A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation
The growth arrest and DNA damage-inducible 45beta (GADD45beta) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45beta in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45beta as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45beta mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45beta protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45beta(-/-) mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45beta in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45beta stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45beta plays an essential role during chondrocyte terminal differentiation.
Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins that are major inflammatory agents. COX-2 production is triggered by exposure to various cytokines and to bacterial endotoxins. We present here a novel role for the Ets transcription factor ESE-1 in regulating the COX-2 gene in response to endotoxin and other pro-inflammatory stimuli. We report that the induction of COX-2 expression by lipopolysaccharide (LPS) and pro-inflammatory cytokines correlates with ESE-1 induction in monocyte/macrophages. ESE-1, in turn, binds to several E26 transformation specific (Ets) sites on the COX-2 promoter. In vitro analysis demonstrates that ESE-1 binds to and activates the COX-2 promoter to levels comparable to LPS-mediated induction. Moreover, we provide results showing that the induction of COX-2 by LPS may require ESE-1, as the mutation of the Ets sites in the COX-2 promoter or overexpression of a dominant-negative form of ESE-1 inhibits LPS-mediated COX-2 induction. The effect of ESE-1 on the COX-2 promoter is further enhanced by cooperation with other transcription factors such as nuclear factor-kappa B and nuclear factor of activated T cells. Neutralization of COX-2 is the goal of many anti-inflammatory drugs. As an activator of COX-2 induction, ESE-1 may become a target for such therapeutics as well. Together with our previous reports of the role of ESE-1 as an inducer of nitric oxide synthase in endothelial cells and as a mediator of pro-inflammatory cytokines in vascular and connective tissue cells, these results establish ESE-1 as an important player in the regulation of inflammation.
Lack of requirement of osteopontin for inflammation, bone erosion, and cartilage damage in the K/BxN model of autoantibody-mediated arthritis
OBJECTIVE: Osteopontin (OPN) is a secreted glycoprotein involved in a range of physiologic processes, including inflammation, immunity mediated by Th1 cells, and bone remodeling. It is expressed in the joints of rheumatoid arthritis patients and has been the subject of conflicting reports concerning its role in arthritis induced by antibodies against type II collagen. This study assessed the role of OPN in the K/BxN serum-transfer model of autoantibody-induced arthritis.
METHODS: Expression of OPN gene transcripts was assessed by microarray analysis of ankle RNA taken at 6 time points after transfer of K/BxN serum. OPN-sufficient or OPN-deficient littermates backcrossed for 10 generations onto the C57BL/6 genetic background were given K/BxN serum. Arthritis severity was measured by ankle thickening and a clinical index. Hind limb sections were stained with hematoxylin and eosin or toluidine blue and scored for inflammation, cartilage damage, and bone erosion.
RESULTS: OPN messenger RNA transcripts progressively increased in ankle joints during the course of K/BxN serum-transferred arthritis. OPN-deficient mice receiving K/BxN serum developed arthritis with kinetics and clinical severity comparable with those of OPN-sufficient littermates. Histologic assessment of arthritic joints from OPN-deficient mice revealed synovial hyperplasia, pannus formation, mononuclear cell infiltration, bone erosion, cartilage damage at sites adjacent to and distal from pannus invasion, and tartrate-resistant acid phosphatase-positive multinucleated cells at sites of bone erosion. Histopathologic scoring demonstrated comparable levels of inflammation, cartilage damage, and bone erosion in OPN-sufficient and OPN-deficient mice.
CONCLUSION: OPN does not have a required role in inflammation, bone erosion, and cartilage damage in the K/BxN serum-transfer model.
