Collagen II Promoter and Enhancer Interact Synergistically Through Sp1 and Distinct Nuclear Factors

120

Interleukin-1␤ (IL-1␤) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1␤ inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1␤ down-regulates COL2A1 gene transcription through a ؊41/؊33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1␤ effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type ؊50/؉1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1␤ inhibition of a ؊63/؉47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the ؊41/؊33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the ؊63/؉1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1␤ down-regulation of that gene, as we found previously for transforming growth factor-␤1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1␤ increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1␤ decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation.Articular cartilage is a highly specialized tissue composed of a complex extracellular matrix of proteoglycans, collagens, and noncollagenous glycoproteins. Cartilage collagens include type II as the major form and types VI, IX, and XI as minor components (1). Type II collagen is an homotrimer composed of ␣1(II) chains encoded by the COL2A1 gene. Previous studies have delineated minimal sequences in the first intron of human, mouse, and rat COL2A1 genes which are sufficient to direct chondrocyte-specific expression in cultured chondrocytes and transgenic mice (2-5). Several binding sites of the intronic enhancer sequences were shown to interact with transcription factors that form chondrocyte-specific complexes, such as SOX9, L-SOX5, and SOX6 (6, 7), and also with factors having less tissue-specific expression, such as Sp1, Sp3, and C-KROX (5, 8). Indeed, promoter sequences are also implicated through interaction with the intronic enhancer sequence, for tissuespecific expression during in vivo and in vitro chondrogenesis (7, 9, 10). In a 266-bp promoter of the human COL2A1 gene mediating enhanced transcription activity, we identified several binding sites for Sp1, Sp3, and C-KROX (5, 8, 11). Sp1 w...

Section: Sp3/sp1 Ratio Mediates Il-1␤ Inhibition Of Col2a1 Genementioning

confidence: 99%

Section: Sp3/sp1 Ratio Mediates Il-1␤ Inhibition Of Col2a1 Genementioning

confidence: 99%

mentioning

confidence: 99%

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Sp1 and Sp3 Transcription Factors Mediate Interleukin-1β Down-regulation of Human Type II Collagen Gene Expression in Articular Chondrocytes

Chadjichristos¹,

Ghayor²,

Kypriotou³

et al. 2003

120

“…However the immunoglobulin , collagen I, collagen II, ␤-globin, CD13, and gb110 genes contain more distal, functional Sp1 sites (41)(42)(43)(44)(45)(46). We anticipate that further characterization of the myeloid protease enhancer and its interaction with the NE and PR-3 promoters in vivo will contribute to our understanding of early myeloid differentiation and of transcriptional mechanisms which enable coordinate gene activation.…”

Section: Fig 4 Enhancer Segment C1 Binds Sp1 and Ets Factorsmentioning

confidence: 99%

An Enhancer Located between the Neutrophil Elastase and Proteinase 3 Promoters Is Activated by Sp1 and an Ets Factor

Nuchprayoon

Shang

Simkevich

et al. 1999

The adjacent neutrophil elastase, proteinase 3, and azurocidin genes encode serine proteases expressed specifically in immature myeloid cells. Subclones of a 17-kilobase (kb) murine neutrophil elastase genomic clone were assessed for their ability to stimulate the neutrophil elastase promoter in 32D cl3 myeloid cells. Region ؊9.3 to ؊7.3 kb stimulated transcription 7-fold, whereas other genomic segments were inactive. This enhancer is located in the second intron of the proteinase-3 gene and so may regulate more than one gene in the myeloid protease cluster. Deletional analysis of the enhancer identified several segments which activated the neutrophil elastase and thymidine kinase promoters 3-6-fold. The most active segment was a 220-base pair region centered at ؊8.6 kb, which activated transcription 31-fold. This segment contains an Sp1 consensus site, which bound Sp1, flanked by two Ets family consensus sequences, which bound PU.1, GABP, and an Ets factor present in myeloid cell extracts. Mutation of the Sp1-binding site reduced enhancer activity 8-fold in 32D cl3 cells, and mutation of either or both Ets-binding sites reduced activity 3-4-fold. Sp1 activated the distal enhancer 5-fold, GABP 3-fold, and the combination 8-fold in Schneider cells.To identify transcriptional events that determine myeloid cell determination, we have been investigating the regulation of the neutrophil elastase (NE) 1 gene. NE is a microbicidal serine protease present in the primary granules of granulocytes and monocytes (1). In bone marrow, NE mRNA is present mainly in promyelocytes (2), and transcriptional regulation plays a key role in its lineage-specific expression (3). We isolated a 17-kb genomic murine NE clone and found that, like the human NE gene, the murine gene contains five exons and initiates transcription 30 bp downstream from a TATAA homology (4). We demonstrated that the 5Ј-flanking region of the NE gene contains a 60-bp proximal enhancer, located just upstream of the TATAA homology, which is strongly active in 32D cl3 myeloid cells differentiating in response to granulocyte-colony-stimulating factor (G-CSF), but only weakly active in uninduced 32D cl3 cells or in fibroblasts (4). This proximal enhancer is activated cooperatively by C/EBP, c-Myb, CBF, and Ets family members such as PU.1 or GABP (4 -6). The NE gene is located just downstream of the proteinase-3 (PR-3) gene, which in turn is located just downstream of the azurocidin gene (7). These three serine protease genes are expressed specifically and coordinately in immature myeloid cells (7), and the promoter of each contains conserved binding sites for C/EBP, c-Myb, and PU.1 (7,8).To determine whether the murine NE gene contains additional regulatory regions, we assessed the activity of NE genomic subclones functionally after transient transfection into 32D cl3 cells. A 2-kb NE genomic region extending from Ϫ7.3 to Ϫ9.3 kb stimulated the NE promoter 7-fold, and stimulated the herpes TK promoter as well, whereas none of the other subclones, from Ϫ7.3 to ϩ7.5 kb...

