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Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase‐2 (TIMP‐2) in normal and transformed chicken embryo fibroblasts
Author(s) -
Aimes Ronald T.,
Li LingHui,
Weaver Brian,
Hawkes Susan,
HahnDantona Elizabeth A.,
Quigley James P.
Publication year - 1998
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199803)174:3<342::aid-jcp8>3.0.co;2-o
Subject(s) - rous sarcoma virus , tissue inhibitor of metalloproteinase , embryo , metalloproteinase , microbiology and biotechnology , biology , messenger rna , complementary dna , clone (java method) , matrix metalloproteinase , population , cloning (programming) , amino acid , peptide sequence , transformation (genetics) , gene , biochemistry , computer science , programming language , demography , sociology
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 λgt11 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. J. Cell. Physiol. 174:342–352, 1998. © 1998 Wiley‐Liss, Inc.