Improvement of atopic dermatitis in NC/Nga mice by topical application of CpG phosphodiester-ODN.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with pruritic and eczematous lesions characterized by increased total IgE level, inflammatory cell infiltration, and the elevated expression of Th2 cytokines. Synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides are known to have immunostimulatory activities in mice and to convert from Th2 to Th1 immune responses in AD. Previous work has shown clinical effectiveness of CpG phosphorothioate-ODN in AD mice model. However, due to longer in vivo half-life and the possibility of causing unwanted side effects, therapeutic use of CpG phosphorothioate-ODN can be limited. Thus, we investigated the efficacy of CpG phosphodiester-ODN with a novel sequence in NC/Nga mice. Topical application of phosphodiester-ODN penetrated rapidly from epidermis to the lymph nodes, accompanied by reduced infiltration of inflammatory cells and decreased number of cells expressing cytokines such as IL-4, IL-10 and IFN-gamma. Furthermore, the expression of IFN-gamma was reduced in the CpG ODNs-treated NC/Nga mice while the expression of IL-12p40 was increased, suggesting stimulation of Th1 immune response. The expression of IL-10 was strongly reduced, which meant the suppression of Th2 immune response in NC/Nga mice, accompanied by reduced level of IgE and IgG1, but increased level of IgG2a in sera. Since phosphodiester-ODN has been shown to cause minimum side effect comparing its phosphorothioate counterpart, it is proposed to become a new therapeutic modality for AD.

Homocysteine enhances bone resorption by stimulation of osteoclast formation and activity through increased intracellular ROS generation.

UNLABELLED: Hyperhomocystinemia is a modifiable risk factor for osteoporosis and fracture. Physiologic concentrations of Hcy directly activate osteoclast formation and activity through stimulation of p38 MAPK and integrin beta3. The effects of Hcy were mediated by generation of intracellular ROS. INTRODUCTION: Hyperhomocysteinemia is a modifiable risk factor for osteoporosis and its related bone fractures. It has been reported that bone resorption and turnover rate were increased in hyperhomocystinemia. Using mouse bone marrow cells, we examined the direct effects of homocysteine (Hcy) on osteoclast formation and activity. MATERIALS AND METHODS: Osteoclast formation was determined by TRACP staining and TRACP activity. Intracellular reactive oxygen species (ROS) generation was measured using a fluorescent probe, dichlorodihydrofluorescein diacetate. Intracellular signaling cascades of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-kappaB were measured by Western blotting. Integrin beta3 mRNA levels were measured by RT-PCR. Actin ring formation and bone resorption assays were also performed. RESULTS: Physiologic concentrations of Hcy upregulated TRACP+ multinucleated cells and TRACP activity, stimulated actin ring formation, and increased the number of nuclei per cell and the level of expression of integrin beta3 mRNA. In addition, Hcy increased bone resorption and stimulated p38 MAPK activity and intracellular reactive oxygen species (ROS) generation. All of these Hcy-induced changes were blocked by pretreatment with the antioxidant, N-acetyl cysteine. CONCLUSIONS: Hcy directly activates osteoclast formation and activity through increased generation of intracellular ROS. These findings suggest that, in individuals with mild to moderate hyperhomocystinemia, increased bone resorption by osteoclasts may contribute to osteoporosis and that an antioxidant may attenuate bone loss in these individuals.

Homocysteine enhances apoptosis in human bone marrow stromal cells.

INTRODUCTION: High plasma homocysteine (Hcy) levels have been associated with increased risk of fracture. Since Hcy has been shown to induce apoptosis in many cell types, including vascular endothelial cells, we hypothesized that Hcy would have a similar apoptotic effect on osteoblasts, leading to osteoporosis by reducing bone formation. MATERIALS AND METHODS: Using primary human bone marrow stromal cells (hBMSC) and HS-5 cell line (human bone marrow stromal cell line), we investigated the effects of Hcy on these cells by cell viability assay and analysis of cytoplasmic histone-associated DNA fragments. Caspase activity assay, Western blots, and electrophoresis mobility shift assay (EMSA) were performed to find the mechanism of apoptosis. Intracellular reactive oxygen species (ROS) were measured by spectrometry using dichlorofluorescein diacetate, and cellular total glutathione level was determined by a commercially available kit. N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) were used as tools for investigating the role of ROS and nuclear factor-kappaB (NF-kappaB), respectively. RESULTS: Hcy induced apoptosis in primary human bone marrow stromal cells and the HS-5 cell line, and this apoptotic effect was caspase-dependent. In addition, Hcy increased cytochrome c release into the cytosol, and activated caspase-9 and caspase-3, but not caspase-8, indicating that Hcy induces apoptosis via the mitochondria pathway. Hcy increased ROS, and NAC inhibited the apoptotic effect of Hcy. Western blot and EMSA showed that Hcy activated the NF-kappaB pathway. PDTC blocked Hcy-induced caspase-3 activation and apoptosis. CONCLUSION: These results suggest that Hcy induces apoptosis via the ROS-mediated mitochondrial pathway and NF-kappaB activation in hBMSCs, and that Hcy may contribute to the development of osteoporosis by reducing bone formation. Antioxidants may have a role in preventing bone loss in individuals with hyperhomocysteinemia.

AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts.

INTRODUCTION: AMP-activated protein kinase (AMPK) has been reported to stimulate differentiation and proliferation of osteoblasts, but the role of AMPK in the physiology of osteoclasts has not been investigated. METHOD: Osteoclasts were differentiated from mouse BMMϕs. TRAP-positive multinucleated cells were considered to be osteoclasts using TRAP staining, and resorption area was determined by incubation of cells on dentine discs. Signaling pathways were investigated using Western blotting and RT-PCR. RESULTS: RANKL induced phosphorylation/activation of AMPK-α in BMMϕs and stimulated formation of TRAP-positive multinucleated cells. Pharmacological inhibition of AMPK with compound C and siRNA-mediated knockdown of AMPK-α1, the predominant α-subunit isoform in BMMϕs, increased RANKL-induced formation of TRAP-positive multinucleated cells and bone resorption via activation of the downstream signaling elements p38, JNK, NF-κB, Akt, CREB, c-Fos, and NFATc1. STO-609, an inhibitor of CaMKK, completely blocked the RANKL-induced activation of AMPK-α, but KN-93, an inhibitor of CaMK, did not. siRNA-mediated TAK1 knockdown also blocked RANKL-induced activation of AMPK-α. The AMPK activators metformin, (-)-epigallocatechin-3-gallate, berberine, resveratrol, and α-lipoic acid dose-dependently suppressed formation of TRAP-positive multinucleated cells and bone resorption. CONCLUSION: AMPK negatively regulates RANKL, possibly by acting through CaMKK and TAK1. Thus, the development of AMPK activators may be a useful strategy for inhibiting the resorption of bone that is stimulated under RANKL-activated conditions.

Increased circulating heat shock protein 60 induced by menopause, stimulates apoptosis of osteoblast-lineage cells via up-regulation of toll-like receptors.

Postmenopausal osteoporosis is a heterogeneous disorder characterized by accelerated bone loss after natural or surgical menopause and an increased risk of fractures. The bone loss in estrogen deficiency results from the increased bone resorption and impaired ability of osteoblastic bone formation. Previous studies have reported that the HSP60 stimulates osteoclast formation and bone resorption. Here we found that plasma HSP60 levels were significantly higher in postmenopausal (median 1152.4 ng/ml; range 724.7-2123.4 ng/ml) than in premenopausal (median 316.3 ng/ml; range 164.6-638.4 ng/ml) women. In primary human bone marrow stromal cells (hBMSC) and the HS-5 hBMSC cell line, HSP60 significantly reduced cell viability and increased caspase-dependent apoptosis. Consistent with these observations, HSP60 activated caspase-3 and -9, but not caspase-8 in HS-5 cells, and increased the release of mitochondrial cytochrome c into the cytosol. In addition, HSP60 activated p38 and NFkappaB, but not ERK or JNK; importantly, inhibitors of p38 (SB203580) and NFkappaB (PDTC) abolished HSP60-induced apoptosis. Furthermore, Western blotting showed that HSP60 up-regulated TLR-2 and TLR-4 expression, and pretreatment with blocking antibodies for TLR-2 and TLR-4 almost completely eliminated the effects of HSP60 on apoptosis, caspase-3 and -9 activation, and activation of NFkappaB and p38 MAPK. Most notably, ovariectomy-induced bone loss was attenuated in TLR-2 KO mice. In conclusion, up-regulation of TLR-2 by HSP60 may play a critical role in promoting bone loss in the estrogen-deficient state.

Heat shock protein 60 causes osteoclastic bone resorption via toll-like receptor-2 in estrogen deficiency.

Estrogen deficiency leads to marked increases in osteoclastic bone resorption, but the exact mechanism is unclear. Proteomic analysis was performed on the femur and tibia of ovariectomy (OVX) and sham-operated Sprague-Dawley rats using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer (MS). Among the nine proteins differentially expressed between OVX and sham-operated rats, heat shock protein 60 (HSP60) was upregulated by 2.6-fold in the bones of OVX rats, and the plasma concentration of HSP60 was also significantly increased in OVX rats. Estrogen deficiency increases in secretions of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha in T cell and osteoclasts (OCs) lineages, IL-1beta and TNF-alpha stimulated the production and secretion of HSP60 from OCs lineages. IL-1 receptor antagonist (ra), TNF-blocking antibody (Ab), and estradiol (E(2)) significantly suppressed the OVX-induced increase in plasma concentrations of HSP60 in mice. HSP60 potentiated OC formation and bone resorption, and pretreatment with HSP60-blocking Ab markedly reduced the potentiation of OC formation and bone resorption by IL-1beta- and TNF-alpha. HSP60 upregulated the expression levels of toll-like receptor (TLR)-2 in bone marrow macrophage (BMMvarphi), and pretreatment with a TLR-2-blocking Ab almost completely inhibited HSP60- or cytokine-induced potentiation of OC formation and/or bone resorption. In conclusion, HSP60 and TLR-2 are novel mediators of estrogen-deficiency-induced bone loss.