ESE-1 is a novel transcriptional mediator of angiopoietin-1 expression in the setting of inflammation
Angiogenesis is a critical component of the inflammatory response associated with a number of conditions. Angiopoietin-1 (Ang-1) is an angiogenic growth factor that promotes the chemotaxis of endothelial cells and facilitates the maturation of new blood vessels. Ang-1 expression is up-regulated in response to tumor necrosis factor-alpha (TNF-alpha). To begin to elucidate the underlying molecular mechanisms by which Ang-1 gene expression is regulated during inflammation, we isolated 3.2 kb of the Ang-1 promoter that contain regulatory elements sufficient to mediate induction of the promoter in response to TNF-alpha, interleukin-1beta, and endotoxin. Surprisingly, sequence analysis of this promoter failed to reveal binding sites for transcription factors that are frequently associated with mediating inflammatory responses, such as NF-kappaB, STAT, NFAT, or C/EBP. However, putative binding sites for ETS and AP-1 transcription factor family members were identified. Interestingly, among a panel of ETS factors tested in a transient transfection assay, only the ETS factor ESE-1 was capable of transactivating the Ang-1 promoter. ESE-1 binds to specific ETS sites within the Ang-1 promoter that are functionally important for transactivation by ESE-1. ESE-1 and Ang-1 are induced in synovial fibroblasts in response to inflammatory cytokines, with ESE-1 induction slightly preceding that of Ang-1. Mutation of a high-affinity ESE-1 binding site leads to a marked reduction in Ang-1 transactivation by ESE-1, inducibility by inflammatory cytokines, and DNA binding to the ESE-1 protein. Transcriptional profiling of cells transiently transfected with an ESE-1 expression vector demonstrates that the endogenous Ang-1 gene is directly inducible by ESE-1. Finally, Ang-1 and ESE-1 exhibit a similar and strong expression pattern in the synovium of patients with rheumatoid arthritis. Our results support a novel paradigm for the ETS factor ESE-1 as a transcriptional mediator of angiogenesis in the setting of inflammation.
Responses to the proinflammatory cytokines interleukin-1 and tumor necrosis factor alpha in cells derived from rheumatoid synovium and other joint tissues involve nuclear factor kappaB-mediated induction of the Ets transcription factor ESE-1
OBJECTIVE: To investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor kappaB (NF-kappaB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines.
METHODS: ESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase-polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor kappaB (IkappaB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-kappaB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-kappaB family were conducted using the NF-kappaB site from the ESE-1 promoter as a probe.
RESULTS: Immunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1beta and TNFalpha in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1beta requires activation of NF-kappaB and binding of p50 and p65 family members to the NF-kappaB site in the ESE-1 promoter. Overexpression of IkappaB using an adenoviral vector blocked IL-1beta-induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-kappaB binds to the ESE-1 promoter in vivo.
CONCLUSION: ESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1beta, TNFalpha, or LPS. This induction relies on the translocation of the NF-kappaB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-kappaB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.
Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor alpha
OBJECTIVES: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis.
METHODS: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha.
RESULTS: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha.
CONCLUSIONS: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.
Increase in expression of receptor activator of nuclear factor kappaB at sites of bone erosion correlates with progression of inflammation in evolving collagen-induced arthritis
OBJECTIVE: The receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL) pathway is critical in osteoclastogenesis and bone resorption and has been implicated in the process of focal bone erosion in arthritis. This study was undertaken to identify in vivo the hitherto-unknown origin and localization of RANK-expressing osteoclast precursor cells at sites of bone erosion in arthritis.
METHODS: DBA-1 mice were immunized with bovine type II collagen/Freund's complete adjuvant and were given an intraperitoneal booster injection of type II collagen on day 21. Arthritis was monitored visually, and joint pathology was examined histologically. RANK and RANKL expression were analyzed using specific immunohistochemistry, and tartrate-resistant acid phosphatase (TRAP) staining was performed. In addition, TRAP and cathepsin K messenger RNA expression were analyzed by in situ hybridization.
RESULTS: A marked increase in the number of cells expressing RANK correlated with the progression of synovial inflammation and clinical disease severity in evolving collagen-induced arthritis (CIA). Interestingly, RANK expression demonstrated a gradient pattern with increased numbers of RANK-positive cells within the synovial infiltrate in areas closer to periosteum and cortical bone. Cells expressing RANK included cells in synovial tissue, bone lining cells on the surface of trabecular bone at sites of erosion, and cells in periosteal areas adjacent to synovial inflammation. In areas where RANK-positive cells were abundant, TRAP-positive, multinucleated osteoclast-like cells were also present at sites of focal bone erosion, suggesting differentiation of synovially derived RANK-positive osteoclast precursor cells into osteoclasts. In addition, TRAP- and cathepsin K-double-positive osteoclast-like cells were detected on the synovial side of cortical bone at sites of early and advanced cortical bone erosion. Sites of RANK expression also correlated well with sites of RANKL expression, and there was a close correlation of the temporal expression of the receptor-ligand pair.
CONCLUSION: Cells expressing RANK increased in abundance with the progression of arthritis in evolving CIA, and sites of RANK-expressing cells correlated with sites of TRAP-positive, multinucleated osteoclast-like cells as well as with sites of RANKL expression. These data support the hypothesis that the RANK/RANKL pathway plays an important role in the process of bone erosion in CIA.
In spontaneous inflammatory arthritis of K/BxN T cell receptor transgenic mice, the effector phase of the disease is provoked by binding of immunoglobulins (Igs) to joint surfaces. Inflammatory cytokines are known to be involved in human inflammatory arthritis, in particular rheumatoid arthritis, although, overall, the pathogenetic mechanisms of the human affliction remain unclear. To explore the analogy between the K/BxN model and human patients, we assessed the role and relative importance of inflammatory cytokines in K/BxN joint inflammation by transferring arthritogenic serum into a panel of genetically deficient recipients. Interleukin (IL)-1 proved absolutely necessary. Tumor necrosis factor (TNF)-alpha was also required, although seemingly less critically than IL-1, because a proportion of TNF-alpha-deficient mice developed robust disease. There was no evidence for an important role for IL-6. Bone destruction and reconstruction were also examined. We found that all mice with strong inflammation exhibited the bone erosion and reconstruction phenomena typical of K/BxN arthritis, with no evidence of any particular requirement for TNFalpha for bone destruction. The variability in the requirement for TNF-alpha, reminiscent of that observed in treated rheumatoid arthritis patients, did not appear genetically programmed but related instead to subtle environmental changes.
OBJECTIVE: To evaluate the safety and efficacy of etanercept in the treatment of adult patients with Still's disease.
METHODS: Twelve adult patients who met criteria for Still's disease and had active arthritis were enrolled in a 6-month open-label trial of etanercept given in biweekly doses of 25 mg. The mean disease duration at study entry was 10.7 years. All patients had been treated unsuccessfully with other disease-modifying antirheumatic drugs. Efficacy was evaluated according to American College of Rheumatology (ACR) improvement criteria, and adverse events were recorded.
RESULTS: Ten patients successfully completed the study; 2 withdrew due to disease flare. In 4 patients, the dosage of etanercept was increased from 25 mg biweekly to 25 mg 3 times per week. Seven patients met ACR 20% response criteria. Of these 7 responders, 4 met ACR 50% response criteria and 2 met ACR 70% response criteria. Among the 3 patients with systemic features of Still's disease (fever and rash), improvement in these features was seen in 1; the arthritis did not improve in any of these 3 patients. Except in the 2 patients who withdrew due to disease flare (rash, fever, and arthritis), no other significant adverse events occurred.
CONCLUSION: In this initial study of etanercept therapy for Still's disease in the adult, this treatment resulted in improvement in the arthritis and was well tolerated. Additional trials should be performed to elucidate the effects of tumor necrosis factor inhibitors in Still's disease.
OBJECTIVE: To determine and compare the expression of cathepsins K and S proteins in joints with rheumatoid arthritis (RA) and osteoarthritis (OA) and to determine the effect of interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) on the expression of cathepsin K in fibroblast-like synoviocytes.
