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Sphingosine Kinase-1 Is Regulated by MicroRNA-495 in Intestinal Epithelial Cells
ALIMENTARY TRACT I stem cells to maintain homeostasis, barrier function and control repair after injury.
Human Small Intestine Stem Cells Respond to Withdrawal of Luminal Nutrients by Decreasing Proliferation and Increasing Self-Renewal in the Crypt Christa N Grant, MD*, Salvador Garcia, MS, Ryan G Spurrier, MD, Tracy C Grikscheit, MD, FACS Children’s Hospital Los Angeles, Los Angeles, CA
METHODS: Quantitative real-time polymerase chain reaction was used to evaluate proteins in resealing colonic biopsy induced mucosal wounds. Protein expression was analyzed by western blotting, immunofluorescence labeling and confocal microscopy. Model intestinal epithelial cell lines were used to analyze WISP1 effects on wound closure in vitro. Statistical analysis was performed by Student’s two-tailed t-test as appropriate. p<0.05 was considered significant.
INTRODUCTION: Global calorie restriction has been shown to affect intestinal stem cell (ISC) behavior in animal models. Little is known about the effect of luminal nutrients on ISCs in the calorie replete organism. We found that villi were shorter and crypts were less deep and less proliferative in unfed human intestine. We hypothesized that without luminal nutrient, human ISCs would remain in the crypt compartment in lieu of differentiating and migrating.
RESULTS: Increased WNT1-inducible secreted protein 1 (WISP1) mRNA was observed in resealing intestinal mucosal wounds (fold increase compared to non wounded tissue ¼ 9.34; p<0.001). Additionally, WISP1 protein and mRNA expression was up-regulated in the intestinal mucosa of patients with active inflammatory bowel disease. Recombinant WISP1 protein promoted wound closure of model intestinal epithelial cell lines in vitro (ctl ¼ 76.080.33% vs WISP1 ¼ 91.621.07% p<0.0001). The pro-repair effects of WISP 1 were mediated by increased signaling of focal adhesion kinase and c-src that regulate cell matrix adhesion, cell migration and therefore wound closure.
METHODS: This study was approved and monitored by the IRB. 5 paired human ileum samples were collected from children undergoing ileostomy reversal at least 6 weeks after intestinal division with minimal resection. The segment contiguous with enteral feeding was denoted “fed” and the excluded segment “unfed.” Apoptotic cells were quantified by cleaved-caspase 3 immunofluorescence. Microarray analysis was performed on 3 paired samples, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) on 5 paired samples for quantification of ISC markers Bmi1 and Lgr5, representing slowly and rapidly cycling ISCs respectively.
CONCLUSIONS: WISP1 promotes intestinal mucosal wound closure. Thus, WISP1 could potentially be used as a therapeutic agent to promote healing of intestinal mucosal wounds.
RESULTS: There was increased epithelial apoptosis (p<0.02), and crypt width in unfed intestine (p<0.01). Microarray analysis demonstrated a 44-fold increase in Lgr5 (p¼0.002), a 15-fold increase in the Paneth cell marker lysozyme (p¼0.008), and a 1.74-fold increase in secretory cell progenitor marker Atoh1 (p¼0.04). RT-qPCR analysis confirmed the increase in Lgr5 expression (p¼0.013).
Sphingosine Kinase-1 Is Regulated by MicroRNA-495 in Intestinal Epithelial Cells Daniel Mansour, MD, Natasha Hansraj, MD, Alexis D Smith, MD, Ruiyun Li, PhD, Rao Jaladanki, PhD, James M Donahue, MD, FACS, Jian-Ying Wang, MD, PhD, Douglas J Turner, MD, FACS Baltimore VA Medical Center, Baltimore, MD, University of Maryland Medical Center, Baltimore, MD
CONCLUSIONS: Human ileum ISCs respond to lack of luminal nutrients by decreasing differentiation, leading to increased villus epithelial apoptosis. Decreased crypt proliferation, increased crypt width, and increased expression of Paneth cell and undifferentiated cell markers suggest decreased migration out of the crypt in which are lodged more possible progenitors.
