Plasma Membrane Of Epithelial Cells Research Articles

Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.

Regressing amphibian tail as a model for the cadherin/β-catenin complex disruption and glycosylation alteration during epithelial apoptosis

Epidermis is one of the many tissues that are resorbed during metamorphosis in the regressing tail of amphibian tadpoles. Apoptotic mechanisms play an important role in this process. In this study, loss of intercellular contacts and alterations in plasma membrane glycosylation were observed during apoptosis. The cadherin/beta-catenin complex represents one of the major adhesive systems in multiple epithelial tissues. Here, we analysed the fate of cadherin/beta-catenin complex and alterations of plasma membrane glycoconjugate compositions in apoptotic epithelial cells. Our results showed that the cadherin molecules were cleaved into extracellular and beta-catenin associated cytosolic domains by an intracellular mechanism. However, the extracellular domains were probably removed completely by matrix metalloproteinases. Lectin histochemistry studies suggested that mannose and alpha(2-->6) linked (but not alpha(2-->3) linked) sialic acids were major sugar motifs in plasma membranes of apoptotic tadpole epithelial cells. Although previous studies indicated reduced levels of sialic acid residues during apoptosis, elevated Sambucus nigra agglutinin (SNA) reactivity might be due to the degradation of high molecular weight glycoproteins (probably including cadherin) that masked the SNA-binding residues of the plasma membrane prior to apoptosis.

The localization of PMCA1b in epithelial cells and aposomes of the rat coagulating gland is influenced by androgens

Rat coagulating gland epithelial cells export proteins by an apocrine secretion mode within membrane blebs arising from the apical plasma membrane. Using a pan-PMCA antibody, we have recently shown the plasma membrane Ca(2+)-ATPase (PMCA) being part of the apical plasma membrane of epithelial cells and incorporated into the aposomal membrane. The mRNA of PMCA isoforms 1 and 4 respectively, have been detected by RT-PCR in rat coagulating gland. In order to identify which PMCA isoform is integrated into aposomes during apocrine secretion and whether or not PMCA export is influenced by androgens RT-PCR, in situ hybridization, Western blotting, and immunofluorescence experiments were performed. PMCA1b is the isoform which is expressed and located in the apical plasma membrane of coagulating gland epithelial cells and is integrated into the aposomal membrane. In contrast, PMCA4 mRNA and protein are restricted to the stroma. Androgen deprivation by castration within 14 days leads to an accumulation of PMCA1b in coagulating gland epithelium, while aposomes are not detected anymore. We showed for the first time that PMCA isoform 1b is released via aposomes of the epithelial cells of the rat coagulating gland and that the localization of PMCA1b in the epithelial cells is influenced by androgens.

Expression and localisation of apical junctional complex proteins in lens epithelial cells

The lens epithelium possesses an apical junctional complex (AJC) comprising adherens and tight junctions (AJs and TJs) and yet several key structural components and associated regulatory proteins have not been identified or localised in these cells. Here we determine the subcellular distribution of the archetypal TJ markers (ZO-1, claudin-1, and occludin) and TJ-associated cell polarity proteins (aPKC, Par3 and Par6β) with AJ markers, E- and N-cadherin. As seen in other polarised epithelia, all these markers were located by confocal immunofluorescence microscopy to the apical ends of the lateral plasma membranes of bovine lens epithelial cells at sites of cell–cell interaction. Using immunoelectron microscopy, we show that ZO-1 concentrated at “kissing points” between neighbouring cells and these data, when taken in the context of our confocal immunofluorescence microscopy and blotting data, suggest the presence of TJs within the AJC. Likewise, immunogold labelling for E-cadherin identified AJs within these AJCs. We also report aPKC immunogold labelling localised to the AJC. These data show that the AJC of lens epithelial cells are a composite of TJs and AJs.

Claudin‐5 is Restricted to the Tight Junction Region of Uterine Epithelial Cells in the Uterus of Pregnant/Gravid Squamate Reptiles

Claudin-5, a tight junctional protein associated with ion and size selectivity, has been found in the uterus of skinks. This study has generated critical information about the molecular assembly of the tight junction at various stages of the reproductive cycle in the skink uterus. Recent studies looking at tight junctional proteins found occludin expression in the tight junction region of uterine epithelial cells in the skink uterus; however, occludin did not disclose any further information about the ions and size of ions permeating across the paracellular pathway. A approximately 22-kDa claudin-5 band was detected in the uterus of the skinks present in this study and immunohistochemistry revealed that claudin-5 redistributes to the tight junction region of the lateral plasma membrane of uterine epithelial cells in late stage pregnancy/gravidity. This finding indicates that the tight junction becomes more assembled to precisely regulate ion and solute permeation in late stage pregnancy/gravidity. Claudin-5 with its functional role as a molecular sieve due to the formation of ion and size selective pores suggests that permeation of ions smaller than 0.8 kDa are restricted when claudin-5 is redistributed to the tight junction region of the later plasma membrane. This report is the first description of the molecular mechanisms that may be involved in regulating nutrient provision in the reptilian uterus.

