Arginine Consumption by the Intestinal Parasite Giardia intestinalis Reduces Proliferation of Intestinal Epithelial Cells

In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the host´s production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that citrulline especially should gain further attention in future treatment strategies.     

Lineage-Specific Expression of Bestrophin-2 and Bestrophin-4 in Human Intestinal Epithelial Cells

Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3^- or Cl^-. Bestrophin genes represent a newly identified group of calcium-activated Cl^- channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.     

Molecular Model of the Microvillar Cytoskeleton and Organization of the Brush Border

Background

Brush border microvilli are ∼1-µm long finger-like projections emanating from the apical surfaces of certain, specialized absorptive epithelial cells. A highly symmetric hexagonal array of thousands of these uniformly sized structures form the brush border, which in addition to aiding in nutrient absorption also defends the large surface area against pathogens. Here, we present a molecular model of the protein cytoskeleton responsible for this dramatic cellular morphology.

Methodology/Principal Findings

The model is constructed from published crystallographic and microscopic structures reported by several groups over the last 30+ years. Our efforts resulted in a single, unique, self-consistent arrangement of actin, fimbrin, villin, brush border myosin (Myo1A), calmodulin, and brush border spectrin. The central actin core bundle that supports the microvillus is nearly saturated with fimbrin and villin cross-linkers and has a density similar to that found in protein crystals. The proposed model accounts for all major proteinaceous components, reproduces the experimentally determined stoichiometry, and is consistent with the size and morphology of the biological brush border membrane.

Conclusions/Significance

The model presented here will serve as a structural framework to explain many of the dynamic cellular processes occurring over several time scales, such as protein diffusion, association, and turnover, lipid raft sorting, membrane deformation, cytoskeletal-membrane interactions, and even effacement of the brush border by invading pathogens. In addition, this model provides a structural basis for evaluating the equilibrium processes that result in the uniform size and structure of the highly dynamic microvilli.     

Salmonella Infection Upregulates the Leaky Protein Claudin-2 in Intestinal Epithelial Cells

Background

Tight junctions seal the space between adjacent epithelial cells. Mounting evidence suggests that tight junction proteins play a key role in the pathogenesis of human disease. Claudin is a member of the tight junction protein family, which has 24 members in humans. To regulate cellular function, claudins interact structurally and functionally with membrane and scaffolding proteins via their cytoplasmic domain. In particular, claudin-2 is known to be a leaky protein that contributes to inflammatory bowel disease and colon cancer. However, the involvement of claudin-2 in bacterial infection in the intestine remains unknown.

Methods/Principal Findings

We hypothesized that Salmonella elevates the leaky protein claudin-2 for its own benefit to facilitate bacterial invasion in the colon. Using a Salmonella-colitis mouse model and cultured colonic epithelial cells, we found that pathogenic Salmonella colonization significantly increases the levels of claudin-2 protein and mRNA in the intestine, but not that of claudin-3 or claudin-7 in the colon, in a time-dependent manner. Immunostaining studies showed that the claudin-2 expression along the crypt-villous axis postinfection. In vitro, Salmonella stimulated claudin-2 expression in the human intestinal epithelial cell lines SKCO15 and HT29C19A. Further analysis by siRNA knockdown revealed that claudin-2 is associated with the Salmonella-induced elevation of cell permeability. Epithelial cells with claudin-2 knockdown had significantly less internalized Salmonella than control cells with normal claudin-2 expression. Inhibitor assays demonstrated that this regulation is mediated through activation of the EGFR pathway and the downstream protein JNK.

Conclusion/Significance

We have shown that Salmonella targets the tight junction protein claudin-2 to facilitate bacterial invasion. We speculate that this disruption of barrier function contributes to a new mechanism by which bacteria interact with their host cells and suggests the possibility of blocking claudin-2 as a potential therapeutic strategy to prevent bacterial invasion.     

