The RB-IL-6 axis controls self-renewal and endocrine therapy resistance by fine-tuning mitochondrial activity.

Retinoblastoma (RB) protein inactivation during tumor progression is often associated with acquisition of immature phenotypes and resistance to therapy. Determination of an RB inactivation signature in a context of gaining undifferentiated phenotype in a p53-null sarcoma system revealed a critical role for interleukin (IL)-6. Using a Gene Set Enrichment Analysis (GSEA), we discovered that poorly differentiated breast cancers are enriched for this RB inactivation signature. Accelerated IL-6 secretion following RB inactivation in an RB-intact luminal-type breast cancer cell line MCF-7 promoted a positive feed forward loop between IL-6 and STAT3 driving tumor growth and endocrine therapy resistance. In addition, some of RB-intact basal-like type breast cancer cell lines exhibited a similar phenotype following RB depletion. The mechanism whereby RB inactivation increases IL-6 production in MCF-7 cells appeared to involve fatty acid oxidation (FAO)-dependent mitochondrial metabolism and c-Jun NH(2)-terminal kinase (JNK). In addition, IL-6, via STAT3-mediated feedback to mitochondria, autonomously adjusts mitochondrial superoxide to levels suitable to maintain stem cell-like activity. The gene expression profile of luminal-type breast cancer patients with low RB expression revealed high enrichment of genes involved in mitochondrial respiration and downstream targets of IL-6. These findings unveiled an unexpected strategy whereby RB suppresses malignant features of cancer cells through metabolic reprogramming and cell-autonomous inflammation.

Relationship of Porphyromonas gingivalis with glycemic level in patients with type 2 diabetes following periodontal treatment.

INTRODUCTION: The aim of this study was to assess the relationship between serum glycemic levels and subgingival microbial profile alteration following periodontal treatment in patients with type 2 diabetes mellitus. METHODS: We studied 30 periodontitis patients with type 2 diabetes mellitus who received full-mouth subgingival debridement by analyzing their subgingival microbial profiles using a polymerase chain reaction method at baseline and various time-points for 12 months following treatment. Concurrently, probing pocket depth, bleeding on probing, and metabolic parameters, including glycated hemoglobin A1c (HbA1c), blood sugar level, C-reactive proteins, total cholesterol, triglyceride, and high-density and low-density lipoprotein cholesterol, were recorded. RESULTS: Periodontal conditions were significantly improved after treatment, and the occurrence rates of periodontal bacterial species, including Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, and Prevotella intermedia, were also reduced. Interestingly, P. gingivalis was detected more frequently in subjects with increased HbA1c values after periodontal treatment than in those patients with decreased HbA1c values. Furthermore, P. gingivalis with type II fimbriae was detected only in HbA1c-increased subjects, while improvements in HbA1c values were observed only in subjects without type II clones. CONCLUSIONS: These results suggest that glycemic level in diabetes is affected by the persistence of P. gingivalis, especially clones with type II fimbriae, in periodontal pockets.

Signaling pathways in osteoblast proinflammatory responses to infection by Porphyromonas gingivalis.

INTRODUCTION: We recently investigated global gene expression in ST2 mouse stromal cells infected by the periodontal pathogen Porphyromonas gingivalis using microarray technology, and found that the bacterium induces a wide range of proinflammatory gene expression. Here, we reported the signaling pathways involved in those proinflammatory responses. METHODS: ST2 cells and primary calvarial osteoblasts from C3H/HeN, C57BL/6, and MyD88-deficient (MyD88(-/-)) mice were infected with P. gingivalis ATCC33277 and its gingipain-deficient mutant KDP136. Expression of the chemokines CCL5 and CXCL10, and matrix metalloproteinase-9 (MMP9) were quantified by real-time polymerase chain reaction, while phosphorylation of protein kinases and degradation of an inhibitor of nuclear factor-kappaB, IkappaB-alpha, were detected by Western blotting, and activation of transcriptional factors was determined by a luciferase reporter assay. The effects of inhibitors of transcriptional factors and protein kinases were also investigated. RESULTS: Infection by P. gingivalis elicited gene expression of CCL5, CXCL10, and MMP9 in both ST2 cells and osteoblasts. Western blot and reporter assay results revealed activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 transcription factors. The NF-kappaB inhibitor suppressed the expression of CCL5 and MMP9, but not that of CXCL10, whereas P. gingivalis infection induced significant CCL5 expression in MyD88(-/-) osteoblasts. In addition, activation of protease-activated receptors by trypsin elicited significant induction of CXCL10. CONCLUSION: Our results suggest that various proinflammatory responses in P. gingivalis-infected stromal/osteoblast cells are NF-kappaB-dependent, but not always dependent on the Toll-like receptor/MyD88 pathway, while some responses are related to the activation of protease-activated receptors. Thus, P. gingivalis does not fully utilize well-established pathogen recognition molecules such as Toll-like receptors.

Porphyromonas gingivalis gingipains cause G(1) arrest in osteoblastic/stromal cells.

INTRODUCTION: The program for mammalian cell growth and division consists of four successive phases; G(1), S, G(2), and M. Porphyromonas gingivalis may manipulate the host cell cycle to benefit bacterial virulence expression, which likely causes the cell and tissue tropism observed in chronic periodontal infections. We examined P. gingivalis for its effects on cell-cycle modulation in mouse ST2 osteoblastic/stromal cells. METHODS: Synchronized ST2 cells were infected with P. gingivalis ATCC33277 (wild-type, WT), gingipain-mutants [KDP136 (DeltargpADeltargpBDeltakgp), KDP129 (DeltargpADeltargpB), and KDP133 (Deltakgp)], and a fimbria-deficient mutant (KDP150) for 24 h, then the cell cycle was evaluated using flow cytometry. Cell-cycle-related molecule expression was examined with a microarray, as well as with quantitative real-time polymerase chain reaction and Western blotting assays. RESULTS: Both the WT and KDP150 strains significantly inhibited cellular proliferation and arrested the cell cycle in the G(0)/G(1) phase, while the expression levels of the cell-cycle regulatory molecules cyclin D and cyclin E were also decreased. In contrast, KDP136 did not show any effects. G(1) arrest was also clearly induced by KDP129 and KDP133, with KDP129 being more effective. CONCLUSION: The present findings suggest that P. gingivalis gingipains reduce cyclin expression and cause early G(1) arrest, leading to the inhibition of cellular proliferation.

Effect of phenytoin on collagen accumulation by human gingival fibroblasts exposed to TNF-alpha in vitro.

OBJECTIVE: Tumor necrosis factor (TNF)-alpha is associated with chronic gingival inflammation and reported to induce gingival overgrowth (GO), while phenytoin (PHT) is also known to be a causative agent of GO. We examined the synergistic effect of PHT and TNF-alpha on collagen metabolism in human gingival fibroblasts (HGFs). MATERIALS AND METHODS: HGFs were cultured with TNF-alpha and PHT. Quantitative real-time RT-PCR was employed to determine the mRNA levels for collagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrin subunits. Cellular collagen endocytosis was determined using a flow-cytometry. RESULTS: The proliferation of HGFs was not affected by TNF-alpha or PHT individually, whereas both synergistically increased collagen accumulation in HGFs. Further, collagen mRNA expression was not increased by TNF-alpha or PHT, although together they markedly prevented cellular collagen endocytosis, associated with the suppression of alpha2beta1-integrin mRNA expression. The mRNA expression of MMP-1 and-2 was suppressed by PHT, while TIMP-1 mRNA expression was enhanced by both TNF-alpha and PHT. CONCLUSION: Our results suggest that TNF-alpha and PHT together cause impaired collagen metabolism by suppression of enzymatic degradation with MMPs/TIMP-1 and integrin-mediated endocytosis. These synergistic effects may also be involved in TNF-alpha- and PHT-induced collagen accumulation, leading to GO.

Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes.

Quantitative analysis, with identification of periodontopathic bacteria, is important for the diagnosis, therapeutic evaluation and risk assessment of periodontal disease. We developed a highly sensitive and specific method using real-time polymerase chain reaction (PCR) to detect and quantify six periodontal bacteria: Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Prevotella nigrescens. Species-specific TaqMan probe/primer sets were designed according to 16S ribosomal RNA gene sequences. Plaque and tongue debris specimens were collected from 10 patients with advanced periodontitis and 10 periodontal healthy individuals and analyzed. All species, except for P. nigrescens, were detected in samples from diseased sites in significantly greater numbers than in those from healthy sites, whereas greater numbers of P. nigrescens were found in the controls. These results suggest that the present real-time PCR method with the designed probe/primer sets enabled sensitive detection of the six periodontal bacteria, and may also assist future microbial studies of periodontal diseases.

Comparison of inflammatory changes caused by Porphyromonas gingivalis with distinct fimA genotypes in a mouse abscess model.

The fimA gene of Porphyromonas gingivalis, encoding fimbrillin (a subunit protein of fimbriae) has been classified into six genotypes (types I-V and Ib). The genotypic variation was previously suggested to be related to the severity of adult periodontitis in the general population. In this study, we compared inflammatory changes caused by bacterial infection to study pathogenic heterogeneity among the different fimA strains in a mouse abscess model. Bacterial suspensions of 13 P. gingivalis strains representing the six fimA types were subcutaneously injected into female BALB/c mice, and serum sialic acid concentrations were assayed as a quantitative host inflammatory parameter. Type II fimA organisms caused the most significant induction of serum sialic acid, as well as other infectious symptoms, followed by types Ib, IV and V. In contrast, types I and III caused weak inflammatory changes. In addition, fimA mutants of type II strains clearly lost their infectious ability. These findings suggest that fimA genotypic variation affects expression of P. gingivalis virulence.

Activation of the p21(CIP1/WAF1) promoter by bone morphogenetic protein-2 in mouse B lineage cells.

BMPs exert a negative growth effect on various types of cells. We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21(CIP1/WAF1) expression. In the present study, we demonstrated that BMP-2 activated the mouse p21(CIP1/WAF1) promoter in HS-72 cells, and that a 29-base pair (b) region of the promoter (-1928/-1900 relative to the TATA box), conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors. Furthermore, an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2-stimulated HS-72 cells. These results suggested that BMP-2 might activate p21(CIP1/WAF1) transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells.

Possible involvement of protein kinases and Smad2 signaling pathways on osteoclast differentiation enhanced by activin A.

Bone tissues reportedly contain considerable amounts of activin A and follistatin, an activin A-binding protein. In the present study, we found that follistatin strongly inhibited osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts induced by 1alpha,25 dihydroxyvitamin D(3), prostaglandin E(2), and interleukin-1alpha. Antibody aganist activin A also inhibited the osteoclast formation. Furthermore, activin A synergistically stimulated osteoclast differentiation mediated by receptor activator NF-kappaB ligand (RANKL). RT-PCR analysis revealed that osteoblasts produced not only activin A but also follistatin. Western blot analysis of a panel of phosphorylated proteins revealed that activin A stimulated the phosphorylation of p44/42 mitogen activated protein (MAP) kinase (ERK1/2) and p38 MAP kinase in macrophage colony-stimulating factor-dependent bone marrow macrophages (M-BMMPhis). In addition, phosphorylation of Smad2 was observed in M-BMMPhis stimulated with activin A. These findings indicate that the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and Smad2 is involved in activin A-enhanced osteoclast differentiation induced by RANKL. Taken together, these results suggest that both activin A and follistatin produced by osteoblasts may play an important role in osteoclast differentiation through MAP kinases and Smad2 signaling pathways.

Involvement of caspases in apoptotic cell death of murine macrophages infected with Actinobacillus actinomycetemcomitans.

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), inhibited apoptotic cell death of murine macrophage cell line J774.1 infected with A. actinomycetemcomitans. During the process of apoptosis, interleukin-1beta (IL-1beta) was detected in the culture supernatants of J774.1 cells. IL-1beta secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1beta secretion from A. actinomycetemcomitans-infected J774.1 cells. Immunoblot analysis revealed that the infection of A. actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein (Rb), suggesting that caspase-3 was activated by A. actinomycetemcomitans infection. The cytosol from A. actinomycetemcomitans-infected J774.1 cells induced Rb proteolysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK. Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12 h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK. These findings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A. actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A. actinomycetemcomitans-infected macrophages.

Osteoclast differentiation is associated with transient upregulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1).

Osteoclasts, bone-resorbing multinucleated cells, develop from monocyte-macrophage lineage cells in the presence of osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) and macrophage colony-stimulating factor (M-CSF). M-CSF-dependent bone marrow macrophages (M-BMMPhis) from mouse bone marrow cells have been shown to differentiate into osteoclast-like multinucleated cells (OCLs) in the presence of soluble ODF/RANKL (sODF/RANKL) and M-CSF within 3 days. In this study, we found that stimulation of M-BMMPhis with sODF/RANKL induced a transient expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21(WAF1/CIP1) and p27(KIP1) by 24 h. The CDK inhibitor proteins disappeared by 48 h. Tumor necrosis factor alpha (TNF-alpha), which is reported to stimulate OCL differentiation, stimulated p21(WAF1/CIP1) and p27(KIP1) expression in M-BMMPhis as well. However, M-CSF alone did not stimulate the expression of the two CDK inhibitors. To clarify the role of p21(WAF1/CIP1) and p27(KIP1) in osteoclastogenesis, accumulation of these CDK inhibitors was aborted by antisense oligonucleotides. Treatment with p21(WAF1/CIP1) antisense oligonucleotide alone, or p27(KIP1) antisense oligonucleotide alone, showed a limited inhibitory effect on OCL formation. However, treatment with a mixture of these two antisense oligonucleotides strongly inhibited OCL formation. These results suggest that a combined modulation of the CDK inhibitors p21(WAF1/CIP1) and p27(KIP1) may be involved in osteoclast differentiation induced by ODF/RANKL.

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7.

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.

Inhibition of experimental bone resorption and osteoclast formation and survival by 2-aminoethanesulphonic acid.

It is known that bone resorption is mediated by osteoclasts, and lipopolysaccharide (LPS) and inflammatory mediators such as interleukin-1 (IL-1) and prostaglandin E2 (PGE2) induce osteoclast differentiation from haemopoietic cells, 2-aminoethanesulphonic acid, which is known as taurine, is an important nutrient and is added to most synthetic human infant milk formulas. In this study, it was found that 2-aminoethanesulphonic acid inhibits the stimulation of bone resorption mediated by LPS of the periodontopathic microorganism Actinobacillus actinomycetemcomitans Y4 in organ cultures of newborn mouse calvaria. The effect of 2-aminoethanesulphonic acid on the development and survival of osteoclast-like multinucleated cells produced in a mouse bone-marrow culture system was also examined. 2-aminoethanesulphonic acid (100 microg/ml) suppressed the formation of these osteoclast-like cells in the presence of LPS of A. actinomycetemcomitans Y4, IL-1alpha or PGE2 in mouse marrow cultures. On the other hand, 2-aminoethanesulphonic acid did not inhibit 1alpha, 25-dihydroxyvitamin D3-mediated osteoclast differentiation. Although IL-1alpha elongated the survival of the osteoclast-like cells, 2-aminoethanesulphonic acid blocked the supportive effect of IL-1alpha on osteoclast survival. 2-aminoethanesulphonic acid showed no effect on the growth of mouse osteoblasts. Finally, it was found that 2-aminoethanesulphonic acid inhibited alveolar bone resorption in experimental periodontitis in hamsters. These results suggest that 2-aminoethanesulphonic acid is an effective agent in preventing inflammatory bone resorption in periodontal diseases.

Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function.

Osteoclast differentiation factor (ODF), a novel member of the TNF ligand family, is expressed as a membrane-associated protein by osteoblasts/stromal cells. The soluble form of ODF (sODF) induces the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here, the effects of sODF on the survival, multinucleation, and pit-forming activity of murine osteoclasts were examined in comparison with those of M-CSF and IL-1. Osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts and bone marrow cells expressed mRNA of RANK (receptor activator of NF-kappaB), a receptor of ODF. The survival of OCLs was enhanced by the addition of each of sODF, M-CSF, and IL-1. sODF, as well as IL-1, activated NF-kappaB and c-Jun N-terminal protein kinase (JNK) in OCLs. Like M-CSF and IL-1, sODF stimulated the survival and multinucleation of prefusion osteoclasts (pOCs) isolated from the coculture. When pOCs were cultured on dentine slices, resorption pits were formed on the slices in the presence of either sODF or IL-1 but not in that of M-CSF. A soluble form of RANK as well as osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF, blocked OCL formation and prevented the survival, multinucleation, and pit-forming activity of pOCs induced by sODF. These results suggest that ODF regulates not only osteoclast differentiation but also osteoclast function in mice through the receptor RANK.

Bone morphogenetic protein-2 enhances osteoclast formation mediated by interleukin-1alpha through upregulation of osteoclast differentiation factor and cyclooxygenase-2.

Bone morphogenic protein-2 (BMP-2) is a member of the transforming growth factor beta (TGF-beta) superfamily. While BMP-2 is capable of inducing bone formation ectopically, little is known about its role on osteoclastogenesis. In this study, we examined the effect of BMP-2 on osteoclast-like multinucleated cell (OCL) formation in cocultures of osteoblast-like cells and hematopoietic cells of bone marrow origin. BMP-2 alone did not stimulate OCL formation in this culture system; however, it strongly enhanced OCL formation in a dose-dependent fashion in the presence of interleukin-1alpha (IL-1alpha). Western blot analysis showed that a simultaneous addition of BMP-2 and IL-1alpha synergistically enhanced cyclooxygenase-2 (COX-2) expression in osteoblast-like cells. Moreover, Northern blot analysis revealed that the level of osteoclast differentiation factor (ODF) mRNA increased by treatment with BMP-2 and IL-1alpha in osteoblast-like cells. It is noted that BMP-2 alone did cause an increase in the expression of both COX-2 and ODF genes. The stimulatory effect of BMP-2 was abolished by adding nonsteroidal anti-inflammatory drugs, such as indomethacin and a selective COX-2 inhibitor NS-398. Addition of NS-398 inhibited the expression of the ODF gene in osteoblast-like cells treated with BMP-2 and IL-1alpha. These results indicated that the combination of BMP-2 and IL-1alpha stimulated osteoblast-like cells to elevate the expression of both COX-2 and ODF genes, resulting in an enhanced OCL formation. Since BMP-2 alone induced the expression of COX-2 and ODF genes in osteoblast-like cells, it appears to be one of the regulating factors of osteoclastogenesis.

Actinobacillus actinomycetemcomitans toxin induces both cell cycle arrest in the G2/M phase and apoptosis.

We found that the culture supernatant of the periodontopathic bacterium Actinobacillus actinomycetemcomitans had a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant of A. actinomycetemcomitans Y4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin from A. actinomycetemcomitans induces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin from A. actinomycetemcomitans could mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue.

Activin A regulates the production of mature interleukin-1beta and interleukin-1 receptor antagonist in human monocytic cells.

Activin A, a member of the transforming growth factor-beta (TGF-beta) family, is produced by a variety of cells and implicated in the regulation of the reproductive endocrine system, mesoderm induction, and erythropoiesis. In the present study, we showed that activin A inhibited the production of interleukin-1beta (IL-1beta), a potent proinflammatory cytokine, and enhanced the production of IL-1 receptor antagonist (IL-1ra), in activated THP-1 and U-937 human monocytic cells, resulting in the reduction of IL-1 biologic activity. Northern blot analysis revealed that activin A had no effect on mRNA accumulation of IL-1beta and IL-1ra, indicating that activin A regulates IL-1beta and IL-1ra production at a posttranscriptional level. As it is well known that an inactive precursor form of IL-1beta (pro-IL-1beta) is converted to an active mature form (mature IL-1beta), we examined the expression levels of pro-IL-1beta and mature IL-1beta by immunoblot analysis. Although activin A inhibited the production of mature IL-1beta in activated U-937 cells, the relative protein expression of pro-IL-1beta was unaltered by activin A, suggesting that activin A inhibits IL-1beta production by blocking proteolytic conversion of pro-IL-1beta into mature IL-1beta. Taken together, these findings suggest that activin A may function as an anti-inflammatory cytokine by modulating mature IL-1beta and IL-1ra production in inflammatory sites.

Caspases (interleukin-1beta-converting enzyme family proteases) are involved in the regulation of the survival of osteoclasts.

Osteoclast-like multinucleated cells (OCLs) were prepared on collagen gels in a coculture system of mouse bone marrow cells and osteoblasts, and purified by collagenase and a subsequent pronase treatment. More than 80% of the purified OCLs were found to undergo apoptotic cell death by 48 h during the culture in a culture medium containing 10% fetal bovine serum (FBS). Withdrawal of FBS from the culture medium accelerated the cell death, which induced more than 80% of OCLs to undergo apoptotic cell death by as early as 18 h. Two peptide inhibitors of caspases (interleukin-1beta-converting enzyme family proteases), benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), extended the survival time of OCLs in the presence and absence of 10% FBS, but the effect was rather limited in the absence of FBS. Because interleukin-1alpha (IL-1alpha) and the macrophage colony stimulating factor (M-CSF) are known to promote the survival of osteoclasts, we examined the effect of the peptide inhibitors and these cytokines. Combinations of the peptide inhibitors and IL-1alpha, or the peptide inhibitors and M-CSF, were more effective than the inhibitors alone. When endogenous caspase activities of OCLs were analyzed using fluorescence peptide substrates, the activities, in particular, caspase-3 (CPP32)-like activity, were markedly increased in OCLs by the withdrawal of FBS from the culture medium. IL-1alpha and M-CSF suppressed the activation of the caspases. In addition, western blot analysis revealed that the expression of Bcl-2, which inhibits the activation of caspases, was very weak or even negligible in OCLs. Taken together, these results suggest that the caspases are involved in the regulation of survival and apoptotic cell death of osteoclasts.

Increase in Bcl-2 level promoted by CD40 ligation correlates with inhibition of B cell apoptosis induced by vacuolar type H(+)-ATPase inhibitor.

We have previously demonstrated that cell death of WEHI-231 cells induced by specific inhibitors of vacuolar type H(+)-ATPase (V-ATPase) occurs through apoptosis. CD40 is involved in regulating activation, differentiation, and apoptosis of B cells. Here we show that the CD40 ligation rescues WEHI-231 cells from apoptotic cell death induced by a specific V-ATPase inhibitor, concanamycin A. CD40 signaling with anti-CD40 antibody resulted in the induction of Bcl-2 and Bcl-XL proteins in WEHI-231 cells. Constitutive expression of Bcl-2 but not Bcl-XL inhibited concanamycin A-induced apoptosis. These findings suggest that the expression of Bcl-2 mediated through CD40 signaling rescues the apoptotic cell death induced by blockade of V-ATPase. Interestingly, the acidification of intracellular acidic compartments was completely inhibited when WEHI-231 cells were cultured with concanamycin A, even in the presence of anti-CD40 antibody. In addition, apoptosis in WEHI-231 cells induced by concanamycin A was strongly suppressed when cultured with imidazole, a cell-permeable base, suggesting that apoptosis induced by concanamycin A is preceded by intraacidification.