Article

Bone Morphogenetic Proteins 2 and 6, Expressed in Arthritic Synovium, are Regulated by Proinflammatory Cytokines and Differentially Modulate Fibroblast-Like Synoviocyte Apoptosis

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Abstract

To examine the expression, regulation, and potential roles of bone morphogenetic proteins (BMPs) in arthritic synovium. Expression of BMPs in arthritic synovium from patients with rheumatoid arthritis (RA) or spondylarthropathy (SpA) and in noninflamed synovium from patients undergoing diagnostic or therapeutic arthroscopies was studied by reverse transcription–polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and 2-color immunofluorescence. In vitro regulation of gene expression in fibroblast-like synoviocytes (FLS) was determined by real-time quantitative RT-PCR and immunohistochemistry. We used 3H-thymidine incorporation after serum deprivation–induced growth arrest to examine effects on FLS proliferation. FLS apoptosis was evaluated by flow cytometry cell cycle analysis. Apoptotic cells in synovial tissue were detected by TUNEL staining. Transcripts of different BMPs, most strikingly BMP-2 and BMP-6, were detected in synovial tissues. By Western blot, BMP-2 and BMP-6 precursor protein was found in RA and SpA synovial tissue extracts, but not in extracts of noninflamed synovial tissue. By immunohistochemistry, BMP-2 and BMP-6 were detected in the hyperplastic lining and the sublining layer of synovium from RA and SpA patients, both in CD90+ cells (FLS) and in some CD68+ cells (macrophages). Proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor α, but not interferon-γ, enhanced the expression of BMP-2 and BMP-6 transcripts in FLS in vitro. Neither BMP-2 nor BMP-6 affected FLS proliferation. BMP-2 promoted FLS apoptosis, whereas BMP-6 protected against nitric oxide–induced FLS apoptosis. BMP-2–positive apoptotic cells were found in arthritic synovium. BMP-2 and BMP-6 are expressed in arthritic synovium and are strongly up-regulated by proinflammatory cytokines. Although BMP signaling has been proposed to be involved in cartilage and bone repair in arthritis, this pathway may be equally important in modulating FLS cell populations in inflamed synovium.

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... Involvement of bone morphogenetic protein (BMP) has also been reported [194,195]. BMP, a member of the TGF-β superfamily, is known as a key regulator of bone formation, strongly inducing the expression of osteogenic transcription factors, such as Runx2 and osterix. Endochondral ossification is observed in bone formation sites of SpA patients, and BMP-2, BMP-6, and TGF-β are highly expressed there [194,195]. ...
... BMP, a member of the TGF-β superfamily, is known as a key regulator of bone formation, strongly inducing the expression of osteogenic transcription factors, such as Runx2 and osterix. Endochondral ossification is observed in bone formation sites of SpA patients, and BMP-2, BMP-6, and TGF-β are highly expressed there [194,195]. When IL-1β or TNF-α acts on synovial fibroblasts derived from patients with SpA, the expression of BMP-2 or BMP-6 is induced [195]. ...
... Endochondral ossification is observed in bone formation sites of SpA patients, and BMP-2, BMP-6, and TGF-β are highly expressed there [194,195]. When IL-1β or TNF-α acts on synovial fibroblasts derived from patients with SpA, the expression of BMP-2 or BMP-6 is induced [195]. In animal experiments, Noggin, which is a non-specific BMP antagonist, suppressed bone formation in sites of ankylosing enthesis [196]. ...
Article
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Arthritis is inflammation of the joints accompanied by osteochondral destruction. It can take many forms, including osteoarthritis, rheumatoid arthritis, and psoriatic arthritis. These diseases share one commonality—osteochondral destruction based on inflammation. The background includes a close interaction between osseous tissues and immune cells through various inflammatory cytokines. However, the tissues and cytokines that play major roles are different in each disease, and as a result, the mechanism of osteochondral destruction also differs. In recent years, there have been many findings regarding not only extracellular signaling pathways but also intracellular signaling pathways. In particular, we anticipate that the intracellular signals of osteoclasts, which play a central role in bone destruction, will become novel therapeutic targets. In this review, we have summarized the pathology of arthritis and the latest findings on the mechanism of osteochondral destruction, as well as present and future therapeutic strategies for these targets.
... Cell viability. Cell viability was determined by MTT assay as described previously 23,24 . ...
... Photo images of APOPercentage dye-labeled cells, which stained pink under a light microscope, were used to quantify the extent of apoptosis. In some experiments, FLS apoptosis was assessed in the presence of BMP-2 (100 ng/ml; R&D), a GREM1 antagonist 24 . ...
... GREM1 is a well-known antagonist of BMP 7 , so we investigated whether GREM1 action on FLS originates from its antagonism to BMP. We focused particularly on BMP-2 among the BMP family, since BMP-2 is dominantly expressed in the RA synovium and promotes FLS apoptosis 24 . As shown in Supplementary Figure 2A (www.ribjd.com/base/data/data01.php?com), cotreat ment of rhGREM1 blocked the BMP-2-induced apoptosis of RA-FLS, suggesting that GREM1 antagonizes proapoptotic 6 The ...
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Objective: To investigate the expression of Gremlin 1 (GREM1), an antagonist of bone morphogenetic protein, in rheumatoid arthritis (RA) synovia and its involvement in the hyperplasia and invasiveness of fibroblast-like synoviocytes of RA (RA-FLS). Methods: Computational analysis was introduced to identify FLS-predominant regulators. GREM1 expression was examined by immunohistochemistry, real-time PCR, and ELISA. FLS proliferation and apoptosis were determined using tetrazolium-based colorimetric assay and APOPercentage assay, respectively. FLS migration and invasion were evaluated by wound migration and Matrigel invasion assay, respectively. Expressions of Bax, Bcl2, pErk1/2, and pAkt were detected by Western blot analysis. Results: Through global transcriptome profiling, we identified a GREM1 gene predominantly expressed in RA-FLS. Indeed, the GREM1 expression was higher in synovia, synovial fluids, and FLS of patients with RA than in those of patients with osteoarthritis, and its levels correlated well with proinflammatory cytokine concentrations. Knockdown of GREM1 transcripts using short interfering RNA (siRNA) reduced the proliferation and survival of RA-FLS along with downregulation of pErk1/2, pAkt, and Bcl2 expressions, whereas it induced Bax expression. Conversely, the addition of recombinant GREM1 to RA-FLS showed the opposite results. Moreover, GREM1 siRNA decreased the migratory and invasive capacity of RA-FLS, whereas exogenous GREM1 increased it. The GREM1-induced FLS survival, migration, and invasion were completely blocked by neutralizing antibodies to ανβ3 integrin on RA-FLS, suggesting that ανβ3 integrin mediates the antiapoptotic and promigratory effects of GREM1. Conclusion: GREM1 is highly expressed in RA joints, and functions as a regulator of survival, proliferation, migration, and invasion of RA-FLS.
... BMP4 and 5 are downregulated in chronically inflamed tissue of RA patients compared to normal individuals, particularly in the synovial lining layer (272). In contrast, BMP2 and 6 are present in macrophages and fibroblasts in the hyperplastic and lining layers of the synovium (273). TNF and IL-1β upregulate BMP2 and 6 in RA and normal synovial fibroblasts, to regulate synovial fibroblast apoptosis (273). ...
... In contrast, BMP2 and 6 are present in macrophages and fibroblasts in the hyperplastic and lining layers of the synovium (273). TNF and IL-1β upregulate BMP2 and 6 in RA and normal synovial fibroblasts, to regulate synovial fibroblast apoptosis (273). ...
... however, similar to the Wnt agonists, their activity has not been associated with bone anabolic activity in this setting (273,274). Chapter IV of this dissertation demonstrates that inflammation induces expression of BMP3, a negative regulator of the BMP signaling pathway and osteoblast activity. TNF-stimulated synovial fibroblasts and IL-17-stimulated mature osteoblasts express BMP3 mRNA. ...
Article
Osteoclast-mediated focal articular bone erosion is a hallmark of rheumatoid arthritis, a disease of inflammation-induced bone loss. Inflammation in the bone microenvironment enhances osteoclast differentiation leading to bone erosion. Simultaneously, inflammation also inhibits osteoblast-mediated bone formation, further contributing to the net loss of bone. Previous studies have shown a paucity of mature osteoblasts at eroded bone surfaces correlating with suppression of bone formation and upregulation of antagonists of the Wnt pathway, a signaling cascade essential for osteoblast lineage commitment. Despite these observations, the exact pathogenesis of impaired bone formation in the setting of inflammation is not clearly understood. This dissertation aims to delineate the mechanisms by which inflammation suppresses osteoblast differentiation and activity in inflammatory arthritis. Specifically, this research elucidates how inflammation-induced alterations in the Wnt and bone morphogenetic protein (BMP) osteogenic signaling pathways contribute to bone loss and formation at distinct inflammatory microenvironments within the bone. Secondly, the means by which cellular mediators, including lymphocytes and macrophages, facilitate bone erosion and formation was addressed. Taken together, the research in this dissertation underscores the relationship between inflammation-induced bone loss and alterations in osteogenic signaling. Using an innovative murine inflammatory arthritis model, this study definitively demonstrates that resolving inflammation promotes osteoblast-mediated bone formation. Repair of erosions correlates with upregulation of synovial expression of Wnt10b, a Wnt agonist, and downregulation of sFRP1 and sFRP2, Wnt antagonists. This work also directly evaluates the contribution of sFRP1 to inflammation-induced bone destruction. Furthermore, this research demonstrates that expression of BMP3, a negative regulator of BMP signaling, is upregulated in osteoblasts by IL-17, a pro-inflammatory cytokine. BMP3-expressing osteoblasts are also observed at erosion sites in murine arthritis. Lastly, evaluation of the mediators of inflammation-induced periosteal bone formation implicates BMP2 as a means by which inflammation may positively regulate osteoblast function. This dissertation further elucidates the role of T cells and macrophages in the erosion and formation processes, respectively. In the absence of lymphocytes, bone erosion occurred normally, demonstrating that RANKL-expressing lymphocytes are not absolutely required for the bone erosion. Preliminary studies also suggest that M2 macrophages are potential mediators of bone formation via the expression of BMP2. In conclusion, this dissertation explores the ability of inflammation to act as a rheostat, which controls the fate of bone by modulating not only osteoclast differentiation, but also osteogenic signaling pathways and cellular mediators in the bone microenvironment. The soluble mediators and cell types identified in this research highlight novel mechanisms by which inflammation may regulate osteoblast activity within the bone microenvironment. Collectively, these data imply that strict control of inflammation may be necessary in order to create an anabolic environment that preserves bone architecture in diseases of inflammation-induced bone loss.
... Our result showing that BMP-2 content is particularly elevated in SpA SF seems thus coherent, especially because a correlation between serum and SF BMP-2 levels was expected [31]. In accordance, an increase in BMP-2 protein expression has already been detected in the inflamed synovial tissue of SpA patients [36]. Of note, BMP-2 seems almost absent from the healthy synovium, tendon, or articular cartilage [36][37][38], but its expression drastically increases in fibroblasts, chondrocytes, and osteoblasts during OA osteophyte formation [37,39], as well as in fibroblasts and chondrocytes during ossification of various ligaments [38]. ...
... In accordance, an increase in BMP-2 protein expression has already been detected in the inflamed synovial tissue of SpA patients [36]. Of note, BMP-2 seems almost absent from the healthy synovium, tendon, or articular cartilage [36][37][38], but its expression drastically increases in fibroblasts, chondrocytes, and osteoblasts during OA osteophyte formation [37,39], as well as in fibroblasts and chondrocytes during ossification of various ligaments [38]. In addition to the previously observed correlation of BMP-2 levels with the Bath Ankylosing Spondylitis Indexes [14,15], we noticed a correlation with OPG and CRP markers. ...
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Background: Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process. Methods: BMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4. Results: In SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes. Conclusions: Our results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.
... During inflammation, BMPs do not participate only in altered homeostasis of the skeletal cells, but affect other cells, such as FLSs and macrophages [177]. Lories et al. reported increased BMP-2 and -6 expression in different cell types of RA hyperplastic synovia including CD68 + macrophages and CD90 + FLS [178]. TNF-α and IL-1β upregulated both BMPs in FLSs, which, in turn, had opposite autocrine effects, with BMP-2 acting as a proapototic and BMP-6 as a protective factor [178]. ...
... Lories et al. reported increased BMP-2 and -6 expression in different cell types of RA hyperplastic synovia including CD68 + macrophages and CD90 + FLS [178]. TNF-α and IL-1β upregulated both BMPs in FLSs, which, in turn, had opposite autocrine effects, with BMP-2 acting as a proapototic and BMP-6 as a protective factor [178]. Varas et al. confirmed the expression of BMP ligands (BMP-2, BMP-6, and BMP-7), receptors (mainly BMPRIA, ACTRIA, and BMPRII), and signal transducers (Smad1/5 and co-Smad4) in FLSs [179]. ...
Article
Osteochondroprogenitors are crucial for embryonic bone development and postnatal processes such as bone repair in response to fracture injury, and their dysfunction may contribute to insufficient repair of structural damage in inflammatory arthritides. In the fracture healing, the early inflammatory phase is crucial for normal callus development and new bone formation. This process involves a complex interplay of many molecules and cell types, responsible for recruitment, expansion and differentiation of osteochondroprogenitor populations. In inflammatory arthritides, inflammation induces bone resorption and causes insufficient bone formation, which leads to local and systemic bone loss. While bone loss is a predominant feature in rheumatoid arthritis, inflammation also induces pathologic bone formation at enthesial sites in seronegative spondyloarthropaties. Bone morphogenetic proteins (BMP) are involved in cell proliferation, differentiation and apoptosis, and have fundamental roles in maintenance of postnatal bone homeostasis. They are crucial regulators of the osteochondroprogenitor pool and drive their proliferation, differentiation and lifespan during bone regeneration. In this review, we summarize the effects of inflammation on osteochondroprogenitor populations during fracture repair and in inflammatory arthritides, with special focus on inflammation-mediated modulation of BMP signaling. We also present data in which we describe a population of murine synovial osteochondroprogenitor cells, which are reduced in arthritis, and characterize their expression of genes involved in regulation of bone homeostasis, emphasizing the up-regulation of BMP pathways in early progenitor subset. Based on the presented data, it may be concluded that during an inflammatory response, innate immune cells induce osteochondroprogenitors by providing signals for their recruitment, by producing BMPs and other osteogenic factors for paracrine effects, and by secreting inflammatory cytokines that may positively regulate osteogenic pathways. On the other hand, inflammatory cells may secrete cytokines that interfere with osteogenic pathways, proapoptotic factors that reduce the pool of osteochondroprogenitor cells, as well as BMP and Wnt antagonists. The net effect is strongly context-dependent and influenced by the local milieu of cells, cytokines, and growth factors. Further elucidation of the interplay between inflammatory signals and BMP-mediated bone formation may provide valuable tools for therapeutic targeting.
... They also detected elevated expression of TGFb 2 messenger RNA in close proximity to the sites of new bone formation. Further evidence implicating BMP/TGF-b in the pathogenesis of the enhanced bone formation in AS was provided by Lories et al. [12] who demonstrated high levels of expression of BMP-2 and -6 in synovial fibroblasts and macrophages in synovial biopsies from patients with AS. They observed similar elevations of BMPs in animal models of AS and showed that gene transfer of noggin (a BMP antagonist) to sites of inflammation reduced bone formation [13]. ...
... They observed similar elevations of BMPs in animal models of AS and showed that gene transfer of noggin (a BMP antagonist) to sites of inflammation reduced bone formation [13]. Using an in vitro cell culture model they showed that treatment of synovial fibroblasts from patients with AS with TNF or IL-1 increased BMP-2 and BMP-6 expression [12]. They also noted elevated expression of BMP-2 and -6 in RA specimens and speculated that the suppression of bone repair could be related to the production of inhibitors of osteoblast-mediated bone formation in the RA synovium. ...
Article
The inflammatory arthropathies share in common their tendency to produce marked alterations in skeletal remodelling and architecture. This review will focus on RA and the seronegative spondyloarthopathies (SpA), which share common features with respect to their tendency to produce localized bone destruction at sites of articular and peri-articular inflammation. However, there are significant differences in the skeletal pathology in these conditions, which include the unique involvement of the axial skeleton and the presence of inflammation in the extra-articular entheses in SpA. There also are differences in the pattern of bone formation and repair associated with the articular and peri-articular inflammation. This review will highlight the molecular and cellular processes that are involved in the pathogenesis of the skeletal pathology in these two forms of inflammatory arthritis with specific focus on the pathogenic mechanisms underlying the differential patterns of bone formation and repair.
... Furthermore, BMP signalling has been proposed to be involved in cartilage and bone repair in arthritis. Moreover, they may be equally important in modulating synoviocytes in inflamed synovium as well [23]. However, their role in degenerative joint diseases is still insufficiently understood. ...
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BMPs regulate synovial quiescence and adult neurogenesis in the hippocampus in non-stress conditions. However, changes in BMP expression that are induced by inflammation during rheumatoid arthritis (RA) have not yet been reported. Here, we show that signalling with synovial BMPs (BMP-4 and -7) mediates the effect of systemic inflammation on adult neurogenesis in the hippocampus during pristane-induced arthritis (PIA) in Dark Agouti (DA) rats, an animal model of RA. Moreover, we show gender differences in BMP expressions and their antagonists (Noggin and Gremlin) during PIA and their correlations with the clinical course and IL-17A and TNF-α levels in serum. Our results indicate gender differences in the clinical course, where male rats showed earlier onset and earlier recovery but a worse clinical course in the first two phases of the disease (onset and peak), which correlates with the initial increase of serum IL-17A level. The clinical course of the female rats worsened in remission. Their prolonged symptoms could be a reflection of an increased TNF-α level in serum during remission. Synovial inflammation was greater in females in PIA-remission with greater synovial BMP and antagonist expressions. More significant correlations between serum cytokines (IL-17A and TNF-α), and synovial BMPs and their antagonists were found in females than in males. On the other hand, males showed an increase in hippocampal BMP-4 expression during the acute phase, but both genders showed a decrease in antagonist expressions during PIA in general. Both genders showed a decrease in the number of Ki-67+ and SOX-2+ and DCX+ cells and in the ratio of DCX+ to Ki67+ cells in the dentate gyrus during PIA. However, in PIA remission, females showed a faster increase in the number of Ki67+, SOX-2+, and DCX+ cells and a faster increase in the DCX/Ki67 ratio than males. Both genders showed an increase of hippocampal BMP-7 expression during remission, although males constantly showed greater BMP-7 expression at all time points. Our data show that gender differences exist in the BMP expressions in the periphery–hippocampus axis and in the IL-17A and TNF-α levels in serum, which could imply differences in the mechanisms for the onset and progression of the disease, the clinical course severity, and adult neurogenesis with subsequent neurological complications between genders.
... BMP-6, a member of the BMP subfamily, has been identified as a multifunctional cytokine capable of regulating cell proliferation, differentiation and apoptosis of multiple cell types, including mesenchymal stem cells (17), hematopoietic stem cells (18) and neural stem cells (19). Previous studies reported that BMP-6 could block low potassium-mediated apoptosis in cultured cerebellar granule cells (20), inhibit oxidative stress-induced injury in retinal pigment epithelial cell (21) and protect fibroblast-like synoviocytes against nitric oxide-induced apoptosis (22). However, the effects of BMP-6 on NSC survival in HIBI has not been fully elucidated. ...
Article
Background and objectives: Neural stem cells (NSCs) remain in the mammalian brain throughout life and provide a novel therapeutic strategy for central nervous system (CNS) injury. Bone morphogenetic protein-6 (BMP-6) had shown a protective effect in different types of cells. However, the role of BMP-6 in NSCs is largely unclear. The present study was aimed to investigate whether BMP-6 could protect human NSCs (hNSCs) against the oxygen and glucose deprivation (OGD)-induced cell death. Methods and results: Upon challenge with OGD treatment, cell viability was significantly decreased in a time-dependent manner, as indicated by the CCK-8 assay. BMP-6 could attenuate the OGD-induced cell injury in a dose-dependent manner and decrease the number of TUNEL-positive cells. Moreover, BMP-6 markedly weakened the OGD-induced alterations in the expression of procaspase-8/9/3 and reversed the expression of cleaved-caspase-3. Interestingly, noggin protein (the BMP-6 inhibitor) attenuated the neuroprotective effect of BMP-6 in cultured hNSCs. Furthermore, the p38 MAPK signaling pathway was activated by OGD treatment and BMP-6 markedly inhibited the phosphorylation of p38 in a concentration-dependent manner. Pretreatment with noggin abolished the effect of BMP-6 on p38 activation. SB239063, a selective p38 inhibitor, exerted similar effects with BMP-6 in protecting hNSCs against the OGD-induced apoptosis. These results indicated that blocking the phosphorylation of p38 might contribute to the neuroprotective effect of BMP-6 against the OGD-induced injury in hNSCs. Conclusions: These findings suggested that BMP-6 might be a therapeutic target in the OGD-induced cell death, which provides a novel therapeutic strategy for enhancing host and graft NSCs survival in hypoxic-ischemic brain injury.
... Scientists have uncovered the significant role of the receptor activator of nuclear factor kappa B ligand (RANK ligand or RANKL)/RANK/osteoprotegerin (OPG) axis in osteoimmunomodulation in the past few years (Okamoto et al., 2017;Tsukasaki and Takayanagi, 2019). Besides, there are an increasing number of novel studies aiming to reveal more promising key cytokines and signaling pathways that connect the skeletal and immune systems and are involved in the research scope of osteoimmunology, such as tumor necrosis factor-α (TNF-α) (Bertolini et al., 1986), interferon-γ (IFN-γ) (Smith et al., 1987), interleukin-1 (IL-1) (Gowen et al., 1983), IL-4 (Shioi et al., 1991), IL-6 (Löwik et al., 1989), IL-17 (Kotake et al., 1999), IL-20 (Hsu et al., 2011), bone morphogenetic protein (BMP) (Lories et al., 2003), sphingosine-1-phosphate (S1P)-S1P receptor-1 (S1PR1) signaling (Xiao et al., 2019), Notch signaling (Liotta et al., 2008), mitogen-activated protein kinase (MAPK) pathway (Chae et al., 2001), dependent or independent on RANKL/RANK/OPG signaling pathway, which further enriches our understanding of osteoimmunology. ...
Article
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The skeletal system and immune system seem to be two independent systems. However, there in fact are extensive and multiple crosstalk between them. The concept of osteoimmunology was created to describe those interdisciplinary events, but it has been constantly updated over time. In this review, we summarize the interactions between the skeletal and immune systems in the co-development of the two systems and the progress of certain typical bone abnormalities and bone regeneration on the cellular and molecular levels according to the mainstream novel study. At the end of the review, we also highlighted the possibility of extending the research scope of osteoimmunology to other systemic diseases. In conclusion, we propose that osteoimmunology is a promising perspective to uncover the mechanism of related diseases; meanwhile, a study from the point of view of osteoimmunology may also provide innovative ideas and resolutions to achieve the balance of internal homeostasis.
... In DBA/1 mice, new bone formation was driven by BMP signaling, which was inhibited by Noggin, a BMP antagonist (60). Studies in humans using immunohistochemistry of synovial tissue showed higher expression of BMP-2 and BMP-6 in inflamed SpA tissue, but not non-inflamed tissue (61). These genes were up-regulated in fibroblast-like synoviocytes by IL-1β and TNFα, which are important cytokines in SpA pathogenesis. ...
Article
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Heritability of Spondyloarthritis (SpA) is highlighted by several familial studies and a high association with the presence of human leukocyte antigen (HLA)-B * 27. Though it has been over four decades since the association of HLA-B * 27 with SpA was first determined, the pathophysiological roles played by specific HLA-B * 27 allotypes are not fully understood. Popular hypotheses include the presentation of arthritogenic peptides, triggering of endoplasmic reticulum (ER) stress by misfolded HLA-B * 27, and the interaction between free heavy chains or heavy chain homodimers of HLA-B * 27 and immune receptors to drive IL-17 responses. Several non-HLA susceptibility loci have also been identified for SpA, including endoplasmic reticulum aminopeptidases (ERAP) and those related to the IL-23/IL-17 axes. In this review, we summarize clinical aspects of SpA including known characteristics of gut inflammation, enthesitis and new bone formation and the existing models for understanding the association of HLA-B * 27 with disease pathogenesis. We also examine newer insights into the biology of HLA class I (HLA-I) proteins and their implications for expanding our understanding of HLA-B * 27 contributions to SpA pathogenesis.
... TGF-β and BMPs have been proposed as potential key players of the enhanced bone formation in AS. The endochondral bone formation during development and postnatal fracture repair are induced by TGF-β and BMPs and have been implicated in this process 141,142 . TGF-β and BMPs signal through the wingless (Wnt) pathway which is a main regulator of skeletal patterning and post-natal bone remodelling 143 . ...
