb, and lies in chromosome22q12.2, which consists of8exons (6are coding exons while2are noncoding), the gene encodes a423amino acid protein. It is a kind of type Ⅱ transmembrane protein in Golgi complex, with two potential N-glycosylation sites, and is the terminal rate-limiting enzyme in sulfated cerebroside synthesis. It is reported in our previous study that cerebroside sulfotransferase (CST) is related to cancer metastasis. Expression level of CST is higher in highly metastatic tumor cells, while lower level in lowly metastatic tumor cells. In addition, sulfated cerebroside is able to bind crucial tumor metastatic related adhesion molecules, such as integrin, midkine, thromboxane, hepatocyte growth factor. Previous results demonstrated that CST promote adhesion of hepatoma carcinoma cell to laminin and vitronectin, and hence metastasis of HCC. Besides, gal:3-O-sulfotransferase-2(Ga13ST-2), highly homologous to CST, and its catalysate-sulfo-Lewis is also involved in metastasis of HCC by exerting impact on integrin expression. We wonder whether sulfation has specific effects on tumor metastasis and how CST and exogenous sulfated lactosyl ceramide affect metastasis of cancer. So we mainly did studies on influence of sulfated lactosyl ceramide, the product of CST, on integrin aV subunit and possible molecular mechanisms. There are two parts in our research.Part1Expression of integrin aV subunit is regulated by sulfation of lactosyl ceramide We treated and incubated SMMC-7721cells with exogenous sulfated lactosyl ceramide and lactosyl ceramide and examine cell adhesive ability to extracellular matrix,(fibronection, fibronogen, vitronectin and collagen type I). We observed that sulfated lactosyl ceramide could specifically enhance adhesive ability of SMMC-7721to vitronectin or laminin which is the binding ligands of integrin αV. Integrin is an important, cancer close-related kind of cell adhesion molecule. We then examine the effect of sulfation of lactosyl ceramide on the expression of integrin aV subunit. Firstly, we added exogenous sulfated lactosyl ceramide and its precursor, lactosyl ceramide to SMMC-7721. By RT-PCR and western blotting, we found that sulfation of lactosyl ceramide could distinctly upregulate integrin aV subunit expression, while the precursor, lactosyl ceramide could not. Furthermore, regulation of integrin aV subunit by exogenous sulfated lactosyl ceramide is time and dose dependent. At the concentration2μmol/L and time point24h, upregulation is most obvious. Sulfated lactosyl ceramide has a sulfate group, so it become negatively charged and acidic glycolipids and then changes the affinity to other ligands. To eliminate the influence of charge, we use exogenous ManN propanyl perac and cyclo ManN propanyl perac, which has similar negative charge with sulfated lactosyl ceramide as controls. Sulfated lactosyl ceramide specifically increase integrin aV subunit, while control groups have no change. This indicates that exogenous sulfated lactosyl ceramide is able to specifically upregulate expression of integrin aV subunit. Next, we studied on the endogenous sulfated glycolipids. We examined integrin aV subunit expression level in CST overexpression stablely transfected SMMC-7721cell lines (CST1and CST8) and CST interfering stablely transfected SMMC-7721cell lines (Chp2and Chp5). Integrin aV subunit expression level is high, when CST is overexpressed. And CST interfered cells express low integrin aV subunit level. This suggests that no matter exogenous or CST catalyzed endogenous sulfated lactosyl ceramide can upregulate integrin aV subunit expression level.Part2Sulfo-lacto-cer upregulates integrin aV subunit expression level by transcription factor Spl Transcription factor is an important factor in mRNA expression regulation. It regulates target gene by changing amount or activity of transcription factor itself. For the purpose of studying potential mechanisms of sulfated lactosyl ceramide upregulating integrin aV subunit expression level, we analyze promoter of integrin aV subunit by informatics. Promoter of the integrin aV subunit owns many transcription factor binding sites, including transcription factor Spl, Sp3V1, Sp3V2, ETS, Egrl, Egr2, AP2and etc. We use realtime-PCR to examine different effects of exogenous sulfo-Lac-Cer on these transcription factors. Results showed that the expression of transcription factor Spl was obviously upregulated. However, other transcription factor remained unchanged or change slightly. So we hypothesize that Spl may be a bridge between sulfation and integrin aV subunit. We checked sulfated lactosyl ceramide impacts on Spl mRNA and protein level. We add exogenous Sulfo-Lacto-Cer, Lacto-Cer, ManN propanyl perac, and cyclo-ManN propanyl perac to the medium of SMMC-7721cells, and observe that Sulfo-Lacto-Cer can specifically increase Sp1expression in mRNA and protein level. Next, we study on whether endogenous sulfation has the same effects as exogenous. We examined Spl expression level in CST overexpressed and CST interfered cell lines. Spl expression level is higher in CST overexpressed cell lines, but is lower in CST interfered cell lines. It suggested that endogenous sulfo-Lacto-Cer is able to upregulate Spl experssion. Sulfo-Lacto-Cer not only upregulate integrin aV subunit expression level, but also Spl. Then we constructed Sp1overexpressing plasmid pcDNA3.0-Spl and Spl interfering plasmid Si-Spl. We transfected pcDNA3.0-Spl to CST interefered cell lines, then check the expression of integrin aV subunit. Transfection with pcDNA3.0-Sp1up-regulates integrin aV subunit expression level. Si-Spl was transfected into CST overexpressed cell lines, and integrin aV subunit expression was inhibited. If we transfect Si-Spl into SMMC-7721cells and add exogenous sulfated lactosyl ceramide at the same time, expression of integrin aV subunit could not be upregulated by exogenous sulfated lactosyl ceramide in Spl interfered cells anymore. It shows that sulfated lactosyl ceramide indeed upregulate integrin aV subunit by Spl transcription factor. The promoter of integrin aV subunit is rich GC box, which is the potential binding site of Spl. We then construct promoter fragments of integrin aV subunit which contains Spl binding sites to check effects on integrin aV subunit promoter activity. Sp1can positively regulate integrin aV subunit promoter activity, and sulfated lactosyl ceramide promote integrin aV subunit promoter activity by increasing Spl expression. Whether Sulfo-Lacto-Cer promote Spl binding to integrin aV subunit promoter, and then promote integrin αV subunit expression level? Furtherly, by EMS A and ChIP methods, we proved that Sp1could directly bind to integrin aV subunit promoter and sulfo-lacto-cer promotes that binding.