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At least 469 records · Page 26Linked to original sources
PubMed2003

Visualizing caveolin-1 and HDL in cholesterol-loaded aortic endothelial cells.

Caveolae are vesicular invaginations of the plasma membranes that regulate signal transduction and transcytosis, as well as cellular cholesterol homeostasis. Our previous studies indicated that the removal of cholesterol from aortic endothelial cells and smooth muscle cells in the presence of HDL is associated with plasmalemmal invaginations and plasmalemmal vesicles. The goal of the present study was to investigate the location and distribution of caveolin-1, the main structural protein component of caveolae, in cholesterol-loaded aortic endothelial cells after HDL incubation. Confocal microscopic analysis demonstrated that the caveolin-1 appeared to colocalize with HDL-fluorescein 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) conjugates on the cell surface. No free HDL-DiI conjugates were revealed in the cytoplasm. Immunoelectron microscopy further demonstrated that caveolin-1 gold (15 nm) conjugates colocalized with HDL gold (10 nm) conjugates in the plasmalemmal invaginations. These morphological results indicated that caveolae are the major membrane domains facilitating the transport of excess cholesterol to HDL on the cell surface of aortic endothelial cells.

Animals↗
PubMed2005

Expression of serum amyloid A, in normal, dysplastic, and neoplastic human colonic mucosa: implication for a role in colonic tumorigenesis.

Serum amyloid A (SAA) is an acute phase reactant, whose level in the blood is elevated in response to trauma, infection, inflammation, and neoplasia. Elevated levels of SAA in the serum of cancer patients were suggested to be of liver origin rather than a tumor cell product. The role of SAA in human malignancies has not been elucidated. We investigated the expression of SAA at various stages of human colon carcinoma progression. Nonradioactive in situ hybridization applied on paraffin tissue sections from 26 colon cancer patients revealed barely detected SAA mRNA expression in normal looking colonic epithelium. Expression was increased gradually as epithelial cells progressed through dysplasia to neoplasia. Deeply invading colon carcinoma cells showed the highest levels of SAA. Expression was also found in colon carcinoma metastases. Cells of lymphoid follicles of the intestinal wall, inflammatory cells, ganglion cells, and endothelial cells, also expressed SAA mRNA. Immunohistochemical staining revealed SAA protein expression that colocalized with SAA mRNA expression. RT-PCR analysis confirmed the expression of the SAA1 and SAA4 genes in colon carcinomas, expression that was barely detectable in normal colon tissues. These findings indicate local and differential expression of SAA in human colon cancer tissues and suggest its role in colonic tumorigenesis.

Adenocarcinoma↗
PubMed2003

FUS/ERG gene fusions in Ewing's tumors.

Ewing's tumors are rare pediatric neoplasms that are characterized by specific chromosomal translocations and gene rearrangements. All of the fusion genes reported to date in Ewing's tumors juxtapose the EWS gene at 22q12 to an ETS-related gene, the most common of which are FLI1 at 11q24 and ERG at 21q22. We present here four cases of Ewing's tumor, which showed no evidence of a EWS gene rearrangement, but instead contained translocations involving 16p11 and 21q22. A rearrangement involving the same chromosome bands, t(16;21)(p11;q22), is found in rare cases of acute myeloid leukemia and fuses the FUS gene at 16p11 to the ERG gene at 21q22. In two of our Ewing's tumor cases, we were able to show at the sequence level that the translocation between chromosomes 16 and 21 similarly results in a FUS/ERG fusion. In one case, exons 1-5 and most of exon 6 of FUS were fused in-frame to exon 9 of ERG; in the other case, FUS exons 1-7 were fused in-frame to ERG exons 8-9. The functional fusion transcript is expected to be expressed from the der(21)t(16;21) derivative. In the two other t(16;21)-positive Ewing's cases, we performed bacterial artificial chromosome fluorescence in situ hybridization analysis on metaphases and interphase nuclei to demonstrate colocalization of bacterial artificial chromosomes containing FUS and ERG genes, also highly suggestive of fusion gene formation. These represent the first four cases where FUS, rather than EWS, is rearranged with an ETS-family transcription factor in Ewing's tumors. Our data provide additional evidence that the transactivation domains of the TET family of RNA-binding proteins (such as EWS and FUS) are interchangeable, and suggests a novel mechanism of oncogenesis in Ewing's tumors.

