Cytochalasin D-mediated hyperinduction of the substrate-associated 52-kilodalton protein p52 in rat kidney fibroblasts.
Regulation of certain differentiated and housekeeping functions in cultured mammalian cells is significantly influenced by cell shape. The shape-modulating agent cytochalasin D (CD) was used, therefore, to elucidate potential cytoarchitectural influences affecting synthesis of a major 52 kDa secreted/substrate-associated protein (p52) of normal rat kidney (NRK) fibroblasts. Biosynthetic labeling experiments indicated that treatment of NRK cells with CD increased, by 10-18-fold, the medium content of an Mr 52,000 protein. Two-dimensional gel electrophoresis and peptide fragment mapping confirmed that the 52 kDa protein produced in abundance as a consequence of CD treatment was identical to p52 constitutively expressed by NRK cells. A lower mw protein (p50; Mr 50,000) was also resolved which, based on pl microheterogeneity, protease fragmentation profile, and sensitivity to tunicamycin, could be identified as a less-glycosylated form of p52. p50 and p52 were both detected in the matrix and medium compartments of NRK and NRK/CD cells. The matrix p52 content of CD-induced and uninduced cells, however, was significantly greater (by 200-500-fold) than the corresponding medium levels. This differential compartmentalization, the time course of p52 accumulation in the matrix of NRK/CD cells compared to its appearance in the medium, and the kinetics of p52 pulse-chase from the matrix collectively indicated that the matrix is the initial site of p52 deposition. Low levels of CD (1 microM) produced extensive disruptions of cellular microfilaments but did not result in an overall cell shape change nor a hyperinduction of p52. Morphologic rounding (seen in 10-100 microM CD) coincided with augmented p52 production. Transition from a flat to a round phenotype in NRK cells, or at least the generation of sufficient microfilament fragmentation to compromise cell-substrate adhesivity, appears to be an essential aspect of CD-mediated p52 hyperinduction.
Higgins
P J
PJ
Laboratory of Cell and Molecular Biology, Veterans Administration Medical Center, Albany, New York.
Ryan
M P
MP
Chaudhari
P
P
D000818:Animals; D002478:Cells, Cultured; D015638:Cytochalasin D; D003572:Cytochalasins / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008970:Molecular Weight; D014176:Protein Biosynthesis; D051381:Rats
D003572:Cytochalasins; D015638:Cytochalasin D