Lires-Deán M, Caramés B, Cillero-Pastor B, Galdo F, López-Armada M J, Blanco F J
Osteoarticular and Aging Research Laboratory, Biomedical Research Center, Rheumatology Division, CH Universitario Juan Canalejo, Coruña, Spain.
Osteoarthritis Cartilage. 2008 Nov;16(11):1370-8. doi: 10.1016/j.joca.2008.04.001. Epub 2008 May 20.
To study whether transforming growth factor-beta1 (TGF-beta1) is able to protect human chondrocytes from apoptosis and to analyze the role of phosphatases in the possible anti-apoptotic effect of TGF-beta1.
Cartilage was obtained from patients with osteoarthritis (OA) who were undergoing joint replacement; normal cartilage was obtained from cadavers who had no history of joint disease. Chondrocytes stimulated with tumor necrosis factor-alpha (TNF-alpha) plus Ro 31-8220 (a specific inhibitor of mitogen-activated kinase phosphatase-1 - MKP-1) were employed as an in vitro model of apoptosis. Apoptosis was assessed by flow cytometry and a cell death immunoassay. Protein phosphatase 2A (PP2A) activity was estimated by measuring the absorbance of a molybdate:malachite green:phosphate reaction complex. MKP-1, bcl-2 and bax expressions were quantified by western blot.
In OA cells, TGF-beta1 significantly reduced the percentage of hypo-diploid chondrocytes, as well as the percentage of internucleosomal DNA breakage. However, in normal chondrocytes, TGF-beta1 did not reduce apoptosis, as assessed by both the percentage of hypo-diploid chondrocytes and internucleosomal DNA breakage. MKP-1 expression did not show significant modulation in OA or normal chondrocytes. However, PP2A activity was differentially modulated in normal and OA chondrocytes. In OA chondrocytes, PP2A activity was not altered by TGF-beta1 stimulation; however in normal chondrocytes PP2A activity was significantly activated by TGF-beta1. The preincubation of normal chondrocytes with TGF-beta1 plus the PP2A inhibitor protein, IPP2A, reduced internucleosomal DNA breakage when compared with TGF-beta1 stimulation alone. The bcl-2/bax protein ratio was significantly higher in TGF-beta1 plus IPP2A preincubated normal chondrocytes than in cells stimulated with TGF-beta1 alone.
By manipulating the degree of PP2A activity, these results show the major role that PP2A plays in the outcome of TGF-beta1 signal transduction. These data suggest that PP2A could be a pivotal regulator of anti-apoptotic TGF-beta1-induced effects.
研究转化生长因子-β1（TGF-β1）是否能够保护人软骨细胞免于凋亡，并分析磷酸酶在TGF-β1可能的抗凋亡作用中的作用。
从正在接受关节置换的骨关节炎（OA）患者获取软骨；从无关节疾病史的尸体获取正常软骨。用肿瘤坏死因子-α（TNF-α）加Ro 31-8220（丝裂原活化激酶磷酸酶-1 - MKP-1的特异性抑制剂）刺激的软骨细胞用作凋亡的体外模型。通过流式细胞术和细胞死亡免疫测定评估凋亡。通过测量钼酸盐：孔雀石绿：磷酸盐反应复合物的吸光度来估计蛋白磷酸酶2A（PP2A）活性。通过蛋白质印迹法定量MKP-1、bcl-2和bax的表达。
在OA细胞中，TGF-β1显著降低了亚二倍体软骨细胞的百分比以及核小体间DNA断裂的百分比。然而，在正常软骨细胞中，通过亚二倍体软骨细胞百分比和核小体间DNA断裂评估，TGF-β1并未降低凋亡。MKP-1表达在OA或正常软骨细胞中未显示出明显调节。然而，PP2A活性在正常和OA软骨细胞中受到不同调节。在OA软骨细胞中，TGF-β1刺激未改变PPPA活性；然而在正常软骨细胞中，TGF-β1显著激活了PP2A活性。与单独的TGF-β1刺激相比，用TGF-β1加PP2A抑制剂蛋白IPP2A预孵育正常软骨细胞可减少核小体间DNA断裂。在TGF-β1加IPP2A预孵育的正常软骨细胞中，bcl-2/bax蛋白比率显著高于单独用TGF-β1刺激的细胞。
通过操纵PP2A活性程度，这些结果表明PP2A在TGF-β1信号转导结果中起主要作用。这些数据表明PPPA可能是抗凋亡TGF-β1诱导效应的关键调节因子。
