Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy
Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy
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DOI：
10.1073/pnas.0914360107
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发表时间：
2010-03-02
影响因子：
11.1
通讯作者：
Gregory, Carl A.
中科院分区：
文献类型：
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作者：
Krause, Ulf;Harris, Sean;Gregory, Carl A.
人骨髓间充质干细胞(HMSCs)在体内被认为是成骨细胞的前体细胞，在体外可分化为成骨细胞。规范无翼(Canonical Wingless，WNT)途径的阳性信号是MSCs向成骨细胞分化的关键。相反，过氧化物酶体增殖物激活受体-伽马(PPAR-γ)介导的途径激活导致脂肪生成。因此，我们比较了糖原合成酶-激酶-3β(GSK3β)抑制剂和PPARγ抑制剂对hMSCs成骨的影响。这两种化合物都改变了β-连环素和GSK3β在细胞内的分布，其方式与激活Wnt信号的方式一致。通过hMSCs和转录组分析，GSK3β抑制剂6-溴-靛玉红-3‘-肟(BIO)和PPARγ抑制剂GW9662(GW)促进了早期成骨标志物、碱性磷酸酶(ALP)和骨保护素(OPG)的表达，并证实了骨相关结构蛋白编码基因的上调。在较高剂量的抑制剂，碱性磷酸酶水平减弱，但地塞米松诱导的生物矿化加速。当hMSCs被BIO或GW预处理并植入实验诱导的非自愈性颅骨缺损处时，GW处理显著增加了细胞修复骨损伤的能力，而BIO处理没有显著效果。进一步的研究表明，与GW不同，BIO在体外诱导细胞周期抑制。此外，我们发现GW治疗显著减少了趋化因子的表达，这些趋化因子可能会加剧中性粒细胞和巨噬细胞介导的细胞排斥反应。这些数据表明，在制备hMSCs的过程中使用PPARγ抑制剂可以增强细胞的成骨细胞治疗能力，而腺嘌呤类似物如BIO在体外和体内都会对hMSC制剂的活性产生不利影响。
Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-gamma (PPAR gamma)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3 beta (GSK3 beta) inhibitors and PPAR gamma inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of beta-catenin and GSK3 beta in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3 beta inhibitor 6-bromo-indirubin-3'-oxime (BIO) and the PPAR gamma inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil-and macrophage mediated cell rejection. These data suggest that use of PPAR gamma inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo.