Hammerhead ribozyme-mediated silencing of the mutant fibrillin-1 of tight skin mouse: insight into the functional role of mutant fibrillin-1.
The tight skin (Tsk/+) mouse is a model for fibrotic disorders. The genetic defect in the Tsk/+ is an in-frame duplication between exons 17 and 40 of the fibrillin-1 gene which gives rise to a large transcript and protein. Mice homozygous for the mutation die in utero, whereas heterozygotes survive and spontaneously develop connective tissue disease. In this study, we generated hammerhead ribozymes directed against the mutant fibrillin-1 transcript. A partially mispairing ribozyme was the most effective vehicle to cleave the mutant transcript without undesired cleavage of wild type transcripts, as shown by cell-free RNA cleavage and cleavage in cell lines harboring the ribozyme, by RT-PCR, Northern and Western Blotting. Global gene expression profiling using oligonucleotide microarrays showed the expected reduction in fibrillin-1 mRNA, and down-regulation of several gene cohorts in ribozyme harboring TskR1 cells compared to Tsk/+ cells. Two of the functional clusters included genes regulating extracellular matrix such as connective tissue growth factor, serpine-1 (plasminogen activator inhibitor-1) and TIMP-1 and TIMP-3, and those involved in cytoskeletal organization and myofibroblast formation including calponins and transgelin. Ribozyme-mediated inhibition was confirmed by Western Blot and functional analysis using cell-reporter systems and remodeling of three dimensional collagen gels. Our results underline the therapeutic potential of hammerhead ribozymes in dominant negative defects and suggest that changes in microfibril architecture brought about by fibrillin-1 mutation lead to a complex disease phenotype.
Menon
Rajesh P
RP
Centre for Rheumatology, Royal Free and University College Medical School, Division of Medicine, University College London (Hampstead Campus), Rowland Hill Street, London NW3 2PF2, UK. rmenon@nimr.mrc.ac.uk
Menon
Malini R
MR
Shi-Wen
Xu
X
Renzoni
Elisabetta
E
Bou-Gharios
George
G
Black
Carol M
CM
Abraham
David J
DJ
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics; D002522:Chlorocebus aethiops; D003094:Collagen / Q000378:metabolism; D055513:Connective Tissue Growth Factor; D003598:Cytoskeletal Proteins / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D020869:Gene Expression Profiling; D020868:Gene Silencing; D017874:Immediate-Early Proteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D008840:Microfilament Proteins / Q000235:genetics* / Q000502:physiology; D009154:Mutation / Q000235:genetics*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D054392:Adipokines
D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; D003094:Collagen; D055513:Connective Tissue Growth Factor; D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D005353:Fibronectins; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D017395:Plasminogen Activator Inhibitor 1; D016337:RNA, Catalytic; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C526318:CCN2 protein, mouse; C000605724:Fbn1 protein, mouse; D054392:Adipokines; C508961:Tgfb1 protein, mouse; C084612:fibulin 2; C427204:hammerhead ribozyme