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Objective: To elucidate the effects of resistin on human articular chondrocytes and to generate a picture of their regulation at the transcriptional and posttranscriptional levels., Methods: Human articular chondrocytes were cultured with resistin. Changes in gene expression were analyzed at various doses and times. Cells were also treated with the transcription inhibitor actinomycin D after resistin treatment or with the NF-kappaB inhibitor IKK-NBD before resistin treatment. Gene expression was tested by quantitative real-time polymerase chain reaction. Computational analysis for transcription factor binding motifs was performed on the promoter regions of differentially expressed genes. TC-28 chondrocytes were transfected with CCL3 and CCL4 promoter constructs, pNF-kappaB reporter, and NF-kappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) expression vectors with or without resistin., Results: Resistin-treated human articular chondrocytes increased the expression of cytokines and chemokines. Levels of messenger RNA (mRNA) for matrix metalloproteinase 1 (MMP-1), MMP-13, and ADAMTS-4 also increased, while type II collagen alpha1 (COL2A1) and aggrecan were down-regulated. The cytokine and chemokine genes could be categorized into 3 groups according to the pattern of mRNA expression over a 24-hour time course. One pattern suggested rapid regulation by mRNA stability. The second and third patterns were consistent with transcriptional regulation. Computational analysis suggested the transcription factors NF-kappaB and C/EBPbeta were involved in the resistin-induced up-regulation. This prediction was confirmed by the cotransfection of NF-kappaB and C/EBPbeta and the IKK-NBD inhibition., Conclusion: Resistin has diverse effects on gene expression in human chondrocytes, affecting chemokines, cytokines, and matrix genes. Messenger RNA stabilization and transcriptional up-regulation are involved in resistin-induced gene expression in human chondrocytes.

Background: More than 450,000 rotator cuff repairs are performed annually, yet healing of tendon to bone often fails. This failure is rooted in the fibrovascular healing response, which does not regenerate the native attachment site. Better healing outcomes may be achieved by targeting inflammation during the early period after repair. Rather than broad inhibition of inflammation, which may impair healing, the current study utilized a molecularly targeted approach to suppress IKKβ, shutting down only the inflammatory arm of the nuclear factor κB (NF-κB) signaling pathway. Purpose: To evaluate the therapeutic potential of IKKβ inhibition in a clinically relevant model of rat rotator cuff repair. Study Design: Controlled laboratory study. Methods: After validating the efficacy of the IKKβ inhibitor in vitro, it was administered orally once a day for 7 days after surgery in a rat rotator cuff repair model. The effect of treatment on reducing inflammation and improving repair quality was evaluated after 3 days and 2, 4, and 8 weeks of healing, using gene expression, biomechanics, bone morphometry, and histology. Results: Inhibition of IKKβ attenuated cytokine and chemokine production in vitro, demonstrating the potential for this inhibitor to reduce inflammation in vivo. Oral treatment with IKKβ inhibitor reduced NF-κB target gene expression by up to 80% compared with a nontreated group at day 3, with a subset of these genes suppressed through 14 days. Furthermore, the IKKβ inhibitor led to enhanced tenogenesis and extracellular matrix production, as demonstrated by gene expression and histological analyses. At 4 weeks, inhibitor treatment led to increased toughness, no effects on failure load and strength, and decreases in stiffness and modulus when compared with vehicle control. At 8 weeks, IKKβ inhibitor treatment led to increased toughness, failure load, and strength compared with control animals. IKKβ inhibitor treatment prevented the bone loss near the tendon attachment that occurred in repairs in control. Conclusion: Pharmacological inhibition of IKKβ successfully suppressed excessive inflammation and enhanced tendon-to-bone healing after rotator cuff repair in a rat model. Clinical Relevance: The NF-κB pathway is a promising target for enhancing outcomes after rotator cuff repair. [ABSTRACT FROM AUTHOR]

Mutated NLRP3 assembles a hyperactive inflammasome, which causes excessive secretion of interleukin (IL)-1β and IL-18 and, ultimately, a spectrum of autoinflammatory disorders known as cryopyrinopathies of which neonatal-onset multisystem inflammatory disease (NOMID) is the most severe phenotype. NOMID mice phenocopy several features of the human disease as they develop severe systemic inflammation driven by IL-1β and IL-18 overproduction associated with damage to multiple organs, including spleen, skin, liver, and skeleton. Secretion of IL-1β and IL-18 requires gasdermin D (GSDMD), which—upon activation by the inflammasomes—translocates to the plasma membrane where it forms pores through which these cytokines are released. However, excessive pore formation resulting from sustained activation of GSDMD compromises membrane integrity and ultimately causes a pro-inflammatory form of cell death, termed pyroptosis. In this study, we first established a strong correlation between NLRP3 inflammasome activation and GSDMD processing and pyroptosis in vitro. Next, we used NOMID mice to determine the extent to which GSDMD-driven pyroptosis influences the pathogenesis of this disorder. Remarkably, all NOMID-associated inflammatory symptoms are prevented upon ablation of GSDMD. Thus, GSDMD-dependent actions are required for the pathogenesis of NOMID in mice. [ABSTRACT FROM AUTHOR]

The article presents a study which examined the role of resistin in human articular chondrocytes. According to the authors, resistin-treated human articular chondrocytes increased the expression of cytokines and chemokines. They indicate the involvement of the messenger RNA (mRNA) stabilization and transcriptional up-regulation in resistin-induced gene expression in human chondrocytes. The three patterns of mRNA expression are discussed.

