Extracellular matrix containing mutated fibrillin-1 (Fbn1) down regulates Col1a1, Col1a2, Col3a1, Col5a1, and Col5a2 mRNA levels in Tsk/+ and Tsk/Tsk embryonic fibroblasts.

It is known that the extracellular matrix (ECM) is able to signal to cells and thereby direct or modulate the transcription of certain mRNAs. This signaling plays an important role in tumor invasion and metastasis, wound healing, remodeling of the ECM and cell differentiation. There are several mechanisms whereby the ECM signals cells to change their metabolism: (1) receptor molecules binding to specific domains in the ECM, (2) direct phagocytosis of the ECM molecules or domains into the cell, (3) structural changes of the ECM domains. We report the effect of an ECM containing either mutant or normal Fbn1 on the transcription levels of several collagen mRNAs. Tsk/Tsk, Tsk/+ and +/+ mouse embryonic fibroblast cell lines were used. Tsk/Tsk cells produce only mutated fibrillin-1 which arises from mRNA containing an in-frame duplication of exons 17-40. To test the effect of the ECM containing mutant Fbn1, cells of the Tsk/Tsk, Tsk/+ and the wild-type (+/+) genotype were each grown on an ECM produced by either Tsk/Tsk, Tsk/+ cells or by wild-type cells (+/+). The embryonic cells were genotyped by Northern analyses for Fbn1 and grown to confluence. The cultures were then harvested and the cells removed, leaving the matrix in the flasks. Matrices produced from Tsk/Tsk, Tsk/+ and from +/+ cells were reseeded with Tsk/Tsk cells, Tsk/+ cells or +/+ cells. The cells were plated at a confluent concentration and incubated on the matrices for 48 h, after which total RNA was harvested and cDNA generated. Real-time PCR using cDNA or Northern analyses using RNA were performed for Fbn1 and Types I, III and V collagens. The PCR and Northern results were normalized using beta-actin and GAPDH, respectively. The Northern analyses showed that the steady state levels of mRNA for Col1a1 were depressed in both Tsk/Tsk and +/+ cells when grown on the matrix produced by Tsk/Tsk cells. Real-time PCR was then performed with primers specific for Col1a2, Col3a1, Col5a1 and Col5a2. The results showed that cells with the Tsk/Tsk, Tsk/+, and +/+ genotype all had lower steady-state levels of the above 4 collagen mRNAs when grown on the matrix produced by homozygous Tsk/Tsk cells or the matrix produced by heterozygous Tsk/+ cells compared with those grown on a matrix produced by +/+ cells. We hypothesize that the mutated Fbn1 molecules with many additional EGF-calcium binding regions and TGF-beta binding domains may (1) change the homeostasis of the ECM by binding additional growth factors and/or (2) present a radically different ECM 3-dimensional architecture. Either or both of these changes could signal the cell to produce less collagen.

Increased numbers of microchimeric cells of fetal origin are associated with dermal fibrosis in mice following injection of vinyl chloride.

OBJECTIVE: To develop a murine model for use in examining the role of microchimeric cells and certain chemical exposures in the pathogenesis of systemic sclerosis (SSc). METHODS: Female BALB/cJ retired breeder mice were bled before and after vinyl chloride injection. The DNA from their white blood cells was obtained, and the number of microchimeric cell equivalents was determined by quantitative polymerase chain reaction using DNA primers specific for the H-2Kb gene, a sequence not found in BALB/cJ mice. Skin was obtained at autopsy, embedded in paraffin, sectioned, and stained with Masson's trichrome. Hydroxyproline analyses were performed on 4-mm skin biopsy samples. RESULTS: Microchimeric cells were identified and quantitated before and after 20 daily intraperitoneal injections of vinyl chloride. The number of microchimeric cells in the peripheral blood increased an average of 48-fold after treatment with vinyl chloride. Histologic examination of the skin of these same mice (which had an increased number of microchimeric cells) showed inflammation, with abundant fibroblasts and a heavy mononuclear infiltration in the dermis. The collagen fibers appeared densely packed and disorganized. Histologic examination of the skin of untreated retired breeder mice and treated virgin mice appeared normal. Quantitative assays to determine the collagen content of skin biopsy samples obtained from treated microchimeric mice compared with nontreated microchimeric or with treated nonmicrochimeric mice showed a 2-3-fold increase in collagen content in the treated microchimeric mice. Extraordinary splenomegaly was present in the vinyl chloride-treated microchimeric mice, accompanied by cellular infiltration and fibrosis. CONCLUSION: The results suggest that vinyl chloride injections into BALB/cJ retired breeder mice lead to activation of microchimeric cells, which causes the cells to divide and multiply. The correlation between the 48-fold increase in microchimeric cells and the appearance of dermal inflammation and fibrosis similar to that of graft-versus-host disease suggests that activated microchimeric cells may be a necessary factor in the pathogenesis of autoimmune diseases such as SSc.

Transcriptional activation of the alpha1(I) procollagen gene and up-regulation of alpha1(I) and alpha1(III) procollagen messenger RNA in dermal fibroblasts from tight skin 2 mice.