Adolescent↗
PubMed1997

Synaptojanin forms two separate complexes in the nerve terminal. Interactions with endophilin and amphiphysin.

Endophilin is a recently discovered src homology 3 domain-containing protein that is a major in vitro binding partner for synaptojanin. To further characterize endophilin, we generated an antipeptide antibody. Endophilin is enriched in the brain, and immunofluorescence analysis reveals a high concentration of the protein in synaptic terminals, where it colocalizes with synaptojanin. In vitro binding assays demonstrate that endophilin binds through its src homology 3 domain to synaptojanin, and immunoprecipitation analysis with the antiendophilin antibody reveals that endophilin is stably associated with synaptojanin in the nerve terminal. Immunoprecipitation with an antibody against amphiphysin I and II, which interact through their src homology 3 domains with dynamin and synaptojanin at sites distinct from those for endophilin, reveals a second stable complex, which includes dynamin and synaptojanin but excludes endophilin. These data demonstrate that synaptojanin is present in two separate complexes in the nerve terminal and support an important role for endophilin in the regulation of synaptojanin function.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Targeting of tissue plasminogen activator into the regulated secretory pathway of neuroendocrine cells.

Plasma levels of tissue plasminogen activator (tPA) increase rapidly in response to specific vasoactive agents, trauma, and neural stimulation. This response has been attributed to acute release of tPA from stored pools within the vascular endothelium and from catecholamine storage vesicles of chromaffin cells. We have tested directly whether tPA can be sorted into the regulated secretory pathway using the murine pituitary-derived neuroendocrine cell line AtT-20 transfected with tPA cDNA. Clones of AtT-20 cells expressing tPA were isolated, and targeting of tPA into the regulated secretory pathway was demonstrated by (1) stimulation of tPA secretion with 8-bromo-cAMP, the secretagogue which promotes the release of dense granule contents; (2) colocalization with ACTH, an endogenous protein that is stored in dense core granules; and (3) retention of newly synthesized tPA in the cell for prolonged periods of time. Laser scanning confocal microscopy analysis of cells immunostained with antibodies to tPA and ACTH showed colocalization at the tips of the neuritic processes under the cytoplasmic membrane, a region where dense granules are known to migrate after maturation. Treatment of the cells with 5 mM 8-bromo-cAMP for 30 min resulted in a 2.41+/-0.36-fold increase in tPA secretion. Both the magnitude of the stimulatory effect and the fraction of the intracellular tPA released were the same regardless of the tPA expression level in the various clones. Pulse-chase experiments showed that a portion of newly synthesized tPA is retained in the cell for at least 4 h and is released into the culture medium in response to 8-bromo-cAMP. These studies indicate that tPA, under the appropriate conditions, can be targeted into the regulated secretory pathway and can be stored for later release by cellular stimuli.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2006

The histone chaperone ASF1 localizes to active DNA replication forks to mediate efficient DNA replication.

The packaging of the eukaryotic genome into chromatin is likely to regulate all processes that occur on the DNA template. The assembly and disassembly of chromatin structures from histone proteins and DNA are mediated by histone chaperones, including the histone H3/H4 chaperone anti-silencing function 1 (ASF1). To address the function of ASF1 in metazoan cells, we used RNA interference-mediated knockdown of Drosophila melanogaster ASF1 (dASF1). Cells lacking dASF1 accumulate in S phase of the cell cycle as determined by flow cytometry analysis of DNA content and quantitation of the proportion of cells with replication foci. In agreement, bromodeoxyuridine (BrdU) pulse-chase analysis demonstrates that the absence of ASF1 leads to delayed progression through S-phase. Furthermore, the absence of ASF1 leads to a reduced ability to incorporate the nucleoside analog BrdU, indicating that ASF1 is required for efficient DNA replication. We have also found that dASF1 colocalizes with DNA replication foci throughout S phase by immunofluorescence analysis and that these dASF1 foci are disrupted upon inhibition of DNA replication by treatment of cells with hydroxyurea. As such, these results demonstrate that dASF1 is present at active, but not stalled, replication forks. We propose that dASF1 has a direct role in modifying chromatin structure during DNA replication and that this function of dASF1 is important for the processivity of the replication machinery.

