Biomedical subjects

James R Goldenring

Publications and source records attributed to James R Goldenring.

At least 19 recordsLinked to original sources

PubMed2006

A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2 shuttle.

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.

Animals↗

PubMed2006

Secretory COPII coat component Sec23a is essential for craniofacial chondrocyte maturation.

An increasing number of human disorders have been linked to mutations in genes of the secretory pathway. The chemically induced zebrafish crusher variant results in malformed craniofacial skeleton, kinked pectoral fins and a short body length. By positional cloning, we identified a nonsense mutation converting leucine to a stop codon (L402X) in the sec23a gene, an integral component of the COPII complex, which is critical for anterograde protein trafficking between endoplasmic reticulum and Golgi apparatus. Zebrafish crusher mutants develop normally until the onset of craniofacial chondrogenesis. crusher chondrocytes accumulate proteins in a distended endoplasmic reticulum, resulting in severe reduction of cartilage extracellular matrix (ECM) deposits, including type II collagen. We demonstrate that the paralogous gene sec23b is also an essential component of the ECM secretory pathway in chondrocytes. In contrast, knockdown of the COPI complex does not hinder craniofacial morphogenesis. As SEC23A lesions cause the cranio-lenticulo-sutural dysplasia syndrome, crusher provides the first vertebrate model system that links the biology of endoplasmic reticulum to Golgi trafficking with a clinically relevant dysmorphology.

Animals↗

PubMed2006

Expression and prognostic significance of prothymosin-alpha and ERp57 in human gastric cancer.

PURPOSE: Prothymosin-alpha and ERp57 were previously identified as markers for gastric metaplasia in a mouse model of Helicobacter-induced gastric metaplasia and neoplasia. In this paper we assess whether the expression of these putative biomarkers in humans is correlated with gastric metaplasia and adenocarcinoma and clinical outcomes. METHODS: Eight tissue microarrays, containing 749 paraffin-embedded tissue cores from 164 gastric cancer patients, were stained for prothymosin-alpha and ERp57 by horseradish peroxidase immunohistochemical techniques. The proportion of stained cells per core was quantitated using the Ariol SL-50 automated image analysis system. RESULTS: Prothymosin-alpha stained a significantly higher percentage of nuclei in cancer and metastases compared with normal gastric mucosa. ERp57 staining was significantly decreased in cancer and metastases compared with both normal gastric mucosa and metaplasias. ERp57 expression also correlated with greater depth of tumor invasion and advanced stage of disease. Kaplan-Meier survival analysis determined that tumors with the highest quartile of ERp57 expression were statistically associated with longer postoperative survival. A Cox proportional hazard analysis showed that maintenance of ERp57 expression was associated with longer postoperative survival. CONCLUSIONS: These results suggest that although prothymosin-alpha is overexpressed in gastric adenocarcinoma, it is not associated with alterations in survival. In contrast, loss of ERp57 expression correlated with more aggressive disease and could provide useful prognostic information for gastric cancer patients.

Adenocarcinoma↗

PubMed2006

Expression of Pdx-1 in human gastric metaplasia and gastric adenocarcinoma.

Metaplastic lineages represent critical putative preneoplastic precursors for gastrointestinal metaplasia. Two metaplastic processes are associated with gastric cancer: intestinal metaplasia (the presence of intestinal goblet cell containing lineages in the stomach) and spasmolytic polypeptide-expressing metaplasia (SPEM; antralization of the gastric fundus). The transcription factor Pdx-1 is expressed in the adult pancreatic islet cells as well as the gastric antrum and duodenum. We have previously noted the increase in Pdx-1 expression in models of TGFalpha overexpression in mice but not in other models of SPEM in rodents. We have therefore sought to examine the presence of Pdx-1 expression in gastric metaplasias and gastric adenocarcinoma in humans. Tissue microarrays containing gastric cancers from the fundus and antrum and samples of SPEM and intestinal metaplasia were immunostained for Pdx-1. Nuclear Pdx-1 expression was observed in only 50% of antral-derived cancers and was present in 40% of fundic tumors. Pdx-1 expression did not correlate with clinical outcome. Although SPEM lineages did not show any staining for Pdx-1, intestinal metaplasia showed strong nuclear staining for Pdx-1. Thus, Pdx-1 expression is not associated with antralizing metaplasia (SPEM) but is associated with intestinal metaplasia. Given the pattern of normal Pdx-1 expression in the duodenum, goblet cell metaplasia in the stomach may reflect the adoption of a duodenal lineage paradigm.

