Negative regulation of osteoclastogenesis and bone resorption by cytokines and transcriptional repressors

Metallothioneins (MTs) are cysteine-rich, metal-sequestering cytosolic proteins that play a key role in maintaining metal homeostasis and detoxification. We had previously characterized NmtA, a MT from the heterocystous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of Anabaena NmtA by AzuR (Alr0831) belonging to the SmtB/ArsR family of transcriptional repressors. There is currently no experimental evidence for any functional role of AzuR. It is observed that azuR is located within the znuABC operon but in the opposite orientation and remotely away from the nmtA locus. Sequence analysis of AzuR revealed a high degree of sequence identity with Synechococcus SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in Escherichia coli and purified it by chitin affinity chromatography. Far-UV circular dichroism spectroscopy indicated that the recombinant AzuR protein possessed a properly folded structure. Glutaraldehyde cross-linking and size-exclusion chromatography revealed that AzuR exists as a dimer of ∼28 kDa in solution. Analysis of its putative promoter region [100 bp upstream of nmtA open reading frame (ORF)] identified the presence of a 12–2–12 imperfect inverted repeat as the cis-acting element important for repressor binding. Electrophoretic mobility shift assays (EMSAs) showed concentration-dependent binding of recombinant dimeric AzuR with the promoter indicating that NmtA is indeed a regulatory target of AzuR. Binding of AzuR to DNA was disrupted in the presence of metal ions like Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Pb2+, and Mn2+. The metal-dependent dissociation of protein–DNA complexes suggested the negative regulation of metal-inducible nmtA expression by AzuR. Overexpression of azuR in its native strain Anabaena 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of Anabaena MT by an α5 SmtB/ArsR metalloregulator AzuR.

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TNF-induced osteoclastogenesis and inflammatory bone resorption are inhibited by transcription factor RBP-J

Journal of Experimental Medicine ◽

10.1084/jem.20111566 ◽

2012 ◽

Vol 209 (2) ◽

pp. 319-334 ◽

Cited By ~ 109

Author(s):

Baohong Zhao ◽

Shannon N. Grimes ◽

Susan Li ◽

Xiaoyu Hu ◽

Lionel B. Ivashkiv

Keyword(s):

Transcription Factor ◽

Bone Resorption ◽

B Lymphocyte ◽

Osteoclast Differentiation ◽

Negative Regulator ◽

Transcriptional Repressors ◽

Recognition Sequence ◽

Activated T Cells ◽

Maturation Protein ◽

Rank Signaling

Tumor necrosis factor (TNF) plays a key role in the pathogenesis of inflammatory bone resorption and associated morbidity in diseases such as rheumatoid arthritis and periodontitis. Mechanisms that regulate the direct osteoclastogenic properties of TNF to limit pathological bone resorption in inflammatory settings are mostly unknown. Here, we show that the transcription factor recombinant recognition sequence binding protein at the Jκ site (RBP-J) strongly suppresses TNF-induced osteoclastogenesis and inflammatory bone resorption, but has minimal effects on physiological bone remodeling. Myeloid-specific deletion of RBP-J converted TNF into a potent osteoclastogenic factor that could function independently of receptor activator of NF-κB (RANK) signaling. In the absence of RBP-J, TNF effectively induced osteoclastogenesis and bone resorption in RANK-deficient mice. Activation of RBP-J selectively in osteoclast precursors suppressed inflammatory osteoclastogenesis and arthritic bone resorption. Mechanistically, RBP-J suppressed induction of the master regulator of osteoclastogenesis (nuclear factor of activated T cells, cytoplasmic 1) by attenuating c-Fos activation and suppressing induction of B lymphocyte–induced maturation protein-1, thereby preventing the down-regulation of transcriptional repressors such as IRF-8 that block osteoclast differentiation. Thus, RBP-J regulates the balance between activating and repressive signals that regulate osteoclastogenesis. These findings identify RBP-J as a key upstream negative regulator of osteoclastogenesis that restrains excessive bone resorption in inflammatory settings.

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