A TRAF2 binding independent region of TNFR2 is responsibl for TRAF2 depletion and enhancement of cytotoxicity driven b TNFR1

Tumor Necrosis Factor (TNF) interacts with two receptors known as TNFR1 and TNFR2. TNFR1 activation may result in either cell proliferation or cell death. TNFR2 activates Nuclear Factor-kappaB (NF-kB) and c-Jun N-terminal kinase (JNK) which lead to transcriptional activation of genes related to cell proliferation and survival. This depends on the binding of TNF Receptor Associated Factor 2 (TRAF2) to the receptor. TNFR2 also induces TRAF2 degradation. In this work we have investigated the structural features of TNFR2 responsible for inducing TRAF2 degradation and have studied the biological consequences of this activity. We show that when TNFR1 and TNFR2 are co-expressed, TRAF2 depletion leads to an enhanced TNFR1 cytotoxicity which correlates with the inhibition of NF-kB. NF-kB activation and TRAF2 degradation depend of different regions of the receptor since TNFR2 mutants at amino acids 343-349 fail to induce TRAF2 degradation and have lost their ability to enhance TNFR1-mediated cell death but are still able to activate NF-kB. Moreover, whereas NF-kB activation requires TRAF2 binding to the receptor, TRAF2 degradation appears independent of TRAF2 binding. Thus, TNFR2 mutants unable to bind TRAF2 are still able to induce its degradation and to enhance TNFR1-mediated cytotoxicity. To test further this receptor crosstalk we have developed a system stably expressing in cells carrying only endogenous TNFR1 the chimeric receptor RANK-TNFR2, formed by the extracellular region of RANK (Receptor activator of NF-kB) and the intracellular region of TNFR2.This has made possible to study independently the signals triggered by TNFR1 and TNFR2. In these cells TNFR1 is selectively activated by soluble TNF (sTNF) while RANK-TNFR2 is selectively activated by RANKL. Treatment of these cells with sTNF and RANKL leads to an enhanced cytotoxicity

FHL2 inhibits the activated osteoclast in a TRAF6-dependent manner

TNF receptor–associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-κB (RANK). This event is central to normal osteoclastogenesis. We discovered that TRAF6 also interacts with FHL2 (four and a half LIM domain 2), a LIM domain–only protein that functions as a transcriptional coactivator or corepressor in a cell-type–specific manner. FHL2 mRNA and protein are undetectable in marrow macrophages and increase pari passu with osteoclast differentiation in vitro. FHL2 inhibits TRAF6-induced NF-κB activity in wild-type osteoclast precursors and, in keeping with its role as a suppressor of TRAF6-mediated RANK signaling, TRAF6/RANK association is enhanced in FHL2(–/–) osteoclasts. FHL2 overexpression delays RANK ligand–induced (RANKL-induced) osteoclast formation and cytoskeletal organization. Interestingly, osteoclast-residing FHL2 is not detectable in naive wild-type mice, in vivo, but is abundant in those treated with RANKL and following induction of inflammatory arthritis. Reflecting increased RANKL sensitivity, osteoclasts generated from FHL2(–/–) mice reach maturation and optimally organize their cytoskeleton earlier than their wild-type counterparts. As a consequence, FHL2(–/–) osteoclasts are hyperresorptive, and mice lacking the protein undergo enhanced RANKL and inflammatory arthritis–stimulated bone loss. FHL2 is, therefore, an antiosteoclastogenic molecule exerting its effect by attenuating TRAF6-mediated RANK signaling

Cthrc1 Is a Positive Regulator of Osteoblastic Bone Formation

Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis

Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice

TRAF6 expression is enhanced during muscle atrophy and induces activation of signal transduction cascades that promote muscle wasting

General acid/base catalysis by a histidine residue of mammalian and bacterial HMG-CoA reductase

By investigating the pH-variation of kinetic parameters, Veloso, Cleland, and Porter (Biochemistry 20, 887-894, 1981) postulated that a histidine residue participates in catalysis by yeast 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Histidine modifying reagents also inactivate yeast HMG-CoA reductase (Dugan and Katiyar, Biochem. Biophys. Res. Commun. 141, 278-284, 1986). I therefore used chemical modification and site-directed mutagenesis to identify a catalytic site histidine of HMG-CoA reductase of Pseudomonas mevalonii (Ps. HMGR) and of the catalytic domain of the Syrian hamster enzyme (R\sb{\rm cat}). Ps. HMGR and R\sb{\rm cat} were overexpressed in Escherichia coli, purified to homogeneity, and characterized. Diethyl pyrocarbonate (DEPC) inactivated both enzymes, and hydroxylamine partially restored activity. Sequence comparisons revealed that only His\sp{381} (Ps. HMGR) and His\sp{865} (R\sb{\rm cat}) are totally conserved among the catalytic domains of all known HMG-CoA reductases. The codon for His\sp{381} was changed to the codons for alanine, lysine, asparagine, and glutamine, and that for His\sp{865} to the codons for lysine and glutamine. Following overexpression in E. coli, all mutant enzymes were purified to homogeneity. While all mutant enzymes exhibited less than 1.5% of wild-type catalytic activity, all chromatographed on substrate affinity supports like wild-type enzyme, and K\sb{\rm m} values approximated those for wild-type enzyme. In addition, Ps. HMGR mutant enzymes exhibited wild-type crystal morphology. I therefore infer that the low catalytic activity of these mutant enzyme was not a result of gross conformational changes. Whereas His\sp{381} mutant enzymes were not inactivated by DEPC, His\sp{865} mutant enzymes were sensitive to DEPC. pK\sb1 values were determined for all Ps. HMGR mutant enzymes. Mutant enzyme Ps. HMGR H381K exhibited an elevated pK\sb1 of 10.2, consistent with lysine acting as a general base at high pH. Exogenous amines enhanced the activity of Ps. HMGR H381A in a pH-dependent manner, suggesting that the unprotonated amine acts as the general base in catalysis. His\sp{381} of Ps. HMGR and His\sp{865} of R\sb{\rm cat}, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xxx-Xxx-Asn-Arg-Ser motif of the catalytic domain of all eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis

TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-Mediated Activation by the Tumor Necrosis Factor Receptor Family Member Latent Membrane Protein 1▿

We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6−/− mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IκB kinase ɛ. In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor