Post-transcriptional regulation of Wnt co-receptor LRP6 and RNA-binding protein HuR by miR-29b in intestinal epithelial cells.

MicroRNAs (miRNAs) control gene expression by binding to their target mRNAs for degradation and/or translation repression and are implicated in many aspects of cellular physiology. Our previous study shows that miR-29b acts as a biological repressor of intestinal mucosal growth, but its exact downstream targets remain largely unknown. In the present study, we found that mRNAs, encoding Wnt co-receptor LRP6 (low-density lipoprotein-receptor-related protein 6) and RNA-binding protein (RBP) HuR, are novel targets of miR-29b in intestinal epithelial cells (IECs) and that expression of LRP6 and HuR is tightly regulated by miR-29b at the post-transcriptional level. miR-29b interacted with both Lrp6 and HuR mRNAs via their 3'-UTRs and inhibited LRP6 and HuR expression by destabilizing Lrp6 and HuR mRNAs and repressing their translation. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-29b through a single binding site in the Lrp6 or HuR 3'-UTR, whereas deletion mutation of this site prevented miR-29b-induced repression of LRP6 and HuR expression. Repression of HuR by miR-29b in turn also contributed to miR-29b-induced LRP6 inhibition, since ectopic overexpression of HuR in cells overexpressing miR-29b restored LRP6 expression to near normal levels. Taken together, our results suggest that miR-29b inhibits expression of LRP6 and HuR post-transcriptionally, thus playing a role in the regulation of IEC proliferation and intestinal epithelial homoeostasis.

JunD enhances miR-29b levels transcriptionally and posttranscriptionally to inhibit proliferation of intestinal epithelial cells.

Through its actions as component of the activating protein-1 (AP-1) transcription factor, JunD potently represses cell proliferation. Here we report a novel function of JunD in the regulation of microRNA expression in intestinal epithelial cells (IECs). Ectopically expressed JunD specifically increased the expression of primary and mature forms of miR-29b, whereas JunD silencing inhibited miR-29b expression. JunD directly interacted with the miR-29b1 promoter via AP-1-binding sites, whereas mutation of AP-1 sites from the miR-29b1 promoter prevented JunD-mediated transcriptional activation of the miR-29b1 gene. JunD also enhanced formation of the Drosha microprocessor complex, thus further promoting miR-29b biogenesis. Cellular polyamines were found to regulate miR-29b expression by altering JunD abundance, since the increase in miR-29b expression levels in polyamine-deficient cells was abolished by JunD silencing. In addition, miR-29b silencing prevented JunD-induced repression of IEC proliferation. Our findings indicate that JunD activates miR-29b by enhancing its transcription and processing, which contribute to the inhibitory effect of JunD on IEC growth and maintenance of gut epithelium homeostasis.

miR-195 competes with HuR to modulate stim1 mRNA stability and regulate cell migration.

Stromal interaction molecule 1 (Stim1) functions as a sensor of Ca2+ within stores and plays an essential role in the activation of store-operated Ca2+ entry (SOCE). Although lowering Stim1 levels reduces store-operated Ca2+ entry and inhibits intestinal epithelial repair after wounding, the mechanisms that control Stim1 expression remain unknown. Here, we show that cellular Stim1 abundance is controlled posttranscriptionally via factors that associate with 3'-untranslated region (3'-UTR) of stim1 mRNA. MicroRNA-195 (miR-195) and the RNA-binding protein HuR competed for association with the stim1 3'-UTR and regulated stim1 mRNA decay in opposite directions. Interaction of miR-195 with the stim1 3'-UTR destabilized stim1 mRNA, whereas the stability of stim1 mRNA increased with HuR association. Interestingly, ectopic miR-195 overexpression enhanced stim1 mRNA association with argonaute-containing complexes and increased the colocalization of tagged stim1 RNA with processing bodies (P-bodies); the translocation of stim1 mRNA was abolished by HuR overexpression. Moreover, decreased levels of Stim1 by miR-195 overexpression inhibited cell migration over the denuded area after wounding but was rescued by increasing HuR levels. In sum, Stim1 expression is controlled by two factors competing for influence on stim1 mRNA stability: the mRNA-stabilizing protein HuR and the decay-promoting miR-195.

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

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.

Chk2-dependent HuR phosphorylation regulates occludin mRNA translation and epithelial barrier function.

