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

Abstract

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). ...

Hyaluronan oligosaccharide–induced activation of transcription factors in bovine articular chondrocytes

“…Type II collagen and COMP have several Sp-1 sites in their promoter regions, and Sp-1 specifically activates type II collagen and COMP synthesis (23,24). In RT-PCR analysis, the gene expressions of type II collagen and COMP were up-regulated by the treatment with HA 6 .…”

“…OHNO ET AL have several Sp-1 binding sites, and overexpression of Sp-1 has been shown to stimulate type II collagen and COMP protein synthesis (23,24). The addition of 250 g/ml of HA 6 to bovine articular chondrocytes increased the expression of both type II collagen and COMP mRNA by 2.5-fold at 24 hours and by ϳ4-fold at 48 hours ( Figures 4C and D, respectively) as compared with untreated control chondrocytes.…”

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

“…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 was found to activate COL2A1 transcription through the promoter binding sites, whatever the differentiation state of chondrocytes, suggesting that this factor may be capable of restoring the altered chondrocyte phenotype (8).…”

“…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)(3)(4)(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).…”

“…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)(3)(4)(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).…”