Detection and Characterization of Sp1 Binding Activity in Human Chondrocytes and Its Alterations during Chondrocyte Dedifferentiation

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). ...

Section: Discussioncontrasting

confidence: 56%

mentioning

confidence: 50%

mentioning

confidence: 98%

See 1 more Smart Citation

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

Ghayor¹,

Chadjichristos²,

Herrouin³

et al. 2001

127

“…One of interesting findings of this study is that c-Jun/AP-1 signaling appears to mediate dedifferentiation of chondrocytes caused by IL-1␤, RA, or EGF, whereas dedifferentiation of chondrocytes caused by the serial monolayer subculture on plastic was found to be independent of c-Jun/AP-1. Indeed, it has been suggested that activation of the Sp-1 transcription factor is associated with subculture-induced dedifferentiation of chondrocytes (41), suggesting that Sp-1 and AP-1 may regulate different pathways of chondrocyte dedifferentiation.…”

Section: C-jun In Chondrocyte Dedifferentiationmentioning

confidence: 99%

c-Jun/Activator Protein-1 Mediates Interleukin-1β-induced Dedifferentiation but Not Cyclooxygenase-2 Expression in Articular Chondrocytes

Hwang

Poo

et al. 2005

“…Silencer elements are located in the regions Ϫ300 to Ϫ400 and Ϫ620 to Ϫ700 bp 1 that are believed to function in inhibiting synthesis of type II collagen in nonchondrocytes (16). In addition, Sp1 binding sites in the proximal promoter are believed to be important for efficient enhancer-mediated transcription (17) and Sp1 binding activity differs between differentiated and dedifferentiated chondrocytes (18). Although it has been reported that a fragment from the first intron enhancer can direct chondrocyte-specific expression through a heterologous promoter (12), it is likely that in vivo, the endogenous Col2a1 promoter also participates in gene regulation during certain developmental stages and in response to specific regulatory signals.…”

mentioning

confidence: 99%

The Transcription Factor δEF1 Is Inversely Expressed with Type II Collagen mRNA and Can Repress Col2a1 Promoter Activity in Transfected Chondrocytes

Murray¹,

Precht²,

Balakir³

et al. 2000

The regulation of Col2a1, which encodes type II collagen, likely results from a balance of both positive and negative proteins. Here we present evidence that the transcription factor ␦EF1 participates in the negative regulation of Col2a1 transcription. A deletion analysis suggested that a region between ؊100 and ؊307 of the rat Col2a1 gene was required for activity in differentiating chick limb bud mesenchymal cells; however, mutation of a conserved E2 box site in this region actually increased promoter activity. Supershift analysis demonstrated that ␦EF1, a known transcriptional repressor, bound to the E2 box in a sequence-dependent manner. Chick limb bud mesenchymal cells, which do not express type II collagen, expressed abundant ␦EF1 mRNA, but, following differentiation in micromass culture, ␦EF1 mRNA expression was lost. Primary embryonic chick sternal chondrocytes, which express abundant type II collagen, displayed minimal levels of ␦EF1 mRNA. The inhibition of Col2a1 transcription following treatment of chick sternal chondrocytes with growth factors was accompanied by increased ␦EF1 expression. Overexpression of ␦EF1 in differentiated chondrocytes resulted in decreased expression of a reporter construct containing a collagen II promoter/enhancer insert; however, this negative regulation was not dependent on the proximal E2 box. This is the first report of a specific transcription factor involved in the negative regulation of Col2a1.Type II collagen, a major structural protein of the cartilage extracellular matrix, is encoded by the Col2a1 gene (1, 2), which is transcribed at a high level in chondrocytes (3-6). In addition to the activation of Col2a1 transcription during chondrogenesis, a variety of factors have been reported to either stimulate or inhibit type II collagen synthesis in differentiated chondrocytes, such as specific growth factors (7, 8) and retinoic acid (9). The developmental pattern of expression of the Col2a1 gene and the fact that its transcription rate is subject to modulation by many factors suggest that the steady-state expression of this gene results from a balance between positive and negative regulatory proteins interacting with several different cis-acting DNA sequences.