GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2006) 11, 1039-1049. doi:10.1111/j.1365-2443.2006.00999.x
© 2006 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takashima, N.
Right arrow Articles by Shigeyoshi, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takashima, N.
Right arrow Articles by Shigeyoshi, Y.

Gq/11-induced intracellular calcium mobilization mediates Per2 acute induction in Rat-1 fibroblasts

Naoyuki Takashima1,2, Atsuko Fujioka1, Naoto Hayasaka1, Ayako Matsuo1,3, Jun Takasaki3 and Yasufumi Shigeyoshi1,*

1 Department of Anatomy and Neurobiology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan
2 Course of Medical Biosignaling, The Public Relations Committee, Graduate School of Medicine, Faculty of Medicine, Osaka University, 2-2 Yamadaoka Suita, Osaka 565-0871 Japan
3 Molecular Medicine Laboratories, Institute for Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Phase resetting is one of the essential properties of circadian clocks that is required for the adjustment to a particular environment and the induction of Per1 and Per2 clock genes is believed to be a primary molecular event during this process. Although the intracellular signal transduction pathway underlying Per1 gene activation has been well characterized, the mechanisms that control Per2 up-regulation have not yet been elucidated. In our present study, we demonstrate that Gq/11 coupled receptors mediate serum-induced immediate rat Per2 (rPer2) transactivation in Rat-1 fibroblasts via intracellular Ca2+ mobilization. Stimulation of these cells with a high concentration of serum was found to rapidly increase the intracellular Ca2+ levels and strongly up-regulated rPer2 gene. rPer2 induction by serum stimulation was abrogated by intracellular Ca2+ chelation and depletion of intracellular Ca2+ store, which suggests that the calcium mobilization is necessary for the up-regulation of rPer2 gene. In addition, suppression of Gq/11 function was observed to inhibit both Ca2+ mobilization and rPer2 induction. Further, we demonstrated that endothelin-induced acute rPer2 transactivation via Gq/11-coupled endothelin receptors is also suppressed by a Gq/11 specific inhibitor. These findings together suggest that serum and endothelin utilize a common Gq/11-PLC mediated pathway for the transactivation of rPer2, which involves the mobilization of calcium from the intracellular calcium store.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The suprachiasmatic nucleus (SCN) is the center for the mammalian circadian rhythm that comprises stable circadian oscillations of clock genes. The endogenous circadian clock of the mammalian SCN can be adjusted by light exposure which synchronizes the clock with the external environment. Although the molecular mechanisms underlying this process are not yet fully understood, a number of recent findings now suggest that Per1/Per2 expression during the night is the principal event that produces long-term state changes, i.e. a phase shift in the circadian rhythm. The Per1 and Per2 genes are induced in the SCN following light exposure during the night (Albrecht et al. 1997; Shigeyoshi et al. 1997; Takumi et al. 1998; Zylka et al. 1998) and the PER1 protein has been shown to respond to such light stimuli in conditions of night, suggesting that it plays a role in photic entrainment (Field et al. 2000). Furthermore, anti-sense oligonucleotides directed against either mouse Per1 or mouse Per2 have been shown to attenuate the extent of the phase shift produced by light exposure in mouse (Akiyama et al. 1999; Wakamatsu et al. 2001; Tischkau et al. 2003). These studies together suggest that Per1 and Per2 are fundamentally involved in the resetting of the circadian clock.

