ABSTRACT
OBJECTIVE: Emilin-1 is a versatile protein abundant in tissues where resilience and elastic recoil are prominent and interacting with components of the extracellular matrix. Still, little is known about Emilin-1 in the skin. Therefore, we investigated Emilin-1 in the skin, its localization, its fate upon ageing, its interactions with other proteins and the effect of its knockdown. METHODS: Skin explants from young or old Caucasian women, immunofluorescently labelled by anti-Emilin-1, anti-Fibrillin-1 and anti-Elastin antibodies, were analysed using confocal microscopy. Skin explants subjected to UV-induced skin ageing were also analysed. Colocalization of Emilin-1 with Collagen IV, Fibrillin-1 and Elastin was studied by multiphoton microscopy and co-immunoprecipitation. Finally, the effect of Emilin-1 extinction was studied by producing small interfering RNA (siRNA) knockdown fibroblasts and by analysing the outcome on selected genes. RESULTS: In skin sections from young donors, Emilin-1 localizes similarly to Elastin and Fibrillin-1. In the papillary dermis, it shows clear and ramified structures, perpendicular to the dermo-epidermal junction that are reminiscent of the oxytalan fibres. In the reticular dermis, Emilin-1 signal appears identical to that of the elastic fibres network. Upon intrinsic or UV-induced ageing, the signal associated with Emilin-1 is drastically reduced and disorganized. Multiphoton microscopy study shows that, as expected, Emilin-1 colocalizes with Elastin. It also colocalizes with Collagen IV in the basement membrane and within dermal fibroblasts. Interaction of Emilin-1 with Elastin and Collagen IV was also found by co-immunoprecipitation. It also reveals interaction with Laminin-5. Finally, siRNA-mediated knockdown of EMILIN-1 show little effect on the expression level of the 61 genes we studied. The most striking change is a downregulation of fibroblast growth factor receptor 2 that show a decrease similar to that of EMILIN-1 itself and after 8 days a downregulation of COL6A1. CONCLUSION: In skin, Emilin-1 locates in the dermis, up to the basement membrane, interacting with components of the extracellular matrix but also with the anchoring complex. These interactions are important for cell adhesion, migration, proliferation and would suggest that Emilin-1 might be important for maintaining the 3D structure of the extracellular matrix.
OBJECTIF: Emilin-1 est une protéine polyvalente, abondante dans les tissus où résilience et élasticité sont importantes et qui interagit avec la matrice extracellulaire. Pourtant, la protéine Emilin-1 a été peu étudiée dans la peau. Nous avons donc étudié sa localisation, son devenir lors du vieillissement, ses interactions avec d'autres protéines et l'effet de son inhibition dans la peau. MÉTHODES: Des explants de peau de femmes caucasiennes jeunes ou âgées, marqués par immunofluorescence avec des anticorps anti-Emilin-1, anti-Fibrilline-1 et anti-Élastine, ont été analysés par microscopie confocale. Des explants cutanés soumis au soumis aux UV pour mimer un photo-vieillissement ont également été analysés. La co-localisation de l'Emilin-1 avec le collagène IV, la fribrilline-1 et l'élastine a été étudiée par microscopie multiphotonique et par co-immunoprécipitation. Enfin, l'effet de l'inhibition de l'expression de la protéine Emilin-1 par interférence ARN a été étudié sur 61 gènes. RÉSULTATS: Dans les coupes de peau de jeunes donneurs, Emilin-1 est localisée comme l'élastine et la fibrilline-1. Dans le derme papillaire, elle présente des structures claires et ramifiées, perpendiculaires à la jonction dermo-épidermique, qui font penser aux fibres d'oxytalane. Dans le derme réticulaire, le signal de l'Emilin-1 apparaît identique à celui du réseau de fibres élastiques. Lors du vieillissement intrinsèque ou induit par les UV, le signal associé à Emilin-1 est considérablement réduit et désorganisé. Une étude en microscopie multiphotonique montre que l'Emilin-1 est co-localisée avec de l'élastine. Elle est aussi co-localisée avec le collagène IV dans la membrane basale et dans les fibroblastes du derme. La co-immunoprécipitation montre l'existence d'interactions entre l'Emilin-1 et l'Elastine ou le collagène IV. Une interaction avec la laminine-5 a aussi été mise en évidence. Enfin, l'inhibition de l'expression de l'EMILIN-1 n'a que peu d'effet sur l'expression des 61 gènes étudiés. Le changement le plus frappant est une diminution de l'expression de FGFR2 (récepteur 2 du facteur de croissance des fibroblastes), diminution similaire à celle d'EMILIN-1, et, après 8 jours, une diminution de l'expression de COL6A1. CONCLUSION: Dans la peau, Emilin-1 se localise dans le derme, jusqu'à la membrane basale, et interagit avec les composants de la matrice extracellulaire mais aussi avec les complexes d'ancrage. Ces interactions sont importantes pour l'adhésion, la migration, la prolifération cellulaire et suggéreraient qu'Emilin-1 pourrait être important pour le maintien de la structure de la matrice extracellulaire.
