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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 29, 2005 as doi:10.1096/fj.05-4216fje. |
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* Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality, The Hebrew University of Jerusalem, Rehovot, Israel; and
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel
1 Correspondence: E-mail: froy{at}agri.huji.ac.il
SPECIFIC AIMS
The goal of this study was to determine whether the biological clock plays a role in cryptdin expression under healthy conditions.
PRINCIPAL FINDINGS
1. FVB/N mice exhibit molecular oscillation of clock genes
To study mouse cryptdin regulation by the biological clock, we first verified that FVB/N mice displayed a functional clock. Although FVB/N mice exhibit physiological circadian rhythms, this strain was shown to display a fragmented wheel-running activity pattern with increased activity during the light phase compared with other mouse strains. Therefore, we first wanted to establish that FVB/N mice displayed a functional biological clock at the molecular levels. Mice were maintained at 12 h light and 12 h dark (LD) conditions for two weeks for their biological clocks to entrain. After two weeks, the expression of two liver clock genes, mPer1 and mBmal1 (data not shown), was determined using Northern blot analyses. Both genes oscillated in the liver (One-way variance, P<0.0001) in opposite phases, in accordance with their opposing roles in the core clock mechanism and similarly to previous reports. Thus, although FVB/N mice may display noncanonical wheel-running behavior, they exhibit a functional clock at the molecular level as well as output physiological mechanisms.
2. Mouse cryptdins oscillate throughout the circadian cycle
After having established clock functionality in FVB/N mice, we set out to study whether cryptdin expression was an output mechanism of the biological clock. After the two week entrainment, ileum and jejunum were collected every 3 h around the circadian cycle in the first day of total darkness (DD conditions) and tested by quantitative real time PCR for cryptdin expression. As cryptdin 1, cryptdin 2, and cryptdin 3 share high sequence similarity, but differ significantly from cryptdin 4, we analyzed the levels of cryptdin 1 and cryptdin 4 with Gapdh as the reference gene. The levels of both cryptdin 1 and cryptdin 4 exhibited 2-fold oscillation between peak and trough (one-way variance, P<0.01) around the circadian cycle, peaking at the end of the subjective night (Fig. 1
). Similar results were obtained by Northern blot analyses using ß-actin as the reference gene (data not shown). Cryptdin oscillation was also found in C57BL/6 (data not shown), a mouse strain that has been extensively studied in circadian rhythms, reiterating the generality of cryptdin control by the biological clock. To eliminate the possibility that the food induces cryptdin expression in the intestine, we studied cryptdin expression after 24 h of fast. The results showed similar oscillation of cryptdin 1 and cryptdin 4 (one-way variance, P<0.001) although with a three-hour phase-shift (Fig. 1)
. This phase shift is reminiscent of the clock phase shift achieved under total darkness (DD) vs. LD conditions in insects or mammals. Indeed, restriction of food to a specific time of the circadian cycle has been shown to have the ability to reset the clock in peripheral organs even after ablation of the master clock in the SCN. Thus, it is possible that food is a zeitgeber (synchronizer) in the small intestine similarly to the role of light in the SCN.
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3. Cryptdin expression level in mice
To determine the expression levels of mouse cryptdins, we analyzed the levels of cryptdin 1 and cryptdin 4 in C57BL/6 and FVB/N mice. The C57BL/6 strain has several variants of genes that are similar to Cryptdin 4. Therefore, the primers tested were in the same region as those for FVB/N, but with complete identity to C57BL/6 sequence. Cryptdin 1 level of expression in the jejunum vs. the ileum in C57BL/6 mice was not significantly different (Student’s t test, P=0.26). In contrast, in FVB/N mice levels of cryptdin 1 were >2-fold higher in the jejunum compared with the ileum (Fig. 2
). However, the levels of cryptdin 4 in the ileum of C57BL/6 were 50-fold higher than those in the jejunum. In FVB/N, cryptdin 4 levels were only 2-fold higher in the ileum vs. the jejunum. The levels of cryptdin 1 and cryptdin 4 in C57BL/6 were on average 10-fold and 7- to 200-fold higher, respectively, compared with those of FVB/N (Student’s t test, P<0.01) (Fig. 2)
. As cryptdin 4 was found to be the most potent antibacterial defensin in mouse small intestine and 
-defensins were found to be crucial for survival, it is expected that C57BL/6 mice will be more resistant to enteric pathogens than other mouse strains. However, C57BL/6 mice have mutations in the sequence of the pro-region preventing cleavage site recognition by the protease to render cryptdin 4 active. Therefore, although cryptdin 4 is expressed to a higher level, its posttranslational processing is impaired and, as a result, C57BL mice (C57BL/Fa, C57BL/6, and C57BL/10 congenic line B10.D2-n) are highly susceptible to infection by the digestive system pathogens Salmonella typhimurium strain C5 and Helicobacter felis.
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CONCLUSIONS AND SIGNIFICANCE
Although mouse enteric cryptdins are induced in inflammatory bowel diseases, their basal level of expression is dictated by the biological clock and their circadian expression can be entrained by food (Fig. 3
). Further study will be required in order to delineate how the biological clock regulates cryptdin expression. With the assumption that human enteric defensin (HD-5 and HD-6) expression is also regulated by the biological clock, we expect their oscillation to peak toward the end of the light period.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4216fje;
This article has been cited by other articles:
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  O. Froy, N. Chapnik, and R. Miskin Long-lived {alpha}MUPA transgenic mice exhibit pronounced circadian rhythms Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E1017 - E1024. [Abstract] [Full Text] [PDF]
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 O Froy, G Levkovich, and N Chapnik Immunonutrition enhances the expression and secretion of mouse intestinal defensins. Gut, June 1, 2006; 55(6): 900 - 901. [Full Text] [PDF]
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