Branched oligomerization of cell-permeable peptides markedly enhances the transduction efficiency of adenovirus into mesenchymal stem cells.

Cell-permeable peptides (CPPs) promote the transduction of nonpermissive cells by recombinant adenovirus (rAd) to improve the therapeutic efficacy of rAd. In this study, branched oligomerization of CPPs significantly enhanced the transduction of human mesenchymal stem cells (MSCs) by rAd in a CPP type-independent manner. In particular, tetrameric CPPs increased transduction efficiency at 3000-5000-fold lower concentrations than did monomeric CPPs. Although branched oligomerization of CPPs also increases cytotoxicity, optimal concentrations of tetrameric CPPs required for maximum transduction are at least 300-1000-fold lower than those causing 50% cytotoxicity. Furthermore, although only approximately 60% of MSCs were maximally transduced at 500 muM of monomeric CPPs, >95% of MSCs were transduced with 0.1 muM of tetrameric CPPs. Tetrameric CPPs also significantly increased the formation and net surface charge of CPP/rAd complexes, as well as the binding of rAd to cell membranes at a greater degree than did monomeric CPPs, followed by rapid internalization into MSCs. In a critical-size calvarial defect model, the inclusion of tetrameric CPPs in ex vivo transduction of rAd expressing bone morphogenetic protein 2 into MSCs promoted highly mineralized bone formation. In addition, MSCs that were transduced with rAd expressing brain-derived neurotrophic factor in the presence of tetrameric CPPs improved functional recovery in a spinal cord injury model. These results demonstrated the potential for tetrameric CPPs to provide an innovative tool for MSC-based gene therapy and for in vitro gene delivery to MSCs.

IL-17 stimulates the proliferation and differentiation of human mesenchymal stem cells: implications for bone remodeling.

Interleukin-17 (IL-17) is a cytokine secreted primarily by T(H)-17 cells. Although IL-17 is primarily associated with the induction of tissue inflammation, the other biological roles of IL-17, including non-immune functions, have yet to be thoroughly explored. Here, we report that T-cell-produced IL-17 can induce proliferation of human bone marrow-derived mesenchymal stem cells (hMSCs) in a manner dependent on the generation of reactive oxygen species (ROS). Rac1 GTPase and NADPH oxidase 1 (Nox1) are activated by IL-17 to produce ROS, which in turn stimulates hMSC proliferation. The activation of the MEK-ERK pathway is also crucial for IL-17-dependent hMSC proliferation. TRAF6 and Act1 are required to activate Nox 1 and to phosphorylate MEK on IL-17 stimulation. Interestingly, IL-17 not only accelerates the proliferation of hMSCs, but also induces their migration, motility, and osteoblastic differentiation. Furthermore, IL-17 induces the expression of M-CSF and receptor activator of NF-kappaB ligand (RANKL) on hMSCs, thereby supporting osteoclastogenesis both in vivo and in vitro. On the basis of these results, we suggest that IL-17 can function as a signal to induce extensive bone turnover by regulating hMSC recruitment, proliferation, motility, and differentiation.

DLX3 mutation in a new family and its phenotypic variations.

Tricho-dento-osseous syndrome (TDO) is an autosomal-dominant disease characterized by curly hair at birth, enamel hypoplasia, taurodontism, and a thick cortical bone. A common DLX3 gene mutation (c.571_574delGGGG) has been identified in multiple families with variable clinical phenotypes. Recently, another DLX3 gene mutation (c.561_562delCT) was reported to cause amelogenesis imperfecta with taurodontism (AIHHT). We identified a Korean family with overlapping phenotypes of TDO and AIHHT. We performed mutational analysis to discover its genetic etiology. The identified mutation was c.561_562delCT mutation in the DLX3 gene. The enamel was hypomature and hypoplastic. The characteristic taurodontic features were not identified. Increased bone density or thickness could not be revealed by cephalometric, hand-wrist, and panoramic radiographs. Affected individuals reported that their nails were brittle, and they had curly hair at birth. This study clearly showed that the c.561_562delCT mutation had not only enamel defects, but also other clinical phenotypes resembling those of TDO syndrome.

Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NF-kappaB transactivation by RANKL.

Osteoclast (Oc) differentiation is fundamentally controlled by receptor activator of nuclear factor kappaB ligand (RANKL). RANKL signalling targets include mitogen-activated protein kinases (MAPKs), nuclear factor kappaB (NF-kappaB), and nuclear factor of activated T cells (NFAT)c1. In this study, we found that p38 MAPK upstream components transforming growth factor-beta-activated kinase 1 (TAK1), MKK3, and MKK6 increased by RANKL in an early stage of osteoclastogenesis from primary bone marrow cells, which led to enhanced p38 activation. Retroviral transduction of dominant-negative (DN) forms of TAK1 and MKK6, but not that of MKK3, reduced Oc differentiation. Transduction of TAK1-DN and MKK6-DN and treatment with the p38 inhibitor SB203580 attenuated NFATc1 induction by RANKL. TAK1-DN, MKK6-DN, and SB203580, but not MKK3-DN, also suppressed RANKL stimulation of NF-kappaB transcription activity in a manner dependent on p65 phosphorylation on Ser-536. These results indicate that TAK1 and MKK6 constitute the p38 signalling pathway to participate to Oc differentiation by RANKL through p65 phosphorylation and NFATc1 induction, and that MKK6 and MKK3 have differential roles in osteoclastogenesis from bone marrow precursors.

Participation of protein kinase C beta in osteoclast differentiation and function.

Protein kinase C (PKC) proteins have been shown to be involved in diverse cellular responses of various cell types. In experiments to identify genes regulated during osteoclast differentiation by a cDNA microarray approach, we found that the gene expression of PKC-betaII was upregulated in differentiated cells. Reverse transcription-polymerase chain reaction and Western blotting analyses also showed an increase in PKC-betaI as well as PKC-betaII during osteoclast formation in mouse bone marrow cell cultures in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL). Use of an antisense oligonucleotide to PKC-betaII resulted in a reduction in the RANKL-driven osteoclastogenesis. Pharmacological intervention with PKC-beta activity by the specific inhibitor CG53353 suppressed cellular differentiation and fusion processes during osteoclastogenesis and inhibited bone-resorbing function of mature osteoclasts. PKC-beta inhibition abolished the ERK and MEK activation by macrophage-colony stimulating factor and RANKL in osteoclast precursor cells whereas the cytokine-induced NF-kappaB activation was not hampered by the PKC-beta inhibition. Our findings indicate that PKC-beta has a role in regulation of osteoclast formation and function potentially by participating in the ERK signaling pathway of M-CSF and RANKL.

The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation.

Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinases (MAPKs) have been implicated in diverse cellular functions, including proliferation, migration, and survival. In this study, we examined the involvement of these kinases in osteoclast differentiation by employing specific inhibitors of the kinases. The osteoclast differentiation was assessed in three different culture systems: a coculture of mouse bone marrow cells with mouse calvarial osteoblasts, a mouse bone marrow cell culture in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and a culture of bone-resident osteoclast precursor cells driven by RANKL and M-CSF. LY294002, a specific inhibitor of PI 3-kinase, potently inhibited osteoclast differentiation in all culture systems when assessed by both tartrate-resistant acid phosphatase (TRAP) staining and dentine resorption assays. Inhibition of p38 MAPK by SB202190 resulted in a strong suppression in the exogenous RANKL dependent mouse bone marrow and bone resident precursor cell cultures. Another MAPK pathway inhibitor (PD98059), which blocks the activation of extracellular signal-regulated kinase (ERK) by inhibiting the upstream kinase MAPK-ERK kinase (MEK) 1, exerted an inhibitory effect on osteoclast differentiation only at the highest concentration tested (30 micromol/L) in many cases. Whether the signaling pathways involving these kinases are activated by RANKL was also examined. The RANKL-stimulated phosphorylation of Akt, a downstream target of PI 3-kinase, and that of ERK were observed. RANKL also stimulated the activity of p38. These results suggest that PI 3 kinase, p38, and ERK play roles in osteoclast differentiation, at least in part, by participating in RANKL signaling.

