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

Abstract

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.

Transcription factors related to chondrogenesis in pleomorphic adenoma of the salivary gland: a mechanism of mesenchymal tissue formation

“…[18][19][20][21][22] Among these, δEF1, AP-2α, and Twist1 are expressed by chondrocyte progenitors and maintain their undifferentiated state. 18,20,21,23 Their expression in the salivary gland was therefore compared with their expression in pleomorphic adenoma. The mRNAs of δEF1 and AP-2α were detected in both salivary glands and pleomorphic adenomas.…”

“…17 Negative regulators include δEF1, AP-2α, Snail, Slug, Twist1, and C/EBPβ. [18][19][20][21][22] Snail, Slug, and C/EBPβ are expressed by hypertrophic chondrocytes, which indicates that they control terminal chondrocyte differentiation and promote endochondral ossification; 11,19,22 δEF1, AP-2α, and Twist1 are expressed by chondroprogenitors, which indicates that they control early-phase chondrogenesis. 18,20,21,23 The aim of our study was to clarify the mechanisms behind chondrogenesis in pleomorphic adenoma epithelial cells.…”

Mouse Snail Family Transcription Repressors Regulate Chondrocyte, Extracellular Matrix, Type II Collagen, and Aggrecan

“…The strong de-suppressive effect of disrupton of E-box 2 and 4 indicated the importance of these two conserved sites. It has been reported that mutation in E-box 4 de-suppressed the activity of the reporter construct containing Ϫ307 to ϩ110 of Col2a1 in chicken limb mesenchyme cells and transregulatory factors that bind to this site remained unknown (13). Therefore to study the mechanism, we focused on E-box 4 located at Ϫ167 to Ϫ162.…”

“…Reporter analysis using the Col2a1 promoter revealed that Snail and Slug suppress the activity. We found that E-boxes, which are binding motifs for the Snail family, are involved in the negative regulation of Col2a1 transcription (13). Reporter analysis showed Snail suppressed the transcriptional activity of Col2a1 through the Eboxes.…”

“…Two elements for the regulation of Col2a1 transcription are known; one is the enhancer located within the first intron that mediates cartilage-specific expression of Col2a1, and the other is in the promoter region that has both silencers and promoter elements (8 -12). One of the E-boxes (CAGGTG) in the promoter region is involved in the negative regulation of Col2a1, but the transcription factors that bind to that E-box remain unknown (13).…”