Occludin is a transmembrane tight junction (TJ) protein that plays an important role in TJ assembly and regulation of the epithelial barrier function, but the mechanisms underlying its post-transcriptional regulation are unknown. The RNA-binding protein HuR modulates the stability and translation of many target mRNAs. Here, we investigated the role of HuR in the regulation of occludin expression and therefore in the intestinal epithelial barrier function. HuR bound the 3'-untranslated region of the occludin mRNA and enhanced occludin translation. HuR association with the occludin mRNA depended on Chk2-dependent HuR phosphorylation. Reduced HuR phosphorylation by Chk2 silencing or by reduction of Chk2 through polyamine depletion decreased HuR-binding to the occludin mRNA and repressed occludin translation, whereas Chk2 overexpression enhanced (HuR/occludin mRNA) association and stimulated occludin expression. In mice exposed to septic stress induced by cecal ligation and puncture, Chk2 levels in the intestinal mucosa decreased, associated with an inhibition of occludin expression and gut barrier dysfunction. These results indicate that HuR regulates occludin mRNA translation through Chk2-dependent HuR phosphorylation and that this influence is crucial for maintenance of the epithelial barrier integrity in the intestinal tract.

Activation of Wnt3a signaling stimulates intestinal epithelial repair by promoting c-Myc-regulated gene expression.

In response to mucosal injury, epithelial cells modify the patterns of expressed genes to repair damaged tissue rapidly. Our previous studies have demonstrated that the transcription factor c-Myc is necessary for stimulation of epithelial cell renewal during mucosal healing, but the up-stream signaling initiating c-Myc gene expression after injury remains unknown. Wnts are cysteine-rich glycoproteins that act as short-range ligands to locally activate receptor-mediated signaling pathways and correlate with the increased expression of the c-Myc gene. The current study tested the hypothesis that Wnt3a signaling is implicated in intestinal epithelial repair after wounding by stimulating c-Myc expression. Elevated Wnt3a signaling in intestinal epithelial cells (IEC-6 line) by coculturing with stable Wnt3a-transfected fibroblasts or ectopic overexpression of the Wnt3a gene enhanced intestinal epithelial repair after wounding. This stimulatory effect on epithelial repair was prevented by silencing the Wnt coreceptor LRP6 or by c-Myc silencing. Activation of the Wnt3a signaling pathway increased ß-catenin nuclear translocation by decreasing its phosphorylation and stimulated c-Myc expression during epithelial repair after wounding. In stable Wnt3a-transfected IEC-6 cells, increased levels of c-Myc were associated with an increase in expression of c-Myc-regulated genes cyclcin D1 and cyclin E, whereas c-Myc silencing inhibited expression of cyclin D1 and cyclin E and delayed epithelial repair. These results indicate that elevated Wnt3a signaling in intestinal epithelial cells after wounding stimulates epithelial repair by promoting c-Myc-regulated gene expression.

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis.

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.

Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca²+ signaling by differentially modulating STIM1 and STIM2.

Early epithelial restitution occurs as a consequence of intestinal epithelial cell (IEC) migration after wounding, and its defective regulation is implicated in various critical pathological conditions. Polyamines stimulate intestinal epithelial restitution, but their exact mechanism remains unclear. Canonical transient receptor potential-1 (TRPC1)-mediated Ca(2+) signaling is crucial for stimulation of IEC migration after wounding, and induced translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane activates TRPC1-mediated Ca(2+) influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca(2+) signaling by altering the ratio of STIM1 to STIM2. Increasing cellular polyamines by ectopic overexpression of the ornithine decarboxylase (ODC) gene stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines by inhibiting ODC activity decreased STIM1 but increased STIM2 levels. Induced STIM1/TRPC1 association by increasing polyamines enhanced Ca(2+) influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca(2+) influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca(2+) influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca(2+) signaling and influencing cell migration after wounding.

An Ultrasensitive Picric Acid Sensor Based on a Robust 3D Hydrogen-Bonded Organic Framework.

Hydrogen-bonded organic frameworks (HOFs), as a newly developed porous material, have been widely used in various fields. To date, several organic building units (OBUs) with tri-, tetra-, and hexa-carboxylic acid synthons have been applied to synthesize HOFs. To our knowledge, di-carboxylic acids have rarely been reported for the construction of HOFs, in particular, di-carboxylic acid-based HOFs with fluorescence sensing properties have not been reported. In this study, a rare example of a di-carboxylic acid-based, luminescent three-dimensional hydrogen-bonded organic framework has been successfully constructed and structurally characterized; it has a strong electron-rich property originated from its organic linker 9-phenylcarbazole-3,6-dicarboxylic acid. It represents the first example of HOF-based sensors for the highly selective and sensitive detection of PA (Picric acid) with reusability; the LOD is less than 60 nM. This work thus provides a new avenue for the fabrication of fluorescent HOFs sensing towards explosives.

Post-transcriptional regulation of MEK-1 by polyamines through the RNA-binding protein HuR modulating intestinal epithelial apoptosis.

MEK-1 [MAPK (mitogen-activated protein kinase) kinase-1] is an important signal transducing enzyme that is implicated in many aspects of cellular functions. In the present paper, we report that cellular polyamines regulate MEK-1 expression at the post-transcriptional level through the RNA-binding protein HuR (Hu-antigen R) in IECs (intestinal epithelial cells). Decreasing the levels of cellular polyamines by inhibiting ODC (ornithine decarboxylase) stabilized MEK-1 mRNA and promoted its translation through enhancement of the interaction between HuR and the 3'-untranslated region of MEK-1 mRNA, whereas increasing polyamine levels by ectopic ODC overexpression destabilized the MEK-1 transcript and repressed its translation by reducing the abundance of HuR-MEK-1 mRNA complex; neither intervention changed MEK-1 gene transcription via its promoter. HuR silencing rendered the MEK-1 mRNA unstable and inhibited its translation, thus preventing increases in MEK-1 mRNA and protein in polyamine-deficient cells. Conversely, HuR overexpression increased MEK-1 mRNA stability and promoted its translation. Inhibition of MEK-1 expression by MEK-1 silencing or HuR silencing prevented the increased resistance of polyamine-deficient cells to apoptosis. Moreover, HuR overexpression did not protect against apoptosis if MEK-1 expression was silenced. These results indicate that polyamines destabilize the MEK-1 mRNA and repress its translation by inhibiting the association between HuR and the MEK-1 transcript. Our findings indicate that MEK-1 is a key effector of the HuR-elicited anti-apoptotic programme in IECs.

Stabilization of XIAP mRNA through the RNA binding protein HuR regulated by cellular polyamines.

The X chromosome-linked inhibitor of apoptosis protein (XIAP) is the most potent intrinsic caspase inhibitor and plays an important role in the maintenance of intestinal epithelial integrity. The RNA binding protein, HuR, regulates the stability and translation of many target transcripts. Here, we report that HuR associated with both the 3'-untranslated region and coding sequence of the mRNA encoding XIAP, stabilized the XIAP transcript and elevated its expression in intestinal epithelial cells. Ectopic HuR overexpression or elevated cytoplasmic levels of endogenous HuR by decreasing cellular polyamines increased [HuR/XIAP mRNA] complexes, in turn promoting XIAP mRNA stability and increasing XIAP protein abundance. Conversely, HuR silencing in normal and polyamine-deficient cells rendered the XIAP mRNA unstable, thus reducing the steady state levels of XIAP. Inhibition of XIAP expression by XIAP silencing or by HuR silencing reversed the resistance of polyamine-deficient cells to apoptosis. Our findings demonstrate that HuR regulates XIAP expression by stabilizing its mRNA and implicates HuR-mediated XIAP in the control of intestinal epithelial apoptosis.

STIM1 translocation to the plasma membrane enhances intestinal epithelial restitution by inducing TRPC1-mediated Ca2+ signaling after wounding.

Early epithelial restitution is an important repair modality in the gut mucosa and occurs as a consequence of epithelial cell migration. Canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel (SOCs) in intestinal epithelial cells (IECs) and regulates intestinal restitution, but the exact upstream signals initiating TRPC1 activation after mucosal injury remain elusive. Stromal interaction molecule 1 (STIM1) is a single membrane-spanning protein and is recently identified as essential components of SOC activation. The current study was performed to determine whether STIM1 plays a role in the regulation of intestinal epithelial restitution by activating TRPC1 channels. STIM1 translocation to the plasma membrane increased after wounding, which was followed by an increase in IEC migration to reseal wounds. Increased STIM1 levels at the plasma membrane by overexpressing EF-hand mutant STIM1 enhanced Ca2+ influx through SOCs and stimulated IEC migration after wounding. STIM1 interacted with TRPC1 and formed STIM1/TRPC1 complex, whereas inactivation of STIM1 by STIM1 silencing decreased SOC-mediated Ca2+ influx and inhibited epithelial restitution. In cells overexpressing EF-hand mutant STIM1, TRPC1 silencing also decreased STIM1/TRPC1 complex, reduced SOC-mediated Ca2+ influx, and repressed cell migration after wounding. Our findings demonstrate that induced STIM1 translocation to the plasma membrane promotes IEC migration after wounding by enhancing TRPC1-mediated Ca2+ signaling and provide new insight into the mechanism of intestinal epithelial restitution.

