DeWitt D L, Meade E A
Department of Biochemistry, Michigan State University, East Lansing 48824.
Arch Biochem Biophys. 1993 Oct;306(1):94-102. doi: 10.1006/abbi.1993.1485.
Mitogenic stimulation has been shown to increase both prostaglandin H (PGH) synthase-1 (PGHS-1) and PGH synthase-2 (PGHS-2) mRNA levels, although the time course and magnitude of induction are different for the two genes. To investigate the mechanism for mRNA induction, we conducted nuclear run-off assays of these two genes in 3T3 cells and correlated mitogen-induced changes in PGHS gene transcription with changes in PGHS mRNA and PGHS isozyme expression. We also examined the mechanism for glucocorticoid inhibition of PGHS mRNA expression and the effects of glucocorticoids on PGHS isozyme expression. Serum stimulation of quiescent 3T3 cells led to a sequential increase in PGHS-2 gene transcription, PGHS-2 mRNA, and PGHS-2 enzyme levels. PGHS-2 gene transcription increased over 25-fold within 30 min of serum addition resulting in an over 70-fold increase in PGHS-2 mRNA by 1 h, and maximal PGHS-2 enzyme expression by 2 h. Increased PGHS-2 isozyme expression thus appears to depend on transcriptional activation of the gene. Transcription of the PGHS-2 gene declined after 30 min, and PGHS-2 mRNA levels declined similarly after 1 h, leading to a return of PGHS-2 levels to near basal levels by 6 h. Glucocorticoids, which previously have been shown to inhibit mitogen-stimulated increases in PGHS-2 levels, were found to inhibit serum-stimulated increases in PGHS-2 gene transcription by 70%, resulting in a 70% reduction in peak serum-stimulated PGHS-2 mRNA levels also. Western blotting with PGHS-2 specific antisera demonstrated that while dexamethasone simply reduced PGHS-2 mRNA levels, it completely suppressed expression of PGHS-2 protein. The coincidental reduction in PGHS-2 transcription, PGHS-2 mRNA, and enzyme levels by dexamethasone, provides further support for the hypothesis that control of transcription is one primary control mechanism for regulating PGHS-2 expression. That complete suppression of PGHS-2 enzyme expression occurs following partial suppression of PGHS-2 mRNA, however, suggests that other mechanisms may also contribute to the glucocorticoid effect. A small, but reproducible, increase in transcription of the PGHS-1 gene occurred 3 h following serum stimulation, coincident with a three- to fourfold increase in PGHS-1 mRNA; PGHS-1 mRNA remained elevated for at least 3 h. Dexamethasone reduced, but did not completely inhibit, the serum-stimulated increases in PGHS-1. However, changes in PGHS-1 mRNA were not accompanied by detectable changes in PGHS-1 protein in the presence or absence of dexamethasone.
有丝分裂原刺激已被证明可增加前列腺素H（PGH）合酶-1（PGHS-1）和PGH合酶-2（PGHS-2）的mRNA水平，尽管这两个基因诱导的时间进程和幅度有所不同。为了研究mRNA诱导的机制，我们在3T3细胞中对这两个基因进行了核转录分析，并将有丝分裂原诱导的PGHS基因转录变化与PGHS mRNA和PGHS同工酶表达的变化相关联。我们还研究了糖皮质激素抑制PGHS mRNA表达的机制以及糖皮质激素对PGHS同工酶表达的影响。血清刺激静止的3T3细胞导致PGHS-2基因转录、PGHS-2 mRNA和PGHS-2酶水平依次增加。血清添加后30分钟内，PGHS-2基因转录增加超过25倍，导致1小时内PGHS-2 mRNA增加超过70倍，2小时时PGHS-2酶表达达到最大值。因此，PGHS-2同工酶表达的增加似乎依赖于该基因的转录激活。PGHS-2基因转录在30分钟后下降，PGHS-2 mRNA水平在1小时后也类似下降，导致PGHS-2水平在6小时时恢复到接近基础水平。糖皮质激素此前已被证明可抑制有丝分裂原刺激引起的PGHS-2水平升高，现发现其可抑制血清刺激引起的PGHS-2基因转录增加70%，导致血清刺激的PGHS-2 mRNA峰值水平也降低70%。用PGHS-2特异性抗血清进行的蛋白质印迹分析表明，地塞米松虽然只是降低了PGHS-2 mRNA水平，但它完全抑制了PGHS-2蛋白的表达。地塞米松同时降低PGHS-2转录、PGHS-2 mRNA和酶水平，进一步支持了转录控制是调节PGHS-2表达的一种主要控制机制的假说。然而，PGHS-2 mRNA部分抑制后PGHS-2酶表达完全被抑制，这表明其他机制可能也参与了糖皮质激素的作用。血清刺激3小时后，PGHS-1基因转录出现小幅但可重复的增加，与PGHS-1 mRNA增加三到四倍同时发生；PGHS-1 mRNA至少3小时保持升高。地塞米松减少但未完全抑制血清刺激引起的PGHS-1增加。然而，无论有无地塞米松，PGHS-1 mRNA的变化都未伴随PGHS-1蛋白的可检测变化。
