Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

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Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

10.1101/2021.02.23.432439 ◽

2021 ◽

Author(s):

Theresa Casey ◽

Aridany Suarez-Trujillo ◽

Shelby Cummings ◽

Katelyn Huff ◽

Jennifer Crodian ◽

...

Keyword(s):

Transcription Factor ◽

Circadian Clock ◽

Mammary Epithelial Cells ◽

Transcriptional Start Site ◽

Prolactin Receptor ◽

Mammary Development ◽

Mammary Epithelial ◽

Pcr Analysis ◽

Protein Coding ◽

Lower Expression

ABSTRACTThe role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). Q-PCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

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Socs2 and Elf5 Mediate Prolactin-Induced Mammary Gland Development

Molecular Endocrinology ◽

10.1210/me.2005-0473 ◽

2006 ◽

Vol 20 (5) ◽

pp. 1177-1187 ◽

Cited By ~ 102

Author(s):

Jessica Harris ◽

Prudence M. Stanford ◽

Kate Sutherland ◽

Samantha R. Oakes ◽

Matthew J. Naylor ◽

...

Keyword(s):

Transcription Factor ◽

Mammary Epithelial Cells ◽

Prolactin Receptor ◽

Mammary Epithelium ◽

Mammary Development ◽

Negative Regulator ◽

Serum Prolactin ◽

Cytokine Signaling ◽

Wild Type ◽

Prolactin Signaling

Abstract The proliferative phase of mammary alveolar morphogenesis is initiated during early pregnancy by rising levels of serum prolactin and progesterone, establishing a program of gene expression that is ultimately responsible for the development of the lobuloalveoli and the onset of lactation. To explore this largely unknown genetic program, we constructed transcript profiles derived from transplanted mammary glands formed by recombination of prolactin receptor (Prlr) knockout or wild-type mammary epithelium with wild-type mammary stroma. Comparison with profiles derived from prolactin-treated Scp2 mammary epithelial cells produced a small set of commonly prolactin-regulated genes that included the negative regulator of cytokine signaling, Socs2 (suppressor of cytokine signaling 2), and the ets transcription factor, E74-like factor 5 (Elf5). Homozygous null mutation of Socs2 rescued the failure of lactation and reduction of mammary signal transducer and activator of transcription 5 phosphorylation that characterizes Prlr heterozygous mice, demonstrating that mammary Socs2 is a key regulator of the prolactin-signaling pathway. Reexpression of Elf5 in Prlr nullizygous mammary epithelium restored lobuloalveolar development and milk production, demonstrating that Elf5 is a transcription factor capable of substituting for prolactin signaling. Thus, Socs2 and Elf5 are key members of the set of prolactin-regulated genes that mediate prolactin-driven mammary development.

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Nuclear Jak2 and Transcription Factor NF1-C2: a Novel Mechanism of Prolactin Signaling in Mammary Epithelial Cells

Molecular and Cellular Biology ◽

10.1128/mcb.02095-05 ◽

2006 ◽

Vol 26 (15) ◽

pp. 5663-5674 ◽

Cited By ~ 41

Author(s):

Jeanette Nilsson ◽

Gunnar Bjursell ◽

Marie Kannius-Janson

Keyword(s):

Transcription Factor ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Alternative Pathway ◽

Prolactin Receptor ◽

Mammary Epithelial ◽

Janus Kinase ◽

Prolactin Signaling ◽

Nuclear Factor 1 ◽

Classical Mechanism

ABSTRACT The classical mechanism by which prolactin transduces its signal in mammary epithelial cells is by activation of cytosolic signal transducer and activator of transcription 5 (Stat5) via a plasma membrane-associated prolactin receptor-Janus kinase 2 (Jak2) complex. Here we describe an alternative pathway through which prolactin via Jak2 localized in the nucleus activates the transcription factor nuclear factor 1-C2 (NF1-C2). Previous reports have demonstrated a nuclear localization of Jak2, but the physiologic importance of nuclear Jak2 has not been clear. We demonstrate that nuclear Jak2 regulates the amount of active NF1-C2 through tyrosine phosphorylation and proteasomal degradation. Our data also demonstrate a link between prolactin and p53 as well as the milk gene carboxyl ester lipase through nuclear Jak2 and NF1-C2. Hence, we describe a novel pathway through which nuclear Jak2 is subject to regulation by prolactin in mammary epithelial cells.

