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Prostaglandin E2 signals through PTGER2 to regulate sclerostin expression - PubMed

  • ️Sat Jan 01 2011

Prostaglandin E2 signals through PTGER2 to regulate sclerostin expression

Damian C Genetos et al. PLoS One. 2011.

Abstract

The Wnt signaling pathway is a robust regulator of skeletal homeostasis. Gain-of-function mutations promote high bone mass, whereas loss of Lrp5 or Lrp6 co-receptors decrease bone mass. Similarly, mutations in antagonists of Wnt signaling influence skeletal integrity, in an inverse relation to Lrp receptor mutations. Loss of the Wnt antagonist Sclerostin (Sost) produces the generalized skeletal hyperostotic condition of sclerosteosis, which is characterized by increased bone mass and density due to hyperactive osteoblast function. Here we demonstrate that prostaglandin E(2) (PGE(2)), a paracrine factor with pleiotropic effects on osteoblasts and osteoclasts, decreases Sclerostin expression in osteoblastic UMR106.01 cells. Decreased Sost expression correlates with increased expression of Wnt/TCF target genes Axin2 and Tcf3. We also show that the suppressive effect of PGE(2) is mediated through a cyclic AMP/PKA pathway. Furthermore, selective agonists for the PGE(2) receptor EP2 mimic the effect of PGE(2) upon Sost, and siRNA reduction in Ptger2 prevents PGE(2)-induced Sost repression. These results indicate a functional relationship between prostaglandins and the Wnt/β-catenin signaling pathway in bone.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PGE2 decreases Sost expression.

(A) Human PTH(1–34) (100 nM) or PGE2 (5 nM–5 µM) or vehicle control (0.05% DMSO) was added to UMR 106.01 cells for 3 hours. Total RNA was collected and analyzed for Sost and Rpl32 expression by qPCR. n = 6–10 samples per treatment. a indicates p<0.05 versus Control; b indicates P<0.05 versus 5 nM PGE2. (B) Sost mRNA expression was quantified in UMR 106.01 cells after 0, 1, 2, 3, 6, or 24 hours treatment with 5 µM PGE2 or vehicle control. n = 4 samples per treatment. For PGE2, each time point is significantly different (p<0.05) from Control, while for PTH, every time point except 1 hr is significantly different (p<0.05) from Control.

Figure 2
Figure 2. PGE2 increases Wnt signaling without affecting Dkk1.

(A) PGE2 (5 nM–5 µM) or vehicle control (0.05% DMSO) was added to UMR 106.01 cells for 24 hours. Total RNA was collected and analyzed for Axin2, Tcf3, and Rpl32 expression by qPCR. n = 4 samples. Compared to vehicle control, a indicates p<0.05. (B) Human PTH(1–34) (100 nM) or PGE2 (50 nM–5 µM) or vehicle control (0.05% DMSO) was added to UMR 106.01 cells for 3 hours. Total RNA was collected and analyzed for Dkk1 and Rpl32 expression by qPCR. n = 8 samples. Compared to vehicle control, a indicates p<0.05.

Figure 3
Figure 3. PGE2 receptor expression and influence of PGE2 selective agonists upon Sost expression.

(A) UMR106.01 cells analyzed for Ptger1, Ptger2, Ptger3, and Ptger4 transcript expression by qPCR. Data are normalized to Rpl32. n = 4 samples. (B) UMR106.01 cells were cultured with DMSO as vehicle control, 100 nM hPTH(1–34), 5 µM PGE2 in the presence and absence of inhibitors of protein kinase A (H-89, 2.5 µM) or phospholipase C (U73122, 10 µM), for 3 hours. Total RNA was analyzed for Sost and normalized to Rpl32. n = 4–8 samples. Compared to solvent control, a indicates p<.001 and b indicates p<0.05; c indicates p<.001. (C) UMR106.01 cells were cultured with DMSO as vehicle control, 100 nM hPTH(1–34), the cell-permeant cyclic AMP analogue 8-br-cAMP (1 mM) or the calcium ionophore ionomycin (1.3 µM) for 3 hours, after which total RNA was collected and analyzed for Sost and Rpl32. n = 4–8 samples. Versus Control, a indicates p<0.05, b indicates p<0.01, and c indicates p<0.001.

