Aberrant expression of the polarity complex atypical PKC and non-muscle myosin IIA in active and inactive inflammatory bowel disease

Epithelial barrier function is contingent on appropriate polarization of key protein components. Work in intestinal epithelial cell cultures and animal models of bowel inflammation suggested that atypical PKC (aPKC), the kinase component of the Par3–Par6 polarity complex, is downregulated by pro-inflammatory signaling. Data from other laboratories showed the participation of myosin light chain kinase in intestinal inflammation, but there is paucity of evidence for assembly of its major target, non-muscle myosin II, in inflammatory bowel disease (IBD). In addition, we showed before that non-muscle myosin IIA (nmMyoIIA) is upregulated in intestinal inflammation in mice and TNFα-treated Caco-2 cells. Thus far, it is unknown if a similar phenomena occur in patients with IBD. Moreover, it is unclear whether aPKC downregulation is directly correlated with local mucosal inflammation or occurs in uninvolved areas. Frozen sections from colonoscopy material were stained for immunofluorescence with extensively validated specific antibodies against phosphorylated aPKC turn motif (active form) and nmMyoIIA. Inflammation was scored for the local area from where the material was obtained. We found a significant negative correlation between the expression of active aPKC and local inflammation, and a significant increase in the apical expression of nmMyoIIA in surface colon epithelia in inflamed areas, but not in non-inflamed mucosa even in the same patients. Changes in aPKC and nmMyoIIA expression are likely to participate in the pathogenesis of epithelial barrier function in response to local pro-inflammatory signals. These results provide a rationale for pursuing mechanistic studies on the regulation of these proteins

Biochemical and molecular characterization of the autocatalytic glycosyltransferase udp-glucose : protein transglucosylase