INTRODUCTION: The bioactive sphingolipid, Sphingosine-1-phophate (S1P), plays a significant role in intestinal epithelial cell renewal. We have shown that cells overexpressing sphingosine kinase-1 (SphK1), the rate limiting enzyme for S1P production, show increased cell proliferation; however, the regulation of SphK1 is currently not known. MicroRNAs are small, noncoding RNAs that regulate gene expression; microRNA-495 (miR-495) has been reported to participate in cell differentiation. However, the role of miR-495 in intestinal epithelial cells (IECs) and the mechanisms that regulate SphK1 remain elusive. We hypothesized that miR-495 regulates IEC proliferation through the direct inhibition of SphK1.
WNT1-Inducible Secreted Protein 1 Regulates Intestinal Mucosal Wound Resealing Philipp-Alexander Neumann, MD, Hikaru Nishio, PhD, Giovanna Leoni, PhD, Miguel Quiros, PhD, Gabriela Bernal, Mingli Feng, Norbert Senninger, MD, FACS, Charles A Parkos, MD, PhD, Asma Nusrat, MD Emory University, Atlanta, GA, University of Muenster, Muenster, Germany
METHODS: Cdx2/IEC-6 cells derived from rat intestinal crypts were used. Protein expression, RNA levels, proliferation assay and miRNA over expression were determined by standard methods.
INTRODUCTION: Mucosal wound closure after inflammation, ischemia or surgery is coordinated by a plethora of signaling proteins. WNT signaling is known to regulate intestinal epithelial
ª 2014 by the American College of Surgeons Published by Elsevier Inc.
S13
http://dx.doi.org/10.1016/j.jamcollsurg.2014.07.018 ISSN 1072-7515/14
S14
J Am Coll Surg
Surgical Forum Abstracts
RESULTS: MiR-495 and miR-206 were found to be present in Cdx2 cells and to have proposed binding sites on SphK1 mRNA. MiR-495 and 206 overexpression via transfection with pre-miR-495 and 206 resulted in a dramatic decrease in protein expression of SphK1. Interestingly, only miR-495 overexpression showed a significant reduction in Sphk1 mRNA levels. In addition, during biotin pull down experiments, again, only miR-495 showed specific binding to Sphk1 mRNA. Additionally, to determine whether the effects on intracellular SphK1 would affect cell proliferation, overexpressed miR-495 in Cdx2 cells were used for cell proliferation assay. Results show that miR-495 overexpression leads to decreased levels of proliferation vs control cells. CONCLUSIONS: Our findings identify miR-495 as a novel negative regulator of SphK1 and define a novel role of miR-495 as a modulator of intestinal epithelial proliferation. Role of Portal Nutrient Sensing in the Regulation of PostPrandial Glucose Atanu Pal, MB BCHIR, MRCS, Ali Tavakkoli, MD, FACS, FRCS Center for Weight Management and Metabolic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA INTRODUCTION: Absorbed nutrients enter circulation through the portal vein. Portal nutrient sensing may therefore be critical in anticipating a nutrient load and preparing the liver for it. We used portal glucose infusions to investigate the role of SGLT3, a glucose-sensitive membrane protein, as a putative portal sensor and its effect on systemic glucose. METHODS: In anesthetized rats, catheters were placed in the portal vein for infusion, and the jugular vein for systemic blood sampling. 90-minute portal glucose infusions were used to simulate a post-prandial glucose load, with co-infusion of saline (control; n¼5) or alpha-methyl-glucopyranoside (aMG; n¼5), an SGLT3 agonist. Hepatic mRNA expression of enzymes involved in gluconeogenesis, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), were also measured. To determine whether a neural pathway was involved in portal sensing, in Table 1. Peak systemic glucose and area-under-curve (AUC) for different groups
Portal infusion
Saline-glucose co-infusion (control) aMG-glucose co-infusion Vagotomy then aMG-glucose co-infusion Portal denervation then aMG-glucose co-infusion
n
5 5
5
Systemic glucose AUC Peak (mg/dL min) (mg/dL)
p Value (vs control)
11517
193.4
N/A
9036
157.6
<0.05
8341
151.2
<0.05
5 11635
191.4
Not significant
