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the Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Japan.
Abstract
Circadian rhythms are regulated by a set of clock genes that form transcriptional feedback loops and generate circadian oscillation with a 24-hour cycle. Aging alters a broad spectrum of physiological, endocrine, and behavioral rhythms. Although recent evidence suggests that cellular aging contributes to various age-associated diseases, its effects on the circadian rhythms have not been examined. We report here that cellular senescence impairs circadian rhythmicity both in vitro and in vivo. Circadian expression of clock genes in serum-stimulated senescent cells was significantly weaker compared with that in young cells. Introduction of telomerase completely prevented this reduction of clock gene expression associated with senescence. Stimulation by serum activated the cAMP response element-binding protein, but the activation of this signaling pathway was significantly weaker in senescent cells. Treatment with activators of this pathway effectively restored the impaired clock gene expression of senescent cells. When young cells were implanted into young mice or old mice, the implanted cells were effectively entrained by the circadian rhythm of the recipients. In contrast, the entrainment of implanted senescent cells was markedly impaired. These results suggest that senescence decreases the ability of cells to transmit circadian signals to their clocks and that regulation of clock gene expression may be a novel strategy for the treatment of age-associated impairment of circadian rhythmicity.
Key Words: senescence clock gene aging CREB ERK
Introduction
Cellular senescence is a limited ability of primary human cells to divide when cultured in vitro and is accompanied by a specific set of phenotypic changes in morphology and gene expression and function. These phenotypic changes have been suggested to play a role in human aging and age-associated diseases.1 This hypothesis of cellular aging was established by Hayflick2 and is supported by the evidence that the replicative potential of primary cultured human cells is dependent on donor age and that the growth potential of cultured cells is correlated well with the mean maximum lifespan of the species of origin.2 We have previously reported that senescent vascular cells are predominately localized in the plaque of human atherosclerosis but not in normal lesions and that vascular cell senescence results in vascular dysfunction.3 Recently, we also demonstrated that atherogenic stimulation induces vascular cell senescence and vascular inflammation, thereby contributing to the development of atheroma.4 There is also evidence indicating that progressive telomere shortening, a biomarker of cellular aging, occurs in human blood vessels, which may be related to age-associated vascular diseases.5eC10 Thus, vascular cell senescence in vivo may contribute to the pathogenesis of vascular aging.11
Aging is associated with a variety of alterations of circadian rhythms.12,13 These include impairment of the rhythms for blood pressure, locomotor activity, core body temperature, and the sleep/wake cycle. In mammals, circadian rhythmicity is under the control of a molecular pacemaker that is composed of clock gene products.14eC16 These gene products constitute an oscillatory mechanism that is based on self-sustained transcriptional/translational feedback loops. The regulatory feedback loops can be divided into positive and negative limbs. The positive limb consists of the PAS helix-loop-helix transcription factors CLOCK and BMAL1, which form heterodimers and bind to E-box enhancer element, thus regulating transcription of the period genes and the cryptochrome genes. PER and CRY proteins are components of the negative limb, which attenuates activation of their own genes by CLOCK/BMAL1, thereby generating negative feedback. It has been shown that disruption of these genes such as Per2 and Bmal1 in mice not only affects behavior rhythmicity but also promotes the development of malignant tumors and metabolic syndrome.17,18 In mammals, the master pacemaker controlling the circadian rhythm is located in the suprachiasmatic nuclei (SCN). Several lines of evidence indicate that various peripheral tissues such as the heart and blood vessels as well as isolated cells including cardiovascular cells, also possess circadian oscillators and suggest that impaired functions of such peripheral clocks may contribute to the development of cardiovascular diseases.19eC27
It has been reported that the circadian rhythm of blood pressure is often impaired with advancing age, resulting in the lack of a decrease at night (nondipper), and this change is known to increase the risk of cardiovascular disease.28,29 Given the important role of vascular cell senescence in vascular aging, vascular cell senescence might impair circadian expression of clock genes, thereby promoting various cardiovascular disorders. In the present study, we found that circadian expression of clock genes was markedly impaired in senescent cells as compared with young cells both in vitro and in vivo. This impairment was associated with decreased responsiveness of cAMP response element-binding protein (CREB)-dependent signaling. Telomere lengthening or activation of CREB restored clock gene expression, suggesting a novel target for the treatment of age-associated alterations of circadian rhythms.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Cell Culture
Primary cultured human aortic vascular smooth muscle cells (HSMC) were purchased from Cambrex and were grown according to the instructions manufacturer. We used cells from passages 5 to 7 as young HSMC and cells from passages 15 to 16 as senescent HSMC. Mouse embryonic fibroblasts (MEF) were prepared from C57/BL6 mouse embryos at embryonic day 13.5 and cultured in DMEM plus 10% FBS. We defined senescent cells as cultures that did not show an increase of cell number and remained subconfluent for 2 weeks.
