(-)-Epigallocatechin gallate but not chlorogenic acid upregulates osteoprotegerin synthesis through regulation of bone morphogenetic protein-4 in osteoblasts.

Chlorogenic acid (CGA) is a primary phenolic component of coffee and (-)-epigallocatechin gallate (EGCG) is a primary flavonoid component of green tea, both of which have been documented to possess beneficial health properties. A previous study by the present authors demonstrated that p38 mitogen-activated protein kinase (MAPK) may be associated with osteoprotegerin synthesis stimulated by bone morphogenetic protein-4 (BMP-4) in osteoblast-like MC3T3-E1 cells. In the present study, the effects of CGA and EGCG on BMP-4-stimulated osteoprotegerin synthesis in MC3T3-E1 cells were investigated. It was observed that CGA had no effect on osteoprotegerin release stimulated by BMP-4, whereas EGCG significantly enhanced BMP-4-stimulated osteoprotegerin release (P=0.003). Levels of osteoprotegerin mRNA expression induced by BMP-4 were also significantly increased by EGCG (P=0.03). By contrast, EGCG had no significant effect on phosphorylation of Smad1 or p38 MAPK induced by BMP-4. In addition, EGCG had little effect on BMP-induced phosphorylation of p70 S6 kinase; however rapamycin, as an inhibitor of p70 S6 kinase, significantly suppressed osteoprotegerin release (P=0.007). These data suggest that EGCG but not CGA may upregulate the synthesis of osteoprotegerin induced by BMP-4 in osteoblasts.

Attenuation of prostaglandin E1­induced osteoprotegerin synthesis in osteoblasts by normoxic HIF inducers.

Mimosine, which is a natural plant amino acid present in the Leucaena genus, is able to induce hypoxia­inducible factors (HIFs). Previous evidence has indicated that HIF regulates angiogenesis­osteogenesis coupling in bone metabolism, and it has previously been reported that mimosine inhibits prostaglandin (PG)F2α­induced osteoprotegerin (OPG) synthesis without affecting interleukin­6 (IL­6) production in osteoblast­like MC3T3­E1 cells. In addition, PGE1 has been demonstrated to induce OPG synthesis via activation of p38 mitogen­activated protein (MAP) kinase and stress­activated protein kinase/c­Jun N­terminal kinase (SAPK/JNK) in these cells, and PGE1 stimulates IL­6 production via the activation of protein kinase A. In the present study, the effects of mimosine on the PGE1­stimulated synthesis of OPG and IL­6 were investigated in osteoblast­like MC3T3­E1 cells. The concentrations of OPG and IL­6 were measured using relevant ELISA kits. OPG mRNA was measured by semi­quantitative reverse transcription polymerase chain reaction. The phosphorylation of p38 MAP kinase and SAPK/JNK was analyzed by western blotting. Mimosine significantly reduced PGE1­induced release of OPG and OPG mRNA expression levels without affecting the release of IL­6. In addition, deferoxamine, which is also a normoxic HIF inducer, significantly inhibited PGE1­induced OPG release and OPG mRNA expression levels; however, it had little effect on IL­6 release. Furthermore, mimosine and deferoxamine failed to affect PGE1­stimulated phosphorylation of p38 MAP kinase or SAPK/JNK. These results strongly suggest that normoxic HIF inducers attenuate PGE1­stimulated OPG synthesis without affecting IL­6 production in osteoblasts.

(-)-Epigallocatechin gallate synergistically potentiates prostaglandin E2-stimulated osteoprotegerin synthesis in osteoblasts.

(-)-Epigallocatechin gallate (EGCG), the most abundant flavonoid in green tea, and chlorogenic acid, the main polyphenol found in coffee, attract significant attention owing to health benefits. We have previously demonstrated that prostaglandin E2 (PGE2) stimulates osteoprotegerin synthesis through the activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of EGCG or chlorogenic acid on the PGE2-stimulated osteoprotegerin synthesis in MC3T3-E1 cells. EGCG significantly amplified the PGE2-induced release. EGCG markedly enhanced the expression levels of osteoprotegerin mRNA induced by PGE2. On the contrary, chlorogenic acid had no effect on the PGE2-stimulated release of osteoprotegerin. EGCG significantly strengthened the PGE2-induced phosphorylation of p38 MAP kinase and SAPK/JNK, whereas chlorogenic acid failed to affect them. BIRB0796 and SP600125, a p38 MAP kinase inhibitor and a SAPK/JNK inhibitor, respectively, markedly reduced the amplification by EGCG of the PGE2-stimulated osteoprotegerin release. These results strongly suggest that EGCG synergistically enhances the PGE2-stimulated osteoprotegerin synthesis via potentiation of p38 MAP kinase and SAPK/JNK in osteoblasts. Our present findings could present a new significant aspect in the favorable effect of EGCG on the prevention of osteoporotic bone loss and fracture especially in elderly people since osteoprotegerin secreted from osteoblasts is well-recognized to act as a suppressor of osteoclastic bone resorption.