another group vagotomy (n¼5) or portal denervation using the neurotoxin capsaicin (n¼5) was performed before performing the above portal infusions. RESULTS: Peak systemic glucose level was lower during aMG coinfusion compared to saline co-infusion (p<0.05), as was the 90minute area-under-the-curve (p<0.05; Table 1). This effect was abolished by portal denervation but not by vagotomy. aMG coinfusion reduced the expression of hepatic G6Pase and PEPCK. CONCLUSIONS: These data suggest that portal glucose is sensed by SGLT3. Stimulation of this sensor leads to a decrease in systemic glucose by lowering hepatic gluconeogenesis and improves glucose tolerance through a neural but non-vagal pathway. This highlights a critical role for portal nutrient sensing in post-prandial glucose homeostasis that could be a therapeutic target in type-2 diabetes. Mechanisms of Bile Acid-Induced Intestinal Epithelial Cell Death Anne S Roberts, MD, Stephanie C Papillon, MD, Avafia Y Roberts, MD, MS, Mark R Frey, PhD, Henri R Ford, MD, FACS, Christopher P Gayer, MD, PhD Children’s Hospital Los Angeles, Los Angeles, CA INTRODUCTION: Intestinal bacteria convert conjugated, primary bile acids into deconjugated, secondary bile acids. Increases in secondary metabolites are seen in some gastrointestinal diseases, such as necrotizing enterocolitis. While secondary bile acids are known to be more toxic than primary metabolites, the mechanisms by which they induce cellular injury have not been elucidated. We hypothesize that secondary bile acids mediate cell death via induction of apoptosis. METHODS: We assessed IEC-6 cell survival using an MTS-based assay. LDH release was used as a measure of cell necrosis. Z-VADFMK, a pan-caspase inhibitor, or necrostatin-1, a RIP kinase inhibitor, was used to inhibit apoptosis and necroptosis, respectively. Caspase-3 cleavage was quantified by Western blotting. RESULTS: Secondary bile acids were more toxic than their primary counterparts, while conjugated forms were more toxic than unconjugated. Conjugated bile acid treatment led to increased LDH release (up to 3-fold), indicating cell necrosis. Unconjugated metabolites generated minimal LDH release. Treatment with Z-VAD-FMK protected IEC-6 cells from secondary bile acid-induced cell death (survival increased 43% for deoxycholic, and 31% for lithocholic acid), but did not prevent primary bile acid-induced death. Western blotting of lysates from cells exposed to secondary bile acids showed increased caspase-3 cleavage vs controls. Necrostatin-1 was partially protective against primary bile acid-induced death. CONCLUSIONS: Our data demonstrate that conjugated bile acids cause intestinal cell death via necrosis. In contrast, unconjugated, secondary metabolites induce apoptosis, while necroptosis contributes to primary bile acid-induced cell death. This is the first report
Human Small Intestine Stem Cells Respond to Withdrawal of Luminal Nutrients by Decreasing Proliferation and Increasing Self-Renewal in the Crypt Christa N Grant, MD*, Salvador Garcia, MS, Ryan G Spurrier, MD, Tracy C Grikscheit, MD, FACS Children’s Hospital Los Angeles, Los Angeles, CA
METHODS: Quantitative real-time polymerase chain reaction was used to evaluate proteins in resealing colonic biopsy induced mucosal wounds. Protein expression was analyzed by western blotting, immunofluorescence labeling and confocal microscopy. Model intestinal epithelial cell lines were used to analyze WISP1 effects on wound closure in vitro. Statistical analysis was performed by Student’s two-tailed t-test as appropriate. p<0.05 was considered significant.
INTRODUCTION: Global calorie restriction has been shown to affect intestinal stem cell (ISC) behavior in animal models. Little is known about the effect of luminal nutrients on ISCs in the calorie replete organism. We found that villi were shorter and crypts were less deep and less proliferative in unfed human intestine. We hypothesized that without luminal nutrient, human ISCs would remain in the crypt compartment in lieu of differentiating and migrating.
RESULTS: Increased WNT1-inducible secreted protein 1 (WISP1) mRNA was observed in resealing intestinal mucosal wounds (fold increase compared to non wounded tissue ¼ 9.34; p<0.001). Additionally, WISP1 protein and mRNA expression was up-regulated in the intestinal mucosa of patients with active inflammatory bowel disease. Recombinant WISP1 protein promoted wound closure of model intestinal epithelial cell lines in vitro (ctl ¼ 76.080.33% vs WISP1 ¼ 91.621.07% p<0.0001). The pro-repair effects of WISP 1 were mediated by increased signaling of focal adhesion kinase and c-src that regulate cell matrix adhesion, cell migration and therefore wound closure.
