Global expression profiling reveals regulation of CTGF/CCN2 during lactogenic differentiation

Abstract

Mammary epithelial cells go through a series of developmental changes during pregnancy and lactation including proliferation, differentiation, secretion and apoptosis. HC11 mouse mammary epithelial cells, which undergo lactogen-induced differentiation in cell culture, were used to follow the changes in gene expression during this process. The expression profiles of over 20,000 genes were compared in HC11 cells undergoing lactogenic differentiation to non-differentiated cells using DNA microarray analysis. Greater than two fold changes were detected in 998 genes in the differentiated cells versus growth controls. Several genes including CTGF/CCN2 exhibited greater than five-fold increase. Validation of the gene expression pattern for more than twenty genes was performed. The results indicate the involvement of numerous genes and pathways in the differentiation of mouse mammary epithelial cells in culture and they identify genetic pathways associated with specific transcriptional regulation. In addition, the expression of a subset of genes regulated by lactogenic differentiation in HC11 cells, including CTGF/CCN2 and osteopontin, was examined in mouse mammary glands revealing expression during pregnancy and lactation that declined during involution of the glands. To probe the mechanism by which epidermal growth factor (EGF), a known inhibitor of lactogenic differentiation in HC11 cells, blocks lactogenesis, the HC11 cells stimulated with lactogenic hormone in the presence of EGF were profiled. This data revealed EGF regulation of a specific subset of genes including important cell cycle regulators. The studies confirm the value of expression profiling in defining gene transcription associated with differentiation of mammary epithelial cells.

Fig. 1

Northern blotting of genes exhibiting elevated expression in HC11 cells undergoing lactogenic differentiation. a. HC11 mouse mammary epithelial cells were cultured in complete medium with EGF for 4 days post confluence followed by incubation in the media without EGF for 24 h. The cells were then stimulated with differentiation media containing dexamethasone (10⁻⁶ M), insulin(5 µg/ml) and prolactin (5 µg/ml) for 0, 12, 24, 48, and 72 h. RNA was extracted and used for northern blots. The probes are described in Materials and Methods. The fold changes of gene expression of each probe normalized to β-actin is shown. b. Northern blotting of genes exhibiting elevated expression in HC11 cells hybridized to RNA from mouse mammary glands. The probes used in part A were hybridized to northern blots of RNA extracted form mouse mammary glands at various stages of pregnancy and lactation. The RNAs are from pregnancy days 10, 12 and 16; lactation days 1 and 3; involution days 1 and 7; and NP represents RNA from non-pregnant mammary glands of adult female mice. The blots were hybridized to β-actin as a loading control. C. HC11 cells undergoing lactogenic differentiation. Control = untreated cells, DIP = cells exposed to DIP for 5 days. Top panel: cells at magnification 200X; bottom panel: cells at magnification of 500X

Fig. 2

CTGF/CCN2 expression is regulated by dexamethasone in HC11 cells. HC11 mammary epithelial cells were grown to confluence and stimulated with dexamethasone (DEX)(1 μM) in serum-containing media in the presence of insulin. RU486 at varying concentrations or vehicle (ethanol) was added to cells alone or in combination with dexamethasone. RNA was isolated and levels of CTGF and actin RNA were determined by realtime PCR. The results indicate the induction of CTGF/CCN2 normalized to actin. *These Dex+Ru486 values represent statistically significant difference (p-value .001) from the Dex alone condition

Fig. 3

Northern blot results of selected on RNA from HC11 mouse mammary epithelial cells undergoing lactogenic differentiation in the presence and absence of EGF. HC11 cells were grown as described in Fig. 1A and then incubated in differentiation media containing dexamethasone (10⁻⁶ M), insulin (5 µg/ml) and prolactin (5 µg/ml) with or without EGF (10 ng/ml) for 0 or 72 h. RNAs were extracted and used for Northern blot with the probes indicated. The blots were hybridized to β-actin as a loading control