Figure 4
Figure 4. PGE2 signals through Ptger2 to decrease Sost expression.

(A) Cells were cultured for 3 hours in the presence of PTH (100 nM), PGE2, EP2 agonist butaprost, or EP4 agonist CAY10580 (each 500 nM). Sost expression was analyzed by qPCR and normalized to Rpl32. n = 5 samples. Compared to vehicle control, b indicates p<0.01 and c indicates p<0.001; a indicates p<0.01 versus CAY10580. (B) UMR106.01 cells were cultured with 50 nM of scrambled or Ptger2 siRNA for 48 hours, after which Ptger2 expression was examined by qPCR. n = 4 samples. Compared to vehicle control, a indicates p<0.05. (C) UMR106.01 cells were cultured with 50 nM of scrambled or Ptger4 siRNA for 48 hours, after which Ptger4 expression was examined by qPCR. n = 4 samples. Compared to vehicle control, a indicates p<0.05. (D) UMR106.01 cells were cultured with 50 nM of scrambled, Ptger2, or Ptger4 siRNA for 48 hours, then with 5 µM PGE2 for 3 hours, after which time total RNA was collected and analyzed for Sost and Rpl32. n = 5 samples. Compared to vehicle control, b indicates p<0.01.

Figure 5
Figure 5. Effects of actinomycin D or cycloheximide upon PGE2 suppression of Sclerostin.

(A) UMR106.01 cells were serum-starved for 1 hour, treated with 2.5 µg/mL actinomycin D with or without 5 µM PGE2, and collected 3 hours later. cDNA was prepared for qPCR analysis of Sost and Rpl32. n = 4 samples. (B) UMR 106.01 cells were treated with combinations of 10 µg/mL cycloheximide and 5 µM PGE2 for 3 hours. Samples were analyzed by qPCR for Sost and Rpl32. n = 4 samples. Compared to vehicle control, a indicates p<0.05 and b indicates p<0.001; compared to 5 µM PGE2, c indicates p<0.001.

Figure 6
Figure 6. Reductions in MEF2 expression do not impair PGE2 decrease of Sost.

(A) UMR106.01 cells were cultured with 50 nM of scrambled or Ptger4 siRNA for 48 hours, after which Mef2c expression was examined by qPCR. n = 4 samples. Compared to scrambled control, c indicates p<0.001. (B) UMR106.01 cells were cultured with 50 nM of scrambled or Ptger4 siRNA for 48 hours, after which Mef2d expression was examined by qPCR. n = 4 samples. Compared to scrambled control, c indicates p<0.01. (C) UMR106.01 cells were cultured with 50 nM of scrambled, Mef2c, or Mef2d siRNA for 48 hours, then with 5 µM PGE2 for 3 hours, after which time total RNA was collected and analyzed for Sost and Rpl32. n = 5 samples. Compared to target siRNA control, b indicates p<0.01 and c indicates p<0.001.

Figure 7
Figure 7. PGE2 decreases Sost without affecting BMP signaling.

(A) Cells were treated with BMP-2 (0–500 ng/mL) in the presence or absence of 5 µM PGE2 for 3 hours, after which (A) Sost or (B) Id1 expression was monitored. Compared to vehicle control, a indicates p<0.05 and b indicates p<0.01; compared to BMP-2 without PGE2; , c indicates p<0.001.

Figure 8
Figure 8. PTH decreases Sost expression independent of prostaglandins.

Cells were exposed to 1 µM indomethacin for 24 hours, then treated with 100 nM hPTH(1–34) or vehicle control for 24 further hours. Sost expression was analyzed by qPCR and normalized to Rpl32. Compared to vehicle or indomethacin control, a indicates p<0.05.

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