Expression of CCAAT/enhancer binding protein C/EBP alpha, beta and delta in rat adipose stromal-vascular cells in vitro

Stromal-vascular (S-V) cells from rat inguinal fat depots were isolated and cultured in medium containing fetal bovine serum (FBS) and differentiated in defined medium until lipid accumulation was apparent. C/EBP alpha, beta and delta levels were evaluated for different growth conditions and at diff erent times using Western blots. Immediately after isolation C/EBP alpha, b eta and delta could not be detected in S-V cells. After seeding for 24 h in Dulbecco's modified Eagle's medium (DMEM) with FBS, C/EBP alpha, beta and delta could all be detected. Cells at day 1 of culture in insulin, transfer rin, triiodothyronine and selenium (ITTS) had increased levels of C/EBP alp ha and continued steady high levels to day 6 of culture. Cultures grown in DMEM alone, with no ITTS, showed C/EBP alpha levels similar to ITTS culture s at day 1 and day 3; however, levels diminished after day 3. DMEM cultures also showed lipid accumulation at day 6; however, the number of cells and the amount of lipid cell were reduced from levels observed in ITTS cultures . C/EBP beta was expressed uniformly throughout the culture period in eithe r DMEM or ITTS cultures while C/EBP delta expression was higher with DMEM t reatment than with ITTS. Treatment of 2 day DMEM cultures with FBS increase d levels of C/EBP beta and delta but significantly reduced levels of C/EBP alpha. Immunocytochemical analysis of S-V cells at day 1 of culture showed a similar percentage of cells stained in DMEM cultures and ITTS cultures. H owever, by day 6 of culture the percentage of cells staining positively for C/EBP alpha in DMEM had been reduced by one half while in ITTS the percent positive cells remained about the same. Our results indicate that ITTS is not necessary for the induction of C/EBP alpha and accumulation of lipid in S-V cells. However, ITTS is responsible for maintaining C/EBP alpha and en hanced lipid accumulation. Because C/EBP alpha, beta and delta expression o ccurs very early in cell culture and C/EBP alpha and delta expression conti nues to increase in DMEM without any apparent inducing agents, our results suggest that these factors may be expressed by the same cells in vivo befor e being placed in culture. Thus, a large fraction of S-V cells may be furth er along in the differentiation program than 3T3 cells are when they begin differentiation. (C) 1999 Elsevier Science B.V. All rights reserved.