Leukemia/lymphoma-related factor (LRF) or osteoclast zinc finger protein (OCZF) overexpression promotes osteoclast survival by increasing Bcl-xl mRNA: A novel regulatory mechanism mediated by the RNA binding protein SAM68.

RANKL induces NFATc1, a key transcriptional factor to induce osteoclast-specific genes such as cathepsin K, whereas transcriptional control of osteoclast survival is not fully understood. Leukemia/lymphoma-related factor (LRF) in mouse and osteoclast zinc finger protein (OCZF) in rat are zinc finger and BTB domain-containing protein (zBTB) family of transcriptional regulators, and are critical regulators of hematopoiesis. We have previously shown that differentiation and survival were enhanced in osteoclasts from OCZF-Transgenic (Tg) mice. In the present study, we show a possible mechanism of osteoclast survival regulated by LRF/OCZF and the role of OCZF overexpression in pathological bone loss. In the in vitro cultures, LRF was highly colocalized with NFATc1 in cells of early stage in osteoclastogenesis, but only LRF expression persisted after differentiation into mature osteoclasts. LRF expression was further enhanced in resorbing osteoclasts formed on dentin slices. Osteoclast survival inhibitor such as alendronate, a bisphosphonate reduced LRF expression. Micro CT evaluation revealed that femurs of OCZF-Tg mice showed significantly lower bone volume compared to that of WT mice. Furthermore, OCZF overexpression markedly promoted bone loss in ovariectomy-induced osteolytic mouse model. The expression of anti-apoptotic Bcl-xl mRNA, which is formed by alternative splicing, was enhanced in the cultures in which osteoclasts are formed from OCZF-Tg mice. In contrast, the expression of pro-apoptotic Bcl-xs mRNA was lost in the culture derived from OCZF-Tg mice. We found that the expression levels of RNA binding splicing regulator, Src substrate associated in mitosis of 68 kDa (Sam68) protein were markedly decreased in OCZF-Tg mice-derived osteoclasts. In addition, shRNA-mediated knockdown of Sam68 expression increased the expression of Bcl-xl mRNA, suggesting that SAM68 regulates the expression of Bcl-xl. These results indicate that OCZF overexpression reduces protein levels of Sam68, thereby promotes osteoclast survival, and suggest that LRF/OCZF is a promising target for regulating pathological bone loss.

Prostate transmembrane protein androgen induced 1 is induced by activation of osteoclasts and regulates bone resorption.

Osteoclasts derived from hematopoietic cells are activated on bone surface. To resorb bone, osteoclasts release acid and lysosome acid hydrolase via membrane transport. Prostate transmembrane protein androgen induced 1 (Pmepa1) is a type I transmembrane protein that regulates proliferation, migration, and metastasis of cancer cells. Because recent reports showed that Pmepa1 is involved in membrane transport in cancer cells, we investigated the role of Pmepa1 in osteoclast function. Pmepa1 expression was barely detected in osteoclasts formed on plastic surfaces in vitro, but was markedly increased in activated osteoclasts formed on calcified matrix. Inhibitors of bone resorption, such as alendronate, bafilomycin A1, and the PI3K inhibitor LY294002, suppressed the expression of Pmepa1 in osteoclasts. Knockdown of Pmepa1 expression impaired bone resorption activity and inhibited formation of a ring-like, actin-rich podosome belt that is essential for osteoclast function. Pmepa1 protein localized to lysosomes in osteoclasts. In addition, in sites of bone destruction observed in rats with adjuvant-induced arthritis, a marked high level of Pmepa1 expression was associated with the osteoclasts' resorbing bone. Our results suggest that Pmepa1 is a critical regulator of bone resorption and is a promising marker for activated osteoclasts and a potential therapeutic target in pathologic bone destruction.-Xu, X., Hirata, H., Shiraki, M., Kamohara, A., Nishioka, K., Miyamoto, H., Kukita, T., Kukita, A. Prostate transmembrane protein androgen induced 1 is induced by activation of osteoclasts and regulates bone resorption.

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state.

A possible suppressive role of galectin-3 in upregulated osteoclastogenesis accompanying adjuvant-induced arthritis in rats.

Galectin-3 is a beta-galactoside-binding animal lectin having pleiotropic effects on cell growth, differentiation, and apoptosis. This lectin has been shown to be involved in phagocytosis by macrophages and in inflammation. Here we investigated an involvement of galectin-3 in the regulatory process of inflammatory bone resorption in rats with adjuvant-induced arthritis (AA rats) accompanying severe bone destruction in the ankle joints. The protein level of galectin-3 in the ankle-joint extracts was markedly augmented at week 3 after adjuvant injection, at the time when severe bone destruction was observed. Immunohistochemical analysis revealed an extremely high expression of galectin-3 in macrophages and granulocytes infiltrated in the area of severe bone destruction. To estimate the role of galectin-3 in osteoclastogenesis and osteoclastic bone resorption, recombinant galectin-3 was added to in vitro culture systems. Galectin-3 markedly inhibited the formation of osteoclasts in cultures of murine osteoclast precursor cell line as well as in rat bone marrow culture systems. This inhibition was not observed by heat-inactivated galectin-3 or by galectin-7. Although recombinant galectin-3 did not affect signaling through mitogen-activated protein kinase (MAPK) or nuclear factor-kappaB (NF-kappaB), it specifically suppressed the induction of nuclear factor of activated T-cells c1 (NFATc1). Galectin-3 significantly inhibited dentine resorption by mature osteoclasts in vitro. Furthermore, in vivo studies clearly showed a significant suppression of bone destruction and osteoclast recruitment accompanying arthritis, when galectin-3 was injected into the cavity of ankle joint of AA rats. Thus, abundant galectin-3 observed in the area of severe bone destruction may act as a negative regulator for the upregulated osteoclastogenesis accompanying inflammation to prevent excess bone destruction.

