Visualizing CD4 T-cell migration into inflamed skin and its inhibition by CCR4/CCR10 blockades using in vivo imaging model.

BACKGROUND: Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo. OBJECTIVES: To visualize directly T-cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy. MATERIALS AND METHODS: A mouse model of allergic contact dermatitis was used. T cells from oxazolone-sensitized and -challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c-SCID mice. The recipient mice were challenged with oxazolone and CD4 T-cell homing into inflamed skin was visualized. RESULTS: T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T-cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T-cell migration and skin inflammation. CONCLUSIONS: Our data indicate that these tissue-selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T-cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.

Genetic segregation of spontaneous erosive arthritis and generalized autoimmune disease in the BXD2 recombinant inbred strain of mice.

The BXD2 strain of mice is one of approximately 80 BXD recombinant inbred (RI) mouse strains derived from an intercross between C57BL/6J (B6) and DBA/2J (D2) strains. We have discovered that adult BXD2 mice spontaneously develop generalized autoimmune disease, including glomerulonephritis (GN), increased serum titres of rheumatoid factor (RF) and anti-DNA antibody, and a spontaneous erosive arthritis characterized by mononuclear cell infiltration, synovial hyperplasia, and bone and cartilage erosion. The features of lupus and arthritis developed by the BXD2 mice segregate in F2 mice generated by crossing BXD2 mice with the parental B6 and D2 strains. Genetic linkage analysis of the serum levels of anti-DNA and RF by using the BXD RI strains shows that the serum titers of anti-DNA and RF were influenced by a genetic locus on mouse chromosome (Chr) 2 near the marker D2Mit412 (78 cm, 163 Mb) and on Chr 4 near D4Mit146 (53.6 cm, 109 Mb), respectively. Both loci are close to the B-cell hyperactivity, lupus or GN susceptibility loci that have been identified previously. The results of our study suggest that the BXD2 strain of mice is a novel model for complex autoimmune disease that will be useful in identifying the mechanisms critical for the immunopathogenesis and genetic segregation of lupus and erosive arthritis.

Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration.

Nitrotyrosine formation is a hallmark of vascular inflammation, with polymorphonuclear neutrophil-derived (PMN-derived) and monocyte-derived myeloperoxidase (MPO) being shown to catalyze this posttranslational protein modification via oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (NO(2)(*)). Herein, we show that MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism and serves as a catalyst of ECM protein tyrosine nitration. Purified MPO and MPO released by intraluminal degranulation of activated human PMNs avidly bound to aortic endothelial cell glycosaminoglycans in both cell monolayer and isolated vessel models. Cell-bound MPO rapidly transcytosed intact endothelium and colocalized abluminally with the ECM protein fibronectin. In the presence of the substrates hydrogen peroxide (H(2)O(2)) and NO(2)(-), cell and vessel wall-associated MPO catalyzed nitration of ECM protein tyrosine residues, with fibronectin identified as a major target protein. Both heparin and the low-molecular weight heparin enoxaparin significantly inhibited MPO binding and protein nitrotyrosine (NO(2)Tyr) formation in both cultured endothelial cells and rat aortic tissues. MPO(-/-) mice treated with intraperitoneal zymosan had lower hepatic NO(2)Tyr/tyrosine ratios than did zymosan-treated wild-type mice. These data indicate that MPO significantly contributes to NO(2)Tyr formation in vivo. Moreover, transcytosis of MPO, occurring independently of leukocyte emigration, confers specificity to nitration of vascular matrix proteins.

Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease.

