Jnk2 deletion disrupts intestinal mucosal homeostasis and maturation by differentially modulating RNA-binding proteins HuR and CUGBP1

Abstract

Homeostasis and maturation of the mammalian intestinal epithelium are preserved through strict regulation of cell proliferation, apoptosis, and differentiation, but the exact mechanism underlying this process remains largely unknown. c-Jun NH₂-terminal kinase 2 (JNK2) is highly expressed in the intestinal mucosa, and its activation plays an important role in proliferation and also mediates apoptosis in cultured intestinal epithelial cells (IECs). Here, we investigated the in vivo function of JNK2 in the regulation of intestinal epithelial homeostasis and maturation by using a targeted gene deletion approach. Targeted deletion of the jnk2 gene increased cell proliferation within the crypts in the small intestine and disrupted mucosal maturation as indicated by decreases in the height of villi and the villus-to-crypt ratio. JNK2 deletion also decreased susceptibility of the intestinal epithelium to apoptosis. JNK2-deficient intestinal epithelium was associated with an increase in the level of the RNA-binding protein HuR and with a decrease in the abundance of CUG-binding protein 1 (CUGBP1). In studies in vitro, JNK2 silencing protected intestinal epithelial cell-6 (IEC-6) cells against apoptosis and this protection was prevented by inhibiting HuR. Ectopic overexpression of CUGBP1 repressed IEC-6 cell proliferation, whereas CUGBP1 silencing enhanced cell growth. These results indicate that JNK2 is essential for maintenance of normal intestinal epithelial homeostasis and maturation under biological conditions by differentially modulating HuR and CUGBP1.

the epithelium of mammalian intestinal mucosa is among the fastest self-renewing tissues in the body and undergoes a continual renewal process, characterized by active proliferation of stem cells localized near the base of the crypts, progression of these cells up the crypt-villus axis with cessation of proliferation, and subsequent differentiation and apoptosis (36). The epithelium of the human small intestine undergoes ∼10¹¹ mitoses per day, and this dynamic turnover rate is highly regulated at multiple levels by numerous intracellular and extracellular factors, including the presence of luminal food (39, 43), gut peptides (17), cellular polyamines (6, 12, 24, 44), RNA-binding proteins (RBPs) (47, 53), and several microRNAs (5, 9, 45, 46). In response to stress, rapid changes in gene expression patterns in intestinal epithelial cells (IECs) control cell division and survival, thereby preserving the epithelial homeostasis (36, 43). Inhibition of intestinal mucosal growth occurs commonly in various critical disorders, particularly in patients who undergo massive surgical operations and are then supported with total parenteral nutrition, leading to mucosal atrophy, delayed healing, and barrier dysfunction (13, 30, 37). However, the exact mechanism underlying control of intestinal epithelial homeostasis and maturation, especially at the molecular level, remains to be fully elucidated.

c-Jun NH₂-terminal kinases (JNKs) are members of the superfamily of mitogen-activated protein kinase family and comprise three main types: JNK1, JNK2, and JNK3. JNK2 isoforms are encoded by the jnk2 gene and are highly homologous with JNK1 and JNK3 that are transcribed from jnk1 and jnk3 genes, respectively (10). The first two enzymes are ubiquitously distributed, but the third is confined to the central nervous system and cardiac myocytes. JNKs bind to the NH₂-terminal activation domain of the AP-1 transcription factor c-Jun and regulate its transcriptional activity (10, 16). Increasing evidence has demonstrated that JNK1, JNK2, and JNK3 have distinct biological functions and are implicated in many aspects of cellular activities such as proliferation, differentiation, migration, transformation, and apoptosis (10, 16, 34, 40). JNK1 and JNK2 are shown to play distinct roles in the regulation of JNK activity and c-Jun-dependent cell proliferation (34); loss of JNK1, but not JNK2, causes spontaneous intestinal tumor formation in mice (40). Mice harboring JNK1 or JNK2 inactivation exhibit opposite susceptibility to tumor formation induced by 12–0-tetradecanoyl-phorbol-13-acetate (7, 38), and loss of JNK2 increases intestinal tumor susceptibility in Apc1638^+/− mice with dietary modulation (3). JNK2 also regulates expression of proinflammatory cytokinesis in intestinal mucosa and is involved in the pathogenesis of inflammatory bowel diseases (8, 32, 42). Interestingly, JNKs were found to cross talk with Wnt/β-catenin signaling in a variety of biological processes (33) and JNK2 interacts with and negatively regulates Wnt/β-catenin signaling activity (15).