Osteoclasts, the primary bone-resorbing cells, play a crucial role in periprosthetic bone loss in response to implant-derived wear debris. Differentiation and activation of osteoclasts at the implant-bone interface are fueled by elevated levels of locally secreted inflammatory cytokines that heighten the osteolytic response. Among these cytokines are members of the TNF superfamily, including TNF and RANKL, which primarily act through activation of the transcription factor NF-κB. Activation of NF-κB is required for osteoclast formation, and its inhibition hampers osteoclastogenesis and bone loss. Activation of NF-κB is permitted following its dissociation from the inhibitory protein IκBα, an event subsequent to phosphorylation of the latter protein by the upstream IκBα kinase (IKK) complex. Our recent findings show that attenuating IKK complex assembly, by using a short peptide termed NEMO-binding domain (NBD) peptide, that blocks binding of IKK2 and IKK1 to IKKγ/NEMO, inhibits NF-κB activation, and arrests RANKL-induced osteoclastogenesis. In this study, we examined if NBD is capable of blocking inflammatory osteolysis by PMMA particles. Our findings indicate that NBD peptide inhibits PMMA-induced IKK2 and NF-κB activation. More importantly, this peptide potently arrests PMMA-stimulated osteoclastogenesis and alleviates PMMA-induced inflammatory and osteolytic responses in mice. Thus, NBD peptide is considered as a promising modality to regulate inflammatory osteolysis. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1358-1365, 2006 [ABSTRACT FROM AUTHOR]

RANKL is a TNF superfamily member and an essential cytokine mediator of developmental osteoclastogenesis. We examined the role of RANKL in PMMA particle-induced osteoclastogenesis in vitro. In murine whole bone marrow cultures, PMMA particles stimulate a 2.5 fold increase in secreted RANKL, a 5–8 fold increase in osteoclast number and induce the formation of giant multinuclear osteoclasts. RANKL and TNF, potential cytokine mediators of PMMA, had similar osteoclastogenic effects. The RANKL inhibitor OPG was utilized to define the role of RANKL in mediating the PMMA response and was found to inhibit basal and PMMA particle-induced osteoclastogenesis. Additionally, particles stimulate osteoclast formation in RANKL-primed osteoclast precursor cells (devoid of supporting stromal cells) while RANKL untreated osteoclast precursors demonstrate no osteoclastogenic response to particles. Since TNF can potentiate RANKL action and is thought to mediate implant osteolysis we analyzed TNF−/− whole bone marrow cultures to elucidate the role of this cytokine. In TNF−/− cultures basal osteoclastogenesis remains intact, yet the PMMA effect is blunted. Finally, we show that PMMA, RANKL and TNF all activate the NF-kB and c-jun/AP-1 signaling pathways which are both fundamental to osteoclast formation and are potential sites of signal convergence in RANKL-mediated particle osteoclastogenesis. [Copyright &y& Elsevier]

Tumor necrosis factor-α (TNF) is a potent osteoclastogenic cytokine that has a fundamental role in the pathogenesis of implant particle-induced osteolysis. The nuclear transcription factor NF-κB mediates TNF signaling and this transcription complex is necessary for osteoclastogenesis. Because polymethylmethacrylate (PMMA) particles cause osteolysis, we reasoned the PMMA would induce NF-κB activation. In fact, we find that exposure of osteoclast precursors, in the form of colony stimulating factor-1 (CSF-1) dependent murine bone marrow macrophages, to PMMA particles prompts nuclear translocation and activation of NF-κB. Supershift assays confirm the presence of the p50 and p65 NF-κB subunits in the activated transcription factor. Particle-induced NF-κB activation is equal in both wild type and LPS- hyporesponsive cells indicating that the phenomenon does not represent endotoxin contamination. A soluble, competitive inhibitor of TNF (huTNFr:Fc) dampens particle-directed NF-κB activation and this response is also abrogated in TNF−/− osteoclast precursors. Thus, PMMA particle activation of NF-κB is a secondary event resulting from enhanced TNF expression and is independent of LPS contamination. [Copyright &y& Elsevier]