Article
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Spondyloarthritis (SpA) is an umbrella term describing a family of chronic inflammatory rheumatic diseases. These diseases are characterised by inflammation of the axial skeleton, peripheral joints, and entheseal insertion sites throughout the body which can lead to structural joint damage including formation of axial syndesmophytes and peripheral osteophytes. Genetic evidence, preclinical and clinical studies indicate a clear role of interleukin (IL)- 23 and IL-17 as mediators in SpA pathogenesis. Targeting the IL-23/−17 pathways seems an efficient strategy for treatment of SpA patients, and despite the remaining challenges the pathway holds great promise for further advances and improved therapeutic opportunities. Much research is focusing on serological markers and imaging strategies to correctly diagnose patients in the early stages of SpA. Biomarkers may facilitate personalised medicine tailored to each patient's specific disease to optimise treatment efficacy and to monitor therapeutic response. This narrative review focuses on the IL-17 pathway in SpA-related diseases with emphasis on its role in pathogenesis, current approved IL-17 inhibitors, and the need for biomarkers reflecting core disease pathways for early diagnosis and measurement of disease activity, prognosis, and response to therapy.
... Initial BRC activation and associated increased bone histomorphometric measurements at 8 weeks resulted from the release of BMP-2 from CPM 32,42,43 and BMP and factors produced by inflammatory cells, synovial cells, endothelial cells, and marrow-lining cells resulting from CPM-induced synovitis 34,35,[38][39][40][47][48][49][50][51][52][53] . Continued bone formation in BMP-2/ CPM-treated knees between 8 and 16 weeks resulted from the Preexisting anastomosis between capsular and epiphyseal and metaphyseal circulation [15][16][17][18][57][58][59][60] and capsular vascular channels penetrating the metaphyseal cortex 15 , observed in animals and humans, provide the potential for an induced venous portal circulation. ...
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Background: Synovial membrane-derived factors are implicated in arthritis-related bone changes. The route that synovial factors use to access subchondral bone and the mechanisms responsible for these bone changes remain unclear. A safety study involving intra-articular injection of bone morphogenetic protein-2 (BMP-2)/calcium phosphate matrix (CPM) or CPM addresses these issues. Methods: Knee joints in 21 monkeys were injected with CPM or 1.5 or 4.5 mg/mL BMP-2/CPM and were evaluated at 1 and 8 weeks. Contralateral joints were injected with saline solution. Knee joints in 4 animals each were injected with 1.5 or 4.5 mg/mL BMP-2/CPM. Contralateral joints were injected with corresponding treatments at 8 weeks. Both joints were evaluated at 16 weeks. Harvested joints were evaluated grossly and with histomorphometry. Knee joints in 3 animals were injected with 125I-labeled BMP-2/CPM and evaluated with scintigraphy and autoradiography at 2 weeks to determine BMP-2 distribution. Results: All treatments induced transient synovitis and increased capsular vascularization, observed to anastomose with metaphyseal venous sinusoids, but did not damage articular cartilage. Both treatments induced unanticipated activation of vascular-associated trabecular bone remodeling compartments (BRCs) restricted to injected knees. Bone volume increased in BMP-2/CPM-injected knees at 8 and 16 weeks. Scintigraphy demonstrated metaphyseal 125I-labeled BMP-2 localization restricted to injected knees, confirming local rather than systemic BMP-2 release. Autoradiography demonstrated that BMP-2 diffusion through articular cartilage into the metaphysis was blocked by the tidemark. The lack of marrow activation or de novo bone formation, previously reported following metaphyseal BMP-2/CPM administration, confirmed BMP-2 and synovial-derived factors were not free in the marrow. The 125I-labeled BMP-2/CPM, observed within venous sinusoids of injected knees, confirmed the potential for capsular and metaphyseal venous portal communication. Conclusions: This study identifies a synovitis-induced venous portal circulation between the joint capsule and the metaphysis as an alternative to systemic circulation and local diffusion for synovial membrane-derived factors to reach subchondral bone. This study also identifies vascular-associated BRCs as a mechanism for arthritis-associated subchondral bone changes and provides additional support for their role in physiological trabecular bone remodeling and/or modeling. Clinical Relevance: Inhibition of synovitis and accompanying abnormal vascularization may limit bone changes associated with arthritis.
... One study found an imbalance between Noggin and BMP2 which could cause abnormal osteogenic differentiation of mesenchymal stem cells, and indicates excessive osteoblast differentiation as a mechanism behind the pathological bone formation in AS [221]. Activation of BMP-2 and 6 was also found in synovial tissue of RA and AS patients [222], which could mean that the differing bone phenotypes between the two diseases results from the anatomical site of inflammation rather than the mechanism itself (entheses vs joint) [7]. ...
Article
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Osteoarthritis, rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis, all have one clear common denominator; an altered turnover of bone. However, this may be more complex than a simple change in bone matrix and mineral turnover. While these diseases share a common tissue axis, their manifestations in the area of pathology are highly diverse, ranging from sclerosis to erosion of bone in different regions. The management of these diseases will benefit from a deeper understanding of the local versus systemic effects, the relation to the equilibrium of the bone balance (i.e., bone formation versus bone resorption), and the physiological and pathophysiological phenotypes of the cells involved (e.g., osteoblasts, osteoclasts, osteocytes and chondrocytes). For example, the process of endochondral bone formation in chondrocytes occurs exists during skeletal development and healthy conditions, but also in pathological conditions. This review focuses on the complex molecular and cellular taxonomy of bone in the context of rheumatological diseases that alter bone matrix composition and maintenance, giving rise to different bone turnover phenotypes, and how biomarkers (biochemical markers) can be applied to potentially describe specific bone phenotypic tissue profiles.
... BMPs promote proliferation and differentiation in chondrocytes (Ch) [13]. Levels of BMPs are elevated in the synovium and synovial fluid of patients with RA while phosphorylation of SMAD1/5/8 is increased in RA synovial biopsies, indicating BMP activation persists despite inflammation control with treatment [14][15][16][17]. Smad2/3 and Smad1/5/8 respond to activated TGFβ and BMP receptors, respectively, by forming complexes with Smad4 and translocating to the nucleus to regulate target gene transcription [18,19]. ...
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Background To examine critical interactions between juvenile idiopathic arthritis synovial fibroblasts (JFLS) and chondrocytes (Ch), and their role in bony overgrowth seen in patients with juvenile idiopathic arthritis (JIA). Methods Control (CFLS) and JFLS were cultured in synoviocyte media containing recombinant BMP4. Ch were cultured in either CFLS or JFLS conditioned-media without stimulation. Media supernatants were analyzed by ELISA. RNA from conditioned media experiment was analyzed by ClariomS microarray. Results As expected, genes expressed in untreated JFLS and CFLS cultured in synoviocyte media were similar to each other and this expression differed from untreated Ch cultured in chondrocyte media. JFLS favor BMP ligand gene expression while downregulating TGFβ receptors’ expression. Noggin and chordin, antagonists with high affinity for BMP4, are JFLS- but not Ch-preferred regulators of BMP signaling. Compared to Ch, JFLS overexpress collagen X (COLX), a marker of chondrocyte hypertrophy. Exogenous BMP4 causes JFLS to significantly decrease expression of noggin and collagen II (COL2), a marker of chondrocyte proliferation, and causes overexpression of COLX and alkaline-phosphatase (ALP). Chondrocytes cultured in JFLS-conditioned media (Ch-JFLS) express BMP genes and favor chordin protein expression over other antagonists. Ch-JFLS have significantly increased expression of COL2 and significantly decreased expression of COLX. Conclusions These data suggest JFLS, in the presence of BMP4, undergo hypertrophy and that JFLS-conditioned media influence chondrocytes to become highly proliferative. To the authors’ knowledge, no prior study has shown that JFLS and chondrocytes play a direct role in the bony overgrowth in joints of patients with JIA and that BMPs or regulation of these growth factors influence the interaction between two prominent synovial cell types.
... BMP promotes osteoblast formation by stimulating expression of the osteogenic transcription factors, such as RUNX2 and osterix [30]. The expression of BMP is induced by the IL-1β and TNF-α [44]. On the other hand, IL-17A inhibits osteoblast differentiation, inducing the expression of secreted Frizzled-Related Protein 1 which is an inhibitor of the Wnt/β-catenin pathway. ...
Article
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Psoriatic arthritis (PsA) is a chronic, progressive, inflammatory arthropathy associated with psoriasis as well as a complex pathogenesis. Genetic and environmental factors trigger the development of the immune-mediated auto-inflam-matory response in different sites: skin, bone marrow, entheses and synovial tissues. Studies of the last two decades have changed the view of PsA from a mild, non-progressive arthritis to an inflammatory systemic disease with serious health consequences, not only associated with joint dysfunction, but also with an increased risk of cardiovascular disease and socioeconomic consequences with significantly reduced quality of life. The joint damage starts early in the course of the disease, thus early recognition and treatment with modern biological treatments, which may modify the natural history and slow down progression of this debilitating disease, is essential for the patient long-term outcome.
... In entheses of patients with axSpA, Laloux et al. [41] described persistent oedema with an inflammatory infiltrate of lymphocytic CD8 þ . Lories et al. [42] demonstrated in arthritic synovium from SpA and RA patients a striking up-regulation of BMP-2 and BMP-6 by the proinflammatory cytokines IL-1 and TNF. Modulation between pro-inflammatory cytokines and these BMPs suggests a function for BMPs as autocrine or paracrine peptides in the pathologic microenvironment of chronic arthritis. ...
Article
Objective: The hallmark of advanced axial SpA (axSpA) is spine ankylosis due to excessive ectopic bone formation. This prospective study aimed to describe the changes in serum levels of different regulators [sclerostin, dickkopf-1 (DKK-1)] and markers of bone formation [bone morphogenetic protein 7 (BMP-7)] over 5 years in early axSpA patients and to assess determinants of such changes. Methods: The DEvenir des Spondyloarthropathies Indifférenciées Récentes cohort is a prospective, multicentre French study of 708 patients with early (>3 months-<3 years) inflammatory back pain suggestive of axSpA. Serum levels of BMP-7, sclerostin and DKK-1 were assessed at baseline and after 2 and 5 years. Changes in bone formation regulators over time were analysed using mixed linear models. Results: Serum BMP-7 significantly increased over time, with a median relative change of 223.7% [interquartile range (IQR) 0-10 700 (0.17 pg/ml/month), P < 0.001]. Serum sclerostin significantly increased over time, with a median relative change of 14.8% [IQR -7.9-41.4% (0.001 ng/ml/month), P < 0.001]. Serum DKK-1 did not significantly change over time. Serum BMP-7 increased over time in active disease (Ankylosing Spondylitis Disease Activity Score with CRP ≥1.3, P = 0.01), but the increase was less pronounced with TNF inhibitor (TNFi) use (P < 0.001). No determinant was associated with serum sclerostin change. Conclusion: Serum BMP-7 change over 5 years was related with inflammation; it was increased in active disease, but the increase was low with TNFi use. Serum sclerostin levels significantly increased over time, but to a lesser degree than for serum BMP-7. Clinical trial registration: https://clinicaltrials.gov/, NCT01648907.
... Although proinflammatory cytokines such as IL-1β can induce BMP-2 expression in chondrocytes [39], few studies have explored the expression and function of BMPs in OA progression [32,79]. In patients with rheumatoid arthritis, stimulation with IL-1β induced BMP-2 and BMP-6, but not BMP-4, BMP-5, or BMP-7 in fibroblast-like synoviocytes [80,81]. In the OA cartilage, IL-1β was found to stimulate the expression of BMP-2, but not BMP-4 or BMP-6 in the cell and tissue explant culture [39]. ...
Article
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Osteoarthritis (OA) is a chronic inflammatory and progressive joint disease that results in cartilage degradation and subchondral bone remodeling. The proinflammatory cytokine interleukin 1 beta (IL-1β) is abundantly expressed in OA and plays a crucial role in cartilage remodeling, although its role in the activity of chondrocytes in cartilage and subchondral remodeling remains unclear. In this study, stimulating chondrogenic ATDC5 cells with IL-1β increased the levels of bone morphogenetic protein 2 (BMP-2), promoted articular cartilage degradation, and enhanced structural remodeling. Immunohistochemistry staining and microcomputed tomography imaging of the subchondral trabecular bone region in the experimental OA rat model revealed that the OA disease promotes levels of IL-1β, BMP-2, and matrix metalloproteinase 13 (MMP-13) expression in the articular cartilage and enhances subchondral bone remodeling. The intra-articular injection of Noggin protein (a BMP-2 inhibitor) attenuated subchondral bone remodeling and disease progression in OA rats. We also found that IL-1β increased BMP-2 expression by activating the mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase (ERK), and specificity protein 1 (Sp1) signaling pathways. We conclude that IL-1β promotes BMP-2 expression in chondrocytes via the MEK/ERK/Sp1 signaling pathways. The administration of Noggin protein reduces the expression of IL-1β and BMP-2, which prevents cartilage degeneration and OA development.
... BMPs promote proliferation and differentiation in chondrocytes (Ch) (13). Levels of BMPs are elevated in the synovium and synovial fluid of patients with RA while phosphorylation of SMAD1/5/8 is increased in RA synovial biopsies, indicating BMP activation persists despite inflammation control with treatment (14)(15)(16)(17). Smad2/3 and Smad1/5/8 respond to activated TGFβ and BMP receptors, respectively, by forming complexes with Smad4 and translocating to the nucleus to regulate target gene transcription (18,19). ...
