Transcriptional Regulation of BMP2 Expression by the PTH-CREB Signaling Pathway in Osteoblasts

Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts

"Immunoregulatory effects of extracts from the stem bark of Albizzia julibrissin Durazz

Although there are many disease modifying antirheumatic drugs (DMARDs) available, more safe and effective reagents have yet to be developed. Activated T cells make good targets for DMARD therapy. Therefore, we screened over fifty herbal extracts, and identified that water extract from the stem bark of Albizzia julibrissin selectively killed activated T cells but not naive splenocytes, in vitro. Wb3-3, obtained by further extraction and column chromatography to maximize selective activity, was used for further investigation. Annexin-V and caspase activity assay revealed that WB3-3 killed activated T cells by inducing apoptosis, which was mediated through mainly caspase-8 rather than caspase-9. The effect of WB3-3 in vivo was conlirmed by the elimination of V8- but no V6-T cells following activation with Streptococcal Enterotoxin B (SEB). Furthermore, WB3-3 inhibited neither CD25 upregulationnor proliferative response, and rather increased IL-2 secretion during Con A activation of naive T cells.Our results imply the potential of WB3-3 to include a lead compound for a new immunoregulatory drug

Suppressive Effect of AIF Extract, a water extract of three herb, on Collagen-induced Arthritis in Mice.

AIF has been formulated using three herbs known to have anti-inflammatory and anti-osteolytic effects. In this study, the potential therapeutic effects of AIF for rheumatoid arthritis were assessed in vitro and in vivo. The effects of AIF on the inflammation (TNF-α, IL-1, iNO), cartilage protection (MMP-13), and selective killing of activated T cells were examined, in vitro. In addition, the therapeutic effect of AIF was evaluated using a collagen-induced arthritis (CIA) mouse model. DBA/1 mice were immunized with type II collagen. Following booster immunization, mice were treated with the oral administration of 276 mg/kg/d AIF once a day for 18 days, then, the severity of CIA was evaluated by macroscopic scoring and histopathological assessment. AIF significantly inhibited the production of TNF-α, IL-1, iNO, and MMP-13 in a dose dependent manner in vitro. Also, AIF killed activated T cells selectively, conserving naïve T cells. The oral administration of AIF in CIA mice suppressed the progression of CIA significantly and decreased synovial hyperplasia, cartilage destruction, and bone erosion. AIF showed potent anti-inflammatory effects in vitro and substantial protective effect for the progression of CIA in vivo. These results suggest that AIF contains effective compound(s) which may modify the progression of rheumatoid arthritis

Anti-inflammatory effects of BT-201, an n-butanol extract of Panax notoginseng, observed in vitro and in a collagen-induced arthritis model

Background & aims Although there has been some success with protein-based anti-tumor necrosis factor α (TNF-α) therapeutics, the problems associated with protein-based drugs demand alternative approaches. We screened various herbal extracts for their ability to inhibit TNF-α secretions and found that BT-201, an n-butanol extract of Panax notoginseng (Burk.) F. H. Chen (P. notoginseng) has such an ability. Methods The purpose of this study has been to evaluate the anti-inflammatory and anti-rheumatic effects of BT-201. The anti-inflammatory effects were evaluated by measuring the effects of BT-201 on the production of TNF-α, interleukin (IL)-1β, inducible nitric oxide (iNO), and matrix metalloproteinase-13 (MMP-13), in vitro. The anti-rheumatic effects were evaluated by treating mice with collagen-induced arthritis (CIA) using a daily oral administration of BT-201 at 15 mg/kg/day. In addition, the effects on NF-κB and mitogen-activated protein kinase (MAPK) pathways were evaluated by Western blotting using phospho-specific antibodies. Results BT-201 significantly inhibited all the inflammatory parameters evaluated in vitro and delayed the onset and progression of CIA. BT-201 inhibited the activation of NF-κB, ERK, p38, and JNK pathways. Conclusions Our results demonstrated that BT-201 can modulate various aspects of inflammation in vitro and that it has disease-modifying, anti-rheumatic effects in vivo, suggesting that it can be a potential alternative to the current anti-TNF-α therapeutics for rheumatoid arthritis and other inflammatory disease.This work was supported by a grant from the Korean Ministry of Commerce, Industry and Energy. Some of this work was performed using facilities of the Oscotec Inc. Dr. Youngnim Choi contributed to writing of the manuscript and analysis of data. Other authors (Dr. Seon-Yle Ko, Dr. Se-Won Kim, and Dr. Jung-Keun Kim) contributed to analysis of data and provision of significant advice

Inhibition of Microtubule Assembly in Osteoblasts Stimulates Bone Morphogenetic Protein 2 Expression and Bone Formation through Transcription Factor Gli2▿

Bone morphogenetic protein 2 (BMP-2) is essential for postnatal bone formation and fracture repair. By screening chemical libraries for BMP-2 mimics using a cell-based assay, we identified inhibitors of microtubule assembly as stimulators of BMP-2 transcription. These microtubule inhibitors increased osteoblast differentiation in vitro, stimulated periosteal bone formation when injected locally over murine calvaria, and enhanced trabecular bone formation when administered systemically in vivo. To explore molecular mechanisms mediating these responses, we examined effects of microtubule inhibitors on the hedgehog (Hh) pathway, since this pathway is known to regulate BMP-2 transcription in osteoblasts and microtubules have been shown to be involved in Hh signaling in Drosophila. Here we show that in osteoblasts, inhibition of microtubule assembly increased cytoplasmic levels and transcriptional activity of Gli2, a transcriptional mediator of Hh signaling that we have previously shown to enhance BMP-2 expression in osteoblasts (M. Zhao et al., Mol. Cell. Biol. 26:6197-6208, 2006). Microtubule inhibition blocked β-TrCP-mediated proteasomal processing of Gli2 in osteoblasts. In summary, inhibition of microtubule assembly enhances BMP-2 gene transcription and subsequent bone formation, in part, through inhibiting proteasomal processing of Gli2 and increasing intracellular Gli2 concentrations