Induced ATF-2 represses CDK4 transcription through dimerization with JunD inhibiting intestinal epithelial cell growth after polyamine depletion.

Intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated by numerous factors including polyamines. Decreased levels of cellular polyamines increase activating transcription factor (ATF)-2, but the exact role and mechanism of induced ATF-2 in the regulation of intestinal epithelial cell (IEC) growth remain elusive. Cyclin-dependent kinase (CDK) 4 is necessary for the G1-to-S phase transition during the cell cycle, and its expression is predominantly controlled at the transcription level. Here, we reported that induced ATF-2 following polyamine depletion repressed CDK4 gene transcription in IECs by increasing formation of the ATF-2/JunD heterodimers. ATF-2 formed complexes with JunD as measured by immunoprecipitation using the ATF-2 and JunD antibodies and by glutathione S-transferase (GST) pull-down assays using GST-ATF-2 fusion proteins. Studies using various mutants of GST-ATF-2 revealed that formation of the ATF-2/JunD dimers depended on the COOH-terminal basic region-leucine zipper domain of ATF-2. Polyamine depletion increased ATF-2/JunD complex and inhibited CDK4 transcription as indicated by a decrease in the levels of CDK4-promoter activity and its mRNA. ATF-2 silencing not only prevented inhibition of CDK4 transcription in polyamine-deficient cells but also abolished repression of CDK4 expression induced by ectopic JunD overexpression. ATF-2 silencing also promoted IEC growth in polyamine-depleted cells. These results indicate that induced ATF-2/JunD association following polyamine depletion represses CDK4 transcription, thus contributing to the inhibition of IEC growth.

Polyamines regulate the stability of activating transcription factor-2 mRNA through RNA-binding protein HuR in intestinal epithelial cells.

Maintenance of intestinal mucosal epithelial integrity requires polyamines that modulate the expression of various genes involved in cell proliferation and apoptosis. Recently, polyamines were shown to regulate the subcellular localization of the RNA-binding protein HuR, which stabilizes its target transcripts such as nucleophosmin and p53 mRNAs. The activating transcription factor-2 (ATF-2) mRNA encodes a member of the ATF/CRE-binding protein family of transcription factors and was computationally predicted to be a target of HuR. Here, we show that polyamines negatively regulate ATF-2 expression posttranscriptionally and that polyamine depletion stabilizes ATF-2 mRNA by enhancing the interaction of the 3'-untranslated region (UTR) of ATF-2 with cytoplasmic HuR. Decreasing cellular polyamines by inhibiting ornithine decarboxylase (ODC) with alpha-difluoromethylornithine increased the levels of ATF-2 mRNA and protein, whereas increasing polyamines by ectopic ODC overexpression repressed ATF-2 expression. Polyamine depletion did not alter transcription via the ATF-2 gene promoter but increased the stability of ATF-2 mRNA. Increased cytoplasmic HuR in polyamine-deficient cells formed ribonucleoprotein complexes with the endogenous ATF-2 mRNA and specifically bound to 3'-UTR of ATF-2 mRNA on multiple nonoverlapping 3'-UTR segments. Adenovirus-mediated HuR overexpression elevated ATF-2 mRNA and protein levels, whereas HuR silencing rendered the ATF-2 mRNA unstable and prevented increases in ATF-2 mRNA and protein. Furthermore, inhibition of ATF-2 expression prevented the increased resistance of polyamine-deficient cells to apoptosis induced by treatment with tumor necrosis factor-alpha and cycloheximide. These results indicate that polyamines modulate the stability of ATF-2 mRNA by altering cytoplasmic HuR levels and that polyamine-modulated ATF-2 expression plays a critical role in regulating epithelial apoptosis.

Polyamines modulate the subcellular localization of RNA-binding protein HuR through AMP-activated protein kinase-regulated phosphorylation and acetylation of importin alpha1.