METHOD: Expression and localization of cathepsins K and S were determined by immunohistochemistry in the synovium of 10 RA- and 8 OA-affected joints. Northern and Western blot analyses were performed to analyze cathepsin K and S expression in primary fibroblast-like synoviocyte cultures from RA and OA patients. The effect of IL-1 beta and TNF alpha on the expression and secretion of cathepsin K in primary cultures of synoviocytes was determined by real-time polymerase chain reaction and Western blot analysis. Staining of in situ activity was used to identify active cathepsin K enzyme in primary synovial fibroblast cultures.
RESULTS: Cathepsin K and S protein expression was identified in the synovium from patients with RA and OA. Cathepsin K protein was localized in synovial fibroblasts, stromal multinucleated giant cells, and, to a lesser degree, in CD68+ macrophage-like synoviocytes. Of note is the expression of cathepsin K in synovial fibroblasts and mononuclear macrophage-like cells at sites of cartilage erosion in RA and in interdigitating cells of lymphocyte-rich areas. In contrast, cathepsin S expression was restricted to CD68+ macrophage-like synoviocytes, interdigitating cells, and endothelial cells of blood vessels. Cathepsin K protein expression in the interstitial areas and perivascular regions of RA-derived synovial specimens was 2-5 times higher than in OA samples (P < 0.001), whereas the expression of cathepsin S did not significantly differ in these diseases. Cathepsin K expression levels in normal synovium were low and restricted to fibroblast-like cells. Of note, cathepsin K also was expressed in repairing fibrocartilage in 1 OA specimen. Primary cell cultures of RA- and OA-derived synovial fibroblasts expressed comparable amounts of cathepsin K at the transcript and protein levels. Both cell cultures secreted mature cathepsin K as well as procathepsin K, and expressed active cathepsin K in cytosolic vesicles. In contrast, neither RA- nor OA-derived fibroblasts expressed detectable levels of cathepsin S. IL-1 beta and TNF alpha stimulated the transcript (7-8-fold) and protein expression (2-fold) of cathepsin K (P < 0.05) in primary synovial fibroblast cultures, without differences in expression between RA- and OA-derived synovial fibroblasts.
CONCLUSION: The presence of cathepsin K polypeptide in synovial fibroblasts and macrophage-like cells in normal, OA, and RA synovia suggests a constitutive expression of this protease and a role in synovial remodeling. The comparable increase in cathepsin K expression after stimulation of RA- and OA-derived synovial fibroblasts with IL-1 beta and TNF alpha further suggests that the expression of cathepsin K is independent of cellular alterations leading to the invasive phenotype of RA-synovial fibroblasts. However, the overexpression of cathepsin K in RA synovia due to an increase in the number of cathepsin K-expressing cells identifies this enzyme as a candidate protease for the pathologic degradation of articular cartilage. Cathepsin S expression in macrophage-like synoviocytes suggests dual activity in antigen presentation and matrix degradation in the inflamed synovia.
The contribution of osteoclasts to the process of bone loss in inflammatory arthritis has recently been demonstrated. Studies in osteoclast biology have led to the identification of factors responsible for the differentiation and activation of osteoclasts, the most important of which is the receptor activator of NF-kappa B ligand/osteoclast differentiation factor (RANKL/ODF), a tumor necrosis factor (TNF)-like protein. The RANKL/ODF receptor, receptor activator of NF-kappa B (RANK), is a TNF-receptor family member present on both osteoclast precursors and mature osteoclasts. Like other TNF-family receptors and the IL-1 receptor, RANK mediates its signal transduction via TNF receptor-associated factor (TRAF) proteins, suggesting that the signaling pathways activated by RANK and other inflammatory cytokines involved in osteoclast differentiation and activation are interconnected.
