Characterization and purification of a secreted plasminogen activator inhibitor (PAI-1) induced by transforming growth factor-beta 1 in normal rat kidney (NRK) cells: decreased PAI-1 expression in transformed NRK cells.
Type 1 transforming growth factor-beta (TGF beta 1) was found to be a potent inducer of the secretion of a 49,000 mol wt protein by normal rat kidney (NRK) cells. This protein was related to type 1 plasminogen activator inhibitor (PAI-1) on the basis of molecular mass, activity in the presence of sodium dodecyl sulfate, immunoprecipitation by antibodies to PAI-1, and N-terminal sequence analysis. PAI-1 levels in the conditioned medium of NRK cells were increased 5- to 11-fold when cells were incubated with picomolar concentrations of TGF beta 1 for 24 h, reaching a concentration of approximately 0.3 microgram/ml. The secreted PAI-1 was deposited in the NRK extracellular matrix as well as released into the culture medium. A spontaneously transformed NRK cell line was found to secrete 3-4 times less PAI-1, in the absence or presence of TGF beta 1, compared to the parent cell line, while PAI-1 secretion in Kirsten sarcoma virus-transformed NRK cells was almost completely abrogated. A novel purification procedure was established, which results in the isolation of highly active and detergent-free TGF beta 1-induced PAI-1.
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
M J
MJ
Newman
E A
EA
Lane
A M
AM
Iannotti
M A
MA
Nugent
R B
RB
Pepinsky
J
J
Keski-Oja
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002472:Cell Transformation, Viral; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005109:Extracellular Matrix / Q000378:metabolism; D007163:Immunosorbent Techniques; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D007708:Kirsten murine sarcoma virus; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015849:Plasminogen Inactivators / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D015291:Transforming Growth Factors / Q000008:administration & dosage / Q000494:pharmacology*
D015849:Plasminogen Inactivators; D003609:Dactinomycin; D012967:Sodium Dodecyl Sulfate; D015291:Transforming Growth Factors