Up-regulation of per mRNA Expression by Parathyroid Hormone through a Protein Kinase A-CREB-dependent Mechanism in Chondrocytes

Abstract

In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h in both cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5 cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage.Recent studies have revealed that the endogenous circadian rhythmicity generated at the cellular level by circadian core oscillators resides not only in the hypothalamic suprachiasmatic nucleus of the anterior hypothalamus (1, 2), which is recognized as the mammalian central clock, but also in various peripheral tissues including heart (3), adipose tissue (4), pancreas (5), and liver (2). The suprachiasmatic nucleus is not essential for driving peripheral oscillations but rather acts as a synchronizer of peripheral oscillators, whereas the physiological rhythmicity may be under the direct control by their own local clock genes in peripheral tissues (7). In mice, the rhythmic transcription of two orthologs of the Drosophila Period (per) gene appears to be essential for circadian rhythms. Expression of mouse per (mper) genes is known to be positively regulated by other clock proteins belonging to the basic helix-loop-helix period/aryl hydrocarbon receptor nuclear translocator/single minded class, which are Clock and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), 2 respectively. In addition, mPer proteins constitute multimeric complexes with products of the cryptochrome (cry) genes, mcry1 and mcry2, which in turn negatively regulate the gene transcription mediated ...

Noradrenaline Induces Clock Gene Per1 mRNA Expression in C6 Glioma Cells Through β2-Adrenergic Receptor Coupled With Protein Kinase A – cAMP Response Element Binding Protein (PKA–CREB) and Src-Tyrosine Kinase – Glycogen Synthase Kinase-3β (Src–GSK-3β)

“…However, the hypothesis that α 1/2 -or β 1 -adrenergic systems might indirectly regulate NA-induced Per1 mRNA expression in other cells and tissues should not be entirely ruled out. The PKA-CREB signaling cascade coupled with β 2 adrenoceptors has been shown to play an important role in regulation of clock genes including Per1 in cerebellar granule cells and chondrocytes (32,33). In our study, this successive cascade was one of the regulatory components in NA-induced Per1 expression.…”

Up-regulation of Per1 expression by estradiol and progesterone in the rat uterus

“…In addition, other two important up-regulatory elements, the cAMP response element (CRE) and glucocorticoid response element (GRE), have been clearly defined by in vitro studies. In response to various resetting cues (Balsalobre et al 2000b, Tsuchiya et al 2005, Yamamoto et al 2005, Hinoi et al 2006, the CRE-binding protein (CREB) or glucocorticoid receptor (GR) was activated, which, in turn, transactivates the transcription of Per1 in the nucleus by binding to the CRE or GRE respectively (Travnickova-Bendova et al 2002, Yamamoto et al 2005, He et al 2007. It is important to note that both CREB and GR act to induce an acute and robust enhancement of Per1 transcription with a peak at 1 h. In contrast, estrogen and P 4 tend to induce a chronic up-regulation of Per1 expression.…”

“…So far, the identification of resetting cues and their regulatory effects on clockwork are mostly based on molecular biological studies in cultured cell lines in vitro. A variety of factors have been unraveled having the potential to reset circadian rhythms in peripheral cells, such as cAMP analog, forskolin, phorbol-12-myristate-13-acetate, endothelin, prostaglandin estradiol (E 2 ), fibroblast growth factor, calcium ionophores, retinoic acid, glucocorticoids, and parathyroid hormone (Akashi & Nishida 2000, Balsalobre et al 2000a,b, Tsuchiya et al 2005, Hinoi et al 2006, Nakahata et al 2006, Shirai et al 2006. All these factors could induce an acute activation of Per1 expression, and thus synchronize or reset the circadian rhythms in cultured cells.…”

A Negative Correlation Between Per1 and Sox6 Expression During Chondrogenic Differentiation in Pre-chondrocytic ATDC5 Cells

“…We first examined whether PTH affects the mRNA expression of endogenous factors essential for chondrogenic differentiation, including Runx2, Sox5, Sox6, and Sox9, at the concentration effective for inducing Per1 upregulation (6). Cells were exposed to 10 nM PTH for different periods up to 48 h, followed by determination of the mRNA levels by real time-based RT-PCR.…”

“…Amongst these endogenous effectors, PTH is believed to negatively regulate chondrogenesis along with positive regulation of osteoblastogenesis to promote osteoclastogenesis required for the elevated levels of circulating Ca 2+ (2), whereas exposure to PTH led to transient upregulation of the clock gene Per1 expression through the cAMP/protein kinase A signaling pathway in mouse pre-chondrocytic cell line ATDC5 cells and in organotypic cultured mouse metatarsals isolated before vascularization (6). These previous findings prompted us to elucidate the possible involvement of essential transcription factors such as Sox9 allies and Runx2 in mechanisms underlying the suppression of chondrogenesis by Per1 up-regulated in response to PTH in ATDC5 cells.…”