Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene

Abstract

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.

Androgen effects on bone metabolism: recent progress and controversies

“…In addition, treatment of hyperandrogenic adolescent women with flutamide did not affect BMD, although biochemical markers were not determined in this study (159). Of interest, OHF which in vitro antagonized all effects of 5a-DHT on the hFOB/AR-6 cell line, including AR activation (43), proliferation (44) and differentiation (44), suppressed cytokine-induced IL-6 production to the same extent as 5a-DHT and, when co-administered with 5a-DHT, was not able to prevent 5a-DHT-induced IL-6 suppression (143). Thus, OHF appears to act as an AR agonist for effects on IL-6 production by osteoblasts.…”

“…Most studies (see Table 1) have been performed in osteoblastic cell systems which have 500 to 6000 AR/cell (18,31,33,34,37,40,43,44), which is in the physiological range as present in other androgen target organs, including the prostate (34), while some studies report less than 500 AR/cell (19,32,34). Osteoblasts with less than 200 AR/cell appear to lack androgen responsiveness (43,44). However, one study failed to detect androgen responsiveness, as assessed by expression of androgenresponsive genes and an ARE reporter construct in three osteosarcoma cell lines (SaOS-2, TE85, and U-2 OS), although these cells have 3600 to 5800 AR/cell (40).…”

“…DHEA also stimulated TGF-b activity by primary human osteoblastic cells in a manner similar to testosterone and 5a-DHT (127) but, in contrast to testosterone and 5a-DHT, inhibited steady-state mRNA levels of the proto-oncogene c-fos (127), suggesting a different mechanism of transcriptional activation of genes. In the hFOB/AR-6 cell line, however, DHEA, unlike 5a-DHT, failed to activate an androgen-responsive element reporter construct (43), to inhibit proliferation (44), to modulate the IGF/IGFBP system (136), or to suppress cytokine-stimulated IL-6 production (143).…”

The role of androgens in fetal growth: observational study in two genetic models of disordered androgen signalling

“…The AR is ubiquitously expressed as a single, common receptor. This includes amniotic cells,16 the fetal urogenital tract17 and the fetal skeleton 18. Thus androgen resistance in CAIS is not only specific to the external genitalia, but affects all tissues related to somatic growth.…”

Regulation of osteoprotegerin production by androgens and anti-androgens in human osteoblastic lineage cells

“…At 33.5 8C, the ts-SV40LTA is active and the hFOB cells proliferate rapidly, whereas at 39.5 8C, the ts-SV40LTA is inactive and the cells differentiate and display the phenotype of mature osteoblasts. The hFOB/AR-6 cell line was derived by stably transfecting the hFOB cells with an expression vector containing the human wild-type AR cDNA (16). The hFOB/AR-6 cell line expresses osteoblastic differentiation markers, including alkaline phosphatase, osteocalcin, and type I collagen, and is capable of forming mineralized nodules under appropriate conditions.…”