Napimoga Marcelo Henrique, Demasi Ana Paula Dias, Jarry Christian Rado, Ortega Mauricio Cardoso, de Araújo Vera Cavalcanti, Martinez Elizabeth Ferreira
Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil.
Laboratory of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil.
Int Immunopharmacol. 2015 Jun;26(2):378-83. doi: 10.1016/j.intimp.2015.04.033. Epub 2015 Apr 24.
Osteoclastogenesis is regulated by osteoblasts especially through the production of receptor activator of nuclear factor kappa-B ligand (RANKL). Immune cells present in inflamed tissues markedly increase this process by upregulating RANKL directly or by secreting proinflammatory cytokines, which stimulate RANKL expression by osteoblasts. A novel T-cell-secreted cytokine, termed secreted osteoclastogenic factor of activated T cells (SOFAT) was recently described. To better understand how SOFAT affects bone metabolism, we investigated its effect on osteoblastic cells. We demonstrate here that SOFAT did not influence MC3T3 cells viability and proliferation, evaluated by trypan blue exclusion and MTT tests, respectively. SOFAT stimulated the secretion of IL-6, IL-10 and GM-CSF in MC3T3 cells, as shown by the analysis of an inflammatory cytokines ELISA array. The upregulation of the corresponding genes was checked by qPCR. Both RANKL mRNA and protein levels did not significantly change in the presence of SOFAT, evaluated by qPCR and western blotting, respectively. In addition, analysis of a PCR array for IL6/STAT3 pathway demonstrated that SOFAT induced the expression of BCL2, IL1B, IL10, IL22, IL2RA, IL4, IL6, TNFSF10 and PIAS3, while IL2, IL21, CD4, CSF3R and TNF were repressed. Our results confirm that the SOFAT mechanism of action is RANKL-independent and indicate that, by co-opting osteoblasts to increase the production of osteoclastogenic cytokines, SOFAT may exacerbate inflammation and support osteoclast formation and bone destruction.
破骨细胞生成受成骨细胞调控，尤其是通过核因子κB受体激活剂配体（RANKL）的产生来调控。存在于炎症组织中的免疫细胞通过直接上调RANKL或分泌促炎细胞因子显著增强这一过程，促炎细胞因子可刺激成骨细胞表达RANKL。最近描述了一种新的T细胞分泌细胞因子，称为活化T细胞分泌的破骨细胞生成因子（SOFAT）。为了更好地理解SOFAT如何影响骨代谢，我们研究了它对成骨细胞的作用。我们在此证明，通过台盼蓝排斥试验和MTT试验分别评估，SOFAT不影响MC3T3细胞的活力和增殖。如炎症细胞因子ELISA阵列分析所示，SOFAT刺激MC3T3细胞分泌IL-6、IL-10和GM-CSF。通过qPCR检测相应基因的上调情况。通过qPCR和蛋白质印迹分别评估，在有SOFAT存在的情况下，RANKL mRNA和蛋白质水平均无显著变化。此外，对IL6/STAT3途径的PCR阵列分析表明，SOFAT诱导BCL2、IL1B、IL10、IL22、IL2RA、IL4、IL6、TNFSF10和PIAS3的表达，而IL2、IL21、CD4、CSF3R和TNF受到抑制。我们的结果证实，SOFAT的作用机制不依赖RANKL，并表明，通过利用成骨细胞增加破骨细胞生成细胞因子的产生，SOFAT可能会加剧炎症并支持破骨细胞形成和骨破坏。
