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Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase-2 (TIMP-2) in normal and transformed chicken embryo fibroblasts.
- Source :
-
Journal of cellular physiology [J Cell Physiol] 1998 Mar; Vol. 174 (3), pp. 342-52. - Publication Year :
- 1998
-
Abstract
- Rous sarcoma virus-transformed chicken embryo fibroblasts (RSVCEF), when compared to normal CEF, produce elevated levels of matrix metalloproteinase-2 (MMP-2) that exists in a form free of complexed tissue inhibitor of metalloproteinase-2 (TIMP-2). In order to ascertain whether the increased levels of TIMP-free MMP-2 in RSVCEF cultures are due to diminished expression of TIMP-2 or alterations in TIMP-2 that diminish its MMP-2 binding ability, it was necessary to clone, characterize, and express chicken TIMP-2 cDNA. The TIMP-2 cDNA was cloned from a chick embryo lambda gt11 library by RT-PCR using primers based on amino-acid sequences determined from isolated TIMP-2. The deduced amino acid sequence for chicken TIMP-2 is 81% identical to human TIMP-2; most of the sequence differences lie in the carboxyl terminal portion of chicken TIMP-2. Northern analysis of mRNA levels in CEF and RSVCEF demonstrates that TIMP-2 mRNA levels are increased in RSVCEF. However, TIMP-2 protein levels, relative to proMMP-2 levels, appear to decrease upon transformation and suggest additional control of TIMP-2 at the post-transcriptional level. Addition of recombinantly expressed TIMP-2 to RSVCEF cultures causes a disappearance of TIMP-free (TF) proMMP-2 with a corresponding increase in the TIMP-complexed (TC) proMMP-2 levels, demonstrating that TF proMMP-2 is capable of converting to TC pro-MMP-2 when free TIMP-2 is available. Surprisingly, RSVCEF cultures manifest a TIMP-2 population that is not complexed to MMP-2, despite the coexistence of TIMP-free proMMP-2. Gel-filtration analysis indicates that this uncomplexed TIMP-2 exhibits an apparent molecular weight of 50 kDa, indicating it is not free TIMP-2 and that it exists in transformed cultures in a noncovalent complex with an undefined molecule. Thus transformed cells can alter the TIMP-2/MMP-2 balance by transcriptional and post-translational modifications, yielding a population of inhibitor-free, proteolytically active MMP2.
- Subjects :
- Amino Acid Sequence
Animals
Avian Sarcoma Viruses
Base Sequence
Cell Line, Transformed
Cells, Cultured
Chick Embryo
Culture Media, Conditioned metabolism
DNA, Complementary isolation & purification
Enzyme Precursors biosynthesis
Fibroblasts cytology
Fibroblasts enzymology
Gelatinases metabolism
Gene Expression
Humans
Matrix Metalloproteinase 2
Metalloendopeptidases metabolism
Mice
Molecular Sequence Data
Multienzyme Complexes metabolism
Recombinant Proteins biosynthesis
Recombinant Proteins isolation & purification
Tissue Inhibitor of Metalloproteinase-2 chemistry
Cell Transformation, Viral genetics
Cloning, Molecular
Tissue Inhibitor of Metalloproteinase-2 biosynthesis
Tissue Inhibitor of Metalloproteinase-2 genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9541
- Volume :
- 174
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Journal of cellular physiology
- Publication Type :
- Academic Journal
- Accession number :
- 9462696
- Full Text :
- https://doi.org/10.1002/(SICI)1097-4652(199803)174:3<342::AID-JCP8>3.0.CO;2-O