Eric D. Strauch

Glypican-3 expression in Wilms tumor and hepatoblastoma.

Background Glypican-3 (GPC3) is a heparan sulfate proteoglycan. When it is disrupted, it causes the X-linked gigantism-overgrowth Simpson-Golabi-Behmel syndrome. Its involvement in growth control is consistent with recent reports that it can bind to growth factors, possibly including insulin-like growth factor 2. Further, it has been hypothesized that it may function as a tumor suppressor gene in breast and ovarian carcinomas and mesotheliomas. Patients and Methods RNA and protein were extracted from Wilms tumor and hepatoblastoma tissue samples and GPC3 levels were measured in these extracts by Northern blotting, reverse transcription polymerase chain reaction, and immunoblotting. Results In contrast to published results with carcinomas, high levels of GPC3 expression were found in Wilms tumor and hepatoblastoma. Low or undetectable expressions of this gene were found in normal tissue surrounding the tumor. Conclusions Increased expression of GPC3 in Wilms tumor and hepatoblastoma suggests a growth-promoting or neutral activity for this gene product rather than a growth-suppressive effect.

Deoxycholic acid causes DNA damage while inducing apoptotic resistance through NF-κB activation in benign Barrett's epithelial cells.

Gastroesophageal reflux is associated with adenocarcinoma in Barretts esophagus, but the incidence of this tumor is rising, despite widespread use of acid-suppressing medications. This suggests that refluxed material other than acid might contribute to carcinogenesis. We looked for potentially carcinogenetic effects of two bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on Barretts epithelial cells in vitro and in vivo. We exposed Barretts (BAR-T) cells to DCA or UDCA and studied the generation of reactive oxygen/nitrogen species (ROS/RNS); expression of phosphorylated H2AX (a marker of DNA damage), phosphorylated IkBα, and phosphorylated p65 (activated NF-κB pathway proteins); and apoptosis. During endoscopy in patients, we took biopsy specimens of Barretts mucosa before and after esophageal perfusion with DCA or UDCA and assessed DNA damage and NF-κB activation. Exposure to DCA, but not UDCA, resulted in ROS/RNS production, DNA damage, and NF-κB activation but did not increase the rate of apoptosis in BAR-T cells. Pretreatment with N-acetyl-l-cysteine (a ROS scavenger) prevented DNA damage after DCA exposure, and DCA did induce apoptosis in cells treated with NF-κB inhibitors (BAY 11-7085 or AdIκB superrepressor). DNA damage and NF-κB activation were detected in biopsy specimens of Barretts mucosa taken after esophageal perfusion with DCA, but not UDCA. These data show that, in Barretts epithelial cells, DCA induces ROS/RNS production, which causes genotoxic injury, and simultaneously induces activation of the NF-κB pathway, which enables cells with DNA damage to resist apoptosis. We have demonstrated molecular mechanisms whereby bile reflux might contribute to carcinogenesis in Barretts esophagus.

Acid and Bile Salt–Induced CDX2 Expression Differs in Esophageal Squamous Cells From Patients With and Without Barrett's Esophagus

BACKGROUND & AIMS It is not clear why only a minority of patients with gastroesophageal reflux disease (GERD) develop Barretts esophagus. We hypothesized that differences among individuals in molecular pathways activated when esophageal squamous epithelium is exposed to reflux underlie the development of Barretts metaplasia. METHODS We used esophageal squamous cell lines from patients who had GERD with Barretts esophagus (normal esophageal squamous [NES]-B3T and NES-B10T) and without Barretts esophagus (NES-G2T and NES-G4T) to study effects of acid and bile salts on expression of the CDX2 gene. Bay 11-705, Ad5 inhibitor kappaB(IkappaB)alpha-SR, and site-directed mutagenesis were used to explore effects of nuclear factor-kappaB (NF-kappaB) inhibition on CDX2 promoter activity; DNA binding of the NF-kappaB subunits p50 and p65 was assessed by chromatin immune-precipitation. RESULTS Acid and bile salts increased CDX2 messenger RNA (mRNA), protein, and promoter activity in NES-B3T and NES-B10T cells, but not in NES-G2T or NES-G4T cells. Inhibition of NF-kappaB abolished the increase in CDX2 promoter activity. Increased CDX2 promoter activity was associated with nuclear translocation of p50, which bound to the promoter. We found CDX2 mRNA in 7 of 10 esophageal squamous biopsy specimens from patients with Barretts esophagus, but in only 1 of 10 such specimens from patients who had GERD without Barretts esophagus. CONCLUSIONS Acid and bile salts induce CDX2 mRNA and protein expression in esophageal squamous cells from patients with Barretts esophagus, but not from GERD patients without Barretts esophagus. We speculate that these differences in acid- and bile salt-induced activation of molecular pathways may underlie the development of Barretts metaplasia.