Adenocarcinoma↗

PubMed2006

The Rab11-FIP1/RCP gene codes for multiple protein transcripts related to the plasma membrane recycling system.

Rab11a is a member of the Rab11 small GTPase family, and plays an important role in plasma membrane recycling. Rab11-Family Interacting Protein 1 (Rab11-FIP1) binds to Rab11 through a carboxyl-terminal amphipathic alpha helix. We have identified eight alternatively spliced Rab11-FIP1 gene transcripts from human chromosome 8. Among them, Rab11-FIP1A-D have carboxyl terminal Rab11 binding domains, while Rab11-FIP1E-H do not contain the Rab11 binding domain. While Rab11-FIP1B and F gene transcripts are ubiquitous, other Rab11-FIP1 transcripts demonstrate more limited patterns of expression in human tissue cDNAs. EGFP-Rab11-FIP1A-D proteins over-expressed in HeLa cells targeted to Rab11a-containing membranes, while EGFP-Rab11-FIP1E/F and H proteins did not localize with recycling system membranes. However, transferrin trafficking was not significantly altered in HeLa cells over-expressing expressing any of the EGFP-Rab11-FIP1 proteins. Rabbit polyclonal antibodies specific for Rab11-FIP1B and Rab11-FIP1C/RCP demonstrated that Rab11-FIP1B and Rab11-FIP1C/RCP are expressed endogenously. Strikingly, endogenous staining for Rab11-FIP1C/RCP only partially co-localized with EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, and EGFP-Rab11a in the perinuclear region, indicating that Rab11-FIP1C/RCP resides in a differentiable subcellular compartment within the plasma membrane recycling system compared with Rab11-FIP1A and Rab11-FIP1B. These data suggest that Rab11-FIP1 proteins may play coordinated roles in regulating plasma membrane recycling with regional specificity within the Rab11a-containing recycling system.

Adaptor Proteins, Signal Transducing↗

PubMed2006

MARK2/EMK1/Par-1Balpha phosphorylation of Rab11-family interacting protein 2 is necessary for the timely establishment of polarity in Madin-Darby canine kidney cells.

Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1Balpha (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.

Adherens Junctions↗

PubMed2006

CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25.

SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25-binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway.

Animals↗

PubMed2006

Omeprazole treatment ameliorates oxyntic atrophy induced by DMP-777.

Atrophic gastritis, characterized as parietal cell loss or oxyntic atrophy, is the primary event in the evolution of the spectrum of metaplastic and hyperplastic lineage changes thought to predispose to gastric neoplasia. A number of animal models have provided insights into the lineage changes induced by oxyntic atrophy. Recently, we have reported a model for pharmacological induction of oxyntic atrophy with DMP-777. DMP-777 ablates parietal cells selectively and leads to the gastric cell lineage changes including foveolar hyperplasia and spasmolytic polypeptide expressing metaplasia (SPEM). Previous investigations showed that DMP-777 dissipated a gastric tubulovesicle proton gradient without impairing the H/K-ATPase activity, consistent with its pharmacological action as a parietal cell-specific protonophore which could induce parietal cell necrosis through backwash of luminal acid into actively secreting cells. We hypothesized that, if DMP-777 was acting as a parietal cell protonophore, then suppression of acid secretion should protect parietal cells from the toxic effects of the drug. In this study, we pretreated and coadministered the proton pump inhibitor omeprazole with DMP-777 to determine the effect of active acid secretion inhibition on the DMP-777-induced histologic changes in the stomachs of male rats. Omeprazole pretreatment ameliorated DMP-777-induced parietal cell loss as well as foveolar hyperplasia. These results indicate that active acid secretion is required for DMP-777 cytotoxicity, consistent with its suggested behavior as a parietal cell-specific protonophore.

Analysis of Variance↗

PubMed2006

Gastric graft-versus-host disease revisited: does proton pump inhibitor therapy affect endoscopic gastric biopsy interpretation?

