Circadian mechanisms in murine and human bone marrow mesenchymal stem cells following dexamethasone exposure

Abstract

A core group of regulatory factors control circadian rhythms in mammalian cells. While the suprachiasmatic nucleus in the brain serves as the central core circadian oscillator, circadian clocks also exist within peripheral tissues and cells. A growing body of evidence has demonstrated that >20% of expressed mRNAs in bone and adipose tissues oscillate in a circadian manner. The current manuscript reports evidence of the core circadian transcriptional apparatus within primary cultures of murine and human bone marrow-derived mesenchymal stem cells (BMSCs). Exposure of confluent, quiescent BMSCs to dexamethasone synchronized the oscillating expression of the mRNAs encoding the albumin D binding protein (dbp), brain-muscle arnt-like 1 (bmal1), period 3 (per3), rev-erb alpha (Rev A), and rev-erb beta (Rev B). The genes displayed a mean oscillatory period of 22.2 to 24.3 h. The acrophase or peak expression of mRNAs encoding "positive" (bmal1) and "negative" (per3) components of the circadian regulatory apparatus were out of phase with each other by approximately 8-12 h, consistent with in vivo observations. In vivo, phosphyrylation by glycogen synthase kinase 3beta (GSK3beta) is known to regulate the turnover of per3 and components of the core circadian regulatory apparatus. In vitro addition of lithium chloride, a GSK3beta inhibitor, significantly shifted the acrophase of all genes by 4.2-4.7 h oscillation in BMSCs; however, only the male murine BMSCs displayed a significant increase in the length of the period of oscillation. We conclude that human and murine BMSCs represent a valid in vitro model for the analysis of circadian mechanisms in bone metabolism and stem cell biology.

MaternalUbe3aLoss Disrupts Sleep Homeostasis But Leaves Circadian Rhythmicity Largely Intact

“…All data are plotted relative to Gapdh as a loading control (DiTacchio et al, 2011). Dbp, RevErb␣, and Bmal1 primers were described in (Wu et al, 2008), and the Per1 and Cry1 primers were described previously (Anand et al, 2013). Rhythms in Bmal1-Luc U2OS cells were monitored using a Lumicycle (Actimetrics) starting 48 h after transfecting cells with 10 pmol of AllStars negative control (Qiagen) or siRNAs directed against Ube3a (Qiagen, catalog #GS7337) as described previously (Yamazaki and Takahashi, 2005;Baggs et al, 2009).…”

“…Previous work had established that, due to silencing of the paternal allele, Ube3a expression in mature neurons arises from the maternal allele (Rougeulle et al, 1997;Yamasaki et al, 2003;Judson et al, 2014). We therefore expected UBE3A protein to also be absent from the SCN of Ube3a mϪ/pϩ mice.…”

“…The paternally inherited UBE3A allele is epigenetically silenced in almost all neurons Rougeulle et al, 1997;Yamasaki et al, 2003); thus, the loss of a functional maternal UBE3A allele causes a nearly complete loss of UBE3A protein in most regions of the mammalian brain (Gustin et al, 2010;Judson et al, 2014). In mice, this epigenetic silencing of the paternal allele is found in neurons by the first postnatal week, while glial cells preserve biallelic expression of Ube3a into adulthood (Judson et al, 2014).…”

“…The UBE3A protein (also known as E6-AP) is an E3 ubiquitin ligase that targets substrate proteins for proteasomal degradation (Scheffner et al, 1993). In Drosophila, loss of the UBE3A protein disrupts circadian patterns of locomotor activity (Wu et al, 2008;Gossan et al, 2014). In mammals, maternal deletion or RNAi-mediated knockdown of Ube3a can cause subtle alterations in circadian rhythmicity, presumably by stabilizing the core clock protein BMAL1 (Gossan et al, 2014;Shi et al, 2015).…”

Circadian Rhythms in Stem Cell Biology and Function

“…Mesenchymal stem cells (MSCs), adult stem cells that can differentiate into cells of the mesoderm, such as osteoblasts, chondrocytes, and adipocytes, provide a good example of this. When MSCs are cultured in vitro, 24-h oscillations in gene expression of core clock genes such as Bmal1 , Cry1 , Per2/3 , Rev-erbα/β , and Dbp are found, indicating that the peripheral circadian clock is present in these cells, independent of the presence of the central clock (Wu et al 2008 ). Differences in expression between MSCs of old and young animals are observed (Yu et al 2011 ).…”