Jose Greenspon

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.

Sphingosine-1-Phosphate Regulates the Expression of Adherens Junction Protein E-Cadherin and Enhances Intestinal Epithelial Cell Barrier Function

BackgroundThe regulation of intestinal barrier permeability is important in the maintenance of normal intestinal physiology. Sphingosine-1-phosphate (S1P) has been shown to play a pivotal role in enhancing barrier function in several non-intestinal tissues. The current study determined whether S1P regulated function of the intestinal epithelial barrier by altering expression of E-cadherin, an important protein in adherens junctions.MethodsStudies were performed upon cultured differentiated IECs (IEC-Cdx2L1 line) using standard techniques.ResultsS1P treatment significantly increased levels of E-cadherin protein and mRNA in intestinal epithelial cells (IECs) and also led to E-cadherin localizing strongly to the cell–cell border. S1P also improved the barrier function as indicated by a decrease in 14C-mannitol paracellular permeability and an increase in transepithelial electrical resistance (TEER) in vitro.ConclusionsThese results indicate that S1P increases levels of E-cadherin, both in cellular amounts and at the cell–cell junctions, and leads to improved barrier integrity in cultured intestinal epithelial cells.

Sphingosine-1-Phosphate Protects Intestinal Epithelial Cells from Apoptosis Through the Akt Signaling Pathway

Objective The regulation of apoptosis of intestinal mucosal cells is important in maintenance of normal intestinal physiology. Summary Sphingosine-1-phosphate (S1P) has been shown to play a critical role in cellular protection to otherwise lethal stimuli in several nonintestinal tissues. Methods The current study determines whether S1P protected normal intestinal epithelial cells (IECs) from apoptosis and whether Akt activation was the central pathway for this effect. Results S1P demonstrated significantly reduced levels of apoptosis induced by tumor necrosis factor-alpha (TNF-α)/cycloheximide (CHX). S1P induced increased levels of phosphorylated Akt and increased Akt activity, but did not affect total amounts of Akt. This activation of Akt was associated with decreased levels of both caspase-3 protein levels and of caspase-3 activity. Inactivation of Akt by treatment with the PI3K chemical inhibitor LY294002 or by overexpression of the dominant negative mutant of Akt (DNMAkt) prevented the protective effect of S1P on apoptosis. Additionally, silencing of the S1P-1 receptor by specific siRNA demonstrated a lesser decrease in apoptosis to S1P exposure. Conclusion These results indicate that S1P protects intestinal epithelial cells from apoptosis via an Akt-dependent pathway.

Gut microbiota, tight junction protein expression, intestinal resistance, bacterial translocation and mortality following cholestasis depend on the genetic background of the host

Failure of the intestinal barrier is a characteristic feature of cholestasis. We have previously observed higher mortality in C57BL/6J compared with A/J mice following common bile duct ligation (CBDL). We hypothesized the alteration in gut barrier function following cholestasis would vary by genetic background. Following one week of CBDL, jejunal TEER was significantly reduced in each ligated mouse compared with their sham counterparts; moreover, jejunal TEER was significantly lower in both sham and ligated C57BL/6J compared with sham and ligated A/J mice, respectively. Bacterial translocation to mesenteric lymph nodes was significantly increased in C57BL/6J mice vs. A/J mice. Four of 15 C57BL/6J mice were bacteremic; whereas, none of the 17 A/J mice were. Jejunal IFN-γ mRNA expression was significantly elevated in C57BL/6J compared with A/J mice. Western blot analysis demonstrated a significant decrease in occludin protein expression in C57BL/6J compared with A/J mice following both sham operation and CBDL. Only C57BL/6J mice demonstrated a marked decrease in ZO-1 protein expression following CBDL compared with shams. Pyrosequencing of the 16S rRNA gene in fecal samples showed a dysbiosis only in C57BL/6J mice following CBDL when compared with shams. This study provides evidence of strain differences in gut microbiota, tight junction protein expression, intestinal resistance and bacterial translocation which supports the notion of a genetic predisposition to exaggerated injury following cholestasis.

Substance P regulates migration in rat intestinal epithelial cells

Objective:The current study examined the effect of substance P (SP) upon intestinal epithelial cells and the mechanistic details of this interaction. Summary Background Data:Intestinal epithelial cells must be capable of migration to reseal mucosal wounds for several vital intestinal functions. This process is incompletely understood; however, recent evidence implicates the neurotransmitter SP in this process. Methods:Normal rat intestinal epithelial cells (IEC-6 cells) were studied to identify the presence of the SP receptor (NK-1 subtype) and then exposed to physiologic doses of SP and antagonists to assess for increased migration. Results:Examination IEC-6 cells revealed the presence of the SP receptor. Wounding of these cells followed by subsequent exposure to SP (10−9 mol/L) resulted in increased migration. Similarly, SP-induced increases in intracellular calcium concentration and actomyosin stress fiber formation. These effects were all blocked through specific NK-1 receptor antagonists. Conclusions:These results indicate that SP stimulates intestinal epithelial migration and increases in calcium concentration. These data support a beneficial role for SP in the maintenance of intestinal mucosal homeostasis.