Tumor necrosis factor-alpha supports the survival of osteoclasts through the activation of Akt and ERK.

Differentiated osteoclasts have a short life span. We tested various cytokines and growth factors for the effects on the survival of purified mature osteoclasts. In the absence of any added factors, osteoclasts exhibited the survival rate of less than 25% after a 24-h incubation. Among the tested factors, tumor necrosis factor-alpha (TNF-alpha) was found to increase the survival rate to approximately 80%. The TNF-alpha-enhanced survival of osteoclasts appeared to be associated with reduction in apoptosis and suppression of caspase activation. The antiapoptotic signaling pathways involved in the TNF-alpha-induced osteoclast survival were investigated. TNF-alpha treatment increased the phosphorylation of Akt in osteoclasts, which was suppressed by a phosphatidylinositol 3-kinase inhibitor LY294002 and an Src family kinase-selective inhibitor PP1. These inhibitors also attenuated the TNF-alpha stimulation of osteoclast survival. In addition an increase in the phosphorylation of ERK was observed upon TNF-alpha stimulation. PD98059, a specific inhibitor of the ERK-activating kinase MEK-1, abolished the TNF-alpha-induced ERK phosphorylation and osteoclast survival, and in these responses the involvement of Grb2 and ceramide was observed. These results suggest that TNF-alpha promotes the survival of osteoclasts by engaging the phosphatidylinositol 3-kinase Akt and MEK/ERK signaling pathways.

Blockade of the p38 mitogen-activated protein kinase pathway inhibits inducible nitric oxide synthase and interleukin-6 expression in MC3T3E-1 osteoblasts.

Treatment of MC3T3E-1 osteoblast cultures with combined interferon- gamma(IFN- gamma), lipopolysaccharide (LPS) and tumor necrosis factor- alpha(TNF- alpha) induces expressions of inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6), resulting in sustained releases of large amounts of nitric oxide and IL-6. However IFN- gamma, LPS and TNF- alpha individually induces non-detectable or small amounts of NO and IL-6 in MC3T3E-1 osteoblasts. The role of mitogen-activated protein kinase (MAPK) activation in the early intracellular signal transduction involved in iNOS and IL-6 transcription in the combined agents-stimulated osteoblasts has been investigated. The p38 MAPK pathway is specifically involved in the combined agents-induced NO and IL-6 release, since NO and IL-6 release in the presence of a specific inhibitor of p38 MAPK, 4-(4-fluorophenyl)-2-(4-metylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580), are significantly diminished. In contrast, PD98059, a specific inhibitor of MEK1, had no effect on NO and IL-6 release. Northern blot analysis showed that the p38 MAPK pathway controlled iNOS and IL-6 transcription levels. These data suggest that p38 MAPK plays an important role in the secretion of NO and IL-6 in LPS/IFN- gamma or TNF- alpha /IFN- gamma -treated MC3T3E-1 osteoblasts.

Cyclic-AMP inhibits nitric oxide-induced apoptosis in human osteoblast: the regulation of caspase-3, -6, -9 and the release of cytochrome c in nitric oxide-induced apoptosis by cAMP.

Nitric oxide (NO) induces apoptotic cell death and cAMP has a significantly protective effect on NO-induced cytotoxicity in human osteoblasts, MG-63 cells. Treatment with S-nitroso-N-acetylpenicillamine (SNAP) (0.6 mM) resulted in genomic DNA fragmentation, characteristic of apoptosis. However, concomitant incubation of the cells with either DBcAMP or forskolin markedly inhibited SNAP-induced apoptosis in a dose-dependent manner. Furthermore, pretreatment of MG-63 cells with H-89 or KT5720, which is known to inhibit cAMP-dependent protein kinase (PKA), abolished the protective effect of DBcAMP and forskolin on SNAP-induced apoptosis. In this study, we explored the involvement of caspases in the regulatory mechanism of SNAP-induced apoptosis by cAMP. Our data show that DBcAMP or forskolin blocked SNAP-induced caspase-3-like cysteine protease activation and that H-89, a PKA inhibitor, reversed the cAMP-induced regulatory effect of caspase-3 like protease. Consistent with the results, cAMP inhibited the proteolytic cleavage of caspase-3, -6, -9 and cytochrome c release to cytoplasm. The inhibition of caspase-3 activation did not block SNAP-induced cytochrome c release to cytoplasm, suggesting that caspase-3 activation may occur downstream of cytochrome c release. In summary, these findings show that the exposure of MG-63 cells to cAMP analogs renders them more resistant to NO-induced damage and suggests the presence of regulatory mechanisms of the cell death pathway by cAMP in which caspase-3, -6, and -9 and cytochrome c release serves to mediate NO-induced apoptosis.

