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. 2008 Apr;149(4):1728-35.
doi: 10.1210/en.2007-0826. Epub 2008 Jan 10.

CBP/p300-interacting protein CITED1 modulates parathyroid hormone regulation of osteoblastic differentiation

Dehong Yang  1 , Jun Guo, Paola Divieti, Toshi Shioda, F Richard Bringhurst
Affiliations

CBP/p300-interacting protein CITED1 modulates parathyroid hormone regulation of osteoblastic differentiation

Dehong Yang et al. Endocrinology. 2008 Apr.

Abstract (VSports)

PTH regulates osteoblastic differentiation and activity and exerts different overall skeletal effects in vivo, depending on the schedule and dose of administration. In clonal Wt9 murine osteoblastic cells, mRNA and protein levels of CITED1 transcriptional coactivator were strongly up-regulated by human (h) PTH(1-34). Stimulation of CITED1 mRNA by PTH was transient, peaking at 4 h, concentration dependent, and blocked by actinomycin D but not cycloheximide. The stimulation was mimicked by forskolin, phorbol ester, and the cAMP-selective PTH analog [G(1),R(19)] hPTH (1-28) and inhibited completely by the protein kinase A inhibitor, H89 and partially by phorbol ester-induced protein kinase C depletion. Increased CITED1 expression was not maintained during persistent (24 h) PTH exposure. Cultured primary calvarial osteoblasts from neonatal homozygous or hemizygous CITED1-knockout (KO) mice achieved 2-fold greater mineralized nodule formation in comparison with wild type (WT) osteoblasts. This effect was blocked by restoration of CITED1 expression via adenoviral gene transfer. Intermittent administration of hPTH(1-34) (10 nm, for 4 h every 48 h) for 3-6 wk increased mineralization up to 2-fold over basal levels in both WT and CITED1 KO mouse calvarial cell cultures. Whereas the cAMP-selective [G(1),R(19)]hPTH(1-28) analog [at 100 nm, equivalent to 10 nm hPTH(1-34)] did not stimulate mineralization in WT cultures, it was twice as effective as hPTH(1-34) in CITED1 KO cultures. Thus, CITED1 negatively regulates osteoblastic differentiation in vitro and inhibits the cAMP-dependent stimulation of differentiation by intermittent PTH VSports手机版. We conclude also that PTH receptor signaling pathways independent of cAMP restrain osteoblastic differentiation, an effect normally obscured in the presence of CITED1 but revealed in its absence. .