Inhibition of IKKβ/NF-κB in stromal cells prevents degeneration and improves healing, representing a potential therapeutic for chronic tendinopathy. Targeting tendinopathy: Inflammation and NF-κB signaling contribute to tendon degeneration and injury, such as rotator cuff injury. Abraham et al. investigated how this signaling pathway causes tendinopathy and potential therapeutic effects of inhibiting the pathway. Clinical tendon samples of human rotator cuff disease showed up-regulation of NF-κB, which was mimicked in mouse tendon fibroblasts overexpressing IKKβ. Genetic deletion of IKKβ in tendon in mice prevented maladaptive tendon remodeling in a treadmill running–induced overuse tendinopathy model and in a surgical model of tendon injury and repair, and human tendon stromal cells treated with an IKKβ inhibitor showed repressed NF-κB target gene transcription. These results suggest that there may be therapeutic potential in blocking IKKβ. Tendon disorders represent the most common musculoskeletal complaint for which patients seek medical attention; inflammation drives tendon degeneration before tearing and impairs healing after repair. Clinical evidence has implicated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway as a correlate of pain-free return to function after surgical repair. However, it is currently unknown whether this response is a reaction to or a driver of pathology. Therefore, we aimed to understand the clinically relevant involvement of the NF-κB pathway in tendinopathy, to determine its potential causative roles in tendon degeneration, and to test its potential as a therapeutic candidate. Transcriptional profiling of early rotator cuff tendinopathy identified increases in NF-κB signaling, including increased expression of the regulatory serine kinase subunit IKKβ, which plays an essential role in inflammation. Using cre-mediated overexpression of IKKβ in tendon fibroblasts, we observed degeneration of mouse rotator cuff tendons and the adjacent humeral head. These changes were associated with increases in proinflammatory cytokines and innate immune cells within the joint. Conversely, genetic deletion of IKKβ in tendon fibroblasts partially protected mice from chronic overuse–induced tendinopathy. Furthermore, conditional knockout of IKKβ improved outcomes after surgical repair, whereas overexpression impaired tendon healing. Accordingly, targeting of the IKKβ/NF-κB pathway in tendon stromal cells may offer previously unidentified therapeutic approaches in the management of human tendon disorders. [ABSTRACT FROM AUTHOR]

Activation of NF-κB leads to expression of ample genes that regulate inflammatory and osteoclastogenic responses. The process is facilitated by induction of IκB kinase (IKK) complex that phosphorylates IκB and leads to its dissociation from the NF-κB complex, thus permitting activation of NF-κB. The IKK complex contains primarily IKKα, IKKβ, and the regulatory kinase IKKγ, also known as NEMO. NEMO regulates the IKK complex activity through its binding to carboxyl-terminal region of IKKα and IKKβ, termed NEMO-binding domain (NBD). In this regard, a cell-permeable NBD peptide has been shown to block association of NEMO with the IKK complex and inhibit activation of NF-κB. Given the pivotal role of cytokine-induced NF-κB in osteoclastogenesis and inflammatory bone loss, we deduced that cell-permeable TAT-NBD peptide may hinder osteoclastogenesis and bone erosion in inflammatory arthritis. Using NBD peptides, we show that wild type, but not mutant, NBD blocks IKK activation and reduces cytokine-induced promoter and DNA binding activities of NF-κB and inhibits cytokine-induced osteoclast formation by osteoclast precursors. Consistent with the key role of NF-κB in osteo-inflammatory responses in vivo, wild type TAT-NBD administered into mice prior to induction of inflammatory arthritis efficiently block in vivo osteoclastogenesis, inhibits focal bone erosion, and ameliorates inflammatory responses in the joints of arthritic mice. The mutant NBD peptide fails to exert these functions. These results provide strong evidence that IKKs are potent regulators of cytokine-induced osteoclastogenesis and inflammatory arthritis. More importantly, blockade of NEMO assembly with the IKK complex is a viable strategy to avert inflammatory osteolysis. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α (TNF) is a pro-inflammatory cytokine with a profound role in many skeletal diseases. The cytokine has been described as a mediator of bone loss in osteolysis and other inflammatory bone diseases. In addition to its known bone resorptive action, TNF reduces bone formation by inhibiting osteoblast differentiation. Using primary and transformed osteoblastic cells, we first document that TNF inhibits expression of alkaline phosphatase and matrix deposition, both considered markers of osteoblast differentiation. The effects are dose- and time-dependent. Core-binding factor A1 (cbfa1) is a transcription factor critical for osteoblast differentiation, and we show here that it is activated by the osteoblast differentiation agent, β-glycerophosphate. Therefore, we investigated whether the inhibitory effects of TNF were associated with altered activity of this transcription factor. Using retardation assays, we show that TNF significantly inhibits cbfa1 activation by β-glycerophosphate, manifested by reduced DNA-binding activity. Next, we turned to determine the signaling pathway by which TNF inhibits osteoblast differentiation. Utilizing animals lacking individual TNF receptors, we document that TNFr1 is required for transmitting the cytokine''s inhibitory effect. In the absence of this receptor, TNF failed to impact all osteoblast differentiation markers tested.In summary, TNF blocks expression of osteoblast differentiation markers and inhibits β-glycerophosphate-induced activation of the osteoblast differentiation factor cbfa1. Importantly, these effects are mediated via a mechanism requiring the TNF type-1 receptor. [Copyright &y& Elsevier]