Bone morphogenetic protein and transforming growth factor beta inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin-1beta

OBJECTIVE: Bone morphogenetic protein (BMP) and transforming growth factor beta (TGFbeta) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFbeta signaling, inhibitory Smad6 (I-Smad6) and I-Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin-1beta (IL-1beta) stimulation in vitro. METHODS: RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I-Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen. RESULTS: Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I-Smads was found. In cultured articular chondrocytes, stimulation with IL-1beta showed up-regulation of Smad7, whereas Smad6 was down-regulated. CONCLUSION: Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I-Smads in articular cartilage in vivo. No evidence was found that up-regulation or down-regulation of I-Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL-1beta suggests a potentially important role of IL-1beta signaling in chondrocytes, via indirect influencing of the BMP/TGFbeta signaling cascade.     

Expression of the tissue inhibitor of metalloproteinases (TIMP) gene family in normal and osteoarthritic joints

The pathophysiological and biological significance of tissue inhibitor of the metalloproteinases-3 (TIMP-3) gene compared to other TIMPs was investigated in osteoarthritic (OA) human and normal bovine joint tissues. Human OA synovial fibroblasts in culture constitutively expressed TIMP mRNAs. TIMP-3, TIMP-1 and gelatinase A mRNAs were elevated in most human OA synovia over controls, while TIMP-2 expression was similar. TIMP-3 and TIMP-1 mRNAs present in bovine cartilage were inducible by serum factors. Transforming growth factor beta (TGF-β1) induced TIMP-3 RNA and protein in human OA and normal bovine chondrocytes. TIMP mRNAs were low (TIMP-1) or undetectable in human fetal chondrocytes but were expressed at all other ages. Thus, the two main joint tissues, synovial membranes and cartilage, express TIMP genes. Due to their matrix protecting activities, the presence of multiple TIMPs may be beneficial for normal joints, while increased TIMP-3 and TIMP-1 expression in arthritic joints may be associated with pathological remodeling. Received: 13 October 1998 / Accepted: 18 February 1999     

Analysis of spatial and temporal expression patterns of bone morphogenetic protein family members in the rat uterus over the estrous cycle

Recent studies have demonstrated that bone morphogenetic proteins (BMPs) play fundamental roles in female fertility. This is particularly evident in terms of the ovary. One major question that is just beginning to be addressed is the role of BMPs in the non-pregnant uterus. To help fill this gap, we used in situ hybridization to investigate the expression of BMP family members in the rat uterus over the estrous cycle. We found that the endometrial/uterine cycle is accompanied by the expression of several components of the BMP pathway - including ligands, receptors and antagonists. The mRNAs encoding BMP receptors are expressed in the epithelial (BMP-RIA, -RIB and -RII), periluminal stroma (BMP-RIA and -RII) and smooth muscle cells (BMP-RIA and -RII). The expression of all three receptors showed clear cyclic variations. The mRNAs encoding BMP ligands were highly expressed in the periluminal stroma (BMP-2 and -7) and glandular epithelium (BMP-7). The expression of BMP-2, but not BMP-7, was cyclical. Notably, the periluminal stroma expressed noggin mRNA. In the blood vascular system, BMP-4, -6 and -RII mRNAs were expressed in myometrial endothelial cells. Interestingly, follistatin, noggin, and BMP-4, -6 and -7 mRNAs were expressed in eosinophilic leukocytes, suggesting unexpected roles for eosinophil-derived BMPs in uterine function. We conclude that BMP ligands, receptors and antagonists are expressed in spatially and temporally restricted patterns that are consistent with a physiological role for these regulatory molecules in promoting uterine cellular processes including cell proliferation, differentiation and apoptosis during the cycle.     

Differential regulation of the bone morphogenic protein antagonist chordin in human normal and osteoarthritic chondrocytes