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Figures

Figure 1
Figure 1
CITED1 expression in osteoblasts. A, Wt9 cells were treated with 100 nm hPTH(1–34) for 4 h before extraction of RNA and subsequent measurement of CITED1, CITED2, and CITED4 mRNA expression by quantitative RT-PCR, as described in Materials and Methods. Results are expressed as fold over control. B, Primary osteoblasts isolated from neonatal C57BL6 mice were treated with 100 nm hPTH(1–34) for 4 h before RNA extraction and measurement of CITED1 mRNA expression, as in A. C, Wt9 cells were treated with 30 nm hPTH(1–34), or vehicle alone (control), for 6 h or 24 h before simultaneous extraction of proteins in radioimmunoprecipitation assay buffer and assessment of CITED1 expression by Western blot analysis. D, Intensities of CITED1 and β-actin protein bands from five experiments performed as in C were assessed densitometrically (see Materials and Methods) and are presented as fold over the corresponding 6 h control in each experiment. Data are shown as mean ± sd for groups of three cultures (A and B). *, P < 0.01 vs. control (CON).
Figure 2
Figure 2
CITED1 is a primary response gene transcriptionally regulated by PTH. Wt9 cells were treated with actinomycin D (ActD; 5 μg/ml) or CHX (5 μg/ml) for 1 h before addition of hPTH(1–34) (30 nm). After 4 h of PTH exposure, total RNA was extracted from the cells and the expression levels of CITED1 and RAMP3 mRNA were measured by quantitative RT-PCR and shown as fold over control (CON). Experiments were performed three times with similar results. Data shown are mean ± sd for groups of two cultures each. a, P < 0.01 vs. control; b, P < 0.01 vs. PTH; c, P < 0.05 vs. CHX.
Figure 3
Figure 3
PTH increases CITED1 expression in a dose- and time-dependent manner in osteoblasts. Wt9 cells were treated with 100 nm hPTH(1–34) for indicated times (A) or 4 h with hPTH(1–34) or G1R19(1–28) at the indicated concentrations (B). Total RNA was extracted and the expression of CITED1 was measured by quantitative RT-PCR and expressed as fold over control. Experiments were performed three times with similar results. Data are shown as mean ± sd for groups of three cultures. *, P < 0.01 vs. control (CON).
Figure 4
Figure 4
Signaling pathways mediating increased CITED1 expression by PTH(1–34). Wt9 cells were exposed to the indicated agonists for 4 h, ± pretreatment with inhibitors for 1 h, before extraction of total RNA and measurement of CITED1 and RAMP3 mRNA expression by quantitative RT-PCR (see Materials and Methods). A, Cells were treated with vehicle (CON), 100 nm hPTH(1–34) PTH(1–34)), 10−7 m forskolin (FSK), or 10−7 m TPA for 4 h. B, Cells were treated with hPTH(1–34) or forskolin as in A ± pretreatment with the PKA inhibitor H89 (20 μm). C, Cells were stimulated with hPTH(1–34) as in A with or without prolonged (16 h) pretreatment with 10−6 m TPA (Pre-TPA). Experiments were performed three times with similar results. Data are shown as mean ± sd for groups of three cultures. a, P < 0.01 vs. control; b, P < 0.01 vs. PTH; c, P < 0.05 vs. Fsk; d, P < 0.01 vs. pre-TPA.
Figure 5
Figure 5
Regulation of osteoblastic mineralization by CITED1. WT and CITED1 KO primary calvarial osteoblasts isolated from neonatal mice (A) or CITED1 KO primary calvarial osteoblasts, either uninfected (CON) or infected with Ad-CITED1 or Ad-Lac-Z at 100 ifu/cell (B) were cultured in mineralization medium for 4 wk. Mineralized nodules in the cultures then were assessed by staining with Alizarin Red S and imaged by either plate scanning (A, top panel, and B) or microscopy (A, bottom panel, at magnification ×40). Calcium content was measured in acid extracts (see Materials and Methods). Experiments were performed three times with similar results. Calcium content (milligrams per well) is expressed as mean ± sd for groups of three cultures. *, P < 0.01 vs. WT (A) or uninfected controls (B).
Figure 6
Figure 6
Role of CITED1 in regulation of osteoblastic differentiation by intermittent PTH in 3-wk cultures. Primary osteoblastic cells from WT or CITED1 KO mice were plated in type I collagen-coated 24-well plates and cultured until 90% confluent before initiation of cyclic (4 h every 48 h) PTH treatment (see Materials and Methods), using the peptides and concentrations indicated. After 3 wk, mineralization was assessed by Alizarin Red-S staining (top panel) and calcium content was measured in acid extracts of the cultures (bottom panel). Bars depict means ± se of three independent experiments. a, P < 0.01 vs. same-genotype control; b, P < 0.01 vs. G1R19(1–28) in same genotype; c, P < 0.01 vs. WT control.
Figure 7
Figure 7
Role of CITED1 in regulation of osteoblastic differentiation by intermittent PTH in 6-wk cultures. Primary osteoblastic cells were isolated and treated with PTH peptides as described in Fig. 6. Histochemical staining for ALP activity (upper photograph), staining with Alizarin Red-S (lower photograph), and measurements of ALP activity (upper graph) and calcium content (lower graph) were performed after 6 wk of PTH treatment. a, P < 0.01 vs. same-genotype control; b, P < 0.01 vs. G1R19(1–28) in same genotype; c, P < 0.01 vs. WT control. Note interrupted ordinate scale in upper graph.
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