Human COL2A1 Research Articles

A mutation-independent therapeutic strategem for osteogenesis imperfecta.

Given the genetically heterogeneous nature of many dominantly inherited disorders, it will be imperative to design mutation-independent therapeutic strategies to circumvent such heterogeneity. Intragenic polymorphism represents a genomic resource that may be harnessed in the development of allele-specific mutation-independent therapeutics. A hammerhead ribozyme, Rzpol1a1, selectively cleaves a common single-nucleotide polymorphism (SNP) of the human COL1A1 transcript (heterozygosity frequency of 2 pq = 0.4032, from Hardy-Weinberg equilibrium). One SNP variant contains a hammerhead ribozyme cleavage site, and the other does not. Kinetic evaluation shows Rzpol1a1 to be both specific and extremely efficient in vitro. Thus, a single efficient ribozyme has been characterized that should be valuable in the development of a gene therapy suitable for up to 1 in 5 dominant-negative osteogenesis imperfecta (OI) patients, where over 150 different mutations have been identified to date. Given the increasing characterization of intragenic SNP, it is predicted that such a mutation-independent strategy, based on selective silencing of mutant alleles at SNP, may become increasingly important in future genomics-driven drug development for many heterogeneous dominant disorders and complex traits.

Restoration of normal bone development by human homologue of collagen type II (COL2A1) gene in Col2a1 null mice.

Development of the vertebrate skeleton is a highly complex process in which collagen type II plays a vital role in the formation of long bones via endochondral ossification. Collagen type II, which is encoded by a single COL2A1/ Col2a1 gene, is the most abundant structural protein in the cartilage matrix, where it undergoes complex interactions with several other proteins. The sequence of mature collagen type II chains, each with about 1,100 amino acids, is conserved between different mammalian species. There are 37 amino acid positions that are different between mouse and human collagen type II. Previously, we have demonstrated that transgenic mice, in which Col2a1 gene is knocked out, exhibit a lethal phenotype due to the absence of endochondral bone formation. To investigate whether the biological role of collagen type II is conserved between the species, human COL2A1 gene was expressed in Col2a1 null mice by crossing with transgenic mice in which human COL2A1 gene was integrated. The collagen type II from human gene rescued the lethal phenotype in null mice, indicating that the biological function of collagen type II is conserved between human and mouse. The animals exhibited normal endochondral bone formation and a normal growth plate in tibio-tarsal joint. Chondrocytes isolated from the cartilage of these mice secreted human protein, suggesting that the animals incorporated heterologous protein to form cartilage which is essentially "humanized." The animals reached puberty and produced normal progeny. A completely normal phenotype in newborns indicates that human COL2A1 gene is expressed properly both temporally and spatially. These animals may be useful to generate models to study the effect of COL2A1 mutations on skeletal development in humans by introducing mutated gene constructs either into embryos or by crossing with transgenic animals with COL2A1 mutations.

Stimulation of Type I Collagen Transcription in Human Skin Fibroblasts by TGF-β: Involvement of Smad 3

Transforming growth factor-beta (TGF-beta) stimulates the transcription of the alpha2(I) procollagen gene (COL1A2). The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-beta. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-beta. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-beta response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-beta. Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-beta-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-beta-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-beta treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-beta-treated fibroblasts by antibody supershifting. These results demonstrate that (i) Smad 3 transmits TGF-beta signals from the receptor to the COL1A2 promoter in human fibroblasts, and is likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-beta; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-beta.

Long- and short-term D-alpha-tocopherol supplementation inhibits liver collagen alpha1(I) gene expression.

We analyzed the role of oxidative stress on liver collagen gene expression in vivo. Long- and short-term supplementation with the lipophilic antioxidant D-alpha-tocopherol (40 IU/day for 8 wk or 450 IU for 48 h) to normal C57BL/6 mice selectively decreased liver collagen mRNA by approximately 70 and approximately 60%, respectively. In transgenic mice, the -0.44 kb of the promoter and the first intron of the human collagen alpha1(I) gene were sufficient to confer responsiveness to D-alpha-tocopherol. Inhibition of collagen alpha1(I) transactivation in primary cultures of quiescent stellate cells from these transgenic animals by D-alpha-tocopherol required only -0.44 kb of the 5' regulatory region. This regulation resembled that of the intact animal following D-alpha-tocopherol treatment and indicates that D-alpha-tocopherol may act directly on stellate cells. Transfection of stellate cells with collagen-LUC chimeric genes allowed localization of an "antioxidant"-responsive element to the -0.22 kb of the 5' region excluding the first intron. These findings suggest that oxidative stress, independently of confounding variables such as tissue necrosis, inflammation, cell activation, or cell proliferation, modulates in vivo collagen gene expression.

Characterization of two distinct families of transcription factors that bind to the CCAAT box region of the human COL1A2 gene

Characterization of two distinct families of transcription factors that bind to the CCAAT box region of the human COL1A2 gene

Characterization of two distinct families of transcription factors that bind to the CCAAT box region of the human COL1A2 gene

Both the mouse and human alpha2(I) procollagen promoters contain an inverted CCAAT box at -80, but only the human promoter contains an additional regulatory element, the collagen modulating element (CME), immediately downstream of the CCAAT box [Collins et al. (1997): Biochem J 322:199-206]. In this study, the transcription factors that bind to the G/CBE and CME within the human promoter were characterized in SVWI-38 and CT-1 nuclear extracts. Two distinct proteins bind to the CME, and both were identified as heat-labile factors that were sensitive to high ionic strengths and required Zn2+ for DNA-binding activity. These proteins had Stokes radii of 4.12 and 3.15 nm, sedimentation coefficients of 3.9 and 3.2 S and native molecular weights of 66 and 41 kDa, respectively. On the basis of biochemical and DNA-binding properties, the CME binding proteins are probably novel factors involved in the regulation of the human alpha2(I) procollagen gene. By contrast, the G/CBE binding proteins were more resistant to heat, ionic strength, and divalent metal ion chelators, demonstrating that the G/CBE and CME binding proteins had distinct DNA-binding properties. The above properties suggest that this factor is a member of the previously characterized family of CCAAT box-binding factors, CBF, NF-Y, CP-1 and alpha-CP1. Taken together, these physicochemical properties of the COL1A2 CCAAT box and CME-binding proteins demonstrated that they were distinct unrelated transcription factors. These results also suggest that there is a distinct difference in the DNA-binding activity between the equivalent region of the mouse and human alpha2(I) procollagen promoters.

Sequence of Canine COL1A2 cDNA: Nucleotide Substitutions Affecting the Cyanogen Bromide Peptide Map of the α2(I) Chain

The α2 chain of canine type I collagen was characterized with both sequence analysis of COL1A2 cDNA and chemical analysis of α2(I) chains. The complete sequence of canine COL1A2 cDNA was determined from reverse-transcribed and polymerase chain reaction-amplified total RNA from cultured skin fibroblasts. Pepsin-digested and cyanogen bromide-digested type I collagen peptides were analyzed with chromatography, gel electrophoresis, and mass spectrometry. Identity between the sequences of canine and human COL1A2 cDNA was 90.9%, predicting conservation of the 3 cysteine residues required for C-propeptide registration and of cleavage sites for signal peptidase, procollagenN-proteinase, vertebrate collagenase, and procollagenC-proteinase. Conservation of all 50 lysine residues was also predicted, including preservation of the 1:2 asymmetry in the X:Y distribution of the 31 lysine residues in the α2(I) triple helix. The human and canine sequences differed in the location of Y-position proline residues and the presence of two unique canine cyanogen bromide peptides, α2 CB3b and α2 CB3c,5. Knowledge of the conserved and unique features of canine COL1A2 will be valuable for analysis of the expression, synthesis, and structure of type I collagen as well as studies of canine osteogenesis imperfecta.

Non calcifying pilomatricoma: Ultrasound imaging of an early morphological stage

Type I collagen is the primary component of the skin dermis. Both the quantity and quality of extracellular collagen are primarily related to skin ageing. In this study, we investigated the possibility that Camellia japonica oil (CJ oil) may be introduced as an anit-wrinkle agent. As a first step to this end, human COL1A2 promoter luciferase assay was performed in human dermal fibroblast cells. CJ oil was determined to activate human COL1A2 promoter in a concentration-dependent manner. In consistency with this result, while matrix metalloproteinase (MMP)-1 activity was inhibited by CJ oil, human type I procollagen synthesis was also induced by CJ oil. These results suggest the possibility that CJ oil may be involved in the skin ageing. For the evaluation of CJ oil's safety and efficiency on human skin, human skin primary irritation test and trans-epidermal water loss (TEWL) were performed. Transepidermal water loss (TEWL) was measured before treatment then, 1 h and 2 h after treatment; the forearm site was selected to measure TEWL. Also, a human skin primary irritation test was performed on the normal skin (upper back) in 30 volunteers to see if a certain material included in CJ oil has irritation or sensitization potential. In these assays, CJ oil reduced trans-epidermal water loss (TEWL) and did not induce any adverse reactions. Therefore, based on these results, we suggest the possibility that CJ oil may be considered as possible wrinkle-reducing candidates for topical application.