Mimosine suppresses the PGF2α-induced synthesis of osteoprotegerin but not interleukin-6 in osteoblasts.

Mimosine, a plant amino acid, is known to act as a normoxic inducer of hypoxia-inducible factor (HIF). Previous research has suggested that HIF plays important roles in angiogenesis-osteogenesis coupling and bone metabolism. We previously reported that prostaglandin F2α (PGF2α) induced osteoprotegerin synthesis through p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. We have also demonstrated that PGF2α induced the synthesis of interleukin-6 (IL-6) via p38 MAP kinase and p44/p42 MAP kinase but not SAPK/JNK in these cells. In the present study, we investigated the effects of mimosine on the PGF2α-induced synthesis of osteoprotegerin or IL-6 in MC3T3-E1 cells. We found that deferoxamine, another inducer of HIF, as well as mimosine, upregulated the protein levels of HIF-1α. Both mimosine and deferoxamine significantly suppressed the PGF2α-induced release of osteoprotegerin, and the mRNA expression level, without markedly affecting PGF2α-induced IL-6 release. Both mimosine and deferoxamine, by themselves, induced the release of vascular endothelial growth factor. The phosphorylation of p38 MAP kinase, p44/p42 MAP kinase or SAPK/JNK induced by PGF2α was not markedly affected by either mimosine or deferoxamine. Thus, the results of the present study strongly suggest that mimosine, a normoxic inducer of HIF, inhibits the PGF2α­induced osteoprotegerin synthesis without affecting the IL-6 synthesis in osteoblasts.

Amplification by (-)-epigallocatechin gallate and chlorogenic acid of TNF-α-stimulated interleukin-6 synthesis in osteoblasts.

Polyphenolic compounds in foods and beverages have beneficial effects on human health. (-)-Epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), a major flavonoid in green tea and a major phenolic acid in coffee, respectively, have potent properties, including antioxidative effects. Our previous study demonstrated that p70 S6 kinase acts as a negative regulator in tumor necrosis factor-α (TNF-α)-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, the effects of EGCG and CGA on the TNF-α-stimulated interleukin­6 synthesis were investigated in MC3T3­E1 cells. EGCG and CGA significantly enhanced TNF-α-stimulated interleukin-6 release. In addition, the interleukin-6 mRNA expression levels induced by TNF­α were supported by EGCG, as well as CGA. EGCG markedly attenuated the TNF-α-induced phosphorylation of p70 S6 kinase whereas CGA failed to affect the phosphorylation. These results strongly suggest that EGCG and CGA enhance the TNF-α-stimulated interleukin-6 synthesis in osteoblasts, and that the amplifying effect of EGCG, but not CGA, is exerted via inhibiting p70 S6 kinase.

PGD2 stimulates osteoprotegerin synthesis via AMP-activated protein kinase in osteoblasts: Regulation of ERK and SAPK/JNK.

AMP-activated protein kinase (AMPK), a key enzyme sensing cellular energy metabolism, is currently known to regulate multiple metabolic pathways. Osteoprotegerin plays a pivotal role in the regulation of bone metabolism by inhibiting osteoclast activation. We have previously reported that prostaglandin D2 (PGD2) stimulates the synthesis of osteoprotegerin through the activation of p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. On the basis of these findings, we herein investigated the implication of AMPK in PGD2-stimulated osteoprotegerin synthesis in these cells. PGD2 induced the phosphorylation of AMPKα (Thr-172) and AMPKß (Ser-108), and the phosphorylation of acetyl-coenzyme A carboxylase, a direct AMPK substrate. Compound C, an AMPK inhibitor, which suppressed the phosphorylation of acetyl-coenzyme A carboxylase, significantly attenuated both the release and the mRNA levels of osteoprotegerin stimulated by PGD2. The PGD2-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK but not p38 MAP kinase were markedly inhibited by compound C. These results strongly suggest that AMPK regulates the PGD2-stimulated osteoprotegerin synthesis at a point upstream of p44/p42 MAP kinase and SAPK/JNK in osteoblasts.