OBJECTIVE: To investigate the levels of expression of type I and type III collagen genes in dermal fibroblasts from tight skin 2 (Tsk2) and normal mice and to examine the transcriptional regulation of the alpha1(I) procollagen gene (COL1A1) in these cells. METHODS: Dermal fibroblasts from Tsk2 mice and from normal age- and sex-matched control mice were studied. Steady-state levels of alpha1(I) and alpha1(III) procollagen messenger RNA (mRNA) were evaluated by Northern and dot-blot hybridization analyses. The transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs containing portions of the COL1A1 promoter ligated to the chloramphenicol acetyltransferase reporter gene. To identify DNA binding proteins that interact with regulatory elements within the COL1A1 promoter, gel mobility shift assays were performed with nuclear extracts prepared from normal and Tsk2 dermal fibroblasts. RESULTS: Synthesis of collagen was almost 100% higher in Tsk2 dermal fibroblasts than in control fibroblasts. Up-regulation of mRNA for 2 extracellular matrix proteins was observed in the Tsk2 dermal fibroblasts compared with the normal cells: the alpha1(I) procollagen mRNA steady-state levels were 50% higher, and those of the alpha1(III) procollagen mRNA 100% higher, in Tsk2 cells. The results of transient transfection experiments with COL1A1 promoter constructs demonstrated that the elevated levels of alpha1(I) collagen mRNA in Tsk2 cells were largely due to increased transcriptional activity of the corresponding gene. Electrophoretic mobility shift assays performed with a probe encompassing a relevant COL1A1 promoter region revealed increased DNA-protein binding activities in nuclear extracts prepared from Tsk2 fibroblasts compared with normal fibroblasts. Competition experiments using consensus Spl and nuclear factor 1 (NF-1) oligonucleotides and supershift experiments using anti-Sp1 and anti-NF-1 antibodies indicated that at least 2 transcription factors, Sp1 and NF-1, or their homologs are involved in the up-regulated transcriptional activity of the COL1A1 promoter in Tsk2 fibroblasts. CONCLUSION: Dermal fibroblasts from Tsk2 mice display increased collagen synthesis and up-regulation of alpha1(I) and alpha1(III) procollagen mRNA in vitro. The data also directly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from Tsk2 mice and suggest that the transcription factors Sp1 and NF-1 or their homologs play an important role in the upregulated expression of this gene in Tsk2 fibroblasts. These findings are similar to those described for fibroblasts from humans with systemic sclerosis and validate the use of Tsk2 as a model for the study of the connective tissue alterations in this disease.

T cells infiltrating the skin of Tsk2 scleroderma-like mice exhibit T cell receptor bias.

The T cell repertoire expressed by Tsk2 mice, a novel experimental model of systemic sclerosis, was examined to determine whether cells infiltrating the areas of involved skin exhibit a T cell receptor (TCR) bias. Reverse transcription-polymerase chain reactions (RT-PCR) were conducted using RNA extracted from lymph nodes and skin from TSk2 mice and from normal mice, with an oligonucleotide primer library specific for the variable region of the TCR (beta) chain. RT-PCR signals were observed in all lymph node cell (LNC) samples from both Tsk2 mice and control mice, with eighteen of the twenty-one Vbeta types present. In contrast, cDNA extracted from areas of involved skin from Tsk2 mice exhibited a restricted pattern, with positive Vbeta signals corresponding to eight T cell subtypes (Vbeta1, 6, 8.1, 8.2, 10, 11, 16, and 18). Band strength analysis revealed that three Vbeta subtypes dominated within this restricted pattern (Vbeta8.1, 11, and 18). Moreover, this pattern of Vbeta bias was consistent among the four skin samples from different Tsk2 mice. These data suggest that a restricted T cell population participates in the inflammatory cell infiltrate of Tsk2 skin.

A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation.

Mice carrying the Tight skin (Tsk) mutation have thickened skin and visceral fibrosis resulting from an accumulation of extracellular matrix molecules. These and other connective tissue abnormalities have made Tskl + mice models for scleroderma, hereditary emphysema, and myocardial hypertrophy. Previously we localized Tsk to mouse chromosome 2 in a region syntenic with human chromosome 15. The microfibrillar glycoprotein gene, fibrillin 1 (FBN1), on human chromosome 15q, provided a candidate for the Tsk mutation. We now demonstrate that the Tsk chromosome harbors a 30- to 40-kb genomic duplication within the Fbn1 gene that results in a larger than normal in-frame Fbn1 transcript. These findings provide hypotheses to explain some of the phenotypic characteristics of Tskl + mice and the lethality of Tsk/Tsk embryos.

The tight skin 2 mouse. An animal model of scleroderma displaying cutaneous fibrosis and mononuclear cell infiltration.

