skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Collagen expression in fibroblasts with a novel LMNA mutation

Abstract

Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.

Authors:
 [1];  [1];  [2];  [2];  [2];  [2];  [1];  [3]
  1. Department of Pathology, University of Washington, Seattle, WA 98195 (United States)
  2. Memorial University of Newfoundland, St. Johns, Nfld, A1B 3V6 (Canada)
  3. Department of Pathology, University of Washington, Seattle, WA 98195 (United States). E-mail: picard@u.washington.edu
Publication Date:
OSTI Identifier:
20857963
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 352; Journal Issue: 3; Other Information: DOI: 10.1016/j.bbrc.2006.11.070; PII: S0006-291X(06)02517-4; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AGING; COLLAGEN; FIBROBLASTS; MORPHOLOGY; MUTANTS; MUTATIONS; PATHOGENESIS; PATIENTS; SKIN

Citation Formats

Nguyen, Desiree, Leistritz, Dru F., Turner, Lesley, MacGregor, David, Ohson, Kamal, Dancey, Paul, Martin, George M., and Oshima, Junko. Collagen expression in fibroblasts with a novel LMNA mutation. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2006.11.070.
Nguyen, Desiree, Leistritz, Dru F., Turner, Lesley, MacGregor, David, Ohson, Kamal, Dancey, Paul, Martin, George M., & Oshima, Junko. Collagen expression in fibroblasts with a novel LMNA mutation. United States. doi:10.1016/j.bbrc.2006.11.070.
Nguyen, Desiree, Leistritz, Dru F., Turner, Lesley, MacGregor, David, Ohson, Kamal, Dancey, Paul, Martin, George M., and Oshima, Junko. Fri . "Collagen expression in fibroblasts with a novel LMNA mutation". United States. doi:10.1016/j.bbrc.2006.11.070.
@article{osti_20857963,
title = {Collagen expression in fibroblasts with a novel LMNA mutation},
author = {Nguyen, Desiree and Leistritz, Dru F. and Turner, Lesley and MacGregor, David and Ohson, Kamal and Dancey, Paul and Martin, George M. and Oshima, Junko},
abstractNote = {Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.},
doi = {10.1016/j.bbrc.2006.11.070},
journal = {Biochemical and Biophysical Research Communications},
number = 3,
volume = 352,
place = {United States},
year = {Fri Jan 19 00:00:00 EST 2007},
month = {Fri Jan 19 00:00:00 EST 2007}
}
  • Transforming growth factor-{beta} (TGF-{beta}), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-{gamma} compared to heterozygous control MEFs. Treatment with the PPAR-{gamma} ligand 15d-PGJ{sub 2} failed to down-regulate collagen gene expression in PPAR-{gamma} null MEFs, whereas reconstitution of these cells with ectopic PPAR-{gamma} resulted in their normalization. Compared to control MEFs, PPAR-{gamma} null MEFs displayed elevated levels of the Type I TGF-{beta} receptor (T{beta}RI),more » and secreted more TGF-{beta}1 into the media. Furthermore, PPAR-{gamma} null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-{beta}, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-{gamma} null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-{beta} responses. Taken together, these results indicate that loss of PPAR-{gamma} in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-{beta} stimulation.« less
  • In an 8-year-old girl and her father, both of whom have severe type III OI, we have previously used RNA/RNA hybrid analysis to demonstrate a mismatch in the region of {alpha}1(I) mRNA coding for aa 558-861. We used SSCP to further localize the abnormality to a subregion coding for aa 579-679. This region was subcloned and sequenced. Each patient`s cDNA has a deletion of the sequences coding for the last residue of exon 34, and all of exons 35 and 36 (aa 604-639), followed by an insertion of 156 nt from the 3{prime}-end of intron 36. PCR amplification of leukocytemore » DNA from the patients and the clinically normal paternal grandmother yielded two fragments: a 1007 bp fragment predicted from normal genomic sequences and a 445 bp fragment. Subcloning and sequencing of the shorter genomic PCR product confirmed the presence of a 565 bp genomic deletion from the end of exon 34 to the middle of intron 36. The abnormal protein is apparently synthesized and incorporated into helix. The inserted nucleotides are in frame with the collagenous sequence and contain no stop codons. They encode a 52 aa non-collagenous region. The fibroblast procollagen of the patients has both normal and electrophoretically delayed pro{alpha}(I) bands. The electrophoretically delayed procollagen is very sensitive to pepsin or trypsin digestion, as predicted by its non-collagenous sequence, and cannot be visualized as collagen. This unique OI collagen mutation is an excellent candidate for molecular targeting to {open_quotes}turn off{close_quotes} a dominant mutant allele.« less
  • Hereditary methemoglobinemia with generalized deficiency of NADH-cytochrome b{sub 5} reductase (b{sub 5}R) (type II) is a rare disease characterized by severe developmental abnormalities, which often lead to premature death. Although the molecular relationship between the symptoms of this condition and the enzyme deficit are not understood, it is thought that an important cause is the loss of the lipid metabolizing activities of the endoplasmic reticulum-located reductase. However, the functions of the form located on outer mitochondrial membranes have not been considered previously. In this study, we have analyzed the gene of an Italian patient and identified a novel G{r_arrow}T transversionmore » at the splice-acceptor site of the 9th exon, which results in the complete absence of immunologically detectable b{sub 5}R in blood cells and skin fibroblasts. In cultured fibroblasts of the patient, NADH-dependent cytochrome c reductase, ferricyanide reductase, and semidehydroascorbate reductase activities were severely reduced. The latter activity is known to be due to b{sub 5}R located on outer mitochondrial membranes. Thus, our results demonstrate that the reductase in its two membrane locations, endoplasmic reticulum and outer mitochondrial membranes, is the product of the same gene and suggest that a defect in ascorbate regeneration may contribute to the phenotype of hereditary methemoglobinemia of generalized type. 37 refs., 5 figs., 2 tabs.« less
  • Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contract the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reductionmore » of pro{alpha}{sub 1} (I), pro{alpha}{sub 2} (I), and pro{alpha}{sub 1} (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.« less
  • Colchicine administered intravenously depolymerized microtubules and disrupted the normal organization of the Golgi apparatus in periodontal ligament fibroblasts. Radioautography with /sup 3/H-proline indicated that collagen secretion was completely inhibited during a period of approximately 4 hours following the onset of the colchicine effect. During this period of secretory inhibition, labeled collagen precursors were present within a variety of dense bodies, primarily located in a juxtanuclear location replacing the normal Golgi complex. The time course of /sup 3/H-proline labeling from 2 to 8 hours suggested that small, newly formed dense bodies fused to form larger dense bodies and pleomorphic structures (zebramore » bodies), within which collagen precursors appeared to undergo partial polymerization. Autophagosomes, many labeled with /sup 3/H-proline, also increased in number after colchicine administration. A gradual decline in /sup 3/H-proline label occurred from 4 to 24 hours, presumably due to exocytosis of dense bodies or by the digestion of labeled collagen precursors within autophagosomes. These results support the concept that an intact microtubular network is essential for the organized transport of collagen precursors, from the rough endoplasmic reticulum to the Golgi apparatus, and the eventual transport and exocytosis of collagen secretory granules.« less