Plasmids and Retroviral Infection
Retroviral vectors were prepared as described in online data supplement available at http://circres.ahajournals.org. Retroviral stocks were generated by transient transfection of a packaging cell line (PT67, Clontech).
Luciferase Assay
The luciferase assay was performed using a dual luciferase reporter assay system according to the instructions of the manufacturer (Promega). The Per1 promoter luciferase reporter gene construct was a gift from Dr S. Yamazaki (University of Virginia).
Statistical Analysis
All values were expressed as the mean±SEM. Comparison of results between different groups was performed by 1-way ANOVA or the unpaired t test using StatView 5.0 (Abacus Concepts).
Results
Circadian Expression of Clock Genes by Senescent Vascular Cells
We first investigated whether cellular senescence had an effect on circadian expression of clock genes in vitro. We used serum-shocked cells, a well-known in vitro model of circadian expression of clock genes.30 HSMC were deprived of serum for 48 hours, transferred to medium containing 50% serum for 2 hours, and returned to serum-free medium. We subsequently harvested the cells at several time points and examined the expression of clock genes by Northern blot analysis. In young HSMC, serum stimulation induced a transient increase of PER2 expression followed by downregulation and sequential upregulation. Expression of PER2 returned to the initial level at 20 to 24 hours after treatment (Figure 1). Expression of BMAL1 exhibited complementary changes. BMAL1 expression increased to reach a peak at 12 hours and declined to the basal level at 20 to 24 hours after treatment (Figure 1A). In contrast, the response of clock gene expression to serum stimulation was markedly attenuated in senescent HSMC. The amplitude of the circadian oscillations of BMAL1 and PER2 was significantly lower in senescent HSMC compared with young HSMC (Figure 1A). Similar expression profiles were observed during 2448 hours after serum stimulation (Figure 1A; supplemental Figure I).
As a consequence of semiconservative DNA replication, the extreme termini of chromosomes are not duplicated completely, resulting in successive shortening of the telomeres with each cell division.31 Critically short telomeres are thought to trigger the onset of cellular senescence by inducing expression of cyclin-dependent kinase inhibitors such as p21Waf1/Cip1 and p16Ink4a. Telomerase is an enzyme that adds telomeric repeats to the ends of chromosomes.32,33 As expected, expression of cyclin-dependent kinase inhibitors was increased, and telomere length was shorter in senescent HSMC compared with young HSMC (Figure 1B and 1C). Senescent-associated -galactosidase activity was significantly increased (supplemental Figure IIA) and telomerase activity was decreased with cellular aging (Figure 1D). Introduction of telomerase catalytic component (TERT) markedly induced telomerase activity (Figure 1D). Induction of telomerase activity prevented telomere shortening as well as accumulation of cyclin-dependent kinase inhibitors despite extensive replication and thereby prolonged the lifespan of HSMC (Figure 1B and 1C and data not shown).34 Therefore, we examined whether inhibition of cellular senescence by telomerase prevented impairment of the oscillation of clock gene expression in HSMC. As shown in Figure 1A, the circadian rhythms and amplitude of clock genes were completely preserved in TERT-infected HSMC (passages 50 to 80). These results suggest that inhibition of telomere shortening could prevent senescent phenotypic changes in HSMC including impaired circadian rhythmicity.