Subject(s)
Membrane Glycoproteins/metabolism , Skin/metabolism , Adult , Aged , Extracellular Matrix Proteins/metabolism , Female , Humans , Membrane Glycoproteins/genetics , Middle Aged , Protein BindingABSTRACT
AIM OF THE STUDY: Cartilage has a limited capacity for healing after trauma. Autologous chondrocyte implantation is widely used for the treatment of patients with focal damage to articular cartilage. Chondrocytes are isolated from biopsy specimen, cultured in monolayers on plastic then transplanted over the cartilage defect. However, chondrocyte amplification on plastic triggers their dedifferentiation. This phenomenon is characterized by loss of expression of type II collagen, the most abundant cartilage protein. The challenge for autologous chondrocyte implantation is to provide patients with well-differentiated cells. The aim of the present study was to test the capability of bone morphogenetic protein (BMP)-2 to promote redifferentiation of human chondrocytes after their expansion on plastic. MATERIALS AND METHODS: Chondrocytes extracted from nasal cartilage obtained after septoplasty were serially cultured in monolayers. After one, two or three passages, BMP-2 was added to the culture medium. The cellular phenotype was characterized at the gene level by using RT-PCR. The expression of genes coding for type II procollagen with the ratio of IIB/IIA forms, aggrecan, Sox9, osteocalcin and type I procollagen was monitored. RESULTS: Our results show that BMP-2 can stimulate chondrogenic expression of the chondrocytes amplified on plastic, without inducing osteogenic expression. However, this stimulatory effect decreases with the number of passages. CONCLUSION: The efficiency of autologous chondrocyte implantation could be improved by using chondrocytes treated with BMP-2 during their in vitro preparation.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Chondrocytes/drug effects , Extracellular Matrix Proteins/biosynthesis , Adolescent , Adult , Aggrecans/biosynthesis , Aggrecans/genetics , Cell Dedifferentiation/drug effects , Cell- and Tissue-Based Therapy/methods , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Chondrocytes/cytology , Chondrocytes/metabolism , Collagen Type II/biosynthesis , Collagen Type II/genetics , Extracellular Matrix Proteins/genetics , Female , Gene Expression Regulation/drug effects , Humans , Male , Middle Aged , Osteocalcin/biosynthesis , Osteocalcin/genetics , Procollagen/biosynthesis , Procollagen/genetics , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor/biosynthesis , SOX9 Transcription Factor/genetics , Young AdultABSTRACT
OBJECTIVES: To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. METHODS: Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). RESULTS: 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. CONCLUSIONS: 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.
Subject(s)
Chondrocytes/drug effects , Estradiol/pharmacology , Uridine Diphosphate Glucose Dehydrogenase/drug effects , Uridine Diphosphate Glucose Dehydrogenase/metabolism , Animals , Animals, Newborn , Blotting, Western , Cartilage, Articular/cytology , Cells, Cultured , Chondrocytes/metabolism , Cytokines/pharmacology , Disease Models, Animal , Gene Expression Regulation , Male , RNA, Messenger/analysis , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Transforming Growth Factor beta/pharmacology , Up-Regulation , Uridine Diphosphate Glucose Dehydrogenase/geneticsABSTRACT
We previously showed that interleukin-1beta (IL-1beta) down-regulation of type II TGFbeta receptor (TbetaRII) involves NFkappaB pathway and requires de novo synthesis of a yet unknown protein. Here, we demonstrate that this effect is mediated through Sp1 site located at position -25 of human TbetaRII promoter. Inhibition of transcription factors binding (decoy oligonucleotides or mithramycin) abolished IL-1beta effect. EMSA and ChIP revealed that this treatment induced Sp3 binding to cis-sequence whereby IL-1beta exerts its transcriptional effects whereas it decreased that of Sp1. Moreover, although the cytokine did not modulate Sp1 expression, it increased that of Sp3 via NFkappaB pathway. Experiments of gain and loss of function clearly showed that Sp3 inhibited TbetaRII expression whereas its silencing abolished IL-1beta effect. In addition, both Sp1 and Sp3 were found to interact with NFkappaB, which therefore may indirectly interact with TbetaRII pro moter. Altogether, these data suggest that IL-1beta decreases TbetaRII expression by inducing Sp3 via NFkappaB and its binding on core promote at the expense of Sp1, which could explain the loss of cell responsiveness in certain conditions. These findings bring new insights in the knowledge of the interference between two antagonistic transduction pathways involved in multiple physiopathological processes.