A high resolution SEM study of the effects of RU486, used as a postcoital contraceptive, on the rat uterus during early pregnancy

During the window of receptivity, a narrow range of time under the control of the ovarian hormones progesterone and oestrogen, when a blastocyst can attach to the uterine surface, the plasma membrane of the uterine epithelial cells undergoes a remarkable change in structure, known as 'the plasma membrane transformation' of early pregnancy. RU486, the controversial abortion drug (Mifegyne), acts as a progesterone receptor antagonist, resulting in transcriptionally inactive progesterone receptors. In view of this, a change in the well-documented sequences of the plasma membrane transformation is postulated. This study therefore aims to investigate the effects of RU486 on this sequence of events in the implantation and non-implantation sites of the rat uterus. In both RU486 treated and control animals, on days 4.5, 5.5 and 6.5 of pregnancy, scanning electron microscopy revealed a distinct pattern of folding of the uterine surface in non-implantation sites. In contrast, folding was not observed within the implantation sites. These results indicate that surface alterations are probably not under the control of progesterone signalling. The lack of folding at the implantation sites possibly ensures maximum close contact between the blastocyst and the maternal tissue thus promoting implantation. During early pregnancy, specifically on day 5.5, the microvilli of the uterine epithelial cells in the treated animals were more dense than those in the untreated animals. Such microvilli are characteristic of the uterine epithelial cells of a uterus under-stimulated by hormones. Flattening of the apical cell borders usually seen at the time of blastocyst attachment and implantation was not observed following RU486 treatment. Large apical protrusions were observed in the RU486 treated animals only, possibly linked in some way to apoptosis. The antiprogestin properties of RU486 may further elucidate the progesterone effects associated with early pregnancy.

Screening, cloning and sequence analysis ofgBTN1A1gene in mammary gland of Xinong Saanen goat

AbstractButyrophilin 1A1 (BTN1A1), which is a member of the Ig superfamily, is highly expressed in the lactating mammary gland and is secreted into milk in association with lipid droplets, and concentrated in the apical plasma membranes of mammary epithelial cells. Because butyrophilin is expressed only during lactation, it appears to be essential for milk fat globule production. However, there is little information about its properties in the dairy goat. The objective of this study was to confirm whether butyrophilin was related to milk changes in different lactation periods of Xinong Saanen goats. Using suppression subtractive hybridization analysis, we identified a significant increase in butyrophilin mRNA in Xinong Saanen goat mammary gland at middle lactation when compared to that of late lactation. The gBTN1A1 complementary DNA has an open reading frame of 1581 nucleotides encoding a putative protein of 526 amino acids. Amino acids 1–26 were the signal peptide domain. The homologies of nucleotide and peptide sequences of goat butyrophilin with bovine (NM_174508), human (NM_001732) and mouse (AK145168) butyrophilin were 97 and 96%, 88 and 84%, 84 and 70%, respectively. The secondary structure, transmembrane domain and hydrophilicity of goat butyrophilin were similar to the those of the bovine, human and mouse protein. In conclusion, we confirmed that the goat mammary gland mRNA level of gBTN1A1 was responsible for goat milk fat globule secretion.

Soft substrate up-regulates the interaction of STIM1 with store-operated Ca2+ channels that lead to normal epithelial cell apoptosis.