Considerable progress has been made in identifying the transcription factors involved in the early specification of the B-lymphocyte lineage. However, little is known about factors that control the transition of mature activated B cells to antibody-secreting plasma cells. Here we report that the transcription factor XBP-1 is required for the generation of plasma cells. XBP-1 transcripts were rapidly upregulated in vitro by stimuli that induce plasma-cell differentiation, and were found at high levels in plasma cells from rheumatoid synovium. When introduced into B-lineage cells, XBP-1 initiated plasma-cell differentiation. Mouse lymphoid chimaeras deficient in XBP-1 possessed normal numbers of activated B lymphocytes that proliferated, secreted cytokines and formed normal germinal centres. However, they secreted very little immunoglobulin of any isotype and failed to control infection with the B-cell-dependent polyoma virus, because plasma cells were markedly absent. XBP-1 is the only transcription factor known to be selectively and specifically required for the terminal differentiation of B lymphocytes to plasma cells.
Tumor biology: use of tiled images in conjunction with measurements of cellular proliferation and death in response to drug treatments
Tumor growth is dependent on the balance between cell proliferation and cell death, and these events occur heterogenously within an individual tumor. We present a methodology that provides integrative information about cell kinetics, cell death, and cell growth within individual tumors in animals treated with cytotoxic chemotherapeutic agents. Using HCT-116 and NCI-H460 cells, human colonic adenocarcinoma and non-small cell lung cells, respectively, traditional xenograft studies were performed. The tumor-bearing animals were treated with cyclophosphamide (Cytoxan), gemcitabine (Gemzar), or mitomycin C, and extensive analysis of the tumors was studied. Cell kinetics were evaluated by measuring the apoptotic and proliferation indices. The ability to image an entire tumor section using "tiling" by creating a large montage from many high-resolution images makes it possible to identify regional differences within areas of tumor and to demonstrate differences in these tumor regions after treatment with selected chemotherapeutic agents. Two specific areas within tumors have been identified: (a) areas of viable cells within the cell cycle, determined by bromodeoxyuridine and/or morphological characteristics determined by hematoxylin staining; and (b) areas of necrosis determined by the absence of bromodeoxyuridine and proliferating cell nuclear antigen-labeled cells coupled with morphological changes. By standardizing the tumor size to 100 mm2, different patterns of tumor responses to chemotherapeutic agents were determined. By creating such tiled images and by quantitating cell cycle kinetics, it is possible to gain a more complete understanding of tumor growth and response to treatment, leading to the development of more reliable methods for assessing the clinical behavior of anticancer drugs.
OBJECTIVE: Osteoclast differentiation factor (ODF; also known as osteoprotegerin ligand, receptor activator of nuclear factor kappaB ligand, and tumor necrosis factor-related activation-induced cytokine) is a recently described cytokine known to be critical in inducing the differentiation of cells of the monocyte/macrophage lineage into osteoclasts. The role of osteoclasts in bone erosion in rheumatoid arthritis (RA) has been demonstrated, but the exact mechanisms involved in the formation and activation of osteoclasts in RA are not known. These studies address the potential role of ODF and the bone and marrow microenvironment in the pathogenesis of osteoclast-mediated bone erosion in RA.
METHODS: Tissue sections from the bone-pannus interface at sites of bone erosion were examined for the presence of osteoclast precursors by the colocalization of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K in mononuclear cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify mRNA for ODF in synovial tissues, adherent synovial fibroblasts, and activated T lymphocytes derived from patients with RA.
RESULTS: Multinucleated cells expressing both TRAP and cathepsin K mRNA were identified in bone resorption lacunae in areas of pannus invasion into bone in RA patients. In addition, mononuclear cells expressing both TRAP and cathepsin K mRNA (preosteoclasts) were identified in bone marrow in and adjacent to areas of pannus invasion in RA erosions. ODF mRNA was detected by RT-PCR in whole synovial tissues from patients with RA but not in normal synovial tissues. In addition, ODF mRNA was detected in cultured adherent synovial fibroblasts and in activated T lymphocytes derived from RA synovial tissue, which were expanded by exposure to anti-CD3.
