Mac Namara P, Loughrey H C
Center for Experimental Therapeutics, University of Pennsylvania, 804 Stellar Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA.
Calcif Tissue Int. 1998 Jul;63(1):39-46. doi: 10.1007/s002239900487.
We have previously shown that human progesterone receptors (PR) are expressed in human osteosarcoma cells and in primary human osteoblast cultures. The aim of this study was to examine PRa and PRb isoform expression in human osteosarcoma cells. In addition, the effect of beta-estradiol on PR promoter activity in three human osteosarcoma cell lines was analyzed. Rapid amplification of 5'cDNA ends (5'RACE) were used to detect PR mRNA transcripts coding for both PR isoforms in HOS-TE85, an early progenitor human osteosarcoma cell line. Analogous 5'RACE products were detected in the PR-positive breast-cancer cell line MCF-7. Southern blot analysis confirmed that the amplified products were PR specific. It was shown that the larger of two RACE products coded specifically for B isoform mRNA and that of the smaller product corresponded to a PRa specific transcript. No RACE products were detected in the PR-negative HeLa cell line. To determine if both PR promoters were active in osteoblasts, chimeric recombinants bearing the PRa (+464, +1105) and PRb (-711, +31) promoter regions subcloned into minimal pBLCAT vectors, were transiently expressed in three human osteosarcoma cell lines-HOS-TE85, MG-63, and SAOS-2. It was shown that beta-estradiol induced both PRa and PRb promoter activity in all of the osteosarcoma cell lines examined. The finding that PRa and PRb mRNA transcripts are expressed in human osteoblasts, and that promoters for both isoforms are estrogen responsive provides further evidence that bone-forming cells are physiologically influenced by progesterone.
我们之前已经表明，人孕激素受体（PR）在人骨肉瘤细胞和原代人成骨细胞培养物中表达。本研究的目的是检测人骨肉瘤细胞中PRa和PRb亚型的表达。此外，还分析了β-雌二醇对三种人骨肉瘤细胞系中PR启动子活性的影响。采用5' cDNA末端快速扩增（5'RACE）技术检测早期祖代人骨肉瘤细胞系HOS-TE85中编码两种PR亚型的PR mRNA转录本。在PR阳性的乳腺癌细胞系MCF-7中检测到类似的5'RACE产物。Southern印迹分析证实扩增产物具有PR特异性。结果表明，两种RACE产物中较大的一种特异性编码B亚型mRNA，较小的产物对应于PRa特异性转录本。在PR阴性的HeLa细胞系中未检测到RACE产物。为了确定两个PR启动子在成骨细胞中是否活跃，将携带PRa（+464，+1105）和PRb（-711，+31）启动子区域的嵌合重组体亚克隆到最小pBLCAT载体中，并在三种人骨肉瘤细胞系HOS-TE85、MG-63和SAOS-2中瞬时表达。结果表明，β-雌二醇在所有检测的骨肉瘤细胞系中均诱导了PRa和PRb启动子活性。PRa和PRb mRNA转录本在人成骨细胞中表达，且两种亚型的启动子均对雌激素有反应，这一发现进一步证明成骨细胞在生理上受到孕激素的影响。
