James R. Goldenring

Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.

BACKGROUND & AIMS Hypergastrinemia occurs frequently in association with acid suppression and Helicobacter infection, but its role in the progression to gastric atrophy and gastric cancer has not been well defined. METHODS The effects of hypergastrinemia, and possible synergy with Helicobacter felis infection, were investigated in insulin-gastrin (INS-GAS) transgenic mice. RESULTS INS-GAS mice initially showed mild hypergastrinemia, increased maximal gastric acid secretion, and increased parietal cell number but later progressed to decreased parietal cell number and hypochlorhydria. Development of gastric atrophy was associated with increased expression of growth factors, heparin-binding epidermal growth factor and transforming growth factor alpha. At 20 months of age, INS-GAS mice showed no evidence of increased enterochromaffin-like cell number, but instead exhibited gastric metaplasia, dysplasia, carcinoma in situ, and gastric cancer with vascular invasion. Invasive gastric carcinoma was observed in 6 of 8 INS-GAS mice that were >20 months old. Helicobacter felis infection of INS-GAS mice led to accelerated (< or = 8 mo) development of intramucosal carcinoma (85%), with submucosal invasion (54%) and intravascular invasion (46%; P < or = 0.05). CONCLUSIONS These findings support the unexpected conclusion that chronic hypergastrinemia in mice can synergize with Helicobacter infection and contribute to eventual parietal cell loss and progression to gastric cancer.

Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm

Sox2 is expressed in developing foregut endoderm, with highest levels in the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is expressed ventrally, in the future trachea. In humans, heterozygosity for SOX2 is associated with anopthalmia-esophageal-genital syndrome (OMIM 600992), a condition including esophageal atresia (EA) and tracheoesophageal fistula (TEF), in which the trachea and esophagus fail to separate. Mouse embryos heterozygous for the null allele, Sox2EGFP, appear normal. However, further reductions in Sox2, using Sox2LP and Sox2COND hypomorphic alleles, result in multiple abnormalities. Approximately 60% of Sox2EGFP/COND embryos have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry or western blot. The mutant esophagus morphologically resembles the trachea, with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very few p63+ basal cells. By contrast, the abnormal foregut of Nkx2.1-null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2EGFP/COND embryos in which Sox2 levels are ∼18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63+ cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In all hypomorphic embryos the forestomach has an abnormal phenotype, with reduced keratinization, ectopic mucus cells and columnar epithelium. These findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach.

Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

cAMP‐dependent protein kinase (A‐kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A‐kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A‐kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin‐associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin‐related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14‐amino‐acid amphipathic α‐helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409–438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RIIimmunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A‐kinase to a region near the secretory canaliculus.

Targeted disruption of the Kvlqt1 gene causes deafness and gastric hyperplasia in mice.

The KvLQT1 gene encodes a voltage-gated potassium channel. Mutations in KvLQT1 underlie the dominantly transmitted Ward-Romano long QT syndrome, which causes cardiac arrhythmia, and the recessively transmitted Jervell and Lange-Nielsen syndrome, which causes both cardiac arrhythmia and congenital deafness. KvLQT1 is also disrupted by balanced germline chromosomal rearrangements in patients with Beckwith-Wiedemann syndrome (BWS), which causes prenatal overgrowth and cancer. Because of the diverse human disorders and organ systems affected by this gene, we developed an animal model by inactivating the murine Kvlqt1. No electrocardiographic abnormalities were observed. However, homozygous mice exhibited complete deafness, as well as circular movement and repetitive falling, suggesting imbalance. Histochemical study revealed severe anatomic disruption of the cochlear and vestibular end organs, suggesting that Kvlqt1 is essential for normal development of the inner ear. Surprisingly, homozygous mice also displayed threefold enlargement by weight of the stomach resulting from mucous neck cell hyperplasia. Finally, there were no features of BWS, suggesting that Kvlqt1 is not responsible for BWS.

