Lamuedra Ana, Gratal Paula, Calatrava Lucía, Ruiz-Perez Víctor Luis, Palencia-Campos Adrián, Portal-Núñez Sergio, Mediero Aránzazu, Herrero-Beaumont Gabriel, Largo Raquel
Bone and Joint Research Unit, Service of Rheumatology, IIS-Fundación Jiménez Díaz, Autonomous University of Madrid, Madrid, Spain.
Instituto de Investigaciones Biomédicas 'Alberto Sols', CSIC-UAM, Madrid, Spain.
FASEB J. 2022 Apr;36(4):e22258. doi: 10.1096/fj.202101791RR.
Chondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2 genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (Evc ) model of OA. For this purpose, OA was induced by surgical knee destabilization in wild-type (WT) and Evc adult mice, and healthy WT mice were used as controls (n = 10 knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1 beta as an inflammatory insult. Our results showed that tamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-Evc mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. We found that hypertrophic-IHH-and inflammatory-COX-2-markers co-localized in OA cartilage samples. We concluded that tamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-Evc mice, but it did not ameliorate cartilage damage. Overall, our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.
骨关节炎（OA）软骨中的软骨细胞呈现出类似肥大的表型，其中刺猬信号通路（Hh）起着关键作用。Hh过度表达会导致类似OA的软骨损伤，而其下调则可防止小鼠模型中的关节破坏。EVC和EVC2基因的突变会破坏Hh信号通路，并导致埃利斯-范克里弗德综合征骨骼发育异常。由于预计埃利斯-范克里弗德综合征蛋白（Evc）缺失会阻碍Hh靶基因的表达，我们推测它也能防止OA进展并避免软骨细胞肥大。我们的目的是研究Evc作为OA的新治疗靶点，以及在Evc他莫昔芬诱导敲除（Evc ）OA模型中，Evc缺失是否能抑制软骨细胞肥大并预防关节损伤。为此，通过手术使野生型（WT）和Evc成年小鼠膝关节不稳定来诱导OA，健康的WT小鼠用作对照（每组10个膝关节）。通过qRT-PCR测量肥大标志物和Hh基因，并通过蛋白质印迹法评估金属蛋白酶（MMP）水平。从接受膝关节置换手术的患者中获取人OA软骨细胞和软骨样本。环杷明（CPA）用于Hh的药理学抑制，白细胞介素-1β用作炎症刺激物。我们的结果表明，他莫昔芬诱导的Evc失活抑制了OA期间Hh的过度表达，并部分预防了软骨细胞肥大，尽管它并未改善DMM-Evc小鼠的软骨损伤。Hh通路抑制并未改变白细胞介素-1β在培养的人OA软骨细胞中诱导的促炎介质的表达。我们发现肥大相关的IHH标志物和炎症相关的COX-2标志物在OA软骨样本中共定位。我们得出结论，他莫昔芬诱导的Evc失活部分预防了DMM-Evc小鼠的软骨细胞肥大，但并未改善软骨损伤。总体而言，我们的结果表明，软骨细胞肥大本身并非OA进展中的致病事件。