CREB Protein Activation Reverses Circadian Rhythm Impairment Associated With Cellular Senescence
Because activation of CREB has been implicated in the generation of circadian rhythms in cells as well as in the SCN,35eC39 we examined CREB phosphorylation in HSMC after serum stimulation. In young HSMC, phosphorylation of CREB started to increase as early as 5 minutes after treatment. Activation of CREB by serum stimulation was significantly weaker in senescent HSMC compared with young HSMC but was preserved in TERT-infected HSMC (Figure 2A; supplemental Figure IIIA and IIIB). It has been reported that external stimuli such as light directly induces PER transcription by activating CREB in the SCN.35eC38 A rapid induction of PER subsequently generates circadian expression of clock genes including BMAL1. To test whether CREB activation was essential for the rhythmic expression of clock genes induced by serum stimulation, we examined the effect of CREB inhibition on PER transcription by using the PER gene reporter system. Introduction of a dominant-negative form of CREB (DNCREB) significantly suppressed PER transcription (Figure 2B). To further investigate the role of CREB activation, we infected young HSMC with a retroviral vector encoding DNCREB and analyzed clock gene expression after serum stimulation. As shown in Figure 2C, a rapid induction of the PER2 gene was significantly decreased, and circadian expression of BMAL1 was significantly lower in DNCREB-infected HSMC compared with mock-infected HSMC. These results suggest that CREB activation is an important pathway for the induction of circadian expression of clock genes in HSMC.
We next investigated whether the activation of CREB could restore impaired circadian expression of clock genes in senescent cells. Senescent HSMC were treated with a serum-rich medium containing forskolin, a well-known protein kinase A (PKA) activator, after which CREB phosphorylation and clock gene expression were analyzed. Treatment with forskolin significantly restored the increase of phospho-CREB levels after serum stimulation in senescent HSMC (Figure 3A). This improvement was significantly inhibited by treatment with the PKA inhibitor H-89, suggesting that the effect of forskolin was mediated by PKA activity (Figure 3A). Moreover, serum-induced clock gene expression was significantly improved by forskolin treatment of senescent HSMC (Figure 3B). These results suggest that cellular senescence interferes with the signal transduction pathway from extracellular stimulation to CREB activation and thereby impairs oscillation of clock gene expression.
Reduced Responsiveness of Extracellular Signal-Regulated Kinase in Senescent HSMC
Recent reports have demonstrated that the mitogen-activated protein kinase (MAPK) cascades are key regulators of the circadian clocks.40eC44 Treatment with high concentrations of serum also increased the level of phospho-extracellular signal-regulated kinase (ERK) in young HSMC, whereas this increase was significantly smaller in senescent HSMC (Figure 4A). The reduced response of ERK to serum stimulation in senescent HSMC was restored by the addition of phorbol 12-myristrate 13-acetate (PMA). Moreover, treatment with PMA caused an increase of phospho-CREB and thus improved the circadian rhythm of clock gene expression induced by serum stimulation (Figure 4B and 4C). This increase of CREB activity was attenuated by additional treatment with U0126, a MAPK kinase (MEK) inhibitor (Figure 4B), suggesting that serum-induced CREB activation was partly mediated via the MEK/ERK pathway and that impaired circadian rhythmicity of senescent HSMC could be attributed to the reduced responsiveness of this pathway to external stimulation. In contrast, p38 MAPK was only slightly phosphorylated to a similar extent after serum stimulation of either young HSMC or senescent HSMC (Figure 4A). SB209030, an inhibitor of p38 MAPK, did not alter the circadian expression of clock genes by young HSMC (data not shown). These results suggest that p38 MAPK does not play a critical role in the regulation of clock gene expression in this setting.