Transgenic Expression of Human Lysophosphatidic Acid Receptor LPA2 in Mouse Intestinal Epithelial Cells Induces Intestinal Dysplasia

Lysophosphatidic acid (LPA) acts on LPA₂ receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA₂ attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA₂ alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA₂ in intestinal epithelial cells (IECs) under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG) human LPA₂; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA₂ colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA₂ compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA₂ overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA₂ alone can lead to intestinal dysplasia.     

Capsaicin-enhanced Ribosomal Protein P2 Expression in Human Intestinal Caco-2 Cells

On the basis of transepithelial electrical resistance (TER) measurements, we found that capsaicin (100 μM)-treated human intestinal Caco-2 cells show a momentary increase in tight-junction (TJ) permeability (decrease in TER) followed by a complete recovery. We used proteome analysis to search for proteins that are associated with the recovery of TJ permeability in capsaicin-treated Caco-2 cells. A protein with a relative molecular mass of 14 kDa was found to be expressed more highly in capsaicin-treated cells than in nontreated cells. Mass spectrometry and sequence analyses revealed that the protein that is expressed significantly upon capsaicin treatment is the ribosomal protein P2; its cDNA sequence was identical to that found in the human genome database. An increase in the amount of cellular filamentous actin (F-actin) was shown after 8 h of incubation with capsaicin. It has been reported that P2 activates elongation factor 2, which stabilizes F-actin filaments, and that the depolymerization of F-actin is associated with the increase in TJ permeability (decrease in TER). Consequently, these results suggest that P2 plays an important role in the recovery of the TJ permeability in capsaicin-treated human intestinal cells. An erratum to this article is available at .     

The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na⁺– K⁺ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b ). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a ,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a ).It is known that a network of intermediate filament (IF)¹, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells.     

Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors.     

Rotavirus Infection of Cells in Culture Induces Activation of RhoA and Changes in the Actin and Tubulin Cytoskeleton

Rotavirus infection induces an increase in [Ca²⁺]_cyto, which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca²⁺ chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca²⁺ homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.     

Human Cysteine-Rich Intestinal Protein: cDNA Cloning and Expression of Recombinant Protein and Identification in Human Peripheral Blood Mononuclear Cells

Cysteine-rich intestinal protein (CRIP) is a small, 8.5-kDa protein with one double zinc-finger motif called a LIM domain. It is very abundant in intestine and some immune cells in rodents, and expression is influenced by development and the immune response. We have cloned a human CRIP cDNA from human small intestine poly(A)⁺RNA by RT-PCR. Through sequencing, we found that the human intestinal CRIP protein (hCRIP) differed from the previously cloned rat CRIP by two amino acids (residues 8 and 58). hCRIP was expressed with the pET vector/bacterial system and isolated by gel filtration and ion-exchange chromatography. The protein was purified to homogeneity as confirmed by PAGE, Western blotting, and immunodetection. Recombinant hCRIP has a molecular mass of 8390 Da based on mass spectrum analysis. Southern analysis suggests that there are three copies of the CRIP gene in the human genome. hCRIP mRNA was detected by RT-PCR in human monocytes purified from peripheral blood and THP-1 cells, a human monocytic cell line. Incubation of THP-1 cells with⁶⁵Zn and chromatography of the cytosol show that a significant amount of the radioactivity is associated with CRIP as was shown previously for rat intestine. The results are consistent with a functional role for CRIP in proliferation/differentiation of specific cell types, particularly those associated with host defense.     

Autophagy Enhances Intestinal Epithelial Tight Junction Barrier Function by Targeting Claudin-2 Protein Degradation

Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2.     