Serum stimulation and exposure to chemical compounds or peptides can induce Per1 and/or Per2 genes in fibroblasts (Balsalobre et al. 1998, 2000b; Akashi & Nishida 2000; Yagita & Okamura 2000; Yagita et al. 2001) and mimic light-induced immediate early gene expression in the SCN. Therefore, the investigation on Per1 and Per2 induction pathways in the fibroblast would lead to the understanding of entrainment processes in the SCN. In the fibroblasts, previous studies have revealed that many agents, such as Dex (dexamethasone), TPA (Phorbol esters, PMA), FK (Forskolin), TG (Thapsigargin), endothelin and high concentration of serum can rapidly induce Per1 and/or Per2 (Balsalobre et al. 1998, 2000b; Akashi & Nishida 2000; Yagita & Okamura 2000; Yagita et al. 2001) and generate a circadian oscillation of clock genes. It is noteworthy in this regard that Per1 mRNA is strongly induced by all of these stimuli and previous studies have also demonstrated that many pathways transactivate this gene. Dex induces Per1 expression via the glucocorticoid receptor (Yamamoto et al. 2005), whereas TPA or TG induce Per1 via the MAP kinase pathways (Akashi & Nishida 2000; Oh-hashi et al. 2002). FK induces Per1 via the PKA/CREB pathways (Yagita & Okamura 2000). However, these stimulations do not increase Per2 mRNA levels. Only serum stimulation and exposure to endothelin have been so far shown to strongly up-regulate the Per2 gene (Balsalobre et al. 1998; Akashi & Nishida 2000; Yagita & Okamura 2000; Yagita et al. 2001) and the intracellular signal transduction system that mediates immediate Per2 up-regulation has not yet been substantially investigated. In our present study, we tried to reveal which intracellular signal transduction pathways are utilized for Per2 induction by serum and endothelin in Rat-1 cells.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
In Rat-1 fibroblasts, Dex, TPA, FK and horse serum have been shown to induce Per1 gene (Akashi & Nishida 2000; Balsalobre et al. 1998, 2000a,b; Yagita & Okamura 2000). We therefore investigated whether these agents would also induce rPer2 in the same cells (Fig. 1). At timepoints corresponding to 1, 2, and 4 h after treatments with 50% horse serum, 100 nM Dex, 10 nM TPA, 10 nM FK and vehicle (0.1% DMSO), the cells were harvested and total RNA was extracted. Our results showed that after 1 h, a high concentration of horse serum increases the levels of rPer2 mRNA significantly (about five-fold; P < 0.01; student's t-test) but that there was no significant induction of this gene by any of the other treatments (Fig. 1B). We then tried to identify the intracellular signal transduction pathway underlying this transactivation of rPer2. Fifteen minutes prior to horse serum stimulation of the Rat-1 fibroblasts, we administered a number of selective inhibitors including GF109203X (Bis), a selective PKC inhibitor, H89, a selective PKA inhibitor, Mifepristone (GR-I), a selective Glucocorticoid receptor inhibitor and BAPTA/AM, an intracellular Ca2+ chelator. One hour after stimulation, the levels of rPer2 transcripts were compared with the control (pretreatment of vehicle) and it was observed that BAPTA/AM almost completely suppresses the serum-dependent rPer2 mRNA increase (Fig. 2A). In contrast, there were no detectable effects upon rPer2 up- regulation following the use of Bis, H89 and GR-I. rPer2 induction is in fact suppressed in a concentration dependent manner by intracellular Ca2+ chelators (Fig. 2B). In contrast to rPer2, BAPTA/AM do not suppress serum induced rPer1 expression, which was found to be suppressed by treatment with a glucocorticoid receptor inhibitor only (P < 0.01) (Fig. 2C). The findings suggest that intracellular Ca2+ is required for the induction of rPer2 by serum stimulation.


Figure 1
View larger version (26K):
[in this window]
[in a new window]
  		Figure 1  Measurement of rPer2 mRNA levels after treatment with various stimuli. (A) Timecourse determination of rPer2 mRNA levels before and after treatments including 50% horse serum, Dex (100 nM), TPA (10 nM), FK (10 nM) and DMSO (0.1%, vehicle). The levels of rPer2 mRNA at time point 0 (before treatment) was set at 1. (B) rPer2 mRNA levels measured 1 h after each treatment. Each value represents the mean ± S.E.M. of three to six samples. **P < 0.01.

 

Figure 2
View larger version (15K):
[in this window]
[in a new window]
  		Figure 2  The effects of different inhibitors upon rPer2 up-regulation in Rat-1 fibroblasts by 50% horse serum. (A) Each inhibitor was added 15 min prior to serum stimulation. mRNA was extracted 1 h after stimulation and transcript levels were measured by Q-PCR. rPer2 mRNA levels induced by serum in cells pretreated with vehicle (0.1% DMSO) were set at 1.00. The effects of treatments with different chemical inhibitors upon the rPer2 mRNA induction levels, 15 min before serum stimulation, are shown for vehicle (0.1% DMSO, Control), GF109203X (Bis, 5 nM), H89 (10 µM), Mifepristone (GR-I, 10 µM) and BAPTA/AM (50 µM). The rightmost bar shows rPer2 mRNA level without serum stimulation (–). Each value represents the mean ± S.E.M. of three to six samples. **P < 0.01. (B) Concentration dependent repression of rPer2 induction by intracellular Ca2+ chelators. Fifteen minutes before serum stimulation, intracellular Ca2+ chelators (10 µM or 50 µM BAPTA/AM) were added to the cells. The relative values of the measured rPer2 mRNA levels are shown. rPer2 induction by serum was inhibited in a concentration dependent manner by BAPTA/AM. rPer2 mRNA levels induced by serum in cells pretreated with vehicle (0.1% DMSO) were set at 1.00. The rightmost bar shows rPer2 mRNA level without serum stimulation (–). Each value represents the mean ± S.E.M. of three to six samples. *P < 0.05, **P < 0.01. (C) The relative values for rPer1 mRNA levels after pretreatment, 15 min before serum stimulation, with vehicle (0.1% DMSO), BAPTA/AM(50 µM) or Glucocorticoid receptor inhibitor (GR-I, 10 µM). rPer1 mRNA levels induced by serum in cells pretreated with vehicle (0.1% DMSO) were set at 1.00. The rightmost bar shows rPer1 mRNA level without serum stimulation (–). Each value represents the mean ± S.E.M. of three to six samples. **P < 0.01.