La enzima UDP-glucosa: proteína transglucosilasa (UPTG) es una glicosiltransferasa autocatalítica descripta tanto en tubérculo de papa (Solanum tuberosum L.) como en endosperma de maíz (Zea mays), involucrada en la síntesis de polisacáridos. Encontramos diferentes isoformas de UPTG de papa las cuales se expresaron en E. coli y se caracterizaron bioquímicamente. Las isoformas UPTG1 y UPTG2 no sólo presentan características cinéticas diferentes sino que además se expresan diferencialmente en los diversos tejidos de la planta. Se encontró una alta expresión de los transcriptos de UPTGl en estolones, tubérculos y raíces mientras que la expresión de los mensajeros de UPTG2 era elevada en todos los tejidos analizados, principalmente en hojas y tallos. El análisis del ADN genómico de papa sugiere la existencia de nuevas isoformas de UPTG, lo que sumado a la presencia de proteínas homólogas a la UPTG en otras especies vegetales, muestran que dichas glicosiltransferasas autocatalíticas constituirían una pequeña familia multigénica presente sólo en plantas superiores. Los estudios bioquímicos de la reacción de glicosilación de la UPTG mostraron que un único residuo de glucosa, xilosa o galactosa se une a la enzima en configuración β, con un tipo de unión que se comportaria más bien como las del tipo N. Además, el análisis de la reversibilidad de la reacción de glicosilación mostró que la UPTG catalizaría la ruptura del enlace glicosídico. El estudio de la naturaleza del inhibidor de la UPTG en maíz reveló que éste seria la isoforma SS1 de la sacarosa sintetasa presente principalmente en el endosperma. La isoforma SS1 ha sido relacionada recientemente con la biosíntesis de polisacáridos de pared celular y su asociación con la UPTG de maíz aporta una nueva evidencia a favor de dicha hipótesis. Los resultados de esta Tesis muestran que la UPTG de papa podía ser fosforilada in vitro. El tratamiento con fosfatasa de la proteína UPTG recombinante mostró que la defosforilación de la UPTG incrementa su glicosilación y que a su vez, la glicosilación de la enzima favorece la aparición de formas de mayor tamaño molecular inmunológicamente relacionadas con la UPTG. Se piensa que la defosforilación de UPTG incrementaría su glicosilación promoviendo la asociación de la proteína a otras proteínas o a sí misma. Se propone que la proteína UPTG se asociaría funcionalmente a elementos de anclaje, desconocidos hasta el momento, presentes en la membrana del Golgi facilitando el transporte de los UDP-azúcares sustratos de la biosíntesis de pectinas y hemicelulosas. Así, no podemos descartar que la defosforilación de la UPTG favorezca su asociación a membranas lo que podria involucrar un mecanismo de localización de la enzima. Los resultados con respecto al análisis de la expresión de ARNm de UPTG, sugieren que la enzima se expresaría principalmente en tejidos cuyas células estarían en estado de activo crecimiento y/o elongación. Basados en el hecho que en tejidos en elongación hay una activa síntesis principalmente de pectinas y hemicelulosas, los datos mostrados constituyen una fuerte evidencia que apoya la hipótesis que propone que la UPTG estaría involucrada en la síntesis de los polisacáridos no celulósicos que componen la pared celular vegetal. Más aún, la existencia de isoenzímas redundantes de UPTG sugiere que la enzima formaría pane de un sistema complejo esencial para la viabilidad de la célula vegetal.The enzyme UDP-glucose: protein transglucosylase (UPTG) is an autocatalytic glycosyltransferase involved in the biosynthesis of polysaccharides, which was described in potato tubers (Solanum tuberosum L.) and in maize endosperm (Zea mays). We found at least two different UPTG isoforms from potato, we expressed the recombinant proteins in E. coli and their biochemical characteristics were determined. UPTG1 and UPTG2 isoforms not only showed different kinetic properties but also a differential mRNA expression in potato plants was observed. While UPTG1 expression was found predominantly in stolons, tubers and roots, UPTG2 expression was high in all the analyzed tissues but mainly in stems and leaves. The analysis of the potato genomic DNA suggested the existence of new isoforms of UPTG, what in addition to the presence of homologue UPTG proteins showed that these autocatalytic glycosyltransferases constitute a small multigenic family present only in higher plants. The biochemical analysis of the UPTG glycosylation reaction showed that only one residue of glucose, xylose or galactose was bound to the enzyme in β configuration and that the carbohydrate behaved like N-glycosidically linked. Furthermore, when we analyzed the reversibility of the glycosylation reaction we found that in the presence of UDP, the glycosidic bond between UPTG and glucose was broken. The nature of the UPTG inhibitor in maize was studied. We found that the SS1 isoform of sucrose synthase that is expressed predominantly in the endosperm, was responsible for UPTG inhibition. This isoform was recently related to cell wall polysaccharide biosynthesis and its association with UPTG provides a new evidence that supports this hypothesis. The Thesis work showed that the potato UPTG can be phosphorylated in vitro. After phosphatase treatment, an increase in UPTG glycosylation was observed. Furthermore, the enzyme glycosylation promotes the appearance of high molecular weight polypeptides, which are immunologically related to UPTG. We think that UPTG dephosphorylation could stimulate its glycosylation favoring its association with other proteins or with itself. We suggest that UPTG may functionally associates to anchoring elements, yet unknown, that are present in Golgi membranes facilitating the UDP-sugars transport from the cytosol for the synthesis of pectins and hemicelluloses. We cannot rule out that UPTG dephosphorylation could favor its association to membranes what may involve a localization mechanism of the enzyme. The results related to the expression of UPTG mRNA, suggested that the enzyme might be mainly expressed in actively growing or elongating tissues. The fact that in elongating tissues there is an active synthesis of pectins and hemicelluloses in addition to the results presented here, represent a strong evidence that supports the hypothesis that UPTG is involved in the synthesis of noncellulosic polysaccharides that constitute the plant cell wall. Moreover, the existence of redundant UPTG isozymes points out that the enzyme may form part of a complex system essential for the viability of the plant cell.Fil:Wald, Flavia A.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Par-complex aPKC and Par3 cross-talk with innate immunity NF-κB pathway in epithelial cells

Summary Components of the Par-complex, atypical PKC and Par3, have been found to be downregulated upon activation of NF-κB in intestinal epithelial cells. To determine their possible role in pro-inflammatory responses we transduced Caco-2 human colon carcinoma cells with constitutively active (ca) PKCι or anti-Par3 shRNA-expressing lentiviral particles. Contrary to previous reports in other cell types, ca-PKCι did not activate, but rather decreased, baseline NF-κB activity in a luminiscence reporter assay. An identical observation applied to a PB1 domain deletion PKCι, which fails to localize to the tight-junction. Conversely, as expected, the same ca-PKCι activated NF-κB in non-polarized HEK293 cells. Likewise, knockdown of Par3 increased NF-κB activity and, surprisingly, greatly enhanced its response to TNFα, as shown by transcription of IL-8, GRO-1, GRO-2 and GRO-3. We conclude that aPKC and Par3 are inhibitors of the canonical NF-κB activation pathway, although perhaps acting through independent pathways, and may be involved in pro-inflammatory responses