Little is known about the biological function of JNK2 in the regulation of normal intestinal epithelial homeostasis and maturation. For example, whether JNK2 has a proapoptotic or antiapoptotic role is still a controversial issue (20, 28). The reasons for this controversy may be from the selective interaction of JNK protein kinase isoforms with different transcriptional factors (4, 14). In addition, most of our knowledge about JNK functions comes from studies conducted in cultured cells and/or experimental conditions in which the levels of JNKs are artificially increased through the use of transgenic mice. Here we have studied the role of JNK2 in intestinal epithelium by using a conditional gene-targeting approach and demonstrated that JNK2 deficiency disrupts mucosal epithelial homeostasis and maturation in the small intestine by differentially modulating RNA-binding proteins (RBPs) HuR and CUG-binding protein 1 (CUGBP1).

MATERIALS AND METHODS

Animal studies.

C57BL/6J mice and JNK2 knockout (JNK2-KO) mice in C57BL/6J background were purchased from the Jackson Laboratory (Bar Harbor, ME), and all experiments were approved according to animal experimental ethics committee guidelines by the University of Maryland Baltimore Institutional Animal Care and Use Committee. Mice were housed and handled in a specific pathogen-free breeding barrier and cared for by trained technicians and veterinarians. Animals were euthanized by CO₂ asphyxiation. Bromodeoxyuridine (BrdU) was incorporated in intestinal mucosa by intraperitoneal injection of 50 mg/kg BrdU (Sigma, St. Louis, MO) in phosphate-buffered saline. A 4-cm small intestinal segment taken 0.5 cm distal to the ligament of Trietz was collected 1 h postinjection. Each tissue sample was divided into two portions, one for extraction of protein and RNA, and the other for histological examination. In experiments with apoptosis, mice were intraperitoneally injected with tumor necrosis factor-α (TNF-α) at a dose of 25 μg/kg body wt, and the mucosal tissues were harvested for measurement of apoptotic cell death at 5 h postinjection.

Cell cultures and plasmid construction.

The IEC-6 cell line, derived from normal rat intestinal crypt cells, was used at passages 15–20 in experiments (6, 13). Tissue culture medium and fetal bovine serum were obtained from Invitrogen (Carlsbad, CA), and biochemicals were obtained from Sigma (St. Louis, MO). Antibodies recognizing JNK2, HuR, CUGBP1, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling (Danvers, MA), and the secondary antibody conjugated to horseradish peroxidase was obtained from Sigma. CUGBP1 expression vector was purchased from Origene (Rockville, MD). Transient transfections were performed using the Lipofectamine Reagent as recommended by the manufacturer.

RNA interference.

The siRNAs that were designed to specifically target the coding regions of JNK2 (siJNK2), HuR, (siHuR), or CUGBP1 (siCUGBP1) mRNAs were synthesized and purchased from Dharmacon (Lafayette, CO). Scrambled control siRNA (C-siRNA), which had no sequence homology to any known genes, was used as the control. The siJNK2, siHuR, siCUGBP1, and C-siRNA were transfected into cells as described in previously (6). Briefly, for each 60-mm cell culture dish, 20 μl of the 5 μM stock siJNK2, siHuR, siCUGBP1, or C-siRNA were mixed with 500 μl of Opti-MEM medium (Invitrogen). This mixture was added to a solution containing LipofectAMINE 2000 in 500 μl of Opti-MEM. The solution was incubated for 20 min at room temperature and gently overlaid onto monolayers of cells in 3 ml of medium, and cells were harvested for various assays after 48 h incubation.

Western blot analysis.

Whole cell lysates were prepared using 2% SDS, sonicated, and centrifuged at 4°C for 15 min. The supernatants were boiled for 5 min and size fractionated by SDS-PAGE. After proteins were transferred onto nitrocellulose filters, the blots were incubated with primary antibodies recognizing JNK2, HuR, and CUGBP1 proteins; following incubations with secondary antibodies, immunocomplexes were developed by using chemiluminescence.