OBJECTIVE: To describe the histopathologic and biochemical characteristics of skin from the Tsk2/+ mouse, a mutation with phenotypic features resembling those of systemic sclerosis (SSc), and to report the initial genetic mapping of the Tsk2 locus. METHODS: Histologic examination was performed and collagen content and type I collagen messenger RNA (mRNA) levels were determined in skin biopsy specimens from Tsk2/+ mice and normal mice. An intersubspecific backcross was conducted as a first step toward identifying the position of the Tsk2 locus on mouse chromosome 1. RESULTS: Histologic examination of Tsk2/+ mouse skin revealed marked accumulation of collagen and infiltration with mononuclear cells in the dermis and adipose tissue. Biochemical studies of Tsk2/+ mouse skin showed increased collagen content and elevated steady-state levels of alpha 1 (I) procollagen mRNA. Tsk2 was mapped to a 15.3-centimorgan interval on mouse chromosome 1. CONCLUSION: Tsk2 is a novel mutation which displays histopathologic and biochemical abnormalities similar to those present in the skin of patients with SSc, including increased collagen content and expression of type I collagen genes. This mutation has been mapped to a 15.3-cM region on mouse chromosome 1. Further study of this novel mutation will allow the identification of previously undescribed mechanisms involved in the regulation of normal and pathologic collagen gene expression.

Immunological characterization of (tight skin/NZB)F1 hybrid mice with connective tissue and autoimmune features resembling human systemic sclerosis.

A new murine model of Systemic Sclerosis (SSc) has been developed by breeding the Tsk+/+pa tight skin mouse (TSK) and the autoimmune disease-prone NZB strain to produce an F1 hybrid displaying the connective tissue abnormalities of the TSK parent and the autoimmune abnormalities of the NZB parent. The interscapular skin thickness in the (TSK/NZB)F1 was significantly greater than in the (+pa/NZB)F1 litter mates. Protein biosynthesis in skin punch biopsies was over 3.5 times higher in the (TSK/NZB)F1 mice than in controls. Hydroxyproline analyses confirmed that the increase in protein synthesis was primarily in collagen. These (TSK/NZB)F1 mice were tested for a number of cellular and humoral autoimmune manifestations. Autoantibodies, including antinuclear antibodies (ANA) and anti-DNA, were present in their sera, and the proliferative response to autologous lymphocyte stimulation (AMLR) was decreased as is commonly observed in murine and human autoimmune disorders. These results indicate that the (TSK/NZB)F1 mice display connective tissue and immunologic abnormalities resembling those present in human SSc and, therefore, these mice may be a valuable model for the study of the disease.

Monoclonal antibodies to different epitopes in amelogenins from fetal bovine teeth recognize high-molecular-weight components.

Enamel proteins were extracted and partitioned into amelogenin and enamelin fractions. Although several attempts were made to raise monoclonal antibodies to each protein fraction, monoclonal antibodies were only obtained against the amelogenin fraction. Six monoclonal antibodies were generated, and these could be classified into three groups recognizing different epitopes by a competitive enzyme-linked immuno-absorbent assay. A model for the arrangement of the epitopes is proposed. In Western-blotting experiments, all six monoclonal antibodies recognized amelogenin components of approx. 45,000 and 60,000 daltons as well as lower molecular-weight components of 10,000 to 30,000. It is proposed that the 45,000 and 60,000 dalton components are precursors of the lower molecular-weight components.

Production of a monoclonal antibody to bovine tooth enamel proteins.

Several monoclonal antibodies to bovine enamel proteins were produced using the mouse myeloma system. Each antibody recognized the same two protein bands on gel electrophoresis. The clones were tested in situ and clone 185 localized specifically in the enamel layer. Clones 185 and 121 were shown to recognize different antigenic determinants.

Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel.

A sequential dissociative extraction scheme is described in which tooth matrix proteins are extracted first in 4 M guanidine HCl, pH 7.4, and then in 4 M guanidine HCl, 0.5 M EDTA, pH 7.4, both with protease inhibitors present. The latter step dissolves the mineralized portion of the tissue and extracts noncollagenous proteins closely associated with hydroxyapatite crystallites in the mineralized matrix. In fetal bovine enamel, the initial dissociative extraction step completely removes proline-rich amelogenins from the tissue without dissolving the enamel apatite. The amelogenin proteins consist of several species on polyacrylamide gel electrophoresis with sodium dodecyl sulfate, but display anomalous migration behavior relative to conventional marker proteins in this technique. Subsequent extraction of fetal bovine enamel with guanidine HCl/EDTA removes matrix enamelins, acidic glycoproteins that are tightly bound to the enamel hydroxyapatite. This latter fetal protein type has not been isolated previously. The enamelins are adsorbed strongly by DEAE-cellulose in 7 M urea and totally adsorb to synthetic apatite, even in 4 M guanidine HCl. The enamelins display normal behavior on polyacrylamide gels and stain positively for sialic acid/phosphate and carbohydrate. With advancing tooth maturation, amelogenins disappear while enamelins are conserved. Gel filtration chromatography in 4 M guanidine HCl showed amelogenin components at apparent molecular weights of approximately 25,000, 15,000, 9,500, 7,500, and 6,000, while the enamelins eluted at Mr positions of approximately 72,000, 56,000, 42,000, 30,000, 21,000, 13,000, and 8,000. The gel filtration data showed a clear shift in molecular size population from higher to lower components for both amelogenins and enamelins with progressive enamel maturation.