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Background: To examine critical interactions between juvenile idiopathic arthritis synovial fibroblasts (JFLS) and chondrocytes (Ch), and their role in bony overgrowth seen in patients with juvenile idiopathic arthritis (JIA). Methods: Control (CFLS) and JFLS were cultured in media containing recombinant BMP4. Ch were cultured in either CFLS or JFLS conditioned-media. Media supernatants were analyzed by ELISA. RNA was analyzed by ClariomS microarray. Results: As expected, genes expressed in JFLS and CFLS were similar to each other and this expression differed from Ch. JFLS favor BMP ligand gene expression while downregulating TGFβ receptors’ expression. Noggin and chordin, antagonists with high affinity for BMP4, are JFLS- but not Ch-preferred regulators of BMP signaling. Compared to Ch, JFLS overexpress collagen X (COLX), a marker of chondrocyte hypertrophy. Exogenous BMP4 causes JFLS to significantly decrease expression of noggin and collagen II (COL2), a marker of chondrocyte proliferation, and causes overexpression of COLX and alkaline-phosphatase (ALP). Chondrocytes cultured in JFLS-conditioned media (Ch-JFLS) express BMP genes and favor chordin protein expression over other antagonists. Ch-JFLS have significantly increased expression of COL2 and significantly decreased expression of COLX. Conclusions: These data suggest JFLS, in the presence of BMP4, undergo hypertrophy and that JFLS-conditioned media influence chondrocytes to become highly proliferative. To the authors’ knowledge, no prior study has shown that JFLS and chondrocytes play a direct role in the bony overgrowth in joints of patients with JIA and that BMPs or regulation of these growth factors influence the interaction between two prominent synovial cell types.
... Par ailleurs, une augmentation significative des taux sériques de BMP-2 et BMP-7 a été observée chez des patients atteints de SA et présentant une fusion vertébrale, en comparaison avec des patients atteints de SA sans fusion vertébrale [331]. De plus, le niveau des ligands BMP-2 et BMP-6 est augmenté dans la synovie arthritique de patients atteints de SA, et est fortement régulé par les cytokines pro-inflammatoires [332]. Par ailleurs, une étude d'association génétique sur une cohorte de 417 patients SA coréens a identifié deux SNPs dans le gène BMP-6 qui sont significativement associés à la sévérité radiologique des patients atteints de SA, ce qui souligne le lien entre la voie BMP et la SA [333]. ...
Thesis
Spondyloarthritis (SpA) is a chronic inflammatory rheumatic disorder characterized by joint manifestations affecting the spine, peripheral joints and entheses, as well as extra-articular manifestations such as psoriasis, uveitis, or intestinal inflammation. This complex disorder has a strong genetic component dominated by the HLA-B27 antigen of the major histocompatibility complex class I (MHC-I), which is present in more than 80% of SpA patients. Discovered 45 years ago, the association between HLA-B27 and SpA development remains unexplained. Several hypotheses have been proposed to explain this association at the molecular level, but all face experimental inconsistencies that seem to invalidate them. Therefore, it appeared to us essential to elaborate new and yet unexplored approaches in order to better understand the molecular role of HLA-B27 in SpA development. Drosophila melanogaster is a powerful genetic model that has led to considerable advances in understanding numerous functions of metazoan cells, as well as in describing the cellular and molecular processes of many human pathologies. To elucidate the molecular pathogenic mechanisms associated with HLA-B27, we have established several transgenic Drosophila lines for SpA-associated and non-associated of HLA-B alleles, as well as for the MHC-I invariant chain, the human 2-microglobulin (hβ2m). Expression of the HLA-B27 alleles, in the presence of hβ2m, in the Drosophila wing and eye led to two specific phenotypes. The crossveinless wing phenotype is due to a disturbance in the Bone Morphogenetic Protein (BMP) signaling pathway. Interestingly, this misregulation is associated with a co-localization of HLA-B27 and the BMP type I receptor named Sax. Our preliminary results obtained in SpA patient cells suggest that HLA-B27 also colocalizes with ALK2 receptor, which is ortholog to Sax. Altogether, our results suggest that the pathogenic role of HLA-B27 in SpA may depend on a BMP signaling misregulation at the crosstalk between ossification and inflammation.
... RANKL was found to be present at sites of bone erosion in RA and is known to be produced by RASF [143]. TNF-α and IL-1β induce osteoclastogenesis via upregulation of bone morphogenetic protein-2 and -6 in RASF [144]. Since RASF have the potential to drive cartilage and bone degradation by producing MMPs and stimulating osteoclastogenesis, it is therefore suggested that they could be driving the destruction of the joints in RA. ...
... At present, understanding of its pathogenesis is limited, and therefore the design of targeted drugs has not been available. The high morbidity rates of AS are a common cause of the burden to families and society (24). Musculoskeletal enthesitis is one of the primary features of the pathogenesis of AS, which involves ligaments in the joint capsule connected to the bone. ...
Article
Ankylosing spondylitis (AS) is an autoimmune condition characterized by chronic inflammation and abnormal ossification as the primary features of the disease. The aim of the present study was to investigate the role of C‑X‑C chemokine receptor type 4 (CXCR4) in ossification from patients with AS. CXCR4 expression was assessed by western blot analysis and immunohistochemistry analysis of tissues obtained from patients with AS and controls. Fibroblasts were isolated, cultured and incubated with AMD 3100 and stromal cell‑derived factor‑1 to inhibit and promote CXCR4 levels, respectively. CXCR4 was upregulated in hip synovial tissues from patients with AS compared with that observed in controls. AS fibroblasts exhibited increased proliferation and growth rates. Inhibition of CXCR4 increased the phosphorylation of β‑catenin and downregulated the expression of β‑catenin, v‑myc avian myelocytomatosis viral oncogene homolog, cyclin D1 and osteocalcin. Alizarin red staining demonstrated a decrease in biomineralization activity following the inhibition of CXCR4. These data support the hypothesis that inhibiting CXCR4 in patients with AS may suppress the ossification of fibroblasts.
... BMP2 and BMP6 are expressed in synovium from RA patients, and expression in patient-derived fibroblast-like synoviocytes in vitro becomes strongly up-regulated in response to pro-inflammatory cytokines IL-1b, TNF-a, or IL-17. 118,119 However, BMP4 expression in synoviocytes decreases in response to TNF-a, 118 aligning with clinical studies observing lower BMP4 mRNA levels in the synovium of RA patients relative to controls. 120 The pro-inflammatory cytokines IL-17 and TNF-a induce a pro-inflammatory phenotype within synoviocytes marked by increased expression of pro-inflammatory cytokines IL-6 and GM-CSF, increased expression of the chemokine IL-8, and increased production of metalloproteinases MMP2 and MMP3. ...
Article
Bone morphogenetic protein signaling has long been established as a crucial pathway during embryonic development. In recent years, our knowledge of the function of bone morphogenetic protein signaling has expanded dramatically beyond solely its important role in development. Today, the pathway is known to have important homeostatic functions across multiple different tissues in the adult. Even more importantly, bone morphogenetic protein signaling is now known to function as a driver of diseases in the adult spanning different organ systems. In this review, we will explore the functions of bone morphogenetic protein signaling in diseases of inflammation. Through this exploration, we will highlight the value and challenges in targeting bone morphogenetic protein signaling for therapeutic interventions. Impact statement By compiling findings from recent studies, this review will garner novel insight on the dynamic and complex role of BMP signaling in diseases of inflammation, highlighting the specific roles played by both individual ligands and endogenous antagonists. Ultimately, this summary will help inform the high therapeutic value of targeting this pathway for modulating diseases of inflammation.
... BMP6 is expressed in arthritic synovium of rheumatoid arthritis (RA) patients and strongly upregulated by proinflammatory cytokines. 9 We previously identified several candidate genes, including gamma-aminobutyric acid receptor pi subunit (GABRP), epithelial stromal interaction 1 (EPSTI1), and BMP6, associated with SLE and RA in our pilot study using a customized 3K single-nucleotide polymorphism (SNP) chip, which revealed that polymorphisms in GABRP and EPSTI1 might be associated with susceptibility to SLE, and that haplotypes of GABRP and EPSTI1 SNPs are useful genetic markers for SLE. 10,11 Therefore, in this study, we aimed to investigate whether BMP6 SNP is associated with susceptibility to SLE. ...
Article
Objectives: This study aims to investigate whether bone morphogenetic protein 6 (BMP6) single-nucleotide polymorphism (SNP) is associated with susceptibility to systematic lupus erythematosus (SLE). Patients and methods: We analyzed the genotype and allele frequencies of BMP6 SNPs using genomic deoxyribonucleic acid isolated from 119 SLE patients (9 males, 110 females; mean age 36.4 years; range 19 to 59 years) and 509 healthy controls (323 males, 186 females; mean age 42.1 years; range 19 to 61 years). Genomic deoxyribonucleic acid was extracted from peripheral blood leukocytes using a standard phenol-chloroform method or by using a genomic deoxyribonucleic acid extraction kit. Erythrocyte sedimentation rate, C-reactive protein, and antinuclear antibody levels of SLE patients were recorded. Results: Our results showed that the genotype frequencies of rs17557 and rs9505273 for BMP6 in SLE patients significantly differed from those of the control group (p=0.01 and p=0.04, respectively). The genotype frequencies of the rs17557 and rs9505273 for BMP6 in female SLE patients were also significantly different from those in female healthy controls (p=0.04 and p=0.03, respectively). We also revealed that the distribution of the main haplotypes of BMP6 SNPs in SLE patients was significantly different from their distribution in healthy controls. Conclusion: These results suggested that SNPs in BMP6 might be associated with susceptibility to SLE and that haplotypes of BMP6 polymorphisms might represent useful genetic markers for SLE.
... These regions of bone formation contained Runx2 and type I collagen-expressing cells, markers that are consistent with immature cells of osteoblast lineage. Lories et al (46) have also analyzed inf lamed synovial tissues from patients with AS versus RA and identified the induction of bone morphogenetic proteins BMP-2 and -6 in tissues from patients with both diseases. They speculated that one of the major differences in AS and RA that could account for differential effects on bone is the anatomic site of inflammation, with inflammatory infiltrates in RA being exclusively within the joint, and in AS being located both within joints and also at entheses. ...
The hallmark of rheumatoid arthritis is synovitis, or inflammation of synovial tissues lining joints. Synovitis in rheumatoid arthritis promotes destruction of articular bone by inducing the differentiation and function of osteoclasts, leading to significant patient morbidity. The cell types and pathways mediating articular bone destruction have now been elucidated and the critical role of receptor activator of nuclear factor-kappa B ligand has been recognized, leading to the identification of new targets for the protection of articular bone. Synovitis not only promotes bone destruction, but also inhibits the ability of bone-forming osteoblasts to repair bone. In stark contrast, inflammation in spondyloarthritis, including ankylosing spondylitis, promotes bone formation at periosteal sites, resulting in pain and decreased motion of the spine and joints. Local anatomic factors contribute to these distinct outcomes for bone and anabolic pathways regulating bone formation are now being investigated to identify novel targets for prevention of abnormal bone formation.
... However, not much is known about BMPs in synovial tissue. BMP2, BMP4, BMP6 and BMP7 are expressed in the synovial membrane of patients with RA and BMP2 and BMP6 are induced in fibroblast-like synoviocytes [31]. The action of BMPs is mediated intracellularly by receptor-associated Smads. ...
Article
Synovial inflammation plays a critical role in the symptoms and structural progression of arthritis which leads to irreversible damage of the adjacent cartilage and bone. Activation of complement system is strongly implicated as a factor in the pathogenesis of chronic synovitis in human rheumatoid arthritis (RA). In this study, we show that the depletion of functional complement activity at the time of the initiation of zymosan-induced arthritis, significantly reduced the expression of TGF-beta1/3, BMP2 and pSmad2 and decreased the number of Sudan Black B positive cells in the synovium. Also, the excessive synthesis of proteoglycans and glycosaminoglycans was diminished. The appearance of apoptotic and senescent cells among the adherent bone marrow cells cultivated in vitro was not observed in complement depleted mice. Therefore, the lack of functional complement prevented the development of chronic synovitis, osteophyte formation and the generation of pathologic senescent arthritic cells.
... Ex vivo, BMP7 increased the proliferation of lamina propia mononuclear cells, which highly expressed pSMAD1/5/8 upon BMP7 challenge (Pluchino et al., 2009). In rheumatoid arthritis (RA) patients, BMP2 and BMP6, which are present in the synovium/synovial membrane, were found to be upregulated by pro-inflammatory cytokines, although a clear role for BMPs was not shown (Lories et al., 2003). In a TNF transgenic mouse model of RA, mice presented infiltrates in the bone marrow principally consisting of B cells at sites near the lesions. ...