Tumor Necrosis Factor Alpha and Inflammation Disrupt the Polarity Complex in Intestinal Epithelial Cells by a Posttranslational Mechanism▿

Inflammatory processes disrupt the barrier function in epithelia. Increased permeability often leads to chronic of inflammation. Important among other cytokines, tumor necrosis factor alpha (TNF-α) initiates an NF-κB-mediated response that leads to upregulation of myosin light chain kinase (MLCK), a hallmark of the pathogenesis of inflammatory bowel disease. Here, we found that two components of the evolutionarily conserved organizer of tight junctions and polarity, the polarity complex (atypical protein kinase C [aPKC]-PAR6-PAR3) were downregulated by TNF-α signaling in intestinal epithelial cells and also in vivo during intestinal inflammation. Decreases in aPKC levels were due to decreased chaperoning activity of Hsp70 proteins, with failure of the aPKC rescue machinery, and these effects were rescued by NF-κB inhibition. Comparable downregulation of aPKC shRNA phenocopied effects of TNF-α signaling, including apical nonmuscle myosin II accumulation and myosin light chain phosphorylation. These effects, including ZO-1 downregulation, were rescued by overexpression of constitutively active aPKC. We conclude that this novel mechanism is a complementary effector pathway for TNF-α signaling

p34cdc2-mediated phosphorylation mobilizes microtubule-organizing centers from the apical intermediate filament scaffold in CACO-2 epithelial cells

We have shown previously that centrosomes and other microtubule-organizing centers (MTOCs) attach to the apical intermediate filament (IF) network in CACO-2 cells. In this cell line, intermediate filaments do not disorganize during mitosis. Therefore, we speculated that the trigger of the G(2)-M boundary may also detach MTOCs from their IF anchor. If that was the case, at least one of the proteins involved in the attachment must be phosphorylated by p34(cdc2) (cdk1). Using confocal microscopy and standard biochemical analysis, we found that p34(cdc2)-mediated phosphorylation indeed released MTOCs from IFs in permeabilized cells. In isolated, immunoprecipitated multiprotein complexes containing both gamma-tubulin and cytokeratin 19, p34(cdc2) phosphorylated only one protein, and phosphorylation released cytokeratin 19 from the complexes. We conclude that this as yet unidentified protein is a part of the molecular mechanism that attaches MTOCs to IFs in interphase

Regulation of self-glycosylation of reversibly glycosylated polypeptides from Solarum tuberosum

Reversibly glycosylated polypeptides (RGPs) belong to a family of self-glycosylating proteins believed to be involved in plant polysaccharide synthesis. The precise function of these enzymes remains to be elucidated. Our results showed that the RGP 38-kDa subunit is phosphorylated in potato extracts (Solanum tuberosum L.). An increase in the self-glycosylation of Solanum tuberosum RGP (StRGP) 38-kDa subunit was observed after alkaline phosphatase (AP) treatment. Our results suggest that phosphorylation of StRGP appears to regulate its self-glycosylation. It was determined that when the StRGP reaction was carried out in the presence of UDP-[14C]Glc as the sugar donor and then 1 mM UDP was added in a chase-out experiment, radioactive UDP-Glc was obtained indicating that StRGP reaction seems to be reversible. The anomeric configuration of transferred sugars to StRGP protein was also studied.Fil: Testasecca, Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Wald, Flavia A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Cozzarin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Moreno, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Ezrin connects the apical F-actin scaffold to membrane proteins in the apical brush border of intestinal epithelial cells. Yet, the mechanisms that recruit ezrin to the apical domain remain obscure. Using stable CACO-2 transfectants expressing keratin 8 (K8) antisense RNA under a tetracycline-responsive element, we showed that the actin-ezrin scaffold cannot assemble in the absence of intermediate filaments (IFs). Overexpression of ezrin partially rescued this phenotype. Overexpression of K8 in mice also disrupted the assembly of the brush border, but ezrin distributed away from the apical membrane in spots along supernumerary IFs. In cytochalasin D-treated cells ezrin localized to a subapical compartment and coimmunoprecipitated with IFs. Overexpression of ezrin in undifferentiated cells showed a Triton-insoluble ezrin compartment negative for phospho-T567 (dormant) ezrin visualized as spots along IFs. Pulse-chase analysis showed that Triton-insoluble, newly synthesized ezrin transiently coimmunoprecipitates with IFs during the first 30 min of the chase. Dormant, but not active (p-T567), ezrin bound in vitro to isolated denatured keratins in Far-Western analysis and to native IFs in pull-down assays. We conclude that a transient association to IFs is an early step in the polarized assembly of apical ezrin in intestinal epithelial cells

