Macrophages are important mediators of chronic inflammation and are prominent in the synovial lining and sublining of patients with rheumatoid arthritis (RA). Recently, we demonstrated increased TLR2 and TLR4 expression and increased response to microbial TLR2 and TLR4 ligands in macrophages from the joints of RA. The current study characterized the expression of the 96-kDa heat shock glycoprotein (gp96) in the joints of RA and its role as an endogenous TLR ligand to promote innate immunity in RA. gp96 was increased in RA compared with osteoarthritis and arthritis-free control synovial tissues. The expression of gp96 strongly correlated with inflammation and synovial lining thickness. gp96 was increased in synovial fluid from the joints of RA compared with disease controls. Recombinant gp96 was a potent activator of macrophages and the activation was mediated primarily through TLR2 signaling. The cellular response to gp96 was significantly stronger with RA synovial macrophages compared with peripheral blood monocytes from RA or healthy controls. The transcription of TLR2, TNF-α, and IL-8, but not TLR4, was significantly induced by gp96, and the induction was significantly greater in purified RA synovial macrophages. The expression of TLR2, but not TLR4, on synovial fluid macrophages strongly correlated with the level of gp96 in the synovial fluid. The present study documents the potential role of gp96 as an endogenous TLR2 ligand in RA and provides insight into the mechanism by which gp96 promotes the chronic inflammation of RA, identifying gp96 as a potential new therapeutic target.

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21 (WAF1/CIP1) , an established suppressor of cell cycle progression, is a inhibitor of IL-1b synthesis in macrophages. Mice deficient in p21 (p21 À/À ) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1b. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21 À/À mice. Analysis of isolated macrophages, which are one of the central producers of IL-1b, reveals that deficiency for p21 led to more IL-1b mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1b pro-protein is associated with elevated secretion of active IL-1b by p21 À/À macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1b secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1b by p21 À/À macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21 (WAF1/CIP1) in inhibiting inflammation. IntroductionThe pleiotropic cytokine IL-1b is required for the progression of numerous inflammatory disorders [1]. As key components of the innate immune response, monocytes and macrophages play an important role in the initiation and/or progression of inflammatory disease, largely through secretion of IL-1b, which contributes significantly to pathogenesis. Current treatments for diseases such as rheumatoid arthritis, gout, and periodic fever syndromes include the IL-1 receptor antagonist anakinra (Kineret s , Amgen) SHORT COMMUNICATIONÃ These authors contributed equally to this work. 820[2]; yet this therapy is ineffective in a cohort of patients [3]. Therefore, a better understanding of the mechanisms by which IL-1b production is regulated remains crucial for the development of new therapies to treat inflammatory diseases.Here, we investigate the role that p21 (WAF1/CIP1) (p21) plays in the production of IL-1b and development of inflammatory disease. As a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, p21 induces blockade of cell cycle progression via inhibition of the activity of CDK/cyclin complexes as well as that of proliferating cell nuclear antigen (PCNA) [4]. p21 binds to CDK and cyclins through two distinct domains on the N-terminus and to PCNA via its C-terminus [5]. Mice deficient in p21 show no developmental or reproductive abnormalities, although the mice display sensitivity to radiation [6] and develop a form of lupus-like disease [6][7][8]. Furthermore, a paucity of p21 expression is observed in synovial tissue from rheumatoid arthritis patients compared with osteoarthritis controls [9]. Because of the implication of p21 in the pathogenesis of lupus and rheumatoid arthritis and the importance of IL-1b in these and other inflamm...

Monocytes recruited into tissues from peripheral blood differentiate into macrophages, which are critical in the pathogenesis of many diseases. There is limited data concerning the global changes in the expression of genes during monocyte to macrophage differentiation, and how the patterns of change identify the mechanism contributing to macrophage differentiation or function. Employing microarray technology, we examined the transcriptional profile of in vitro adherence-induced differentiation of primary human monocytes into macrophages. We found the significant up regulation of genes contributing to the functions of macrophages, including those regulating to immunity and defense; lipid, fatty acid and steroid metabolism; cell adhesion, carbohydrate metabolism; amino acid metabolism and endocytosis. In contrast, the vast majority of transcription factors affected were down regulated during monocyte to macrophage differentiation, suggesting that transcriptional repression may be important for the transition from monocytes to macrophages. However, a limited number of transcription factors were up regulated, among these was C/EBPα, which may contribute to differentiation by regulating down stream genes, which are a characteristic of differentiated macrophages. These observations suggest that examination of the transcriptional profile in monocytes and macrophages in patients may identify relevant therapeutic targets in diseases mediated by macrophages.

MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNAbinding domain that is found in several tRNA and rRNA modification enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/ DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysomeassociated mRNAs, we showed that up-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis. (Cancer Res 2006; 66(18): 8994-9001)

MicroRNA-132 (miR‑132) has been reported to play a tumor suppressive role in different human malignancies. However, its role and underling mechanism in hepatocellular carcinoma (HCC) remains poorly defined due to lack of target gene information. In the present study, we demonstrated that the mean level of miR‑132 in hepatocellular carcinoma (HCC) tissues was significantly lower than that in matched tumor-adjacent tissues, and its expression negatively correlated with tumor differentiation (P<0.01), TNM stage (P<0.01) and lymph node metastasis (P<0.01). Similarly, the expression of miR‑132 was obviously reduced in HCC cell lines as compared with a normal hepatic cell line. Ectopic expression of miR‑132 inhibited cell proliferation, colony formation, migration and invasion, and induced apoptosis in HepG2 cells. In vivo studies showed that miR‑132 inhibited tumor growth of HCC and decreased tumor volume and weight. In addition, phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) was identified as a direct target of miR‑132 by a luciferase reporter assay. Western blot and qRT-PCR analysis indicated that PIK3R3 was significantly downregulated by miR‑132 in HCC cells. miR‑132 expression inversely correlated with PIK3R3 mRNA expression in clinical HCC tissues. Investigations into possible mechanisms revealed that miR‑132 inactivated the AKT/mTOR signaling pathway, which may contribute to inhibition of proliferation, migration, and invasion of HCC. These findings suggested that miR‑132 may serve as a potential target in the treatment of human HCC.