title: CLOCK regulates mammary epithelial cell growth and differentiation.
abstract: Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63 qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.
authors.affiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana; theresa-casey@purdue.edu.
authors.forename: Theresa
authors.identifier: 0000-0002-8835-3550
authors.initials: T
authors.lastname: Casey
authors.affiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Crodian
authors.affiliation: Department of Animal Science, Universidad de Las Palmas de Gran Canaria, Arucas, Canary Islands, Spain.
authors.forename: Aridany
authors.initials: A
authors.lastname: Suárez-Trujillo
authors.affiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana.
authors.forename: Emily
authors.initials: E
authors.lastname: Erickson
authors.affiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana.
authors.forename: Bethany
authors.initials: B
authors.lastname: Weldon
authors.affiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana.
authors.forename: Kristi
authors.initials: K
authors.lastname: Crow
authors.affiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana.
authors.forename: Shelby
authors.initials: S
authors.lastname: Cummings
authors.affiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana.
authors.forename: Yulu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Ruminant, Agriculture Research Organization, Volcani Center, Bet Dagan, Israel; and.
authors.forename: Avi
authors.initials: A
authors.lastname: Shamay
authors.affiliation: Department of Ruminant, Agriculture Research Organization, Volcani Center, Bet Dagan, Israel; and.|Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
authors.forename: Sameer J
authors.initials: SJ
authors.lastname: Mabjeesh
authors.affiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana.
authors.forename: Karen
authors.initials: K
authors.lastname: Plaut
mesh_terms: D000818:Animals; D056926:CLOCK Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology*; D002478:Cells, Cultured; D002940:Circadian Rhythm / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D008297:Male; D008321:Mammary Glands, Animal / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D056926:CLOCK Proteins; C538944:Clock protein, mouse
keywords: CLOCK; circadian; lactation; mammary development