Cloning and Expression of Collagen-Derived Protein in E. Coli And Baculovirus System

Using appropriately designed primers we amplified a 783 bp frag ment of the triple helical portion of human α1-collagen (I) by PCR methods. The expression of this biosynthetic collagen gene and a modified one containing cell adhesion sequences was carried out in a prokaryotic (E. coli) and a eukaryotic (baculovirus) system. The expression products were purified by affinity chromatography.

Human COL9A1 and COL9A2 genes. Two genes of 90 and 15 kb code for similar polypeptides of the same collagen molecule.

Here we report the complete structure for the human COL9A1 and the complete sequence for the human COL9A2 genes. The COL9A1 gene is about 90 kb and consists of 38 exons. The COL9A2 gene is only about 15 kb, and it contains 32 exons. Sequence analysis of the promoter regions for the human COL9A2, the mouse Col9a2 and the human COL2A1 genes identified a conserved 14 bp sequence. The data also indicated that the alternative exon 1* found in intron 6 of the COL9A1 gene is separated from exon 7 only by a short intron in the chick, human, mouse and rat genes probably explaining why transcripts from exon 1* are spliced directly to exon 8.

Different cis-regulatory DNA elements mediate developmental stage- and tissue-specific expression of the human COL2A1 gene in transgenic mice.

Expression of the type II collagen gene (human COL2A1, mouse Col2a1) heralds the differentiation of chondrocytes. It is also expressed in progenitor cells of some nonchondrogenic tissues during embryogenesis. DNA sequences in the 5' flanking region and intron 1 are known to control tissue-specific expression in vitro, but the regulation of COL2A1 expression in vivo is not clearly understood. We have tested the regulatory activity of DNA sequences from COL2A1 on the expression of a lacZ reporter gene in transgenic mice. We have found that type II collagen characteristic expression of the transgene requires the enhancer activity of a 309-bp fragment (+2, 388 to +2,696) in intron 1 in conjunction with 6.1-kb 5' sequences. Different regulatory elements were found in the 1.6-kb region (+701 to +2,387) of intron 1 which only needs 90-bp 5' sequences for tissue-specific expression in different components of the developing cartilaginous skeleton. Distinct positive and negative regulatory elements act together to control tissue-specific transgene expression in the developing midbrain neuroepithelium. Positive elements affecting expression in the midbrain were found in the region from -90 to -1,500 and from +701 to +2,387, whereas negatively acting elements were detected in the regions from -1,500 to -6,100 and +2,388 to +2,855.

COL1A1 Transgene Expression in Stably Transfected Osteoblastic Cells: RELATIVE CONTRIBUTIONS OF FIRST INTRON, 3′-FLANKING SEQUENCES, AND SEQUENCES DERIVED FROM THE BODY OF THE HUMAN COL1A1MINIGENE

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

Molecular cloning and chromatin structure analysis of the murine α1(I) collagen gene domain

We have isolated molecular clones of genomic mouse DNA spanning 55 kb, including the entire coding region of the murine α1(I) collagen ( Col1a1) gene and 24 kb of 5′ and 13 kb of 3′-flanking sequences, and have performed a detailed chromatin structure analysis of these sequences. Several new DNase-I-hypersensitive sites were identified. The distal 5′-flanking region contains two clusters of DNase-I-hypersensitive sites located between 7 and 8 kb and between 15 and 20 kb upstream of the start site of transcription, respectively. Several of these sites were shown to be present in collagen-producing, but not in non-producing cells, indicating that they are associated with transcription of the gene and may function in its regulation. One strong constitutive DNase-I-hypersensitive site at −18.5 kb was also cleaved by endogenous nucleases. The 3′-flanking region of the gene contains a DNase-I-hypersensitive site located 6 kb downstream of the end of the gene, as well as sequences that can induce a non-B DNA structure. Because these latter sequences coincide with DNase-I-hypersensitive sites in the homologous human gene, our results suggest that some regulatory elements may play a role in gene regulation, not by specific protein–DNA interactions but by virtue of their ability to induce a non-B DNA structure and/or an alternate chromatin conformation. A comparison of the murine and human Col1a1 domains shows a similar, although not identical, distribution of DNase-I-hypersensitive sites, indicating a conserved arrangement of regulatory elements. Our results strongly suggest that these new sites constitute regulatory elements which are involved in the transcriptional regulation and/or chromatin loop organization of the Col1a1 gene, and they are now amenable for functional analyses.