RT followed by PCR analysis.

Total RNA was isolated by using RNeasy mini kit (Qiagen, Valencia, CA) and used in reverse transcription and PCR amplification reactions as described (9). Equal amounts of total RNA (5 μg) were transcribed to synthesize single-strand cDNA with an RT kit (Invitrogen, Carlsbad, CA). The specific sense and antisense primers for JNK2 included 5′-GGTCCTCCCTCCTGAGCCTAGAT-3′ and 5′-GTGTTCTGAGGCCGCT-CTCCTAC-3′, and the expected size of JNK2 fragments was 368 bp. Reverse transcription and PCR was performed as described in our earlier publications (5).

Immunofluorescence staining.

Immunofluorescence was performed as described previously (46, 52). Mucosal tissues were fixed using 3.7% formaldehyde, and the rehydrated samples were incubated overnight at 4°C with primary antibody anti-BrdU diluted 1:300 in blocking buffer and then incubated with secondary antibody conjugated with Alexa Fluor-488 (Molecular Probes, Eugene, OR) for 2 h at room temperature. After being rinsed, slides were incubated with 1 μM TO-PRO3 (Molecular Probes) for 10 min to stain nuclei, rinsed again, mounted, and viewed through a Zeiss confocal microscope (model LSM700). Images were processed using Photoshop software (Adobe, San Jose, CA).

Measurement of intestinal permeability.

Intestinal permeability was determined by examining the appearance in blood of FITC-dextran administered by gavage as described previously (49). Briefly, mice were gavaged with FITC-dextran at a dose of 60 mg/100-g wt 4 h before harvest. Blood sample was collected by cardiac puncture. The serum concentration of the FITC-dextran was determined using a fluorescence plate reader with an excitation wavelength at 490 nm and an emission wavelength of 530 nm.

Statistics.

Results are expressed as means ± SE from three to six samples. The significance of the difference between means was determined by Student's t-test; P < 0.05 was considered significant.

RESULTS

Jnk2 deletion increases cell proliferation within the crypts but reduces the height of villi.

To define the in vivo function of JNK2 in the regulation of intestinal mucosal homeostasis and maturation, JNK2^−/− mice were used in this study. As described previously (11, 19), heterozygous JNK2^+/− mice appeared phenotypically normal and were subsequently intercrossed for the generation of homozygous JNK2^−/− mice. Generally, JNK2^−/− mice looked normal; there were no significant differences in body weight, gastrointestinal gross morphology, ability to reproduce, and general appearances between JNK2^−/− mice and littermate controls (data not shown). Age-matched JNK2^−/− mice and littermate control mice (3 or 4-mo-old) were used for phenotype and showed that JNK2 mRNA and protein in the small intestinal mucosa were undetectable in JNK2^−/− mice, whereas JNK2 expression levels in the intestinal mucosa of control littermate mice were normal (Fig. 1A). Interestingly, JNK2^−/− mice exhibited a significant increase in the length of crypts in the small intestinal mucosa but displayed shorter villi, thus leading to a decrease in the villus-to-crypt ratio (Fig. 1, B and C). Abnormal histological features of the mucosa were also observed in JNK2^−/− mice as shown by cells with enlarged cytoplasm and filled with unknown granules at the base of crypts. As shown in Fig. 2, A and B, the proliferating crypt cell population, marked by BrdU (S-phase), increased remarkably in the small intestine of JNK2^−/− mice compared with those from littermates. We also examined changes in colonic mucosal growth in JNK2^−/− mice and found that JNK2 deletion did not alter mucosal growth in the colon. There were no significant decreases in the lengths of villi and crypts and BrdU-labeled cell proliferation in the colonic mucosa of JNK2^−/− mice compared with those observed in control littermates (data not shown). In addition, JNK2 deletion did not affect basal level of gut permeability as measured by FITC-dextran tracer flux assays (Fig. 2C). These results indicate that JNK2 deletion increases crypt cell proliferation in the small intestine and it also disrupts villus maturation.

Fig. 1.

