Abstract
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.
Highlights
NF-κB signaling entails transmission of receptor-activating signals leading to assembly and activation of a signalosome complex that includes adaptor proteins and kinases
Genotyping PCR and Western blot analyses confirmed efficient deletion of NEMO evident by minimal expression of NEMO in NM-cKO mice (Fig. 1B, top panel). This was further supported by failure to activate IKK2 (Fig. 1B, middle panel) and subsequent phosphorylation of IκB (p-IκB) or phosphorylation of exogenous GST-IκB substrate (Fig. 1C) following treatments with receptor activator of nuclear factor kappa-B ligand (RANKL or RL), confirming that NEMO is required for RANKL-induced activation of NF-κB pathway
The results indicate that NM-cKO-derived marrow cells exhibit impaired osteoclast differentiation potential evident by significantly less osteoclast formation in vitro (Fig. 3B) and by reduced expression of molecular markers of osteoclast differentiation, including tartrate-resistant acid phosphatase (TRAP), Cathepsin-K (CTSK), Matrix metallopeptidase 9 (MMP9), and Nuclear Factor of Activated T-Cells Calcineurin-Dependent 1 (NFATc1) (Fig. 3C)
Summary
NF-κB signaling entails transmission of receptor-activating signals leading to assembly and activation of a signalosome complex that includes adaptor proteins and kinases. Despite this massive effort, decoding the precise repertoire of NF-κB action under physiologic and pathologic conditions remains an overwhelming challenge In this regard, a relentless effort continues to decipher the molecular role of IKKƴ/NEMO as a signal integrator and a scaffold protein pairing upstream cellular responses with kinase-targeted selection of appropriate substrates in a cell-specific manner[26]. A relentless effort continues to decipher the molecular role of IKKƴ/NEMO as a signal integrator and a scaffold protein pairing upstream cellular responses with kinase-targeted selection of appropriate substrates in a cell-specific manner[26] Supporting this contention is a recent report indicating that NEMO determined stimulus-specific transduction by directing the IKK complex hub toward IκBα27. This defect arises from dysregulation of NF-κB-dependent transcriptional machinery in the myeloid lineage
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