Polyamines are required for maintenance of intestinal epithelial integrity, and a decrease in cellular polyamines increases the cytoplasmic levels of RNA-binding protein HuR stabilizing p53 and nucleophosmin mRNAs, thus inhibiting IEC (intestinal epithelial cell) proliferation. The AMPK (AMP-activated protein kinase), an enzyme involved in responding to metabolic stress, was recently found to be implicated in regulating the nuclear import of HuR. Here, we provide evidence showing that polyamines modulate subcellular localization of HuR through AMPK-regulated phosphorylation and acetylation of Impalpha1 (importin alpha1) in IECs. Decreased levels of cellular polyamines as a result of inhibiting ODC (ornithine decarboxylase) with DFMO (D,L-alpha-difluoromethylornithine) repressed AMPK activity and reduced Impalpha1 levels, whereas increased levels of polyamines as a result of ODC overexpression induced both AMPK and Impalpha1 levels. AMPK activation by overexpression of the AMPK gene increased Impalpha1 but reduced the cytoplasmic levels of HuR in control and polyamine-deficient cells. IECs overexpressing wild-type Impalpha1 exhibited a decrease in cytoplasmic HuR abundance, while cells overexpressing Impalpha1 proteins bearing K22R (lacking acetylation site), S105A (lacking phosphorylation site) or K22R/S105A (lacking both sites) mutations displayed increased levels of cytoplasmic HuR. Ectopic expression of these Impalpha1 mutants also prevented the increased levels of cytoplasmic HuR following polyamine depletion. These results indicate that polyamine-mediated AMPK activation triggers HuR nuclear import through phosphorylation and acetylation of Impalpha1 in IECs and that polyamine depletion increases cytoplasmic levels of HuR as a result of inactivation of the AMPK-driven Impalpha1 pathway.

Induced JunD in intestinal epithelial cells represses CDK4 transcription through its proximal promoter region following polyamine depletion.

Maintenance of intestinal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. In prior studies, depletion of cellular polyamines has been shown to stabilize JunD, a member of the AP-1 (activator protein-1) family of transcription factors, leading to inhibition of intestinal epithelial cell proliferation, but the exact downstream targets of induced JunD remain elusive. CDK4 (cyclin-dependent kinase 4) is essential for the G1- to S-phase transition during the cell cycle and its expression is primarily controlled at the transcriptional level. In the present study, we show that induced JunD in IECs (intestinal epithelial cells) is a transcriptional repressor of the CDK4 gene following polyamine depletion. Increased JunD in polyamine-deficient cells was associated with a significant inhibition of CDK4 transcription, as indicated by repression of CDK4-promoter activity and decreased levels of CDK4 mRNA and protein, all of which were prevented by using specific antisense JunD oligomers. Ectopic expression of the wild-type junD also repressed CDK4-promoter activity and decreased levels of CDK4 mRNA and protein without any effect on CDK2 expression. Gel shift and chromatin immunoprecipitation assays revealed that JunD bound to the proximal region of the CDK4-promoter in vitro as well as in vivo, while experiments using different CDK4-promoter mutants showed that transcriptional repression of CDK4 by JunD was mediated through an AP-1 binding site within this proximal sequence of the CDK4-promoter. These results indicate that induced JunD in IECs represses CDK4 transcription through its proximal promoter region following polyamine depletion.

Polyamine-modulated c-Myc expression in normal intestinal epithelial cells regulates p21Cip1 transcription through a proximal promoter region.

Maintenance of intestinal mucosal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. Our previous studies have shown that polyamines are essential for expression of the c-myc gene and that polyamine-induced c-Myc plays a critical role in stimulation of normal IEC (intestinal epithelial cell) proliferation, but the exact downstream targets of induced c-Myc are still unclear. The p21Cip1 protein is a major player in cell cycle control, which is primarily regulated at the transcriptional level. The current study was designed to determine whether induced c-Myc stimulates normal IEC proliferation by repressing p21Cip1 transcription following up-regulation of polyamines. Overexpression of the ODC (ornithine decarboxylase) gene increased levels of cellular polyamines, induced c-Myc expression and inhibited p21Cip1 transcription, as indicated by repression of p21Cip1 promoter activity and a decrease in p21Cip1 protein levels. In contrast, depletion of cellular polyamines by inhibiting ODC enzyme activity with alpha-difluoromethylornithine decreased c-Myc, but increased p21Cip1 transcription. Ectopic expression of wild-type c-myc not only inhibited basal levels of p21Cip1 transcription in control cells, but also prevented increased p21Cip1 in polyamine-deficient cells. Experiments using different p21Cip1 promoter mutants showed that transcriptional repression of p21Cip1 by c-Myc was mediated through Miz-1- and Sp1-binding sites within the proximal region of the p21Cip1 promoter in normal IECs. These findings confirm that p21Cip1 is one of the direct mediators of induced c-Myc following increased polyamines and that p21Cip1 repression by c-Myc is implicated in stimulation of normal IEC proliferation.

