Choi Hyeonhae, Choi Yuri, Kim Jisook, Bae Jaeman, Roh Jaesook
Department of Anatomy & Cell Biology, College of Medicine, Hanyang University, Seoul, South Korea.
Department of Pathology, College of Medicine, Hanyang University, Seoul, South Korea.
J Anat. 2017 Jan;230(1):117-127. doi: 10.1111/joa.12530. Epub 2016 Aug 3.
We showed previously that caffeine adversely affects longitudinal bone growth and disrupts the histomorphometry of the growth plate during the pubertal growth spurt. However, little attention has been paid to the direct effects of caffeine on chondrocytes. Here, we investigated the direct effects of caffeine on chondrocytes of the growth plate in vivo and in vitro using a rapidly growing young rat model, and determined whether they were related to the adenosine receptor signaling pathway. A total of 15 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg day for 4 weeks. After sacrifice, the tibia processed for the analysis of the long bone growth and proliferation of chondrocytes using tetracycline and BrdU incorporation, respectively. Caffeine-fed animals showed decreases in matrix mineralization and proliferation rate of growth plate chondrocytes compared with the control. To evaluate whether caffeine directly affects chondrocyte proliferation and chondrogenic differentiation, primary rat chondrocytes were isolated from the growth plates and cultured in either the presence or absence of caffeine at concentrations of 0.1-1 mm, followed by determination of the cellular proliferation or expression profiles of cellular differentiation markers. Caffeine caused significant decreases in extracellular matrix production, mineralization, and alkaline phosphatase activity, accompanied with decreases in gene expression of the cartilage-specific matrix proteins such as aggrecan, type II collagen and type X. Our results clearly demonstrate that caffeine is capable of interfering with cartilage induction by directly inhibiting the synthetic activity and orderly expression of marker genes relevant to chondrocyte maturation. In addition, we found that the adenosine type 1 receptor signaling pathway may be partly involved in the detrimental effects of caffeine on chondrogenic differentiation, specifically matrix production and mineralization, as evidenced by attenuation of the inhibitory effects of caffeine by blockade of this receptor. Thus, our study provides novel information on the integration of caffeine and adenosine receptor signaling during chondrocyte maturation, extending our understanding of the effect of caffeine at a cellular level on chondrocytes of the growth plate.
我们之前的研究表明，咖啡因会对青春期生长突增期间的纵向骨骼生长产生不利影响，并扰乱生长板的组织形态计量学。然而，咖啡因对软骨细胞的直接影响却很少受到关注。在此，我们使用快速生长的幼鼠模型，在体内和体外研究了咖啡因对生长板软骨细胞的直接影响，并确定这些影响是否与腺苷受体信号通路有关。总共15只雄性大鼠（21日龄）被随机分为三组：对照组和两组通过灌胃给予咖啡因，剂量分别为120和180毫克/千克/天，持续4周。处死后，分别用四环素和BrdU掺入法处理胫骨，用于分析长骨生长和软骨细胞增殖。与对照组相比，喂食咖啡因的动物生长板软骨细胞的基质矿化和增殖率降低。为了评估咖啡因是否直接影响软骨细胞增殖和软骨形成分化，从生长板中分离出原代大鼠软骨细胞，在有或无浓度为0.1 - 1毫摩尔的咖啡因存在的情况下进行培养，随后测定细胞增殖或细胞分化标志物的表达谱。咖啡因导致细胞外基质产生、矿化和碱性磷酸酶活性显著降低，同时软骨特异性基质蛋白如聚集蛋白聚糖、II型胶原蛋白和X型胶原蛋白的基因表达也降低。我们的结果清楚地表明，咖啡因能够通过直接抑制与软骨细胞成熟相关的合成活性和标记基因的有序表达来干扰软骨诱导。此外，我们发现1型腺苷受体信号通路可能部分参与了咖啡因对软骨形成分化的有害影响，特别是基质产生和矿化，这一点通过阻断该受体可减弱咖啡因的抑制作用得到证明。因此，我们的研究提供了关于软骨细胞成熟过程中咖啡因与腺苷受体信号整合的新信息，扩展了我们对咖啡因在细胞水平上对生长板软骨细胞影响的理解。
