Transcription In Chondrocytes Research Articles

The chick type III collagen gene contains two promoters that are preferentially expressed in different cell types and are separated by over 20 kb of DNA containing 23 exons.

Type III collagen is present in prechondrogenic mesenchyme, but not in cartilages formed during endochondral ossification. However, cultured chick chondrocytes contain an unusual transcript of the type III collagen gene in which exons 1-23 are replaced with a previously undescribed exon, 23A; this alternative transcript does not encode type III collagen. This observation suggested that, although production of type III collagen mRNA is repressed in chondrocytes, transcription of the type III collagen gene may continue from an alternative promoter. To test this prediction, we isolated and characterized both the upstream and internal promoters of this gene and tested their ability to direct transcription in chondrocytes and skin fibroblasts. The upstream promoter is active in fibroblasts, but inactive in chondrocytes, indicating that repression of type III collagen synthesis during chondrogenesis is transcriptionally mediated. Additionally, sequences in intron 23, preceding exon 23A, function as a highly active promoter in chondrocytes; transcription from this promoter is repressed in fibroblasts. Thus transcriptional control of the type III collagen gene is highly complex, with two promoters separated by at least 20 kb of DNA that are preferentially expressed in different cell types and give rise to RNAs with different structures and functions.

Cell-type specific promoters of the chick α2(I) collagen gene in chondrocytes and fibroblasts

Two different promoter regions responsible for the cell-type specific transcripts of the collagen alpha 2(I) gene in chick embryonic fibroblasts and chondrocytes are described. A region including part of intron 1 of the fibroblast alpha 2(I) gene is able to promote transcription in chondrocytes. This promoter region is located 5' to the chondrocyte-specific transcript, from -2205 to +42, relative to the chondrocyte transcription start site. When it is placed upstream of a luciferase reporter gene in transiently transfected chondrocytes, luciferase activity is stimulated 78-fold. The region between -2205 and -1298 is most important for the activity of the chondrocyte promoter, since a truncated promoter from -1298 through +42 gives only 15% as much activity. A similar low level of activity is given by the sequence -96 through +42. In transfected fibroblasts, a region from -1600 through +46, relative to the fibroblast transcription start site, stimulates transcription of a luciferase reporter gene by more than 1,000-fold. Deletions from -1185 through -807 and from -707 through -90 reduce promoter activity to 22% and 32%, respectively, of that given by the intact promoter. The chondrocyte and fibroblast promoters are each cell-type specific and are relatively inactive in the other cell type. Subregions within the fibroblast promoter, from -1154 through -1055, from -712 through -85, and the sequence containing the inverted CCAAT motif from -93 through -67, differentially bind factors in fibroblast but not chondrocyte nuclear extracts.

The control of expression of type II collagen: relevance to cartilage disease.

Cartilage is a unique tissue containing only one cell type, the chondrocyte, surrounded by an extensive extracellular matrix. One of the principal components of the cartilage matrix is type II collagen. The gene coding for type II collagen is relatively large and contains several distinct sequences that function to both up-regulate and down-regulate expression by interacting with chondrocyte transcription factors. Also, there appears to be regulation of collagen II expression by differential splicing of the collagen II mRNA to form different forms of the protein. Finally, the gene is a target for mutations that result in diseases of cartilage such as chondrodysplasias and some forms of osteoarthritis.

Two silencers regulate the tissue-specific expression of the collagen II gene.

Collagen II, the major component of cartilage, is synthesized primarily by chondrocytes and by certain cells in the eye. Previously, we have studied the regulatory regions of the collagen II gene by DNA transfection assays (Horton, W., Miyashita, T., Kohno, K., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8864-8868). These studies show that both the promoter and an enhancer sequence in the first intron are required for high transcriptional activity in chondrocytes. These elements do not show significant activity in cells which do not synthesize collagen II, such as in muscle cells and fibroblasts. In this report, we have constructed plasmids containing various deletions of the promoter of the collagen II gene, fused to a reporter gene for chloramphenicol acetyltransferase (CAT) and transfected them into both chick embryonic fibroblasts and HeLa cells. We have found that silencer elements in the collagen II promoter region reduce CAT activity 11-fold in fibroblasts, while not affecting the enhancer-mediated transcription in chondrocytes. Deletions in the promoter showed that most of the silencing activity was localized in two sites, between -360 and -460 base pairs and between -620 and -700 base pairs. Furthermore, a fragment containing these two sequences in a thymidine kinase promoter CAT construct reduced the activity of the promoter in an orientation independent fashion. Sequence analysis revealed that the two silencer regions are homologous and contain consensus motifs for silencer elements found in other genes. Gel retardation experiments showed that nuclear factors from HeLa cells bind specifically to a DNA fragment containing the silencer, whereas chondrocyte nuclear extracts did not show any activity. Thus, our study indicates that the expression of the collagen II gene is controlled by both negative and positive elements to ensure that the gene is only expressed in suitable cells.

Retinoic acid rapidly reduces cartilage matrix synthesis by altering gene transcription in chondrocytes

Retinoic acid can alter the differentiation of a variety of cell types, including chondrocytes. This action may explain the high incidence of craniofacial and limb defects resulting from exposure to retinoic acid during development, and may be the basis for the compound's inhibition of a chondrosarcoma tumor in vivo. In order to understand the mechanism of action of retinoic acid, we studied the expression of chondrocyte-specific proteins as well as other proteins that indicate a shift in the differentiated phenotype of the cell following exposure to retinoic acid. After 48 hr of exposure to retinoic acid chondrocytes stopped synthesizing the chondrocyte-specific pro α1 (II) chain of collagen II and a 370-kDa precursor protein of a cartilage-specific proteoglycan. Instead, the cells synthesized increased amounts of fibronectin and the pro α1 chain of collagen III. These changes could be detected as early as 12 hr after treatment. In addition, the steady-state levels of specific mRNA transcripts coding for these differentiation markers correlated with their protein synthesis levels. Also, nuclear runoff experiments indicated that retinoic acid down regulated transcription of the collagen II gene, while stimulating collagen III gene transcription. These observations suggest that retinoic acid may alter the expression of the chondrocyte phenotype by selectively changing the normal pattern of gene expression.