FoxO4 is the main forkhead transcriptional factor localized in the gastrointestinal tracts of pigs

FoxO4 is the main forkhead transcriptional factor localized in the gastrointestinal tracts of pigs first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:fxshi@njau.edu.cn †E-mail:fxshi@njau.edu.cn fxshi@njau.edu.cn College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Forkhead box (Fox) proteins play critical roles in the regulation of differentiation, proliferation, immunity and aging of cells. Most studies on Fox proteins are limited to cultured cells and rodent. The aim of the current study is to detect by immunohistrochemistry whether FoxO1, FoxO3a and FoxO4 proteins are localized in the stomach and intestine of the pig. The results showed that FoxO4 exists in the mucosa in all parts of the stomach and intestine; FoxO3a exists mainly in the lamina propria and muscularis of some parts. However, FoxO1 is not detectable in all parts of the stomach and intestine. Collectively, the results of the present study indicate that there exists a distinct expression pattern of Fox proteins, and that FoxO4 is a primary forkhead transcriptional factor localized in the gastrointestinal tracts of the pig. Forkhead FoxO Gastrointestinal tract Immunohistochemistry Swine INTRODUCTION 1 Pigs are omnivorous animals and constitute a good animal model for studying human digestive processes. The gastrointestinal tracts of pigs are similar to that of the human in size and weight and are the ideal organs to transplant to human. Recently, pigs are widely used in the fields of study of oncology, immunology and for some disease models, for example, pig’s virosis gastroenteritis as an infantile virural diarrhea model, swine mycoplasma arthritis as a human arthritis model and double albuminemia, which has only been observed in humans and pigs (Wang et al., 2005 2005 ; Zhang et al., 2001 2001 ). Forkhead box (Fox) proteins are one kind of transcriptional factors and tumor suppressors which play a critical role in many physiological processes, including cell apoptosis, development and proliferation which may all impact with the generation of the tumor (Accili and Arden, 2004 2004 ). Studies of Fox proteins constitute a very exciting field since they have been nomenclature formally in 2000, but most of them are limited to cell culture, invertebrate and rodent studies including caenorhabditis elegans, fruit fly, mouse and rat (Liu and Lehmann, 2006 2006 ; Skurk et al., 2005 2005 ; Weinkove et al., 2006 2006 ). Fox proteins induce cell death mainly through two downstream pathways of FasL and Bim, and connect with cell cycle through the downstream pathways of p27 kip1 , cyclin G2, cyclin B, p130, and are related to DNA repairs through the downstream pathway of GADD45 (Barthélémy et al., 2004 2004 ; Martinez-Gac et al., 2004 2004 ; Kobayashi et al., 2005 2005 ; Yamamura et al., 2006 2006 ). This is why Fox proteins are called the “survival and death switch” (Burgering and Kops, 2002 2002 ). In the past decade, scientists engaged in the expression and mechanism of PI3K/PKB, upstream of FoxO in the gastrointestinal cancers (Michl and Downward, 2005 2005 ). So, researches about FoxO, downstream of PI3K/PKB, on gastrointestinal cancers therapy are immediately important. However, data on Fox proteins in the gastrointestinal tracts of pigs are lacking. Three members of FoxO family, i.e., FoxO1 (FKHR), FoxO3a (FKHRL1) and FoxO4 (AFX) are mostly studied in vertebrates (Arden and Biggs, 2002 2002 ). Recently, a novel FoxO member, FoxO6 was found in the mouse brain (Jacobs et al., 2003 2003 ; Hoekman et al., 2006 2006 ). However, there are only antisera against FoxO1, FoxO3a and FoxO4 ready available to date. Therefore, the aim of the present study is to examine whether these three FoxO members are specifically localized in the gastrointestinal tracts of the pig using immunohistochemistry. MATERIALS AND METHODS 1 Reagents 2 Antibodies for FoxO1 (Cat. No. 9462, Lot. 2), FoxO3a (Cat. No. 06-951, Lot. 27567) and FoxO4 (Cat. No. 9472, Lot. 1) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). All other chemicals were reagent grade and were obtained from Fisher Scientific (Pittsburgh, PA, USA). Animals 2 Four commercial pigs of Landrace-Duroc F1 cross breed commercial pigs including two castrated males and two other were females from Dingshan Hopgen Farm (Nanjing, China) were used in the current experiment. The experimental pigs were about 4 months old with 87~90 kg of body weights. The gastrointestinal samples were obtained within 30 min after sacrifice, fixed in 4% paraformaldehyde, and processed for immunohistochemical analyses of FoxO protein levels. The experimental protocol was approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institutional Animal Care and Use Committee, Nanjing Agricultural University. Immunohistochemistry 2 After fixation, the stomach and intestine samples were embedded in