Molecular Mechanisms Of Sulfatide Regulation On Integrin αV Expression

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 (SM3) or galactosylcermide sulfate (SM4). Cerebroside, which belongs to membrane lipid molecules, has an important biological role in cellular membrane signaling. Our previous results demonstrated that sulfatide promoted integrin aV subunit gene expression via Spl and Stat3, adhesion of hepatocellular carcinoma cells (HCC) and hence metastasis of HCC. However, the mechanism of sulfatide-regulated integrin aV subunit expression was still elusive. Based on our previous study, the main point in this study focuses on the molecular mechanism of sulfatide regulation of integrin aV expression. The thesis contains two parts:Part1Sulfatide Suppresses miR-223Gene ExpressionIntegrin aV is an important adhensive receptor, mediated cell migration. Here we showed that integrin aV expressions were most frequently up-regulated in HCC (48/57,84%) and integrin aV subunit expression level was significantly positively correlated with that of sulfatide in103HCC specimens (r=0.425, P＜0.01). We further found that miR-223inhibited the expression of integrin aV and miR-223gene was down-regulated in HCC (45/57,79%). In vivo tumorigenicity and metastasis assays demonstrated that miR-223was proved to effectively inhibit HCC growth and metastasis in both subcutaneous and orthotopic mouse model. miR-223has crucial functions in hepatocarcinogenesis although the supporting evidence is still contradictory. Whether deregulated miR-223expression was associated with hepatoma migration and transcriptional regulation of sulfatide in HCC remained elusive. Intriguingly, sulfatide reduced the recruitment of acetylated histone H3and the transcription factor C/EBPa to the promoter of miR-223gene, which maybe related with MOZ and HDAC1in miR-223promoter, suggesting that sulfatide was responsible for transcriptional suppression of miR-223gene. We also detected the methylation status of miR-223gene promoter, but the methylation was not significantly regulated by sulfatide, indicating that sulfatide regulated miR-223gene not through methylation. Cells transfected with cerebroside sulfatetransferase showed enhanced metastasis potential in the nude mice experiments and inhibited miR-223expression. Through predictions and experiments, we identified transcription factor Spl was a direct target of miR-223and stimulated the mRNA synthesis from integrin aV subunit gene whose promoter contains functional recognition sites for Spl. These data support differential expression of miR-223, sulfatide and integrin aV in HCC and demonstrate the correlation between sulfatide and miR-223in HCC though acetylated histone H3and the transcription factor C/EBPa.Part2Sulfatide Interacts with Bromodomain-Containing Protein1Sulfatide is abnormally overexpressed in hepatocellular carcinoma (HCC), but the relationship between the elevated sulfatide and the initiation and promotion of HCC has not been known. Here we showed that sulfatide enhanced transcription factor Spl acetylation and promoted integrin aV expression. Intriguingly, histone deacetylase inhibitor (TSA) obviously increased the expression of Sp1and integrin aV subunit genes. A strengthened expression of integrin aV subunit by sulfatide was associated with enhancing acetylated histone H3and Sp1recruitment to the promoter of integrin aV subunit gene. To elucidate the molecular mechanism of the regulation by sulfatide on the acetylation, sulfatide was bound to the agarose beads and incubated these beads with cell lysate. After elusion and electrophoresis, a unique band at130KD was identified to be bromodomain-containing protein1(BRD1) and Paired amphipathic helix protein (SIN3B) by Mass Spectrometry as compared to the Gal-Cer control. Both BRD1and SIN3B appeared to be colocalized with sulfatide in HCC cells in immunofluoresent observation and complexed with Sp1, which were directly recruited to the integrin aV gene promoter. BRD1was translocated from the cytosol to the nucleus in response to sulfatide treatment and bound with acetyltransferases MOZ, forming multiprotein complex to mediate acetylation modification of histone H3and Spl. Spl-BRD1complex was required for transcriptional activation of integrin aV subunit gene, while SIN3B bound weakly to sulfatide, and was dissociated from the Spl protein complex following sulfatide treatment. Moreover, there were also other components including HBO1, HDAC1and p300in this complex. Herein this study demonstrated that sulfatide interacted with BRD1which was important for histone H3and Spl acetylation, and played a pivotal role in Spl-dependent transcriptional activation of the integrin aV promoter.