Li Ying-Hua, Tardif Ginette, Hum David, Kapoor Mohit, Fahmi Hassan, Pelletier Jean-Pierre, Martel-Pelletier Johanne
Osteoarthritis Research Unit, University of Montreal Hospital Research Centre (CRCHUM), 900 Saint-Denis, R11.412B, Montreal, QC, H2X 0A9, Canada.
Division of Genetics and Development, Toronto Western Research Institute, University Health Network (UHN), Toronto, ON, Canada.
Arthritis Res Ther. 2016 Jul 19;18:172. doi: 10.1186/s13075-016-1070-6.
The unfolded protein response (UPR) is activated following an endoplasmic reticulum (ER) stress. The aim of this study was to investigate the global expression of UPR genes in human OA chondrocytes in induced (I)-UPR conditions, and to explore the regulation and role of the UPR genes in homeostatic (H)-UPR conditions in human normal and OA chondrocytes.
Gene expression was determined by PCR array and qPCR. Protein production in cartilage was determined by immunohistochemistry, gene silencing by specific siRNAs, and gene regulation by treating chondrocytes with cytokines and growth factors associated with cartilage pathobiology.
Several UPR genes, among them ERN1, PERK, and CREB3L2 were downregulated in OA compared to normal chondrocytes at both the mRNA and protein levels, but the ER stress response triggered by thapsigargin or tunicamycin treatment was similar in normal and OA chondrocytes. The activation of ER stress sensors (phosphorylated PERK, cleavage of ATF6B, and the spliced mRNA forms of XBP1) was not significantly increased in OA chondrocytes/cartilage. PDGF-BB and IL-6 significantly downregulated the expression of ERN1, PERK, and CREB3L2, but not that of ATF6B. Silencing experiments done under conditions of no ER stress (physiological conditions) revealed that decreasing ERN1 expression led to decreased COL2a1, MMP-13, ADAMTS4 and ADAMTS5 expression, while decreasing CREB3L2 and ATF6B led to decreased ADAMTS5 and ADAMTS4 expression, respectively. Importantly, the downregulation of PERK expression increased COL1a1 and suppressed COL2a1 expression.
Although the level of ER stress is not significantly increased in OA chondrocytes, these cells respond strongly to an acute ER stress despite the decreased expression of ERN1, PERK, and CREB3L2. Emerging findings revealed for the first time that these genes play a role in cartilage biology in conditions where an acute ER stress response is not triggered and OA is not characterized by an overall basal activation of the ER stress response. Importantly, these findings identify PERK as a potential target for new OA treatment avenues.
内质网（ER）应激后未折叠蛋白反应（UPR）被激活。本研究的目的是调查在诱导性（I）-UPR条件下人骨关节炎（OA）软骨细胞中UPR基因的整体表达情况，并探讨UPR基因在人正常和OA软骨细胞稳态（H）-UPR条件下的调控及作用。
通过PCR阵列和定量PCR（qPCR）测定基因表达。通过免疫组织化学测定软骨中的蛋白质生成，通过特异性小干扰RNA（siRNA）进行基因沉默，并通过用与软骨病理生物学相关的细胞因子和生长因子处理软骨细胞来进行基因调控。
与正常软骨细胞相比，在OA中，几个UPR基因，包括ERN1、PERK和CREB3L2，在mRNA和蛋白质水平均下调，但毒胡萝卜素或衣霉素处理引发的ER应激反应在正常和OA软骨细胞中相似。OA软骨细胞/软骨中ER应激传感器（磷酸化的PERK、ATF6B的裂解以及XBP1的剪接mRNA形式）的激活没有显著增加。血小板衍生生长因子-BB（PDGF-BB）和白细胞介素-6（IL-6）显著下调ERN1、PERK和CREB3L2的表达，但不影响ATF6B的表达。在无ER应激（生理条件）下进行的沉默实验表明，降低ERN1表达会导致Ⅱ型胶原α1（COL2a1）、基质金属蛋白酶-13（MMP-13）、含血小板反应蛋白基序的解聚蛋白样金属蛋白酶4（ADAMTS4）和含血小板反应蛋白基序的解聚蛋白样金属蛋白酶5（ADAMTS5）表达降低，而降低CREB3L2和ATF6B表达则分别导致ADAMTS5和ADAMTS4表达降低。重要的是，PERK表达的下调增加了Ⅰ型胶原α1（COL1a1）的表达并抑制了COL2a1的表达。
尽管OA软骨细胞中ER应激水平没有显著升高，但这些细胞对急性ER应激仍有强烈反应，尽管ERN1、PERK和CREB3L2的表达降低。新出现的研究结果首次揭示，在未引发急性ER应激反应且OA不以ER应激反应的整体基础激活为特征的情况下，这些基因在软骨生物学中发挥作用。重要的是，这些发现确定PERK是OA新治疗途径的潜在靶点。
