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First published online 28 September 2005
doi: 10.1242/dev.02050

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C/EBP is a crucial regulator of pro-apoptotic gene expression during mammary gland involution

Muthusamy Thangaraju^1,*, Martina Rudelius², Brian Bierie^3,, Mark Raffeld², Shikha Sharan¹, Lothar Hennighausen³, A-Mei Huang^1, and Esta Sterneck^1,

¹ Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA
² Laboratory of Pathology, National Cancer Institute, National Institute of Health, Bethesda MD 20892, USA
³ Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 20892, USA

View larger version (133K): [in a new window]   Fig. 1. C/EBP is expressed in luminal epithelial cells. Immunohistochemistry of C/EBP in wild-type and C/EBP null mammary glands at the indicated time points of involution after 7 days of lactation. Representative sections at 400 x and 50 x original magnification are shown (insets). M, myoepithelial cells; solid arrows, positive stromal cells; open arrows, negative stromal cells.

View larger version (42K): [in a new window]   Fig. 2. C/EBP and MMP3 expression in the mammary gland are dependent on STAT3. Northern analysis of the indicated genes in total RNA from mammary glands of mice with a floxed Stat3 allele and with or without the WAP-cre (WC) transgene during lactation (involution day 0) and 1-3 days after removal of the pups (two to four independent mice per group).

View larger version (129K): [in a new window]   Fig. 3. Involution is delayed in C/EBP null mammary glands. Comparison of mammary gland histology during involution in wild-type (WT) and C/EBP null (KO) mice. (A) Representative photographs of sections from two mice per indicated time points (original magnification 50x). (B) Quantification of the areas occupied by adipocytes in mammary gland sections at days 1-4 of involution (three to four mice per time point). **P<0.01, ***P<0.01, *****P<0.00001, by two-way ANOVA with follow-up tests.

View larger version (105K): [in a new window]   Fig. 4. Pro-apoptotic protein expression and activity are delayed in C/EBP null mammary glands. Western blot analysis of the indicated proteins in total protein extracts (50 µg each) from mammary glands of wild-type and C/EBP null mice on days 0-4 and 8 of involution.

View larger version (60K): [in a new window]   Fig. 5. Involution-associated gene expression patterns are significantly altered in the absence of C/EBP. (A) Multiprobe ribonuclease protection assay of the indicated genes in total RNA from mammary glands of two mice per genotype and time point. Connecting lines were drawn as a visual aid. Shaded boxes indicate the time points of most significant differences between wild-type and C/EBP null glands. (B) Northern analysis of the indicated genes in total RNA from mammary glands during lactation (Inv d0) and 1-4 days after removal of the pups (two mice per group). (C) A selection of the data from panel B quantified by phosphoimaging and normalized to cyclophilin (CK18) or CK18 (IGFBP5, SGP2, p53) expression. (D) Northern analysis of the indicated genes in total RNA from mammary glands on days 0 and 1 of involution. Data shown are the phosphoimage quantifications of expression levels in four to five mice per data point normalized to cyclophilin expression. a.u., arbitrary units.

View larger version (23K): [in a new window]   Fig. 6. C/EBP induces involution-related genes in HC11 cells and cooperates with p53 in inducing apoptosis. The non-transformed mouse mammary epithelial cell line HC11 was transiently transfected with constructs expressing the indicated proteins, and harvested after 2 days. (A) Northern analysis of the indicated genes in total RNA from representative samples are shown. (B) The flow cytometric quantification of arrested (G1 DNA content) and (C) apoptotic cells (sub-G1 DNA content) represents the means ± s.d. of six to eight samples from three to four independent experiments.

View larger version (13K): [in a new window]   Fig. 7. Schematic representation of the gene expression events following C/EBP induction by the STAT3 pathway in involuting mouse mammary glands. Dashed lines indicate interactions that may be either direct or indirect. Solid lines indicate probable direct interactions.