Bile salts regulate intestinal epithelial cell migration by nuclear factor–κB–induced expression of transforming growth factor–β☆

Abstract Background Mucosal restitution is an important repair modality in the gastrointestinal tract. We have shown that taurodeoxycholate increases intestinal epithelial cell migration by increasing TGF-β expression, and that the transcription factor NF-κB regulates TDCA induced cell migration after injury. The objectives of this study were to determine if this is a property shared by other bile salts or an effect specific to TDCA, and to determine if NF-κB regulates TGF-β expression. Study design Studies were conducted in IEC-6 cells. Cell migration was examined using an in vitro model. TGF-β protein and mRNA expression was determined by ELISA and Northern blot analysis. Sequence-specific NF-κB binding activity was measured by gel shift assays. Results Taurocholate and deoxycholate at physiologic concentrations significantly increased intestinal epithelial cell migration 6 hours after wounding (p Conclusions We conclude that bile salts at physiologic conditions increase cell migration after injury, an effect regulated by NF-κB. Further, NF-κB elicits TGF-β gene transcription during cell migration. These data support a physiologic role of bile salts in the maintenance of intestinal mucosal integrity.

Bile salts induce resistance to apoptosis through NF-κB-mediated XIAP expression

Apoptosis plays a critical role in intestinal mucosal homeostasis. We previously showed that the bile salt taurodeoxycholate has a beneficial effect on the intestinal mucosa through an increase in resistance to apoptosis mediated by nuclear factor (NF)-&kgr;B. The current study further characterizes the effect of bile salts on intestinal epithelial cell susceptibility to apoptosis and determines if the X-linked inhibitor of apoptosis protein (XIAP) regulates bile salt-induced resistance to apoptosis. Exposure of normal intestinal epithelial cells (IEC-6) to the conjugated bile salts taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) resulted in an increase in resistance to tumor necrosis factor (TNF)-&agr; and cycloheximide (CHX)-induced apoptosis, and NF-&kgr;B activation. Treatment with TDCA and TCDCA resulted in an increase in XIAP expression. Specific inhibition of NF-&kgr;B by infection with an adenoviral vector that expresses the I&kgr;B&agr; super-repressor (I&kgr;BSR) prevented the induction of XIAP expression and the bile salt-mediated resistance to apoptosis. Treatment with the specific XIAP inhibitor Smac also overcame this increase in enterocyte resistance to apoptosis. Bile salts inhibited formation of the active caspase-3 from its precursor procaspase-3. Smac prevented the inhibitory effect of bile salts on caspase-3 activation. These results indicate that bile salts increase intestinal epithelial cell resistance to apoptosis through NF-&kgr;B-mediated XIAP expression. Bile salt-induced XIAP mediates resistance to TNF-&agr;/CHX-induced apoptosis, at least partially, through inhibition of caspase-3 activity. These data support an important beneficial role of bile salts in regulation of mucosal integrity. Decreased enterocyte exposure to luminal bile salts, as occurs during starvation and parenteral nutrition, may have a detrimental effect on mucosal integrity.

NF-κB Regulates Intestinal Epithelial Cell and Bile Salt-Induced Migration After Injury

ObjectiveTo determine if NF-&kgr;B regulates intestinal epithelial cell migration and if it has a role during bile salt-induced migration. Summary Background DataMucosal restitution is an important repair modality in the gastrointestinal tract. The authors have shown that taurodeoxycholate (TDCA) increases intestinal epithelial cell migration. NF-&kgr;B regulates activation of a number of genes involved in inflammatory responses. MethodsStudies were conducted in IEC-6 cells. I&kgr;B protein expression was determined by Western blot analysis. Sequence-specific NF-&kgr;B binding activity was measured by EMSA shift assays and nuclear localization by immunohistochemistry. Cell migration was examined by using an in vitro model that mimics the early cell division-independent stages of epithelial restitution. ResultsThe process of cell migration over the wounded area was associated with a significant increase in NF-&kgr;B binding activity in IEC-6 cells. Immunohistochemistry revealed translocation of NF-&kgr;B into the nucleus. Western blot analysis showed that injury decreased I&kgr;B protein expression. Inhibition of the binding activity by treatment with a specific NF-&kgr;B inhibitor, MG-132, inhibited cell migration during restitution. Further, exposure to TDCA at the physiologic concentration that induces intestinal epithelial cell migration increased NF-&kgr;B binding activity, induced NF-&kgr;B translocation into the nucleus, and decreased I&kgr;B protein expression. MG-132 also inhibits bile salt-induced cell migration. ConclusionsNF-&kgr;B regulates intestinal epithelial cell migration. Bile salts at physiologic concentrations increase cell migration by activation of NF-&kgr;B. These data show that bile salts may have a role in the maintenance of intestinal mucosal integrity.

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.

Cutaneous ciliated cyst: a case report with focus on mullerian heterotopia and comparison with eccrine sweat glands.

Cutaneous ciliated cyst is an exceedingly rare, benign lesion most commonly found in the dermis or subcutis of the lower extremities of young female patients in their second and third decades. The pathogenesis of the cyst is unknown. We report a cutaneous ciliated cyst in the lower extremity of a 13-year-old female patient. On histologic examination, clusters of eccrine sweat glands were observed adjacent to the cyst. Upon comparison of the immunohistochemical profile of the cutaneous ciliated cyst and the eccrine sweat glands, they appeared almost completely unrelated. The histologic, immunohistochemical, and ultrastructural findings of this case and the literature provide evidence in favor of the Mullerian heterotopia theory.

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.