4675 Introduction Hormonal stimuli during pregnancy lead to extensive proliferation of the epithelial compartment, culminating in the differentiation of the milk-secreting alveolar units, while adipocytes lose their lipid stores. During lactation, the tissue is relatively static and produces large quantities of milk. Involution, experimentally initiated by removal of the pups at mid-lactation, is caused by accumulation of milk and changes in hormone levels, which trigger cascades of molecular events leading to dramatic tissue reorganization (Green and Streuli, 2004). Our current knowledge on these processes has been summarized in a recent, comprehensive review (Green and Streuli, 2004). Involution can be divided into two morphologically recognizable stages. The first reversible phase constitutes the initiation of mammary epithelial cell (MEC) apoptosis and the second phase includes extensive tissue remodeling (Lund et al., 1996). Furthermore, adipocytes refill during the process of involution. Microarray expression analyses lead to the characterization of several distinct clusters of gene expression patterns, suggesting a number of sub-stages with intricate regulation and communication of multiple signaling pathways occurring in several cell types that fine- tune the tissue remodeling process (Clarkson et al., 2004; Stein et al., 2004). Several hundred genes are dynamically regulated during involution. However, a specific role has been established by gene targeting approaches in mice for only a few. According to such studies, the signals contributing to initiation of epithelial cell apoptosis include death-receptor activation and TGF3/SMAD3 signaling (Green and Streuli, 2004). Well characterized is the crucial role of the signal transducer and activator of transcription 3 (STAT3), which is activated by the gp130 cytokine receptor with leukemia inhibitory factor (LIF) as a critical activating ligand (Kritikou et al., 2003; Zhao et al., 2004). Stat3 null mice are embryonic lethal (Takeda et al., 1997). However, conditional deletion of the Stat3 gene in secretory MEC leads to a severe delay in involution with expanded, lactation-competent alveoli present for several days. Stat3 null epithelial cells exhibit significantly less apoptosis than controls and protease activity in the second phase of involution is delayed (Chapman et al., 1999; Humphreys et al., 2002). STAT3 mediates MEC apoptosis in part by inducing expression of regulatory PI(3) kinase subunits and IGFBP5, which lead to inhibition of the pro-survival signals from AKT and IGF1 (Abell et al., 2005; Chapman et al., 1999). Another gene with reduced expression in both LIF and STAT3-deficient mammary glands encodes the transcription factor C/EBP (CEBPD, CRP3, CELF, NF-IL6) (Kritikou et al., 2003). C/EBP is a member of the C/EBP family of transcription factors, which dimerize and bind DNA through a The STAT3 transcription factor is an important initiator of mammary gland involution in the mouse. This work shows that the STAT3 target gene CCAAT/enhancer binding protein delta (C/EBP) is a crucial mediator of pro- apoptotic gene expression events in mammary epithelial cells. In the absence of C/EBP, involution is delayed, the pro-apoptotic genes encoding p53, BAK, IGFBP5 and SGP2/clusterin are not activated, while the anti-apoptotic genes coding for BFL1 and Cyclin D1 are not repressed. Consequently, p53 targets such as survivin, BRCA1, BRCA2 and BAX are not regulated appropriately and protease activation is delayed. Furthermore, expression of MMP3 and C/EBP during the second phase of involution is perturbed in the absence of C/EBP. In HC11 cells, C/EBP alone is sufficient to induce IGFBP5 and SGP2. It also suppresses Cyclin D1 expression and cooperates with p53 to elicit apoptosis. This study places C/EBP between STAT3 and several pro- and anti-apoptotic genes promoting the physiological cell death response in epithelial cells at the onset of mammary gland involution. Key words: Mammary gland, Involution, C/EBP, Apoptosis, Mouse Summary C/EBP is a crucial regulator of pro-apoptotic gene expression during mammary gland involution Muthusamy Thangaraju 1, *, Martina Rudelius 2 , Brian Bierie 3,† , Mark Raffeld 2 , Shikha Sharan 1 , Lothar Hennighausen 3 , 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 *Present address: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-0006, USA † Present address: Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA ‡ Present address: Department of Biochemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan § Author for correspondence (e-mail: sterneck@ncifcrf.gov) Accepted 19 August 2005 Development 132, 4675-4685 Published by The Company of Biologists 2005 doi:10.1242/dev.02050 Research article Development