Saminathan Muthusamy

N-glycosylation is essential for ileal ASBT function and protection against proteases

The bile acid transporter ASBT is a glycoprotein responsible for active absorption of bile acids. Inhibiting ASBT function and bile acid absorption is an attractive approach to lower plasma cholesterol and improve glucose imbalance in diabetic patients. Deglycosylation of ASBT was shown to decrease its function. However, the exact roles of N-glycosylation of ASBT, and how it affects its function, is not known. Current studies investigated the roles of N-glycosylation in ASBT protein stability and protection against proteases utilizing HEK-293 cells stably transfected with ASBT-V5 fusion protein. ASBT-V5 protein was detected as two bands with molecular mass of ~41 and ~35 kDa. Inhibition of glycosylation by tunicamycin significantly decreased ASBT activity and shifted ASBT bands to ~30 kDa, representing a deglycosylated protein. Treatment of total cellular lysates with PNGase F or Endo H glycosidases showed that the upper 41-kDa band represents a fully mature N-acetylglucosamine-rich glycoprotein and the lower 35-kDa band represents a mannose-rich core glycoprotein. Studies with the glycosylation deficient ASBT mutant (N10Q) showed that the N-glycosylation is not essential for ASBT targeting to plasma membrane. However, mature glycosylation significantly increased the half-life and protected ASBT protein from digestion with trypsin. Incubating the cells with high glucose (25 mM) for 48 h increased mature glycosylated ASBT along with an increase in its function. These results unravel novel roles for N-glycosylation of ASBT and suggest that high levels of glucose alter the composition of the glycan and may contribute to the increase in ASBT function in diabetes mellitus.

PKCδ-dependent activation of ERK1/2 leads to upregulation of the human NHE2 transcriptional activity in intestinal epithelial cell line C2BBe1

The apical Na+/H+ exchanger (NHE) isoform NHE2 is involved in transepithelial Na+ absorption in the intestine. Our earlier studies have shown that mitogenic agent phorbol 12-myristate 13-acetate (PMA) induces the expression of NHE2 through activation of transcription factor early growth response-1 (Egr-1) and its interactions with the NHE2 promoter. However, the signaling pathways involved in transcriptional stimulation of NHE2 in response to PMA in the intestinal epithelial cells are not known. Chemical inhibitors and genetic approaches were used to investigate the signaling pathways responsible for the stimulation of NHE2 expression by PMA via Egr-1 induction. We show that, in response to PMA, PKCδ, a member of novel PKC isozymes, and MEK-ERK1/2 pathway of mitogen-activated protein kinases stimulate the NHE2 expression in C2BBe1 intestinal epithelial cells. PMA rapidly and transiently induced activation of PKCδ. Small inhibitory RNA-mediated knockdown of PKCδ blocked the stimulatory effect of PMA on the NHE2 promoter activity. In addition, blockade of PKCδ by rottlerin, a PKCδ-specific inhibitor, and ERK1/2 by U0126, a MEK-ERK inhibitor, abrogated PMA-induced Egr-1 expression. Immunofluorescence studies revealed that inhibition of ERK1/2 activation prevents translocation of PMA-induced Egr-1 into the nucleus. Consistent with these data, PMA-induced Egr-1 interaction with the NHE2 promoter region was prevented in nuclear extracts from U0126-pretreated cells. In conclusion, our data provide the first evidence that the stimulatory effect of PMA on NHE2 expression is mediated through the initial activation of PKCδ, subsequent PKCδ-dependent activation of MEK-ERK1/2 signaling pathway, and stimulation of Egr-1 expression. Furthermore, we show that transcription factor Egr-1 acts as an intermediate effector molecule that links the upstream signaling cues to the long-term stimulation of NHE2 expression by PMA in C2BBe1 cells.

Extracellular Acidosis Stimulates NHE2 Expression through Activation of Transcription Factor Egr-1 in the Intestinal Epithelial Cells

