pH-sensing G protein-coupled orphan receptor GPR68 is expressed in human cartilage and correlates with degradation of extracellular matrix during OA progression.
pH-sensing G protein-coupled orphan receptor GPR68 is expressed in human cartilage and correlates with degradation of extracellular matrix during OA progression.
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pH 感应 G 蛋白偶联孤儿受体 GPR68 在人类软骨中表达，与 OA 进展过程中细胞外基质的降解相关。
作者：
Khan NM;Diaz-Hernandez ME;Martin WN;Patel B;Chihab S;Drissi H
骨关节炎(OA)是一种使人衰弱的关节疾病，在全球范围内影响着数百万人。随着骨性关节炎的进展，软骨细胞经历了高度的分解代谢活动，通常伴随着细胞外环境的渗透压和酸度的改变。然而，软骨细胞感知和响应酸性压力的确切机制仍不清楚。近年来，肌肉骨骼组织中的pH敏感G蛋白偶联受体(GPCRs)，如GPR68，受到越来越多的关注。然而，GPR68在骨性关节炎进展过程中在软骨中的功能仍不清楚。本研究旨在利用模拟骨性关节炎分解代谢过程的体外模型，确定GPR68在分解代谢基因表达调控中的作用。我们通过分析从健康供者和骨关节炎患者分离的人软骨高通量RNA-Seq数据来检测GPCRs的表达。从关节置换术中获取去鉴定和废弃的骨性关节炎软骨，并用酶消化法制备软骨细胞。用GPR68激动剂Ogerin刺激软骨细胞，用Trizol法提取β。用cDNA合成试剂盒进行逆转录，用SYBR®Green方法定量检测GPR68和OA相关分解代谢基因的表达。转录组分析表明，人软骨中表达了pH敏感的GPCRs，其中GPR68在骨性关节炎软骨中的表达明显增加，提示GPR68在骨性关节炎的发病机制中具有潜在的作用。免疫组织化学(IHC)和定量聚合酶链式反应(QPCR)分析表明，随着OA分级的增加，软骨中GPR68的表达逐渐增加，强调了GPR68的表达与OA的严重程度之间的相关性。此外，对手术诱导的骨性关节炎小鼠软骨中GPR68的IHC分析表明，在膝关节软骨和半月板中GPR68的水平升高。利用IL-1β刺激的OA体外分解代谢模型，我们的定量聚合酶链式反应显示，在IL-1β刺激下，GPR68的表达呈时间依赖性增加，这与基质降解蛋白的表达有关，提示GPR68在软骨细胞分解代谢和基质退变中起作用。使用GPR68的药理激活剂，我们的结果进一步表明GPR68的激活抑制了人软骨细胞中MMPs的表达。我们的结果表明，GPR68在人和小鼠的软骨中都有很强的表达，并且它的表达与人和手术模型中的基质退变和骨性关节炎进展的严重程度有关。人软骨细胞中GPR68的激活进一步抑制了骨关节炎病理状态下MMPs的表达。这些结果表明，GPR68可能是调节骨性关节炎过程中基质降解的治疗靶点。
Osteoarthritis (OA) is a debilitating joints disease affecting millions of people worldwide. As OA progresses, chondrocytes experience heightened catabolic activity, often accompanied by alterations in the extracellular environment’s osmolarity and acidity. Nevertheless, the precise mechanism by which chondrocytes perceive and respond to acidic stress remains unknown. Recently, there has been growing interest in pH-sensing G protein-coupled receptors (GPCRs), such as GPR68, within musculoskeletal tissues. However, function of GPR68 in cartilage during OA progression remains unknown. This study aims to identify the role of GPR68 in regulation of catabolic gene expression utilizing an in vitro model that simulates catabolic processes in OA. We examined the expression of GPCR by analyzing high throughput RNA-Seq data in human cartilage isolated from healthy donors and OA patients. De-identified and discarded OA cartilage was obtained from joint arthroplasty and chondrocytes were prepared by enzymatic digestion. Chondrocytes were treated with GPR68 agonist, Ogerin and then stimulated IL1β and RNA isolation was performed using Trizol method. Reverse transcription was done using the cDNA synthesis kit and the expression of GPR68 and OA related catabolic genes was quantified using SYBR® green assays. The transcriptome analysis revealed that pH sensing GPCR were expressed in human cartilage with a notable increase in the expression of GPR68 in OA cartilage which suggest a potential role for GPR68 in the pathogenesis of OA. Immunohistochemical (IHC) and qPCR analyses in human cartilage representing various stages of OA indicated a progressive increase in GPR68 expression in cartilage associated with higher OA grades, underscoring a correlation between GPR68 expression and the severity of OA. Furthermore, IHC analysis of Gpr68 in murine cartilage subjected to surgically induced OA demonstrated elevated levels of GPR68 in knee cartilage and meniscus. Using IL1β stimulated in vitro model of OA catabolism, our qPCR analysis unveiled a time-dependent increase in GPR68 expression in response to IL1β stimulation, which correlates with the expression of matrix degrading proteases suggesting the role of GPR68 in chondrocytes catabolism and matrix degeneration. Using pharmacological activator of GPR68, our results further showed that GPR68 activation repressed the expression of MMPs in human chondrocytes. Our results demonstrated that GPR68 was robustly expressed in human cartilage and mice and its expression correlates with matrix degeneration and severity of OA progression in human and surgical model. GPR68 activation in human chondrocytes further repressed the expression of MMPs under OA pathological condition. These results identify GPR68 as a possible therapeutic target in the regulation of matrix degradation during OA.
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DOI：
10.1016/j.clim.2012.12.011
发表时间：
2013-03
期刊：
Clinical immunology (Orlando, Fla.)
影响因子：
--
作者：
Haseeb A;Haqqi TM
通讯作者：
Haqqi TM
影响因子：
4.9
作者：
de Vallière C;Wang Y;Eloranta JJ;Vidal S;Clay I;Spalinger MR;Tcymbarevich I;Terhalle A;Ludwig MG;Suply T;Fried M;Kullak-Ublick GA;Frey-Wagner I;Scharl M;Seuwen K;Wagner CA;Rogler G
通讯作者：
Rogler G
影响因子：
27.4
作者：
Goldring MB;Otero M;Tsuchimochi K;Ijiri K;Li Y
通讯作者：
Li Y
影响因子：
14.9
作者：
Nguyen DT;Mathias S;Bologa C;Brunak S;Fernandez N;Gaulton A;Hersey A;Holmes J;Jensen LJ;Karlsson A;Liu G;Ma'ayan A;Mandava G;Mani S;Mehta S;Overington J;Patel J;Rouillard AD;Schürer S;Sheils T;Simeonov A;Sklar LA;Southall N;Ursu O;Vidovic D;Waller A;Yang J;Jadhav A;Oprea TI;Guha R
通讯作者：
Guha R
影响因子：
4.8
作者：
Diaz-Hernandez, Martha Elena;Kinter, Christopher W.;Drissi, Hicham
通讯作者：
Drissi, Hicham