Caspase-mediated cleavage of TRAF3 in FasL-stimulated Jurkat-T cells.

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) proteins play a central role in the early steps of signal transduction by TNFR superfamily proteins, which induce various cellular responses, including apoptosis. Influences of TRAF proteins on the regulation of cell death and physical interactions between TRAFs and caspases have been reported. In this study, we demonstrate that TRAF3 is proteolyzed during cell death in a caspase-dependent manner. TRAF3 was found to be cleaved by incubation with caspase3 in vitro and by Fas- or CD3-triggering in Jurkat-T cells. The Fas- or CD3-induced cleavage of TRAF3 was blocked by caspase inhibitors and by introduction of alanine substitutions for D347 and D367 residues. Furthermore, the amino-terminal fragment of TRAF3 showed a different intracellular localization from the full-length TRAF3 with preferential distribution to particulate fractions and the nucleus. These findings suggest that TRAF3 may be regulated by caspases during apoptosis of T cells.

Characterization of mouse brain-specific angiogenesis inhibitor 1 (BAI1) and phytanoyl-CoA alpha-hydroxylase-associated protein 1, a novel BAI1-binding protein.

Previously, PAHX-AP1 (PAHX-associated protein 1) was isolated as a novel protein to interact with Refsum disease gene product (phytanoyl-CoA alpha-hydroxylase, PAHX) and specifically expressed in mouse brain. PAHX-AP1 is also suggested to be involved in the development of the central neurologic deficits of Refsum disease. To clarify its function, we have searched for proteins that associate with PAHX-AP1 via yeast two-hybrid system. We found that PAHX-AP1 interacts with the cytoplasmic region of human brain-specific angiogenesis inhibitor 1 (hBAI1), and isolated murine homolog of hBAI1. Structural analysis of the PAHX-AP1 with three reported hBAI-associated proteins (BAP) revealed no homology among them, and we designated PAHX-AP1 as BAP4. The ability of BAP4 to interact with BAI1 was confirmed by pulling-down BAI1 with GST-BAP4 protein and immunoprecipitation study using brain lysate. Northern and Western blot analyses demonstrated a unique pattern of BAI1 expression in the brain. The peak level of BAI1 was observed 10 days after birth. In situ hybridization analyses of the brain showed the same localization of BAI1 as BAP4, such as most neurons of cerebral cortex, hippocampus, and V, VI, VII, VIII, and XII nuclei. Because BAI1 possessed thrombospondin-type 1 repeats in its extracellular region, changes of BAI1 expression were examined in the focal cerebral ischemia model. The BAI1 expression decreased on the ischemic side after 24 h but BAP4 was not changed after the time-course of ischemia. Our results indicate that expression and localization of BAI1 in the brain is correlated with BAP4, and that BAI1 is involved in inhibition of angiogenesis and neuronal differentiation.

The p38 mitogen-activated protein kinase pathway regulates interleukin-6 synthesis in response to tumor necrosis factor in osteoblasts.

The induction of interleukin-6 (IL-6), using a proinflammatory cytokine (tumor necrosis factor-alpha), was studied in a human osteoblast cell line (MG-63) in relation to p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB transcription factor. When added to MG-63 cells, tumor necrosis factor-alpha (TNF-alpha) had a stimulatory effect on the production of IL-6, and this elevation was significantly reduced by SB203580, a specific p38 MAPK inhibitor. In addition, the stimulation of IL-6 release was also reduced by pyrrolidine dithiocarbamate (PDTC) or NF-kappaB SN50, which has been reported to be a potent NF-kappaB inhibitor. Both the NF-kappaB inhibitors in the presence of SB203580 had a more inhibitory effect on IL-6 release. In this study, TNF-alpha stimulated NF-kappaB binding affinity as well as p38 MAP kinase activation, leading to the release of IL-6. However, the specific inhibitor of p38 MAPK, SB203580, had no effect on TNF-alpha-induced NF-kappaB activation and both NF-kappaB inhibitors failed to reduce the p38 MAPK activation in the TNF-alpha-stimulated osteoblasts. In addition, inhibition of p38 MAPK partially, but significantly, impaired TNF-alpha-regulated release of osteocalcin, an important differentiation marker in osteoblasts. These results strongly suggest that both p38 MAPK and NF-kappaB are required in TNF-alpha-induced IL-6 synthesis and that these two TNF-alpha-activated pathways can be primarily dissociated. Furthermore, p38 MAPK may play a significant role in differentiation in MG-63 cells.

A novel leucine-rich repeat protein (LRR-1): potential involvement in 4-1BB-mediated signal transduction.

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is induced on CD4+ and CD8+ T cells upon engagement of the T cell receptor (TCR)/ CD3 complex with the antigen bound to MHC. 4-1BB plays an important role in transmitting costimulatory signal during T cell activation. However, 4-1BB-mediatded signal transduction pathways have remained elusive. We conducted the yeast two-hybrid screening to identify intracellular signaling molecules that associate with 4-1BB. A novel leucine-rich repeat (LRR)-containing protein, herein named LRR-1, was found to specifically interact with the cytoplasmic domain of 4-1BB. Overexpression of LRR-1 suppressed the activation of NF-KB induced by 4-1BB or TNF receptor-associated factor (TRAF) 2. In addition, LRR-1 down-regulated JNK1 activity was induced by 4-1BB. These results indicate that LRR-1 negatively regulates the 4-1BB-mediated signaling cascades which result in the activation of NF-kappaB and JNK1.

Activation of c-Jun N-terminal kinase and activator protein 1 by receptor activator of nuclear factor kappaB.

Receptor activator of nuclear factor kappaB (RANK), a lately identified member of the tumor necrosis factor receptor superfamily, plays important roles both in osteoclastogenesis and in lymph node development. Previously, we and others showed that RANK could stimulate the activity of c-Jun N-terminal kinase (JNK). In this study, we investigated the mechanism by which RANK activates JNK. We found that N-terminal deletion mutants of tumor necrosis factor receptor-associated factor 2 and 6 were inhibitory to RANK activation of JNK. The JNK activation by RANK was also reduced by cotransfection of kinase-inactive mutants of apoptosis signal-regulating kinase 1, MAPK/ERK kinase kinase 1, and nuclear factor kappaB-inducing kinase. In addition, dominant negative mutants of Rac and Ras decreased the RANK stimulation of JNK activity. Furthermore, we determined whether the RANK engagement of JNK signaling pathways could lead to the activation of the activator protein 1 (AP-1) transcription factor, one of the potential downstream targets of activated JNK. RANK was found to activate AP-1 in a manner dependent on the signaling molecules involved in the JNK activation by this receptor. Furthermore, the activation of JNK and ERK, but not that of p38, appeared to be involved in the AP-1 activation by RANK. Thus, RANK may use both JNK and ERK pathways to signal to the AP-1 transcription factor.

Activation of c-jun N-terminal kinase by 4-1BB (CD137), a T cell co-stimulatory molecule.