Animals↗
PubMed2002

Connexin family members target to lipid raft domains and interact with caveolin-1.

Lipid rafts are cholesterol-sphingolipid-rich microdomains that function as platforms for membrane trafficking and signal transduction. Caveolae are specialized lipid raft domains that contain the structural proteins known as the caveolins. Connexins are a family of transmembrane proteins that self-associate to form cell-cell connections known as gap junctions and that are linked to cytosolic proteins, forming a protein complex or Nexus. To determine the extent to which these intracellular compartments intersect, we have systematically evaluated whether connexins are associated with lipid rafts and caveolin-1. We show that connexin 43 (Cx43) colocalizes, cofractionates, and coimmunoprecipitates with caveolin-1. A mutational analysis of Cx43 reveals that the hypothesized PDZ- and presumptive SH2/SH3-binding domains within the Cx43 carboxyl terminus are not required for this targeting event or for its stable interaction with caveolin-1. Furthermore, Cx43 appears to interact with two distinct caveolin-1 domains, i.e., the caveolin-scaffolding domain (residues 82-101) and the C-terminal domain (135-178). We also show that other connexins (Cx32, Cx36, and Cx46) are targeted to lipid rafts, while Cx26 and Cx50 are specifically excluded from these membrane microdomains. Interestingly, recombinant coexpression of Cx26 with caveolin-1 recruits Cx26 to lipid rafts, where it colocalizes with caveolin-1. This trafficking event appears to be unique to Cx26, since the other connexins investigated in this study do not require caveolin-1 for targeting to lipid rafts. Our results provide the first evidence that connexins interact with caveolins and partition into lipid raft domains and indicate that these interactions are connexin specific.

3T3 Cells↗
PubMed2003

Molecular biology and electrophysiology of neuronal nicotinic receptors of rat chromaffin cells.

Neuronal nicotinic acetylcholine receptors of chromaffin cells in the adrenal medulla are physiologically activated by acetylcholine to mediate catecholamine release into the bloodstream. The present study examined the subunit composition and functional properties of rat chromaffin cell neuronal nicotinic acetylcholine receptors using molecular biology, immunocytochemistry and whole-cell patch-clamp. Reverse transcription-polymerase chain reaction analysis indicated the presence of alpha2, alpha3, alpha4, alpha5, alpha7, beta2 and beta4 transcripts (alpha6 and beta3 could not be detected). Immunocytochemistry revealed most cells positive for alpha3, beta2, beta4 and alpha5 proteins. Few cells were immunoreactive for alpha2 and alpha4, while none was for alpha7. At single-cell level, colocalization could be demonstrated for alpha3alpha5 and alpha4beta2. Western blot analysis confirmed antibody specificity for alpha3, alpha4, alpha5, beta2 and beta4 subunits. Inward currents elicited by nicotine pulses were insensitive to alpha-bungarotoxin and low doses of methyllycaconitine, demonstrating lack of functional alpha7 receptors. Partial block of nicotine currents was observed with either AuIB alpha-conotoxin (selective against alpha3beta4 receptors) or MII alpha-conotoxin (selective against alpha3beta2 receptors). With high concentrations of co-applied toxins, antagonism occlusion developed, suggesting loss of subunit selectivity. Antagonism by dihydro-beta-erythroidine summated nonlinearly with AuIB and MII inhibition, confirming heterogeneity of neuronal nicotinic acetylcholine receptor block. The present results suggest that the most frequently encountered receptors of rat chromaffin cells should comprise alpha3beta4, alpha3beta2 with the addition of alpha5 subunits. Because of the prevailing subunit composition, rat chromaffin cell neuronal nicotinic acetylcholine receptors are suitable models, particularly for the alpha3beta4 subclasses of mammalian brain receptors recently demonstrated in discrete cerebral areas.

Adrenal Glands↗
PubMed2002

Plasminogen activator inhibitor-1 promotes formation of endothelial microparticles with procoagulant potential.

