Beyond epithelial-to-mesenchymal transition: Common suppression of differentiation programs underlies epithelial barrier dysfunction in mild, moderate, and severe asthma.

BACKGROUND: Epithelial barrier dysfunction is a central feature in the pathogenesis of allergic disease. Epithelial-to-mesenchymal transition (EMT) has been proposed as one mechanism afflicting barrier in asthma. However, genes and pathways involved in aberrant epithelial-mesenchymal signaling, and their relationship to asthma severity, are poorly understood. METHODS: We used unbiased gene network analysis to evaluate functional convergence in epithelial gene expression signatures across multiple public access transcriptomics datasets of human asthma, followed by text mining to evaluate functional marker relevance of discovered genes. We objectively confirmed these findings in epithelial brushings and primary asthmatic epithelial cells cultured in different biological contexts. RESULTS: We found a striking suppression of epithelial differentiation in asthma, overrepresented by insufficiency in insulin and Notch signaling, but with the absence of conventional EMT markers. We identified EFNB2, FGFR1, FGFR2, INSR, IRS2, NOTCH2, TLE1, and NTRK2 as novel markers central to dysregulation of epithelial-mesenchymal signaling, but surprisingly overlooked in asthma research. We found that this "core" signature of asthma is shared by mild, moderate, and severe forms of disease, progressing with severity. Loss of epithelial differentiation induced by insulin deprivation in normal human bronchial epithelial cells cultured in organotypic conditions closely approximated gene expression in asthmatic epithelial brushings. CONCLUSIONS: The comparative analysis of publically available transcriptomes demonstrated that epithelial barrier dysfunction in asthma is characterized by persistent underlying de-differentiation program with complex etiology. The lasting alteration of the asthmatic epithelial cell transcriptome implicates regulation involving metabolism and epigenetics, beyond EMT driven by injury and repair in chronic inflammation.

Cadherins and cancer: how does cadherin dysfunction promote tumor progression?

It has long been recognized that the cell-cell adhesion receptor, E-cadherin, is an important determinant of tumor progression, serving as a suppressor of invasion and metastasis in many contexts. Yet how the loss of E-cadherin function promotes tumor progression is poorly understood. In this review, we focus on three potential underlying mechanisms: the capacity of E-cadherin to regulate beta-catenin signaling in the canonical Wnt pathway; its potential to inhibit mitogenic signaling through growth factor receptors and the possible links between cadherins and the molecular determinants of epithelial polarity. Each of these potential mechanisms provides insights into the complexity that is likely responsible for the tumor-suppressive action of E-cadherin.

Adhesion signaling: how beta-catenin interacts with its partners.

The multi-functional protein beta-catenin plays essential roles in cell-cell adhesion and nuclear signaling. Elucidation of the structures of beta-catenin complexes is beginning to clarify how beta-catenin uses the same surface to bind its various partners, and provides insights into how these interactions might be regulated.

E-cadherin suppresses cellular transformation by inhibiting beta-catenin signaling in an adhesion-independent manner.

E-cadherin is a tumor suppressor protein with a well-established role in cell-cell adhesion. Adhesion could contribute to tumor suppression either by physically joining cells or by facilitating other juxtacrine signaling events. Alternatively, E-cadherin tumor suppressor activity could result from binding and antagonizing the nuclear signaling function of beta-catenin, a known proto-oncogene. To distinguish between an adhesion- versus a beta-catenin signaling-dependent mechanism, chimeric cadherin constructs were expressed in the SW480 colorectal tumor cell line. Expression of wild-type E-cadherin significantly inhibits the growth of this cell line. Growth inhibitory activity is retained by all constructs that have the beta-catenin binding region of the cytoplasmic domain but not by E-cadherin constructs that exhibit adhesive activity, but lack the beta-catenin binding region. This growth suppression correlates with a reduction in beta-catenin/T cell factor (TCF) reporter gene activity. Importantly, direct inhibition of beta-catenin/TCF signaling inhibits the growth of SW480 cells, and the growth inhibitory activity of E-cadherin is rescued by constitutively activated forms of TCF. Thus, the growth suppressor activity of E-cadherin is adhesion independent and results from an inhibition of the beta-catenin/TCF signaling pathway, suggesting that loss of E-cadherin expression can contribute to upregulation of this pathway in human cancers. E-cadherin-mediated growth suppression was not accompanied by overall depletion of beta-catenin from the cytosol and nucleus. This appears to be due to the existence of a large pool of cytosolic beta-catenin in SW480 cells that is refractory to both cadherin binding and TCF binding. Thus, a small pool of beta-catenin that can bind TCF (i.e., the transcriptionally active pool) can be selectively depleted by E-cadherin expression. The existence of functionally distinct pools of cytosolic beta-catenin suggests that there are mechanisms to regulate beta-catenin signaling in addition to controlling its level of accumulation.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells.