It has been widely accepted that T cell activation requires two signals; one from the binding of the antigen/major histocompatibility complex to the T-cell receptor (TCR)/CD3 complex and the other from the interaction between a surface molecule on antigen presenting cells and its receptor on T cells. The second signal is considered as co-stimulatory and the B7/CD28 pair has been well studied as a prototype. Recently 4-1BB (CD137) has been characterized as another co-stimulatory molecule for T cell activation. However, unlike the CD28/B7 pair, 4-1BB and its ligand 4-1BBL constitute a member of the tumor necrosis factor (TNF) receptor/TNF pair superfamily. The signaling mechanism of 4-1BB has not been revealed in detail. To investigate whether 4-1BB takes the signaling pathways analogous to those for TNF receptors, we generated polyclonal antibodies against human 4-1BB and 4-1BBL and established stable transfectants of the receptor and the ligand with a high level of cell surface expression. Over-expression of h4-1BB was found to result in the activation of c-Jun N-terminal kinase (JNK) in the human embryonic kidney cell line 293. In T cells, it has been previously demonstrated that JNK activation requires dual signals such as the ligation of TCR/CD3 complex plus CD28 co-stimulation or PMA plus ionomycin. The JNK activation by 4-1BB in Jurkat T cells was also found to require stimulation of the TCR/CD3 complex, consistent with the notion that 4-1BB functions as a co-stimulatory molecule for T cell activation.

Receptor activator of NF-kappaB recruits multiple TRAF family adaptors and activates c-Jun N-terminal kinase.

Receptor activator of NF-kappaB (RANK) is a recently cloned member of the tumor necrosis factor receptor (TNFR) superfamily, and its function has been implicated in osteoclast differentiation and dendritic cell survival. Many of the TNFR family receptors recruit various members of the TNF receptor-associated factor (TRAF) family for transduction of their signals to NF-kappaB and c-Jun N-terminal kinase. In this study, the involvement of TRAF family members and the activation of the JNK pathway in signal transduction by RANK were investigated. TRAF1, 2, 3, 5, and 6 were found to bind RANK in vitro. Association of RANK with each of these TRAF proteins was also detected in vivo. Expression of RANK in cultured cells also induced the activation of JNK, which was blocked by a dominant-negative form of JNK. Furthermore, by employing various C-terminal deletion mutants of RANK, the regions responsible for TRAF interaction and JNK activation were identified. TRAF5 was determined to bind to the C-terminal 11 amino acids and the other TRAF members to a region N-terminal to the TRAF5 binding site. The domain responsible for JNK activation was localized to the same region where TRAF1, 2, 3, and 6 bound, which suggests that these TRAF molecules might mediate the RANK-induced JNK activation.

TR1, a new member of the tumor necrosis factor receptor superfamily, induces fibroblast proliferation and inhibits osteoclastogenesis and bone resorption.

A newly identified member of the tumor necrosis factor receptor (TNFR) superfamily shows activities associated with osteoclastogenesis inhibition and fibroblast proliferation. This new member, called TR1, was identified from a search of an expressed sequence tag database, and encodes 401 amino acids with a 21-residue signal sequence. Unlike other members of TNFR, TR1 does not contain a transmembrane domain and is secreted as a 62 kDa glycoprotein. TR1 gene maps to chromosome 8q23-24.1 and its mRNA is abundantly expressed on primary osteoblasts, osteogenic sarcoma cell lines, and primary fibroblasts. The receptors for TR1 were detected on a monocytic cell line (THP-1) and in human fibroblasts. Scatchard analyses indicated two classes of high and medium-high affinity receptors with a kD of approximately 45 and 320 pM, respectively. Recombinant TR1 induced proliferation of human foreskin fibroblasts and potentiated TNF-induced proliferation in these cells. In a coculture system of osteoblasts and bone marrow cells, recombinant TR1 completely inhibited the differentiation of osteoclast-like multinucleated cell formation in the presence of several bone-resorbing factors. TR1 also strongly inhibited bone-resorbing function on dentine slices by mature osteoclasts and decreased 45Ca release in fetal long-bone organ cultures. Anti-TR1 monoclonal antibody promoted the formation of osteoclasts in mouse marrow culture assays. These results indicate that TR1 has broad biological activities in fibroblast growth and in osteoclast differentiation and its functions.

Human 4-1BB (CD137) signals are mediated by TRAF2 and activate nuclear factor-kappa B.