METHODS: This study was approved and monitored by the IRB. 5 paired human ileum samples were collected from children undergoing ileostomy reversal at least 6 weeks after intestinal division with minimal resection. The segment contiguous with enteral feeding was denoted “fed” and the excluded segment “unfed.” Apoptotic cells were quantified by cleaved-caspase 3 immunofluorescence. Microarray analysis was performed on 3 paired samples, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) on 5 paired samples for quantification of ISC markers Bmi1 and Lgr5, representing slowly and rapidly cycling ISCs respectively.
CONCLUSIONS: WISP1 promotes intestinal mucosal wound closure. Thus, WISP1 could potentially be used as a therapeutic agent to promote healing of intestinal mucosal wounds.
RESULTS: There was increased epithelial apoptosis (p<0.02), and crypt width in unfed intestine (p<0.01). Microarray analysis demonstrated a 44-fold increase in Lgr5 (p¼0.002), a 15-fold increase in the Paneth cell marker lysozyme (p¼0.008), and a 1.74-fold increase in secretory cell progenitor marker Atoh1 (p¼0.04). RT-qPCR analysis confirmed the increase in Lgr5 expression (p¼0.013).
Sphingosine Kinase-1 Is Regulated by MicroRNA-495 in Intestinal Epithelial Cells Daniel Mansour, MD, Natasha Hansraj, MD, Alexis D Smith, MD, Ruiyun Li, PhD, Rao Jaladanki, PhD, James M Donahue, MD, FACS, Jian-Ying Wang, MD, PhD, Douglas J Turner, MD, FACS Baltimore VA Medical Center, Baltimore, MD, University of Maryland Medical Center, Baltimore, MD
CONCLUSIONS: Human ileum ISCs respond to lack of luminal nutrients by decreasing differentiation, leading to increased villus epithelial apoptosis. Decreased crypt proliferation, increased crypt width, and increased expression of Paneth cell and undifferentiated cell markers suggest decreased migration out of the crypt in which are lodged more possible progenitors.
INTRODUCTION: The bioactive sphingolipid, Sphingosine-1-phophate (S1P), plays a significant role in intestinal epithelial cell renewal. We have shown that cells overexpressing sphingosine kinase-1 (SphK1), the rate limiting enzyme for S1P production, show increased cell proliferation; however, the regulation of SphK1 is currently not known. MicroRNAs are small, noncoding RNAs that regulate gene expression; microRNA-495 (miR-495) has been reported to participate in cell differentiation. However, the role of miR-495 in intestinal epithelial cells (IECs) and the mechanisms that regulate SphK1 remain elusive. We hypothesized that miR-495 regulates IEC proliferation through the direct inhibition of SphK1.
WNT1-Inducible Secreted Protein 1 Regulates Intestinal Mucosal Wound Resealing Philipp-Alexander Neumann, MD, Hikaru Nishio, PhD, Giovanna Leoni, PhD, Miguel Quiros, PhD, Gabriela Bernal, Mingli Feng, Norbert Senninger, MD, FACS, Charles A Parkos, MD, PhD, Asma Nusrat, MD Emory University, Atlanta, GA, University of Muenster, Muenster, Germany
METHODS: Cdx2/IEC-6 cells derived from rat intestinal crypts were used. Protein expression, RNA levels, proliferation assay and miRNA over expression were determined by standard methods.
INTRODUCTION: Mucosal wound closure after inflammation, ischemia or surgery is coordinated by a plethora of signaling proteins. WNT signaling is known to regulate intestinal epithelial
ª 2014 by the American College of Surgeons Published by Elsevier Inc.