CONCLUSION: TRAP-positive, cathepsin K-positive osteoclast precursor cells are identified in areas of pannus invasion into bone in RA. ODF is expressed by both synovial fibroblasts and by activated T lymphocytes derived from synovial tissues from patients with RA. These synovial cells may contribute directly to the expansion of osteoclast precursors and to the formation and activation of osteoclasts at sites of bone erosion in RA.
The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis
Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor.
Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis
Focal resorption of bone at the bone-pannus interface is common in rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) and can result in significant morbidity. However, the specific cellular and hormonal mechanisms involved in this process are not well established. We examined tissue sections from areas of bone erosion in patients with RA and JRA. Multinucleated cells (MNCs) were present in resorption lacunae in areas of calcified cartilage and in subchondral bone immediately adjacent to calcified cartilage, as previously described. mRNA for the calcitonin receptor (CTR) was localized to these MNCs in bone resorption lacunae, a finding that definitively identifies these cells as osteoclasts. These MNCs were also positive for tartrate-resistant acid phosphatase (TRAP) mRNA and TRAP enzymatic activity. Occasional mononuclear cells on the bone surface were also CTR positive. Mononuclear cells and MNCs not on bone surfaces were CTR negative. The restriction of CTR-positive cells to the surface of mineralized tissues suggests that bone and/or calcified cartilage provide signals that are critical for the differentiation of hematopoietic osteoclast precursors to fully differentiated osteoclasts. Some MNCs and mononuclear cells off bone and within invading tissues were TRAP positive. These cells likely represent the precursors of the CTR-TRAP-positive cells on bone. Parathyroid hormone receptor mRNA was present in cells with the phenotypic appearance of osteoblasts, in close proximity to MNCs, and in occasional cells within pannus tissue, but not in the MNCs in bone resorption lacunae. These findings demonstrate that osteoclasts within the rheumatoid lesion do not express parathyroid hormone receptor. In conclusion, the resorbing cells in RA exhibit a definitive osteoclastic phenotype, suggesting that pharmacological agents that inhibit osteoclast recruitment or activity are rational targets for blocking focal bone erosion in patients with RA and JRA.
Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5-17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.
Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc
The NF-AT family of transcription factors activates early immune response genes such as cytokines. In the adult, NF-ATc is expressed exclusively in the lymphoid system and is induced upon lymphocyte activation. NF-ATc null mutant mice die in utero of cardiac failure, precluding analysis of the role of NF-ATc in lymphocyte activation. By using RAG-2-deficient blastocyst complementation, we now demonstrate that young, highly chimeric mice lacking NF-ATc have impaired repopulation of both thymus and peripheral lymphoid organs. Furthermore, NF-ATc deficiency impaired T lymphocyte activation and secretion of IL-4. B lymphocytes displayed reduced proliferation and a selective loss of IL-4-driven immunoglobulin isotypes both in vivo and in vitro. Our data demonstrate that NF-ATc is essential for the optimal generation and function of mature T and B lineage cells, with an especially profound effect on IL-4-driven responses.
Cytokines in murine lyme carditis: Th1 cytokine expression follows expression of proinflammatory cytokines in a susceptible mouse strain
The cardiac infiltrate seen in murine Lyme carditis is composed predominantly of macrophages, but small numbers of T cells are also present. To identify the cytokines present in cardiac lesions from susceptible mice, semiquantitative polymerase chain reaction was done on cardiac tissue from mice infected with Borrelia burgdorferi. The temporal expression of proinflammatory and T cell-derived cytokines was characterized in cardiac tissue at days 0, 3, 7, 14, 21, and 42 after infection with B. burgdorferi. Early in the course of infection, up-regulation of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha was detected. The Th1 cytokine interferon-gamma appeared after the expression of the proinflammatory cytokines and remained elevated throughout the study. Interleukin-4 was not detectable at any time in cardiac lesions. These data are the first to identify cytokines expressed at the lesional level in murine Lyme carditis and to demonstrate a Th1 pattern of cytokine expression in this lesion.