Alexis D. Smith

Regulation of Cyclin-Dependent Kinase 4 Translation through CUG-Binding Protein 1 and microRNA-222 by Polyamines

The findings presented indicate that polyamine-regulated CUG-binding protein 1 and microRNA-222 modulate cyclin-dependent kinase 4 (CDK4) translation at least in part by altering the recruitment of CDK4 mRNA to processing bodies.

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 β-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.

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.

Laparoscopic adrenalectomy for unsuspected unilateral primary adrenal lymphoma.

Laparoscopic surgery for the incidentally enlarged adrenal mass may reveal unexpected findings, such as primary adrenal lymphoma of the large B cell type in this case report. Laparoscopic management proved to be feasible and resulted in minimal surgical morbidity to the patient.

Candidate genes for limiting cholestatic intestinal injury identified by gene expression profiling

The lack of bile flow from the liver into the intestine can have devastating complications including hepatic failure, sepsis, and even death. This pathologic condition known as cholestasis can result from etiologies as diverse as total parenteral nutrition (TPN), hepatitis, and pancreatic cancer. The intestinal injury associated with cholestasis has been shown to result in decreased intestinal resistance, increased bacterial translocation, and increased endotoxemia. Anecdotal clinical evidence suggests a genetic predisposition to exaggerated injury. Recent animal research on two different strains of inbred mice demonstrating different rates of bacterial translocation with different mortality rates supports this premise. In this study, a microarray analysis of intestinal tissue following common bile duct ligation (CBDL) performed under general anesthesia on these same two strains of inbred mice was done with the goal of identifying the potential molecular mechanistic pathways responsible. Over 500 genes were increased more than 2.0‐fold following CBDL. The most promising candidate genes included major urinary proteins (MUPs), serine protease‐1‐inhibitor (Serpina1a), and lipocalin‐2 (LCN‐2). Quantitative polymerase chain reaction (qPCR) validated the microarray results for these candidate genes. In an in vitro experiment using differentiated intestinal epithelial cells, inhibition of MUP‐1 by siRNA resulted in increased intestinal epithelial cell permeability. Diverse novel mechanisms involving the growth hormone pathway, the acute phase response, and the innate immune response are thus potential avenues for limiting cholestatic intestinal injury. Changes in gene expression were at times found to be not only due to the CBDL but also due to the murine strain. Should further studies in cholestatic patients demonstrate interindividual variability similar to what we have shown in mice, then a “personalized medicine” approach to cholestatic patients may become possible.

Tu2060 Sphingosine-1-Phosphate Prevents LPS-Induced Loss of Permeability in Intestinal Epithelial Cells

Intestinal epithelial barrier dysfunction results from a wide variety of pathologic conditions; at the gastrointestinal mucosal layer cells must be capable of maintaining barrier integrity, and do this through the interplay of multiple active processes. Previous reports from our lab have shown that Sphingosine-1-phophate (S1P) promotes intestinal epithelial barrier function in part through regulation of barrier proteins, and S1P has also been found to be protective in various pathologic states. Lipopolysaccharide (LPS) has been shown to increase paracellular permeability, and recently, to also decrease intracellular S1P. In the current study we hypothesized that S1P would decrease paracellular permeability upon LPS exposure, and would act in part through regulation of caveolin-1 expression IEC-Cdx2L1 (Cdx) differentiated intestinal epithelial cells were utilized. Western blot analysis, real-time PCR, immunohistochemical staining, were utilized by standard techniques. Transwell permeability to C14-mannitol, FITC-dextran, and measurement of transepithelial electrical resistance (TEER) were utilized for permeability assessments. Sphingosine Kinase 1 (SphK-1) overexpression stable cell lines were selected in rat intestinal epithelial cells (IECs). SphK-1 activity and S1P production were measured by radioactive isotope assay. LPS-treated (50 mcM) Cdx cells show dramatically increased permeability at 4h, but pretreatment with S1P (0.5 mcM for one hour) was protective of this LPS-induced increase in permeability, and returned permeability to normal levels. S1P also prevented LPS-associated decreases in phosphorylated occludin, and in immunofluorescence studies S1P preserved cortical accumulation of occludin that was disrupted with LPS administration alone. S1P was found to increase levels of toll-like receptor (TLR) 2 in Cdx cells, with no change in levels of TLR4. Similarly, cells stably overexpressing SphK1 demonstrated increased levels of S1P and also increased levels of TLR2 and not TLR4. Cells overexpressing SphK1 and S1P showed dramatically increased plasma membrane levels of Stim1, TRPC1, and the scaffolding protein caveolin-1. Phosphorylated caveolin-1 was significantly decreased with exposure to LPS (5 mcM), however co-treatment with S1P preserved basal caveolin-1 levels. Finally, inhibition of caveolin-1 with siRNA prevented S1P rescue of LPS loss of permeability. Our findings demonstrate that S1P prevents LPS-associated loss of permeability, and this is in part through its ability to prevent LPS-associated loss of caveolin-1.