Accurate diagnosis of gastrointestinal graft-versus-host disease (GvHD) is important, as it contributes significantly to postallogeneic stem cell transplant (SCT) morbidity and mortality. To test the hypothesis that proton pump inhibitor (PPI) therapy may interfere with histologic evaluation of gastric GvHD by inducing apoptosis, we evaluated epithelial apoptotic body counts in antral and fundic biopsies from SCT recipients and control patients, both taking and not taking PPIs at the time of endoscopic biopsy. Hematoxylin and eosin-stained slides of gastric biopsies from 130 patients (75 allogeneic SCT with GvHD on clinical and histologic grounds, and a comparison group of 55 age- and sex-matched nontransplant patients with histologically normal gastric biopsies) were reviewed. The groups were further stratified into patients taking (PPI+) and not taking PPIs (PPI-) at the time of biopsy. Apoptotic bodies (AB)/10 (400 x) high power fields (HPF) were quantified for each case. Mean apoptotic body counts were then calculated for each case group. Seventy antral cases (31 control and 39 transplant) were also evaluated via gastrin immunohistochemistry, and the mean number of gastrin positive cells/400 x HPF calculated. In the PPI- groups, apoptosis was increased in biopsies from transplant patients, compared with controls, both in antral and fundic mucosa. In PPI+ patients, there was significantly more apoptosis in the gastric body in transplant patients than in controls. However, comparing antral biopsies from control and transplant PPI+ patients, there was no significant difference in AB quantitation. More apoptosis was seen in antral biopsies from PPI+ control patients when compared with PPI- control patients (P = 0.009). Mean numbers of gastrin positive cells/400 x HPF were increased in both control and transplant patients taking PPIs (85 and 58, respectively) compared with samples from those patients not taking PPIs (48 and 51, respectively). PPI therapy is associated with increased apoptosis in antral biopsies and may interfere with the evaluation of GvHD in biopsies from this site. A similar increase in apoptosis was not seen in fundic biopsies; biopsy of the gastric fundus rather than antrum may be preferable for the diagnosis of upper gastrointestinal GvHD.

Adolescent↗

PubMed2006

The pericentriolar recycling endosome plays a key role in Vpu-mediated enhancement of HIV-1 particle release.

The HIV-1 accessory gene product Vpu is required for efficient viral particle release from infected human cells. The mechanism by which Vpu enhances particle assembly or release is not yet defined. Here, we identify an intracellular site that is critical for Vpu-mediated enhancement of particle release. Vpu was found to co-localize with markers for the pericentriolar recycling endosome. Expression of dominant negative mutants of Rab11a and myosin Vb that disrupt protein sorting through the recycling endosome abrogated the ability of Vpu to augment particle release. Remarkably, the effects of blocking recycling endosome function on HIV particle release were demonstrable only in human cell lines known to be responsive to Vpu, while no effect on particle release was seen in African green monkey cells. Inhibition of recycling endosome function in human cells also blocked the ability of HIV-2 envelope to enhance particle release. These studies indicate that Vpu and HIV-2 envelope glycoprotein enhance particle release via a common mechanism that requires the activity of the pericentriolar recycling endosome.

Animals↗

PubMed2006

Differentiation of the gastric mucosa III. Animal models of oxyntic atrophy and metaplasia.

Gastric cancer in humans arises in the setting of oxyntic atrophy (parietal cell loss) and attendant hyperplastic and metaplastic lineage changes within the gastric mucosa. Helicobacter infection in mice and humans leads to spasmolytic polypeptide-expressing metaplasia (SPEM). In a number of mouse models, SPEM arises after oxyntic atrophy. In mice treated with the parietal cell toxic protonophore DMP-777, SPEM appears to arise from the transdifferentiation of chief cells. These results support the concept that intrinsic mucosal influences regulate and modulate the appearance of gastric metaplasia even in the absence of significant inflammation, whereas chronic inflammation is required for the further neoplastic transition.

Animals↗

PubMed2005

AKAP350 modulates microtubule dynamics.