JNK2 deletion disrupts intestinal mucosal maturation. A: levels of JNK2 mRNA (a) and protein (b) in small intestinal mucosa in littermate control and JNK2 knockout (JNK2-KO) mice as measured by RT-PCR and Western blotting analyses. B: photomicrographs of hematoxylin and eosin staining of the small intestine. Scale bar = 100 μm. C: changes in the length of villi (left), crypt (middle), and the villus-to-crypt ratio of the mucosa described in B. Values are shown as means ± SE (n = 4). *P < 0.05, compared with littermates.

Fig. 2.

Loss of JNK2 induces cell proliferation in small intestinal crypts. A: proliferating cells in small intestinal crypts as measured by bromodeoxyuridine (BrdU) labeling technique: littermates (a) and JNK2-KO (b) mice. BrdU (1 h postinjection, S-phase) is in green. (B) Summarizing data from mice described in A. Values are the means ± SE (n = 5). *P < 0.05, compared with littermates. C: changes in gut permeability as measured by FITC-dextran tracer flux assays in animals described in A. FITC-dextran was given orally, and blood samples were collected 4 h thereafter for measurement. Values are means ± SE of data from 5 samples.

Jnk2 deletion reduces susceptibility of the intestinal epithelium to apoptosis.

The JNK2-deficient intestinal epithelium showed no spontaneous apoptosis, since there was no apparent cell death in the intestinal epithelium in both littermates and JNK2^−/− mice as examined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining, a classic indicator of apoptotic cells (Fig. 3, Aa and Ab, left). To determine the susceptibility of JNK2-deficient intestinal epithelium to induced apoptosis, mice were exposed to TNF-α for 5 h. In control littermate mice, typical morphological features characteristic of apoptotic cell death were observed in small intestinal mucosa: increased levels of TUNEL-positive cells (Fig. 3, Aa, right, and B, left) and active caspase-3 (Fig. 3C, left). However, JNK2^−/− mice exhibited significant tolerance to TNF-α-induced apoptosis as indicated by decreases in the percentages of apoptotic cells (Fig. 3, Ab and B) and active caspase-3 (Fig. 3C, right) after treatment with TNF-α. These results strongly suggest that JNK2 deletion protects the intestinal epithelium against TNF-α-induced apoptosis.

Fig. 3.

JNK2 deletion protects the intestinal mucosa against apoptosis. A: terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining of apoptotic cells in small intestinal mucosa in littermates and JNK2-KO mice after treatment with TNFα for 5 h. TUNEL-positive cells are shown as green, but nuclei are in purple. B: percentages of apoptotic cells in the mucosa in mice described in A. Values are means ± SE; n = 5. *P < 0.05, compared with mice untreated with TNFα; +P < 0.05, compared with littermates exposed to TNFα. C: changes in levels of caspase-3 in mice described in A. The levels of procaspase-3 and caspase-3 were assessed by Western blot analysis; GAPDH immunoblotting was performed as an internal control for equal loading.

JNK2-deficient intestinal epithelium is associated with increased HuR but decreased CUGBP1.

To investigate the mediators of the JNK2 deletion-elicited effects, we examined changes in the levels of HuR and CUGBP1 in JNK2^−/− mice, because these two RBPs were recently found to play an important role in intestinal epithelial homeostasis (45, 52, 53). As shown in Fig. 4, intestinal mucosal HuR levels increased in JNK2-deficient mice, whereas CUGBP1 abundance decreased. Since JNK2 deletion did not alter the levels of p53 protein, it is suggested that observed changes in expression of HuR and CUGBP1 in JNK2^−/− mice are specific. Immunohistochemical staining assays further revealed that HuR was predominantly located at the crypt region in control littermates, but its immunostaining increased significantly at both villi and crypts in JNK2^−/− mice. On the other hand, immunohistochemical staining of CUGBP1 in small intestinal mucosa decreased at the crypt region in JNK2^−/− mice compared with those observed in control littermates. Consistently, the level of HuR mRNA in the mucosa of JNK2^−/− mice also increased but CUGBP1 mRNA abundance decreased (data not shown). These results indicate that JNK2 deletion induces HuR level but decreases CUGBP1 content in small intestinal mucosal tissue.

Fig. 4.

