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. 2017 May 22;7(1):2243.
doi: 10.1038/s41598-017-02650-y.

Prostaglandin E2 inhibits matrix mineralization by human bone marrow stromal cell-derived osteoblasts via Epac-dependent cAMP signaling

Ali Mirsaidi  1 , André N Tiaden  1 , Peter J Richards  2   3
Affiliations

Prostaglandin E2 inhibits matrix mineralization by human bone marrow stromal cell-derived osteoblasts via Epac-dependent cAMP signaling

Ali Mirsaidi et al. Sci Rep. .

Abstract

The osteoinductive properties of prostaglandin E2 (PGE2) and its signaling pathways have led to suggestions that it may serve as a potential therapeutic strategy for bone loss. However, the prominence of PGE2 as an inducer of bone formation is attributed primarily to findings from studies using rodent models. In the current study, we investigated the effects of PGE2 on human bone marrow stromal cell (hBMSC) lineage commitment and determined its mode of action. We demonstrated that PGE2 treatment of hBMSCs significantly altered the expression profile of several genes associated with osteoblast differentiation (RUNX2 and ALP) and maturation (BGLAP and MGP). This was attributed to the activation of specific PGE2 receptors, and was associated with increases in cAMP production and sustained AKT phosphorylation. Pharmacological inhibition of exchange protein directly activated by cAMP (Epac), but not protein kinase A (PKA), recovered the mineralization functions of hBMSC-derived osteoblasts treated with PGE2 and restored AKT phosphorylation, along with the expression levels of RUNX2, ALP, BGLAP and MGP. Our findings therefore provide insights into how PGE2 influences hBMSC-mediated matrix mineralization, and should be taken into account when evaluating the role of PGE2 in human bone metabolism.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
PGE2 inhibits hBMSC-mediated matrix mineralization. (A) Alizarin Red S staining was used to assess the influence of continuous PGE2 treatment on matrix mineralization in hBMSC cultures at 14 and 16 days post-osteogenic induction. *p < 0.01, **p < 0.001 as compared to untreated hBMSCs using ANOVA. (B,C) RT-qPCR was used to determine expression levels of osteogenic differentiation markers RUNX2 and ALP at day 3, 7 and 17 post-osteogenic induction (B), and markers of osteoblast maturation and/or matrix mineralization BGLAP, MGP and SPP1 at day 17 post-osteogenic induction (C). Data were normalized to GUSB and expressed as fold change as compared to non-induced controls at day 0 (value 1) using the comparative C T method. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to untreated hBMSCs using ANOVA. The data represent triplicate determinations and were replicated at least two times. All values are presented as mean ± S.D.
Figure 2
Figure 2
Effect of PGE2 on hBMSC-mediated matrix mineralization is dependent on differentiation stage of hBMSC osteogenesis and duration of exposure. hBMSCs were treated with PGE2 (10 nM) for varying durations beginning at the initiation of osteogenic induction (Day 0) (A), or at selected time points thereafter (B), and matrix mineralization quantified at day 16 by Alizarin Red S staining. *p < 0.01, **p < 0.001 as compared to hBMSCs induced to undergo osteogenesis for 14 days in the absence of PGE2 (control) using ANOVA. The data represent triplicate determinations and were replicated at least two times. All values are presented as mean ± S.D.
Figure 3
Figure 3
PGE2 enhances hBMSC adipogenesis. (A) Oil Red O staining was used to assess the influence of continuous PGE2 treatment on triglyceride accrual in hBMSC cultures at day 17 post-adipogenic induction. *p < 0.001 as compared to untreated hBMSCs using ANOVA. (B) RT-qPCR was used to determine expression levels of adipogenic markers PPARG, FABP4 and CD36 in hBMSCs at day 17 post-adipogenic induction. Data were normalized to GUSB and expressed as fold change as compared to non-induced controls at day 0 (value 1) using the comparative C T method. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to untreated hBMSCs using ANOVA. The data represent triplicate determinations and were replicated at least two times. All values are presented as mean ± S.D.
Figure 4
Figure 4