AKAP350 is a multiply spliced type II protein kinase A-anchoring protein that localizes to the centrosomes in most cells and the Golgi apparatus in epithelial cells. Multiple studies suggest that AKAP350 is involved in microtubule nucleation at the centrosome. Our previous studies demonstrated that AKAP350 was necessary for the maintenance of Golgi apparatus integrity. These data suggested that AKAP350 might be necessary for normal cytoskeletal interactions with the Golgi. To examine the relationship of AKAP350 with the microtubule cytoskeleton, we analyzed the effect of the depletion of AKAP350 on microtubule regrowth after nocodazole treatment in HeLa cells. The decrease in AKAP350 expression with short interfering RNA induced a delay in microtubule elongation with no effect on microtubule aster formation. In contrast, overexpression of the centrosomal targeting domain of AKAP350 elicited alterations in aster formation, but did not affect microtubule elongation. RNA interference for AKAP350 also induced an increase in cdc42 activity during microtubule regrowth. Our data suggest that AKAP350 has a role in the remodeling of the microtubule cytoskeleton.

A Kinase Anchor Proteins↗

PubMed2005

Involvement of myosin Vb in glutamate receptor trafficking.

Myosin V motors mediate cargo transport; however, the identity of neuronal molecules transported by these proteins remains unknown. Here we show that myosin Vb is expressed in several neuronal populations and associates with the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor subunit GluR1. In developing hippocampal neurons, expression of the tail domain of myosin Vb, but not myosin Va, enhanced GluR1 accumulation in the soma and reduced its surface expression. These changes were accompanied by reduced GluR1 clustering and diminished frequency of excitatory but not inhibitory synaptic currents. Similar effects were observed upon expression of full-length myosin Vb lacking a C-terminal region required for binding to the small GTPase Rab11. In contrast, mutant myosin Vb did not change the localization of several other neurotransmitter receptors, including the glutamate receptor subunit NR1. These results reveal a novel mechanism for the transport of a specific glutamate receptor subunit in neurons mediated by a member of the myosin V family.

Animals↗

PubMed2005

Altered metaplastic response of waved-2 EGF receptor mutant mice to acute oxyntic atrophy.

Metaplastic cell lineages are putative precursors for the development of gastric adenocarcinoma. The loss of parietal cells (oxyntic atrophy) is the initiating step in the evolution of gastric fundic mucosal lineage changes including metaplasia and hyperplasia. However, the intrinsic mucosal factors that promote and modulate the emergence of metaplastic phenotypes remain obscure. Over the past several years, we have studied pharmacologically induced, reversible oxyntic atrophy in rodents treated with DMP-777, a drug that acts as a parietal cell secretory membrane protonophore. DMP-777 elicits a rapid loss of parietal cells followed by the emergence of foveolar hyperplasia and spasmolytic polypeptide (SP)-expressing metaplasia (SPEM). The objective of the present study was to provide further insights into the intrinsic mucosal factors regulating the emergence of SPEM in the setting of oxyntic atrophy. We therefore studied the effects of DMP-777 administration on both SP/trefoil factor (TFF)2-deficient mice, which lack SP/TFF2, a marker of SPEM, and waved-2 mice, which harbor a point mutation in the EGF receptor that attenuates its tyrosine kinase activity. As in wild-type mice, treatment with DMP-777 for 7 days did elicit SPEM in SP/TFF2-deficient mice. These results suggest that SP/TFF2 does not impact on the development of metaplasia after the induction of parietal cell loss. In contrast, waved-2 homozygous mice displayed accelerated SPEM development by 3 days of treatment with DMP-777. These findings indicate that attenuation of EGF receptor signaling in waved-2 mice does elicit a more rapid emergence of SPEM. The results support a role for EGF receptor ligands in the regulation of gastric metaplasia.

Animals↗

PubMed2005

Saturation labeling with cysteine-reactive cyanine fluorescent dyes provides increased sensitivity for protein expression profiling of laser-microdissected clinical specimens.

Laser capture microdissection (LCM) provides the capability to isolate and analyze small numbers of cells from a specific area of a histologic section. LCM has particular value for analysis of early stage tumors, which are often small and intermixed with non-tumor tissue. It has previously been shown that a new generation of cysteine-reactive cyanine dyes can, in principle, provide increased sensitivity for two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) profiling when sample quantitities are limiting. However, the comparative advantage of the new dyes in a clinical setting has not been established. Here, we report that cysteine-reactive dyes allowed the identification of more features than established, lysine-reactive dyes with a given number of cells. This was true both with extracts prepared from human papillomavirus E6 and E7-transduced human keratinocytes, a model for early-stage cervical cancer, and with LCM samples. In an experiment comparing LCM clinical samples of gastric adenocarcinoma versus precancerous, spasmolytic polypeptide expressing metaplasia (SPEM) from the same patient, cysteine labeling allowed the identification of more than 1000 discrete protein spots in samples containing 5000 cells. This is a 5- to 50-fold smaller sample than used in previous studies. Both labeling methods had a comparable success rate for protein identification by mass spectrometry (MS). The proteins associated with more than 40 differentially abundant spots in the clinical samples were identified by MS. In this exploratory analysis, changes in expression levels of cytoskeletal proteins, molecular chaperones, and cell-signaling proteins were seen. The identification of a number of proteins that are potentially relevant to tumor progression suggests that the method holds promise for biomarker discovery.

Adenocarcinoma↗

PubMed2005

Assessment of Rab11-FIP2 interacting proteins in vitro.

Members of the Rab family of small GTPases are involved in multiple trafficking events in both endocytotic and biosynthetic pathways. To understand more fully the regulation of these events, a concerted effort is underway to ascertain the binding partners and regulators of Rabs. Here, we describe methods to assess binding of Rab11a with Rab11-FIP2 and other Rab11-FIPs utilizing a modified far-Western approach. We then broaden this application to assess binding of Rab11-FIP2 with myosin Vb and homodimerization of Rab11-FIP2.

Animals↗

PubMed2005

Interactions of myosin vb with rab11 family members and cargoes traversing the plasma membrane recycling system.

Myosin Vb interacts with Rab11 family members and is the major motor protein identified in association with plasma membrane recycling systems in nonpolarized and polarized cells. Using yeast two-hybrid binary screens, we demonstrated that dominant active Rab25S21V fails to interact with myosin Vb, but does interact with Rab11-FIP2. Transfection of DsRed2-myosin Vb tail in MDCK cell lines stably transfected with wild-type or dominant active forms of Rab11a or Rab25 demonstrated that the distribution of Rab25S21V is only partially altered by expression of the myosin Vb tail. Finally, we demonstrate EGF-dependent sequestration of internalized EGF receptor by EGFP-myosin Vb in A431 cells. Expression of myosin Vb tail represents a powerful provocative test for the trafficking of cargoes through Rab11-containing plasma membrane recycling systems.

Animals↗

PubMed2005

Evidence for repatterning of the gastric fundic epithelium associated with Ménétrier's disease and TGFalpha overexpression.

BACKGROUND & AIMS: Increase of intramucosal transforming growth factor alpha (TGFalpha) levels in the gastric fundus leads to oxyntic atrophy and massive foveolar hyperplasia in both metallothionein (MT)-TGFalpha mice and patients with Ménétrier's disease. We have evaluated the hypothesis that increased levels of TGFalpha in the fundus induces an antral pattern of cell differentiation in fundic glands by studying Pdx1, a transcription factor whose expression normally is confined to the gastric antrum. METHODS: Induction of Pdx1 expression was evaluated in Pdx1(lacZ/+)/MT-TGFalpha bigenic mice treated with zinc. The distribution of Pdx1 in MT-TGFalpha mice and Ménétrier's disease patients was evaluated with anti-Pdx1 antibodies. Transcript levels were evaluated by quantitative polymerase chain reaction in mouse and human tissues and AGS cells. RESULTS: In Pdx1(lacZ/+) mice, Pdx1 was expressed in antral mucosal cells including gastrin cells and TFF2-expressing deep glandular mucous cells. Zinc treatment for 2 to 8 weeks in Pdx1(lacZ/+)/MT-TGFalpha transgenic mice resulted in expression of Pdx1 throughout the fundus. No ectopic fundic Pdx1 expression was observed in either H. felis-infected or DMP777-treated mice. In MT-TGFalpha mice, 8 weeks of zinc treatment elicited nuclear Pdx1 staining throughout the fundic mucosa. TGFalpha treatment in AGS cells led to increases in Pdx1 and gastrin messenger RNA expression. Fundic sections from Ménétrier's disease patients showed nuclear Pdx1 staining throughout the fundic glands. Treatment of a Ménétrier's disease patient with an anti-epidermal growth factor receptor monoclonal antibody reduced fundic expression of both Pdx1 and gastrin. CONCLUSIONS: Overexpression of TGFalpha in MT-TGFalpha mice and Ménétrier's disease patients elicits ectopic expression in the fundus of Pdx1, consistent with the phenotype of antralization.

Animals↗