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Embryonic chick cartilages contain transcripts derived from the ␣2(I) collagen gene, although type I collagen is not normally found in these tissues; most of these RNAs are alternative transcripts initiating within intron 2. Use of the internal start site results in replacement of exons 1 and 2 with a previously undescribed exon and a change in the translational reading frame; thus, the alternative transcript cannot encode ␣2(I) collagen. We have demonstrated that production of the alternative transcript is due to activation of an internal promoter in chondrocytes and have identified a 179-base pair domain that is required for its activity. Furthermore, we have shown that the alternative transcript resulting from activation of the internal promoter turns over relatively rapidly; thus, the steady-state level of this transcript is less than predicted based on the transcription rate. The upstream promoter is only partially repressed in chondrocytes, suggesting that the lack of authentic ␣2(I) collagen mRNA may also be due in part to decreased mRNA stability. Thus, repression of ␣2(I) collagen synthesis in cartilage involves both transcriptional and post-transcriptional mechanisms. In contrast, repression of ␣1(I) collagen synthesis appears to be mediated primarily at the level of transcription.Normal skeletal development requires precisely regulated expression of the genes encoding type I collagen, the major collagen produced by both prechondrogenic mesenchymal cells and osteoblasts. As mesenchymal cells differentiate into cartilage-producing chondrocytes, they stop producing type I collagen and initiate synthesis of several cartilage-specific collagens (reviewed in Refs. 1 and 2). Type I collagen is a heterotrimer containing two ␣1(I) and one ␣2(I) subunits. In cells and tissues that produce type I collagen, the genes encoding these subunits are coordinately regulated (3-6). We previously identified an unusual molecular mechanism that mediates the cessation of ␣2(I) collagen production in cartilage. Embryonic chick chondrocytes contain transcripts derived from the ␣2(I) collagen gene (7), despite the fact that these cells do not synthesize type I collagen. These transcripts initiate at an internal start site within intron 2 (8, 9), rather than at the previously identified site at the beginning of exon 1 (10) (Fig. 1A). Use of this internal start site results in replacement of exons 1 and 2 with a previously undescribed exon (exon A) and a change in the translational reading frame; this unusual RNA cannot encode ␣2(I) collagen, since the potential open reading frames are out of frame with the collagen coding sequence. Hereafter we will refer to the transcript initiating at the internal start site as the alternative transcript, in contrast to the authentic ␣2(I) collagen mRNA, which initiates at the beginning of exon 1 and encodes the ␣2 subunit of type I collagen.We initially predicted (9) that a developmentally programmed change from the upstream promoter to the presumptive internal promoter for transcription of t...

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During endochondral ossification, small flat resting and proliferating chondrocytes mature into large round hypertrophic chondrocytes that synthesize a unique collagen, type X. We have asked whether this change in cell shape during chondrocyte maturation regulates type X collagen gene expression, using immature chick vertebral chondrocytes grown in monolayer or in suspension. The freshly isolated chondrocytes contained no type X collagen RNA, but after 30 days of culture, both attached and suspended cells contained a similar large amount. However, in cells that were grown in monolayer and then resuspended three days before harvest, type X collagen gene expression increased a further 6 fold. These results suggest that the change from a flat to a round shape that occurs during chondrocyte maturation in vivo may be important for maximal expression of the type X collagen gene.

Abstract.Chicken vertebral chondrocytes, which normally grow in suspension, synthesize large amounts of cartilage extracellular matrix proteins, but little fibronectin. We have analyzed the effects of both substrate attachment and transformation with a temperaturesensitive mutant of Rous sarcoma virus on fibronectin gene expression in these cells. Our experiments show that viral transformation increases fibronectin synthesis to a greater extent than substrate attachment. Furthermore, transformed chondrocytes have lost the ability to decrease fibronectin synthesis in response to suspension culture, suggesting that transformation alters the normal attachment-responsive control of fibronectin gene expression. Finally, infected substrate-attached chondrocytes shifted to the nonpermissive temperature for transformation use fibronectin RNA more efficiently in protein synthesis than cells grown under the other conditions, suggesting for the first time a role for translational control of fibronectin gene expression.F IB RON E CTI N, a large glycoprotein that is a component of the surface and extracellular matrix of many types of cells, plays an important role in adhesion and maintenance of morphology. The functions of fibronectin and the control of its synthesis and accumulation have been examined most extensively in cultured fibroblasts, which are anchorage dependent, grow in monolayer, and synthesize large amounts of fibronectin (reviewed in references 23 and 46). Additional information has been obtained, however, by examination of cultured chondrocytes (cartilage-producing cells). These cells are anchorage independent and grow equally well in suspension as round, refractile, isolated cells and, attached to the substrate as polygonal cells. Under both conditions they synthesize large amounts of the cartilage extracellular matrix components, primarily type II collagen (reviewed in reference 41) and chondroitin sulfate proteoglycan (13, 31, 33). They differ, however, in that the substrateattached cells synthesize more fibronectin (9, 12) and accumulate more fibronectin on the cell surface (42) than the cells grown in suspension.With time in culture, substrate-attached chondrocytes often lose the ability to synthesize cartilage matrix proteins (reviewed in reference 41). Furthermore, transformation by Rous sarcoma virus, as well as treatment with the tumor promoter PMA, bromodeoxyuridine, retinoic acid, embryo extract and fibronectin (reviewed in reference 41), interferes with expression of the normal chondrocyte phenotype. These agents reportedly have vastly different effects on fibronectin synthesis; for example, synthesis is increased by viral transformation (1,3,20,47) and PMA (18,19), while retinoic acid has little effect (21,22). However, interpretation of those experiments is complicated by the fact that in some laboratories normal (untreated) chondrocytes were grown in suspension (1,3,18,19) and synthesized little or no fibronectin, while in others they were grown in monolayer (20-22) and synthesized a large amou...