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface. We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodomain of the interleukin-2alpha (IL-2alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK(1) epithelial cells. Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin. Truncation mutants unable to bind beta-catenin were correctly targeted, showing, contrary to current understanding, that beta-catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine-mediated targeting is maintained in LLC-PK(1) cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line. These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

Sorting of P-type ATPases in polarized epithelial cells.

The Na,K-ATPase and the H,K-ATPase are highly homologous members of the P-type family of ion transporting ATPase. Despite their structural similarity, these two pumps are sorted to different destinations in polarized epithelial cells. While the Na,K-ATPase is restricted to the basolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular storage compartment in the parietal cells of the stomach. We have generated molecular chimeras composed of complementary portions of these two pumps' alpha-subunits. By expressing these pump constructs in polarized epithelial cells in culture, we have been able to identify sequence domains which participate in the targetting of the holoenzyme. We find that information embedded within the sequence of the fourth transmembrane domain of the H,K-ATPase is sufficient to account for this protein's apical localization. Stimulation of gastric acid secretion results in insertion of the intracellular H,K-ATPase pool into the apical plasma membrane and inactivation of acid secretion is accompanied by the re-internalization of these pumps. We have identified a tyrosine-based signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the critical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cell surface in parietal cells from these animals, and they constitutively hypersecrete gastric acid. These results demonstrate that the beta-subunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least two sorting signals are required to ensure the proper targetting and regulation of the gastric H,K pump.

Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells.

Tyrosine-dependent sequence motifs are implicated in sorting membrane proteins to the basolateral domain of Madin-Darby canine kidney (MDCK) cells. We find that these motifs are interpreted differentially in various polarized epithelial cell types. The H, K-ATPase beta subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK1 cells. Similarly, HA-Y543, a construct in which a tyrosine-based motif was introduced into the cytoplasmic tail of influenza hemagglutinin, was sorted to the basolateral membrane of MDCK cells and retained at the apical membrane of LLC-PK1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase beta subunit protein was replaced with the analogous region of the Na,K-ATPase beta subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase beta subunit cytoplasmic sequence to an alanine was sufficient to disrupt basolateral localization of this polypeptide. In contrast, these constructs all remain localized to the apical membrane in LLC-PK1 cells. The FcRII-B2 protein bears a di-leucine motif and is found at the basolateral membrane of both MDCK and LLC-PK1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.

A basolateral sorting signal is encoded in the alpha-subunit of Na-K-ATPase.

Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the alpha-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric alpha-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1 cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase alpha-subunit contain a basolateral sorting signal. H-K-ATPase beta-subunit expressed alone in LLC-PK1 cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase beta-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase alpha-subunit masks or is dominant over any apical sorting information present in the beta-polypeptide. In gastric parietal cells, the H-K-ATPase beta-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase beta-subunit complex is directed to a similar compartment in LLC-PK1 cells, we treated transfected cells with drugs that raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase beta-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts.

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.

Biotinylation and assessment of membrane polarity: caveats and methodological concerns.

Studies of epithelial membrane polarity have been greatly facilitated through the use of the N-hydroxysuccinimide-biotin surface labeling technique (M. Sargiacomo, M. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. J. Membr. Biol. 107: 277-286, 1989). We have used this technique in studies on the sorting and targeting of ion-transporting adenosinetriphosphatase molecules in polarized epithelial cells. Through efforts to optimize this technique in our experimental system, we have encountered several experimental conditions and circumstances where biotinylation is extremely inefficient and the assessment of membrane polarity which it provides is misleading. We demonstrate that the pH and ionic strength of the biotinylation buffer can dramatically affect biotin incorporation and that protocol-dependent variations in the recovery of biotinylated proteins can result in misrepresentation of the actual apical/basolateral distribution of a protein. Conditions and protocols that may improve the sensitivity and accuracy of this technique are discussed.

Molecular requirements for the cell-surface expression of multisubunit ion-transporting ATPases. Identification of protein domains that participate in Na,K-ATPase and H,K-ATPase subunit assembly.