Article
Bone morphogenetic proteins (BMPs) are growth factors that represent the largest subgroup of signalling ligands of the transforming growth factor beta (TGF-β) superfamily. Their participation in the proliferation, survival and cell fate of several cell types and their involvement in many pathological conditions are now well known. BMP expression is altered in multiple sclerosis (MS) patients, suggesting that BMPs have a role in the pathogenesis of this disease. MS is a demyelinating and neurodegenerative autoimmune disorder of the central nervous system (CNS). MS is a complex pathological condition in which genetic, epigenetic and environmental factors converge, although its aetiology remains elusive. Multifunctional molecules, such as BMPs, are extremely interesting in the field of MS because they are involved in the regulation of several adult tissues, including the CNS and the immune system. In this review, we discuss the extensive data available regarding the role of BMP signalling in neuronal progenitor/stem cell fate and focus on the participation and expression of BMPs in CNS demyelination. Additionally, we provide an overview of the involvement of BMPs as modulators of the immune system, as this subject has not been thoroughly explored even though it is of great interest in autoimmune disorders. Moreover, we describe the data on BMP signalling in autoimmunity and inflammatory diseases, including MS and its experimental models. Thus, we aim to provide an integrated view of the putative role of BMPs in MS pathogenesis and to open the field for the further development of alternative therapeutic strategies for MS patients.
... The local production of pro-inflammatory cytokines and bone growth factors in the inflamed tissues, including bone morphogenic proteins, interleukin (IL)-22, and transforming growth factor-β is thought to be responsible for the ectopic bone formation described in AS [13][14][15][16][17][18]. These local anabolic effects are enhanced by the up-regulation of a critical player in osteoblast differentiation, the Wnt pathway, as serum levels of its inhibitors Dickkopf-related protein-1 and sclerostin are both reduced in patients with AS [13,[18][19][20][21][22]. ...
Article
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The ankylosed spine is prone to fracture even after minor trauma due to its changed biomechanical properties. The two central features of ankylosing spondylitis (AS) that promote the pathological remodeling of the spine are inflammation and new bone formation. AS is also associated with osteoporosis that is attributed to an uncoupling of the bone formation and bone resorption processes. Therefore, bone resorption occurs and promotes weakening of the spine as well as increased risk of vertebral fractures which can be hugely different in terms of clinical relevance. Even in the presence of symptomatic clinical vertebral fractures, the diagnosis can be overruled by attributing the pain to disease activity. Furthermore, given the highly abnormal structure of the spine, vertebral fracture diagnosis can be difficult on the basis of radiography alone. CT can show the fractures in detail. Magnetic resonance imaging is considered the method of choice for the imaging of spinal cord injuries, and a reasonable option for exclusion of occult fractures undetected by CT. Since it is equally important for radiologists and clinicians to have a common knowledge base rather than a compartmentalized view, the aim of this review article was to provide the required clinical knowledge that radiologists need to know and the relevant radiological semiotics that clinicians require in diagnosing clinically significant injury to the ankylosed spine.
... Although these studies offer new opportunities for molecular analysis of marker-positive cells, the necessity to fix the cells for intracellular flow cytometry precludes downstream live cell experimentation. Furthermore, these mesenchymal lineage-related pathways may be equally active in SFs, in addition to MSCs, as shown earlier by their continuous activation in the inflamed synovium [53,54] . Therefore, their MSC-selectivity, even in the normal synovium, remains to be proven. ...
... FLS at passages 4-7 were identified by flow cytometric analysis as a homogeneous population with the phenotype of <2Á5% CD14, <1% CD68 and >98% vimentin [18] (Supporting information, Fig. S1). Cell migration and invasion are regulated in part by inflammatory cytokines, such as TNF-a, that trigger cellular motility when applied in a uniform concentration. ...
Article
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Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone erosion. Fibroblast-like synoviocytes (FLS) play a pivotal role in RA pathogenesis through aggressive migration and matrix invasion, and certain proinflammatory cytokines may affect synoviocyte invasion. Whether interleukin (IL)-21 influences this process remains controversial. Here, we evaluated the potential regulatory effect of IL-21 on the migration, invasion, and matrix metalloproteinase (MMP) expression in RA-FLS. We found that IL-21 promoted the migration, invasion, and MMP (MMP-2, MMP-3, MMP-9, MMP-13) production in RA-FLS. Moreover, IL-21 induced activation of the PI3K, STAT3, and ERK1/2 pathways, and blockage of these pathways (PI3K/AKT inhibitor LY294002, STAT3 inhibitor STA-21, and ERK1/2 inhibitor PD98059) attenuated IL-21-induced migration and secretion of MMP-3 and MMP-9. In conclusion, our results suggest that IL-21 promotes migration and invasion of RA-FLS. Therefore, therapeutic strategies targeting IL-21 might be effective for the treatment of RA. This article is protected by copyright. All rights reserved.
... A few studies have addressed interactions between TNF-α/IL-17 and the BMPs. Expression of BMPs and regulation of their expression by pro-inflammatory cytokines has been shown in RA synovial tissue [8]. The expression of BMP-2 is enhanced by TNF-α in osteoarthritic chondrocytes and pro-inflammatory T-cell cytokines have been suggested to play a role in the differentiation of mesenchymal stromal cells into the osteoblast phenotype and in BMP-induced heterotopic ossification [9,10]. ...
Article
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Pro-inflammatory cytokines and bone morphogenetic proteins are generally studied separately and considered to be elements of different worlds, immunology and developmental biology. Varas and colleagues report that these factors show cross-talk in rheumatoid arthritis synoviocytes. They show that pro-inflammatory cytokines not only stimulate the production of bone morphogenetic proteins but that these endogenously produced bone morphogenetic proteins interfere with the effects of pro-inflammatory cytokines on synoviocytes.
... BMP-2 and BMP-4 levels are higher in the serum of AS patients who develop spinal fusion in comparison to AS patients who do not develop spinal fusion, and compared to healthy controls [112]. BMP-2 and BMP-6 are expressed at higher levels in synovial fibroblasts and macrophages from AS synovium compared to non-inflamed synovium [113]. Thus, BMPs contribute to bone formation in AS and are potential targets for the prevention of bone formation in SpA. ...
Article
Remodeling of bone is a continuous process that occurs throughout life. Under normal physiologic conditions, bone-resorbing osteoclasts and bone-forming osteoblasts are tightly coupled and regulated to ensure the proper balance, such that there is no net change in bone mass. However, inflammation perturbs normal bone homeostasis. The impact of inflammation on bone is dependent upon the anatomic site affected, cell types, factors and cytokines present in the local microenvironment, and local mechanical forces. Cytokines are central to the pathogenesis of inflammation-induced bone loss and contribute to the uncoupling of osteoclast-mediated bone resorption and osteoblast-mediated bone formation, thereby disrupting normal remodeling. In this review, we will discuss the effects of cytokines on bone in two settings, rheumatoid arthritis (RA) and spondyloarthritis (SpA), a disease category that includes ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, inflammatory bowel disease, and juvenile onset spondyloarthropathy. The outcome for bone in these disease settings is quite different, and an understanding of the pathogenic mechanisms leading to the net impact on bone has been essential in developing new therapeutic approaches to bone health in these diseases.
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Conditions prompting physicians and surgeons first adapting endoscopes to peer into joints were mainly the sort of synovial conditions that would concern today's rheumatologists. Rheumatologists were among the pre-World War II pioneers developing and documenting arthroscopy. The post-War father of modern arthroscopy, Watanabe, found rheumatologists among his early students, who took back the technique to their home countries, teaching orthopedists and rheumatologists alike. Rheumatologists described and analyzed the intra-articular features of their common diseases in the ’60s and ’70s. A groundswell of interest from academic rheumatologists in adapting arthroscopy grew considerably in the ’90s with development of “needle scopes” that could be used in an office setting. Rheumatologists helped conduct the very trials the findings of which reduced demand for their arthroscopic services by questioning the efficacy of arthroscopic debridement in osteoarthritis (OA) and also developing biological compounds that greatly reduced the call for any resective intervention in inflammatory arthropathies. The arthroscope has proven an excellent tool for viewing and sampling synovium and continues to serve this purpose at several international research centers. While cartilage is now imaged mainly by magnetic resonance imaging, some OA features – such as a high prevalence of visible calcinosis – beg further arthroscopy-directed investigation. A new generation of “needle scopes” with far superior optics awaits future investigators, should they develop interest.
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Aim: To investigate whether TNF inhibitors (TNFi) impact spinal radiographic progression in patients with axial spondyloarthritis (axSpA) and whether this is coupled to their effect on inflammation. Methods: Patients with axSpA fulfilling the modified New York criteria were included in this prospective cohort (ALBERTA FORCAST). Spine radiographs, done every 2 years up to 10 years, were scored by 2 central readers, using the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS). The indirect effect of TNFi on mSASSS was evaluated with generalized estimating equations by testing the interaction between TNFi and ASDAS at the start of each 2-year interval (t). If significant, the association between ASDAS at t and mSASSS at the end of the interval (t+1) was assessed in: i. patients treated with TNFi in all visits; ii. some visits and iii. never treated. In addition, the association between TNFi at t and mSASSS at t+1 (adjusting for ASDAS at t) was also tested (direct effect). Results: In total, 314 patients were included. A gradient was seen for the effect of ASDAS at t on mSASSS at t+1 (interaction p-value 0.10), with a higher progression in patients never treated with TNFi [β (95% CI): 0.41 (0.13; 0.68)] compared to those continuously treated [0.16 (0.00;0.31)] (indirect effect). However, TNFi also directly slowed progression as treated patients had on average 0.85 mSASSS-units less on t+1 compared to those not treated [-0.85 (-1.35; -0.35)]. Conclusion: TNFi reduce spinal radiographic progression in patients with radiographic axSpA which might be partially uncoupled from their effects on ASDAS inflammation.
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Background Inflammation is a core element of many different, systemic and chronic diseases that usually involve an important autoimmune component. The clinical phase of inflammatory diseases is often the culmination of a long series of pathologic events that started years before. The systemic characteristics and related mechanisms could be investigated through the multi–omic comparative analysis of many inflammatory diseases. Therefore, it is important to use molecular data to study the genesis of the diseases. Here we propose a new methodology to study the relationships between inflammatory diseases and signalling molecules whose dysregulation at molecular levels could lead to systemic pathological events observed in inflammatory diseases. Results We first perform an exploratory analysis of gene expression data of a number of diseases that involve a strong inflammatory component. The comparison of gene expression between disease and healthy samples reveals the importance of members of gene families coding for signalling factors. Next, we focus on interested signalling gene families and a subset of inflammation related diseases with multi–omic features including both gene expression and DNA methylation. We introduce a phylogenetic–based multi–omic method to study the relationships between multi–omic features of inflammation related diseases by integrating gene expression, DNA methylation through sequence based phylogeny of the signalling gene families. The models of adaptations between gene expression and DNA methylation can be inferred from pre–estimated evolutionary relationship of a gene family. Members of the gene family whose expression or methylation levels significantly deviate from the model are considered as the potential disease associated genes. Conclusions Applying the methodology to four gene families (the chemokine receptor family, the TNF receptor family, the TGF– β gene family, the IL–17 gene family) in nine inflammation related diseases, we identify disease associated genes which exhibit significant dysregulation in gene expression or DNA methylation in the inflammation related diseases, which provides clues for functional associations between the diseases. Electronic supplementary material The online version of this article (10.1186/s12859-018-2413-x) contains supplementary material, which is available to authorized users.
Chapter
The inflammatory joint diseases include a diverse group of disorders that share in common the presence of inflammatory and destructive changes that adversely affect the structure and function of articular and periarticular tissues. This chapter focuses on rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and seronegative spondyloarthritis, which includes ankylosing spondylitis (AS), reactive arthritis, arthritis of inflammatory bowel disease, juvenile‐onset spondyloarthropathy, and psoriatic arthritis. In RA and SLE, the synovial lining is the initial site of the inflammatory process. In patients with RA and SLE, the synovium becomes a site of an intense immune‐mediated inflammatory process that results in synovial proliferation and production of inflammatory cytokines and soluble mediators that are responsible for the clinical signs of joint inflammation. Synovial inflammation is also present in the seronegative spondyloarthropathies. Immobilization and reduced mechanical loading are factors that have been implicated in the pathogenesis of peri‐articular bone loss.