BACKGROUND: Endothelial dysfunction is emerging as a common denominator for diverse and highly prevalent cardiovascular diseases. Increased level of plasminogen activator inhibitor-1 (PAI-1) and procoagulant activity have been recognized as hallmarks of endothelial dysfunction. This study was aimed at investigating cellular actions of PAI-1 and a potential link between PAI-1 and procoagulant state. METHODS AND RESULTS: Human umbilical vein endothelial cells treated with PAI-1 were subjected to laser confocal fluorescence microscopy, immunoprecipitation and Western blotting, and FACS analysis for isolation and identification of endothelial microparticles. PAI-1 treatment resulted in a reduced expression of uPAR, its colocalization with caveolin, and the concomitant increase of uPAR abundance in the culture medium. FACS analysis revealed that PAI-1 rapidly and dose-dependently increased the number of endothelial microparticles expressing uPAR and alpha(V)beta3 integrin. This process was attenuated by pretreatment with neutralizing anti-uPAR antibodies. PAI-1 knockout mice showed a significantly decreased number of circulating endothelial microparticles than wild-type mice; however, PAI-1-deficient animals responded to infusion of PAI-1 with a more pronounced rise in the number of microparticles. PAI-1 treatment increased the number of microparticles stained with Annexin V, evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin. CONCLUSIONS: The data disclose a novel effect of PAI-1 to dose-dependently promote formation of endothelial microparticles with the reduced transmembrane asymmetry of phospholipids. This phenomenon may be responsible for the observed increase in in vitro thrombin generation. These findings could potentially link these hallmarks of endothelial dysfunction-elevated levels of PAI-1 and propensity toward thrombosis.

Animals↗
PubMed2005

Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction.

BACKGROUND: We used a molecular probe activated by protease cleavage to image expression of matrix metalloproteinases (MMPs) in the heart after myocardial infarction. METHODS AND RESULTS: We synthesized and characterized a near-infrared fluorescent (NIRF) probe that is activated by proteolytic cleavage by MMP2 and MMP9. The NIRF probe was injected into mice at various time points up to 4 weeks after myocardial infarction induced by ligation of the left anterior descending coronary artery. NIRF imaging of MMP activity increased in the infarct region, with maximal expression at 1 to 2 weeks, persisting to 4 weeks. Zymography and real-time polymerase chain reaction analysis showed that MMP9 expression is increased at 2 to 4 days, and MMP2 expression is increased at 1 to 2 weeks. Dual-label confocal microscopy showed colocalization of NIRF imaging with neutrophils on day 2, and flow cytometric analysis confirmed that NIRF signal is associated with leukocytes in the infarct zone. CONCLUSIONS: This study demonstrates that the activity of MMPs in the myocardium may be imaged by use of specific activity-dependent molecular probes.

Animals↗
PubMed2026

Genetic overlap between depression and C-reactive protein levels: Evidence from a cross-trait analysis.

Inflammation and depression have been consistently associated, with elevated C-reactive protein (CRP) levels observed in a significant subset of affected individuals. However, the genetic mechanisms underlying this association remain poorly understood. We integrated results from large-scale genome-wide association studies (GWAS) of depression and CRP levels in a cross-trait analysis specifically focusing on identifying horizontally pleiotropic loci. Identified variants were stratified as concordant versus discordant based on their direction of effects on the two traits and followed up using functional annotation, gene set enrichment, and colocalization analyses. We also explored causal relationships using Mendelian Randomization (MR) analysis with extensive sensitivity analyses, including adjustment for body mass index (BMI). We identified 9 novel loci. Functional analyses revealed that concordant loci were enriched in genes linked to immune and inflammatory processes, while discordant loci mostly mapped to metabolic pathways, including lipid regulation. MR provided strong evidence for body mass index driving a causal relationship between the genetic liability of depression on CRP levels. Our findings suggest that the association between depression and CRP levels is partly driven by shared genetic influences, pointing to different biological pathways depending on whether genetic effects are concordant or discordant. These results underscore the importance of considering effect direction when assessing the genetic overlap between depression and inflammatory processes. In addition, they highlight BMI as a key factor in the causal relationship between depression and systemic inflammation.

C-Reactive Protein↗
PubMed2026

Decoding Primary Open-Angle Glaucoma: A Multi-Omics Approach to Identify Druggable Effector Genes.