Plasma xanthine oxidase (XO) activity was defined as a source of enhanced vascular superoxide (O(2)( *-)) and hydrogen peroxide (H(2)O(2)) production in both sickle cell disease (SCD) patients and knockout-transgenic SCD mice. There was a significant increase in the plasma XO activity of SCD patients that was similarly reflected in the SCD mouse model. Western blot and enzymatic analysis of liver tissue from SCD mice revealed decreased XO content. Hematoxylin and eosin staining of liver tissue of knockout-transgenic SCD mice indicated extensive hepatocellular injury that was accompanied by increased plasma content of the liver enzyme alanine aminotransferase. Immunocytochemical and enzymatic analysis of XO in thoracic aorta and liver tissue of SCD mice showed increased vessel wall and decreased liver XO, with XO concentrated on and in vascular luminal cells. Steady-state rates of vascular O(2)( *-) production, as indicated by coelenterazine chemiluminescence, were significantly increased, and nitric oxide (( *)NO)-dependent vasorelaxation of aortic ring segments was severely impaired in SCD mice, implying oxidative inactivation of ( *)NO. Pretreatment of aortic vessels with the superoxide dismutase mimetic manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin markedly decreased O(2)( small middle dot-) levels and significantly restored acetylcholine-dependent relaxation, whereas catalase had no effect. These data reveal that episodes of intrahepatic hypoxia-reoxygenation associated with SCD can induce the release of XO into the circulation from the liver. This circulating XO can then bind avidly to vessel luminal cells and impair vascular function by creating an oxidative milieu and catalytically consuming (*)NO via O(2)( small middle dot-)-dependent mechanisms.

Molecular cloning of pituitary tumor transforming gene 1 from ovarian tumors and its expression in tumors.

Pituitary tumor transforming gene 1 (PTTG1) recently cloned from human testis is a potent oncogene and is highly expressed in all the tumors analyzed to date. However, primary structure of PTTG1 and the cell types that express PTTG1 in tumors remained undescribed. We have used the reverse transcriptase-polymerase chain reaction technique to clone PTTG1 from ovarian tumors. Nucleotide sequencing of the PTTG1 cDNAs from various ovarian tumors showed identity with that of the human testis PTTG1. To determine the cell types that express PTTG1 in normal and tumor tissues, we performed in situ hybridization using digoxigenin-labeled cRNA as a probe. Our studies revealed a high level of expression of PTTG1 mRNA in both seminomatous and non-seminomatous testicular tumors; epithelial, sex-cord and stromal cell, and germ cell tumors of the ovary; and invasive ductal, ductal in situ and infiltrating ductal carcinoma of the breast. In normal tissues, expression of PTTG1 mRNA was very low or undetectable except in testis, where PTTG1 mRNA was found to be localized to spermatocytes and spermatids. Tumors that expressed high levels of PTTG1 mRNA also exhibited high levels of expression of basic fibroblast growth factor (bFGF), suggesting a correlation between PTTG1 and bFGF expression, and further suggesting that the PTTG1 protein may be involved in tumor angiogenesis and mitogenesis.

CXC chemokine receptor 4 expression and function in human astroglioma cells.

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.

Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes.

Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine alpha-ENaC bound to the plasma membrane of normal and Liddle's lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 microM 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddle's cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P(open) was 4.2 +/- 3.9% for patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddle's lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddle's disease induces excessive channel expression.

Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers.

Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl(-) current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O(2) produced similar improvements in Cl(-) current and CFTR protein as air-liquid interface culture, while increasing PO(2) from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking.

Ca(2+)-activated Cl(-) channels: a newly emerging anion transport family.

A new family of chloride transport proteins has recently emerged. These proteins have extensive homology to a protein previously isolated from bovine tracheal epithelium that acts as a Ca(2+)-sensitive Cl(-) channel (CaCC) when heterologously expressed or when reconstituted into planar lipid bilayers. Several new members of this family have been identified in human, murine, and bovine epithelia, in addition to some other tissues, and are associated with Ca(2+)-sensitive conductive chloride transport when heterologously expressed in Xenopus oocytes or HEK 293 cells. The expressed current is also sensitive to inhibitors such as DIDS and niflumic acid. In addition, at least one family member acts as an endothelial cell adhesion molecule. This emerging family may underlie the Ca(2+)-mediated Cl(-) conductance responsible for rescue of the cystic fibrosis (CF) knockout mouse from significant airway disease.

Regulation of epithelial Na(+) channels by actin in planar lipid bilayers and in the Xenopus oocyte expression system.

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.