 
Having observed that Ca2+ is an essential component of the rPer2 induction pathway, we examined the changes in the intracellular Ca2+ levels during this process using GFP-based FRET by YC-3.60 (Nagai et al. 2004). Following 50% horse serum stimulation of Rat-1 fibroblasts, the Ca2+ level increase immediately peaked within 1 s and decreased rapidly to 30% of this peak within 45 s of stimulation (Fig. 3A,B). A phase of gradual decrease in Ca2+ was then maintained for more than 3 min, and at 4 min following serum stimulation, the Ca2+ concentration had almost returned to basal levels. We then examined whether an intracellular Ca2+ chelator would suppress these increases in intracellular Ca2+ by serum stimulation and observed that BAPTA/AM (Fig. 3C) greatly reduce the extent of the Ca2+ concentration peak (Fig. 3C,D). We next investigated whether the application of ionomycin to the rat fibroblasts could induce rPer2 but found no significant effects (Fig. 4A,B). The cameleon sensors revealed that ionomycin increased the intracellular Ca2+ concentration rapidly in these cells and sustained these high levels for more than 5 min (data not shown).


Figure 3
View larger version (27K):
[in this window]
[in a new window]
  		Figure 3  Intracellular Ca2+ concentration profiles after serum stimulation of Rat-1 fibroblasts with and without BAPTA/AM pretreatment. Comparative measurements of Ca2+ dynamics in Rat-1 cells after serum stimulation are also shown. At time point 0 (before treatment), an equal volume of horse serum was added to both vehicle-pretreated cells and BAPTA/AM pretreated cells. (A,C) Panels showing a series of confocal pseudocolored ratio images. (B,D) Timecourse of emission ratios (535/480 nm). Each experiment was performed more than 3 times. Acute increases in Ca2+ levels are shown in (A) and (B). This acute elevation was attenuated by pretreatment with BAPTA/AM (50 µM) (C) and (D).

 

Figure 4
View larger version (17K):
[in this window]
[in a new window]
  		Figure 4  The effects of ionomycin on rPer2 expression in Rat-1 cells. (A) Timecourse of rPer2 mRNA levels after ionomycin exposure. The rPer2 transcript levels at time point 0 (immediately prior to the treatment) was set at 1 (control). (B) rPer2 mRNA levels 1 h after ionomycin treatment. Each value represents the mean ± S.E.M. of three to six samples.

 
BAPTA/AM is a cell membrane permeable compound and is a Ca2+ insensitive form of BAPTA. Once inside the cell, BAPTA/AM molecules are hydrolyzed by ubiquitous intracellular esterases, releasing the Ca2+ sensitive and cell membrane impermeable active form of the compound, which works effectively as a Ca2+ chelator (Strayer et al. 1999). We determined the time required to activate BAPTA/AM in Rat-1 cells and at 0, 3, 5, 10, 15 and 20 min prior to horse serum stimulation, we administrated 50 µM of this agent. When BAPTA/AM was added to Rat-1 cells just before serum stimulation, the immediate Ca2+ spike was unaffected (Fig. 5A). However, when BAPTA/AM was added to the fibroblasts at 3 and 5 min prior to serum treatment, the intracellular Ca2+ increase was partially repressed (data not shown). Treatment with BAPTA/AM from 10 to 20 min before serum stimulation resulted in an almost complete abrogation of intracellular Ca2+ induction (Fig. 5C). These data suggest that approximately 10 min is required for BAPTA/AM to be esterized to its active form. Then, we quantitated the levels of rPer2 mRNA induction by serum following BAPTA/AM treatment at 0 and 10 min prior to stimulation, with a control vehicle (Fig. 5D). When BAPTA/AM was added to Rat-1 cells just before horse serum stimulation, rPer2 mRNA induction was moderately repressed. In contrast, however, when BAPTA/AM was added to the cultures 10 min prior to serum stimulation, rPer2 mRNA induction was significantly repressed. These findings suggest that the immediate spike in Ca2+ levels contributes to the induction of rPer2 and that the long-term Ca2+ increase also contributes to the rPer2 transactivation following serum stimulation.


Figure 5
View larger version (13K):
[in this window]
[in a new window]
  		Figure 5  The intracellular Ca2+ concentration profiles in serum stimulated Rat-1 fibroblasts that have been pretreated with the intracellular Ca2+ chelator BAPTA/AM. Serum was added at time point 0. (A–C) Representative graphs showing the timecourse of relative intracellular Ca2+ levels. (A) BAPTA/AM treatment at time 0. (B) Control (vehicle treatment). (C) BAPTA/AM treatment 10 min prior to serum stimulation. At time point 0, equal volumes of horse serum were added in each case. Each experiment was independently performed at least 3 times. (D) The amount of rPer2 mRNA by serum induction was set at 1 (control). The relative values of the rPer2 mRNA levels are shown. Each value represents the mean ± S.E.M. of three to six samples. **P < 0.01.

