Lambert Cécile, Oury Cécile, Dejardin Emmanuel, Chariot Alain, Piette Jacques, Malaise Michel, Merville Marie-Paule, Franchimont Nathalie
Department of Rheumatology, Center for Biomedical Intergrative Genoproteomics, University of Liège, Liège, Belgium.
J Bone Miner Res. 2007 Sep;22(9):1350-61. doi: 10.1359/jbmr.070508.
The mechanisms of IL-1beta stimulation of OPG were studied in more detail. Whereas p38 and ERK activation was confirmed to be needed, NF-kappaB was not necessary for this regulation. We also found that OPG production after IL-1beta stimulation was not sufficient to block TRAIL-induced apoptosis in MG-63 cells.
Osteoprotegerin (OPG) plays a key role in the regulation of bone resorption and is stimulated by interleukin (IL)-1beta. Herein, we defined the mechanisms of IL-1beta stimulation of OPG focusing on the potential involvement of MAPK and NF-kappaB. We also examined whether OPG production in response to IL-1beta influences TRAIL-induced apoptosis in MG-63 cells.
OPG mRNA levels in MG-63 cells were quantified by real-time RT-PCR and protein levels of OPG and IL-6 by ELISA. Cell viability was assessed using the methyltetrazidium salt (MTS) reduction assay. The role of the MAPK pathway was studied by both Western blotting and the use of specific chemical inhibitors. NF-kappaB function was studied using BAY 11-7085 and by siRNA transfection to inhibit p65 synthesis. Transcription mechanisms were analyzed by transiently transfecting MG-63 cells with OPG promoter constructs. Post-transcriptional effects were examined by using cycloheximide and actinomycin D.
MG-63 cells treatment with IL-1beta resulted in the phosphorylation of c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). The use of the specific inhibitors showed that p38 and ERK but not JNK were needed for IL-1beta-induced OPG production. In contrast, NF-kappaB was not essential for IL-1beta induction of OPG. We also showed a small transcriptional and a possible post-transcriptional or translational regulation of OPG by IL-1beta. Exogenous OPG blocked TRAIL-induced apoptosis, but IL-1beta induction of OPG did not influence TRAIL-induced cell death.
IL-1beta stimulates OPG production by mechanisms dependent on p38 and ERK. In contrast, NF-kappaB was not essential for this regulation. Although the relevance of IL-1beta stimulation of OPG is still not fully understood, our data showed that IL-1beta stimulation of OPG does not modify TRAIL-induced cell death.
对白细胞介素-1β（IL-1β）刺激骨保护素（OPG）的机制进行了更详细的研究。虽然已证实需要激活p38和细胞外信号调节激酶（ERK），但核因子κB（NF-κB）对这种调节并非必需。我们还发现，IL-1β刺激后OPG的产生不足以阻断肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导的MG-63细胞凋亡。
骨保护素（OPG）在骨吸收调节中起关键作用，并受到白细胞介素（IL）-1β的刺激。在此，我们聚焦丝裂原活化蛋白激酶（MAPK）和NF-κB的潜在参与，确定了IL-1β刺激OPG的机制。我们还研究了IL-1β刺激后产生的OPG是否会影响TRAIL诱导的MG-63细胞凋亡。
通过实时逆转录聚合酶链反应（RT-PCR）定量MG-63细胞中OPG信使核糖核酸（mRNA）水平，并用酶联免疫吸附测定（ELISA）法检测OPG和IL-6的蛋白水平。使用甲基噻唑基四唑盐（MTS）还原试验评估细胞活力。通过蛋白质印迹法和使用特异性化学抑制剂研究MAPK途径的作用。使用BAY 11-7085并通过小干扰RNA（siRNA）转染抑制p65合成来研究NF-κB的功能。通过用OPG启动子构建体瞬时转染MG-63细胞来分析转录机制。使用放线菌酮和放线菌素D检查转录后效应。
用IL-1β处理MG-63细胞导致c-Jun氨基末端激酶（JNK）、p38和细胞外信号调节激酶（ERK）磷酸化。使用特异性抑制剂表明，IL-1β诱导OPG产生需要p38和ERK，但不需要JNK。相反，NF-κB对IL-1β诱导OPG并非必需。我们还显示IL-1β对OPG有微小的转录以及可能的转录后或翻译调节。外源性OPG可阻断TRAIL诱导的凋亡，但IL-1β诱导OPG并不影响TRAIL诱导的细胞死亡。
IL-1β通过依赖p38和ERK的机制刺激OPG产生。相反，NF-κB对这种调节并非必需。虽然IL-1β刺激OPG的相关性仍未完全了解，但我们的数据表明，IL-1β刺激OPG不会改变TRAIL诱导的细胞死亡。