We have demonstrated that soft substrate induced apoptosis in polarized cells, but not in transformed cells by disturbance of Ca(2+) homeostasis. This study aims to further investigate the regulatory mechanisms underlying the disruption of Ca(2+)-signaling integrity in soft substrate-induced epithelial apoptosis. Soft substrate up-regulated the store-operated Ca(2+) (SOC) entry across the plasma membrane of normal cervical epithelial cells, which resulted in increased cytosolic Ca(2+) levels. Concomitantly, soft substrate induced the aggregation and translocation of stromal interacting molecule 1 (STIM1) toward the cell periphery to colocalize with Orai1, an essential pore subunit of SOC channel, detected by fluorescence resonance energy transfer approach and confocal image analyses. The disturbed Ca(2+) homeostasis resulted in the activation of mu-calpain, which cleaved alpha-spectrin, induced actin disorganization, and caused apoptosis. In contrast, soft substrate did not disturb Ca(2+) homeostasis or induce apoptosis in cervical cancer cells. Chelating extracellular Ca(2+) by EGTA and down-regulated SOC entry by small interfering RNA targeting STIM1 or inhibitors targeting Ca(2+)-binding site of calpain significantly inhibited soft substrate-induced activation of mu-calpain and epithelial cell apoptosis. Thus, soft substrate up-regulates the interaction of STIM1 with SOC channels, which results in the activation of mu-calpain and subsequently induces normal epithelial cell apoptosis.

Ion Channels

Ion Channels

The Highly Repetitive Region of the Helicobacter pylori CagY Protein Comprises Tandem Arrays of an α-Helical Repeat Module

The cag-pathogenicity-island-encoded type IV secretion system of Helicobacter pylori functions to translocate the effector protein CagA directly through the plasma membrane of gastric epithelial cells. Similar to other secretion systems, the Cag type IV secretion system elaborates a surface filament structure, which is unusually sheathed by the large cag-pathogenicity-island-encoded protein CagY. CagY is distinguished by unusual amino acid composition and extensive repetitive sequence organised into two defined repeat regions. The second and major repeat region (CagYrpt2) has a regular disposition of six repetitive motifs, which are subject to deletion and duplication, facilitating the generation of CagY size and phenotypic variants. In this study, we show CagYrpt2 to comprise two highly thermostable and acid-stable α-helical structural motifs, the most abundant of which (motif A) occurs in tandem arrays of one to six repeats terminally flanked by single copies of the second repeat (motif B). Isolated motifs demonstrate hetero- and homomeric interactions, suggesting a propensity for uniform assembly of discrete structural subunit motifs within the larger CagYrpt2 structure. Consistent with this, CagY proteins comprising substantially different repeat 2 motif organisations demonstrate equivalent CagA translocation competence, illustrating a remarkable structural and functional tolerance for precise deletion and duplication of motif subunits. We provide the first insight into the structural basis for CagYrpt2 assembly that accommodates both the variable motif sequence composition and the extensive contraction/expansion of repeat modules within the CagYrpt2 region.

Expression and Immunohistochemical Localization of Aquaporin‐1 in Male Reproductive Organs of the Mouse

Aquaporin-1 (AQP-1), a six-transmembrane domain protein, is found to be responsible for water transport. The purpose of this study was to investigate the expression of AQP-1 mRNA and protein in the testis, epididymis, vas deferens, ventral prostate, and seminal vesicle from mature mice by using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and the cellular localization of AQP-1 by immunohistochemistry. RT-PCR revealed that AQP-1 mRNA was expressed in all organs we examined. Western blotting displayed a 29-kDa band and a 35- to 45-kDa band corresponding to non-glycosylated and/or glycosylated AQP-1 in those organs. The immunohistochemical evidence showed that AQP-1 was mainly located on the plasma membrane of epithelial cells of the rete testis, vas deferens, ventral prostate, seminal vesicle and the non-ciliated cells of the proximal and distal efferent ducts. However, AQP-1 was absent from spermatogenic cells lining the seminiferous epithelium and from the spermatozoa in the lumen of the distal efferent duct. These findings provide valuable information on the expression of AQP-1 in male reproductive organs and suggest that AQP-1 is involved in water transport to regulate water homeostasis in male reproductive physiology.

Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.

Phosphatidylinositol 4-Phosphate 5-Kinase Reduces Cell Surface Expression of the Epithelial Sodium Channel (ENaC) in Cultured Collecting Duct Cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.

The role of the UPS in cystic fibrosis

CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER). More than 70% of patients harbor the ΔF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of ‘corrector’ drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients.Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; ).

AMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane

APC is a multifunctional tumor suppressor protein that negatively controls Wnt signaling, but also regulates cell adhesion and migration by interacting with the plasma membrane and the microtubule cytoskeleton. Although the molecular basis for the microtubule association of APC is well understood, molecular mechanisms that underlie its plasma membrane localization have remained elusive. We show here that APC is recruited to the plasma membrane by binding to APC membrane recruitment 1 (AMER1), a novel membrane-associated protein that interacts with the ARM repeat domain of APC. The N-terminus of AMER1 contains two distinct phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)]-binding domains, which mediate its localization to the plasma membrane. Overexpression of AMER1 increases APC levels and redirects APC from microtubule ends to the plasma membrane of epithelial cells. Conversely, siRNA-mediated knockdown of AMER1 reduces the overall levels of APC, promotes its association with microtubule ends in cellular protrusions and disturbs intercellular junctions. These data indicate that AMER1 controls the subcellular distribution of APC between membrane- and microtubule-associated pools, and might thereby regulate APC-dependent cellular morphogenesis, cell migration and cell-cell adhesion.

The Calcium-Sensing Receptor Regulates Plasma Membrane Calcium Adenosine Triphosphatase Isoform 2 Activity in Mammary Epithelial Cells: A Mechanism for Calcium-Regulated Calcium Transport into Milk

The calcium-sensing receptor (CaR) regulates transepithelial calcium transport into milk by mammary epithelial cells. Using a genome-wide screening strategy, we identified the plasma membrane calcium ATPase isoform 2 (PMCA2) as a potential downstream target of the CaR. We show that PMCA2 expression in the mouse mammary gland increases during lactation and that PMCA2 is localized solely to the apical plasma membrane of mammary epithelial cells. In milk from deafwaddler mice, which have mutations in the gene encoding PMCA2, calcium concentrations were reduced, confirming its importance in calcium transport into milk. Furthermore, in cultured primary and EpH4 mouse mammary epithelial cells, CaR stimulation up-regulated calcium-dependent ATPase activity in plasma membrane preparations. By small interfering RNA-mediated gene knockdown of PMCA2, we show that PMCA2 accounts for the preponderance of calcium-ATPase activity. We also show that reduction of CaR expression with small interfering RNA eliminates the ability of extracellular calcium to elicit an increase in calcium-dependent ATPase activity in EpH4 cell membranes. These results demonstrate that activation of the CaR increases PMCA2 activity in mouse mammary epithelial cells, providing a mechanism for the regulation of transepithelial calcium transport by calcium in the lactating mouse mammary gland.

Intoxication of epithelial cells by plasmid-encoded toxin requires clathrin-mediated endocytosis

It has been shown that the autotransporter plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) produces cytotoxic and enterotoxic effects. Both effects can be explained by the proteolytic activity of Pet on its intracellular target alpha-fodrin (alphaII spectrin). In addition, Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, and on its internalization into epithelial cells. However, the mechanisms of Pet uptake by epithelial cells are unknown. Here, we show that Pet interacts with the plasma membrane of epithelial cells, and afterwards is detected inside the cells. Furthermore, Pet was internalized via clathrin-mediated endocytosis, since its internalization was inhibited by monodansylcadaverine and sucrose, but not by filipin or methyl-beta-cyclodextrin, which are drugs that interfere with protein entry via a clathrin-independent pathway. Additionally, Pet was immunoprecipitated by anti-clathrin antibodies, but not by anti-caveolin antibodies. Moreover, small interfering RNA (siRNA), designed to knock out clathrin gene expression in HEp-2 cells, prevented Pet internalization, and thereby the Pet-induced cytotoxic effect. However, the use of siRNA to knock out caveolin expression had no effect on Pet internalization, and the cytotoxic effect was clearly observed. Together, these data indicate that Pet secreted by EAEC binds to the cell surface via an unknown receptor, to be taken up by clathrin-mediated endocytosis, and exert its toxic effect in the cytoplasm.

Organic extract of tire debris causes localized damage in the plasma membrane of human lung epithelial cells

The potential toxicity of tire debris organic extracts on human alveolar epithelial cells (A549) was investigated. We analysed time- and dose dependent modifications produced on plasma membrane molecular composition and on lipid microdomains expression (caveolae and lipid rafts) that represent specific signalling platforms. Cells were exposed to increasing organic extract concentrations (10, 60 and 75 μg/ml) for 24, 48 and 72 h. An up to three fold dose and time dependent increase in specific protein markers of lipid microdomains was found, suggesting a corresponding increase in signalling platforms. Since the total pool of these plasma membrane markers was unchanged, we supposed that these proteins were translocated within the plasma membrane as to assemble the newly formed lipid microdomains. Despite no major modifications in lipid bilayer composition, a time- and dose dependent toxic effect was documented at 48 h of exposure by an increase of cells positive to Trypan Blue assay. After 48 h a dose dependent increase in the cell medium of the cytosolic enzyme lactate dehydrogenase was also observed, indicating greater damage of the plasma membrane as prenecrotic sign. The overall ultrastructural morphology of the plasma membrane of treated cells was not greatly modified, suggesting that organic extracts from tire debris cause focalized discontinuities on cell surfaces.