JNK2 deficiency increases HuR but decreases CUG-binding protein 1 (CUGBP1) abundance in small intestinal mucosa. A: changes in the levels of HuR, CUGBP1, and p53 proteins in littermates and JNK2-KO mice. B: immunohistochemical staining of HuR (a) and CUGBP1 (b) in small intestinal mucosa. HuR and CUGBP1 are in brown. Scale bar = 100 μm.

HuR is crucial for induced resistance of JNK2-silenced cells to apoptosis in vitro.

To investigate the consequences of induced HuR upon intestinal epithelial homeostasis in JNK2^−/− mice, we measured changes in apoptosis after silencing JNK2 alone and silencing both JNK2 and HuR in IEC-6 cells. Decreased levels of endogenous JNK2 and HuR by transfection with siRNA specifically targeting JNK2 (siJNK2) or siRNA targeting HuR (siHuR) did not directly induce apoptosis in IEC-6 cells, since there were no apparent differences in cell viability between control cells and cells transfected with siJNK2 alone or cotransfected with siJNK2 and siHuR (Fig. 5B, left). To determine whether JNK2 altered the susceptibility to apoptosis via HuR, cells were exposed to TNF-α plus cycloheximide (CHX). When control cells or cells transfected with control siRNA (C-siRNA) were treated with TNF-α/CHX for 4 h, we observed morphological features characteristic of programmed cell death, elevated annexin V staining (Fig. 5, Ba and Bb, and C, left), and increased levels of active (cleaved) caspase-3 (Fig. 5D, left). However, JNK2 silencing by siJNK2 protected IEC-6 cells against TNF-α/CHX-induced apoptosis as shown by decreases in the percentages of apoptotic cells (Fig. 5C) and in the levels of active caspase-3 (Fig. 5D). Interestingly, this protective effect was lost when HuR expression was silenced by siHuR. The percentages of apoptotic cells and levels of active caspase-3 in JNK2-silenced cells transfected with siHuR were higher than those seen in cells transfected with siJNK2 alone after exposure to TNF-α/CHX. Consistent with our previous findings (47, 50), silencing HuR alone also enhanced TNF-α/CHX-induced apoptosis in IEC-6 cells (data not shown). These results indicate that HuR is necessary for protection of JNK2-silenced cells against apoptosis, suggesting that induced HuR in JNK2-deficient intestinal epithelium contributes to the tolerance to apoptosis in mice.

Fig. 5.

JNK2 silencing protects intestinal epithelial cells (IECs) against apoptosis through endogenous HuR in IEC-6 cells. A: levels of JNK2 and HuR proteins after transfection with siRNA targeting JNK2 (siJNK2) or HuR (siHuR) alone or cotransfection with siJNK2 and siHuR for 48 h. B: TNF-α/cycloheximide (CHX)-induced apoptosis in cells described in A. Apoptosis was measured following 4 h of treatment with TNF-α/CHX: a: untransfected cells (control); b: cells transfected with control siRNA (C-siRNA); c: cells transfected with siJNK2; d: cells cotransfected with siJNK2 and siHuR. C: percentages of apoptotic cells after different treatments as described in B. Values are means ± SE of data from 3 experiments. *P < 0.05, compared with cells untreated with TNF-α/CHX; +P < 0.05, compared with controls or C-siRNA-transfected cells exposed to TNF-α/CHX; #P < 0.05, compared with cells transfected with siJNK2 and then exposed to TNF-α/CHX. D: changes in levels of caspase-3 in cells described in B. Whole cell lysates were harvested, and the levels of procaspase-3 and caspase-3 were assessed by Western blot analysis. GAPDH immunoblotting was performed as an internal control for equal loading.

CUGBP1 represses IEC-6 cell proliferation.

To determine whether the induction in crypt cell proliferation in the small intestine of JNK2^−/− mice results from a decrease in CUGBP1 levels, we examined the effects of increased or decreased CUGBP1 on IEC-6 cell proliferation. As shown in Fig. 6, A and B, ectopic overexpression of CUGBP1 by transfection with the CUGBP1 expression vector repressed cell growth dramatically. The proliferation rate of CUGBP1-transfected cells was much lower than that of cells transfected with control vector. In contrast, CUGBP1 silencing by transfection with siRNA targeting CUGBP1 (siCUGBP1) enhanced cell growth (Fig. 6, C and D). Cell numbers were increased significantly in CUGBP1-silenced cells compared with those obtained from cells transfected with C-siRNA. These results indicate that CUGBP1 inhibits IEC growth in vitro and suggest that decreased levels of CUGBP1 in the JNK2-deficient intestinal epithelium plays an important role in stimulation of crypt cell proliferation in mice.