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The ETS Transcription Factor ESE-1 Transforms MCF-12A Human Mammary Epithelial Cells via a Novel Cytoplasmic Mechanism

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5548-5564.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5548-5564 ◽

Cited By ~ 39

Author(s):

Jason D. Prescott ◽

Karen S. N. Koto ◽

Meenakshi Singh ◽

Arthur Gutierrez-Hartmann

Keyword(s):

Breast Cancer ◽

Transcription Factor ◽

Epithelial Cells ◽

Nuclear Localization ◽

Human Breast Cancer ◽

Mammary Epithelial Cells ◽

Cell Transformation ◽

Mammary Epithelial ◽

Human Breast ◽

Human Mammary Epithelial

ABSTRACT Several different transcription factors, including estrogen receptor, progesterone receptor, and ETS family members, have been implicated in human breast cancer, indicating that transcription factor-induced alterations in gene expression underlie mammary cell transformation. ESE-1 is an epithelium-specific ETS transcription factor that contains two distinguishing domains, a serine- and aspartic acid-rich (SAR) domain and an AT hook domain. ESE-1 is abundantly expressed in human breast cancer and trans-activates epithelium-specific gene promoters in transient transfection assays. While it has been presumed that ETS factors transform mammary epithelial cells via their nuclear transcriptional functions, here we show (i) that ESE-1 protein is cytoplasmic in human breast cancer cells; (ii) that stably expressed green fluorescent protein-ESE-1 transforms MCF-12A human mammary epithelial cells; and (iii) that the ESE-1 SAR domain, acting in the cytoplasm, is necessary and sufficient to mediate this transformation. Deletion of transcriptional regulatory or nuclear localization domains does not impair ESE-1-mediated transformation, whereas fusing the simian virus 40 T-antigen nuclear localization signal to various ESE-1 constructs, including the SAR domain alone, inhibits their transforming capacity. Finally, we show that the nuclear localization of ESE-1 protein induces apoptosis in nontransformed mammary epithelial cells via a transcription-dependent mechanism. Together, our studies reveal two distinct ESE-1 functions, apoptosis and transformation, where the ESE-1 transcription activation domain contributes to apoptosis and the SAR domain mediates transformation via a novel nonnuclear, nontranscriptional mechanism. These studies not only describe a unique ETS factor transformation mechanism but also establish a new paradigm for cell transformation in general.

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Prolactin receptor on dissociated mammary epithelial cells at different stages of development

Molecular and Cellular Endocrinology ◽

10.1016/0303-7207(78)90087-4 ◽

1978 ◽

Vol 12 (3) ◽

pp. 285-298 ◽

Cited By ~ 29

Author(s):

S. Sakai ◽

J. Enami ◽

S. Nandi ◽

M.R. Banerjee

Keyword(s):

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Prolactin Receptor ◽

Mammary Epithelial ◽

Stages Of Development

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Circadian Gene PER2 Silencing Downregulates PPARG and SREBF1 and Suppresses Lipid Synthesis in Bovine Mammary Epithelial Cells

Biology ◽

10.3390/biology10121226 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1226

Author(s):

Yujia Jing ◽

Yifei Chen ◽

Shan Wang ◽

Jialiang Ouyang ◽

Liangyu Hu ◽

...

Keyword(s):

Lipid Metabolism ◽

Epithelial Cells ◽

Circadian Clock ◽

Lipid Droplets ◽

Mammary Epithelial Cells ◽

Lipid Synthesis ◽

Mammary Epithelial ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Bovine Mammary Epithelial Cells

PER2, a circadian clock gene, is associated with mammary gland development and lipid synthesis in rodents, partly via regulating peroxisome proliferator-activated receptor gamma (PPARG). Whether such a type of molecular link existed in bovines was unclear. We hypothesized that PER2 was associated with lipid metabolism and regulated cell cycles and apoptosis in bovine mammary epithelial cells (BMECs). To test this hypothesis, BMECs isolated from three mid-lactation (average 110 d postpartum) cows were used. The transient transfection of small interfering RNA (siRNA) was used to inhibit PER2 transcription in primary BMECs. The silencing of PER2 led to lower concentrations of cellular lipid droplets and triacylglycerol along with the downregulation of lipogenic-related genes such as ACACA, FASN, LPIN1, and SCD, suggesting an overall inhibition of lipogenesis and desaturation. The downregulation of PPARG and SREBF1 in response to PER2 silencing underscored the importance of circadian clock signaling and the transcriptional regulation of lipogenesis. Although the proliferation of BMECs was not influenced by PER2 silencing, the number of cells in the G2/GM phase was upregulated. PER2 silencing did not affect cell apoptosis. Overall, the data provided evidence that PER2 participated in the coordination of mammary lipid metabolism and was potentially a component of the control of lipid droplets and TAG synthesis in ruminant mammary cells. The present data suggested that such an effect could occur through direct effects on transcriptional regulators.

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