Localization of silencer and enhancer elements in the human type X collagen gene.

Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5'-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements.

SOX9 directly regulates the type-II collagen gene.

Mutations in human SOX9 are associated with campomelic dysplasia (CD), characterised by skeletal malformation and XY sex reversal. During chondrogenesis in the mouse, Sox9 is co-expressed with Col2a1, the gene encoding type-II collagen, the major cartilage matrix protein. Col2a1 is therefore a candidate regulatory target of SOX9. Regulatory sequences required for chondrocyte-specific expression of the type-II collagen gene have been localized to conserved sequences in the first intron in rats, mice and humans. We show here that SOX9 protein binds specifically to sequences in the first intron of human COL2A1. Mutation of these sequences abolishes SOX9 binding and chondrocyte-specific expression of a COL2A1-driven reporter gene (COL2A1-lacZ) in transgenic mice. Furthermore, ectopic expression of Sox9 trans-activates both a COL2A1-driven reporter gene and the endogenous Col2a1 gene in transgenic mice. These results demonstrate that COL2A1 expression is directly regulated by SOX9 protein in vivo and implicate abnormal regulation of COL2A1 during, chondrogenesis as a cause of the skeletal abnormalities associated with campomelic dysplasia.

A GT-rich Sequence Binding the Transcription Factor Sp1 Is Crucial for High Expression of the Human Type VII Collagen Gene (COL7A1) in Fibroblasts and Keratinocytes

Type VII collagen is the major component of anchoring fibrils, structural elements that stabilize the attachment of the basement membrane to the underlying dermis. In this study, we have dissected the human type VII collagen gene (COL7A1) promoter to characterize the cis-elements responsible for the expression of the gene in cultured fibroblasts and keratinocytes. Using transient cell transfections with various 5' end deletion COL7A1 promoter/chloramphenicol acetyltransferase reporter gene plasmid constructs, we determined that the region between nucleotides -524 and -456, relative to the transcription start site, is critical for high promoter activity in both cell types studied. Gel mobility shift assays using several DNA fragments spanning this region identified a GT-rich sequence between residues -512 and -505, necessary for the binding of nuclear proteins to this region of the promoter. Point mutations abolished the binding of nuclear proteins in gel shift assays and drastically diminished the activity of the promoter in transient cell transfections. Supershift assays with antibodies against various transcription factors including Sp1, Sp3, c-Jun/AP-1, and AP-2, and competition experiments with oligonucleotides containing consensus sequences for Sp1 and AP-1 binding identified Sp1 as the transcription factor binding to this region of the COL7A1 promoter. Indeed, recombinant human Sp1 was shown to bind the COL7A1 promoter GT-rich element but not its mutated form in gel mobility shift assays. In addition, co-transfection of pPacSp1, an expression vector for Sp1, together with the COL7A1 promoter/chloramphenicol acetyltransferase construct into Sp1-deficient Drosophila Schneider SL2 cells unequivocally demonstrated that Sp1 is essential for high expression of the COL7A1 gene. These data represent the first in-depth analysis of the human COL7A1 promoter transcriptional control.

Functional analysis of the lysyl oxidase promoter in myofibroblast-like clones of 3T6 fibroblast.

