Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2576
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ABSTRACT |
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Very little is known about the circadian regulation of cell entry into the S and M phases of the cell cycle. Yet, in the mouse esophagus, a seven- to ninefold increase in DNA synthesis coincides with nocturnal feeding. The phosphorylation of the cAMP response element binding protein (CREB), a transcriptional factor, may regulate hypothalamic circadian rhythms in the brain. Here, we investigate the circadian regulation of CREB and Ser-133-phospho-CREB (PCREB) in the mouse esophagus by immunocytochemical and biochemical methods. We found that, during the dark phase, coincident with the onset of feeding and increased DNA synthesis, esophageal CREB and PCREB expression decreased. Although CREB-like immunoreactivity (CREB-lir) was expressed in many different cell types, it was concentrated in the mucosa, particularly in the replicating basal cell layer. The injection of epidermal growth factor, at a dosage known to maximally stimulate esophageal DNA synthesis in a 4- to 8-h period, rapidly decreased PCREB levels within 10 min of injection. We speculate that PCREB-lir may be involved in the circadian regulation of cell cycle events in the intact mouse esophagus.
circadian; adenosine 3',5'-cyclic monophosphate response element binding protein; activating transcription factor-1; cell cycle
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INTRODUCTION |
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THE EPITHELIA of the oral cavity, tongue, and esophagus
display pronounced rhythms in cell cycle progression through the S and
M phases of the cell cycle (6, 9-12). Little is known about the
regulation of these rhythms; however, feeding and, to a lesser extent,
photoperiod are involved (9, 10). Because a single injection of
epidermal growth factor (EGF) within a 12-h period causes DNA synthesis
to peak when it is otherwise normally low, we have proposed that
changes in the local concentrations of EGF-like ligands (i.e., EGF,
transforming growth factor-, betacellulin, and heparin-binding EGF)
or in the number or biochemical properties of the EGF receptor regulate
these rhythms (11, 12).
Our proposed model of regulation presupposes that growth factors or their receptors control circadian cell cycle progression in vivo analogous to in vitro cell synchronization cycle by the removal and subsequent replacement of growth factor-rich serum to the culture medium. However, tissue in the intact animal is not deprived of serum under normal physiological conditions. Moreover, some tissues, such as the hypothalamic suprachiasmatic nucleus (SCN) and retina, possess endogenous time-keeping mechanisms (3). The dominant regulator of circadian clock phasing in these tissues is light, which regulates the circadian clock through immediate early genes. For example, considerable attention has focused on the regulatory role of the cAMP response element binding protein (CREB). Photic stimulation at night leads to CREB phosphorylation at its transactivation site, causing lasting phase shifts in the locomotor activity rhythm for days after stimulation. Within 5 min of light exposure, Ser-133-phosphorylated CREB (PCREB) appears in the SCN (3).
Here we investigated the expression of CREB in the mouse esophagus. We wanted to find out whether this tissue expressed CREB. Because the esophagus is histologically complex, we wanted to learn where it was expressed. Given the importance of transcriptional elements in circadian clocks, we also analyzed the circadian rhythmicity of CREB and PCREB expression. We found high levels of CREB-like immunoreactivity (CREB-lir) in basal replicating cells, but only at certain times of day. We speculate that CREB phosphorylation may play a key role in the circadian regulation of esophageal cell cycle progression.
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MATERIALS AND METHODS |
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Materials. BSA, Tris, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), and mouse submaxillary gland receptor-grade EGF were from Sigma (St. Louis, MO). Ammonium persulfate, N',N'-methylene-bis-acrylamide, polyacrylamide, N',N',N',N'-tetraethylethylenediamine, protein standards, and Tween 20 were from Bio-Rad (Richmond, CA). Prestained protein standards were from Amersham Life Sciences (Arlington Heights, IL). Nitro-Pure nitrocellulose was from Micron Separation (Westboro, MA). 5'-Bromo-2'-deoxyuridine (BrdU) labeling reagents were from Boehringer Mannheim (Indianapolis, IN). Antibodies from New England Biolabs included one against CREB and one against PCREB, which recognizes the peptide sequence containing phosphorylated Ser-133. This sequence occurs in the transcriptional activating domain of the CREB family of transcription factors, CREB, cAMP response element modulator, and activating transcription factor-1 (ATF-1) (5). Control SK-N-MC cell extracts were also from New England Biolabs. Anti-rabbit peroxidase-conjugated enzyme was from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence reagent was from New England Nuclear Life Science Products (Boston, MA).
Animals. We obtained B6D2F1 male mice from Jackson Laboratories. The animals for a given experiment were between 8 and 10 wk of age. Mice were standardized for at least 2 wk before the experiment to a 12:12 light-dark cycle (lights on at 0600; lights off at 1800) and provided with food and water ad libitum. We used a dim red light when getting mice from the animal room during the dark period.
Preparation of tissue extracts.