Infection of RANKL-primed RAW-D macrophages with Porphyromonas gingivalis promotes osteoclastogenesis in a TNF-α-independent manner.

Infection of macrophages with bacteria induces the production of pro-inflammatory cytokines including TNF-α. TNF-α directly stimulates osteoclast differentiation from bone marrow macrophages in vitro as well as indirectly via osteoblasts. Recently, it was reported that bacterial components such as LPS inhibited RANKL-induced osteoclastogenesis in early stages, but promoted osteoclast differentiation in late stages. However, the contribution to osteoclast differentiation of TNF-α produced by infected macrophages remains unclear. We show here that Porphyromonas gingivalis, one of the major pathogens in periodontitis, directly promotes osteoclastogenesis from RANKL-primed RAW-D (subclone of RAW264) mouse macrophages, and we show that TNF-α is not involved in the stimulatory effect on osteoclastogenesis. P. gingivalis infection of RANKL-primed RAW-D macrophages markedly stimulated osteoclastogenesis in a RANKL-independent manner. In the presence of the TLR4 inhibitor, polymyxin B, infection of RANKL-primed RAW-D cells with P. gingivalis also induced osteoclastogenesis, indicating that TLR4 is not involved. Infection of RAW-D cells with P. gingivalis stimulated the production of TNF-α, whereas the production of TNF-α by similarly infected RANKL-primed RAW-D cells was markedly down-regulated. In addition, infection of RANKL-primed macrophages with P. gingivalis induced osteoclastogenesis in the presence of neutralizing antibody against TNF-α. Inhibitors of NFATc1 and p38MAPK, but not of NF-κB signaling, significantly suppressed P. gingivalis-induced osteoclastogenesis from RANKL-primed macrophages. Moreover, re-treatment of RANKL-primed macrophages with RANKL stimulated osteoclastogenesis in the presence or absence of P. gingivalis infection, whereas re-treatment of RANKL-primed macrophages with TNF-α did not enhance osteoclastogenesis in the presence of live P. gingivalis. Thus, P. gingivalis infection of RANKL-primed macrophages promoted osteoclastogenesis in a TNF-α independent manner, and RANKL but not TNF-α was effective in inducing osteoclastogenesis from RANKL-primed RAW-D cells in the presence of P. gingivalis.

Development of a novel PCR method to comprehensively analyze salivary bacterial flora and its application to patients with odontogenic infections.

OBJECTIVE: The objective of this study was to develop a novel polymerase chain reaction (PCR) method to comprehensively analyze salivary bacterial flora. STUDY DESIGN: The bacterial flora in the saliva of 10 healthy persons and 11 patients with odontogenic infections were examined using a DNA extraction method with a high level of cell destruction efficiency and a novel universal primer set to amplify approximately 580 bp of the 16S rDNA sequence. RESULTS: Streptococcus (54.5%), Neisseria (14.7%), Actinomyces (8.4%), Gemella (4.1%), Granulicatella (3.8%), and Prevotella (1.4%) were dominant in a total of 1655 clones examined from the saliva of the healthy subjects. The dominant genera differed among the patients with odontogenic infections (a total of 823 clones) and were entirely different from those of the healthy subjects. CONCLUSION: This novel comprehensive salivary bacterial flora analysis method may be a useful supportive method to identify causative agents of odontogenic infections.

Mature and activated osteoclasts exist in the synovium of rapidly destructive coxarthrosis.

We compared histological and functional findings in rapidly destructive coxarthrosis (RDC) and slowly progressive osteoarthritis (OA) to investigate whether osteoclasts contribute to the extensive bone destruction observed in RDC. A histological analysis of tissue specimens from the synovium obtained from 10 cases of RDC and 40 cases with OA of the hip was performed after staining for tartrate-resistant acid phosphatase (TRAP). The cells isolated from these tissue specimens from the synovium were cultured for 24 h, and the numbers of TRAP-positive giant cells were counted. Thereafter, we performed a resorption pit formation assay by isolated cells cultured on dentine slices for 7 days. The number of TRAP-positive multinuclear giant cells present in the synovial membrane obtained from RDC patients was significantly larger than that obtained from OA patients. Large lacunar resorption pits were only seen on the dentin slices in a culture of isolated cells from RDC patients without any stimulators. This is the first report, to our knowledge, to reveal that mature and activated osteoclasts exist only in the synovium of RDC and not in the OA synovium. This result might suggest that the underlying mechanism of RDC is therefore associated with osteoclastogenesis in the synovium.

Extremely high expression of beta-actin mRNA in osteoclasts resorbing alveolar bone located at the distal area of the developing molar tooth germ in newborn rats.

Expression of beta-actin is widely utilized as an internal control of mRNA expression in various cells. Here we show evidence that the expression level of beta-actin mRNA in osteoclasts significantly differs in its intensity according to the position in bone tissues. By use of in situ hybridization, we obtained clear data showing that osteoclasts facing the distal part of a developing molar tooth germ expressed extremely high levels of beta-actin mRNA in comparison with other osteoclasts observed in the mandibular bone surface. No signal was detected when beta-nerve growth factor transcripts were observed. Electron microscopic analysis revealed that osteoclasts localized in these areas were functionally active, estimated from the expression of high levels of the osteoclast membrane antigen, Kat1 antigen, which associated with active osteoclasts. These data suggest that osteoclasts expressing extremely high levels of beta-actin are highly related to active osteoclasts induced by the mechanical stress caused by physiological movement of molar tooth germ towards distal directions in the developing mandible.