Anas Alakkam

Lactobacillus acidophilus upregulates intestinal NHE3 expression and function

A major mechanism of electroneutral NaCl absorption in the human ileum and colon involves coupling of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. Disturbances in these mechanisms have been implicated in diarrheal conditions. Probiotics such as Lactobacillus have been indicated to be beneficial in the management of gastrointestinal disorders, including diarrhea. However, the molecular mechanisms underlying antidiarrheal effects of probiotics have not been fully understood. We have previously demonstrated Lactobacillus acidophilus (LA) to stimulate Cl(-)/HCO3- exchange activity via an increase in the surface levels and expression of the Cl(-)/HCO3- exchanger DRA in vitro and in vivo. However, the effects of LA on NHE3, the Na(+)/H(+) exchanger involved in the coupled electroneutral NaCl absorption, are not known. Current studies were, therefore, undertaken to investigate the effects of LA on the function and expression of NHE3 and to determine the mechanisms involved. Treatment of Caco2 cells with LA or its conditioned culture supernatant (CS) for 8-24 h resulted in a significant increase in Na(+)/H(+) exchange activity, mRNA, and protein levels of NHE3. LA-CS upregulation of NHE3 function and expression was also observed in SK-CO15 cells, a human colonic adenocarcinoma cell line. Additionally, LA treatment increased NHE3 promoter activity, suggesting involvement of transcriptional mechanisms. In vivo, mice gavaged with live LA showed significant increase in NHE3 mRNA and protein expression in the ileum and colonic regions. In conclusion, LA-induced increase in NHE3 expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of probiotics.

Probiotic Bifidobacterium species stimulate human SLC26A3 gene function and expression in intestinal epithelial cells.

SLC26A3, or downregulated in adenoma (DRA), plays a major role in mediating Cl(-) absorption in the mammalian intestine. Disturbances in DRA function and expression have been implicated in intestinal disorders such as congenital Cl(-) diarrhea and gut inflammation. We previously showed that an increase in DRA function and expression by Lactobacillus acidophilus and its culture supernatant (CS) might underlie antidiarrheal effects of this probiotic strain. However, the effects of Bifidobacterium species, important inhabitants of the human colon, on intestinal Cl(-)/HCO3 (-) exchange activity are not known. Our current results demonstrate that CS derived from Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum increased anion exchange activity in Caco-2 cells (∼1.8- to 2.4-fold). Consistent with the increase in DRA function, CS also increased the protein, as well as the mRNA, level of DRA (but not putative anion transporter 1). CS of all three Bifidobacterium sp. increased DRA promoter activity (-1,183/+114 bp) in Caco-2 cells (1.5- to 1.8-fold). Furthermore, the increase in DRA mRNA expression by CS of B. breve and B. infantis was blocked in the presence of the transcription inhibitor actinomycin D (5 μM) and the ERK1/2 MAPK pathway inhibitor U0126 (10 μM). Administration of live B. breve, B. infantis, and B. bifidum by oral gavage to mice for 24 h increased DRA mRNA and protein levels in the colon. These data demonstrate an upregulation of DRA via activation of the ERK1/2 pathway that may underlie potential antidiarrheal effects of Bifidobacterium sp.

Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells.

Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether KGF2 directly regulates Pgp in the human intestine is not known. Therefore, the present studies were undertaken to determine the modulation of Pgp by KGF2 using Caco-2 cells. Short-term treatment of Caco-2 cells with KGF2 (10 ng/ml, 1 h) increased Pgp activity (~2-fold, P < 0.05) as measured by verapamil-sensitive [(3)H]digoxin flux. This increase in Pgp function was associated with an increase in surface Pgp levels. The specific fibroblast growth factor receptor (FGFR) antagonist PD-161570 blocked the KGF2-mediated increase in Pgp activity. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 attenuated the stimulatory effects of KGF2 on Pgp activity. Small-interfering RNA knockdown of Erk1/2 MAPK blocked the increase in surface Pgp levels by KGF2. Long-term treatment with KGF2 (10 ng/ml, 24 h) also significantly increased PgP activity, mRNA, protein expression, and promoter activity. The long-term effects of KGF2 on Pgp promoter activity were also blocked by the FGFR antagonist and mediated by the Erk1/2 MAPK pathway. In conclusion, our findings define the posttranslational and transcriptional mechanisms underlying stimulation of Pgp function and expression by KGF2 that may contribute to the beneficial effects of KGF2 in intestinal inflammatory disorders.

Transcriptional modulation of SLC26A3 (DRA) by sphingosine-1-phosphate

SLC26A3 or Downregulated in adenoma (DRA) is the major Cl(-)/HCO3 (-) exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Alterations in DRA function and expression have been implicated in diarrheal diseases associated with inflammation or infection. Therefore, agents that upregulate DRA activity may serve as potential antidiarrheals. In this regard, sphingosine-1-phosphate (S1P), a member of the bioactive sphingolipid family, has been shown to modulate various cellular processes including improvement of intestinal barrier function. However, the role of S1P in modulating intestinal chloride absorption by regulating DRA is not known. Therefore, the present studies were designed to examine the direct effects of S1P on apical Cl(-)/HCO3 (-) exchange activity and DRA expression. S1P significantly increased Cl(-)/HCO3 (-) exchange activity and also significantly increased DRA mRNA and protein expression. Increased DRA mRNA by S1P was accompanied by enhanced DRA promoter activity, indicating involvement of transcriptional mechanisms. The specific S1P receptor subtype-2 (S1PR2) antagonist JTE-013 blocked the stimulatory effects of S1P on DRA promoter activity, indicating the involvement of S1PR2 S1P-mediated increase in DRA promoter activity involved PI3K/Akt pathway. Progressive deletions of the DRA promoter indicated that the putative S1P-responsive elements are present in the -790/-398 region of the DRA promoter. Furthermore, results obtained from electrophoretic mobility shift assay showed that S1P stimulated DRA promoter activity via increased binding of Ying-Yang1 (YY1) in the S1P-responsive region. In conclusion, transcriptional modulation of DRA expression and function in response to S1P through a PI3/Akt pathway represents a novel role of S1P as a potential proabsorptive agent.

Mo1792 Inhibition of DRA Expression by NF-κβ Pathway in Human Intestinal Epithelial Cells

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

Mo1791 Epigallocatechin-3-Gallate Inhibits DRA (SLC26A3) Expression in Intestinal Epithelial Cells

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

Lactobacillus Acidophilus Attenuates Dysregulation of DRA Function and Expression in Inflammatory Models

Varsha Singh,* Anoop Kumar,* Geetu Raheja, Arivarasu N. Anbazhagan, Shubha Priyamvada, Seema Saksena, Muhammad Nauman Jhandier, Ravinder K. Gill, Waddah A. Alrefai, Alip Borthakur, and Pradeep K. Dudeja Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; and Singhania University, Pacheri Bari, Rajasthan, India