Anti-inflammatory Properties of the Alpha-Melanocyte-Stimulating Hormone in Models of Granulomatous Inflammation.

PURPOSE: Alpha-melanocyte stimulating hormone (α-MSH) is known to have anti-inflammatory effects. However, the anti-inflammatory properties of α-MSH on normal bronchial epithelial cells are largely unknown, especially in the context of in vitro sarcoidosis models. METHODS: We evaluated the anti-inflammatory effects of α-MSH on two different in vitro sarcoidosis models (lung-on-membrane model; LOMM and three-dimensional biochip pulmonary sarcoidosis model; 3D-BSGM) generated from NBECs and an in vivo sarcoidosis mouse model. RESULTS: Treatment with α-MSH decreased inflammatory cytokine levels and downregulated type I interferon pathway genes and related proteins in LOMM and 3D-BSGM models. Treatment with α-MSH also significantly decreased macrophages and cytotoxic T-cells counts in a sarcoidosis mice model. CONCLUSION: Our results confirm the direct role of type I IFNs in the pathogenesis of sarcoid lung granulomas and highlight α-MSH as a potential novel therapeutic agent for treating pulmonary sarcoidosis.

Mycobacterium abscessus-Bronchial Epithelial Cells Cross-Talk Through Type I Interferon Signaling.

Introduction:Mycobacteria are aerobic non-motile organisms with lipid rich, hydrophobic cell walls that render them resistant to antibiotics. While there are over 150 different species of NTM, Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are two of the most common culprits of pulmonary infection. MAB has been found to be most common in southeastern United States (Florida to Texas) and the third most rapidly growing NTM infection. It is responsible for chronic lung infections. Mycobacterial cell wall components initiate the interaction between bacteria and host. The reaction between bronchial epithelia and components in the envelope of mycobacterial cell wall is poorly understood. Methods: A lung-on-membrane model was developed with normal human bronchial epithelial (NHBE) cells re-differentiated at the air-liquid interface (ALI) and human endothelial cells on a transwell® polyester membrane. Microparticles from MAB cell walls were developed by an inhouse protocol and added to the ALI side of lung model. NHBE cells were harvested at day 3. RNA was isolated and analyzed with RNASeq. NHBE cells were lysed and protein assay was performed with western blot. We tested whether lung INF-alpha expression would increase in mice treated with intratracheal MAB cell wall particles. A paired t-test is used to compare two population means using GraphPad Prism 7 software. Results: RNAseq analysis identified 1759 differentially expressed genes between NHBE cells challenged with and without MAB microparticles with FDR < 0.5. 410 genes had a 2.5-fold change (FC) or greater. NHBE cells exposure to MAB microparticles significantly enriched the IFN I signaling pathway. Protein overexpression of IFN I family (2'-5'-Oligoadenylate Synthetase 1, Interferon-induced GTP-binding protein Mx1, Interferon-stimulated gene 15) was found in bronchial epithelial cells following exposure to MAB cell wall microparticles. IFN-α protein and gene expressions were significantly increased in mice lung challenged with microparticles in comparison with controls. Conclusion: These data strongly support the role of Type I IFN in cross-talk between NHBE cells and MAB. They also suggest that initiating immune response by NHBE cells may play a central role in innate immunity. Furthermore, this study underscores the importance of mycobacterial cell wall in initiating innate immune response.

Modulation of Wnt signaling is essential for the differentiation of ciliated epithelial cells in human airways.

Wnt signaling is essential for the differentiation of airway epithelial cells during development. Here, we examined the role of Wnt signaling during redifferentiation of ciliated airway epithelial cells in vitro at the air liquid interface as a model of airway epithelial repair. Phases of proliferation and differentiation were defined. Markers of squamous metaplasia and epithelial ciliation were followed while enhancing ß-catenin signaling by blocking glycogen synthase kinase 3ß with SB216763 and shRNA as well as inhibiting canonical WNT signaling with apical application of Dickkopf 1 (Dkk1). Our findings indicate that enhanced ß-catenin signaling decreases the number of ciliated cells and causes squamous changes in the epithelium, whereas treatment with DDk1 leads to an increased number of ciliated cells.