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Transforming growth factor (TGF)-βs are pluripotent cytokines with stimulatory and inhibitory properties for multiple types of immune cells. Analyses of genetic knockouts of each isoform of TGF-β have revealed differing expression patterns and distinct roles for the three mammalian isoforms of TGF-β. Considerable effort has been focused on understanding the molecular mechanisms of TGF-β1-mediated immune regulation, given its pivotal role in prohibiting systemic autoimmune disease. In recent years, functional similarities and differences between the TGF-β isoforms have delineated their distinct roles in the development of immunopathology and immune tolerance, with increased recent attention being focused on TGF-β3. In addition to the characteristic properties of each TGF-β isoform, recent progress has identified determinants of context-dependent functionality, including various cellular targets, cytokine concentrations, tissue microenvironments, and cytokine synergy, which combine to shape the physiological and pathophysiological roles of the TGF-βs in immunity. Controlling TGF-β production and signaling is being tested as a novel therapeutic strategy in multiple clinical trials for several human diseases. This review highlights advances in the understanding of the cellular sources, activation processes, contextual determinants, and immunological roles of TGF-β3 with comparisons to other TGF-β isoforms.
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Ethnopharmacological relevance: Aconiti Sinomontani Radix is frequently used in the treatment of Bi syndrome in traditional Chinese medicine. Several reports indicate that Aconiti Sinomontani Radix has therapeutic effects for rheumatoid arthritis (RA). However, the cellular mode of action is still unclear. To investigate the effect of alkaloid extracts of Aconiti Sinomontani Radix on proliferation and migration of human synovial sarcoma SW982 cells as well as the molecular mechanism underlying. Materials and methods: SW982 was examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Wound scratch assays were performed to assess the migrated rate of SW982 cells. Quantitative real-time PCR was used to measure the mRNA expression levels of Wnt5a, Runx2, MMP3, and Bmp2. Western blotting was used to measure the phosphorylated levels of JNK and NF-κB as well as the expression of MMP3. Results: The alkaloid extract from Aconiti Sinomontani Radix (MQA) and MQB, which removed lappaconitine from MQA significantly inhibited the proliferation of SW982 in a dose-dependent manner. The proliferation inhibitory effect of MQB was more potent. Incubation with 10μg/ml MQB for 12, 24, and 36h inhibited the migration of SW982 cells by 83%, 58%, and 42%, respectively. Treatment with different concentrations of MQB for 24h inhibited mRNA expression of Wnt5a, Runx2, MMP3, but Bmp2 mRNA expression was elevated by MQB. Further, MQB inhibited phosphorylation of JNK and NF-κB p65 as well as MMP3 expression by Western blotting analysis. Conclusion: The results showed that MQB inhibited proliferation and migration of SW982 cells possibly through suppressing Wnt5a-mediated JNK and NF-κB pathways. These results indicated that MQB might be active extract of Aconiti Sinomontani Radix for targeting fibroblast-like synoviocytes (FLS) and be potential for RA therapy.
Article
We previously reported that synovial fibroblast-like cells (SFs) can be differentiated into chondrocytes through activin receptor-like kinase (ALK) 3 activation. The aim of this study was to clarify the effect and signaling pathways of tumor necrosis factor (TNF)-α on the chondrogenic differentiation of SFs. Primary SFs from patients with rheumatoid arthritis (RA) were treated with recombinant human bone morphogenetic protein-2 or transduced with a constitutively active mutant of the ALK3 gene (ALK3(CA)) with or without TNF-α, and then cultured in pellets. Expression of chondrocyte-specific genes was analyzed by real-time polymerase chain reaction or by histological analysis. Inhibitors of mitogen-activating protein kinase (MAPK) pathways or adenovirus vectors carrying a dominant-negative mutant of the IκB kinase 2 gene (AxIKK2(DN)) were used to analyze the signaling pathways of TNF-α. Expression of chondrocyte-specific genes was induced in SFs either by rhBMP-2 treatment or by ALK3(CA) transduction, which was strongly suppressed by TNF-α treatment. TNF-α markedly increased the p38 MAPK pathways in SFs, and inhibition of p38 MAPK activation partially restored the inhibitory effect of TNF-α on the chondrogenic differentiation of SFs. Combination therapy BMP-2 and anti-TNF-α agents especially targeting p38 MAPK might be a good approach to stimulating neochondrogenesis in the damaged joints in RA.
Chapter
This chapter focuses on rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and the seronegative spondyloarthropathies, which include ankylosing spondylitis (AS), reactive arthritis, the arthritis of inflammatory bowel disease, juvenile-onset spondyloarthropathy, and psoriatic arthritis. Four major forms of pathologic skeletal remodeling can be observed in RA, including focal marginal articular erosions, subchondral bone loss, periarticular osteopenia, and systemic osteoporosis. AS is characterized by inflammation in the entheses, as well as the synovial lining of peripheral joints. Examination of the synovial lesion reveals many of the same features as the RA synovium, including synovial lining hyperplasia, lymphocytic infiltration and pannus formation. In contrast to the pattern of articular bone remodeling in RA, in patients with AS, the inflammatory process may be accompanied by evidence of increased bone formation. SLE, similar to RA, is a systemic inflammatory disease, which, in addition to targeting joint structures, may be associated with widespread extra-articular organ damage. © 2013 American Society for Bone and Mineral Research. All rights reserved.
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Bone morphogenetic proteins are regulators of embryonic development with multiple functions in adult organs and tissues. Here we summarize effects of BMPs outside the musculoskeletal system, focused on their role in inflammatory disorders, e.g., fibrosis, inflammatory bowel disease, anchylosing spondylitis, and rheumatoid arthritis. Additionally, we discuss the interplay between BMPs and vascular disorders leading to atherosclerosis and decipher the key role of BMP in iron metabolism.
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The transforming growth factor-β (TGFβ) superfamily is encoded by 33 genes and includes TGFβ, bone morphogenetic proteins (BMPs) and activins. Although TGFβ is well recognized as a crucial regulator of immune responses, the immunoregulatory functions of other TGFβ family members are less clear. However, recent evidence suggests that BMPs and activins have important roles in regulating immune responses. In this Review, we briefly outline the signalling pathways of the TGFβ superfamily and discuss new insights into the immunoregulatory functions of BMPs and activins in the context of infection, inflammation and cancer.
Article
Objective: To compare the effects of TNF-α and IL-17A on osteogenic differentiation of isolated fibroblast-like synoviocytes (FLS) from healthy donors, osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Methods: FLS were cultured in osteogenic medium, with and without TNF-α and/or IL-17A. Extracellular matrix mineralization was evaluated by alizarin red staining and alkaline phosphatase activity (ALP) measurement. mRNA expression was analyzed by qRT-PCR for Wnt5a, BMP2 and Runx2, genes associated with osteogenesis, for DKK1 and RANKL, genes associated with osteogenesis inhibition and Schnurri-3, a new critical gene in the cross talk with osteoclasts. IL-6 and IL-8 production was measured by ELISA. Results: In osteogenic medium, matrix mineralization and increased ALP activity indicated that FLS can undergo osteogenic differentiation, which was increased with TNF-α and IL-17A. The expression of osteogenesis activators (BMP2 and Wnt5a) was increased with cytokines and that of the osteogenesis inhibitor DKK1 was decreased. There was no difference between all three cell types. In contrast, RA FLS were particularly sensitive to the synergistic increase of Shn3 with TNF-α and IL-17A. Levels of IL-6 and IL-8 were also higher for RA-FLS, compared to healthy and OA FLS. Conclusion: IL-17A and/or TNF-α treatment favor an osteogenesis induction in isolated FLS, independent of their origin. RA-FLS were more sensitive to the synergistic increase of Schnurri-3 expression. Combined with the higher levels of inflammation, this may in turn activate osteoclastogenesis, leading to increased bone destruction seen in destructive arthritis.
Chapter
The rheumatic diseases include a spectrum of disorders that share a propensity to affect the anatomic components of joints and adjacent tissues. The capsule of diarthrodial joints is lined by a specialized membrane, termed synovium, which is the source of lubricants that diminish friction at cartilage surfaces. In patients with inflammatory arthritis, including rheumatoid arthritis (RA) and spondyloarthritis, the synovium becomes inflamed. Cytokines and factors including receptor activator of NF-κB ligand, produced by cells within this inflamed tissue, contribute to articular joint destruction by inducing osteoclastogenesis. Patients with RA typically experience three sites of altered skeletal remodeling: articular erosion, periarticular bone loss, and loss of bone in the skeleton. In spondyloarthritis, articular erosion and skeletal bone loss can also be seen. However, a unique feature of spondyloarthritis is the production of periarticular bone at sites of entheses. This chapter will review the immune mechanisms responsible for these effects on bone.
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Studies of cytokine expression in rheumatoid arthritis have provided key insights into the pathogenesis of disease and have offered clues for effective therapy. Patterns of T-cell products in chronic rheumatoid synovitis suggest that T helper type 1 cells contribute to the perpetuation of disease. However, there is no guarantee that the mechanisms of late disease are identical to very early rheumatoid arthritis. Evaluation of the cytokine profile at the earliest time points after onset of symptoms could identify novel targets that prevent progression to chronic arthritis.
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Bore morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β superfamily, a group of secreted proteins that regulate embryonic development. This review summarizes the effects of BMPs on physiological processes not exclusively linked to the musculoskeletal system. Specifically, we focus on the involvement of BMPs in inflammatory disorders, e.g. fibrosis, inflammatory bowel disease, anchylosing spondylitis, rheumatoid arthritis. Moreover, we discuss the role of BMPs in the context of vascular disorders, and explore the role of these signalling proteins in iron homeostasis (anaemia, hemochromatosis) and oxidative damage. The second and third parts of this review focus on BMPs in the development of metabolic pathologies such as type-2 diabetes mellitus and obesity. The pancreatic beta cells are the sole source of the hormone insulin and BMPs have recently been implicated in pancreas development as well as control of adult glucose homeostasis. Lastly, we review the recently recognized role of BMPs in brown adipose tissue formation and their consequences for energy expenditure and adiposity. In summary, BMPs play a pivotal role in metabolism beyond their role in skeletal homeostasis. However, increased understanding of these pleiotropic functions also highlights the necessity of tissue-specific strategies when harnessing BMP action as a therapeutic target.
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To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1+Ý. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture
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Bone Morphogenetic Protein 2 (BMP-2) and Osteogenic Protein 1 (OP-1, also termed BMP-7) are members of the transforming growth factor beta superfamily. In the present study, we have analyzed the effects of administering them locally at different stages and locations of the chick limb bud using heparin beads as carriers. Our results show that these BMPs are potent apoptotic signals for the undifferentiated limb mesoderm but not for the ectoderm or the differentiating chondrogenic cells. In addition, they promote intense radial growth of the differentiating cartilages and disturb the formation of joints accompanied by alterations in the pattern of Indian hedgehog and ck-erg expression. Interestingly, the effects of these two BMPs on joint formation were found to be different. While the predominant effect of BMP-2 is alteration in joint shape, OP-1 is a potent inhibitory factor for joint formation. In situ hybridizations to check whether this finding was indicative of specific roles for these BMPs in the formation of joints revealed a distinct and complementary pattern of expression of these genes during the formation of the skeleton of the digits. While Op-1 exhibited an intense expression in the perichondrium of the developing cartilages with characteristic interruptions in the zones of joint formation, Bmp-2 expression was a positive marker for the articular interspaces. These data suggest that, in addition to the proposed role for BMP-2 and OP-1 in the establishment of the anteroposterior axis of the limb, they may also play direct roles in limb morphogenesis: (i) in regulating the amount and spatial distribution of the undifferentiated prechondrogenic mesenchyme and (ii) in controlling the location of the joints and the diaphyses of the cartilaginous primordia of the long bones once the chondrogenic aggregates are established.
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The identification of molecules that regulate human hematopoietic stem cells has focused mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs) may play a critical role in the specification of hematopoietic tissue from the mesodermal germ layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified primitive human hematopoietic cells from adult and neonatal sources. Populations of rare CD34(+)CD38(-)Lin- stem cells were isolated from human hematopoietic tissue and were found to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor beta, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34(+)CD38(-) phenotype and repopulation capacity. In contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34(+) CD38(-)Lin- cells, whereas at higher concentrations BMP-4 extended the length of time that repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a new pathway for controlling human stem cell development and a powerful model system for studying the biological mechanism of BMP action using primary human cells.
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Bone morphogenetic protein (BMP)-6 is a member of the transforming growth factor (TGF)-(&bgr;) superfamily, and is most similar to BMP-5, osteogenic protein (OP)-1/BMP-7, and OP-2/BMP-8. In the present study, we characterized the endogenous BMP-6 signaling pathway during osteoblast differentiation. BMP-6 strongly induced alkaline phosphatase (ALP) activity in cells of osteoblast lineage, including C2C12 cells, MC3T3-E1 cells, and ROB-C26 cells. The profile of binding of BMP-6 to type I and type II receptors was similar to that of OP-1/BMP-7 in C2C12 cells and MC3T3-E1 cells; BMP-6 strongly bound to activin receptor-like kinase (ALK)-2 (also termed ActR-I), together with type II receptors, i.e. BMP type II receptor (BMPR-II) and activin type II receptor (ActR-II). In addition, BMP-6 weakly bound to BMPR-IA (ALK-3), to which BMP-2 also bound. In contrast, binding of BMP-6 to BMPR-IB (ALK-6), and less efficiently to ALK-2 and BMPR-IA, together with BMPR-II was detected in ROB-C26 cells. Intracellular signalling was further studied using C2C12 and MC3T3-E1 cells. Among the receptor-regulated Smads activated by BMP receptors, BMP-6 strongly induced phosphorylation and nuclear accumulation of Smad5, and less efficiently those of Smad1. However, Smad8 was constitutively phosphorylated, and no further phosphorylation or nuclear accumulation of Smad8 by BMP-6 was observed. These findings indicate that in the process of differentiation to osteoblasts, BMP-6 binds to ALK-2 as well as other type I receptors, and transduces signals mainly through Smad5 and possibly through Smad1.