 
We next determined whether the intracellular Ca2+ increase is caused by an intracellular release or an extracellular influx. First, we examined whether extracellular Ca2+ is required for the elevation of intracellular Ca2+ after serum stimulation in Rat-1 cells. Fifteen minutes prior to serum stimulation, we supplemented the culture medium with EGTA, which would chelate the extracellular Ca2+. EGTA treatment was found to result in a complete reduction of the late phase elevation of intracellular Ca2+ (Fig. 6A), compared with the control (Fig. 6B), which suggests that extracellular Ca2+ influx contributed to the late phase Ca2+ increase. Further, treatment with TG, 20 min before serum stimulation, which has been shown to deplete intracellular Ca2+ stores (Thastrup et al. 1990; Takuwa et al. 1995), completely blocked the serum-induced elevation of intracellular Ca2+ (Fig. 6C). Furthermore, pretreatment of BTP2 that specifically blocks store operated Ca2+ channel (SOC) (Zitt et al. 2004; Mauban et al. 2006), abrogated the late phase of intracellular Ca2+ (Fig. 6D). The findings suggest that the immediate Ca2+ spike from the intracellular Ca2+ store induced the late phase of extracellular Ca2+ influx via SOC. To determine which phases of Ca2+ elevation generates rPer2 elevation, we quantitated the rPer2 mRNA levels with BAPTA/AM, EGTA, TG, BTP2 and Ryanodine (Ryr). With the introduction of EGTA as well as BTP2, the up-regulation of rPer2 by high concentration of serum was suppressed to 70% of the control levels (Fig. 6E). Furthermore, we examined whether the release from the intracellular Ca2+ store contributes to the rPer2 induction by treatment with TG. TG treatment also suppressed rPer2 induction by serum (Fig. 6E) (35% of control). BAPTA/AM treatment also suppressed rPer2 induction to approximately the same level to TG, more strongly than EGTA treatment (Fig. 6E) (P < 0.01). In contrast, a high concentration (10 µM) of Ryanodine, that has been reported to inhibit the Ryanodine receptor function, did not abrogate rPer2 gene induction (Fig. 6E). These findings suggest that both the immediate spike in Ca2+ levels and the long-term Ca2+ increase in the late phase also contribute to the rPer2 transactivation following serum stimulation.


Figure 6
View larger version (13K):
[in this window]
[in a new window]
  		Figure 6  The intracellular Ca2+ concentration profiles in serum stimulated Rat-1 fibroblasts that have been pretreated with EGTA (5 mM), TG (10 µM), Ryr (10 µM) and BTP2 (50 nM). (A–D) Representative figures showing timecourse measurements of emission ratios (535/480 nm) for EGTA pretreated cells (A), control cells (B), TG pretreated cells (C) and BTP2 pretreated cells (D). Each experiment was independently performed at least 3 times. (E) rPer2 mRNA levels following BAPTA/AM (50 µM), EGTA, TG and ryanodine pretreatments are shown. For TG or BAPTA/AM, the amount of rPer2 mRNA induction was significantly repressed. The amount of rPer2 mRNA by serum induction was set at 1 (control). Each value represents the mean ± S.E.M. of three to six samples. **P < 0.01.

 
It is known that the Gq-coupled GPCR mobilizes intracellular calcium via the PLC-IP3 pathway and the rapid increases and decreases in Ca2+ following serum stimulation of rat fibroblasts is similar to the profile of intracellular Ca2+ mobilization via G-protein coupled receptors (GPCRs) (Evans et al. 1997; Werry et al. 2003). Therefore, we examined whether the increase in Ca2+ and subsequent induction of rPer2 are in fact mediated through GPCRs. We employed a selective PLC inhibitor, U-73122, and a Gq/11 selective inhibitor, YM-254890, to examine the involvement of PLC and Gq/11 during rPer2 gene activation. Our subsequent findings indicated that both U-73122 and YM-254890 suppress rPer2 mRNA induction (Fig. 7A). The involvement of Gq/11 coupled receptors in this activation was further validated by the use of Gq-I, which is a minigene that selectively inhibits receptor-Gq interactions and which was also found to suppress the serum-induced up-regulation of rPer2 (Fig. 7A). On the other hand, the G12/13 signal blocker, Y27362, did not abrogate the serum-induced increases in rPer2 transcript levels (Fig. 7A). Further, we examined whether the increase of Ca2+ was also suppressed by the Gq/11 inhibitor and by the PLC inhibitor. Using the YC-3.60 cameleon sensor of calcium, we found that YM-254890 completely abolishes the serum induced elevation of intracellular Ca2+ levels (Fig. 7B) in both the early and late phases, when compared with the vehicle control (Fig. 7D). Moreover, U-73122 significantly suppressed the serum induced elevation of intracellular Ca2+ levels in both phases (Fig. 7C). The findings suggest that rPer2 is mediated by a Gq/11 coupled receptor-PLC-intracellular Ca2+ pathway.


Figure 7
View larger version (13K):
[in this window]
[in a new window]
  		Figure 7  The effects of Gq/11, PLC and Rho inhibitors on rPer2 induction by serum in Rat-1 fibroblasts. (A) rPer2 mRNA levels after treatment with Gq/11 or PLC inhibitors. The rPer2 induction by serum stimulation following pretreatment with vehicle was set at 1 (control). The relative values of the rPer2 mRNA levels are shown. Each value represents the mean ± S.E.M. of three to six samples. **P < 0.01. (B–D) Timecourse measurements of emission ratios (535/480 nm) for YC-3.60. (B) YM-254890 (1 µM) pretreated cells; (C) U73122 (10 µM) pretreated cells; and (D) Control cells (pretreated with DMSO vehicle). Each experiment was independently performed at least 3 times.