We killed mice in a CO2 chamber at
the indicated times. We removed the esophagus (~1.5 cm between
gastric junction and proximal esophagus) and other tissues, quickly
freezing them in liquid nitrogen and storing them at 75°C
until use. In some experiments, the mucosa was immediately separated
from the underlying muscularis by blunt dissection. Quick-thawed or
fresh tissue was homogenized in 300-700 µl of a homogenization
buffer containing 10 mM Tris, 1 mM EGTA, 500 µM
Na3VO4,
50 µM
Na2Mo4,
500 µM sodium fluoride, 50 mM glycerophosphate, 10 mM tetrasodium
pyrophosphate, 40 µg/ml PMSF, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin (pH 7.4). The sample was homogenized by a Tissue-Tearor
(Biospec Products) for 30 s at setting 2 and then sonicated 15 s at
output 1 using a Branson Sonifier 450. Particulate fractions were
prepared by centrifuging the homogenate for 30 min at 21,000 g and resuspending the pellet in
homogenization buffer. The final protein concentrations for the
homogenate and particulate fractions were between 1 and 5 mg/ml.
Western blotting. Homogenate or particulate protein (40 µg) was separated by SDS-PAGE (8.5%), transferred to nitrocellulose, and probed with the various antibodies. Antibody binding was detected by incubation with an appropriate horseradish peroxidase-conjugated goat anti-rabbit antibody. Enhanced chemiluminescence was performed as described previously (8). Immunoreactive bands were scanned by use of scanning laser densitometry (equipment courtesy of Dr. Fridolin Sulser, Vanderbilt University).
Immunohistochemistry. The esophagi were removed from CO2-killed mice and fixed in fresh 4% paraformaldehyde at 4°C for 4 h. Tissue pieces were rinsed several times in 70% ethanol at 4°C, embedded in paraffin, and cut into 5-µm sections. Immunohistochemistry was carried out as described elsewhere (8). We analyzed at least five esophagi from nine different circadian time points.
BrdU labeling. Mice were injected intraperitoneally with BrdU in 0.2 ml of 0.9% NaCl (50 mg/kg). They were killed 30 min after injection at either 1700 or 0500. The esophagi were fixed in 4% paraformaldehyde. We then prepared paraffin blocks and slides. BrdU was detected by immunohistochemistry. Tissue sections were pretreated with 1 N HCl for 8 min at 60°C to denature the DNA. We calculated the labeling indexes by counting labeled nuclei and total basal cells (n = 5).
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RESULTS |
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Circadian rhythmicity of esophageal DNA synthesis. [3H]thymidine incorporation into the total DNA obtained from the mouse esophagus varies in a circadian manner. To confirm this finding, we determined the in situ incorporation of BrdU into esophageal nuclei obtained from mice killed at either the trough (at 1700) or peak (at 0500) times of DNA synthesis (Fig. 1). As expected, BrdU localization was greatest in the nuclei of basal epithelial cells (cell layer marked by arrows). As shown in Fig. 1, over 30% of the ~500 basal cells were labeled at 0500, but <5% were labeled at 1700. This suggests that circadian variation largely arises from changes in the number of cells in S phase rather than from changes in the rate of DNA synthesis by individual cells.
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Immunocytochemical localization of CREB-lir. To determine the localization of CREB-lir, we carried out immunocytochemistry experiments. We detected CREB-lir in the nuclei of many cells in the esophagus (Fig. 2A). Although we also detected it in the muscularis and some submucosal fibroblasts, the epithelium displayed the strongest staining. Within the epithelium, we detected CREB-lir in cells within the spinous and granular layers. We also observed CREB-lir in an occasional elongated nucleus typical of apoptotic cells. However, the greatest staining was in the basal layer, where cells replicate and divide.
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Immunoblot localization of PCREB and CREB immunoreactivity in esophageal mucosa and muscularis. To confirm the predominant localization of CREB-lir to the mucosa as opposed to the submucosa and muscularis, we bluntly dissected the mucosa free from the underlying tissue and prepared homogenates from total esophagus, mucosa, and muscularis. In some mice, we injected EGF intraperitoneally (during midlight phase at 1400) 10 min before killing to determine whether it affected basal CREB-lir phosphorylation.
Figure 3A shows that CREB-lir resolved into three main specific proteins of 46, 43, and 36 kDa, each of which disappeared in the absence of primary antibody (data not shown). Short (15 s) and long (2 min) exposures are shown to bring out the various immunoreactivites. All three species were concentrated in the mucosa rather than in the muscularis. Scanning densitometry revealed 10-20 times more CREB-lir in the mucosa than in the muscularis. Virtually no 46-kDa band reactivity was found in the muscularis. Moreover, when a replica blot was probed with an anti-PCREB antibody, we again found a concentration of the phosphorylated species in the epithelium. Although this antibody did not work as well for immunohistochemistry as the CREB antibody, the major PCREB signal again localized to the basal cell layer (data not shown). Of the three CREB-lir isoforms, the 43- and 36-kDa species showed the greatest baseline phosphorylation.
