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During endochondral bone formation, chondrocytes undergo terminal differentiation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of collagen X, as well as proteins required for matrix mineralization. This maturation process is responsible for most longitudinal bone growth, both during embryonic development and in postnatal long bone growth plates. Among the major signaling molecules implicated in regulation of this process are the positive regulators thyroid hormone (T3) and bone morphogenetic proteins (BMPs). Both T3 and BMPs are essential for endochondral bone formation and cannot compensate for each other, suggesting interaction of the two signaling pathways. We have analyzed the temporal and spatial expression patterns of numerous genes believed to play a role in chondrocyte maturation. Our results show that T3 stimulates collagen X gene expression in cultured chondrocytres with kinetics and magnitude similar to those observed in vivo. Stimulation of collagen X gene expression by T3 occurs only after a significant delay, implying that this hormone may act indirectly. We show further that T3 rapidly stimulates production of BMP 4, concomitant with a decrease in the BMP inhibitor Noggin, potentially resulting in a net increase in BMP signaling. Finally, inhibition of BMP signaling with exogenous Noggin prevents T3 stimulation of collagen X expression, indicating that BMP signaling is essential for this process. These data position thyroid hormone at the top of a T3/BMP cascade, potentially explaining why both pathways are essential for chondrocyte maturation. J. Cell. Physiol. 219: 595-605, 2009. (c) 2009 Wiley-Liss, Inc.

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...

Retinoids are essential for the terminal differentiation of chondrocytes during endochondral bone formation. This maturation process is characterized by increased cell size, expression of a unique extracellular matrix protein, collagen X, and eventually by mineralization of the matrix. Retinoids stimulate chondrocyte maturation in cultured cells and experimental animals, as well as in clinical studies of synthetic retinoids; furthermore, retinoid antagonists prevent chondrocyte maturation in vivo. However, the mechanisms by which retinoids regulate this process are poorly understood. We and others showed previously that retinoic acid (RA) stimulates expression of genes encoding bone morphogenetic proteins (BMPs), suggesting that retinoid effects on chondrocyte maturation may be indirect. However, we now show that RA also directly stimulates transcription of the collagen X gene promoter. We have identified three RA response element (RARE) half-sites in the promoter, located 2,600 nucleotides upstream from the transcription start site. These three half-sites function as two overlapping RAREs that share the middle half-site. Ablation of the middle half-site destroys both elements, abolishing RA receptor (RAR) binding and drastically decreasing RA stimulation of transcription. Ablation of each of the other two half-sites destroys only one RARE, resulting in an intermediate level of RAR binding and transcriptional stimulation. These results, together with our previously published data, indicate that retinoids stimulate collagen X transcription both directly, through activation of RARs, and indirectly, through increased BMP production.

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.

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Endochondral bone formation is characterized by several transitions in the pattern of collagen gene expression, the best characterized of which occurs during chondrogenesis. Prechondrogenic mesenchymal cells synthesize predominantly type I collagen; during chondrogenesis, type I collagen synthesis ceases and production of cartilage‐characteristic collagens is initiated. We previously identified the molecular mechanism that mediates cessation of α2(I) collagen synthesis in chondrocytes (Bennett and Adams [1990] J. Biol. Chem. 265:2223–2230). This mechanism involves a change in the transcription initiation site, resulting in an alternative transcript that cannot encode α2(I) collagen. In this report we demonstrate that the alternative transcript appears only transiently in cartilage. Its initial appearance is coincident with the onset of high levels of type II collagen synthesis in differentiated chondrocytes. However, it disappears in hypertrophic cartilage, and production of the authentic α2(I) collagen mRNA is reinitiated, contributing to synthesis of a high level of type I collagen in hypertrophic chondrocytes at the chondro‐osseous junction. We also show that the alternative transcript is not restricted to cartilage during embryonic development, since it initially appears in presomite embryos, well before the appearance of cartilage. At early stages of embryogenesis the alternative transcript is restricted to tissues derived from neuroectoderm; its appearance in those tissues is also transient. These data suggest that production of the alternative transcript of the α2(I) collagen gene may be required for cessation of α2(I) collagen synthesis during chondrogenesis, but the alternative transcript may be involved in other important developmental programs as well. © 1996 Wiley‐Liss, Inc.