Intermediate filaments: A role in epithelial polarity

Intermediate filaments have long been considered mechanical components of the cell that provide resistance to deformation stress. Practical experimental problems, including insolubility, lack of good pharmacological antagonists, and the paucity of powerful genetic models have handicapped the research of other functions. In single-layered epithelial cells, keratin intermediate filaments are cortical, either apically polarized or apico-lateral. This review analyzes phenotypes of genetic manipulations of simple epithelial cell keratins in mice and Caenorhabditis elegans that strongly suggest a role of keratins in apico-basal polarization and membrane traffic. Published evidence that intermediate filaments can act as scaffolds for proteins involved in membrane traffic and signaling is also discussed. Such a scaffolding function would generate a highly polarized compartment within the cytoplasm of simple epithelial cells. While in most cases mechanistic explanations for the keratin-null or overexpression phenotypes are still missing, it is hoped that investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments

Membrane repolarization is delayed in proximal tubules after ischemia-reperfusion: possible role of microtubule-organizing centers

We have previously shown that microtubule-organizing centers (MTOCs) attach to the apical network of intermediate filaments (IFs) in epithelial cells in culture and in epithelia in vivo. Because that attachment is important for the architecture of microtubules (MTs) in epithelia, we analyzed whether chemical anoxia in LLC-PK1 and CACO-2 cells or unilateral ischemia-reperfusion in rat kidney (performed under fluorane anesthesia) had an effect on the binding and distribution of MTOCs. In culture, we found that chemical anoxia induces MTOC detachment from IFs by morphological and biochemical criteria. In reperfused rat proximal tubules, noncentrosomal MTOCs were fully detached from the cytoskeleton and scattered throughout the cytoplasm at 3 days after reperfusion, when brush borders were mostly reassembled. At that time, MTs were also fully reassembled but, as expected, lacked their normal apicobasal orientation. Two apical membrane markers expressed in S2 and S3 segments were depolarized at the same stage. At 8 days after reperfusion, membrane polarity, MTOCs, and MTs were back to normal. Na+-K+-ATPase was also found redistributed, not to the apical domain but rather to an intracellular compartment, as described by others (Alejandro VS, Nelson W, Huie P, Sibley RK, Dafoe D, Kuo P, Scandling JD Jr., and Myers BD. Kidney Int 48: 1308–1315, 1995). The prolonged depolarization of the apical membrane may have implications in the pathophysiology of acute renal failure

Par-complex aPKC and Par3 cross-talk with innate immunity NF- B pathway in epithelial cells

Components of the Par-complex, atypical PKC and Par3, have been found to be downregulated upon activation of NF-κB in intestinal epithelial cells. To determine their possible role in pro-inflammatory responses we transduced Caco-2 human colon carcinoma cells with constitutively active (ca) PKCι or anti-Par3 shRNA-expressing lentiviral particles. Contrary to previous reports in other cell types, ca-PKCι did not activate, but rather decreased, baseline NF-κB activity in a luminiscence reporter assay. An identical observation applied to a PB1 domain deletion PKCι, which fails to localize to the tight-junction. Conversely, as expected, the same ca-PKCι activated NF-κB in non-polarized HEK293 cells. Likewise, knockdown of Par3 increased NF-κB activity and, surprisingly, greatly enhanced its response to TNFα, as shown by transcription of IL-8, GRO-1, GRO-2 and GRO-3. We conclude that aPKC and Par3 are inhibitors of the canonical NF-κB activation pathway, although perhaps acting through independent pathways, and may be involved in pro-inflammatory responses