PGE2 effects are mediated through specific PGE2 receptor subtypes. (A) hBMSC were cultured in growth medium (GM) or osteogenic medium (OM), and PGE2 receptor gene expression levels determined at day 7 and 14 using RT-qPCR. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to GM using Student’s t-test. (B) RT-qPCR was used to assess the short-term effects (48 h) of siRNA specific for PTGER2 (siPTGER2), PTGER4 (siPTGER4) or both PTGER2 and PTGER4 (siPTGER2/4) on PTGER2 and PTGER4 gene expression in hBMSCs undergoing osteogenesis. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to cells treated with scrambled control siRNA (siControl) using ANOVA. (C) RT-qPCR was used to assess the long-term effects (3, 7 and 10 days) of siRNA specific for both PTGER2 and PTGER4 (siPTGER2/4) on PTGER2 and PTGER4 gene expression in hBMSCs undergoing osteogenesis. *p < 0.001 as compared to cells treated with scrambled control siRNA (siControl) using Student’s t-test. (D) The effects of continuous PGE2 (10 nM) treatment on matrix mineralization in siRNA-treated hBMSCs was assessed at day 15 by Alizarin Red S staining. *p < 0.01, **p < 0.001, as compared to untreated hBMSCs (−PGE2) using Student’s t-test. The data represent triplicate determinations and were replicated at least two times. All values are presented as mean ± S.D.
Figure 5
Figure 5
Inhibitory effects of PGE2 on hBMSC-mediated matrix mineralization are independent of dexamethasone. (A) hBMSC were cultured in growth medium (GM), or osteogenic medium (OM) supplemented with (+Dex) or without (−Dex) dexamethasone, and PTGER2 and PTGER4 gene expression levels determined at day 10 using RT-qPCR. *p < 0.05, **p < 0.001 as compared to GM; # p < 0.001 as compared to OM (−Dex) using ANOVA. (B) The effect of continuous PGE2 (10 nM) treatment on hBMSC-mediated matrix mineralization in the presence or absence of dexamethasone was determined at day 15 by Alizarin Red S staining. *p < 0.001 as compared to untreated hBMSCs (−PGE2) using Student’s t-test. The data represent triplicate determinations and were replicated at least two times. All values are presented as mean ± S.D.
Figure 6
Figure 6
PGE2 inhibits hBMSC-mediated matrix mineralization via Epac-dependent cAMP signaling. (A) Intracellular cAMP levels were measured in hBMSCs treated with PGE2 (100 nM) for 2, 5 and 15 min. *p < 0.01, **p < 0.001, as compared to untreated cells using ANOVA. (B) hBMSCs undergoing osteogenic differentiation were treated continuously with cAMP analog 8-Br-cAMP, and matrix mineralization quantified at day 14 by Alizarin Red S staining. *p < 0.01, **p < 0.001 as compared to control using ANOVA. (C) hBMSCs were continuously cultured in the absence (control) or presence (PGE2) of PGE2 (10 nM) with or without Epac inhibitor ESI-09 (10 μM) or PKA inhibitor PKI (10 μM), and matrix mineralization quantified at day 14 by Alizarin Red S staining. *p < 0.001 as compared to control using ANOVA. (D) hBMSCs undergoing osteogenic differentiation were treated continuously with cAMP analog 8-pCPT-2-O-Me-cAMP (8-pCPT) (50 μM), and matrix mineralization quantified at day 14 and 16 by Alizarin Red S staining. *p < 0.01, **p < 0.001 as compared to control (−8-pCPT) using Student’s t-test. The data represent triplicate determinations and were replicated at least two times. All values are presented as mean ± S.D.
Figure 7
Figure 7
PGE2 activates AKT in an Epac-dependent manner. (A) hBMSCs were cultured continuously in the absence (−PGE2) or presence (+PGE2) of PGE2 (10 nM), and AKT phosphorylation levels determined by Western blot analysis at day 7, 8 and 9. *p < 0.01 as compared to −PGE2 using Student’s t-test. (B) hBMSCs were cultured continuously in the absence (−PGE2) or presence (+PGE2) of PGE2 (10 nM) with or without Epac inhibitor ESI-09 (10 μM) or PKA inhibitor PKI (10 μM), and AKT phosphorylation levels determined by Western blot analysis at day 9. *p < 0.01, **p < 0.001 as compared to -PGE2 using ANOVA. # p < 0.01, ## p < 0.001 using ANOVA. In both cases, GAPDH served as a loading control and representative cropped blots shown. (C) hBMSCs were cultured continuously in the absence (−PGE2) or presence (+PGE2) of PGE2 (10 nM) with or without Epac inhibitor ESI-09 (10 μM), and RUNX2, ALP, BGLAP and MGP expression levels determined by RT-qPCR at day 17. *p < 0.05, **p < 0.001 as compared to -PGE2 using ANOVA. # p < 0.01, ## p < 0.001 using ANOVA. The data represent triplicate determinations and were replicated at least two times. All values are presented as mean ± S.D.
Figure 8
Figure 8
Proposed mechanism by which PGE2 exerts its influence over hBMSC-mediated matrix mineralization. Based on the findings presented in the current report, we propose that PGE2 increases intracellular cAMP levels via receptors EP2 and EP4, leading to activation of Epac, which in turn acts to sustain AKT phosphorylation levels. Prolonged AKT activation ultimately results in impaired hBMSC-derived osteoblast maturation and matrix mineralization, possibly by inducing temporal changes in the production and activity of RUNX2 and its downstream target genes (e.g. ALP, BGLAP and MGP). The signaling pathways responsible for mediating the stimulatory effects of PGE2 on hBMSC adipogenesis still remain to be determined.
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