Differential expression of platelet-derived growth factor-alpha receptor by Thy-1(-) and Thy-1(+) lung fibroblasts.

Fibroblasts are heterogeneous with respect to surface markers, morphology, and participation in fibrotic responses. This study was undertaken to determine whether Thy-1(-) and Thy-1(+) rat lung fibroblasts, which have distinct and relevant phenotypes, differ in their proliferative responses to platelet-derived growth factor (PDGF) isoforms. Homogeneous populations of Thy-1(-) and Thy-1(+) fibroblasts were found to proliferate equally in the presence of PDGF-BB, but PDGF-AA-mediated proliferation occurred only in Thy-1(-) cells. This differential activity correlated with significantly higher expression of PDGF-alpha receptor in Thy-1(-) fibroblasts as shown by immunoblotting, immunofluorescence, and Northern blotting. There was a rapid increase in c-myc mRNA in Thy-1(-) but not in Thy-1(+) fibroblasts on stimulation with PDGF-AA and PDGF-BB. The PDGF-alpha receptor, which mediates signaling by all PDGF isoforms, has been implicated in numerous clinical and experimental forms of fibrosis and regulates lung morphogenesis. Differential expression of the PDGF-alpha receptor supports distinct roles for Thy-1(-) and Thy-1(+) fibroblast populations in developmental and fibrotic processes in the lung.

The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel constructed from two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBD) and a regulatory (R) domain. The NBDs and R-domain are cytosolic and how they are assembled with the MSDs to achieve the native CFTR structure is not clear. Human DnaJ 2 (Hdj-2) is a co-chaperone of heat shock cognate 70 (Hsc70) which is localized to the cytosolic face of the ER. Whether Hdj-2 directs Hsc70 to facilitate the assembly of cytosolic regions on CFTR was investigated. We report that immature ER forms of CFTR and DeltaF508 CFTR can be isolated in complexes with Hdj-2 and Hsc70. The DeltaF508 mutation is localized in NBD1 and causes the CFTR to misfold. Levels of complex formation between DeltaF508 CFTR and Hdj-2/Hsp70 were approximately 2-fold higher than those with CFTR. The earliest stage at which Hdj-2/Hsc70 could bind CFTR translation intermediates coincided with the expression of NBD1 in the cytosol. Interestingly, complex formation between Hdj-2 and nascent CFTR was greatly reduced after expression of the R-domain. In experiments with purified components, Hdj-2 and Hsc70 acted synergistically to suppress NBD1 aggregation. Collectively, these data suggest that Hdj-2 and Hsc70 facilitate early steps in CFTR assembly. A putative step in the CFTR folding pathway catalyzed by Hdj-2/Hsc70 is the formation of an intramolecular NBD1-R-domain complex. Whether this step is defective in the biogenesis of DeltaF508 CFTR will be discussed.

Characterization of CFTR expression and chloride channel activity in human endothelia.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a low-conductance, cAMP-regulated chloride (Cl-) channel in a variety of cell types, such as exocrine epithelial cells. Our results demonstrate that human primary endothelial cells isolated from umbilical vein (HUVEC) and lung microvasculature (HLMVEC) also express CFTR as determined via RT-PCR and immunohistochemical and immunoprecipitation analyses. Moreover, Cl- efflux and whole cell patch-clamp analyses reveal that HUVEC (n = 6 samples, P < 0.05) and HLMVEC (n = 5 samples, P < 0.05) display cyclic nucleotide-stimulated Cl- transport that is inhibited by the CFTR selective Cl- channel blocker glibenclamide but not by the blocker DIDS, indicative of CFTR Cl- channel activity. Taken together, these findings demonstrate that human endothelial cells derived from multiple organ systems express CFTR and that CFTR functions as a cyclic nucleotide-regulated Cl- channel in human endothelia.

Microtubule disruption inhibits AVT-stimulated Cl- secretion but not Na+ reabsorption in A6 cells.

