Mutual modulation of femarelle and vitamin D analog activities in human derived female cultured osteoblasts

Figures & data

Figure 1. Basal levels of DNA synthesis and CK specific activity in pre-menopausal (pre), in post-menopausal human osteoblasts (post) and male human cultured bone cells.

Notes: Bone cells were cultured, treated, and analyzed for DNA synthesis and CK specific activity as described in Materials. Results are means ± SEM for triplicate cultures from 5 humans/group. Experimental means were compared to control (vehicle alone) means: *p < 0.05, **p < 0.01.

Figure 2. Stimulation of DNA synthesis or CK specific activity by F, D or E₂ as well as JKF in hObs from pre- and post-menopausal women or male.

Notes: Cells were obtained, cultured, treated, and assayed as described in Materials and Methods. They were treated for 24 h with vehicle (C), 200 ng/ml F, 300nM D or 30nM E₂ as well as 1nM JKF. Results are means ± SEM for cultures obtained from 3–5 women or male. Control means were 6,286 ± 675 and 5,824 ± 460 and 5,964 ± 575 dpm/well, for pre- and post-menopausal women and for male respectively. Experimental means compared to control means: *p < 0.01. For CK control means were 28.6 ± 6.5 and 24.6 ± 4.0 and 26.2 ± 5.7 nmol/min/mg protein, for pre- and post-menopausal women and for male respectively. Experimental means compared to control means: *p < 0.01.

Figure 3. Stimulation of DNA synthesis or CK specific activity by F, D, or E₂ in hObs from pre- and post-menopausal women after pre-treatment with JKF (light gray bars).

Notes: Cells were obtained, cultured, treated, and assayed as described in Materials and Methods. Cells were treated for 3 days by daily addition of 1nM JKF and then treated for 24 h with 200 ng/ml F, 300nM D or 30nM E₂. Results are means cultures obtained from 5 women/group for JKF treated cultures and 10 women for control cultures. Control means were 6,286 ± 675 and 5,824 ±4 60 dpm/well, for pre- and post-menopausal women, respectively. Experimental means compared to control means: *p < 0.01; experimental means with JKF compared to experimental means with control: #p < 0.01; experimental means with JKF compared to experimental means with control: #p < 0.01.

Figure 4. The expression of estrogen receptors ERα and ERβ in pre-menopausal human osteoblasts (pre), in post-menopausal human osteoblasts (post) and human males (male).

Notes: Bone cells were cultured, treated, and extracts prepared for mRNA expression as described in Materials and Methods. Results are means ± SEM for cultures from 5 humans/group.

Figure 5. Modulation by pre-treatment with E₂, D, F or JKF on the expression of ER mRNA in hObs obtained from pre- and post-menopausal women.

Notes: Bone cells were obtained, cultured, treated daily for 3 days with 20 ng/ml F, 30nM D or 3nME₂ or 1nM JKF, and extracts prepared for analysis as described in Materials and Methods. Results are SEM for triplicate cultures from 5 donors for each group. Means of hormonal treated compared to means of vehicle-treated cells: *p < 0.01.

Figure 6. The effect of E₂, D, F or JKF on intracellular binding of E₂ in pre- or post-menopausal female hObs, as measured by competition of the binding of ³ [H] E₂. The effects of treatment with JKF (3 days with 1nM) or E₂, D and F on intracellular binding of E₂ in pre- or post-menopausal female hObs, as measured by competition of the binding of ³ [H] E₂.

Notes: Results are means of 4 donors for each group, and are expressed as % modulation of the binding in hormone treated cells compared to untreated cells. **p < 0.01.

Figure 7. Modulation of the specific intracellular binding of ³ [H] E₂ binding in pre-menopausal human osteoblasts (pre-), in post-menopausal human osteoblasts (post-) and males (male) by 30nM E₂, 3,000 nM D or 200 ng/ml F after three daily treatments with 1nM JKF.

Notes: Bone cells were cultured, treated, and extracts prepared for binding activity as described in Materials and Methods. Results are means of 5 donors for each group and are means of experimental compared to control (vehicle alone) means: *p < 0.01.

Figure 8. The expression of VDR or 1OHase and 1,25D production in pre-menopausal human osteoblasts (pre-), in post-menopausal human osteoblasts (post-) and in human male osteoblasts (male).

Notes: Bone cells were cultured, treated, and extracts prepared for mRNA expression as described in Materials ± SEM for cultures from 5 humans group.

Figure 9. The effects of treatment with E₂, D, F, or JKF on 1,25D production (lower panel) and 1OHase mRNA expression (middle panel), as well as on VDR mRNA expression (upper panel) in pre- and post-menopausal and male hObs.

Notes: Cells were incubated for 24 h for the production of 1,25D or for 3 days for VDR and 1OHase mRNA expressions with F (20 ng/ml), D (30nM) or E₂ (3nM) or JKF (1nM). Results are expressed as % change in the concentration of 1,25D (pg/mg protein) or in 1OHase and VDR mRNA expression quantified by real time PCR (n = 4–8 human/group). *p < 0.05; **p < 0.01.