Tramadol enhances PGF2α-stimulated osteoprotegerin synthesis in osteoblasts

Abstract

Osteoprotegerin (OPG) synthesized by osteoblasts is currently considered a crucial regulator to suppress the formation and function of osteoclasts. We previously showed that the synthesis of OPG is stimulated by prostaglandin F_2α (PGF_2α) in the involvement of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. We also found that Rho-kinase is involved in the signaling of PGF_2α upstream of p38 MAPK in these cells. Tramadol is widely used to treat chronic pain, such as low back pain associated with osteoporosis. We investigated whether or not tramadol affects the OPG release induced by PGF_2α in osteoblast-like MC3T3-E1 cells. The levels of OPG in the conditioned medium were measured by an enzyme-linked immunosorbent assay. The mRNA expression of OPG was determined with real-time reverse transcription polymerase chain reaction. The phosphorylation of target protein was determined with a Western blot analysis. PGF_2α induced the release and the mRNA expression of OPG, which tramadol significantly enhanced. Morphine, a selective μ-opioid receptor (MOR) agonist, also enhanced the PGF_2α-induced OPG release. In addition, naloxone, a MOR antagonist, suppressed the enhancement by tramadol or morphine of the PGF_2α-induced OPG synthesis. Tramadol upregulated the phosphorylation of SAPK/JNK and p38 MAPK stimulated by PGF_2α but not that of p44/p42 MAPK or myosin phosphatase targeting protein (MYPT), a substrate of Rho-kinase. The inhibitors of both p38 MAPK and SAPK/JNK, SB203580 and SP600125, respectively, reduced the tramadol amplification of OPG release stimulated by PGF_2α. The present results strongly suggest that tramadol enhances the synthesis of OPG stimulated by PGF_2α through MOR in osteoblasts, and that the amplifying effect is exerted at upstream of p38 MAPK and SAPK/JNK but downstream of Rho-kinase.

Keywords: Biochemistry; Bone; Cell biology; MAP kinase; Osteoblast.; Osteoprotegerin; PGF2α; Pain management; Pharmaceutical chemistry; Pharmaceutical science; Pharmacology; Tramadol; μ-opioid receptor.

Figure 1

Effects of tramadol on the release and mRNA expression of OPG stimulated by PGF_2αin MC3T3-E1 cells. (A) The cells pretreated with 0, 10, 30, 100 and 200 μM of tramadol for 60 min were stimulated by 10 μM PGF_2α or vehicle for 48 h. The concentrations of OPG in the conditioned medium were determined. Each value represents the mean ± S.E.M. of three-fold determinations from three independently prepared cells. ∗p < 0.05, in comparison to the PGF_2α value alone. (B) The cells pretreated with 300 μM tramadol or vehicle for 60 min were stimulated by 10 μM PGF_2α or vehicle for 3 h. Thereafter, the total RNA was isolated in each and transcribed to cDNA. The expression of OPG mRNA and that of GAPDH mRNA were quantified by quantitative RT-PCR. We normalized the OPG mRNA levels to GAPDH mRNA levels. Each value represents the mean ± S.E.M. of three-fold determinations from three independently prepared cells. ∗p < 0.05 compared to the control value. ∗∗p < 0.05 compared to the PGF_2α value alone.

Figure 2

Effects of morphine on the PGF_2α-stimulated OPG release in MC3T3-E1 cells. The cells pretreated with 0, 10, 30, 100 and 300 μM of morphine for 60 min were stimulated by 10 μM PGF_2α or vehicle for 48 h. The concentrations of OPG in the conditioned medium were determined. Each value represents the mean ± S.E.M. of three-fold determinations from three independently prepared cell preparations. ∗p < 0.05 compared to the PGF_2α value alone.