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Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-beta (TGF-beta) superfamily, regulates a variety of cell fates and functions. At present, the molecular mechanism by which BMP2 induces apoptosis has not been fully elucidated. Here we propose a BMP2 signaling pathway that mediates apoptosis in mouse hybridoma MH60 cells whose growth is interleukin-6 (IL-6)-dependent. BMP2 dose-dependently induces apoptosis in MH60 cells even in the presence of IL-6. BMP2 has no inhibitory effect on the IL-6-induced tyrosine phosphorylation of STAT3, and the bcl-2 gene expression which is known to be regulated by STAT3, suggesting that BMP2-induced apoptosis is not attributed to alteration of the IL-6-mediated bcl-2 pathway. We demonstrate that BMP2 induces activation of TGF-beta-activated kinase (TAK1) and subsequent phosphorylation of p38 stress-activated protein kinase. In addition, forced expression of kinase-negative TAK1 in MH60 cells blocks BMP2-induced apoptosis. These results indicate that BMP2-induced apoptosis is mediated through the TAK1-p38 pathway in MH60 cells. We also show that MH60-derived transfectants expressing Smad6 are resistant to the apoptotic signal of BMP2. Interestingly, this ectopic expression of Smad6 blocks BMP2-induced TAK1 activation and p38 phosphorylation. Moreover, Smad6 can directly bind to TAK1. These findings suggest that Smad6 is likely to function as a negative regulator of the TAK1 pathway in the BMP2 signaling, in addition to the previously reported Smad pathway.
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Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor, specifically binds to osteoclast differentiation factor and inhibits osteoclast maturation. Members of the transforming growth factor-β superfamily including bone morphogenetic proteins (BMPs) stimulate OPG mRNA expression. In this study, we have characterized the transcription mechanism of BMP-induced OPG gene expression. Transfection of Smad1 and a constitutively active BMP type IA receptor ALK3 (Q233) stimulated the OPG promoter. Deletion analysis of the OPG promoter identified two Hoxc-8 binding sites that respond to BMP stimulation. Glutathione S-transferase-Hoxc-8 protein binds to these two Hox sites specifically. Consistent with the transfection results of the native promoter, ALK3 or Smad1 linker region, which interacts with Hoxc-8, stimulated the activation of the reporter construct with the two Hox sites. Overexpression of Hoxc-8 inhibited the induced promoter activity. When the two Hox binding sites were mutated, ALK3 or Smad1 linker region no longer activated the transcription. Importantly, Smad1 linker region induced both OPG promoter activity and endogenous OPG protein expression in 2T3 osteoblastic cells. The medium from cells transfected with Smad1 linker region expression plasmid effectively inhibited osteoclastogenesis. Collectively, our data indicate that Hox sites mediate both OPG promoter construct activity and endogenous OPG gene expression in response to BMP stimulation.
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Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-beta) family, regulates osteoblast differentiation and bone formation. Here we show a novel function of BMP-2 in human osteoblasts and identify a signaling pathway involved in this function. BMP-2 promotes apoptosis in primary human calvaria osteoblasts and in immortalized human neonatal calvaria osteoblasts, as shown by terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. In contrast, TGF-beta 2 inhibits apoptosis in human osteoblasts. Studies of the mechanisms of action showed that BMP-2 increases the Bax/Bcl-2 ratio, whereas TG beta-2 has a negative effect. Moreover, BMP-2 increases the release of mitochondrial cytochrome c to the cytosol. Consistent with these results, BMP-2 increases caspase-9 and caspase-3, -6, and -7 activity, and an anti-caspase-9 agent suppresses BMP-2-induced apoptosis. Overexpression of dominant-negative Smad1 effectively blocks BMP-2-induced expression of the osteoblast transcription factor Runx2 but not the activation of caspases or apoptosis induced by BMP-2, indicating that the Smad1 signaling pathway is not involved in the BMP-2-induced apoptosis. The proapoptotic effect of BMP-2 is PKC-dependent, because BMP-2 increases PKC activity, and the selective PKC inhibitor calphostin C blocks the BMP-2-induced increased Bax/Bcl-2, caspase activity, and apoptosis. In contrast, the cAMP-dependent protein kinase A inhibitor H89, the p38 MAPK inhibitor SB203580, and the MEK inhibitor PD-98059 have no effect. The results show that BMP-2 uses a Smad-independent, PKC-dependent pathway to promote apoptosis via a Bax/Bcl-2 and cytochrome c-caspase-9-caspase-3, -6, -7 cascade in human osteoblasts.
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Bone morphogenetic protein (BMP)-2 has been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in non-precardiac mesodermal cells, suggesting that BMP-2 is an inductive signaling molecule that participates in cardiac development. However, direct evidence of the effects of BMP-2 on cardiac myocytes has not been reported. To examine the role of BMP-2 and its receptors, we studied the ability of BMP-2 to promote survival of isolated neonatal rat cardiac myocytes. BMP receptors IA, IB, and II and activin receptor I were found to be expressed in myocytes, and BMP-2 phosphorylated Smad1 and p38 MAPK. Interestingly, BMP-2 promoted survival and inhibited apoptosis of serum-deprived myocytes, although it did not strongly induce hypertrophic growth. To explore the mechanisms for this protective effect, an adenovirus-based vector system was used. Similar to BMP-2, Smad1 promoted survival that was repressed by Smad6. Moreover, BMP-2 and Smad1 enhanced the expression of the anti-apoptotic molecule Bcl-xL. Antisense oligonucleotides to bcl-xL attenuated the survival effected by BMP-2. Overall, our findings suggest that BMP-2 prevents apoptosis of myocytes by induction of Bcl-xLvia a Smad1 pathway and might be a novel survival factor without any hypertrophic effect on myocytes.
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The activation of nuclear factor κB (NF-κ B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IκBα inhibitor of NF-κB expressed in C2C12 cells and IκB kinase β-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-κB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-κB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.
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Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases.
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Rheumatoid arthritis results from a T cell-driven inflammation in the synovial membrane that is frequently associated with the formation of tertiary lymphoid structures. The significance of this extranodal lymphoid neogenesis is unknown. Microdissection was used to isolate CD4 T cells residing in synovial tissue T cell/B cell follicles. CD4 T cells with identical TCR sequences were represented in independent, nonadjacent follicles, suggesting recognition of the same Ag in different germinal centers. When adoptively transferred into rheumatoid arthritis synovium-SCID mouse chimeras, these CD4 T cell clones enhanced the production of IFN-gamma, IL-1beta, and TNF-alpha. In vivo activity of adoptively transferred CD4 T cells required matching of HLA-DRB1 alleles and also the presence of T cell/B cell follicles. HLA-DRB1-matched synovial tissues that were infiltrated by T cells, macrophages, and dendritic cells, but that lacked B cells, did not support the activation of adoptively transferred CD4 T cell clones, raising the possibility that B cells provided a critical function in T cell activation or harbored the relevant Ag. Dependence of T cell activation on B cells was confirmed in B cell depletion studies. Treatment of chimeric mice with anti-CD20 mAb inhibited the production of IFN-gamma and IL-1beta, indicating that APCs other than B cells could not substitute in maintaining T cell activation. The central role of B cells in synovial inflammation identifies them as excellent targets for immunosuppressive therapy.
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Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)–β superfamily, are a group of related proteins that are capable of inducing the formation of cartilage and bone but are now regarded as multifunctional cytokines. We show in this report a novel function of BMPs in hematopoietic cells: BMP-2 induces apoptosis not only in human myeloma cell lines (U266, RPMI 8226, HS-Sultan, IM-9, OPM-2, and KMS-12 cells), but also in primary samples from patients with multiple myeloma. The mechanism of BMP-2–induced apoptosis was investigated with the use of U266 cells, which are dependent on the interleukin-6 autocrine loop. We showed that BMP-2 caused cell-cycle arrest in the G1 phase and the subsequent apoptosis of myeloma cells. BMP-2 up-regulated the expression of cyclin-dependent kinase inhibitors (p21CIP1/WAF1 and p27KIP1) and caused hypophosphorylation of retinoblastoma (Rb) protein. In studies of apoptosis-associated proteins, BMP-2 was seen to down-regulate the expression of Bcl-xL; however, BMP-2 had no effects on the expression of Bcl-2, Bax, or Bad. Therefore, BMP-2 induces apoptosis in various human myeloma cells by means of the down-regulation of Bcl-xL and by cell-cycle arrest through the up-regulation of p21CIP1/WAF1 and p27KIP1 and by the hypophosphorylation of Rb. Further analysis showed that the signal transducer and activator of transcription 3 (STAT3) was inactivated immediately after BMP-2 treatment. We conclude that BMP-2 would be useful as a novel therapeutic agent in the treatment of multiple myeloma both by means of its antitumor effect of inducing apoptotis and through its original bone-inducing activity, because bone lesions are frequently seen in myeloma patients.
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Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-β/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3ex8/ex8) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen–expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-β1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3ex8/ex8 mice. These data suggest that TGF-β/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.
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The revised criteria for the classification of rheumatoid arthritis (RA) were formulated from a computerized analysis of 262 contemporary, consecutively studied patients with RA and 262 control subjects with rheumatic diseases other than RA (non-RA). The new criteria are as follows: 1) morning stiffness in and around joints lasting at least 1 hour before maximal improvement; 2) soft tissue swelling (arthritis) of 3 or more joint areas observed by a physician; 3) swelling (arthritis) of the proximal interphalangeal, metacarpophalangeal, or wrist joints; 4) symmetric swelling (arthritis); 5) rheumatoid nodules; 6) the presence of rheumatoid factor; and 7) radiographic erosions and/or periarticular osteopenia in hand and/or wrist joints. Criteria 1 through 4 must have been present for at least 6 weeks. Rheumatoid arthritis is defined by the presence of 4 or more criteria, and no further qualifications (classic, definite, or probable) or list of exclusions are required. In addition, a “classification tree” schema is presented which performs equally as well as the traditional (4 of 7) format. The new criteria demonstrated 91–94% sensitivity and 89% specificity for RA when compared with non-RA rheumatic disease control subjects.
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Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a pro- gram of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is ar- rested before terminal hypertrophic differentiation. In this study, we showed that TGF- b /Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 ( Smad3 ex8/ex8 ) developed degenerative joint disease resembling human osteoarthritis, as char- acterized by progressive loss of articular cartilage, for- mation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF- b 1 significantly inhibits chondrocyte differentia- tion of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3 ex8/ex8 mice. These data suggest that TGF- b / Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and un- dergo abnormal terminal differentiation, ultimately leading to osteoarthritis.
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Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.
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For the purposes of classification, it should be specified whether osteoarthritis (OA) of the knee is of unknown origin (idiopathic, primary) or is related to a known medical condition or event (secondary). Clinical criteria for the classification of idiopathic OA of the knee were developed through a multicenter study group. Comparison diagnoses included rheumatoid arthritis and other painful conditions of the knee, exclusive of referred or paraarticular pain. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop sets of criteria that serve different investigative purposes. In contrast to prior criteria, these proposed criteria utilize classification trees, or algorithms.
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Classification criteria for most of the disorders belonging to the spondylarthropathy group already exist. However, the spectrum of spondylarthropathy is wider than the sum of these disorders suggests. Sero-negative oligoarthritis, dactylitis or polyarthritis of the lower extremities, heel pain due to enthesitis, and other undifferentiated cases of spondylarthropathy have been ignored in epidemiologic studies because of the inadequacy of existing criteria. In order to define classification criteria that also encompass patients with undifferentiated spondylarthropathy, we studied 403 patients with all forms of spondylarthropathy and 674 control patients with other rheumatic diseases. The diagnoses were based on the local clinical expert's opinion. The 403 patients included 168 with ankylosing spondylitis, 68 with psoriatic arthritis, 41 with reactive arthritis, 17 with inflammatory bowel disease and arthritis, and 109 with unclassified spondylarthropathy. Based on statiscal analysis and clinical reasoning, we propose the following classification criteria for spondylarthropathy inflammatory spinal pain or synovitis (asymmetric or predominantly in the lower limbs), together with at least 1 of the following: positive family history, psoriasis inflammatory bowel disease, urethritis, or acute diarrhea, alternating buttock pain, enthesopathy, or sacroiliitis as determined from radiography of the pelvic region. These criteria resulted in a sensitivity of 87% and a specificity of 87%. The proposed classification criteria are easy to apply in clinical practice and performed well in all 7 participating centers. However, we regard them as preliminary until they have been further evaluated in other settings.