The ion-transporting H,K-ATPase and Na,K-ATPase enzymes are each composed of an alpha and a beta subunit. It is known that assembly of the alpha and beta subunits of the Na,K-ATPase is necessary for the cell-surface delivery of the active enzyme. We have examined the molecular domains involved in the assembly of the H,K-ATPase and Na,K-ATPase alpha and beta subunits by expressing individual subunits and subunit chimeras in transiently transfected COS-1 cells. Our results demonstrate that the H,K-ATPase alpha subunit requires its beta subunit for efficient cell-surface expression, as determined by indirect immunofluorescence. The H,K-ATPase beta protein appears to be able to get to the cell surface unaccompanied by any alpha subunit and appears to localize as well to a population of intracellular vesicles. We find that a transfected chimera encoding the NH2-terminal half of the H,K-ATPase alpha subunit and the COOH-terminal half of the Na,K-ATPase alpha subunit appears to assemble with the endogenous Na,K-ATPase beta subunit and to reach the plasmalemma. Transfection of the complementary alpha chimera requires coexpression with the H,K-ATPase beta subunit in order to attain surface delivery. Thus, it is the COOH-terminal half of the alpha subunit that specifies assembly with a particular beta subunit.

Functional properties of an H,K-ATPase/Na,K-ATPase chimera.

Cultured pig kidney epithelial cells were transfected with a chimeric P-type ATPase catalytic subunit composed of the NH2-terminal half of the rat gastric H,K-ATPase and the COOH-terminal half of the rat Na,K-ATPase (alpha 1 isoform). Low concentrations of ouabain (< or = 0.2 mM) were used to inhibit completely the endogenous pig Na,K-ATPase and high concentrations (5 nM) to test the sensitivity of the chimeric rodent pump. In the presence of a low concentration of ouabain, a small but significant inhibition of residual Rb+(K+) influx by 5 mM ouabain was observed in only the transfected cells. Conditions were found in which a similar component of Rb+ influx was inhibited by the gastric H,K-ATPase inhibitor SCH28080, consistent with SCH28080 binding to the extracellular H1-H2 loop of this enzyme. These experiments demonstrate that this chimera behaves as a functional ion pump and indicate that the protein domains involved in cardiac glycoside binding are not confined to the amino-terminal half of the Na,K-ATPase.

An ion-transporting ATPase encodes multiple apical localization signals.

Epithelial cells accumulate distinct populations of membrane proteins at their two plasmalemmal domains. We have examined the molecular signals which specify the differential subcellular distributions of two closely related ion pumps. The Na,K-ATPase is normally restricted to the basolateral membranes of numerous epithelial cell types, whereas the H,K-ATPase is a component of the apical surfaces of the parietal cells of the gastric epithelium. We have expressed full length and chimeric H,K-ATPase/Na,K-ATPase cDNAs in polarized renal proximal tubular epithelial cells (LLC-PK1). We find that both the alpha and beta subunits of the H,K-ATPase encode independent signals that specify apical localization. Furthermore, the H,K-ATPase beta-subunit possesses a sequence which mediates its participation in the endocytic pathway. The interrelationship between epithelial sorting and endocytosis signals suggested by these studies supports the redefinition of apical and basolateral as functional, rather than simply topographic domains.

Sorting of ion transport proteins in polarized cells.

The plasma membranes of polarized epithelial cells and neurons express distinct populations of ion transport proteins in their differentiated plasma membrane domains. In order to understand the mechanisms responsible for this polarity it will be necessary to elucidate the nature both of sorting signals and of the cellular machinery which recognizes and acts upon them. In our efforts to study sorting signals we have taken advantage of two closely related families of ion transport proteins whose members are concentrated in different epithelial plasmalemmal domains. The H+,K(+)-ATPase and the Na+,K(+)-ATPase are closely related members of the E1-E2 family of ion transporting ATPases. Despite their high degree of structural and functional homology, they are concentrated on different surfaces of polarized epithelial cells and pursue distinct routes to the cell surface in cells which manifest a regulated delivery pathway. We have transfected cDNAs encoding these pumps' subunit polypeptides, as well as chimeras derived from them, in a variety of epithelial and non-epithelial cell types. Our observations suggest that these pumps encode multiple sorting signals whose relative importance and functions may depend upon the cell type in which they are expressed. Recent evidence suggests that the sorting mechanisms employed by epithelial cells may be similar to those which operate in neurons. We have examined this proposition by studying the distributions of ion pumps and neurotransmitter re-uptake co-transporters expressed endogenously and by transfection in neurons and epithelial cells, respectively. We find that one of the classes of proteins we studied obeys the correlation between neuronal and epithelial sorting while another does not.(ABSTRACT TRUNCATED AT 250 WORDS)