Figure 3

Effects of naloxone on the amplification by tramadol or morphine of the PGF_2α-stimulated OPG release in MC3T3-E1 cells. The cells pre-incubated with (A) 200 μM naloxone or vehicle, (B) 300 μM naloxone or vehicle for 60 min were subsequently pretreated with (A) 200 μM tramadol or vehicle, (B) 200 μM morphine or vehicle for 60 min. The treated cells were then stimulated by 10 μM PGF_2α or vehicle for 48 h. The concentrations of OPG in the conditioned medium were determined. Each value represents the mean ± S.E.M. of three-fold determinations from three independently prepared cells. ∗p < 0.05 compared to the control value. ∗∗p < 0.05 compared to the PGF_2α value alone. ∗∗∗p < 0.05 compared to the value of the combination of (A) tramadol and PGF_2α, (B) morphine and PGF_2α.

Figure 4

Effects of tramadol on the PGF_2α-stimulated phosphorylation of p44/p42 MAPK, p38 MAPK, SAPK/JNK and MYPT in MC3T3-E1 cells. The cells pretreated with 0, 100, 200, and 250 μM of tramadol or vehicle for 60 min were stimulated by 10 μM PGF_2α or vehicle for (A) 20 min, (B) 10 min, (C) 20 min or (D) 2 min. The extracts of cells were subjected to SDS-PAGE with a subsequent Western blot analysis with antibodies against (A) phospho-specific p44/p42 MAPK or p44/p42 MAPK, (B) phospho-specific p38 MAPK or p38 MAPK, (C) phospho-specific SAPK/JNK or SAPK/JNK and (D) phospho-specific MYPT or GAPDH. The histogram shows the quantitative representation of the levels of PGF_2α-induced phosphorylation with a laser densitometric analysis. The levels indicate the fold increase from the basal levels (lane 1). Each value represents the mean ± S.E.M. of three-fold determinations from three independently prepared cells. ∗p < 0.05, in comparison to the control value (Lane 1). ∗∗p < 0.05, in comparison to the PGF_2α alone value (Lane 2). Full, non-adjusted images of Figure 4A, Figure 4B, Figure 4C and Figure 4D are provided as supplementary materials.

Figure 5

Effects of SB203580 on the amplification by tramadol or morphine of the PGF_2α-stimulated OPG release in MC3T3-E1 cells. The cells pre-incubated with (A) 5 μM SB203580 or vehicle (B) 7 μM SB203580 or vehicle for 60 min were pretreated with (A) 200 μM tramadol or vehicle (B) 300 μM morphine or vehicle for a further 60 min. The treated cells were subsequently stimulated by 10 μM PGF_2α or vehicle for 48 h. The concentrations of OPG in the conditioned medium were determined. Each value represents the mean ± S.E.M. of three-fold determinations from three independently prepared cells. ∗p < 0.05 compared to the control value. ∗∗p < 0.05 compared to the PGF_2α value alone. ∗∗∗p < 0.05 compared to the value of the combination of (A) tramadol and PGF_2α or (B) morphine and PGF_2α.

Figure 6

Effects of SP600125 on the amplification by tramadol and morphine of the PGF_2α-stimulated OPG release in MC3T3-E1 cells. The cells pre-incubated with 10 μM SP600125 or vehicle for 60 min were pretreated with (A) 200 μM tramadol or vehicle or (B) 200 μM morphine or vehicle for a further 60 min. The treated cells were subsequently stimulated by 10 μM PGF_2α or vehicle for 48 h. The OPG concentrations in the conditioned medium were determined. Each value represents the mean ± S.E.M. of three-fold determinations from three independently prepared cells. ∗p < 0.05 compared to the control value. ∗∗p < 0.05 compared to the PGF_2α alone value. ∗∗∗p < 0.05 compared to the value of the combination of (A) tramadol and PGF_2α or (B) morphine and PGF_2α.

Figure 7

A schematic illustration of the regulatory mechanism of tramadol in PGF2α-stimulated in osteoblasts.

supplementary material