Identification and Characterization of a Family of Rab11-interacting Proteins

Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling systems. We hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. Here we define a family of four Rab11-interacting proteins: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family InteractingProtein 2 (Rab11-FIP2), Rab11-FamilyInteracting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associated with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacted with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a was dependent upon a conserved carboxyl-terminal amphipathic α-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalized with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalized with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also coenriched with Rab11a and H+K+-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocated with Rab11a and the H+K+-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors.

Identification of the Major Postsynaptic Density Protein as Homologous with the Major Calmodulin-Binding Subunit of a Calmodulin-Dependent Protein Kinase

Abstract: The major postsynaptic density protein (mPSDp), comprising >50% of postsynaptic density (PSD) protein, is an endogenous substrate for calmodulin‐dependent phosphorylation as well as a calmodulin‐binding protein in PSD preparations. The results in this investigation indicate that mPSDp is highly homologous with the major calmodulin‐binding subunit (p) of tubulin‐associated calmodulin‐dependent kinase (TACK), and that PSD fractions also contain a protein homologous with the o‐subunit of TACK. Homologies between mPSDp and a 63,000 dalton PSD protein and the p‐ and ó‐subunits of TACK were established by the following criteria: (1) identical apparent molecular weights: (2) identical calmodulin‐binding properties; (3) manifestation of Ca2+ ‐calmodulin‐stimulated autophosphorylation; (4)identical isoelectric points; (5) identical calmodulin binding and autophosphorylation patterns on two‐dimensional gels; (6) homologous two‐dimensional tryptic peptide maps; and (7) similar phosphoamino acid‐specific phosphorylation of tubulin. The results suggest that mPSDp is a calmodulin‐binding protein involved in modulating protein kinase activity in the postsynaptic density and that a tubulin kinase system homologous with TACK exists in a membrane‐bound form in the PSD.

Mice lacking secretory phospholipase A2 show altered apoptosis and differentiation with Helicobacter felis infection

BACKGROUND & AIMS Infection with Helicobacter pylori uniformly leads to a chronic superficial gastritis that may progress to atrophic gastritis, a premalignant process. A mouse model of Helicobacter felis infection was used to study possible genetic determinants of the response to infection. METHODS Three inbred mouse strains with known secretory phospholipase A2 (sPLA2) genotypes [BALB/c (+/+), C3H/HeJ (+/+), and C57BL/6 (-/-)] were orally infected with H. felis and examined longitudinally using routine histology, immunocytochemistry, electron microscopy, proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and Northern and Western blot studies. RESULTS Only the C57BL/6 strain showed increased gastric fundic proliferation and apoptosis in response to infection. In addition, the C57BL/6 mouse showed a marked loss of parietal and chief cells, along with a marked expansion of an aberrant gastric mucous cell lineage that stained positive for spasmolytic polypeptide. In contrast, no significant change in these cell types was observed in BALB/c and C3H/HeJ strains. Increased expression of sPLA2 was observed in BALB/c and C3H/HeJ after H. felis infection, whereas sPLA2 expression was absent in C57BL/6 mice. CONCLUSIONS H. felis infection leads to increased apoptosis and altered cellular differentiation in the C57BL/6 mouse, a strain that lacks gastric sPLA2 expression. Because sPLA2 has been identified recently as the MOM1 (modifier of MIN) locus that influences polyp formation in the colon, these studies suggest that sPLA2 may also influence the gastric epithelial response to Helicobacter infection.