Cellular Senescence Impairs Clock Gene Expression In Vivo
To investigate the profile of clock gene expression for the peripheral clocks, we harvested hearts at the indicated time points from 8-week-old C57/BL6 mice that were maintained under a 12 hour:12 hour light:dark (LD) cycle and kept in complete darkness for 2 days. Circadian time 0 (CT0) under the dark:dark (DD) cycle corresponded to the lights-on time of the LD cycle. Northern blot analysis demonstrated that Per2 displayed robust cyclical expression, peaking at CT12 and CT36 (Figure 5A). Bmal1 expression was also observed to cycle rhythmically but in a pattern antiphase to Per2, peaking at CT0, CT24, and CT48 (Figure 5A). We next attempted to determine whether cellular senescence had an influence on circadian expression of clock genes in vivo. We prepared MEF derived from C57/BL6 mice. Some populations of MEF were passaged until they underwent cellular senescence. These cells exhibited senescent phenotypes such as increased activity of senescent-associated -galactosidase and high levels of p21 and p16 expression (supplemental Figure IIA and IIB). We simultaneously implanted Matrigel containing young MEF or senescent MEF into 8-week-old C57/BL6 mice and maintained the animals for 2 days under the LD cycle. The mice were subsequently kept under the DD cycle for 2 days. On the fifth day, we recovered cells from the gel at CT0 and CT12 and analyzed clock gene expression. Consistent with the expression profile of the peripheral clocks, expression of Per2 in implanted young MEF as well as in the heart of the young recipient was increased at CT12 from the basal level at CT0 (Figure 5B). In contrast, induction of Per2 expression at CT12 was markedly reduced not only in the heart of old mice (70 to 80 weeks old) but also in implanted senescent MEF of the young recipient (Figure 5B). Circadian expression of Bmal1 was also impaired in senescent MEF and the heart of old mice (Figure 5B). No signal was detected in the samples prepared from Matrigel without MEF (data not shown). Vascularization of Matrigel containing young MEF did not differ from that of senescent MEF (supplemental Figure IV). When young MEF was implanted into old mice (70 to 80 weeks old), expression of clock genes was not reduced, suggesting that circulating factor(s) in old mice is not responsible for age-related alterations of clock gene expression. Inhibition of senescence by the introduction of E6 oncoprotein, which binds p53 and facilitates its destruction by ubiquitin-mediated proteolysis, was able to improve the impaired expression of clock genes in implanted MEF (Figure 5B). Finally, we implanted fat tissues from young mice (8 weeks old) or old mice (70 to 80 weeks old) into young recipient (8 weeks old) and examined circadian expression of clock genes. As shown in Figure 5C, levels of clock gene expression were less in the fat tissues from old mice than in those from young mice. These results suggest that cellular senescence in vivo inhibits circadian oscillation of the peripheral clocks, thereby contributing to the pathogenesis of age-associated impairment of circadian rhythms.
Discussion
In this study, we demonstrated that cellular senescence impairs circadian rhythmicity. Treatment with high concentrations of serum induced the circadian expression of clock genes in young HSMC, but this response was significantly weaker in senescent HSMC. Introduction of telomerase prevented this senescence-associated decrease in the oscillation of clock genes. The profile of clock gene expression by young cells implanted into young mice was similar to that of the endogenous peripheral clocks during the DD cycle. However, clock gene expression was still severely impaired when senescent cells were implanted into young recipients. This impairment was improved by inhibition of senescence, suggesting a causal link between cellular senescence and the control of clock gene expression in vivo as well as in vitro. We introduced E6 oncoprotein into MEF because disruption of p53 is able to immortalize rodent cells but not human somatic cells.45,46 There is recent evidence suggesting that cellular senescence occurs in vivo and contributes to the pathogenesis of age-associated diseases.1,11 For example, the existence of senescent cells has been reported in humans with heart failure47 as well as human atheroma.3 Thus, it is possible that the central pacemaker may be unable to regulate peripheral circadian rhythms in patients with atherosclerosis or heart failure because cells in the cardiovascular system undergo senescence. Impaired circadian rhythms of peripheral clocks may lead to dysregulated expression of clock-controlled genes such as plasminogen activator inhibitor-121 and vascular endothelial growth factor,48 both of which have been implicated in the pathogenesis of cardiovascular diseases.
