Institute of Anatomy and Cell Biology, Paracelsus Medical University, Nuremberg, 90419 Nuremberg, Germany.
Faculty of Applied Chemistry, Nuremberg Institute of Technology Georg Simon Ohm, 90489 Nuremberg, Germany.
Cells. 2022 Mar 28;11(7):1134. doi: 10.3390/cells11071134.
Both increased activity of the complement system (CS) and the role of the pituitary hormone prolactin (PRL) are implicated in osteoarthritis (OA) pathogenesis. Besides, Cathepsin D (CatD) activity is increased in the context of OA and can exert not only proteolytic but also non-proteolytic effects on cells. For the first time, possible crosstalk between two separate humoral systems: the CS and the PRL hormone systems in chondrocytes are examined together.
Primary human articular chondrocytes (hAC) were stimulated with complement protein C5 (10 µg /mL), PRL (25 ng/mL), CatD (100 ng/mL), or anaphylatoxin C5a (25 ng/mL) for 24 h or 72 h, while unstimulated cells served as controls. In addition, co-stimulations of C5 or PRL with CatD were carried out under the same conditions. The influence of the stimulants on cell viability, cell proliferation, and metabolic activity of hAC, the chondrosarcoma cell line OUMS-27, and endothelial cells of the human umbilical cord vein (HUVEC) was investigated. Gene expression analysis of C5a receptor (C5aR1), C5, complement regulatory protein CD59, PRL, PRL receptor (PRLR), CatD, and matrix metal-loproteinases (MMP)-13 were performed using real-time PCR. Also, collagen type (Col) I, Col II, C5aR1, CD59, and PRL were detected on protein level using immunofluorescence labeling.
The stimulation of the hAC showed no significant impairment of the cell viability. C5, C5a, and PRL induced cell growth in OUMS-27 and HUVEC, but not in chondrocytes. CatD, as well as C5, significantly reduced the gene expression of CatD, C5aR1, C5, and CD59. PRLR gene expression was likewise impaired by C5, C5a, and PRL+CatD stimulation. On the protein level, CatD, as well as C5a, decreased Col II as well as C5aR1 synthesis.
The significant suppression of the C5 gene expression under the influence of PRL+CatD and that of CD59 via PRL+/-CatD and conversely a suppression of the PRLR gene expression via C5 alone or C5a stimulation indicates an interrelation between the two mentioned systems. In addition, CatD and C5, in contrast to PRL, directly mediate possible negative feedback of their own gene expression.
补体系统（CS）活性增加和垂体激素催乳素（PRL）的作用都与骨关节炎（OA）的发病机制有关。此外，组织蛋白酶 D（CatD）在 OA 中的活性增加，不仅可以发挥蛋白水解作用，还可以对细胞发挥非蛋白水解作用。这是第一次同时研究两种独立的体液系统（CS 和 PRL 激素系统）在软骨细胞中的可能串扰。
用人关节软骨细胞（hAC）为研究对象，用补体蛋白 C5（10μg/ml）、PRL（25ng/ml）、CatD（100ng/ml）或过敏毒素 C5a（25ng/ml）刺激 24 小时或 72 小时，未刺激的细胞作为对照。此外，在相同条件下进行 C5 或 PRL 与 CatD 的共刺激。研究了这些刺激物对 hAC、软骨肉瘤细胞系 OUMS-27 和人脐静脉内皮细胞（HUVEC）的细胞活力、细胞增殖和代谢活性的影响。采用实时 PCR 法检测 C5a 受体（C5aR1）、C5、补体调节蛋白 CD59、PRL、PRL 受体（PRLR）、CatD 和基质金属蛋白酶（MMP）-13 的基因表达。同时，用免疫荧光标记法检测 Col I、Col II、C5aR1、CD59 和 PRL 在蛋白水平的表达。
hAC 的刺激并没有显著损害细胞活力。C5、C5a 和 PRL 诱导 OUMS-27 和 HUVEC 的细胞生长，但不诱导软骨细胞的细胞生长。CatD 以及 C5 显著降低了 CatD、C5aR1、C5 和 CD59 的基因表达。C5、C5a 和 PRL+CatD 刺激也损害了 PRLR 基因的表达。在蛋白水平上，CatD 和 C5a 均降低了 Col II 和 C5aR1 的合成。
PRL+CatD 对 C5 基因表达的显著抑制以及 PRL+/-CatD 对 CD59 的抑制和 C5 或 C5a 刺激对 PRLR 基因表达的抑制表明这两个系统之间存在相互关系。此外，CatD 和 C5 与 PRL 相反，直接介导它们自身基因表达的可能负反馈。