Shoshin beriberi mimicking central line sepsis in a child with short bowel syndrome

BackgroundShoshin beriberi, cardiac failure secondary to a severe deficiency of the vitamin thiamine, can develop in patients following extensive intestinal resection or bypass; however, parenteral supplementation has largely eliminated this complication. Hemodynamic instability resulting from central line sepsis is a far more common complication in these parenteral nutrition-dependent patients. This case report details the diagnosis and treatment of shoshin beriberi in a patient with short bowel syndrome whose presentation mimicked central line sepsis.MethodsA retrospective chart review was performed. Appropriate laboratory data were included.ResultsThe patient was treated unsuccessfully with antibiotics and supportive measures. Resolution of symptoms was achieved only after the empiric administration of thiamine and folate.ConclusionsThis case highlights that life-threatening thiamine deficiency mimicking septic shock can develop in patients with short bowel syndrome, despite oral multivitamin administration. We recommend diligent monitoring of vitamin levels in any total parenteral alimentation dependent patient unable to receive the intravenous multivitamin complex, regardless of oral vitamin supplementation or clinical findings.

Intraabdominal pulmonary sequestration presenting with elevated urinary normetanephrine levels

Carrying a prenatal diagnosis of a left-sided intraabdominal mass, a term female newborn underwent postnatal imaging that confirmed a left suprarenal mass. Urinary normetanephrine levels were elevated. Given a preoperative diagnosis of neuroblastoma, the baby underwent an uneventful resection of the mass en bloc with the left adrenal gland. The pathologic examination returned pulmonary sequestration and a normal adrenal gland. Postoperative urinary catecholamines were normal. To the best of our knowledge, this is the first description of a newborn with an intraabdominal pulmonary sequestration presenting with elevated urinary catecholamines.

M1703 Intestinal Permeability and Bacterial Translocation Are Modulated By the Genetic Background of the Host

G A A b st ra ct s manner with significant inhibition noted at 5.0 and 10.0 μM. 18β-GA washout restored a normal rate of cell migration. No inhibition of migration was noted between 0.625 to 2.5 μM despite reaching near complete inhibition (90-95%) of GJIC at these concentrations. The Cx-43 polypeptide exists in three isoforms under control conditions: two predominant phosphorylated forms (P2 and P1) and one minor non-phosphorylated form (P0). 18β-GA shifts the phosphorylation state of Cx-43 from nearly equal amounts of P2 and P1 to a predominantly P0 isoform at concentrations between 0.625 to 2.5 μM. Confocal microscopy revealed that Cx-43 is not present at the leading-edge lamellipodia extensions of migrating cells, but localizes primarily to the retracting edge at points of contact between cells and along lines of cortical actin filaments. Conclusions: Although a correlation exists between GJIC and Cx-43 phosphorylation state, GJIC and maintenance of the P2 isoform are not required for IEC-6 migration. Based on the specific subcellular localization of Cx-43 in migrating cells, we speculate that regulation of actin cytoskeleton dynamics may be a novel function for gap junction proteins and might regulate epithelial cell migration In Vivo along the intestinal villous. (Supported by an Intramural Research Grant for New Faculty from Michigan State University).

M1731 Sphingosine-1-Phosphate Regulates Barrier Function in Cultured Intestinal Epithelial Cells and Murine Intestinal Tissue

assessed by stable isotope methodology after administration of U-13C-glucose and 1-13Clactose. Concentrations of the EFA linoleic acid (LA), and the enzyme activity and mRNA expression of lactase, were measured in the mucosa of proximal, mid and distal small intestine. Results: Mice fed the EFA-deficient diet were markedly EFA-deficient (triene/ tetraene ratio 0.23±0.06 vs. 0.01±0.00 in controls, p<0.05) with a profound lower fat absorption of dietary fat (81±3 vs. 99±0% in controls, of ingested amount, p<0.05). EFA deficiency did not affect the histology or proliferative capacity of the small intestine as demonstrated by the histological and proliferative staining, and by the morphometrcal measurements of the small intestinal villi. Blood C6-glucose appearance and disappearance were similar in both groups, indicating unaffected monosaccharide absorption. In contrast, blood appearance of 13C-glucose, originating from 13C-lactose, was delayed in EFA-deficient mice. EFA deficiency profoundly reduced lactase activity (-58%, p<0.01) and mRNA expression (-55%, p<0.01) in mid small intestine. Both lactase activity and its mRNA expression strongly correlated with mucosal LA concentrations (r=0.89 and 0.79, resp., p< 0.01). Conclusions: EFA deficiency in mice inhibits the capacity to digest lactose, but does not affect small intestinal histology. These data underscore the observation that EFA deficiency functionally impairs the small intestine, possibly mediated by low LA levels in the enterocytes. This research was funded by the Dutch Digestive Foundation (MWO 04-38).