Induced TRPC1 expression sensitizes intestinal epithelial cells to apoptosis by inhibiting NF-kappaB activation through Ca2+ influx.

Apoptosis occurs within crypts and at the intestinal luminal surface and plays a critical role in mucosal homoeostasis. NF-kappaB (nuclear factor-kappaB) is the central regulator of the transcription of genes involved in apoptosis, and its activity is highly regulated in the intestinal mucosa. We have recently demonstrated that TRPC1 (transient receptor potential canonical-1) is expressed in IECs (intestinal epithelial cells) and functions as a Ca2+ permeable channel activated by Ca2+ store depletion. The present study tests the hypothesis that TRPC1 channels are implicated in the regulation of apoptosis by inhibiting NF-kappaB through the induction of TRPC1-mediated Ca2+ influx in the IEC-6 line. The expression of TRPC1 induced by stable transfection of IEC-6 cells with the wild-type TRPC1 gene (IEC-TRPC1 cells) increased Ca2+ influx after Ca2+ store depletion and repressed NF-kappaB transactivation, which was associated with an increase in susceptibility to apoptosis induced by exposure to TNFalpha (tumour necrosis factor-alpha) plus CHX (cycloheximide) (TNF-alpha/CHX), or STS (staurosporine). By contrast, the induction of endogenous NF-kappaB activity, by the depletion of cellular polyamines, promoted resistance to apoptosis, which was prevented by the ectopic expression of the IkappaBalpha super-repressor. Furthermore, inhibition of TRPC1 expression by transfection with siRNA (small interfering RNA) targeting TRPC1 (siTRPC1) decreased Ca2+ influx, increased NF-kappaB transactivation, and prevented the increased susceptibility of IEC-TRPC1 cells to apoptosis. Decreasing Ca2+ influx by exposure to a Ca2+-free medium also induced NF-kappaB activity and blocked the increased susceptibility to apoptosis of stable IEC-TRPC1 cells. These results indicate that induced TRPC1 expression sensitizes IECs to apoptosis by inhibiting NF-kappaB activity as a result of the stimulation of Ca2+ influx.

Hypermethylation of the p14(ARF) gene in ulcerative colitis-associated colorectal carcinogenesis.

The p14(ARF) protein directly inhibits the MDM-2 oncoprotein, which mediates degradation of the p53 protein. It has been shown that p14(ARF) expression is frequently down-regulated by p14(ARF) gene hypermethylation in colorectal cancer. To determine whether p14(ARF) inactivation was involved in ulcerative colitis (UC)-associated carcinogenesis, the frequency and timing of p14(ARF) methylation was investigated in four different histological stages of UC-associated carcinogenesis. Methylation-specific PCR and bisulfite sequencing were used to determine the prevalence of p14(ARF) gene methylation. p14(ARF) methylation was observed in 19 of 38 (50%) adenocarcinomas, 4 of 12 (33%) dysplasias, and 3 of the 5 (60%) nonneoplastic UC mucosae. In contrast, 3 of 40 (3.7%) normal tissues showed p14(ARF) methylation (chi(2) test: P = 0.0003). Bisulfite sequencing was used to analyze 28 CpGs of p14(ARF) gene in 20 samples. The number of methylated CpGs ranged from 0 to 4, 0 to 20, and 0 to 28 in the normal, dysplastic, and carcinomatous samples, respectively (Kruskall-Wallis test: P = 0.0005). Densely methylated alleles were detected only in carcinomas by bisulfite sequencing. In conclusion, our data suggest that methylation of p14(ARF) is a relatively common early event in UC-associated carcinogenesis. p14(ARF) offers potential as a biomarker for the early detection of cancer or dysplasia in UC. Finally, analyses of p14(ARF) methylation in other organs should explore not only frank cancers but other premalignant lesions.