Hofbauer L C, Hicok K C, Schroeder M J, Harris S A, Robinson J A, Khosla S
Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905, USA.
J Cell Biochem. 1997 Sep 15;66(4):542-51. doi: 10.1002/(sici)1097-4644(19970915)66:4<542::aid-jcb13>3.0.co;2-d.
Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completeness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild-type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5 alpha-dihydrotestosterone (5 alpha-DHT) (10(-8) M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 +/- 73 (mean +/- SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 +/- 395 functional hARs/nucleus in hFOB/AR-2 cells, and 3,987 +/- 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5 alpha-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5 alpha-DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.
雄激素对骨骼具有显著的有益作用。然而，由于缺乏能同时具备成骨细胞表型完整性、快速增殖率以及雄激素受体（AR）稳定表达的合适模型系统，关于雄激素对成骨细胞作用的研究有限。因此，我们用人类野生型AR（hAR）cDNA稳定转染了条件永生化的人类胎儿成骨细胞系（hFOB）。通过半定量逆转录聚合酶链反应（RT-PCR）评估，与未转染的hFOB细胞相比，在三个独立的hAR转染的hFOB克隆（hFOB/AR）中，hAR mRNA的组成性表达在hFOB/AR-16中高15倍，在hFOB/AR-2中高62倍，在hFOB/AR-6细胞中高72倍。通过Northern印迹分析，在hFOB/AR-2和hFOB/AR-6细胞中可检测到hAR mRNA的组成性水平，但在hFOB/AR-16或hFOB细胞中未检测到。用5α-二氢睾酮（5α-DHT）（10⁻⁸ M）处理24小时，并未改变hFOB/AR细胞系中hAR mRNA的稳态水平。核结合研究表明，在未转染的hFOB细胞中，每个细胞核有152±73（平均值±标准误）个功能性hAR，在hFOB/AR-2细胞中为3940±395个功能性hAR/细胞核，在hFOB/AR-6细胞中为3987±823个hAR/细胞核。用5α-DHT处理以剂量和时间依赖性方式增加了hFOB/AR-6细胞中瞬时转染的雄激素反应元件-氯霉素乙酰转移酶（ARE-CAT）报告构建体的表达；在缺乏外源性转染hAR的瞬时转染hFOB细胞中未观察到这种效应。此外，5α-DHT诱导的ARE-CAT表达被选择性雄激素受体拮抗剂羟基氟他胺抑制。总之，我们已开发并鉴定了源自正常人类胎儿骨骼的雄激素反应性成骨细胞系，其表达具有生理水平的功能性hAR。这些细胞系应为进一步研究雄激素对成骨细胞功能的影响提供合适的模型，包括鉴定潜在的雄激素调节生长因子和细胞因子。