PURPOSE: Genomewide association studies (GWAS) have identified numerous primary open angle glaucoma (POAG) risk loci, yet most reside in non-coding regions with unclear function. Mapping these loci to effector genes can elucidate disease mechanisms, identify functionally conserved variants, improve cross-ancestry risk prediction by reducing population-specific noise, and uncover shared therapeutic targets. METHODS: Here, we integrate European POAG GWAS with six types of multi-omics molecular Quantitative Trait Locis (xQTLs) using multi-trait colocalization to identify candidate effector variants and evaluate their cross-population relevance using genetic risk score (GRS) analysis, and their therapeutic potential through drug target prioritization. RESULTS: We identified 25 POAG effector variants colocalized with at least one xQTLs. In non-European populations, effector variants showed stronger effect size correlations with Europeans than non-colocalized variants (Pearson r2 = African 0.85 vs. 0.71; East Asian 0.81 vs. 0.69; and Latin American 0.91 vs. 0.75). Effector variants also had smaller allele frequency variations across populations (average interquartile range [IQR] = 0.15 vs. 0.20). The genetic risk score based on effector variants performed comparably to the genome-wide significant single-nucleotide polymorphism (SNP)-based GRS in non-European populations. Drug prioritization identified zinc, copper, sunitinib, probucol, and astemizole as potential common therapeutic agents for POAG and its subtypes. CONCLUSIONS: Our findings offer deeper insight into the molecular mechanisms underlying glaucoma and effector variants for developing more robust GRS models and broadly effective therapeutic strategies for POAG.

Humans↗
PubMed1996

Pericytes as collagen-producing cells in excessive dermal scarring.

Immunohistochemistry and image analysis were performed on sections from excessive dermal scar formation to investigate the potential of pericytes to differentiate into collagen-producing cells. Expression of the prolyl-4-hydroxylase beta-subunit (P-4-H) was used as a marker for collagen synthesis as the distribution of this protein was identical to the distribution of procollagen type I C-propeptide and similar to the distribution of cells expressing pro alpha 1(I) collagen mRNA. Double immunofluorescence stainings using combinations of monoclonal antibodies specific for activated pericytes in vivo (high molecular weight-melanoma associated antigen (HMW-MAA)), P-4-H, smooth muscle alpha-actin (SMA), endothelial cells (PAL-E), platelet-derived growth factor (PDGF) beta-receptor, and the integrin alpha 5 subunit were performed. Stained sections were analyzed by computerized image analysis allowing for a quantification of the degree of colocalization between pairs of antigens on the same tissue section. Four different subpopulations of HMW-MAA expressing cells were discerned. The first subpopulation corresponded to intramural pericytes, juxtapositioned to the endothelium, that expressed HMW-MAA, SMA, integrin alpha 5 subunit and the PDGF beta-receptor, but not P-4-H. The second subpopulation was partly dissociated from the microvascular wall and exhibited a similar antigen expression except for a decrease in expression of SMA. Cells in the third subpopulation were located in the perivascular space and expressed P-4-H, integrin alpha 5 subunit, the PDGF beta-receptor and, albeit less pronounced, HMW-MAA, but not SMA. The fourth subpopulation expressed integrin alpha 5 subunit, HMW-MAA and the PDGF beta-receptor, no expression of SMA and a strong expression of P-4-H. Moreover, an in vitro analysis of cells derived from isolated microvascular fragments from human dermis revealed a similar pattern of phenotypical change. Taken together the data suggest that a population of intramural pericytes migrate into the perivascular space and develop into collagen-synthesizing fibroblasts during fibrosis.

Basement Membrane↗
PubMed2005

Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

Cell Line, Tumor↗
PubMed1991

Differential mechanisms for the N-acetylation of alpha-melanocyte-stimulating hormone and beta-endorphin in the intermediate pituitary of the frog, Xenopus laevis.

