Prostaglandin-mediated inhibition of PTH-stimulated β-catenin signaling in osteoblasts by bone marrow macrophages

Abstract

Bone marrow macrophages (BMMs), in the presence of cyclooxygenase-2 (Cox2) produced PGE2, secrete an inhibitory factor in response to Rankl that blocks PTH-stimulated osteoblastic differentiation. This study was to determine if the inhibitory factor also blocks PTH-stimulated Wnt signaling. Primary calvarial osteoblasts (POBs) were co-cultured with conditioned medium (CM) from Rankl-treated wild type (WT) BMMs, which make the inhibitory factor, and Cox2 knockout (KO) BMMs, which do not. PTH induced cAMP production was blocked by WT CM but not by KO CM. In the presence of KO CM, PTH induced phosphorylation at β-catenin serine sites, ser552 and ser675, previously shown to be phosphorylated by protein kinase A (PKA). Phosphorylation was blocked by WT CM and by H89, a PKA inhibitor. PTH did not increase total β-catenin. PTH-stimulated transcription factor/lymphoid enhancer-binding factor response element activity in POBs was blocked by WT CM and by serum amyloid A (SAA), the human recombinant analog of murine Saa3, which has recently been shown to be the inhibitory factor. In POBs cultured with Cox2 KO CM, PTH increased expression of multiple genes associated with the anabolic actions of PTH and decreased expression of Wnt antagonists. This differential regulation of gene expression was not seen in POBs cultured with WT CM. These data highlight the ability of PTH to phosphorylate β-catenin directly via PKA and demonstrate the ability of a Cox2-dependent inhibitory factor, secreted by Rankl-stimulated BMMs, to abrogate PTH stimulated β-catenin signaling. Our results suggest that PTH can stimulate a novel negative feedback of its anabolic actions by stimulating Rankl and Cox2 expression.

Keywords: Cyclooxygenase-2; Protein kinase A; Serine 552/675; Serum amyloid A; Wnt genes.

Fig. 1. CM from WT BMMs, but not Cox2 KO BMMs, inhibited PTH-stimulated osteoblast differentiation and cAMP production

For all experiments, CM was collected from either the WT BMMs (WT CM) or the Cox2 KO BMMs (KO CM) that were stimulated with Rankl for 3 days. (A) Cox2 KO POBs were cultured for 14 days in the presence of no CM (differentiation medium), WT CM, or Cox2 KO CM and either vehicle or PTH (10 nM). On day 14 of culture, Alp mRNA expression was measured by qRT-PCR. (B) On day 5 of culture, Cox2 KO POBs were treated with vehicle or PTH (10 nM) for 3 hours. Runx2 expression was measured by qRT-PCR. (C) On day 5 of culture, Cox2 KO POBs were treated with vehicle or PTH (10 nM) for 15 minutes. cAMP was measured by ELISA. (D) On day 5 of culture, Cox2 KO POBs were treated with vehicle or PTH (10 nM) for 3 hours. c-Fos expression was measured by qRT-PCR. Bars represent mean ± SEM, n=3. ^aSignificant effect of PTH, p<0.01. ^bSignificant difference compared to WT CM, p<0.01.

Fig. 2. PTH increased phosphorylation of β-catenin at ser552 and ser675 in the presence of Cox2 KO CM but not WT CM

CM was collected from either the WT BMMs (WT CM) or the Cox2 KO BMMs (KO CM) that were stimulated with Rankl for 3 days. Cox2 KO POBs were treated with vehicle or PTH (10 nM) on day 5 of culture and Western analysis was performed. (A) Time course examining effects of PTH on β-catenin phosphorylation. (B–E) Densitometry of bands was performed and normalized to β-actin. Vehicle-treated WT CM was set to 1. Bars represent means ± SEM, n=4. ^aSignificant effect of PTH, p<0.01; ^bp<0.05. ^cSignificant difference compared to WT CM, p<0.01; ^dp>0.05.

Fig. 3. PTH phosphorylated β-catenin at ser552 and ser675 in a PKA- dependent manner

Cox2 KO POBs were treated with vehicle or 10 nM PTH on day 5 of culture and Western analysis was performed. CM was collected from either the WT BMMs (WT CM) or the Cox2 KO BMMs (KO CM) that were stimulated with Rankl for 3 days. (A) Cultures treated with 10 nM PTH for 15 minutes following WT/KO CM and ± H89 (30 μM) pretreatment. (B) Cultures treated as noted with the addition of ± GF109203X (1 μM). (C–G) Densitometry was performed on KO CM treated groups, values were normalized to β-actin and vehicle-treated groups were set to 1. Bars represent means ± SEM, n=3. ^aSignificant effect of PTH, p<0.05.

Fig. 4. PTH-stimulated TCF/LEF reporter activity is inhibited by WT CM and SAA

(A) Cultures were treated for 6 hours with LiCl (20 mM) or NaCl (20 mM). CM was collected from either the WT BMMs (WT CM) or the Cox2 KO BMMs (KO CM) that were stimulated with Rankl for 3 days. (B, C) Cultures were treated for 2 hours with no CM, WT CM, Cox2 KO CM, or SAA (10 μg/mL) prior to 6 hours of treatment with vehicle or PTH (10 nM). Panels (B) and (C) represent independent transductions. Promoter activity values are expressed as relative firefly luciferase units normalized to Renilla reporter activity. Bars are means ± SEM, n=3. ^aSignificant difference compared to vehicle, p<0.01. ^bSignificant difference compared to WT CM, p<0.01. ^cSignificant effect compared to SAA, p<0.01.

Fig. 5. CM from WT BMMs attenuates PTH-regulated expression of genes associated with Wnt signaling

CM was collected from either the WT BMMs (WT CM) or the Cox2 KO BMMs (KO CM) that were stimulated with Rankl for 3 days. Cox2 KO POBs were treated for 2 hours with WT CM or Cox2 KO CM prior to treatment with vehicle or 10 nM PTH for 3 hours on day 5 of culture. mRNA was measured by qRT-PCR. Genes whose expression was undetectable are marked Und. Bars are means ± SEM, n=3. ^aSignificant effect of PTH, p<0.01; ^bp<0.05. ^cSignificant effect compared to WT CM, p<0.01; ^dp<0.05.

Fig. 6. Proposed means by which PTH inhibits its own osteogenic/anabolic actions

PTH stimulates Rankl and Cox2/PGE₂ in osteoblast (OB) lineage cells. Rankl acts on osteoclast (OC) lineage cells to commit them to become osteoclasts and to stimulate more Cox2/PGE₂. The combination of Rankl and PGE₂ cause cells committed to become osteoclasts to secrete Saa3. In the presence of Saa3, PTH-stimulated cAMP production is abrogated, preventing subsequent PKA activation and β-catenin signaling. When PTH is unable to initiate this negative feedback pathway, either by absence of osteoclasts or Cox2, β-catenin is phosphorylated at Ser552 and Ser675 and can stimulate gene transcription associated with osteoblastic differentiation.