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery.

Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases.

IL-13 Inhibits Multicilin Expression and Ciliogenesis via Janus Kinase/Signal Transducer and Activator of Transcription Independently of Notch Cleavage.

Loss of ciliated cells and increases in goblet cells are seen in respiratory diseases such as asthma. These changes result in part from reduced differentiation of basal progenitor cells to ciliated cells during injury and repair. The T helper 2 cytokine, IL-13, has been shown to inhibit ciliated cell differentiation, but the mechanism is not clearly understood. We recently showed that Notch signaling inhibits ciliated cell differentiation in submerged culture by repressing multicilin and forkhead box J1 (FOXJ1) expression, genes required for ciliogenesis. Using a novel method to study ciliated cell differentiation, we investigated the relationship between IL-13 and Notch signaling pathways. We found that IL-13 inhibits ciliated cell differentiation by repressing multicilin and FOXJ1 expression but does so independent of Notch signaling. In addition, we show that pharmacological inhibition of Janus kinase/signal transducer and activator of transcription, but not mitogen activated protein kinase kinase, signaling rescues multicilin and FOXJ1 expression and ciliated cell differentiation in the presence of IL-13. These findings indicate that regulation of multicilin expression by two distinct signaling pathways affects ciliated cell differentiation. In addition, the requirement for Janus kinase activation in IL-13-induced inhibition of ciliogenesis provides a potential therapeutic target for the treatment of respiratory disease.

A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice.

Ciliary beating is important for effective mucociliary clearance. Soluble adenylyl cyclase (sAC) regulates ciliary beating, and a roughly 50-kD sAC variant is expressed in axonemes. Normal human bronchial epithelial (NHBE) cells express multiple sAC splice variants: full-length sAC; variants with catalytic domain 1 (C1) deletions; and variants with partial C1. One variant, sACex5v2-ex12v2, contains two alternative splices creating new exons 5 (ex5v2) and 12 (ex12v2), encoding a roughly 45-kD protein. It is therefore similar in size to ciliary sAC. The variant increases in expression upon ciliogenesis during differentiation at the air-liquid interface. When expressed in NHBE cells, this variant was targeted to cilia. Exons 5v2-7 were important for ciliary targeting, whereas exons 2-4 prevented it. In vitro, cytoplasmic sACex2-ex12v2 (containing C1 and C2) was the only variant producing cAMP. Ciliary sACex5v2-ex12v2 was not catalytically active. Airway epithelial cells isolated from wild-type mice revealed sAC-dependent ciliary beat frequency (CBF) regulation, analogous to NHBE cells: CBF rescue from HCO3(-)/CO2-mediated intracellular acidification was sensitive to the sAC inhibitor, KH7. Compared with wild type, sAC C2 knockout (KO) mice revealed lower CBF baseline, and the HCO3(-)/CO2-mediated CBF decrease was not inhibited by KH7, confirming lack of functional sAC. Human sACex5v2-ex12v2 was targeted to cilia and sACex2-ex12v2 to the cytoplasm in these KO mice. Introduction of the ciliary sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored functional sAC activity in C2 KO mice. Thus, we show, for the first time, a mammalian axonemal targeting sequence that localizes a sAC variant to cilia to regulate CBF.

Submersion and hypoxia inhibit ciliated cell differentiation in a notch-dependent manner.

