Meszaros Evan C, Malemud Charles J
Division of Rheumatic Diseases, Department of Medicine, Arthritis Research Laboratory, Case Western Reserve University School of Medicine and University Hospitals Case Medical Center, Cleveland, Ohio 44106, USA.
Division of Rheumatic Diseases, Department of Medicine, Arthritis Research Laboratory, Case Western Reserve University School of Medicine and University Hospitals Case Medical Center, Cleveland, Ohio 44106, USA ; Department of Anatomy, Case Western Reserve University School of Medicine and University Hospitals Case Medical Center, Cleveland, Ohio 44106, USA.
J Clin Cell Immunol. 2015 Apr;6(2). doi: 10.4172/2155-9899.1000307.
T/C28a2 immortalized juvenile human chondrocytes were employed to determine the extent to which activation of Signal Transducers and Activators of Transcription-1 (STAT1) occurred in response to recombinant human interleukin-6 (rhIL-6) or rhIL-6 in combination with the soluble IL-6 receptor (sIL-6R). Two forms of STAT1, STAT1A and STAT1B, were identified on SDS-PAGE and western blotting with anti-STAT1 antibody. Western blotting revealed that STAT1 was constitutively phosphorylated (p-STAT1). Although incubation of T/C28a2 chondrocytes with rhIL-6 (50 ng/ml) increased p-STAT1A by Δ=22.3% after 30 min, this percent difference failed to reach significance by Chi-square analysis. Similarly, no effect of rhIL-6 (Δ=+10.7%) on p-STAT1B was seen at 30 min. In contrast, although the combination of rhIL-6 plus sIL-6R had no effect on p-STAT1A, rhIL-6 plus sIL-6R increased p-STAT1B by Δ=73.3% (p<0.0001) after 30 min compared to the control group and by Δ=56.7% (p<0.0001) compared to rhIL-6 alone. Janex-1, a Janus kinase-3-specific inhibitor (100 μM) partially reduced the effect of rhIL-6 on p-STAT1B by Δ=27.7% (p<0.05). The results of this study showed that STAT1A/STAT1B was constitutively activated in T/C28a2 chondrocytes. Although rhIL-6 increased p-STAT1B to a small extent, the combination of rhIL-6 plus sIL-6R was far more effective in stimulating STAT1B phosphorylation compared to controls or rhIL-6 alone. These data support the likelihood that although JAK3-mediated activation of STAT1 in T/C28a2 chondrocytes may involve the IL-6/IL-6R/gp130 pathway, these results indicated that STAT1 activation in response to IL-6 preferentially involved IL-6 -signaling via sIL-6R.
采用T/C28a2永生化幼年人软骨细胞来确定信号转导和转录激活因子1（STAT1）在响应重组人白细胞介素-6（rhIL-6）或rhIL-6与可溶性IL-6受体（sIL-6R）联合使用时的激活程度。在SDS-PAGE上以及用抗STAT1抗体进行蛋白质印迹时鉴定出两种形式的STAT1，即STAT1A和STAT1B。蛋白质印迹显示STAT1组成性磷酸化（p-STAT1）。尽管用rhIL-6（50 ng/ml）孵育T/C28a2软骨细胞30分钟后p-STAT1A增加了Δ=22.3%，但经卡方分析该百分比差异未达到显著水平。同样，在30分钟时未观察到rhIL-6对p-STAT1B有影响（Δ=+10.7%）。相比之下，尽管rhIL-6加sIL-6R对p-STAT1A没有影响，但与对照组相比，rhIL-6加sIL-6R在30分钟后使p-STAT1B增加了Δ=73.3%（p<0.0001），与单独使用rhIL-6相比增加了Δ=56.7%（p<0.0001）。Janex-1，一种Janus激酶-3特异性抑制剂（100 μM），使rhIL-6对p-STAT1B的作用部分降低了Δ=27.7%（p<0.05）。本研究结果表明，STAT1A/STAT1B在T/C28a2软骨细胞中组成性激活。尽管rhIL-6在一定程度上增加了p-STAT1B，但与对照组或单独使用rhIL-6相比，rhIL-6加sIL-6R在刺激STAT1B磷酸化方面效果要显著得多。这些数据支持这样一种可能性，即尽管JAK3介导的T/C28a2软骨细胞中STAT1的激活可能涉及IL-6/IL-6R/gp130途径，但这些结果表明，响应IL-6的STAT1激活优先涉及通过sIL-6R的IL-6信号传导。
