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Proprotein convertases are important mediators of the adipocyte differentiation of mouse 3T3-L1 cells

¹ Diseases of Aging Program, Ottawa Health Research Institute at Ottawa Hospital, University of Ottawa, 725 Parkdale Avenue, Ottawa, Ontario K1Y 4K9, Canada
² Biochemical Neuroendocrinology Laboratory, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec H2W 1R7, Canada

View larger version (31K): [in a new window]   Fig. 1. Levels of PACE4, PC7 and furin transcripts in 3T3-L1 cells during adipocyte conversion. (A) Total RNA extracted from 3T3-L1 preadipocytes and adipocytes was analyzed by semi-quantitative RT-PCR as described in Materials and Methods. RNA from epididymal WAT was also examined for comparison. (B) Ratios between PC and L30 densitometric values obtained by semi-quantitative RT-PCR were determined. They are presented as percent of maximal expression of each PC. The values represent means±s.e.m. of five independent experiments.

View larger version (20K): [in a new window]   Fig. 2. 1-PDX expression inhibits 3T3-L1 adipocyte differentiation. (A) Three control and 16 1-PDX 3T3-L1 independent transfectant cell lines at confluence were treated (or not) with adipogenesis-inducing agents for 2 days. They were subsequently cultured for 5 days, fixed and stained with Oil Red O on day 7. A typical staining of differentiated and undifferentiated cells is shown. Unlike control cells, 1-PDX transfectant cell lines failed to respond to the adipogenic treatment. (B) Expression of 1-PDX transgene by RT-PCR in control and 1-PDX transfectants cell lines. Transcripts were observed in 1-PDX cells and not in control cells.

View larger version (77K): [in a new window]   Fig. 3. Effect of exogenously added serine protease inhibitors on adipocyte conversion. Post-confluent 3T3-L1 cells were incubated for 24 hours before and 48 hours during the adipogenic induction in medium containing 1-AT, 1-PDX or dec-RVKR-CMK. They were then cultured for 5 days in normal medium and stained with Oil Red O on day 7 to assess conversion. (A) Staining was reduced in cells treated with 8 µM 1-PDX, but not in those treated with 1-AT. (B) Treatment with dec-RVKR-CMK reduced the staining in a concentration-dependent manner. The same results were obtained in four separate experiments. (C) By general cellular morphology, blockage of adipocyte conversion was partial at 20 µM dec-RVKR-CMK and complete at 100 µM.

View larger version (33K): [in a new window]   Fig. 4. Expression of PPAR and adipsin transcripts in 3T3-L1 transfectants. Total RNA isolated on days 0, 1, 2, 3, 5 and 7 after adipogenic induction was analyzed by semi-quantitative RT-PCR as described in Materials and Methods. Results are representative of five independent experiments on three individual clones of each transfectant type. The levels of PPAR and adipsin transcripts markedly increased in control cells during differentiation. In 1-PDX cells, the increase was significantly lower for adipsin and not observed for PPAR.

View larger version (40K): [in a new window]   Fig. 5. C/EBPß expression, binding activity and localization in 3T3-L1 transfectants. (A) Control and 1-PDX cells were subjected to an adipogenic treatment as described in Materials and Methods. Cell extracts were prepared on days 0, 1, 2, 3, 5 and 7. Aliquots of 25 µl were fractionated by SDS-PAGE and analyzed by immunoblotting for C/EBPß. The antibody recognized the LAP (liver-enriched activating protein, 32 kDa) and the LIP (liver-enriched inhibitor protein, 18 kDa) C/EBPß isoforms. Control and 1-PDX producing cells carried equivalent amounts of both isoforms. (B) Nuclear extracts were prepared from control or 1-PDX expressing preadipocytes treated with adipogenesis-inducing agents for 24 hours. An EMSA of a radiolabeled C/EBP consensus oligonucleotide probe was conducted using 10 µg of nuclear extract proteins. For supershift assays, the extract was preincubated with an antibody directed against the C-terminus of C/EBPß. In competition assays, a 100-fold molar excess of unlabeled oligonucleotide was supplemented to the binding mixture. (C) The nuclear extracts (50 µg of proteins) analyzed for binding activity in (B) were fractionated by SDS-PAGE and further analyzed by immunoblotting for C/EBPß. Extracts from 1-PDX-expressing cells contained less C/EBPß LAP than those from control cells (lanes 1,2). The upper band in lane 1 corresponded to a hyperphosphorylated form of C/EBPß as demonstrated by its disappearance in CIAP-treated extracts (lanes 3,4). The blots shown are representative of three separate experiments with three control and three 1-PDX clonal lines. (D) Immunohistochemical analysis on 3T3-L1 transfectant cells after adipogenic treatment for 24 hours. Localization of C/EBPß was nuclear in control cells (c) and perinuclear in 1-PDX-expressing cells (d). Panels a and c represent control samples not treated with the anti-C/EBPß primary antibody.

View larger version (26K): [in a new window]   Fig. 6. Effect of PC inhibitors on IGF-1 receptor pathway. (A) Confluent 3T3-L1 preadipocyte cells treated or not with dec-RVKR-CMK (100 µM) for 48 hours (lanes 1,2) as well as control and 1-PDX cells (lanes 3,4) were analyzed by immunoblot for proIGF-1R processing. The arrowhead points to an unknown immuno-crossreactive protein that was unaffected by the inhibitor. (B) IRS-1 phosphorylation was analyzed by sequential probing of a blot carrying proteins from control and 1-PDX cells, stimulated or not with insulin (10 µg/ml). The first probing was conducted with an anti-IRS-1 antibody and the second probing was conducted with an anti-phosphotyrosine antibody. (C) Effect on mitotic clonal expansion. Control and 1-PDX cells were induced or not to differentiate. After 3 days, cells from 6-well culture dishes were trypsinized and cell number was determined by counting. Data shown are the means±s.e. Results are representative of three independent experiments with three individual clones of each cell lines.