S13
http://dx.doi.org/10.1016/j.jamcollsurg.2014.07.018 ISSN 1072-7515/14
S14
J Am Coll Surg
Surgical Forum Abstracts
RESULTS: MiR-495 and miR-206 were found to be present in Cdx2 cells and to have proposed binding sites on SphK1 mRNA. MiR-495 and 206 overexpression via transfection with pre-miR-495 and 206 resulted in a dramatic decrease in protein expression of SphK1. Interestingly, only miR-495 overexpression showed a significant reduction in Sphk1 mRNA levels. In addition, during biotin pull down experiments, again, only miR-495 showed specific binding to Sphk1 mRNA. Additionally, to determine whether the effects on intracellular SphK1 would affect cell proliferation, overexpressed miR-495 in Cdx2 cells were used for cell proliferation assay. Results show that miR-495 overexpression leads to decreased levels of proliferation vs control cells. CONCLUSIONS: Our findings identify miR-495 as a novel negative regulator of SphK1 and define a novel role of miR-495 as a modulator of intestinal epithelial proliferation. Role of Portal Nutrient Sensing in the Regulation of PostPrandial Glucose Atanu Pal, MB BCHIR, MRCS, Ali Tavakkoli, MD, FACS, FRCS Center for Weight Management and Metabolic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA INTRODUCTION: Absorbed nutrients enter circulation through the portal vein. Portal nutrient sensing may therefore be critical in anticipating a nutrient load and preparing the liver for it. We used portal glucose infusions to investigate the role of SGLT3, a glucose-sensitive membrane protein, as a putative portal sensor and its effect on systemic glucose. METHODS: In anesthetized rats, catheters were placed in the portal vein for infusion, and the jugular vein for systemic blood sampling. 90-minute portal glucose infusions were used to simulate a post-prandial glucose load, with co-infusion of saline (control; n¼5) or alpha-methyl-glucopyranoside (aMG; n¼5), an SGLT3 agonist. Hepatic mRNA expression of enzymes involved in gluconeogenesis, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), were also measured. To determine whether a neural pathway was involved in portal sensing, in Table 1. Peak systemic glucose and area-under-curve (AUC) for different groups
Portal infusion
Saline-glucose co-infusion (control) aMG-glucose co-infusion Vagotomy then aMG-glucose co-infusion Portal denervation then aMG-glucose co-infusion
n
5 5
5
Systemic glucose AUC Peak (mg/dL min) (mg/dL)
p Value (vs control)
11517
193.4
N/A
9036
157.6
<0.05
8341
151.2
<0.05
5 11635
191.4
Not significant
another group vagotomy (n¼5) or portal denervation using the neurotoxin capsaicin (n¼5) was performed before performing the above portal infusions. RESULTS: Peak systemic glucose level was lower during aMG coinfusion compared to saline co-infusion (p<0.05), as was the 90minute area-under-the-curve (p<0.05; Table 1). This effect was abolished by portal denervation but not by vagotomy. aMG coinfusion reduced the expression of hepatic G6Pase and PEPCK. CONCLUSIONS: These data suggest that portal glucose is sensed by SGLT3. Stimulation of this sensor leads to a decrease in systemic glucose by lowering hepatic gluconeogenesis and improves glucose tolerance through a neural but non-vagal pathway. This highlights a critical role for portal nutrient sensing in post-prandial glucose homeostasis that could be a therapeutic target in type-2 diabetes. Mechanisms of Bile Acid-Induced Intestinal Epithelial Cell Death Anne S Roberts, MD, Stephanie C Papillon, MD, Avafia Y Roberts, MD, MS, Mark R Frey, PhD, Henri R Ford, MD, FACS, Christopher P Gayer, MD, PhD Children’s Hospital Los Angeles, Los Angeles, CA INTRODUCTION: Intestinal bacteria convert conjugated, primary bile acids into deconjugated, secondary bile acids. Increases in secondary metabolites are seen in some gastrointestinal diseases, such as necrotizing enterocolitis. While secondary bile acids are known to be more toxic than primary metabolites, the mechanisms by which they induce cellular injury have not been elucidated. We hypothesize that secondary bile acids mediate cell death via induction of apoptosis. METHODS: We assessed IEC-6 cell survival using an MTS-based assay. LDH release was used as a measure of cell necrosis. Z-VADFMK, a pan-caspase inhibitor, or necrostatin-1, a RIP kinase inhibitor, was used to inhibit apoptosis and necroptosis, respectively. Caspase-3 cleavage was quantified by Western blotting. RESULTS: Secondary bile acids were more toxic than their primary counterparts, while conjugated forms were more toxic than unconjugated. Conjugated bile acid treatment led to increased LDH release (up to 3-fold), indicating cell necrosis. Unconjugated metabolites generated minimal LDH release. Treatment with Z-VAD-FMK protected IEC-6 cells from secondary bile acid-induced cell death (survival increased 43% for deoxycholic, and 31% for lithocholic acid), but did not prevent primary bile acid-induced death. Western blotting of lysates from cells exposed to secondary bile acids showed increased caspase-3 cleavage vs controls. Necrostatin-1 was partially protective against primary bile acid-induced death. CONCLUSIONS: Our data demonstrate that conjugated bile acids cause intestinal cell death via necrosis. In contrast, unconjugated, secondary metabolites induce apoptosis, while necroptosis contributes to primary bile acid-induced cell death. This is the first report