Modification of mesenchymal stem cells by HMGB1 promotes the activity of Cav3.2 T-type calcium channel via PKA/beta-catenin/gamma-cystathionase pathway

Background: Mesenchymal stem cells (MSC) hold great promise for treating cardiovascular disease. Recently, we genetically modified MSCs with high mobility group box 1 (HMGB1), and these cells demonstrated high mobility by efficient migrating and homing to target neointima. The possible mechanism was investigated in the current study. Methods: Rat MSCs were transfected with lentivirus containing HMGB1 cDNA to yield MSC-H cell line stably overexpressing HMGB1. The MSC-C cells which were transfected with empty lentivirus served as negative control, and the differentially expressed genes were analyzed by microarray. The cell mobility was determined by transwell migration assay. Intracellular free calcium and the expression of Cav3.2 T-type calcium channel (CACNA1H) were assayed to analyze activity of CACNA1H-mediated calcium influx. H2S production and gamma-cystathionase expression were examined to assess the activity of gamma-cystathionase/H2S signaling. The interaction of HMGB1 with gamma-cystathionase in MSC-H cells was analyzed by co-immunoprecipitation. Luciferase reporter assay was performed to determine whether the promoter activity of gamma-cystathionase was regulated by interaction of beta-catenin and TCF/LEF binding site. Intercellular cAMP, PKA activity, phosphorylation of beta-catenin, and GSK3 beta were investigated to reveal cAMP/PKA mediated beta-catenin activation. Result: Microarray analysis revealed that differentially expressed genes were enriched in cAMP signaling and calcium signaling. CACNA1H was upregulated to increase intracellular free calcium and MSC-H cell migration. Blockage of CACNA1H by ABT-639 significantly reduced intracellular free calcium and cell migration. The gamma-cystathionase/H2S signaling was responsible for CACNA1H activation. H2S production was increased with high expression of gamma-cystathionase in MSC-H cells, which was blocked by gamma-cystathionase inhibitor DL-propargylglycine. Upregulation of gamma-cystathionase was not attributed to interaction with HMGB1 overexpressed in MSC-H cells although gamma-cystathionase was suggested to co-immunoprecipitate with oxidized HMGB1. Bioinformatics analysis identified a conserved TCF/LEF binding site in the promoter of gamma-cystathionase gene. Luciferase reporter assay confirmed that the promoter had positive response to beta-catenin which was activated in MSC-H cells. Finally, cAMP/PKA was activated to phosphorylate beta-catenin at Ser657 and GSK313, enabling persisting activation of Wnt/beta-catenin signaling in MSC-H cells. Conclusion: Our study revealed that modification of MSCs with HMGB1 promoted CACNA1H-mediated calcium influx via PKA/beta-catenin/gamma-cystathionase pathway. This was a plausible mechanism for high mobility of MSC-H cell line.