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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. HS production and γ-cystathionase expression were examined to assess the activity of γ-cystathionase/HS signaling. The interaction of HMGB1 with γ-cystathionase in MSC-H cells was analyzed by co-immunoprecipitation. Luciferase reporter assay was performed to determine whether the promoter activity of γ-cystathionase was regulated by interaction of β-catenin and TCF/LEF binding site. Intercellular cAMP, PKA activity, phosphorylation of β-catenin, and GSK3β were investigated to reveal cAMP/PKA mediated β-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 γ-cystathionase/HS signaling was responsible for CACNA1H activation. HS production was increased with high expression of γ-cystathionase in MSC-H cells, which was blocked by γ-cystathionase inhibitor DL-propargylglycine. Upregulation of γ-cystathionase was not attributed to interaction with HMGB1 overexpressed in MSC-H cells although γ-cystathionase was suggested to co-immunoprecipitate with oxidized HMGB1. Bioinformatics analysis identified a conserved TCF/LEF binding site in the promoter of γ-cystathionase gene. Luciferase reporter assay confirmed that the promoter had positive response to β-catenin which was activated in MSC-H cells. Finally, cAMP/PKA was activated to phosphorylate β-catenin at Ser657 and GSK3β, enabling persisting activation of Wnt/β-catenin signaling in MSC-H cells.

CONCLUSION

Our study revealed that modification of MSCs with HMGB1 promoted CACNA1H-mediated calcium influx via PKA/β-catenin/γ-cystathionase pathway. This was a plausible mechanism for high mobility of MSC-H cell line.

BACKGROUND

METHODS

RESULT

CONCLUSION

背景

间充质干细胞（MSC）在治疗心血管疾病方面具有巨大的应用潜力。最近，我们通过基因修饰 MSC 使其高表达高迁移率族蛋白 1（HMGB1），并通过高效迁移和归巢到目标新生内膜来证实这些细胞具有高迁移性。在本研究中，我们对可能的机制进行了探讨。

方法

将含 HMGB1 cDNA 的慢病毒转染大鼠 MSC，得到高表达 HMGB1 的 MSC-H 细胞系。用空慢病毒转染的 MSC-C 细胞作为阴性对照，并通过微阵列分析差异表达基因。通过 Transwell 迁移实验测定细胞迁移能力。测定细胞内游离钙和 Cav3.2 T 型钙通道（CACNA1H）的表达，以分析 CACNA1H 介导的钙内流活性。通过测定半胱氨酸γ-裂解酶（γ-cystathionase）的表达和羟化磺酸（HS）的生成来评估 γ-cystathionase/HS 信号通路的活性。通过共免疫沉淀分析 MSC-H 细胞中 HMGB1 与 γ-cystathionase 的相互作用。通过荧光素酶报告基因检测确定 β-连环蛋白（β-catenin）和 TCF/LEF 结合位点与 γ-cystathionase 启动子活性的相互作用是否受调控。检测细胞间 cAMP、PKA 活性、β-catenin 的磷酸化和 GSK3β 的磷酸化，以揭示 cAMP/PKA 介导的 β-catenin 激活。

结果

微阵列分析显示，差异表达的基因富集在 cAMP 信号和钙信号通路中。CACNA1H 表达上调，导致细胞内游离钙增加和 MSC-H 细胞迁移增加。用 ABT-639 阻断 CACNA1H 可显著减少细胞内游离钙和细胞迁移。γ-cystathionase/HS 信号通路负责 CACNA1H 的激活。MSC-H 细胞中 γ-cystathionase 高表达导致 HS 生成增加，而 γ-cystathionase 抑制剂 DL-丙炔基甘氨酸（DL-propargylglycine）可阻断这种增加。尽管有研究表明 γ-cystathionase 与氧化型 HMGB1 共免疫沉淀，但在 MSC-H 细胞中，γ-cystathionase 的上调并不是与高表达的 HMGB1 相互作用的结果。生物信息学分析发现，γ-cystathionase 基因启动子中存在一个保守的 TCF/LEF 结合位点。荧光素酶报告基因检测证实，β-catenin 在 MSC-H 细胞中被激活，其启动子对 β-catenin 有正向反应。最后，cAMP/PKA 激活使 β-catenin 在 Ser657 和 GSK3β 上磷酸化，使 Wnt/β-catenin 信号通路在 MSC-H 细胞中持续激活。