Lysyl oxidase (LO), an extracellular enzyme catalysing the first step of collagen and elastin cross-linking, is transiently expressed by myofibroblasts during fibrosis. A cell model with features of myofibroblast was thus established for studying the regulation of LO. Two clones of the 3T6 fibroblast cell line were selected because 1) they produced a relatively high steady-state level of the three lysyl oxidase mRNAs with the same relative ratio similar to fibrotic tissue and 2) they stably displayed certain features of myofibroblast (alpha-smooth muscle actin cytoskeleton, bundles of cytoskeletal filaments beneath the cytoplasmic membranes). These clones synthesized predominantly type I collagen fibers and a small amount of type III collagen. Neither type IV collagen nor elastin were observed. The cloning and sequencing of 2,073 bp of the mouse Balb/C LO promoter was performed, allowing the identification around the initiation of transcription of consensus sequences which are found on the COL1 promoters. A series of deletion constructs containing the LO 5'-flanking region ligated to the luciferase gene were transiently transfected into 3T6-5 fibroblasts. The region allowing the maximal activity was found between positions -416 to -192, while the more upstream region negatively regulated the promoter. The -898 to -865 sequence (called LOcol1) displayed 79% of homology with a conserved sequence of murine, rat, and human COL1A1 promoters. This sequence participated to the binding of several nuclear factors within a region (-970 to -784) allowing 50% of inhibition of the LO promoter. Therefore, the level of LO transcription is regulated in 3T6-5 fibroblast by positive and negative cis-acting regulatory elements which might have common features with the COL1A1 promoter.

Functional analysis of the lysyl oxidase promoter in myofibroblast-like clones of 3T6 fibroblast

Lysyl oxidase (LO), an extracellular enzyme catalysing the first step of collagen and elastin cross-linking, is transiently expressed by myofibroblasts during fibrosis. A cell model with features of myofibroblast was thus established for studying the regulation of LO. Two clones of the 3T6 fibroblast cell line were selected because 1) they produced a relatively high steady-state level of the three lysyl oxidase mRNAs with the same relative ratio similar to fibrotic tissue and 2) they stably displayed certain features of myofibroblast (alpha-smooth muscle actin cytoskeleton, bundles of cytoskeletal filaments beneath the cytoplasmic membranes). These clones synthesized predominantly type I collagen fibers and a small amount of type III collagen. Neither type IV collagen nor elastin were observed. The cloning and sequencing of 2,073 bp of the mouse Balb/C LO promoter was performed, allowing the identification around the initiation of transcription of consensus sequences which are found on the COL1 promoters. A series of deletion constructs containing the LO 5'-flanking region ligated to the luciferase gene were transiently transfected into 3T6-5 fibroblasts. The region allowing the maximal activity was found between positions -416 to -192, while the more upstream region negatively regulated the promoter. The -898 to -865 sequence (called LOcol1) displayed 79% of homology with a conserved sequence of murine, rat, and human COL1A1 promoters. This sequence participated to the binding of several nuclear factors within a region (-970 to -784) allowing 50% of inhibition of the LO promoter. Therefore, the level of LO transcription is regulated in 3T6-5 fibroblast by positive and negative cis-acting regulatory elements which might have common features with the COL1A1 promoter.

Increased activity of the α1(I) procollagen promoter in skin fibroblasts from patients with chronic eosinophilia-myalgia syndrome

Eosinophilia-myalgia syndrome (EMS), a novel l-tryptophan-associated disease, which occurred as an epidemic in 1989, is characterized by diffuse fibrosis of the skin. The objective of these studies was to compare the in vitro expression of the gene encoding the human α1(I) procollagen (COL1A1) in dermal fibroblasts derived from patients with long-standing EMS and from healthy controls. Early passage fibroblasts in culture were transiently transfected with a series of progressively 5′ deleted human COL1A1 promoter-CAT reporter gene DNA constructs. Resultant CAT activity was assayed in the cytoplasmic extracts. Following transient transfection, COL1A1 promoter activity in 4/5 fibroblast cell lines derived from patients with EMS was two-to threefold greater than in matched normal fibroblasts. Deletion analysis indicated that CAT activity was highest, and displayed the greatest increase in EMS vs normal fibroblasts, with promoter constructs spanning 174 bp upstream from the COL1A1 transcription start site. The study shows increased CAT activity driven by a 174 bp fragment of COL1A1 in transiently transfected skin fibroblasts from patients with EMS even in the chronic stage of disease. These findings expand on previous observations indicating increased type I collagen gene expression in EMS fibroblasts, and suggest that fibrosis in l-tryptophan-induced EMS is associated with transcriptional activation of type I collagen gene expression, which persists even following cessation of l-tryptophan use.

A 1.9-Kb 5' fragment from the human COL1A1 gene drives inappropriate expression of the human COL2A1 gene in tissues of transgenic mice that normally express only the COL1A1 gene.

A 1.9-Kb 5' fragment from the human COL1A1 gene drives inappropriate expression of the human COL2A1 gene in tissues of transgenic mice that normally express only the COL1A1 gene.