Insulin-like Growth Factor Binding Protein-3 expression in the human corneal epithelium

Insulin-like Growth Factor Binding Protein-3 (IGFBP3) is a high-affinity binding protein shown to regulate cell growth, differentiation, and apoptosis in a variety of cellular systems. The primary aim of this study was to characterize IGFBP3 expression in the human corneal epithelium and in a corneal epithelial cell line and to establish a potential role for IGFBP3-mediated apoptotic signaling in corneal epithelial cells. Using a telomerase-immortalized human corneal epithelial (hTCEpi) cell line cultured in serum-free media and fresh human eye bank donor tissue, expression and localization of IGFBP3 were established in situ and in vitro by indirect immunofluorescence and western blotting. Real-time PCR was used to measure IGFBP3 mRNA levels following Trichostatin A (TSA) treatment and as a function of confluence. IGFBP3 protein levels were assessed in resting human tears and in conditioned media by western blotting as was the ability of recombinant human IGFBP3 protein to associate with the cell surface. Apoptotic signaling was assessed in vitro using TSA and recombinant human (rh)IGFBP3. Apoptosis was measured by Viability/Cytotoxicity, Annexin V, and TUNEL assays. IGFBP3 was localized to the plasma membrane of human corneal epithelial cells in situ and was upregulated in surface cells in the central cornea. IGFBP3 was secreted in conditioned media of growing cells, with a robust upregulation following confluence ( P = 0.014) and differentiation. IGFBP3 was undetectable in human tears. Addition of TSA to the culture media resulted in an upregulation of IGFBP3 mRNA ( P < 0.001) and protein. In addition, TSA treatment led to a significant increase in Annexin V positive cells at 18 and 24 h ( P < 0.001) and TUNEL positive cells at 24 and 48 h ( P < 0.001). The addition of rhIGFBP3 to the cell culture media appeared to induce occasional membrane blebbing, but cells failed to become positive with Annexin V or TUNEL. Taken together, these results demonstrate that cell membrane-associated IGFBP3 is produced by corneal epithelial cells and associates with the plasma membrane of superficial cells in situ and in cultured cells, but not present in human tears. The differential localization and effect(s) on apoptosis suggest that the effects of IGFBP3 are likely tissue compartment and receptor specific and may be regulated by glycosylation.

Liquid movement across the surface epithelium of large airways

The cystic fibrosis transmembrane conductance regulator CFTR gene is found on chromosome 7 [Kerem, B., Rommens, J.M., Buchanan, J.A., Markiewicz, D., Cox, T.K., Chakravarti, A., Buchwald, M., Tsui, L.C., 1989. Identification of the cystic fibrosis gene: genetic analysis. Science 245, 1073-1080; Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.L., et al., 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1073] and encodes for a 1480 amino acid protein which is present in the plasma membrane of epithelial cells [Anderson, M.P., Sheppard, D.N., Berger, H.A., Welsh, M.J., 1992. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263, L1-L14]. This protein appears to have many functions, but a unifying theme is that it acts as a protein kinase C- and cyclic AMP-regulated Cl(-) channel [Winpenny, J.P., McAlroy, H.L., Gray, M.A., Argent, B.E., 1995. Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells. Am. J. Physiol. 268, C823-C828; Jia, Y., Mathews, C.J., Hanrahan, J.W., 1997. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272, 4978-4984]. In the superficial epithelium of the conducting airways, CFTR is involved in Cl(-) secretion [Boucher, R.C., 2003. Regulation of airway surface liquid volume by human airway epithelia. Pflugers Arch. 445, 495-498] and also acts as a regulator of the epithelial Na(+) channel (ENaC) and hence Na(+) absorption [Boucher, R.C., Stutts, M.J., Knowles, M.R., Cantley, L., Gatzy, J.T., 1986. Na(+) transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J. Clin. Invest. 78, 1245-1252; Stutts, M.J., Canessa, C.M., Olsen, J.C., Hamrick, M., Cohn, J.A., Rossier, B.C., Boucher, R.C., 1995. CFTR as a cAMP-dependent regulator of sodium channels. Science 269, 847-850]. In this chapter, we will discuss the regulation of these two ion channels, and how they can influence liquid movement across the superficial airway epithelium.