 
It has been reported that endothelin (ET-1) increases rPer2 levels in Rat-1 fibroblasts (Yagita et al. 2001). The endothelin receptors, ETA and ETB, are GPCRs that are coupled with Gq/11 (Jouneaux et al. 1994; Hilal-Dandan et al. 1997) and we therefore examined whether ET-1 also elevates rPer2 mRNA levels via Gq/11-coupled receptors. ET-1 treatment of Rat-1 cells was indeed observed to significantly increase the rPer2 mRNA levels in Rat-1 fibroblasts by up to four-fold. Moreover, both YM-254890 and BAPTA/AM suppressed this induction of rPer2 by ET-1 (Fig. 8A). ET-1 also increased intracellular Ca2+ concentration (Fig. 8B). These findings suggest that ET-1 also increases rPer2 mRNA by intracellular Ca2+ mobilization mediated by Gq/11-coupled GPCRs. Another signal transduction pathway that is known to involve Gq/11 is the MAPK cascade. However treatment of ET-1 stimulated Rat-1 cells by, U0126, an effective inhibitor of ERK activation by MEK, did not abrogate rPer2 mRNA up-regulation suggesting that the MAPK cascade is not involved in this transactivation pathway.


Figure 8
View larger version (16K):
[in this window]
[in a new window]
  		Figure 8  The effects of YM-254890, BAPTA/AM and U0126 on rPer2 induction by ET-1 in Rat-1 fibroblasts. (A) rPer2 induction by ET-1 following vehicle pretreatment was set at 1 (control). The relative values of the rPer2 expression levels are shown. Each value represents the mean ± S.E.M. of three to six samples **P < 0.01. (B) Representative figures showing time course measurements of emission ratios (535/480 nm) for ET-1 stimulated cells. Each experiment was independently performed at least 3 times.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In our present study, we demonstrate that Gq/11 coupled receptors mediate the rapid serum-induced activation of rPer2 in Rat-1 cells. Treatment of serum-stimulated cells with YM-254890, a selective inhibitor of the Gq/11 protein (Takasaki et al. 2004), represses rPer2 induction. Furthermore, the Gq-I minigene, that selectively inhibits receptor-Gq interaction (Akhter et al. 1998), also inhibits serum induced rPer2 expression, suggesting that the activation of Gq/11 plays an important role in the transactivation of this gene. Our current analyses also suggest that ET-1 shares the same intracellular signaling pathway as it also induces the acute expression of rPer2 through Gq/11 coupled receptors, which can be inhibited with YM254890. Significantly, the Endothelin receptors known to be expressed in Rat-1 cells is ET-A, which is a Gq/11-coupled receptor (Jouneaux et al. 1994; Hilal-Dandan et al. 1997). By utilizing the yellow cameleon-3.60 Ca2+ sensor (Nagai et al. 2004), we further found that high serum treatment and endothelin induces an immediate Ca2+ spike in early phase followed by a low level Ca2+ elevation of comparatively long duration and that the Ca2+ mobilization was abrogated by YM-254890. The significant role of Ca2+ mobilization in the induction of rPer2 by serum and endothelin was evident by the chelation of intracellular calcium that blocked the induction in serum- and endothelin-stimulated cells. This suggests that, among the several pathways that are induced by the activation of Gq/11, calcium mobilization from the intracellular stores by IP3 (Creba et al. 1983; Streb et al. 1983; Taylor et al. 1991; Singer et al. 1997; Werry et al. 2003) is the principal event leading to rPer2 transactivation.

Our present study suggests that the early Ca2+ increase leads to the longer term elevation in its levels. Treatment of serum stimulated Rat-1 cells with TG, which depletes the intracellular Ca2+ store and thus inhibits IP3R receptor-mediated Ca2+ release, was found to repress both the early and late phase of intracellular Ca2+ elevation. On the other hand, EGTA treatment in the same experiment, causing chelation of extracellular Ca2+, inhibits late phase Ca2+ elevation suggesting that these levels are induced by extracellular Ca2+ influx. These data suggest that the early phase of intracellular Ca2+ mobilization is tightly linked to the late phase of intracellular Ca2+ elevation. Previous studies have demonstrated that intracellular Ca2+ release might prompt the extracellular Ca2+ influx via the membrane Ca2+ channels (named store operated Ca2+ channels: SOC) (Hoth & Penner 1992; Parekh & Putney 2005). This was supported by the finding that BTP2, a SOC blocker, inhibits serum induced late phase of intracellular Ca2+ elevation, which suggests that the Ca2+ spike in early phase induced late phase of extracellular Ca2+ influx to control SOC.

