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/en.137.6.2208
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发表时间：
1996-06-01
期刊：
影响因子：
4.8
通讯作者：
Hahn, TJ
中科院分区：
文献类型：
--
作者：
Lowe, GN;Fu, YH;Hahn, TJ
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PGF（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PGF（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，如通过[H-3]胸苷（[H-3]TdR）掺入测量的。PGF（2 α）（0.001-5 μ M）引起[H-3]TdR掺入的剂量相关性增加，在5 μ M时最大增加超过基线值1.6倍，在10 μ M时略微下降至低于最大水平（2）表现出相反的反向双相反应，在0.1 μ M时具有最大的初始小抑制作用，在5 μ M时第二刺激阶段产生相对于对照值的小增加。PGE（1）具有一致的抑制作用，在10 μ M时产生30%的减少。尽管PGE（1）、PGE（2）和PGF（2 α）对第二信使产生和DNA合成的作用不同，但所有三种PG都产生与剂量相关的对angrecan合成的刺激。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，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）（2）cAMP模拟PGE（1）对[H-3]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-十四酰基佛波醇乙酸酯模拟PGF（2 α）对[H-3]TdR掺入和聚集蛋白聚糖合成的刺激，并且PGF（2 α）对这些过程的作用是解锁的bg蛋白激酶C抑制剂。因此。似乎在哺乳动物软骨细胞中，PGE（1）主要激活cAMP-蛋白激酶第二信使系统，PGF（2 α）主要影响Ca 2 +-蛋白激酶C系统，PGE（2）激活两条途径。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 the effects of PG on chondrocyte function remain undefined. We, therefore, examined tile effects of PGE(1), PGE(2), and PGF(2 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. PGE(1) and PGE(2) (0.001-100 mu M) 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 mu M. On the other hand, both PGE(2) and PGF(2 alpha) raised the intracellular Free calcium ([Ca2+](i)) concentration, derived primarily from extracellular sources, whereas PGE(1) was without effect on [Ca2+](i). These three PGs also had divergent effects on DNA synthesis, as measured by [H-3]thymidine ([H-3]TdR) incorporation. PGF(2 alpha) (0.001-5 mu M) produced a dose-related increase in [H-3]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 mu M and a slight decline to below maximal levels at 10 mu M PGE(2) exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 mu M and a secondary stimulatory phase producing a small increase over control values at 5 mu M. PGE(1) had a uniformly suppressive effect, producing a 30% decrease at 10 mu M Despite the divergent effects of PGE(1), PGE(2), and PGF(2 alpha) on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of angrecan synthesis. PGF(2 alpha) was the most potent, producing significant stimulation at 0.001 mu M and a maximal 104% increase at 5 mu M. PGE(1) and PGE(2) were approximately equipotent and approximately 60% as effective as PGF(2 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)(2)cAMP mimicked the suppressive effects of PGE(1) on [H-3]TdR incorporation, as well as the stimulatory effect of PGE(1) on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF(2 alpha) stimulation of [H-3]TdR incorporation and aggrecan synthesis, and the effects of PGF(2 alpha) on these processes were unlocked bg protein kinase C inhibitors. Therefore. it appears that in mammalian chondrocytes, PGE(1) primarily activates the cAMP-protein kinase a second messenger system, PGF(2 alpha) affects primarily the Ca2+-protein kinase C system, and PGE(2) 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.