The SCN rhythm generator can be entrained by a number of external stimulation, such as light. This allows animals to adjust their biological rhythms to changes in the external environment. It is known that brief exposure to light shifts the circadian clock during the subjective night.49 Photic stimulation causes rapid induction of immediate-early genes such as c-fos and induces the period genes in the SCN.50eC53 Recent studies have demonstrated that cAMP response element (CRE)-mediated transcription plays a critical role in the induction of these genes and that activation of the Ca2+ and cAMP pathways induces CRE-mediated transcription.37,54 The MAPK cascades are thought to mediate Ca2+-dependent CREB phosphorylation.36 It has been reported that aging hampers light-induced expression of clock genes as well as c-fos in the SCN.55,56 This impairment is associated with a decrease of the CREB phosphorylation to response to light.57 The molecular changes underlying age-related alterations in the effects of light on the SCN seem to be very similar to those in senescent cells. For example, serum-induced CREB activation as well as clock gene expression was significantly reduced in senescent cells. A loss of serum-induced c-fos expression is known to be one of the hallmarks of cellular senescence.58 Activation of CREB by an increase of cAMP partially restored the impaired expression of clock genes. Likewise, activation of the MAPK cascades partially increased the phosphorylation of CREB in senescent cells, and activation of these pathways seemed to have synergic effects (T.K., T.M., I.K., unpublished data, 2005), suggesting that cellular aging may affect both MAPK-dependent and PKA-dependent CRE-mediated transcription.
Although a body of evidence suggests that aging alters a broad spectrum of physiological, endocrine, and behavioral rhythms, it remains unclear whether these alterations are the result of age-associated effects on the central pacemaker, on peripheral oscillators, or on the mechanisms that mediate synchronization among contributing oscillators. Whereas photic stimulation-induced expression of Per1 and Per2 was reduced in the aged SCN, the cycling of the important clock genes has been shown to be unaffected by aging.59 In transgenic rats with a luciferase reporter driven by the Per1 promoter, Yamazaki et al60 measured the circadian rhythm of Per1 expression in cultured tissues of the free-running period and found that the rhythmicity of some peripheral tissues was severely affected by aging, whereas there was only a small difference in SCN rhythmicity between young and old rats. In our implantation experiments, we showed that senescent implants were not entrained by the circadian rhythm of young recipients. The reduced ability of senescent cells to undergo entrainment was restored by activating CREB in vitro. Consistent with this, Yamazaki et al60 demonstrated that cultured tissues from aged rats became rhythmic after treatment with forskolin. A recent study demonstrated that exposure of old mice to serum factor(s) present in young mice restored the decline of muscle regeneration, suggesting a critical role of systemic factors in age-related changes. In contrast, our results showed that circadian rhythmicity was impaired in senescent implants of the young recipient and that young implants could be entrained by the rhythms of old recipients, excluding the possibility that circulating factors that change with age contribute to impaired rhythmicity in the peripheral clock of old animals. These results suggest that aging affects entrainment of the circadian signals to external stimulation in the peripheral clocks. Thus, cellular senescence in aged peripheral tissues may underlie the mechanism by which aging impairs entrainment of circadian rhythms in the peripheral clocks.
Acknowledgments
This work was supported by grants-in-aid for Scientific Research, Developmental Scientific Research, and Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture; Health and Labor Sciences Research grants (to I.K.); and grants from the Japan Research Foundation for Clinical Pharmacology, the NOVARTIS foundation, and the Ministry of Education, Science, Sports, and Culture of Japan (to T.M.).
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