Na+/H+ exchangers (NHEs) play important roles in regulating internal pH (pHi), cell volume and neutral Na+ absorption in the human intestine. Earlier studies have shown that low extracellular pH (pHe) and metabolic acidosis increases the expression and function of NHE1-3 genes. However, transcriptional mechanisms involved remained unknown. Therefore, we investigated the molecular mechanisms underlying acid-induced NHE2 expression in C2BBe1 and SK-CO15 intestinal epithelial cells. Assessing total RNA and protein by RT-PCR and Western blot analysis, respectively, displayed significant increases in the NHE2 mRNA and protein levels in cells exposed to acidic media (pH 6.5 and 6.7) compared to normal medium. Acid treatment was also associated with a significant enhancement in NHE2 transport activity. Quantification of the heterogeneous nuclear RNA indicated that the rate of NHE2 transcription was increased in response to acid. Furthermore, acid caused a significant increase in NHE2 promoter activity confirming transcriptional upregulation. Through functional and mutational studies the acid-response element was mapped to a 15-nucleotide GC-rich sequence at bp −337 to −323 upstream from the transcription start site. We previously identified this element as an overlapping Egr-1/Sp1/Egr-1 motif that was essential for the NHE2 upregulation by mitogen-induced transcription factor Egr-1. Cells exposed to acid exhibited a temporal increase in Egr-1 mRNA and protein expression. These events were followed by Egr-1 nuclear accumulation, as detected by immunofluorescence microscopy, and potentiated its in vitro and in vivo interaction with the NHE2 promoter. Disruption of ESE motif and knockdown of Egr-1 expression by targeted small interfering RNA abrogated the acid-induced NHE2 transcriptional activity. These data indicate that the acid-dependent NHE2 stimulation is implemented by transcriptional upregulation of NHE2 via acid-induced Egr-1 in the intestinal epithelial cells.

Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

194 N-Glycosylation Is Essential for Ileal ASBT Function and Protection Against Proteases

those patients. Total and surface expression of NHE3 were reduced in rat ileum treated with rapamycin, along with suppressed S6 protein phophorylation and increased ratio of LCII/I, confirming inhibition of mTOR and subsequent activation of autophagy by rapamycin. We further demonstrated that genetic inactivation of autophagy in mouse intestinal epithelium resulted in a marked accumulation of NHE3 and attenuated the rapamycin-induced down-regulation of NHE3, demonstrating a critical role for autophagy in maintaining NHE3 homeostasis. However, in addition to causing autophagic down-regulation of NHE3, we also demonstrate that rapamycin has a second mode of action: at high doses which mimics the sharp rise in serum rapamycin in non-infectious diarrhea, it causes acute inhibition of NHE3 surface expression in mouse ileum and NHE3 transporter activity in fibroblast PS120 cells, lacking endogenous NHE3, when stably transfected with human NHE3. Together, our data implicate two pathophysiological mechanisms of NHE3 down-regulation in the profound non-infectious diarrhea induced by rapamycin: 1) increased autophagic turnover of NHE3 by chronic exposure to rapamycin, and 2) reduced surface expression of NHE3 by acute exposure to a high concentration (spike) of rapamycin. Our findings not only lead to new insights into NHE3 regulation, but also provide new strategies to manage diarrhea in organ transplantations.

Mo1760 Histone Deacetylase Inhibition Decreases the Expression of Intestinal Cholesterol Transporter Niemann Pick-C1-Like 1 (NPC1L1)

regulating CIE; however, its role in asymmetric CCH-stimulated signal transduction is not well understood. PURPOSE: Since PLD generates phosphatidic acid (PA) that regulates endocytosis, we (1) investigated whether CCH activates BB PLD, (2) identified which PLD isoform is necessary for CCH-inhibition of NHE3 activity, and (3) determined whether PA or PA-derived diacylglycerol (DAG) is required for this effect. METHODS: 12 day postconfluent Caco-2/BBe cells grown on filters were transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 hours later. NHE3 activity was measured by fluorimetry using the pH-sensitive dye, BCECF, in Caco-2/BBe cells treated with vehicle, isoform specific PLD inhibitors (EVJ = PLD1; JWJ = PLD2, 5WO = dual), or propranolol (to inhibit conversion of PA to DAG) in the presence or absence of 10μM CCH to elevate [Ca]i. PLD activity was determined in total cell lysates from Caco-2/BBe cells treated with vehicle or CCH in the presence and absence of 10nM (blocks PLD1) or 50nM (blocks PLD2) FIPI. RESULTS: CCH induced rapid (1min) elevation of PLD activity in Caco-2/BBe cells, an effect which was blocked by BB, but not BLM, pretreatment with FIPI. Pretreatment of Caco-2/BBe cells with apical 10μM 5WO abolished CCH-mediated inhibition of NHE3 activity suggesting that BB PLD1 and/or PLD2 are necessary for NHE3 inhibition by CCH. This result was confirmed in studies in which cells were pretreated with both EVJ and JWJ as exposure to each inhibitor alone did not prevent CCH-inhibition of NHE3 activity. Propranolol treatment also prevented CCH-inhibition of NHE3 activity suggesting that PA-derived DAG is required for this effect. CONCLUSIONS: In summary, these data suggest that both PLD1 and PLD2 are activated by CCH signaling and are both necessary to inhibit NHE3 activity, which involves production of PA and DAG. Elevated [Ca]i-mediated signal transduction from the BLMM3 cholingergic receptor activates both BB PLD1 and PLD2 as part of an asymmetric signaling pathway that stimulates production of PA-derived DAG that results in the inhibition of BB NHE3 activity.