Expansion of Pdx1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor α

BACKGROUND & AIMS The progenitor cells responsible for transforming growth factor (TGF)-alpha-induced pancreatic ductal metaplasia and neoplasia remain uncharacterized. During pancreatic development, differentiated cell types arise from ductal progenitor cells expressing the Pdx1 homeodomain transcription factor. The aims of this study were, first, to evaluate the role of Pdx1-expressing stem cells in MT-TGFalpha transgenic mice, and second, to further characterize cell proliferation and differentiation in this model. METHODS To assess Pdx1 gene expression in normal and metaplastic epithelium, we performed in vivo reporter gene analysis using heterozygous Pdx1(lacZ/+) and bigenic Pdx1(lacZ/+)/MT-TGFalpha mice. RESULTS Pdx1(lacZ/+)/MT-TGFalpha bigenics showed up-regulated Pdx1 expression in premalignant metaplastic ductal epithelium. In addition to Pdx1 gene activation, TGF-alpha-induced metaplastic epithelium demonstrated a pluripotent differentiation capacity, as evidenced by focal expression of Pax6 and initiation of islet cell neogenesis. The majority of Pdx1-positive epithelial cells showed no expression of insulin, similar to the pattern observed during embryonic development. CONCLUSIONS Overexpression of TGF-alpha induces expansion of a Pdx1-expressing epithelium characterized by focal expression of Pax6 and initiation of islet neogenesis. These findings suggest that premalignant events induced by TGF-alpha in mouse pancreas may recapitulate a developmental program active during embryogenesis.

Possible role of transforming growth factor α in the pathogenesis of Ménétrier's disease: Supportive evidence from humans and transgenic mice

Ménétriers disease is an uncommon disorder of unknown etiology characterized by enlarged gastric folds with foveolar hyperplasia and cystic dilatation of gastric glands. Biochemical features that are seen frequently include hypoproteinemia, hypochlorhydria, and increased gastric mucus. Because transforming growth factor alpha (TGF alpha) is an epithelial cell mitogen that inhibits gastric acid secretion and increases gastric mucin content, we hypothesized that its altered expression might be involved in the pathogenesis of this disease. Therefore, we characterized TGF alpha immunoreactivity in the gastric mucosa of 4 patients with Ménétriers disease. In contrast to the normal pattern of TGF alpha immunostaining in which TGF alpha appears most concentrated in parietal cells, there was intense staining in the majority of mucous cells in the gastric mucosa of patients with Ménétriers disease. In one patient from whom sufficient fresh tissue was obtained to isolate RNA, expression of TGF alpha and the epidermal growth factor receptor was higher in the gastric mucosa relative to a normal control. In addition, metallothionein-TGF alpha transgenic mice, which overexpress TGF alpha in gastric mucosa, show a number of features characteristic of Ménétriers disease. These include foveolar hyperplasia and glandular cystic dilatation, increased gastric neutral mucin staining, and reduced basal and histamine-stimulated rates of acid production. Taken together, observations derived from the human material and correlation with data from a transgenic mouse model support an important role for TGF alpha in the pathogenesis of Ménétriers disease.

Calcium/Ganglioside‐Dependent Protein Kinase Activity in Rat Brain Membrane

Abstract: The effects of gangliosides on phosphorylation were studied in rat brain membrane. Gangliosides stimulated phosphorylation only in the presence of Ca2+ with major phosphoproteins of 45,000, 50,000, 60,000, and 80,000 daltons and high‐molecular‐weight species. In addition, gangliosides inhibited the phosphorylation of three proteins with molecular weights of 15,000, 20,000, and 78,000 daltons. The two low‐molecular‐weight proteins comigrated with rat myelin basic proteins. Ganglioside stimulation was dependent on the formation of a Ca2+ ganglioside complex since the calcium salt of gangliosides stimulated phosphorylation maximally. Disialo and trisialo gangliosides were more potent stimulators of kinase activity than the monosialo GM1. GD1a was the most potent activator tested. Asialo‐GM1, cerebroside, sialic acid, neuraminyllactose, sulfatide, and the acidic phospholipids phosphatidylserine and phosphatidylinositol did not stimulate kinase activity. The Ca2+ ‐dependent, ganglioside‐stimulated phosphorylation was qualitatively similar to the pattern for calmodulin‐dependent phosphorylation. However, while calmodulin‐dependent kinase activity was inhibited with an IC50 of 10 μM trifluoperazine, ganglioside‐stimulated kinase was inhibited with an IC50 of 200 μM trifluoperazine. These results indicate that gangliosides have complex effects on membrane‐associated kinase activities and suggest that Ca2+ ‐ganglioside complexes are potent stimulators of membrane kinase activity.