Phosphate regulates osteopontin gene transcription

Abstract

Extracellular inorganic phosphate (ePi) is a key regulator of cementoblast behavior, both in vivo and in vitro, and results in a marked increase in osteopontin expression in vitro. To examine the molecular mechanisms involved in ePi induction of osteopontin gene expression, we transfected a series of osteopontin promoter-luciferase constructs into OCCM-30 cementoblasts. Our results demonstrate that ePi can directly induce osteopontin gene transcription. The region responsive to ePi signaling was localized to a 53-bp region of the promoter between -1454 and -1401 that contains a glucocorticoid response element (GRE). Mutation of the GRE abolished the ePi response, suggesting that glucocorticoid receptor (GR) signaling is required for ePi-mediated transcription. In addition, treatment of cells with the GR antagonist RU-486 (Mifepristone) prevented promoter activation by ePi. The results presented support a model demonstrating that inorganic phosphate regulates OPN gene transcription in cementoblasts through a pathway that requires a functional GR.

Figure 1.

Phosphate activation of the OPN gene in cementoblasts. (A) The -1454/+ 5 OPN-luc construct was transfected into OCCM-30 cells, and cells were incubated with ePi for 12 hrs before being harvested. Results are shown normalized to the Renilla luciferase activity ± 1 SD. EV (empty vector) and pGL3 C represent cells transfected with pGL3-Basic and pGL3-Control, respectively. The mean Firefly/Renilla luciferase values (± SE) were: OPN –ePi/EV, 167.6 ± 5; OPN+ 2mM ePi/EV, 370 ± 29.5; and OPN+ ePi 4mM/EV, 388 ± 15.8. (B) Time-course of Pi activation of the OPN promoter. Cells were transfected with -1454/+ 5 OPN-luc and treated with 3 mM ePi (+ Pi) or media (-ePi) for 6, 12, and 24 hrs before being harvested. The mean Firefly/Renilla luciferase values of OPN promoter/EV (± SE) were: 6 hrs, 5.82 ± 0.6, 10.42 ± 1.2 (- Pi, + Pi); 12 hrs, 21.6 ± 1.6, 41.85 ± 1.7 (- Pi, + Pi); and 24 hrs, 22.22 ± 1.2, 9.6 ± 1.02 (- Pi, + Pi). (C) OPN mRNA expression measured by Q-PCR. Cells were treated with 5 mM ePi (P) or media only (NT) for between 1 and 48 hrs and then harvested for RNA analysis. Q-PCR was performed as described in MATERIALS & METHODS. Results are shown normalized to GAPDH. These experiments were performed in triplicate and represent 1 of the 5 experiments. *P ≤ 0.05.

Figure 2.

Deletion mapping to identify the phosphate-responsive region. (A) Diagram of the OPN constructs generated by restriction enzyme digestion or PCR. The distal 300 bp of the mouse OPN promoter sequence contained in the -1454/+5 OPN-luc construct is shown. The GRE motif is underlined in bold. (B,C) Luciferase activity of the OPN promoter constructs in the absence (- Pi) and presence (+ Pi) of 3 mM ePi. (B) Mean Firefly/Renilla values ± SE for the OPN constructs/EV (left to right) were: 33.66 ± 8.8, 82.8 ± 7.4, 57.0 ± 6.2, and 95.4 ± 10.9. (C) Mean Firefly/Renilla values ± SE for the OPN constructs /EV (left to right) were: 20 ± 2.9, 40 ± 7.5, 15 ± 3.2, 20 ± 5.8, 16 ± 5.9, 24 ± 11.4, 20 ± 3.4, and 22 ± 6.4, respectively. EV represents a control transfection with pGL3-Basic. All results are shown normalized to Renilla luciferase activity. These experiments represent the mean of triplicates and represent 1 of the 4 experiments. *P ≤ 0.05.

Figure 3.

Phosphate induction of the OPN promoter requires a Glucocorticoid Receptor Element. OPN-luc promoter constructs were prepared by PCR and ligated into a PGL3-Promoter vector. Constructs were transfected into OCCM-30 cells as described in MATERIALS & METHODS. Cells were treated with 3 mM Pi and analyzed for luciferase activity as described in Figs. 1 and 2. (A) Mean Firefly/Renilla values ± SE for the OPN constructs /EV (left to right) were: 26.15 ± 1, 40.94 ± 2.5, 19.23 ± 0.7, 33.4 ± 6.1, 22.42 ± 2.95, 37.9 ± 2.5, 26 ± 2.9, 24 ± 2.1, 19.4 ± 1.2, and 19.54 ± 5.2, respectively. (B) Mutagenesis of the OPN GRE prevents Pi induction of promoter activity. Cells were transfected with the indicated mutant constructs that contained 2 or 4 base substitutions in the 3’ half-site of the GRE, and then tested for Pi responsiveness. The control construct (denoted TGTTCT) was -1454/-1134 OPN-luc. Mean Firefly/Renilla values ± SE for the OPN constructs /EV (left to right) were: 13.5 ± 3, 28.66 ± 2.8, 55.3 ± 3, 97 ± 5.8, 34.85 ± 4, 42.86 ± 1, 54 ± 7.9, and 46.7 ± 1.5, respectively. Note that the 2 mutant constructs with altered 3’ half-sites are no longer responsive to ePi addition. These experiments were performed in triplicate and represent 1 of 3 experiments. *P ≤ 0.05.

Figure 4.

Effects of dexamethasone and RU-486 on OPN promoter activity. OCCM-30 cells were transfected with the indicated plasmids (4 µg/60 mm dish) and then incubated with Pi (ePi), dexamethasone, and/or RU-486, and luciferase assays were performed as described in MATERIALS & METHODS. Firefly luciferase activity is shown normalized to Renilla luciferase. (A) OPN promoter activity in the presence of dexamethasone and Pi. Cells were transfected with; pGL3-Control (C), empty vector (EV), or -1454/+ 5 OPN-luc (OPN-luc), followed by treatment with 3 mM Pi and/or 1 mM dexamethasone (DEX) as indicated. Mean Firefly/Renilla values ± SE for the OPN constructs/EV (left to right) were: 152 ± 5.2, 221 ± 38.7, 221 ± 38.7, and 228 ± 32.8. (B) GRE-luc promoter activity in the presence of dexamethasone and Pi. Cells were transfected with GR-luc and then treated with Pi, dexamethasone, or dexamethasone and Pi. Mean Firefly/Renilla values ± SE for the GR construct/EV (left to right) were: 7.21 ± 0.75, 5.6 ± 0.13, 61.43 ± 10.08, and 49.9 ± 8.55, respectively. (C) OPN promoter activity in the presence of 3mM Pi and RU-486 (100 nM-1 µM). Cells were transfected with pGL3-Control (C), empty vector (EV), or -1454/+ 5 OPN-luc (OPN-luc) and then treated with Pi, or Pi and RU-486 as indicated. Mean Firefly/Renilla values ± SE for the OPN constructs/EV (left to right) were: 7.7 ± 0.4, 18.46 ± 0.7, 11.57 ± 2.2, and 9.06 ± 0.5, respectively. (D) GRE-luc promoter activity in the presence of dexamethasone and RU-486 (100 nM and 1 mM). Cells were transfected with GR-luc and then treated with dexamethasone, or dexamethasone and RU-486 (100 nM and 1 mM). Mean Firefly/Renilla values ± SE for the GR constructs/EV (left to right) were: 1.47± 0.19, 9.84 ± 0.9, 2.9 ± 0.9, and 1.84 ± 0.5, respectively. *P ≤ 0.05.