The effects of microtubule disruption on arginine vasotocin (AVT)-stimulated Na+ and Cl- transport were studied in A6 cells by measuring short-circuit currents (Isc) across cell layers grown in tissue culture on permeable supports. Microtubule disruption inhibited an AVT-stimulated secretory Cl- current but did not prevent activation of amiloride-sensitive Na+ transport. This AVT-stimulated secretory Cl- current was significantly inhibited by glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). Reverse transcription of RNA isolated from A6 cells followed by polymerase chain reaction (PCR) using primers designed to amplify a portion of the R-domain of CFTR cloned from Xenopus laevis skin and immunocytochemistry demonstrated the presence of CFTR in A6 cells and an apparent recruitment of cytoplasmic CFTR to the apical cell surface after AVT stimulation. In contrast, indirect immunofluorescent labeling of Na+ channels using a polyclonal antibody raised against a biochemically isolated Na+ channel complex from bovine renal medulla labeled the apical plasma membrane but failed to demonstrate intracellular labeling of Na+ channels (except in subconfluent cells) or recruitment of Na+ channels to the apical membrane region after AVT stimulation.

Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. While 70% of CF chromosomes carry a deletion of the phenylalanine residue 508 (deltaF508) of CFTR, roughly 5% of all CF chromosomes carry a premature stop mutation. We reported that the aminoglycoside antibiotics G-418 and gentamicin can suppress two premature stop mutations [a stop codon in place of glycine residue 542 (G542X) and arginine residue 553 (R553X)] when expressed from a CFTR cDNA in HeLa cells. Suppression resulted in the synthesis of full-length CFTR protein and the appearance of a cAMP-activated anion conductance characteristic of CFTR function. However, it was unclear whether this approach could restore CFTR function in cells expressing mutant forms of CFTR from the nuclear genome. We now report that G-418 and gentamicin are also capable of restoring CFTR expression in a CF bronchial epithelial cell line carrying the CFTR W1282X premature stop mutation (a stop codon in place of tryptophan residue 1282). This conclusion is based on the reappearance of cAMP-activated chloride currents, the restoration of CFTR protein at the apical plasma membrane, and an increase in the abundance of CFTR mRNA levels from the W1282X allele.

Regulation of a cloned epithelial Na+ channel by its beta- and gamma-subunits.

Using the Xenopus oocyte expression system, we examined the mechanisms by which the beta- and gamma-subunits of an epithelial Na+ channel (ENaC) regulate alpha-subunit channel activity and the mechanisms by which beta-subunit truncations cause ENaC activation. Expression of alpha-ENaC alone produced small amiloride-sensitive currents (-43 +/- 10 nA, n = 7). These currents increased > 30-fold with the coexpression of beta- and gamma-ENaC to -1,476 +/- 254 nA (n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the alpha-subunit of ENaC. Truncation of the last 75 amino acids of the beta-subunit COOH terminus, as found in the original pedigree of individuals with Liddle's syndrome, caused a 4.4-fold (n = 17) increase of the amiloride-sensitive currents compared with wild-type alpha beta gamma-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human beta-ENaC significantly reduced the amiloride-sensitive currents by 40-50%. These observations suggest a tonic inhibitory role on the channel's open probability (Po) by the COOH terminus of beta-ENaC. We conclude that the changes of current observed with coexpression of the beta- and gamma-subunits or those observed with beta-subunit truncation are likely the result of changes of channel density in combination with large changes of Po.

Apical recruitment of CFTR in T-84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments.

Previous studies from this laboratory have demonstrated that chloride transport induced by forskolin, but not ionomycin, in T84 cells is highly dependent on an intact microtubular network. Using an antibody raised against a region of the R domain of CFTR, we now show by indirect immunofluorescence that forskolin causes relocation of CFTR to the apical domain of T84 cells. T84 cells grown on transparent filters were incubated with agonists and/or cytoskeletal inhibitors prior to fixation, permeabilization, and staining with the antibody. A 30 second stimulation with forskolin (10 microM) caused a twofold increase in relative fluorescence intensity at the apical surface. In contrast, a 30 second exposure to ionomycin (2 microM), had no effect on the distribution of CFTR-related fluorescence. Incubation of the cells with nocodazole (33 microM), a microtubule disrupting agent, prevented the forskolin-induced rise in CFTR fluorescence at the apical surface. However, incubation of the cells with cytochalasin D, an actin inhibitor, was without effect on forskolin-related re-distribution of CFTR-associated fluorescence. In double label experiments using antibodies against both beta-tubulin and actin, CFTR-related fluorescence was found to co-localize with the microtubule network, but not with actin filaments. These observations are consistent with the microtubule-dependent acute recruitment of CFTR to the apical plasma membrane of T84 cells in response to elevations in intracellular cAMP.

