Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27.
Evidence is presented that calpain, a calcium-activated protease, degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation. Calpain activity is required during an early stage of the adipocyte differentiation program. Thus, inhibition of calpain with N-acetyl-Leu-Leu-norleucinal (ALLN) blocks clonal expansion and acquisition of the adipocyte phenotype only when added between 12 and 24 h after the induction of differentiation. Likewise, inhibition of calpain by overexpression of calpastatin, the specific endogenous inhibitor of calpain, prevents 2-day post-confluent preadipocytes from reentering the cell cycle triggered by the differentiation inducers. Inhibition of calpain with ALLN causes preadipocytes to arrest just prior to S phase and prevents phosphorylation of the retinoblastoma gene product, DNA replication, clonal expansion, and subsequent adipocyte differentiation but does not affect the expression of immediate early genes (i.e. fos, jun, C/EBPbeta, and C/EBPdelta). Inhibition of calpain by either ALLN or by overexpression of calpastatin blocks the degradation of p27. p27 is degraded in vitro by cell-free extracts from clonally expanding preadipocytes that contain "active" calpain but not by extracts from pre-mitotic preadipocytes that do not. This action is inhibited by calpastatin or ALLN. Likewise, p27 in preadipocyte extracts is a substrate for purified calpain; this proteolytic action was inhibited by heat inactivation, EGTA, or ALLN. Thus, extracellular signals from the differentiation inducers appear to activate calpain, which degrades p27 allowing density-dependent inhibited preadipocytes to reenter the cell cycle and undergo mitotic clonal expansion.
Patel
Y M
YM
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Lane
M D
MD
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology* / Q000502:physiology*; D000818:Animals; D002154:Calpain / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002999:Clone Cells; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D017781:Genes, Immediate-Early; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008938:Mitosis; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D014280:Triglycerides; D025521:Tumor Suppressor Proteins; C063261:acetylleucyl-leucyl-norleucinal; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases; D002154:Calpain