“…Moreover, Sp1, by interacting with itself when bound on separate Sp1-DNA-binding sites, can induce loop formation between distant regions of a gene (39,40). In the case of the COL2A1 gene, Sp1 and a Sp1-like factor called CIIZFP are involved in the cartilage-specific expression of rat collagen type II, through a looping mechanism between the promoter and the enhancer regions (41). Additionally, an Sp1 binding activity was found in nuclear extracts from chondrocytes and Sp1-DNA binding was shown to increase as chondrocytes were allowed to de-differentiate in vitro (42).…”

mentioning

confidence: 99%

SP3 Represses the SP1-mediated Transactivation of the HumanCOL2A1 Gene in Primary and De-differentiated Chondrocytes

Ghayor¹,

Chadjichristos²,

Herrouin³

et al. 2001

127

Sp1 and Sp3 effects on the transcription of the human ␣1(II) procollagen gene (COL2A1) were investigated in both differentiated and de-differentiated rabbit articular chondrocytes. Transient transfection with constructs of deleted COL2A1 promoter sequences driving the luciferase reporter gene revealed that the region spanning ؊266 to ؉121 base pairs showed Sp1-enhancing effects, whatever the differentiation state. In contrast, Sp3 did not influence COL2A1 gene transcription. Concomitant overexpression of the two Sp proteins demonstrated that Sp3 blocked the Sp1 induction of COL2A1 promoter activity. Moreover, inhibition of Sp1/ Sp3 binding to their target DNA sequence decreased both COL2A1 gene transcription and Sp1-enhancing effects. DNase I footprinting and gel retardation assays revealed that Sp1 and Sp3 bind specifically to cis-sequences of the COL2A1 gene promoter whereby they exert their transcriptional effects. Sp1 and Sp3 levels were found to be reduced in de-differentiated chondrocytes, as revealed by DNA-binding and immunochemical study. Sp1 specifically activated collagen neosynthesis whatever the differentiation state of chondrocytes, suggesting that this factor exerts a major role in the expression of collagen type II. However, our data indicate that type II collagen-specific expression in chondrocytes depend on both the Sp1/Sp3 ratio and cooperation of Sp1 with other transcription factors, the amounts of which are also modulated by phenotype alteration.Differentiation of mesenchymal cells into chondrocytes results in the synthesis and secretion of a series of proteins characteristic of the cartilage matrix, including type II, IX, XI, and X collagens, the proteoglycan aggrecan, link protein, and cartilage matrix protein (1, 2). Type II collagen is considered as a critical phenotypic marker gene for analysis of molecular events involved in chondrogenesis process as well as in chondrocyte phenotype maintenance. Alteration of type II collagen expression in cartilage may be due to a variety of genetic, inflammatory, or degenerative circumstances and may lead to a variety of chondrodysplasias and joint diseases such as osteoarthritis (3-8). In osteoarthritis, chondrocytes undergo dedifferentiation and synthesize types I and III collagens at the expense of type II (9 -11). Similarly, when chondrocytes are subcultured in vitro as monolayers, they progressively reduce their synthesis of type II collagen (12-14), mimicking the behavior of osteoarthritic chondrocytes. However, they can recover the chondrocytic phenotype by transfer to three-dimensional culture systems (14 -16). Therefore, in vitro analysis of the molecular mechanisms that regulate COL2A1 gene expression can be an approach to understand the process of phenotype alteration in chondrocytes, and its impact on joint diseases.A 48-bp 1 minimal DNA element has been identified as an enhancer that directs chondrocyte-specific expression of the COL2A1 gene in transgenic mice (17)(18)(19). Such an element was also found in the rat COL2A1 gene (20, 21). ...