Immunohistochemical analysis of the pituitary of Xenopus laevis revealed the colocalization of alpha-melanocyte-stimulating-hormone (MSH)-related immunoreactivity and N-acetyl-beta-endorphin-related immunoreactivity in the cells of the intermediate pituitary. In order to determine whether the immunoreactive N-acetylated beta-endorphin is released in parallel with the immunoreactive alpha-MSH, intermediate pituitaries were incubated in L-15 medium for 24 h. The medium and an acid extract of the intermediate pituitaries from each incubation were separately fractioned by a combination of gel filtration chromatography, reverse-phase high-performance liquid chromatography, and cation exchange chromatography. In the intermediate pituitary extract, the major form of alpha-MSH had chromatographic properties which corresponded to nonacetylated alpha-MSH (ACTH)(1-13)amide; whereas the major form of beta-endorphin had an apparent molecular weight of 1.2 kDa and was N-acetylated. The 1.2-kDa form of beta-endorphin and ACTH(1-13)amide were present in equimolar amounts. Analysis of the medium indicated that both end products were released in parallel. However, as reported in the literature, there was a significant increase in the N-acetylation of ACTH(1-13)amide during secretion. There was no further processing of beta-endorphin during secretion. Collectively, these observations indicate that in the intermediate pituitary of X. laevis there are separate mechanisms for the N-acetylation of alpha-MSH and beta-endorphin.

Acetylation↗
PubMed2007

Somatic microindels: analysis in mouse soma and comparison with the human germline.

Microindels, defined as mutations that result in a colocalized microinsertion and microdeletion with a net gain or loss of between 1 and 50 nucleotides, may be an important contributor to cancer. We report the first comprehensive analysis of somatic microindels. Our large database of mutations in the lacI transgene of Big Blue((R)) mice contains 0.5% microindels, 2.8% pure microinsertions, and 11.5% pure microdeletions. There appears to be no age, gender, or tissue-type specificity in the frequency of microindels. Of the independent somatic mutations that result in a net in-frame insertion or deletion, microindels are responsible for 13% of protein expansions and 6% of protein contractions. These in-frame microindels may play a crucial role in oncogenesis and evolution via "protein tinkering" (i.e., modest expansion or contraction of proteins). Four characteristics suggest that microindels are caused by unique mechanisms, not just simple combinations of the same mechanisms that cause pure microinsertions and pure microdeletions. First, microinsertions and microdeletions commonly occur at hotspots, but none of the 30 microindels are recurrent. Second, the sizes of the deletions and insertions in microindels are larger and more varied than in pure microdeletions and pure microinsertions. Third, microinsertions overwhelmingly repeat the adjacent base (97%) while the insertions in microindels do so only infrequently (17%). Fourth, analysis of the sequence contexts of microindels is consistent with unique mechanisms including recruitment of translesion DNA synthesis polymerases. The mouse somatic microindels have characteristics similar to those of human germline microindels, consistent with similar causative mechanisms in mouse and human, and in soma and germline.

Age Factors↗
PubMed1994

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

Amino Acid Sequence↗
PubMed2004

Serum amyloid A-activating factor-1 (SAF-1) transgenic mice are prone to develop a severe form of inflammation-induced arthritis.

The transcription factor serum amyloid A-activating factor-1 (SAF-1) has been identified as a regulator of a number of cellular genes. To assess the pleiotropic role of SAF-1 in vivo, we generated SAF-1 transgenic mice, in which CMV immediate-early promoter was used to direct expression of the SAF-1 transgene in multiple organs. Our study shows that overexpression of SAF-1 predisposes animals to arthritis. Although SAF-1 transgenic mice do not spontaneously develop arthritis, they develop a severe form of arthritis when challenged with the Lyme disease agent Borrelia burgdorferi, which is known to promote arthritis development in both humans and mice. CMV-SAF-1 transgenic mice, upon B. burgdorferi infection, showed increased joint swelling and synovial inflammation compared with nontransgenic littermates. Immunohistochemical analysis of joint tissues collected 21 days after B. burgdorferi infection revealed colocalization of matrix metalloproteinase-1, a degradative enzyme that destroys type II collagen, a major architectural component of articular cartilage, and SAF-1 in both SAF-1 transgenic and nontransgenic mice. Further analysis by RNase protection assay and Western immunoblot demonstrated the presence of higher levels of matrix metalloproteinase-1 and SAF-1 in the inflamed joints of SAF-1 transgenic mice compared with their levels in nontransgenic mice. Consistent with these findings, reduced levels of proteoglycans were detected in the inflamed joint cartilage of transgenic mice, indicating damage to the cartilage structure. Together these results suggest a role of SAF-1 in the pathogenesis of inflammation-induced arthritis.

Animals↗