paraffin and 8 μm sections were cut and mounted on slides. The sections were then processed for immunohistochemical analysis similar to the method used in our previous reports (Shi and LaPolt, 2003 2003 ; Li et al., 2005a 2005a ). Briefly, sections were incubated overnight at room temperature with a polyclonal rabbit immunoaffinity-purified antiserum directed against the full length FoxO1, FoxO3a or FoxO4 proteins (0.5 mg/ml IgG). The specific protein immunoreactivity was visualized by the ABC Kit Elite and 0.05% 3,3′-diaminobenzidine tetrachloride (DAB; Sigma Chemical Co., St. Louis, MO, USA) in 10 mmol/L PBS-buffered saline containing 0.01% H 2 O 2 for 5 min. Specificity of the antibody was examined using normal rabbit serum instead of primary antibody. In order to identify structural components and cell types within the stomach and intestine, the section was counter-stained with hematoxylin and mounted with coverslips. Relative levels of immunostaining between animals and cell types were evaluated by three independent observers. Three individuals blinded to the experimental design were asked to examine the pictures and assign the intensity of staining using a scale of 0~10 with 0 indicating lack of brown immunoreactivity for FoxO proteins and 10 reflecting intense dark brown staining. All observers evaluated all slides and observations outside of the 5th to 95th percentile of the remaining observations for that treatment group were considered outlying data and were excluded from analysis. At last, we calculated the mean of them. If the immunostained cells were 1~3 the result was +, 4~6 was ++, more than 7 was +++. RESULTS 1 In order to detect the localization of FoxO family in the gastrointestinal tracts of pigs, we took samples from the fundic gland, non-glandular zones, pyloric gland of stomachs, duodenum, jejunum, ileum of small intestines, colon, cecum, and rectum of large intestines and performed immunohistochemsitry of FoxO1, FoxO3a and FoxO4 proteins. Localization of FoxO4, FoxO1 and FoxO3a in the Stomach 2 1. Fundic part 3 Localizations of FoxO4 in fundic part were distinct, and primarily concentrated in the nuclei of cells in the mucosa (Figs. 1a and 1b 1a and 1b ; Table 1 1 ). However, there was no detectable localization of FoxO1 and FoxO3a in the fundic part (Table 1 1 ). 2 Control FoxO4 FoxO1 FoxO3a 2 Fundic gland − +++ − − 2 Pyloric gland − +++ − − 2 Non-glandular zones − − − − 2 Duodenum Intestinal gland − +++ − − Lamina propria − − − − 2 Jejunum Intestinal gland − ++ − − Lamina propria − + − + 2 Ileum Intestinal gland − +++ − − Lamina propria − − − − 2 Colon Intestinal gland − + − − Lamina propria − − − + 2 Cecum Intestinal gland − + − − Lamina propria − − − + 2 Rectum Intestinal gland − ++ − − Lamina propria − − − + 2. Non-glandular zones 3 Compared with the non-glandular zones as control, FoxO4 (Fig. 1c 1c ; Table 1 1 ) and FoxO1 were not observed, and FoxO3a showed scattered localization in the muscularis layer. 3. Pyloric gland 3 FoxO4 in the pyloric gland was very similar to immunocytochemical localization in the fundus, and the localizations were concentrated in the nuclei of cells in the mucosa (Fig. 1d 1d ; Table 1 1 ). However, there was no FoxO1 and FoxO3a localization observed in this part. Localization of FoxO4, FoxO1 and FoxO3a in the small intestine 2 1. Duodenum 3 FoxO4 staining was very obvious in the intestinal gland of the Duodenum. The localization patterns were all basically in the base of the mucosa, close to the muscularis mucosa. These immunostained positive cells were almost all absorptive cells and goblet cells, but positively staining cells were observed in lamina propria (Figs. 2a and 2b 2a and 2b ; Table 1 1 ). Same as for the above parts, there was no localization of FoxO1 and FoxO3a observed. 2. Jejunum 3 FoxO4 proteins were localized clearly in the intestinal gland of the mucosa, with these localizations possibly all in absorptive cells, little in goblet cells, and little in the lamina propria (Fig. 2c 2c ; Table 1 1 ). Under high magnification, FoxO3a was non-existent in the intestinal gland but was present in the lamina propria. 3. Ileum 3 FoxO4 existed in the nuclei of absorptive cells and goblet cells in the intestinal gland. All of the localized protein was in the gland or near the gland, but not in the lamina propria (Fig. 2d 2d ; Table 1 1 ). FoxO1 and FoxO3a were undetectable in this part of the small intestine; however, FoxO3a was found in the muscularis of blood vessels in this area. Localization of FoxO4, FoxO1 and FoxO3a in the large intestine 2 1. Colon 3 FoxO4 was found mainly in nuclei of cells in the intestinal gland, and around the intestinal gland, with less localization in the lamina propria, and non-existent in the submucosa and muscularis (Fig. 2e 2e ; Table 1 1 ). Strong staining for FoxO3a was found in nuclei of cells in the lamina propria, but not in the intestinal gland cells. Under high magnification, some FoxO3a were observed in the muscularis of blood vessels (Fig. 2f 2f ; Table 1 1 ). 