Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients.
Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.
Arris
Christine E
CE
Department of Rheumatology and Gerentology, University of Newcastle Medical School, Framlington Place, NE2 4HH, Newcastle, UK.
Bevitt
Debra J
DJ
Mohamed
Jeseem
J
Li
Zheng
Z
Langton
Kevin P
KP
Barker
Michael D
MD
Clarke
Michael P
MP
McKie
Norman
N
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D018093:Gelatinases / Q000378:metabolism; D015870:Gene Expression; D005799:Genes, Dominant; D005881:Gingiva / Q000378:metabolism*; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3; D018093:Gelatinases