Article
Objective To characterize mesenchymal stem cells (MSCs) from human synovial membrane (SM).Methods Cell populations were enzymatically released from the SM obtained from knee joints of adult human donors and were expanded in monolayer with serial passages at confluence. Cell clones were obtained by limiting dilution. At different passages, SM-derived cells were subjected to in vitro assays to investigate their multilineage potential. Upon treatments, phenotypes of cell cultures were analyzed by histo- and immunohistochemistry and by semiquantitative reverse transcription–polymerase chain reaction for the expression of lineage-related marker genes.ResultsSM-derived cells could be expanded extensively in monolayer, with limited senescence. Under appropriate culture conditions, SM-derived cells were induced to differentiate to the chondrocyte, osteocyte, and adipocyte lineages. Sporadic myogenesis was also observed. Five independent cell clones displayed multilineage potential. Interestingly, only 1 clone was myogenic. Donor age, cell passaging, and cryopreservation did not affect the multilineage potential of SM-derived cells. In contrast, normal dermal fibroblasts under the same culture conditions did not display this potential.Conclusion Our study demonstrates that human multipotent MSCs can be isolated from the SM of knee joints. These cells have the ability to proliferate extensively in culture, and they maintain their multilineage differentiation potential in vitro, establishing their progenitor cell nature. SM-derived MSCs may play a role in the regenerative response during arthritic diseases and are promising candidates for developing novel cell-based therapeutic approaches for postnatal skeletal tissue repair.
Article
Subcutaneous implantation of demineralized bone matrix initiates a sequence of developmental events, which culminate in endochondral bone formation. During early stages of development of matrix-induced implants, ED1, Ia-positive monocytes-macrophages were observed, suggesting that in the initial phases of the endochondral bone formation cascade, the bone-inductive protein osteogenin and related bone morphogenetic proteins (BMPs) might serve as potent chemoattractants to recruit circulating monocytes. In this investigation, we demonstrate that at concentrations of 10-100 fg/ml (0.3-3 fM), native bovine osteogenin and recombinant human BMP-2B (rhBMP-2B) induce the directed migration of human blood monocytes in vitro. This chemotactic response was associated with expression of BMP binding sites (receptors) on monocytes. About 750 receptors per cell were detected with an apparent dissociation constant of 200 pM. Both osteogenin and rhBMP-2B at higher concentrations (0.1-30 ng/ml) stimulated mRNA expression for an additional regulatory molecule, type beta 1 transforming growth factor (TGF-beta 1) in human monocytes. TGF-beta 1, in turn, is known to induce a cascade of events leading to matrix generation. Monocytes stimulated by TGF-beta are known to secrete a number of chemotactic and mitogenic cytokines that recruit endothelial and mesenchymal cells and promote their synthesis of collagen and associated matrix constituents. TGF-beta 1 in concert with these other cytokines and matrix components regulates chemotaxis, mesenchymal proliferation, differentiation, angiogenesis, and controlled synthesis of extracellular matrix. Our results demonstrate that osteogenin and related BMPs through their profound effects on monocyte recruitment and cytokine synthesis may promote additional successive steps in the endochondral bone formation cascade.
Article
Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.
Article
For the purposes of classification, it should be specified whether osteoarthritis (OA) of the knee is of unknown origin (idiopathic, primary) or is related to a known medical condition or event (secondary). Clinical criteria for the classification of idiopathic OA of the knee were developed through a multicenter study group. Comparison diagnoses included rheumatoid arthritis and other painful conditions of the knee, exclusive of referred or para-articular pain. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop sets of criteria that serve different investigative purposes. In contrast to prior criteria, these proposed criteria utilize classification trees, or algorithms.
Article
Wandering histiocytes, foreign body giant cells, and inflammatory connective-tissue cells are stimulated by degradation products of dead matrix to grow in and repopulate the area of an implant of decalcified bone. Histiocytes are more numerous than any other cell form and may transfer collagenolytic activity to the substrate to cause dissolution of the matrix. The process is followed immediately by new-bone formation by autoinduction in which both the inductor cells and the induced cells are derived from ingrowing cells of the host bed. The inductor cell is a descendant of a wandering histiocyte; the induced cell is a fixed histiocyte or perivascular young connective-tissue cell. Differentiation of the osteoprogenitor cell is elicited by local alterations in cell metabolic cycles that are as yet uncharacterized.
Article
The pattern of skeletal structures and muscles in the branchial region of the head is profoundly influenced by the neural crest, whose cells arise at discrete segmental levels of the chick hindbrain: specifically, rhombomeres (r)1+2, r4 and r6, whereas r3 and r5 are crest-depleted. We have demonstrated that an interaction between even-numbered rhombomeres and r3/r5 effects this depletion of neural crest, resulting in the sculpting of discrete migratory streams of neural crest. This mechanism acts through increased expression of msx2 and the induction of apoptosis in dorsal cells of r3 and r5 (ref. 3) (Fig. 1A). Here we demonstrate that the signalling molecule Bmp4 is expressed in r3 and r5 and is dependent on the neighbouring rhombomeres. Addition of recombinant BMP4 protein to explant cultures of r3 or r5, which produce neural crest when isolated from their neighbouring rhombomeres, upregulates msx2 and reinstates apoptosis in the neural crest population.
Article
Temporal and spatial distribution of a gene encoding murine bone morphogenetic protein 4 (mBMP-4) during fracture repair were investigated in mice by RT-PCR and in situ hybridization. For in situ hybridization, fractured ribs and surrounding tissues were decalcified and hybridized with a mBMP-4-specific complementary RNA probe labeled with digoxigenin-11 UTP. mBMP-4 messenger RNA (mRNA) was not detected in ribs without fracture, whereas it was detected only in the early phase of fracture from 12 to 72 h after the onset of fracture before new cartilage or bone formation. The mBMP-4 mRNAs were present in cells distributed in three distinct regions, namely, the proliferating periosteum, the medullary cavity, and the muscles near the fracture site. These BMP-4-positive cells did not express bone gla protein mRNA, which is a marker of the mature osteogenic cell. RT-PCR also showed a transient increase in the level of BMP-4 mRNA in the early phase of fracture repair. The findings provide us with some new information. (1) The BMP-4 gene is produced by less differentiated osteoprogenitor cells, not by differentiated osteoblasts. (2) The BMP-4 gene is enhanced by the impact of fracture and localized in callus-forming tissue before callus formation. Together with the activities of BMP-4, as was previously described, our results suggest that newly produced BMP-4 gene product is one of the local contributing factors in callus formation in the early phase of fracture healing.
Article
The bone morphogenetic proteins (BMPs) constitute a large family of cytokines related to members of the transforming growth factor-beta superfamily. Recent evidence, in particular from gene targeting experiments in the mouse, indicates that BMPs are required for mesoderm formation and for the development and patterning of many different organ systems. Significant progress has also been made in understanding the role of BMPs in gastrulation and neurulation in Xenopus and in identifying genes regulating BMP expression and components of the downstream signaling pathways. Extracellular modifiers of BMP activity may constitute an opposing morphogenetic system.
Article
To investigate the effects of the cartilage-derived morphogenetic proteins (CDMPs) in an in vitro cartilage explant model that mimics the chondrocytic response to matrix depletion, and to demonstrate their presence in articular cartilage. Adult bovine articular cartilage and postmortem specimens from adult human donors with and without osteoarthritic (OA) lesions were stained by immunohistochemistry using polyclonal antibodies specific for CDMP-1 and CDMP-2. Extracts of bovine articular cartilage were analyzed by Western blotting for the presence of the CDMPs. Bovine articular cartilage explants were depleted of their matrix by trypsin digestion, followed by a 7-day culture period in a chemically defined serum-free basal medium (BM), with or without recombinant CDMPs 1 and 2. The metabolic activity of chondrocytes was measured by 35S-sulfate incorporation into macromolecules. Newly synthesized proteoglycans (PGs) were analyzed using Sephacryl S-500 HR gel chromatography. The expression levels of the messenger RNA (mRNA) for chondrogenic markers were investigated by Northern analysis. CDMP-1 and CDMP-2 were detected in both bovine and human healthy and OA articular cartilage. Treatment of matrix-depleted cartilage explants with CDMPs 1 and 2 increased equally the incorporation of 35S-sulfate into PGs compared with tissue maintained in BM. Gel chromatography analysis indicated that aggrecan was the predominant PG species. Northern blot analysis showed that the expression of link protein, type II collagen, and aggrecan mRNA transcripts was not modulated by CDMP treatment. This study shows the presence of CDMP-1 and CDMP-2 in adult bovine and human articular cartilage. In addition, our in vitro data indicate that CDMPs 1 and 2 stimulate the metabolic activity of articular chondrocytes. Therefore, these signaling molecules may be contributing to the maintenance of the integrity of the joint surface.
Article
The bone morphogenetic proteins are secreted signalling molecules that belong to the transforming growth factor beta family of growth and differentiation factors. Individual bone morphogenetic proteins are prominent at many sites during embryogenesis and are likely to be key regulators of early development and organogenesis. In vertebrates, one of the functions of bone morphogenetic like proteins is to induce formation of bone, cartilage, and connective tissues associated with the skeleton. This osteoinductive ability has led to the use of bone morphogenetic proteins as therapeutic agents for creation of new bone useful in treatment of skeletal injuries and diseases, and in oral and maxillofacial applications.
Article
Rheumatoid arthritis and periodontitis are chronic inflammatory diseases associated with tissue destruction that is mediated in part by elevated levels of cytokines (e.g., interleukin-1 and tumor necrosis factor). Differential screening of a human synovial fibroblast cDNA library for interleukin-1 induced genes revealed a clone identical to the gene encoding human bone morphogenetic protein-2. Northern blot analysis of human synovial fibroblast mRNA confirmed up-regulation of bone morphogenetic protein-2 in the presence of interleukin-1. Utilizing a specific antibody, levels of bone morphogenetic protein-2 protein in conditioned medium from synovial fibroblasts were also up-regulated in the presence of interleukin-1. This is the first report of the production of bone morphogenetic protein-2 by synovial fibroblasts, and the first report of its up-regulation in response to interleukin-1. However, interleukin-1 did not induce bone morphogenetic protein-2 mRNA in human gingival fibroblasts.
Article
To evaluate the efficacy, pharmacokinetics, immunogenicity, and safety of multiple infusions of a chimeric monoclonal anti-tumor necrosis factor alpha antibody (cA2) (infliximab; Remicade, Centocor, Malvern, PA) given alone or in combination with low-dose methotrexate (MTX) in rheumatoid arthritis (RA) patients. In a 26-week, double-blind, placebo-controlled, multicenter trial, 101 patients with active RA exhibiting an incomplete response or flare of disease activity while receiving low-dose MTX were randomized to 1 of 7 groups of 14-15 patients each. The patients received either intravenous cA2 at 1, 3, or 10 mg/kg, with or without MTX 7.5 mg/week, or intravenous placebo plus MTX 7.5 mg/week at weeks 0, 2, 6, 10, and 14 and were followed up through week 26. Approximately 60% of patients receiving cA2 at 3 or 10 mg/kg with or without MTX achieved the 20% Paulus criteria for response to treatment, for a median duration of 10.4 to >18.1 weeks (P < 0.001 versus placebo). Patients receiving cA2 at 1 mg/kg without MTX became unresponsive to repeated infusions of cA2 (median duration 2.6 weeks; P=0.126 versus placebo). However, coadministration of cA2 at 1 mg/kg with MTX appeared to be synergistic, prolonging the duration of the 20% response in >60% of patients to a median of 16.5 weeks (P < 0.001 versus placebo; P=0.006 versus no MTX) and the 50% response to 12.2 weeks (P < 0.001 versus placebo; P=0.002 versus no MTX). Patients receiving placebo infusions plus suboptimal low-dose MTX continued to have active disease, with a Paulus response lasting a median of 0 weeks. A 70-90% reduction in the swollen joint count, tender joint count, and C-reactive protein level was maintained for the entire 26 weeks in patients receiving 10 mg/kg of cA2 with MTX. In general, treatment was well tolerated and stable blood levels of cA2 were achieved in all groups, except for the group receiving 1 mg/kg of cA2 alone, at which dosage antibodies to cA2 were observed in approximately 50% of the patients. Multiple infusions of cA2 were effective and well tolerated, with the best results occurring at 3 and 10 mg/kg either alone or in combination with MTX in approximately 60% of patients with active RA despite therapy with low-dose MTX. When cA2 at 1 mg/kg was given with low-dose MTX, synergy was observed. The results of the trial provide a strategy for further evaluation of the efficacy and safety of longer-term treatment with cA2.