Department of Orthopaedic Surgery, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90033, USA; Division of Nephrology and Hypertension, Department of Medicine and USC/UKRO Kidney Research Center, Keck School of Medicine of the University of Southern California, Los Angeles, CA, 90033, USA.
Department of Orthopaedic Surgery, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90033, USA.
Osteoarthritis Cartilage. 2019 Jan;27(1):158-171. doi: 10.1016/j.joca.2018.08.017. Epub 2018 Sep 8.
Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation.
Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII.
Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFβ1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFβ1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes.
Current study demonstrates a direct role for CaMKII in TGF-β and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.
钙/钙调蛋白依赖性激酶 II（CaMKII）的上调与骨关节炎（OA）的发病机制和关节软骨肥大的再激活有关。然而，CaMKII 的直接抑制出人意料地加剧了动物模型中的 OA 症状。CaMKII 在 OA 中的作用仍不清楚，需要进一步研究。
在正常人和 OA 关节软骨细胞中分析 CaMKII 的表达，并使用 CaMKII 的药理学（KN93）、肽（AC3-I）和小干扰 RNA（siRNA）抑制剂在关节、胎儿和多能干细胞（PSC）衍生的人软骨细胞中评估信号转导机制。
在人 OA 标本中，磷酸化 CaMKII（pCaMKII）的表达水平显著且一致增加。BMP2/4 激活了人成年关节软骨细胞中 pCaMKII 以及 COLII 和 COLX 的表达，并增加了 SMADs1/5/8 的水平和核定位，而 TGFβ1 对成年软骨细胞中的软骨形成程序几乎没有或没有激活作用。用特异性 siRNA 靶向阻断 CaMKII 可降低正常和 OA 成年关节软骨细胞中 pSMADs、COLII、COLX 和蛋白聚糖的水平，同时存在 BMP4 和 TGFβ1。人胎儿和 PSC 衍生的软骨细胞也显示在 CaMKII 的小分子和肽抑制剂存在下软骨分化减少。此外，用 SMADs1/5/8 或 2/3 进行免疫沉淀，然后用 pCaMKII 进行 Western blot 显示了在原代软骨细胞中 SMADs 和 pCaMKII 之间的直接相互作用。
本研究表明 CaMKII 在 TGF-β 和 BMP 介导的原代和 PSC 衍生的软骨细胞中的反应中具有直接作用。这些发现对干细胞来源的软骨组织的组织工程和 OA 的治疗管理具有直接意义。