Our findings suggest that intracellular Ca2+ elevation plays important roles during serum- and endothelin-induced expression of the rPer2 gene. However, we could not reproduce this rPer2 induction by treatment with ionomycin, which causes an increase in intracellular Ca2+ levels. The difference in the amount of rPer2 gene transcription may be attributable to changes in the profiles of intracellular Ca2+. The profiles of intracellular Ca2+ elevation induced by ionomycin were different, however, from the profiles induced by high serum-stimulation, which comprise an early phase spike for 45 s, followed by a moderate late phase elevation for several minutes. On the other hand, ionomycin induces strong and continuous elevation of intracellular Ca2+ but does not induce rPer2. Previous investigations have revealed that intracellular Ca2+ concentration and the frequency of intracellular Ca2+ oscillation regulate the activation of transcription factors such as NFAT, NF-kb and AP-1 (Quintana et al. 2005). A timecourse and/or time-dependent localization of intracellular Ca2+ mobilization might therefore be the chief contributor to the acute induction of rPer2 expression.

In conclusion, we have shown from our current experiments that rPer2 is induced via a Gq/11 coupled receptor-PLC-intracellular Ca2+ pathway by serum stimulation in Rat-1 cells (Fig. 9). Intracellular calcium mobilization via IP3 receptors, as well as an extracellular influx of calcium via SOC contributes to this Per2 mRNA induction. The precise nature of the pathway linking intracellular Ca2+ to Per2 gene expression will need to be further investigated in future studies.


Figure 9
View larger version (24K):
[in this window]
[in a new window]
  		Figure 9  A working model of the mechanisms underlying rPer2 induction. rPer2 mRNA is induced via a Gq/11 coupled receptor-PLC-IP3-intracellular Ca2+ pathway. Intercellular Ca2+ induces the Ca2+ influx. Intracellular calcium mobilization, via IP3 receptors, in addition to an extracellular flux of Ca2+ via SOC also contributes to rPer2 transactivation. The pathway linking intracellular Ca2+ to the induction of Per2 gene expression is not yet investigated. Solid lines indicate the pathways examined in the present study that are involved in the induction of rPer2 in Rat-1 cells. Dashed lines indicate putative pathways involved in the rPer2 induction.

 

   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Drugs

BAPTA/AM (Nacalai, Kyoto, Japan), FK (Forskolin) (Wako, Osaka, Japan), GF109203X (Bisindolyl-maleimide I) (Calbiochem, San Diego, CA, USA), Mifepristone (Sigma, Chicago, IL, USA), H89 (Biomol. Research Laboratories Inc., Plymouth Meeting, PA, USA), Ionomycin (Calbiochem), Ryanodine (Calbiochem), BTP2 (Calbiochem), Thapsigargin (TG, Alomone Laboratories, Ltd, Jerusalem, Israel), TPA (Phorbol 12 Myristate 13 Acetete (Wako)), U0126 (Promega, Madison, WI, USA), U-73122 (Calbiochem), YM-254890 (a gift from Astellas Pharma Inc.) and Y27362 (Wako) were dissolved in dimethylsulfoxide (DMSO) (Wako). EGTA (Nacalai) was dissolved in 1 M NaOH. Endothelin-1 (ET-1, Sigma) was dissolved in distilled water. Dexamethasone (Dex) (ICN, Costa Mesa, CA, USA) was dissolved in ethanol (Nacalai). Vehicles: distilled water, DMSO (Wako) or ethanol (Nacalai) was added to the control.

DNA construction

Receptor-Gq interaction inhibitory "mini gene"(Gq-I), which corresponds to the C-terminal peptide sequence of Gq residues 305-359, was cloned into pcDNA3 (Akhter et al. 1998; Takasaki et al. 2004; Matsuo et al. 2005). The Yellow Cameleon (YC-3.60) was a gift from Dr A. Miyawaki (Nagai et al. 2004).

Cell culture and transfection

Rat-1 cells were grown in Dulbecco's modified Eagle medium (DMEM (Sigma)) with 10% FCS. One day before transfection, 5 x 105 cells were transferred to 35 mm dishes. Transfection was performed with LipofectAmine 2000 regents (Invitrogen, Carlsbad, CA, USA). Each transfection was performed with 1 µg of YC-3.60 or 1 µg of Gq-I. Forty-eight hours post transfection, those cells were transferred 35 mm glass dishes.

Calcium imaging by fluorescence resonance energy transfer (FRET)

Four days after transfection of YC-3.60, Rat-1 cells were replaced in phenol-red free DMEM (Invitrogen). Fluorescence observations were performed on an LSM-510 (Zeiss, Oberkochen, Germany) confocal microscope. Using 458 nm laser excited cells, two images were collected of fluorescence emissions (465–495 for CFP, 535-555 nm for YFP) (Miyawaki et al. 1997; Nagai et al. 2004).