OBJECTIVE: To investigate the distribution of the bone morphogenic protein (BMP) antagonist chordin in normal and osteoarthritic cartilage and synovial membranes, and its regulation in chondrocytes and synovial fibroblasts by inflammatory and growth factors. METHODS: Localisation of chordin in tissues was undertaken by immunohistochemistry and gene regulation was determined by real time polymerase chain reaction. RESULTS: In normal cartilage, chordin was found at low levels (mean (SD), 7.6 (1.3)%), mainly in the very superficial layers. In osteoarthritis, chordin was also found in the superficial layers (8.9 (1.1)%), though at a significantly higher level (24.7 (1.5)%) in the last two thirds of the cartilage. In contrast to normal cells, chordin mRNA and protein levels were significantly downregulated (p<0.01) in osteoarthritic chondrocytes by all the growth factors tested. Interferon gamma stimulated chordin expression in normal but not in osteoarthritic chondrocytes (p<0.0002), while interleukin 1 beta and tumour necrosis factor alpha did not affect the expression level. However, no difference was found in either the distribution or regulation of chordin in normal and osteoarthritic synovial membranes or synovial fibroblasts. CONCLUSIONS: The differential distribution and regulation of chordin in normal and osteoarthritic cartilage and chondrocytes suggests an involvement of this antagonist in the osteoarthritic process.     

Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor beta/bone morphogenetic protein inhibitors during experimental osteoarthritis

OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor beta (TGFbeta) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFbeta and BMP during papain-induced OA-like changes in mice. METHODS: We used adenoviral overexpression of TGFbeta and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan-specific TGFbeta antagonist called murine latency-associated peptide 1 (mLAP-1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFbeta/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss. RESULTS: Intraarticular injection of papain caused increased protein expression of several TGFbeta and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP-1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFbeta inhibitor mLAP-1 increased articular cartilage PG loss. CONCLUSION: Our results indicate a pivotal role of endogenous TGFbeta in the development of osteophytes and synovial thickening, implicating endogenous TGFbeta in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFbeta signifies the protective role of TGFbeta in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.     

Gradients in bone morphogenetic protein-related gene expression across the growth plate

In the growth plate, stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone and then terminally differentiate into the non-dividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ) of 7-day-old male rats. Expression of approximately 29,000 genes was analyzed by microarray and selected genes verified by real-time PCR. The analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were upregulated in HZ compared with RZ and PZ (30-fold each, P < 0.01 and 0.001 respectively). In contrast, BMP signaling inhibitors were expressed early in the differentiation pathway; BMP-3 and gremlin were differentially expressed in RZ (100- and 80-fold, compared with PZ, P < 0.001 and 0.005 respectively) and growth differentiation factor (GDF)-10 in PZ (160-fold compared with HZ, P < 0.001). Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. Since BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes, these findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower RZ, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation. Thus, BMP signaling gradients may be a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in growth plate cartilage.     

Extracellular sulfatases support cartilage homeostasis by regulating BMP and FGF signaling pathways

The balance between anabolic and catabolic signaling pathways is critical in maintaining cartilage homeostasis and its disturbance contributes to joint diseases such as osteoarthritis (OA). A unique mechanism that modulates the activity of cell signaling pathways is controlled by extracellular heparan endosulfatases Sulf-1 and Sulf-2 (Sulfs) that are overexpressed in OA cartilage. This study addressed the role of Sulfs in cartilage homeostasis and in regulating bone morphogenetic protein (BMP)/Smad and fibroblast growth factor (FGF)/Erk signaling in articular cartilage. Spontaneous cartilage degeneration and surgically induced OA were significantly more severe in Sulf-1^−/− and Sulf-2^−/− mice compared with wild-type mice. MMP-13, ADAMTS-5, and the BMP antagonist noggin were elevated whereas col2a1 and aggrecan were reduced in cartilage and chondrocytes from Sulf^−/− mice. Articular cartilage and cultured chondrocytes from Sulf^−/− mice showed reduced Smad1 protein expression and Smad1/5 phosphorylation, whereas Erk1/2 phosphorylation was increased. In human chondrocytes, Sulfs siRNA reduced Smad phosphorylation but enhanced FGF-2-induced Erk1/2 signaling. These findings suggest that Sulfs simultaneously enhance BMP but inhibit FGF signaling in chondrocytes and maintain cartilage homeostasis. Approaches to correct abnormal Sulf expression have the potential to protect against cartilage degradation and promote cartilage repair in OA.     

Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk

Pituitary tumor development involves clonal expansion stimulated by hormones and growth factorscytokines. Using mRNA differential display, we found that the bone morphogenetic protein (BMP) inhibitor noggin is down-regulated in prolactinomas from dopamine D2-receptor-deficient mice. BMP-4 is overexpressed in prolactinomas taken from dopamine D2-receptor-deficient female mice, but expression of the highly homologous BMP-2 does not differ in normal pituitary tissue and prolactinomas. BMP-4 is overexpressed in other prolactinoma models, including estradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types (Western blot analysis of 48 tumors). BMP-4 stimulates, and noggin blocks, cell proliferation and the expression of c-Myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumors. GH3 cells stably transfected with a dominant negative of Smad4 (Smad4dn; a BMP signal cotransducer) or noggin have reduced tumorigenicity in nude mice. Tumor growth recovered in vivo when the Smad4dn expression was lost, proving that BMP-4Smad4 are involved in tumor development in vivo. BMP-4 and estrogens act through overlapping intracellular signaling mechanisms on GH3 cell proliferation and c-myc expression: they had additive effects at low concentrations but not at saturating doses, and their action was inhibited by blocking either pathway with the reciprocal antagonist (i.e., BMP-4 with ICI 182780 or 17beta-estradiol with Smad4dn). Furthermore, coimmunoprecipitation studies demonstrate that under BMP-4 stimulation Smad4 and Smad1 physically interact with the estrogen receptor. This previously undescribed prolactinoma pathogenesis mechanism may participate in tumorigenicity in other cells where estrogens and the type beta transforming growth factor family have important roles.     

Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro

OBJECTIVE: Osteoarthritic (OA) cartilage destruction depends on collagen- and aggrecan-degrading proteases such as collagenases (MMP-1 and MMP-13), stromelysin (MMP-3), MMP-14, as well as the so-called aggrecanases (ADAM-TS4 and ADAM-TS5). In this study, we tried to clarify whether these proteases are expressed in vivo in human normal and OA cartilage (and whether they are up-regulated or down-regulated during the disease process) and in interleukin-1beta (IL-1beta)-stimulated chondrocytes in vitro. METHODS: Quantitative polymerase chain reaction assays were developed and performed on RNA isolated directly from normal and degenerative cartilage tissue as well as from primary human articular chondrocytes cultured with and without IL-1beta. RESULTS: In vivo, MMP-1 was detectable only at very low levels in any condition. MMP-13 expression was low in normal and early degenerative cartilage but was strongly up-regulated in late-stage OA specimens. MMP-1 and MMP-13 were expressed much higher in vitro than in vivo and were up-regulated by IL-1beta. Among all proteases, MMP-3 was by far the most strongly expressed, although it was strongly down-regulated in late-stage OA specimens. Expression of MMP-3 was higher in vitro than in vivo and was up-regulated by IL-1beta. ADAM-TS5 and MMP-14 were expressed in all sample groups. Expression of ADAM-TS4 was very low in vivo and was induced in vitro after stimulation by IL-1beta. CONCLUSION: Our expression data clearly support MMP-13 as the major collagenase in OA cartilage. The most strongly expressed aggrecanase was ADAM-TS5. ADAM-TS4 was expressed only at a very low level in normal cartilage and was only slightly up-regulated in OA cartilage, casting doubt on this enzyme being the relevant aggrecanase of articular cartilage. Results of our study show that expression of many enzymes is significantly different in vitro and in vivo and suggest that IL-1beta stimulation of articular chondrocytes might not be a good model for the matrix catabolism in OA cartilage.     

Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor beta-induced osteophytes: limited role of bone morphogenetic protein in early osteoarthritic osteophyte formation

OBJECTIVE: Osteoarthritis (OA) is characterized by cartilage damage, synovial fibrosis, and osteophyte formation. Both transforming growth factor beta (TGFbeta) and bone morphogenetic protein 2 (BMP-2) can induce the formation of osteophytes during OA, but their specific role in this process is unclear. The purpose of this study was to investigate the respective contributions of TGFbeta and BMP-2 to OA. METHODS: Mouse knee joints injected with adenovirus (Ad-TGFbeta or Ad-BMP-2) were compared histologically with knee joints from murine models of OA (joints injected with collagenase and joints from STR/Ort mice with spontaneous OA). To further investigate the role of BMP during osteophyte formation, adenovirus Ad-Gremlin was injected into knee joints that had previously been injected with Ad-TGFbeta or collagenase. RESULTS: BMP-2 induced early osteophytes, which bulged from the growth plates on the femur and grew on top of the patella, whereas TGFbeta induced early osteophyte formation on the bone shaft beneath the collateral ligament on the femur as well as on top of the patella. The pattern of osteophyte formation during experimental OA closely resembled that of TGFbeta-induced osteophyte formation, but differed from the pattern induced by BMP-2. Ad-Gremlin proved to be able to totally block BMP-2-induced osteophyte formation. However, blocking BMP activity inhibited neither TGFbeta-induced nor experimental OA-associated osteophyte formation. CONCLUSION: Our findings demonstrate that the role of BMP during the onset of TGFbeta-induced and experimental OA-induced osteophyte formation is limited. The latter finding does not rule out a role of BMP during osteophyte maturation.     

