Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers.
Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers.
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DOI：
10.1210/endo.137.6.8641167
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发表时间：
1996-06
期刊：
影响因子：
4.8
通讯作者：
Gina;-N.;Lowe;Y.-HSIEN;Fu;Skye;Mcdougall;Ruben;Polendo;Alexia;Williams;Paul;-D.;Benya;Theodore;-J.;Hahn
中科院分区：
文献类型：
--
作者：
Gina;-N.;Lowe;Y.-HSIEN;Fu;Skye;Mcdougall;Ruben;Polendo;Alexia;Williams;Paul;-D.;Benya;Theodore;-J.;Hahn
PG在正常和病理状态下调节关节软骨细胞功能中起重要作用。然而，PG对软骨细胞功能的影响的机制仍不清楚。因此，我们研究了PGE 1、PGE 2和PGE 2 α对非转化大鼠RCJ 3.1C5.18（RCJ）软骨细胞系中与DNA和聚集蛋白聚糖合成相关的第二信使产生的影响。RCJ细胞在复合胶原-琼脂糖（0.15%/0.8%）凝胶上在最小附着条件下生长以维持分化的表型。PGE 1和PGE 2（0.001-100 μ M）在cAMP积累方面产生了类似的剂量相关性增加，最大增加超过基础值8倍，而PGF 2 α仅在100 μ M时产生了最小的cAMP水平增加1.3倍。另一方面，PGE 2和PGE 2 α均升高细胞内游离钙（[Ca 2 +]i）浓度，主要来源于细胞外来源，而PGE 1对[Ca 2 +]i无影响。这三种PG对DNA合成也有不同的影响，通过[3 H]胸腺嘧啶核苷（[3 H]TdR）掺入测定。PGF 2 α（0.001-5 μ M）使[3 H]TdR掺入量呈剂量相关性增加，在5 μ M时最大增加为基线值的1.6倍，在10 μ M时略微下降至低于最大水平。PGE 2表现出相反的反向双相反应，在0.1 μ M时最大的初始小抑制作用和在5 μ M时产生比对照值小的增加的次级刺激相。PGE 1具有均匀的抑制作用，在10 μ M时产生30%的降低。尽管PGE 1、PGE 2和PGE 2 α对第二信使产生和DNA合成的影响不同，但所有三种PG都产生了与剂量相关的聚集蛋白聚糖合成刺激。PGF 2 α是最有效的，在0.001 μ M时产生显著刺激，在5 μ M时最大增加104%。PGE 1和PGE 2在刺激聚集蛋白聚糖合成方面的效力大致相等，并且约为PGF 2 α的60%。北方分析表明，PG对聚集蛋白聚糖合成的影响并不伴随聚集蛋白聚糖核心蛋白稳态信使RNA水平的变化。因此，PG对RCJ细胞中聚集蛋白聚糖产生的影响似乎是在转录后水平上调节的。Forskolin和（Bu）2cAMP模拟PGE 1对[3 H]TdR掺入的抑制作用，以及PGE 1对聚集蛋白聚糖合成的刺激作用。此外，佛波酯12-O-十四烷酰基佛波乙酸酯模拟PGF 2 α刺激[3 H]TdR掺入和聚集蛋白聚糖合成，PGE 2 α对这些过程的影响被蛋白激酶C抑制剂阻断。因此，似乎在哺乳动物软骨细胞中，PGE 1主要激活cAMP-蛋白激酶A第二信使系统，PGE 2 α主要影响Ca 2（+）-蛋白激酶C系统，PGE 2激活两种途径。此外，PG转录后调节软骨细胞中聚集蛋白聚糖的合成涉及cAMP-蛋白激酶A和Ca 2（+）-蛋白激酶C第二信使系统。
PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.