Analyses on the mechanisms that underlie the chondroprotective properties of calcitonin.
Analyses on the mechanisms that underlie the chondroprotective properties of calcitonin.
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DOI：
10.1016/j.bcp.2014.07.034
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发表时间：
2014-10
影响因子：
5.8
通讯作者：
Karin V. Greco;G. Nalesso;M. Kaneva;J. Sherwood;A. Iqbal;N. Moradi-Bidhendi;F. Dell’Accio;M. Perret
中科院分区：
文献类型：
--
作者：
Karin V. Greco;G. Nalesso;M. Kaneva;J. Sherwood;A. Iqbal;N. Moradi-Bidhendi;F. Dell’Accio;M. Perret
降钙素（Calcitonin，CT）具有软骨保护作用。在这里，我们研究了CT提供这些action. MethodsImmortized C-28/I2细胞或原代成人关节软骨细胞（AHAC）培养在高密度micromasses研究：（i）CT合成代谢的影响，采用qPCR和免疫组化分析;（ii）CT抗凋亡的影响，采用定量Bax/Bcl基因产物比，TUNEL法和caspase-3表达; CT对CREB、COL 2A 1和NFAT转录因子的影响。结果CT（10 - 10-10 - 8 nM）诱导软骨表型标志物（SOX 9、COL 2A 1和ACAN）显著上调，同时在静息和炎症条件下下调分解代谢（MMP 1andMMP 13 andADAMTS 5）基因产物。这反映了增强生产II型胶原和积累的糖胺聚糖和蛋白聚糖丰富的细胞外基质体外。机制分析显示，只有部分参与环磷酸腺苷的形成，这些影响的CT。相应地，使用特定转录因子的报告基因测定，没有CREB激活的迹象，而COL 2A 1启动子是真正的，直接激活细胞暴露于CT。从表型上看，这些机制支持CT在自身无活性的情况下能够抵消IL-1β的促凋亡作用，这一点通过软骨细胞的TUNEL阳性染色和BAX/BCL基因产物的比例得到证明。结论这些数据可能为开发基于CT的软骨保护策略提供了新的线索，这些策略依赖于导致增强软骨细胞合成代谢和抑制软骨细胞凋亡的机制的参与。
IntroductionCalcitonin (CT) has recently been shown to display chondroprotective effects. Here, we investigate the putative mechanisms by which CT delivers these actions.MethodsImmortalized C-28/I2 cells or primary adult human articular chondrocytes (AHAC) were cultured in high-density micromasses to investigate: (i) CT anabolic effects using qPCR and immuhistochemistry analysis; (ii) CT anti-apoptotic effects using quantitation of Bax/Bcl gene products ratio, TUNEL assay and caspase-3 expression; (iii) CT effects on CREB, COL2A1 and NFAT transcription factors.ResultsCT (10−10–10−8nM) induced significant up-regulation of cartilage phenotypic markers (SOX9, COL2A1andACAN), with down-regulation of catabolic (MMP1andMMP13andADAMTS5) gene products both in resting and inflammatory conditions. This was mirrored by an augmented production of type II collagen and accumulation of glycosaminoglycan- and proteoglycan-rich extracellular matrixin vitro. Mechanistic analyses revealed only partial involvement of cyclic AMP formation in these effects of CT. Congruently, using reporter assays for specific transcription factors, there was no indication for CREB activation, whereas the COL2A1 promoter was genuinely and directly activated by cell exposure to CT. Phenotypically, these mechanisms supported the ability of CT, whilst inactive on its own, to counteract the pro-apoptotic effects of IL-1β, demonstrated by TUNEL-positive staining of chondrocytes and ratio ofBAX/BCLgenes products.ConclusionThese data may provide a novel lead for the development of CT-based chondroprotective strategies that rely on the engagement of mechanisms that lead to augmented chondrocyte anabolism and inhibited chondrocyte apoptosis.