The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development.

During the course of differentiation of preadipocytes into adipocytes, several differentiation-linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3-F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP) is an important factor in determining the ability to accumulate lipid droplets in terminally differentiated adipocytes. In one experiment we can suppress C/EBP expression through administration of hydrocortisone to differentiating 3T3-F442A cells, which is accompanied by an inability of the cells to accumulate lipid. In another experiment a C/EBP antisense expression vector has been stably introduced into 3T3-F442A cells and as compared with control cells, a 62% decrease of C/EBP mRNA (p less than 0.01) is demonstrated. This decrease of C/EBP mRNA is accompanied by a change in cellular morphology characterized by a reduced ability to form lipid droplets. We can also demonstrate a correlation between the degree of reduction of C/EBP mRNA and the amount of lipid present in the cells. These findings strongly support the view that C/EBP is a necessary component of terminal adipocyte differentiation.     

Nulliparous CCAAT/enhancer binding proteindelta (C/EBPdelta) knockout mice exhibit mammary gland ductal hyperlasia

CCAAT/Enhancer binding proteins (C/EBPs) are a family of nuclear proteins that function in the control of cell growth, death, and differentiation. We previously reported that C/EBPdelta plays a key role in mammary epithelial cell G(0) growth arrest. In this report, we investigated the role of C/EBPdelta in mammary gland development and function using female mice homozygous for a targeted deletion of C/EBPdelta (C/EBPdelta -/-). C/EBPdelta -/- females develop normally and exhibit normal reproductive and lactational performance. Adult nulliparous C/EBPdelta -/- females, however, exhibit mammary epithelial cell growth control defects. The mean number of mammary ductal branches is significantly higher in adult nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ (wild-type control) females (66.8 +/- 5.2 vs 42.9 +/- 6.3 branch points/field, P < 0.01). In addition, the mean total mammary gland cellular volume occupied by epithelium is significantly higher in adult nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ controls (29.0 +/- 1.4 vs 20.4 +/- 1.3, P < 0.001). Our results showed that the BrdU labeling index was significantly higher in mammary epithelial cells from nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ controls during the proestrus/estrus (4.55 +/- 0.70 vs 2.14 +/- 0.43, P < 0.01) and metestrus/diestrus (6.92 +/- 0.75 vs 3.98 +/- 0.43 P < 0.01) phases of the estrus cycle. In contrast, the percentage of mammary epithelial cells undergoing apoptosis during both phases of the estrus cycle did not differ between C/EBPdelta -/- and C/EBPdelta +/+ females. The increased epithelial cell content and proliferative capacity was restricted to the nulliparous C/EBPdelta -/- females as no differences in mammary gland morphology, ductal branching or total epithelial content were observed between multiparous C/EBPdelta -/- and C/EBPdelta +/+ females. These results demonstrate that C/EBPdelta plays a novel role in mammary epithelial cell growth control that appears to be restricted to the nulliparous mammary gland.