Serum Opens Tight Junctions and Reduces ZO-1 Protein in Retinal Epithelial Cells

Abstract: We have shown previously that serum inhibits tight junction formation in a retinal epithelial cell culture model for the blood-brain barrier. We have now examined in detail the effects of serum on the tight junctions. Our data show that serum induces a breakdown in tight junction function as indicated by decreased transepithelial electrical resistance and increased permeability. Rat serum had effects similar to those of bovine serum, indicating that the activity is species-independent. The effect is concentration-dependent, reversible, and specific for the apical surface, suggesting the involvement of a specific receptor-ligand interaction. Differences in the time course, response magnitude, and structural manifestations between the serum-induced breakdown and that induced by switching the cultures to a low-calcium medium suggest fundamental differences in their mechanisms. The calcium switch results in an immediate and complete junctional breakdown with cell retraction and perinuclear translocation of both actin and the tight junction protein zonula occludens-1. The serum-induced breakdown occurs slowly, is incomplete, and is manifested structurally by decreases in zonula occludens-1 protein, whereas actin organization is unchanged. Thus, serum induces a specific breakdown in retinal epithelial cell tight junctions that may be mediated by effects on the expression of zonula occludens-1.     

Effect of Exogenous Zinc on MsrB1 Expression and Protein Oxidation in Human Lens Epithelial Cells

Biological Trace Element Research - Aluminum (Al), a potentially neurotoxic element, provokes various adverse effects on human health such as dialysis dementia, osteomalacia, and microcytic anemia....     

Sugars Increase Non-Heme Iron Bioavailability in Human Epithelial Intestinal and Liver Cells

Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55) increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl₃ increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.     

SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.     

Hepatocyte Growth Factor Enables Enhanced Integrin–Cytoskeleton Linkage by Affecting Integrin Expression in Subconfluent Epithelial Cells

Hepatocyte growth factor (HGF) exerts mitogenic and motogenic effects in different cell types. In the epithelial cell line mHepR1 we found that HGF induced pronounced alterations in cell morphology and promoted cell adhesion and spreading. To analyze the mechanisms how HGF affects these integrin mediated functions we studied the physical linkage of integrins with the cytoskeleton. First we found that HGF increased the expression of different integrin subunits in subconfluent cells and influenced the distribution of integrins on the cell surface. To address the physical association of integrins with the cytoskeleton we analyzed Triton X-100-extracted cell fractions using flow cytometry. Here we show that cultivation of the cells with HGF for 24 h prior to integrin cross-linking significantly enhanced the cytoskeletal anchorage of integrins. To further find out whether HGF directly induces an integrin–cytoskeleton link without subsequent cross-linking we added HGF to suspended cells but failed to detect cytoskeletally immobilized integrins in the detergent-insoluble cell fraction which could be related to the absence of a calcium response induced by HGF. Overall, the results indicate that HGF promotes the physical linkage of integrins to the cytoskeleton which requires additional stimulation of integrins.     

Targeting of an Intestinal Apical Endosomal Protein to Endosomes in Nonpolarized Cells

Polarized cells such as epithelial cells and neurons have distinct endosomal compartments associated with different plasma membrane domains. The endosomes of the neuronal cell body and the basolateral cytoplasm of epithelial cells are thought to perform cellular “housekeeping” functions such as the uptake of nutrients and metabolites, while the endosomes in the apical cytoplasm or axons are thought to be specialized for the sorting and transcytosis of cell type–specific ligands and receptors. However, it is not known if nonpolarized cells such as fibroblasts contain a specialized endosomal compartment analogous to the specialized endosomes found in neurons and epithelia. We have expressed a protein that is normally found in the apical early endosomes of developing intestinal epithelial cells in normal rat kidney fibroblasts. This apical endosomal marker, called endotubin, is targeted to early endosomes in transfected fibroblasts, and is present in peripheral as well as perinuclear endosomes. The peripheral endosomes that contain endotubin appear to exclude transferrin, fluid phase markers, and the mannose-6-phosphate receptor, although in the perinuclear region colocalization of endotubin and these markers is present. In addition, endotubin positive structures do not tubulate in response to brefeldin A and instead redistribute to a diffuse perinuclear location. Since this endosomal compartment has many of the characteristics of an apical or axonal endosomal compartment, our results indicate that nonpolarized cells also contain a specialized early endosomal compartment.