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Circadian regulation of CREB-lir and PCREB. We further investigated whether CREB varied in a circadian manner by immunoblot analysis. Homogenates were prepared from five esophagi at seven different circadian times. Pooled homogenates from 1700, 2100, 0100, 0500, 0900, 1300, and 1700 were subjected to SDS-PAGE and immunoblotted with antibodies against either CREB or PCREB. Figure 4A shows the immunoblot results for CREB-lir. Both short (15 s) and long (2 min) exposures are presented to display the various immunoreactivites. The short exposure shows that the most abundant CREB-lir expression is seen during the light period. All immunoreactivities fell during the early dark phase, particularly the 46-kDa species and PCREB. All begin to increase with the onset of light, although the increase in the 46-kDa species and particularly PCREB occurred as early as 0500. Scanning densitometry indicated that the trough (dark phase) to peak (light phase) variation was 245 and 210% for the 46- and 43-kDa CREB-lir. Moreover, PCREB showed similar circadian changes. PCREB levels were threefold higher late in the light phase at 1700 than 4 h later early in the dark phase at 2100.
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DISCUSSION |
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Although the investigation of the cell cycle in vitro has been progressing at a rapid rate (4), very little attention has been paid to the normal mechanisms by which cells progress through the S and M phases of the cell cycle under the control of biological clocks (7). This is partly due to difficulties associated with animal work. It is also due to an overriding supposition by many investigators that cell entry into the S and M phase is random in the intact animal. Yet, in the oral cavity and esophagus, DNA synthesis varies by as much as ninefold during a 24-h circadian period, peaking in the late dark phase when most workers are not at work (Fig. 1). DNA synthesis then plummets during the normal working day, unbeknown to many workers.
Recently, phosphorylation of the transcription factor CREB has been shown to be a key step in coupling extracellular stimuli to long-lasting intracellular responses. Phosphorylation of CREB on Ser-133 by cAMP-dependent protein kinase A, Ca2+/calmodulin-dependent protein kinase, or nitric oxide generates PCREB. This leads to a dramatic increase in the transactivating potential of this protein and to increased transcription of cAMP-responsive genes, such as c-fos (3). In the hypothalamic SCN, CREB phosphorylation synchronizes the environmental light cycle with the SCN biological clock (3). Because a parallel mechanism could be operative in other circadian systems, we evaluated whether CREB and PCREB expression in the mouse esophagus varied in a circadian manner.
We found that CREB-lir, including CREB, ATF-1, and a yet to be
identified 46-kDa protein (1), were abundantly expressed in the mouse
esophagus, particularly in the epithelial basal cells. These cells
undergo cell replication, suggesting a role for CREB in cell cycle
events. Although the cell cycle-regulated expression of CREB has not
received much attention in cell culture systems, several reports
suggest such a relationship. For example, CREB phosphorylation in
several cell lines increases cyclin A inducibility via a cAMP response
element at positions 80 to
73 of the cyclin A
transcription initiation site (2). CREB phosphorylation also reportedly
inhibits the proliferation of hepatic stellate cells. In this case,
expression of the trans-dominant
negative CREB-Ala-133 induces quiescent cells to enter the S phase (5).
The latter findings are relevant to the esophagus because cAMP inhibits
both stellate and esophageal cell cycle progression (14). To what extent protein kinase A,
Ca2+/calmodulin-dependent kinase,
or nitric oxide contributes to the circadian variation in CREB
phosphorylation remains to be determined; however, it is interesting
that EGF, which stimulates and phase shifts esophageal DNA synthesis,
inhibited the basal levels of PCREB within 10 min after intraperitoneal
injection (Fig. 3B). Accordingly, it
will be important to identify the CREB serine kinases or phosphatases
affected by activation of the EGF tyrosine kinase receptor and to
define the exact mechanism and signaling pathways through which the EGF
receptor modulates their activities.
The mechanisms responsible for circadian variation in cell cycle progression of replicating cells in the alimentary epithelia are not known. We have previously speculated that changes in the local concentrations of growth factors, such as EGF-like ligands, or their receptors are involved by analogy to the in vitro model in which the importance of serum factors in cell cycle progression is shown by removing serum from the medium and then adding it back (11, 12). However, because serum starvation occurs only in pathological processes leading to an interruption of blood flow, there is no reason to assume a priori that the circadian synchronization of cell cycle progression is growth factor dependent. Although growth factors are certainly required for growth, cyclic oscillations in these factors or their receptors at the membrane level may not control circadian cell entry into the S phase. Intracellular regulation of transcription factors by neural or genetic clocks could play the primary regulatory role in circadian cell cycle progression.
In summary, we have shown circadian variation in the protein levels of CREB, ATF-1, and their phosphoproteins in the esophagus, particularly in the basal replicating cell layer. We speculate that these proteins may have regulatory roles in the circadian variation of cell cycle events in this tissue. Our work also shows the importance of considering time of day when analyzing the expression of transcription factors in animal work (15).
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ACKNOWLEDGEMENTS |
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This work was supported by the Smokeless Tobacco Research Council.
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FOOTNOTES |
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Address for reprint requests: L. A. Scheving, Dept. of Pediatrics, Division of Gastroenterology and Nutrition, Vanderbilt University School of Medicine, 21st and Garland Ave., Nashville, TN 37232-2561.
Received 7 July 1997; accepted in final form 9 December 1997.
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