Expression of interleukin-21 receptor, but not interleukin-21, in synovial fibroblasts and synovial macrophages of patients with rheumatoid arthritis

OBJECTIVE: To determine the role and expression of the cytokine/receptor pair interleukin-21 (IL-21)/IL-21 receptor (IL-21R) in rheumatoid arthritis (RA). METHODS: The expression of IL-21R and IL-21 was analyzed by TaqMan real-time polymerase chain reaction (PCR) and in situ hybridization of synovial biopsy samples from patients with RA and osteoarthritis (OA). Double labeling by immunohistochemistry after in situ hybridization was performed with anti-CD68 antibodies. The expression of IL-21R at the protein level was confirmed by Western blotting. Stimulation experiments were performed with recombinant IL-1beta, tumor necrosis factor alpha (TNFalpha), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGFbeta). The role of IL-21R in cartilage destruction was analyzed in the SCID mouse coimplantation model of RA. RESULTS: IL-21R was found in total RNA extracts and in synovial biopsy samples from RA patients, whereas no expression or only minimal expression was seen in samples from OA patients. Double labeling indicated that both synovial macrophages and synovial fibroblasts expressed IL-21R. Western blotting with anti-IL-21R antibodies confirmed the expression of IL-21R protein in RA synovial fibroblasts (RASFs). Of note, IL-21 was not detectable by real-time PCR and in situ hybridization in the same samples in vivo as in vitro. The level of expression of IL-21R messenger RNA (mRNA) was not altered by stimulation with IL-1beta, TNFalpha, PDGF, or TGFbeta. Interestingly, in the SCID mouse coimplantation model, RASFs did not maintain their expression of IL-21R at sites of invasion into the cartilage. Similarly, IL-21R mRNA was not expressed at sites of invasion into cartilage and bone in RA synovium. CONCLUSION: Our data demonstrate that IL-21R is expressed in RA synovium by RASFs and synovial macrophages. IL-21R is associated with the activated phenotype of RASFs independently of the major proinflammatory cytokines IL-1beta and TNFalpha, but correlates negatively with the destruction of articular cartilage and bone.     

Fibroblast-mediated delivery of growth factor complementary DNA into mouse joints induces chondrogenesis but avoids the disadvantages of direct viral gene transfer

OBJECTIVE: To assess the advantages and disadvantages of a direct adenoviral and a cell-mediated approach to the induction of cartilage formation in joints by transfer of growth factor genes. METHODS: Adenoviral vectors carrying insulin-like growth factor 1 (IGF-1) or bone morphogenetic protein 2 (BMP-2) complementary DNA were constructed and applied to primary human and murine chondrocytes or fibroblasts. Transgene expression was quantified by enzyme-linked immunosorbent assay. Direct injection of these vectors or AdLacZ, a reporter gene vector, into mouse knee joints was compared with the transplantation of syngeneic fibroblasts (infected ex vivo with the same vectors) with respect to virus spread, immune response, and cartilage formation by use of histologic, immunohistochemical, and molecular analyses. RESULTS: AdIGF-1 and AdBMP-2 efficiently infected all cell types tested. Human cells secreted biologically relevant levels of protein over a period of at least 28 days. Direct transfer of AdLacZ into mouse knee joints resulted in positively stained synovial tissues, whereas AdLacZ-infected fibroblasts settled on the surface of the synovial membranes. Inadvertent spread of vector DNA into the liver, lung, and spleen was identified by nested polymerase chain reaction in all mice that had received the vector directly; this rarely occurred following fibroblast-mediated gene transfer. Direct injection of AdBMP-2 induced the synthesis of new cartilage in periarticular mesenchyme, accompanied by extensive osteophyte formation. When AdBMP-2 was administered by injecting ex vivo-infected fibroblasts, cartilage formation was observed only in regions near the injected cells. AdIGF-1 treatment did not lead to morphologic changes. Importantly, fibroblast-mediated gene transfer avoided the strong immune response to adenovirus that was elicited following direct application of the vector. CONCLUSION: Our results indicate that cell-mediated gene transfer provides sufficient BMP-2 levels in the joint to induce cartilage formation while avoiding inadvertent vector spread and immune reactions.