Human 4-1BB (CD137), a member of the tumor necrosis factor receptor (TNFR) superfamily, costimulates T cell activation. No apparent intrinsic kinase activity is seen with 4-1BB, which suggests that 4-1BB-associated molecules may be involved in 4-1BB-mediated signal transduction. We found that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, and TRAF3, all interacted with the cytoplasmic domain of 4-1BB. Mutation analysis showed that TRAF1, TRAF2, and TRAF3 were associated with one of two runs of acidic residues found in the cytoplasmic domain of 4-1BB. In addition, 4-1BB cross-linking with TCR signal in Jurkat cells and overexpression of 4-1BB in 293 cells were able to induce activation of the nuclear factor-kappa B (NF-kappa B). 4-1BB-mediated NF-kappa B activation was inhibited by a dominant negative-TRAF2 or -NF-kappa B-inducing kinase (NIK). These data suggest that 4-1BB functions may be mediated by NF-kappa B activation, which requires a TRAF2/NIK pathway.

A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation.

The tumor necrosis factor receptor (TNFR) superfamily consists of approximately 10 characterized members of human proteins. We have identified a new member of the TNFR superfamily, TR2, from a search of an expressed sequence tag data base. cDNA cloning and Northern blot hybridization demonstrated multiple mRNA species, of which a 1.7-kilobase form was most abundant. However, TR2 is encoded by a single gene which, maps to chromosome 1p36.22-36.3, in the same region as several other members of the TNFR superfamily. The most abundant TR2 open reading frame encodes a 283-amino acid single transmembrane protein with a 36-residue signal sequence, two perfect and two imperfect TNFR-like cysteine-rich domains, and a short cytoplasmic tail with some similarity to 4-1BB and CD40. TR2 mRNA is expressed in multiple human tissues and cell lines and shows a constitutive and relatively high expression in peripheral blood T cells, B cells, and monocytes. A TR2-Fc fusion protein inhibited a mixed lymphocyte reaction-mediated proliferation suggesting that the receptor and/or its ligand play a role in T cell stimulation.

4-1BB is expressed on CD45RAhiROhi transitional T cell in humans.

Murine 4-1BB is an approximately 30-kDa glycoprotein expressed on activated T cells and plays a role in T-cell-mediated proliferative response. To date the majority of work on 4-1BB has been conducted in the mouse. To assess the role of 4-1BB in humans, mAbs were made against the recombinant human (rh) 4-1BB protein. One such mAb 4B4-1 specifically binds SF-21 insect cells expressing rh4-1BB but not irrelevant control protein as measured by flow cytometry (FCM). 4B4-1 mAb stains PMA- and ionomycin-stimulated CEM (human T lymphoma) cells and PHA-stimulated peripheral blood T cells, but not resting cells. 4B4-1 mAb immunoprecipitates both approximately 32 and approximately 80 kDa protein from rh4-1BB expressing SF-21 cells and an approximately 39- and approximately 85-kDa protein from PMA-stimulated CEM cells under reducing conditions by SDS-PAGE. As added proof of its specificity, binding of FITC-labeled 4B4-1 mAb to PHA-stimulated T cells was blocked by rh4-1BB protein. Together these data demonstrate that 4B4-1 is specific for 4-1BB in humans. Unlike in the mouse, 4-1BB is expressed much earlier (within 24 hr) peaking around 2-3 days following PHA stimulation. As in the mouse 4-1BB is induced on both CD4+ and CD8+ T cell subsets. 4-1BB expression is induced upon PHA stimulation in both the naive (CD45RAhi-CD45ROlo/-) and the memory (CD45RAlo/-ROhi) T cell populations. Virtually all CD45RAhiROlo/- cells upon culture in PHA give rise to an intermediate CD45RAhiROhi 4-1BB+ transitional cell and subsequently CD45RAlo/-ROhi 4-1BBlo/- and CD45RAlo/-ROhi 4-1BBhi cells. In contrast, approximately 27% of CD45RAlo/-ROhi 4-1BB- cells when cultured in PHA for 24 hr acquire 4-1BB expression and all remain CD45RAlo/-ROhi.