2. Cecum 3 FoxO4 was found in nuclei of cells in the intestinal gland (Fig. 2g 2g ; Table 1 1 ), while FoxO3a was observed in nuclei of cells in the lamina propria and muscularis of blood vessel. Localization of FoxO1 was not observed. 3. Rectum 3 Localization of FoxO4 in nuclei of cells in the intestinal gland of mucosa was very obvious; and it might exist in absorptive cells, goblet cells, and endocrine cells (Fig. 2h 2h ; Table 1 1 ). FoxO3a staining was observed in the lamina propria, but there was no staining for FoxO1. DISCUSSION 1 To our knowledge this is the first report of localization of FoxO4, FoxO1 and FoxO3a in the stomach and intestine of pigs. The results of our current study clearly showed that FoxO4 exists in the nuclei of cells in the mucosa of the fundic gland, pyloric gland, non-glandular zones, duodenum, jejunum, ileum, colon, cecum, rectum; and that FoxO3a exists mainly in the nuclei of lamina propria cells in the mucosa of jejunum, colon, cecum and rectum, and probably in the muscularis. FoxO1 was not found to be localized immunohistochemically in the stomach and intestine of pigs. This distinct localization pattern of FoxO proteins implies that functions of FoxO proteins, especially FoxO4, are involved in the processes of the digestive system of pigs. Earlier researches showed that expression of FoxO4 blocked cell-cycle progression at phase G1, and is dependent on the cell-cycle inhibitor p27 kip1 (Yang et al., 2005 2005 ). Protein kinase B (PKB) phosphorylates and down-regulates the FoxO4 then enhances the transcription of p27 kip1 (Fukuoka et al., 2003 2003 ). It is known that p27 kip1 inhibits the activity of cyclinE/CDK2, which allows the cell-cycle progression through G1 (Graff et al., 2005 2005 ). Some researchers found that the mutant of FoxO4 (FoxO4A3), which maintains the activity to transactivate p27 kip1 , may be used as an anticancer agent for HER2-overexpressing cancers (Yang et al., 2005 2005 ). In the mammal, the molecular signaling FoxOs are located at the intersection of three important signaling pathways: Smad, PI3K and FoxG1 (Seoane et al., 2004 2004 ). Smad binds to FoxO, and actively inhibits the growth-related gene, p21 cip1 (Li et al., 2005b 2005b ; 2006 2006 ); however, the binding of FoxG1 to the compound FoxO-Smad inhibits the expression of p21 cip1 (Seoane et al., 2004 2004 ). This interaction requires further study to examine the expression patterns of FoxO4 related to metabolism and proliferation. Our previous study showed that FoxO1 protein exists in the rat ovary (Shi and LaPolt, 2003 2003 ; Li et al., 2005a 2005a ). However, the present study did not find any significant localization of FoxO1 in the gastrointestinal tract of pigs. This implies that this member of FoxO family has tissue-specific expression patterns. From the present study, we found different localization patterns of FoxO3a in the different parts of the stomach and intestine in the pig. This may imply some relation with the cell-cycle. Some researches have shown that independent actions of Akt, Ikappa B kinase (IKK-β) can cause proteolysis of FoxO3a via the Ub-dependent proteasome pathway (Hu et al., 2004 2004 ). TGF-β-active Smad protein then binds to FoxO and activates the growth inhibitory gene p21 cip1 (Hu et al., 2004 2004 ). In addition, IKK-β was shown to interact with and phosphorylate FoxO3a and that the IKKs might serve as a potential drug target in anticancer therapy (Finnberg and El-Deiry, 2004 2004 ). Active forms of FoxO3a increase cyclin G2 mRNA levels and then block the cell cycle progression (Martinez-Gac et al., 2004 2004 ). Some other studies proved that active FoxO3a could induce expression of IL-2-regulated genes, such as the cdk inhibitor p27 kip1 and the proapoptotic Bcl-2 family member Bim (Stahl et al., 2002 2002 ). On the other hand, active FoxO3a leads to accumulation of Bcl6 and down-regulation of cyclin D2 at protein and mRNA levels (Fernandez de Mattos et al., 2004 2004 ). More interestingly, in contrast to FoxO4, FoxO3a does not exist in the intestinal gland, which may show that it has no relation to the gland of the intestinal tract. In summary, FoxO4 localization is observed in the nuclei of cells in the mucosa, and FoxO3a localized in the nuclei of cells in the lamina propria. 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Hutz (Department of Biological Sciences, University of Wisconsin-Milwaukee, USA) for reading the original manuscript and for valuable suggestions. 10.1631/jzus.2007.B0039 Project supported by the National Basic Research Program (973) of China (No. 2004CB117500) and the National Natural Science Foundation of China (Nos. 30571335 and 330471253) 2006-04-14 2006-05-30 2006-12-18 2022-01-05 Copyright © 2007, Journal of Zhejiang University Science 2007 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 08