Quantitative analysis of mRNA

One x 106 Rat-1 fibroblast cells were transferred to 35 mm dishes. Cells are confluent after about 1 day under these conditions. These cells were kept over night in serum-free DMEM. At the point of 0 h, each drugs 100 nM dexamethasone (Dex), 10 nM TPA, ionomycin, 10 µM FK and 30 nM Endothelin (ET-1) were added to the medium and the medium was replaced with the serum-free DMEM after 2 h. In case of serum stimulation, these cells were kept DMEM with 10% FCS before the stimulation. At the point of 0 h, the medium was replaced DMEM with 50% horse serum and was replaced with serum free DMEM after 2 h. Each inhibitors or vehicles were added in the medium 15 min before the stimulation. Total RNA was extracted from these cells. cDNAs were synthesized by reverse transcription with ReverTra Ace (TOYOBO, Osaka, Japan). Quantitative PCR (Q-PCR) was carried out using ABI PRISM7700 Sequence detection System (Applied Biosystems, Foster City, CA, USA). Rat rPer1 and rPer2 specific primers and fluorescence-labeled probes used for the quantitation of rPer1 and rPer2 were as follows: rPer2, 5'-GCTCTCAGAGTTTGTGCGATGA-3', and 5'-AAAAGACACAAGCAGTCACACAAATA-3'; rPer2-Probe, 5'-FAM-TTGTTCATGCGCAAACCAAACGTACC-TAMRA-3'; rPer1, 5'-AGCGCATCCACTCTGGTTATG-3' and 5'-GTGTGCCGTGTGGTGAAGAT-3'; rPer1-Probe, 5'-FAM-AGCTCCCCGGATTCCTCCTGACAAG-TAMRA-3'. GAPDH (Applied Biosystems) and 18S Ribosomal RNA (Applied Biosystems) was used as an internal control for cDNA.

Statistics

The data are expressed as the mean ± S.E.M. The signifi-cance of differences between groups was determined by Student's t-test.


   Acknowledgements
 
This work was supported by a High-Tech Research Center Grant from the Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan. We thank Atsushi Miyawaki for providing Yellow Cameleon 3.60 and Hideyuki Mukai for comments and fruitful discussion.


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: shigey{at}med.kindai.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Akashi, M. & Nishida, E. (2000) Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock. Genes Dev. 14, 645–649.[Abstract/Free Full Text]

Akhter, S.A., Luttrell, L.M., Rockman, H.A., Iaccarino, G., Lefkowitz, R.J. & Koch, W.J. (1998) Targeting the receptor–Gq interface to inhibit in vivo pressure overload myocardial hypertrophy. Science 280, 574–577.[Abstract/Free Full Text]

Akiyama, M., Kouzu, Y., Takahashi, S., et al. (1999) Inhibition of light- or glutamate-induced mPer1 expression represses the phase shifts into the mouse circadian locomotor and suprachiasmatic firing rhythms. J. Neurosci. 19, 1115–1121.[Abstract/Free Full Text]

Albrecht, U., Sun, Z.S., Eichele, G. & Lee, C.C. (1997) A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light. Cell 91, 1055–1064.[CrossRef][Medline]

Balsalobre, A., Brown, S.A., Marcacci, L., et al. (2000a) Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 289, 2344–2347.[Abstract/Free Full Text]

Balsalobre, A., Damiola, F. & Schibler, U. (1998) A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93, 929–937.[CrossRef][Medline]

Balsalobre, A., Marcacci, L. & Schibler, U. (2000b) Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts. Curr. Biol. 10, 1291–1294.[CrossRef][Medline]

Creba, J.A., Downes, C.P., Hawkins, P.T., Brewster, G., Michell, R.H. & Kirk, C.J. (1983) Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones. Biochem. J. 212, 733–747.[Medline]

Evans, J.J., Forrest-Owen, W. & McArdle, C.A. (1997) Oxytocin receptor-mediated activation of phosphoinositidase C and elevation of cytosolic calcium in the gonadotrope-derived {alpha}T3-1 cell line. Endocrinology 138, 2049–2055.[Abstract/Free Full Text]

Field, M.D., Maywood, E.S., O’Brien, J.A., Weaver, D.R., Reppert, S.M. & Hastings, M.H. (2000) Analysis of clock proteins in mouse SCN demonstrates phylogenetic divergence of the circadian clockwork and resetting mechanisms. Neuron 25, 437–447.[CrossRef][Medline]

Hilal-Dandan, R., Ramirez, M.T., Villegas, S., et al. (1997) Endothelin ETA receptor regulates signaling and ANF gene expression via multiple G protein-linked pathways. Am. J. Physiol. 272, H130–H137.

Hoth, M. & Penner, R. (1992) Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355, 353–356.[CrossRef][Medline]

Jouneaux, C., Mallat, A., Serradeil-Le Gal, C., Goldsmith, P., Hanoune, J. & Lotersztajn, S. (1994) Coupling of endothelin B receptors to the calcium pump and phospholipase C via Gs and Gq in rat liver. J. Biol. Chem. 269, 1845–1851.[Abstract/Free Full Text]

Matsuo, A., Matsumoto, S., Nagano, M., et al. (2005) Molecular cloning and characterization of a novel Gq-coupled orphan receptor GPRg1 exclusively expressed in the central nervous system. Biochem. Biophys. Res. Commun. 331, 363–369.[CrossRef][Medline]

Mauban, J.R., Wilkinson, K., Schach, C. & Yuan, J.X. (2006) Histamine-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells. Am. J. Physiol. Cell. Physiol. 290, C325–C336.[Abstract/Free Full Text]

