NUMBL Interacts with TAK1, TRAF6 and NEMO to Negatively Regulate NF-κB Signaling During Osteoclastogenesis.

NF-κB signaling is essential for osteoclast differentiation and skeletal homeostasis. We have reported recently that NUMB-like (NUMBL) protein modulates osteoclastogenesis by down regulating NF-κB activation. Herein, we decipher the mechanism underlying this phenomenon. We found that whereas NUMBL mRNA expression decreases upon stimulation of wild type (WT) bone marrow macrophages (BMMs) with RANKL, TAK1 deficiency in these cells leads to increased NUMBL and decreased TRAF6 and NEMO expression. These changes were restored upon WT-TAK1 expression, but not with catalytically inactive TAK1-K63W, suggesting that TAK1 enzymatic activity is required for these events. Forced expression of NUMBL inhibits osteoclast differentiation and function as evident by reduction in all hallmarks of osteoclastogenesis. Conversely, NUMBL-null BMMs, show increased osteoclast differentiation and mRNA expression of osteoclast marker genes. Post-translationally, K48-linked poly-ubiquitination of NUMBL is diminished in TAK1-null BMMs compared to elevated K48-poly-ubiquitination in WT cells, indicating increased stability of NUMBL in TAK1-null conditions. Further, our studies show that NUMBL directly interacts with TRAF6 and NEMO, and induces their K48-poly-ubiquitination mediated proteasomal degradation. Collectively, our data suggest that NUMBL and TAK1 are reciprocally regulated and that NUMBL acts as an endogenous regulator of NF-κB signaling and osteoclastogenesis by targeting the TAK1-TRAF6-NEMO axis.

Myeloid Deletion of Nemo Causes Osteopetrosis in Mice Owing to Upregulation of Transcriptional Repressors.

The transcription factor NF-κB is central to numerous physiologic processes including bone development, and its activation is controlled by IKKγ (also called NEMO), the regulatory subunit of IKK complex. NEMO is X-linked, and mutations in this gene result in Incontinentia Pigmenti in human hemizygous females. In mice, global deficiency causes embryonic lethality. In addition, certain point mutations in the NEMO (IKBKG) human gene manifest skeletal defects implicating NEMO in the regulation of bone homeostasis. To specifically investigate such role, we conditionally deleted Nemo from osteoclast and myeloid progenitors. Morphometric, histologic, and molecular analyses demonstrate that myeloid NEMO deletion causes osteopetrosis in mice. Mechanistically, NEMO deficiency hampered activation of IKK complex in osteoclast precursors, causing arrest of osteoclastogenesis and apoptosis. Interestingly, inhibiting apoptosis by genetic ablation of TNFr1 significantly increased cell survival, but failed to rescue osteoclastogenesis or reverse osteopetrosis. Based on this observation, we analyzed the expression of different regulators of osteoclastogenesis and discovered that NEMO deletion leads to increased RBPJ expression, resulting in a decrease of Blimp1 expression. Consequently, expression of IRF8 and Bcl6 which are targets of Blimp1 and potent osteoclastogenic transcriptional repressors, is increased. Thus, NEMO governs survival and osteoclast differentiation programs through serial regulation of multiple transcription factors.

Depletion of UBC9 Causes Nuclear Defects during the Vegetative and Sexual Life Cycles in Tetrahymena thermophila.

Ubc9p is the sole E2-conjugating enzyme for SUMOylation, and its proper function is required for regulating key nuclear events such as transcription, DNA repair, and mitosis. In Tetrahymena thermophila, the genome is separated into a diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid somatic macronucleus (MAC) that divides amitotically. This unusual nuclear organization provides novel opportunities for the study of SUMOylation and Ubc9p function. We identified the UBC9 gene and demonstrated that its complete deletion from both MIC and MAC genomes is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with CdCl2-dependent expression of green fluorescent protein (GFP)-Ubc9p. Depletion of Ubc9p in vegetative cells resulted in the loss of MICs, but MACs continued to divide. In contrast, expression of catalytically inactive Ubc9p resulted in the accumulation of multiple MICs. Critical roles for Ubc9p were also identified during the sexual life cycle of Tetrahymena. Cell lines that were depleted for Ubc9p did not form mating pairs and therefore could not complete any of the subsequent stages of conjugation, including meiosis and macronuclear development. Mating between cells expressing catalytically inactive Ubc9p resulted in arrest during macronuclear development, consistent with our observation that Ubc9p accumulates in the developing macronucleus.

SUMOylation is developmentally regulated and required for cell pairing during conjugation in Tetrahymena thermophila.

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena, including a dynamic regulation associated with the sexual life cycle.