Fig. 6.

CUGBP1 inhibits IEC-6 cell proliferation. A: immunoblots of CUGBP1 after transfection with the CUGBP1 expression vector for 48 h. B: changes in cell growth after ectopic CUGBP1 overexpression. Values are the means ± SE (n = 3). *P < 0.05, compared with cells transfected with null vector. C: CUGBP1 protein in cells transfected with siRNA targeting CUGBP1 (siCUGBP1) or control siRNA (C-siRNA) for 48 h. D: cell growth after CUGBP1 silencing. *P < 0.05, compared with cells transfected with C-siRNA.

DISCUSSION

JNK2 is identified as a stress-activated kinase and shown to play a critical role in various pathological states in different tissues including intestinal mucosa (1, 8, 32, 34, 40), but little is known about its function in maintenance of normal intestinal epithelial homeostasis and maturation. Although there are several studies available showing the implication of JNK2 in the regulation of IEC apoptosis and epithelial tight junctions (2, 26, 31, 35), all of these studies were conducted in cultured IECs; therefore, the exact function of JNK2 in the intestinal epithelium in vivo remains to be fully defined. Using JNK2^−/− mice, we provided powerful genetic evidence of the physiological role of JNK2 in the control of intestinal mucosal epithelial homeostasis and maturation. Targeted deletion of the jnk2 gene not only induced crypt hyperplasia in the small intestine but also disrupted mucosal maturation as indicated by shorter villi and a decrease in the villus-to-crypt ratio. Experiments aimed at characterizing the mechanism underlying this process suggest that the disruption of intestinal mucosal homeostasis and maturation induced by JNK2 deletion results from the induction in cellular HuR abundance and reduction in CUGBP1 levels. These findings advance our understanding of the biological function of JNK2 in the intestinal epithelium and highlight a novel role of JNK2 in the regulation of intestinal epithelial homeostasis and maturation.

Several recent mouse JNK2 gene-deletion studies have improved our understanding of the roles of JNK2 in various pathological conditions in mammals that, in some circumstances, contradict conventional wisdom of previous in vitro cell biology studies (3, 7, 38, 40). Results from our in vivo studies demonstrate the essential role of JNK2 for normal mucosal epithelial homeostasis and integrity in the small intestine, since significant defects in mucosal growth and maturation occurred in JNK2^−/− mice, as indicated by an increase in cell proliferation within the crypts and shrinkages of villi. The present study was conducted in global JNK2 knockout mice; therefore, there is a possibility that at least some of the effects observed in vivo could reflect JNK2 deficiency in cells other than the small epithelium. This stimulatory phenotype in small intestinal crypt growth and inhibitory effect on villus maturation by JNK2 deletion are consistent with results from others who demonstrated that JNK2 plays an important role in cellular proliferation (10, 18, 25, 51) and is also absolutely required for differentiation (48). It has been reported that JNK2^−/− murine embryonic fibroblasts (MEFs) proliferate more rapidly than wild-type MEFs, although JNK1^−/− MEFs proliferate more slowly than wild-type MEFs (41). Moreover, JNK2^−/− embryonic stem cells isolated from JNK2-deficient mice proliferate more rapidly than wild-type embryonic stem cells and also exhibit major defects in lineage-specific differentiation (48). Although JNK2 levels were also undetectable in the colonic epithelium of JNK2^−/− mice, there were no significant differences in the rates of colonic mucosal growth and maturation between JNK2-deficient mice and littermates. The exact reasons for which JNK2 deletion failed to alter colonic mucosal growth remain unknown, but basal mucosal turnover rate in the colon is lower than that observed in the small intestine (17). This interesting finding should be extensively investigated in the future study.

Our results reported here also indicate that JNK2 functions as a proapoptotic regulator in the intestinal mucosa and that JNK2-deficient intestinal epithelium exhibited an increased resistance to TNFα-induced apoptosis. The importance of apoptosis to the integrity of the intestinal epithelium is well documented (29, 36), and apoptosis is a fundamental event in the control of cell number and tissue homeostasis, which is tightly controlled by numerous factors. Spontaneous apoptosis is also necessary during intestinal development and tissue remodeling and for the prevention of malignancy (27, 29). Although JNK2 appears to be antiapoptotic in certain cells (28), most studies using a conditional gene targeting approach in mice revealed that JNK2 acts as a potent proapoptotic regulator in many cell types and tissues (7, 11, 19, 27, 38, 42). For example, JNK2 is shown to mediate oxidative stress-induced apoptosis in a variety of cellular systems; induced JNK2 activation triggers proapoptotic signaling and apoptosis in embryos, leading to diabetic embryopathy (22, 23). Vandevyer et al. (42) have recently reported that JNK2^−/− mice are significantly protected against TNF-α-induced apoptosis. In cultured IECs, TNF-α/CHX-induced apoptosis is accompanied by the activation of JNK2, whereas inhibition of JNK activity prevents caspase-9 activation and protects cells against TNF-α/CHX-induced apoptosis (2). On the other hand, while camptothecin also induces apoptosis in IEC-6 cells, the increase in JNK2 activity is not proportional to the degree of apoptosis (31), suggesting that the JNK2 function in IEC apoptosis depends on the nature of different apoptotic inducers.

Another significant finding from this study is that JNK2-deficient intestinal epithelium is associated with significant changes in the levels of HuR and CUGBP1; both are recently recognized as critical posttranscriptional modulators in intestinal mucosal homeostasis and maturation. In cultured IECs, JNK2 silencing by transfection with siJNK2 for 48 h did not alter HuR levels, suggesting that complete elimination of JNK2 or a longer treatment with siJNK2 might necessary to increase HuR. Although the exact mechanisms by which JNK2 regulates the expression of HuR and CUGBP1 remain unknown, the specific changes in HuR and CUGBP1 levels in the intestinal mucosa of JNK2^−/− mice may contribute to an increase in crypt depth and a reduction in villus length. Consistent with the findings in vivo, JNK2-silenced IEC-6 cells exhibited resistance to TNFα/CHX-induced apoptosis, but this protection was almost completely prevented by HuR silencing. Moreover, ectopic overexpression of CUGBP1 repressed IEC-6 cell proliferation, while CUGBP1 silencing stimulated cell growth. It is likely that induced resistance of the JNK2-deficient intestinal epithelium to TNFα-induced apoptosis results primarily from the increase in mucosal HuR abundance, whereas increased cell proliferation within the crypts by JNK2 deletion is predominantly due to a decrease in the CUGBP1 levels, specifically at the crypt regions. In support of this notion, HuR is shown to increase expression of several antiapoptotic factors such as ATF2 (47), JunD (21, 54), and XIAP (50) at the posttranscriptional level, and CUGBP1 represses translation of cyclin dependent kinase 4 (CDK4) that controls cells entry into and is committed to progress through the cell cycle (45). Taken together, our results indicate that JNK2 is essential for maintenance of normal intestinal mucosal homeostasis and maturation by differentially modulating RBPs HuR and CUGBP1.

GRANTS

This work was supported by Merit Review Awards (to J.-Y. Wang, D. J. Turner, and J. N. Rao) from the US Department of Veterans Affairs and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57819, DK-61972, and DK-68491 (to J.-Y. Wang). J.-Y. Wang is a Senior Research Career Scientist, Medical Research Service, US Department of Veterans Affairs.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: H.K.C., J.N.R., T.Z., L.L., L.X., and H.G. performed experiments; H.K.C., J.N.R., T.Z., L.X., P.Y., and J.-Y.W. analyzed data; H.K.C., D.J.T., and J.-Y.W. interpreted results of experiments; H.K.C., L.L., D.J.T., and J.-Y.W. prepared figures; P.Y. and J.-Y.W. conception and design of research; J.-Y.W. drafted manuscript; J.-Y.W. edited and revised manuscript; J.-Y.W. approved final version of manuscript.