Down-regulation of secretion of human complement component C2 by the product of an alternatively spliced C2 messenger RNA.

We have previously described alternatively spliced transcripts of the human C2 gene. Among those, C2delta(17), in which exon 17 has been spliced out, encodes a polypeptide that contains the C4b binding and the CIs cleavage sites of C2, but lacks the serine protease active center. To study the possible function of this variant, we constructed C2delta(17) cDNA by deletional mutagenesis and expressed it transiently in COS cells. Transfected COS cells secreted only trace amounts of the C2delta(17) polypeptide, which had no detectable hemolytic activity and could not be cleaved by CIs. Pulse-chase experiments using [35S]methionine demonstrated that the majority of the 88-kDa C2delta(17) remained intracellular. Control wild-type (wt) C2 in the intracellular compartment consisted of two bands of 93 and 99 kDa, the latter corresponding to mature secreted C2. Intracellular C2delta(17) and only the 93-kDa wt C2 were sensitive to endoglycosidase H, a marker for transport from the endoplasmic reticulum (ER) to the Golgi. Experiments using brefeldin A and double label immunofluorescence staining indicated that C2delta(17) exhibited a typical ER distribution pattern, while wt C2 accumulated in the ER-Golgi intermediate compartment. Secretion of C2 by COS cells cotransfected with wt C2 and C2delta(17) cDNA was significantly decreased compared with that by cells transfected with wt C2 alone. These combined results indicate that C2delta(17) is retained in the ER probably because it is incorrectly folded and that it could down-regulate the expression of the wt C2 gene.

Expression and polarized targeting of a rab3 isoform in epithelial cells.

Pathways of polarized membrane traffic in epithelial tissues serve a variety of functions, including the generation of epithelial polarity and the regulation of vectorial transport. We have identified a candidate regulator of polarized membrane traffic in epithelial cells (i.e., rab3B), which is a member of the rab family of membrane traffic regulators. Rab3B is highly homologous to a brain-specific rab3 isoform (rab3A) that targets in a polarized fashion to the presynaptic nerve terminal, where it probably regulates exocytosis. The coding region for human rab3B was cloned from epithelial mRNA using a reverse-transcription polymerase chain reaction strategy. This cDNA clone hybridized to a single mRNA species in Northern blots of poly(A)+ RNA isolated from epithelial cell lines. A rab3B-specific antibody that was raised against recombinant fusion protein recognized a 25-kD band in immunoblots of cell lysates prepared from cultured epithelial cells (e.g., T84 and HT29-CL19A), but not from a variety of nonepithelial cells (e.g., PC12 neuroendocrine cells). Immunofluorescence analysis confirmed that rab3B protein is preferentially expressed in cultured epithelial cells as well as in a number of native epithelial tissues, including liver, small intestine, colon, and distal nephron. Rab3B localized to the apical pole very near the tight junctions between adjacent epithelial cells within all of these cell lines and native epithelial tissues, as determined by immunofluorescence and immunoelectron microscopic analysis. Moreover, this pattern of intracellular targeting was regulated by cell contact; namely, rab3B was reversibly retrieved from the cell periphery as epithelial cell contact was inhibited by reducing the extracellular Ca2+ concentration. Our results indicate that neurons and epithelial cells express homologous rab3 isoforms that target in a polarized fashion within their respective tissues. The pattern and regulation of rab3B targeting in epithelial cells implicates this monomeric GTPase as a candidate regulator of apical and/or junctional protein traffic in epithelial tissues.