Mo1789 Hepatocyte Nuclear Factor 4α Regulates the Human Na+/H+ Exchanger Isoform-3 Expression in Intestine

Background and aims: Chylomicron retention disease (CRD) is caused by mutations in the SARA2 gene that encodes the SAR1B protein, involved in the vesicular coat protein complex II-dependent transport of proteins/lipoproteins from the endoplasmic reticulum to the Golgi apparatus. Given the large spectrum of CRD phenotypes, it appears important to search for SARA2 polymorphisms and probe functional cause-effects resulting from SAR1B knockdown. Methods: To examine the allelic frequencies of the SARA2 genetic variants, SNPs were assessed in the promoter (2kb, 3.5 and 6 kb regions upstream of the transcription start site), exon 4 and exon 8 in CRD and control subjects. In addition, SAR1B was suppressed in HepG2 and Caco-2/15 cell lines by specific siRNA, and lipid transport was determined. Results: No changes were noted in the polymorphisms in the promoter region (7 variants), exon 4 (2 variants) and exon 8 (1 variant), suggesting that they do not play an essential role in the several biochemical and clinical abnormalities characterizing CRD patients. On the other hand, SAR1B silencing resulted in multiple alterations in lipids, apolipoproteins (apo) and lipoproteins in Caco-2/15 and HepG2 cells. It reduced the output of triglycerides (TG) and TG-rich lipoproteins. Furthermore, SAR1B silencing limited the synthesis of apo B-48 and apo B-100 in Caco-2/15 cells and HepG2 cells, respectively. Conclusions: Our functional experiments show that the suppression of SAR1B has a negative impact on apo B synthesis and lipid/lipoprotein secretion. If previous studies have established only an association between SAR1B defects and lipid transport abnormalities, our findings constitute the first direct demonstration illustrating the SAR1B deficiency cause-effects in enterocytes and hepatocytes. Acknowledgment: This study was supported by the Canadian Institutes of Health Research and the J.A. DeSeve Research Chair in Nutrition

5 Acid-Induced Stimulation of the NHE2 Promoter Activity is Mediated by Transcription Factor EGR-1 in the Human Intestinal Epithelial Cell Line C2BBe1

NHE8 is a member of the SLC9 family. This Na+/H+ exchanger is expressed on the apical membrane of intestinal epithelial cells. Although NHE8 has been shown to be important for intestinal sodium absorption early in development, its physiological role in the gastrointestinal tract remains unknown. By using NHE8 knockout (NHE8KO) mouse model, we discovered that NHE8 plays an important role in protecting the gastric mucosa against gastric acid. Stomach tissue was isolated from NHE8KO mice and their wild-type littermates. Localization of NHE8 expression in the stomach was detected using Immunohistochemistry. Mucosal surface pH was measured using a surface pH meter. Histology of the stomach of NHE8KOmice was also analyzed. RNA was isolated and used for PCR to detect the expression of PAT1 and DRA. Total tissue lysate was prepared for Western detection of NHE8, PAT1 and DRA. Immunohistochemistry indicated that NHE8 is expressed on the apical membrane of gastric epithelial cells. Histological evaluation showed no abnormality of the stomach in NHE8KO mice. Interestingly, higher incidence of gastric bleeding was observed in NHE8KO mice. The appearance of ulcers was seen in the glandular stomach. Mucosal surface pH measurement indicated that NHE8KO mice had decreased mucosal surface pH compared to wild-type mice (3.72 ± 0.13 in NHE8KO vs. 4.26 ± 0.09 in wild-type). Lower surface pH of NHE8KO gastric mucosa suggests that HCO3secretion may be altered. Therefore, we studied the expression of PAT1 and DRA in NHE8KO mice. Indeed, PAT1 and DRA mRNA expression in the stomach of NHE8KO mice were significantly reduced by ~30% and ~90%, respectively, in NHE8KO mice compared to their wild-type littermates. At the protein expression level, PAT1 protein abundance was reduced by ~70% and DRA protein abundance was nearly abolished in NHE8KO mice. Thus NHE8 may play an important role in mucosal protection by participating bicarbonate secretion.