Miyawaki, A., Llopis, J., Heim, R., et al. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.[CrossRef][Medline]

Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. (2004) Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 101, 10554–10559.[Abstract/Free Full Text]

Oh-hashi, K., Naruse, Y. & Tanaka, M. (2002) Intracellular calcium mobilization induces period genes via MAP kinase pathways in NIH3T3 cells. FEBS Lett. 516, 101–105.[CrossRef][Medline]

Parekh, A.B. & Putney, J.W. Jr (2005) Store-operated calcium channels. Physiol. Rev. 85, 757–810.[Abstract/Free Full Text]

Quintana, A., Griesemer, D., Schwarz, E.C. & Hoth, M. (2005) Calcium-dependent activation of T-lymphocytes. Pflugers Arch. 450, 1–12.[CrossRef][Medline]

Shigeyoshi, Y., Taguchi, K., Yamamoto, S., et al. (1997) Light-induced resetting of a mammalian circadian clock is associated with rapid induction of the mPer1 transcript. Cell 91, 1043–1053.[CrossRef][Medline]

Singer, W.D., Brown, H.A. & Sternweis, P.C. (1997) Regulation of eukaryotic phosphatidylinositol-specific phospholipase C and phospholipase D. Annu. Rev. Biochem. 66, 475–509.[CrossRef][Medline]

Strayer, D.S., Hoek, J.B., Thomas, A.P. & White, M.K. (1999) Cellular activation by Ca2+ release from stores in the endoplasmic reticulum but not by increased free Ca2+ in the cytosol. Biochem. J. 344, 39–46.

Streb, H., Irvine, R.F., Berridge, M.J. & Schulz, I. (1983) Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature 306, 67–69.[CrossRef][Medline]

Takasaki, J., Saito, T., Taniguchi, M., et al. (2004) A novel Galphaq/11-selective inhibitor. J. Biol. Chem. 279, 47438–47445.[Abstract/Free Full Text]

Takumi, T., Matsubara, C., Shigeyoshi, Y., et al. (1998) A new mammalian period gene predominantly expressed in the suprachiasmatic nucleus. Genes Cells 3, 167–176.[Abstract]

Takuwa, N., Zhou, W., Kumada, M. & Takuwa, Y. (1995) Involvement of intact inositol-1,4,5-trisphosphate-sensitive Ca2+ stores in cell cycle progression at the G1/S boundary in serum-stimulated human fibroblasts. FEBS Lett. 360, 173–176.[CrossRef][Medline]

Taylor, S.J., Chae, H.Z., Rhee, S.G. & Exton, J.H. (1991) Activation of the ß1 isozyme of phospholipase C by {alpha} subunits of the Gq class of G proteins. Nature 350, 516–518.[CrossRef][Medline]

Thastrup, O., Cullen, P.J., Drobak, B.K., Hanley, M.R. & Dawson, A.P. (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA 87, 2466–2470.[Abstract/Free Full Text]

Tischkau, S.A., Mitchell, J.W., Tyan, S.H., Buchanan, G.F. & Gillette, M.U. (2003) Ca2+/cAMP response element-binding protein (CREB)-dependent activation of Per1 is required for light-induced signaling in the suprachiasmatic nucleus circadian clock. J. Biol. Chem. 278, 718–723.[Abstract/Free Full Text]

Wakamatsu, H., Takahashi, S., Moriya, T., et al. (2001) Additive effect of mPer1 and mPer2 antisense oligonucleotides on light-induced phase shift. Neuroreport 12, 127–131.[CrossRef][Medline]

Werry, T.D., Wilkinson, G.F. & Willars, G.B. (2003) Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+. Biochem. J. 374, 281–296.[CrossRef][Medline]

Yagita, K. & Okamura, H. (2000) Forskolin induces circadian gene expression of rPer1, rPer2 and dbp in mammalian rat-1 fibroblasts. FEBS Lett. 465, 79–82.[CrossRef][Medline]

Yagita, K., Tamanini, F., van Der Horst, G.T. & Okamura, H. (2001) Molecular mechanisms of the biological clock in cultured fibroblasts. Science 292, 278–281.[Abstract/Free Full Text]

Yamamoto, T., Nakahata, Y., Tanaka, M., et al. (2005) Acute physical stress elevates mPeriod1 mRNA expression in mouse peripheral tissues via a glucocorticoid responsive element. J. Biol. Chem. 280, 42036–42043.[Abstract/Free Full Text]

Zitt, C., Strauss, B., Schwarz, E.C., et al. (2004) Potent inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2. J. Biol. Chem. 279, 12427–12437.[Abstract/Free Full Text]

Zylka, M.J., Shearman, L.P., Weaver, D.R. & Reppert, S.M. (1998) Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain. Neuron 20, 1103–1110.[CrossRef][Medline]

Received: 23 March 2006
Accepted: 5 June 2006





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takashima, N.
Right arrow Articles by Shigeyoshi, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takashima, N.
Right arrow Articles by Shigeyoshi, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS