Tm4sf19 deficiency inhibits osteoclast multinucleation and prevents bone loss.

BACKGROUND: Multinucleation is a hallmark of osteoclast formation and has a unique ability to resorb bone matrix. During osteoclast differentiation, the cytoskeleton reorganization results in the generation of actin belts and eventual bone resorption. Tetraspanins are involved in adhesion, migration and fusion in various cells. However, its function in osteoclast is still unclear. In this study, we identified Tm4sf19, a member of the tetraspanin family, as a regulator of osteoclast function. MATERIALS AND METHODS: We investigate the effect of Tm4sf19 deficiency on osteoclast differentiation using bone marrow-derived macrophages obtained from wild type (WT), Tm4sf19 knockout (KO) and Tm4sf19 LELΔ mice lacking the large extracellular loop (LEL). We analyzed bone mass of young and aged WT, KO and LELΔ mice by µCT analysis. The effects of Tm4sf19 LEL-Fc fusion protein were accessed in osteoclast differentiation and osteoporosis animal model. RESULTS: We found that deficiency of Tm4sf19 inhibited osteoclast function and LEL of Tm4sf19 was responsible for its function in osteoclasts in vitro. KO and LELΔ mice exhibited higher trabecular bone mass compared to WT mice. We found that Tm4sf19 interacts with integrin αvß3 through LEL, and that this binding is important for cytoskeletal rearrangements in osteoclast by regulating signaling downstream of integrin αvß3. Treatment with LEL-Fc fusion protein inhibited osteoclast function in vitro and administration of LEL-Fc prevented bone loss in an osteoporosis mouse model in vivo. CONCLUSION: We suggest that Tm4sf19 regulates osteoclast function and that LEL-Fc may be a promising drug to target bone destructive diseases caused by osteoclast hyper-differentiation.

Homozygous deletions at 3p22, 5p14, 6q15, and 9p21 result in aberrant expression of tumor suppressor genes in gastric cancer.

Homozygous deletion is a frequent mutational mechanism of silencing tumor suppressor genes in cancer. Therefore, homozygous deletions have been analyzed for identification of tumor suppressor genes that can be utilized as biomarkers or therapeutic targets for cancer treatment. In this study, to elucidate potential tumor suppressor genes involved in gastric cancer (GC), we analyzed the entire set of large homozygous deletions in six human GC cell lines through genome- and transcriptome-wide approaches. We identified 51 genes in homozygous deletion regions of chromosomes and confirmed the deletion frequency in tumor tissues of 219 GC patients from The Cancer Genome Atlas database. We evaluated the effect of homozygous deletions on the mRNA level and found significantly affected genes in chromosome bands 9p21, 3p22, 5p14, and 6q15. Among the genes in 9p21, we investigated the potential tumor suppressive effect of KLHL9. We demonstrated that ectopic expression of KLHL9 inhibited cell proliferation and tumor formation in KLHL9-deficient SNU-16 cell line. In addition, we observed that homozygous focal deletions generated truncated transcripts of TGFBR2, CTNNA1, and STXBP5. Ectopic expression of two kinds of TGFBR2-reverse GADL1 fusion genes suppressed TGF-ß signaling, which may lead to the loss of sensitivity to TGF-ß tumor suppressive activity. In conclusion, our findings suggest that novel tumor suppressor genes that are aberrantly expressed through homozygous deletions may play important roles in gastric tumorigenesis.

Comprehensive genome- and transcriptome-wide analyses of mutations associated with microsatellite instability in Korean gastric cancers.

Microsatellite instability (MSI) is a critical mechanism that drives genetic aberrations in cancer. To identify the entire MS mutation, we performed the first comprehensive genome- and transcriptome-wide analyses of mutations associated with MSI in Korean gastric cancer cell lines and primary tissues. We identified 18,377 MS mutations of five or more repeat nucleotides in coding sequences and untranslated regions of genes, and discovered 139 individual genes whose expression was down-regulated in association with UTR MS mutation. In addition, we found that 90.5% of MS mutations with deletions in gene regions occurred in UTRs. This analysis emphasizes the genetic diversity of MSI-H gastric tumors and provides clues to the mechanistic basis of instability in microsatellite unstable gastric cancers.

Inactivation of glycogen synthase kinase-3ß is required for osteoclast differentiation.

Glycogen synthase kinase-3ß (GSK-3ß) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3ß in osteoblasts is well characterized as a negative regulator of ß-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3ß inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3ß activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3ß (GSK3ß-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3ß (GSK3ß-K85R) or small interfering RNA (siRNA)-mediated GSK-3ß silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3ß further confirmed the negative role of GSK-3ß in osteoclast formation. We also show that overexpression of the GSK3ß-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3ß-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3ß in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.

TRAF6-mediated regulation of the PI3 kinase (PI3K)-Akt-GSK3beta cascade is required for TNF-induced cell survival.

We recently demonstrated that the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) helps maintenance of cell survival by regulating glycogen synthase kinase 3beta (GSK3beta) activity during TNF signaling. However, the molecular linkage between TRAF6 and GSK3beta signaling is unknown. Herein, we showed that TRAF6 positively regulated cell survival by modulating PI3K-Akt-GSK3beta cascades. In 3T3 cells lacking TRAF6, but not those lacking TRAF2, TNF stimulation led to prolonged hyperphosphorylation of Akt, which coincided with the activation of upstream PI3K. Pharmacologically blocking PI3K significantly inhibited Akt and GSK3beta phosphorylation. Importantly, PI3K inhibition rescued cell death in TRAF6-null 3T3 cells. These data suggested TRAF6 regulates TNF-mediated cell survival through PI3K-Akt-GSK3beta cascades.

TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis.

TRAF6 has a key role in the regulation of innate immune responses by mediating signals from both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Here we show that T cell-specific deletion of TRAF6 unexpectedly results in multiorgan inflammatory disease. TRAF6-deficient T cells exhibit hyperactivation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway compared with wild-type T cells and, as a result, become resistant to suppression by CD4+ CD25+ regulatory T cells. These data identify a previously unrecognized role for TRAF6 in the maintenance of peripheral tolerance, and suggest the presence of a T cell-intrinsic control mechanism to render responder T cells susceptible to tolerizing signals.

Direct interaction of Smac with NADE promotes TRAIL-induced apoptosis.

Second mitochondria-derived activator of caspase (Smac) has been implicated in the activation of apoptosis in response to cell stress. We screened for Smac/DIABLO-binding protein for further understanding of Smac-mediated apoptosis. We identified NADE, previously known as p75NTR-associated cell death executor, as a Smac-binding protein. Smac-NADE interaction was mapped to the N-terminal region of Samc and the C-terminal region of NADE. Co-expression of NADE and Smac promotes TRAIL-induced apoptosis in MCF-7 cells. Interestingly, the co-presence of Smac and NADE inhibits XIAP-mediated Smac ubiquitination. In conclusion, our results provide the first evidence that the interaction between Smac and NADE regulates apoptosis through the inhibition of Smac ubiquitination.

PIAS3 suppresses NF-kappaB-mediated transcription by interacting with the p65/RelA subunit.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor critical for key cellular processes, including immune response, apoptosis, and cell cycle progression. A yeast two-hybrid screening, using the Rel homology domain (RHD) of the p65 subunit (RelA) of NF-kappaB as bait, led to the isolation of PIAS3, previously identified as a specific inhibitor of STAT3. We show that PIAS3 can directly associate with p65 using an in vitro pull-down and in vivo coimmunoprecipitation assays. When overexpressed, PIAS3 inhibits NF-kappaB-dependent transcription induced by treatment with tumor necrosis factor alpha (TNF-alpha) or interleukin-1beta or by overexpression of TNF family receptors such as RANK, TNFR1, and CD30 or signal transducers of TNF receptor-associated factors (TRAFs), including TRAF2, TRAF5, and TRAF6. Downregulation of PIAS3 by RNA interference reverses its effect on TNF-alpha-mediated NF-kappaB activation. We found that an N-terminal region of PIAS3 is necessary for both the interaction with p65 and the transcriptional suppression activity. In addition, we found that an LXXLL coregulator signature motif located within the N-terminal region of PIAS3 is the minimal requirement for the interaction with p65. Furthermore, we demonstrate that PIAS3 interferes with p65 binding to the CBP coactivator, thereby resulting in a decreased NF-kappaB-dependent transcription. Taken together, these data suggest that PIAS3 may function in vivo as a modulator in suppressing the transcriptional activity of p65.

TNF-related weak inducer of apoptosis receptor, a TNF receptor superfamily member, activates NF-kappa B through TNF receptor-associated factors.

TNF-related weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family that induces angiogenesis in vivo. The TWEAK receptor (TweakR) is a recently identified member of the TNF receptor (TNFR) superfamily and is expressed on smooth muscle cells (SMCs) and endothelial cells (ECs). In this report we identify the TNF receptor-associated factor (TRAF) family of signal transducers as important components of TweakR-mediated NF-kappa B activation. Coimmunoprecipitation experiments suggested potential interactions between the cytoplasmic tail of TweakR with TRAFs 1, 2, 3, and 5. Dominant negative forms of TRAF2 and TRAF5 substantially inhibited TweakR-mediated NF-kappa B activation, suggesting a role of TRAFs in regulating smooth muscle and endothelial cell function. Using alanine-scanning analysis, we defined a TRAF-binding motif, PIEET, in TweakR that mediates TRAF binding and NF-kappa B activation. Furthermore, TweakR mutations within the TRAF-binding motif abolished TweakR-stimulated SMC migration, revealing a role for TRAFs in TweakR-induced activation events.