The epithelium that lines the conducting airways is composed of several distinct cell types that differentiate from common progenitor cells. The signals that control fate selection and differentiation of ciliated cells, a major component of the epithelium, are not completely understood. Ciliated cell differentiation can be accomplished in vitro when primary normal human bronchial epithelial (NHBE) cells are cultured at an air-liquid interface, but is inhibited when NHBE cells are cultured under submerged conditions. The mechanism by which submersion prevents ciliogenesis is not understood, but may provide clues to in vivo regulation of ciliated cell differentiation. We hypothesized that submersion creates a hypoxic environment that prevents ciliated cell differentiation by blocking the gene expression program required for ciliogenesis. This was confirmed by showing that expression of multicilin and Forkhead box J1, key factors needed for ciliated cell differentiation, was inhibited when NHBE cells were cultured in submerged and hypoxic conditions. Multicilin and Forkhead box J1 expression and ciliated cell differentiation were restored in submerged and hypoxic cells upon treatment with the γ-secretase inhibitor, N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), which suggested that Notch signaling was involved. Overexpression of Notch intracellular domain inhibited differentiation in the presence of DAPT, confirming the role of Notch signaling. These results indicate that submersion and hypoxia prevent ciliated cell differentiation by maintaining Notch signaling, which represses genes necessary for ciliogenesis. These data provide new insights into the molecular mechanisms that control human bronchial differentiation.

Rapid nongenomic actions of inhaled corticosteroids on long-acting ß(2)-agonist transport in the airway.

Corticosteroids inhibit organic cation transporters (OCTs) that play an important role in drug absorption, tissue distribution and elimination. Corticosteroid sensitivity of bronchodilator trafficking in the airway tissue, however, is poorly understood. To assess the effects of inhaled corticosteroids on airway absorption and disposal mechanisms of long-acting ß(2)-agonists, human airway epithelial and smooth muscle cell uptake of tritiated formoterol and salmeterol was measured in vitro. Corticosteroids caused a rapid, concentration-dependent inhibition of uptake of the cationic formoterol by airway smooth muscle cells, but not airway epithelial cells. Uptake of the non-charged lipophilic salmeterol was corticosteroid-insensitive in both cell types. In smooth muscle cells, inhaled corticosteroids inhibited formoterol uptake with a novel potency rank order: des-ciclesonide > budesonide > beclomethasone 17-monopropionate > beclomethasone dipropionate > ciclesonide > fluticasone. The inhibitory action was rapidly reversible, and was not enhanced by prolonged corticosteroid exposure or sensitive to a transcription inhibitor. Suppression of OCT3 expression using lentivirus-mediated production of shRNA reduced corticosteroid sensitivity of formoterol uptake by smooth muscle cells. Our data support a corticosteroid insensitive absorption and a corticosteroid-sensitive disposition mechanism for cationic long-acting ß(2)-agonist bronchodilators in the airway. Potency rank order and other 'classical' features of anti-inflammatory effects do not apply to inhaled corticosteroids' rapid drug transport actions.

Decreased soluble adenylyl cyclase activity in cystic fibrosis is related to defective apical bicarbonate exchange and affects ciliary beat frequency regulation.

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO(3)(-)/CO(2) stimulation to increase ciliary beat frequency (CBF). Because apical HCO(3)(-) exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO(3)(-)/CO(2) exposure in part because of greater intracellular acidification from unbalanced CO(2) influx (estimated by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO(3)(-)/CO(2) perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR(inh)172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO(3)(-)/CO(2) rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.

Reactive oxygen species and hyaluronidase 2 regulate airway epithelial hyaluronan fragmentation.

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALI), treated with PI-specific phospholipase C (PI-PLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported that HA degradation was mediated by reactive oxygen species (ROS) in human airways. Here we show that ROS increase Hyal2 expression and activity in NHBE cells and that the p38MAPK signaling pathway is involved in this effect. Hyal2 induction was confirmed by using small interfering RNA (siRNA) expressing lentivirus. These in vitro findings correlated in vivo with smokers, where increased Hyal2 immunoreactivity in the epithelium was associated with augmented levels of HA and the appearance of low molecular mass HA species in bronchial secretions. In summary, this work provides evidence that ROS induce Hyal2, suggesting that Hyal2 is likely responsible for the sustained HA fragmentation in the airway lumen observed in inflammatory conditions associated with oxidative stress.

Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli.

Epithelia express oxidative antimicrobial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H(2)O(2)), and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H(2)O(2) for use by LPO. To investigate the regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells challenged with Pseudomonas aeruginosa flagellin or IFN-gamma. Flagellin upregulated Duox2 mRNA 20-fold, but upregulated LPO mRNA only 2.5-fold. IFN-gamma increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFN-gamma. Both stimuli increased H(2)O(2) synthesis and LPO-dependent killing of P. aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H(2)O(2) production, whereas Duox2 siRNA markedly reduced basal H(2)O(2) production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2-mediated H(2)O(2) production seem to be coordinated with increases in LPO mRNA and, without increased LPO, H(2)O(2) levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H(2)O(2) synthesis despite the presence of greater amounts of Duox1.

Pannexin 1 contributes to ATP release in airway epithelia.

ATP is a paracrine regulator of critical airway epithelial cell functions, but the mechanism of its release is poorly understood. Pannexin (Panx) proteins, related to invertebrate innexins, form channels (called pannexons) that are able to release ATP from several cell types. Thus, ATP release via pannexons was examined in airway epithelial cells. Quantitative RT-PCR showed Panx1 expression in normal human airway epithelial cells during redifferentiation at the air-liquid interface (ALI), at a level comparable to that of alveolar macrophages; Panx3 was not expressed. Immunohistochemistry showed Panx1 expression at the apical pole of airway epithelia. ALI cultures exposed to hypotonic stress released ATP to an estimated maximum of 255 (+/-64) nM within 1 minute after challenge (n = 6 cultures from three different lungs) or to approximately 1.5 (+/-0.4) microM, recalculated to a normal airway surface liquid volume. Using date- and culture-matched cells (each n > or = 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 microM) and probenecid (1 mM), but not the connexon inhibitor flufenamic acid (100 microM), inhibited ATP release by approximately 60%. The drugs affected Panx1 currents in Xenopus oocytes expressing exogenous Panx1 correspondingly. In addition, suppression of Panx1 expression using lentivirus-mediated production of shRNA in differentiated airway epithelial cells inhibited ATP release upon hypotonic stress by approximately 60% as well. These data not only show that Panx1 is expressed apically in differentiated airway epithelial cells but also that it contributes to ATP release in these cells.

Molecular heterogeneity and alternative splicing of human lactoperoxidase.

Human lactoperoxidase (LPO) exists as two distinct molecules independent of glycosylation. The N-terminus of one form is blocked and has not been identified while the other is proteolytically processed at the N-terminus similar to myeloperoxidase. Our analysis identified alternatively spliced human LPO mRNAs that may explain the observed molecular heterogeneity of LPO. Two mRNAs omit propeptide encoding exons while retaining the 5' exon encoding the secretion signal, consistent with the heterogeneity and suggesting a possible functional role for the propeptide. Two LPO forms were expressed using baculovirus and both showed similar enzyme activity. LC/MS/MS analysis of trypsin digested, partially purified, salivary LPO confirmed the larger unprocessed LPO is present in saliva. To compare variant expression patterns, antisera were raised against purified recombinant (rhLPO) as well as against an antigenic peptide sequence within the exons encoding the propeptide region. Immunohistochemistry demonstrated proLPO was differently localized within gland cells compared to other forms of LPO. The data suggested splice variants may contribute to LPO molecular heterogeneity and its regulation by intracellular compartmental localization.

Epigenetic regulation of HYAL-1 hyaluronidase expression. identification of HYAL-1 promoter.

HYAL-1 (hyaluronoglucosaminidase-1) belongs to the hyaluronidase family of enzymes that degrade hyaluronic acid. HYAL-1 is a marker for cancer diagnosis and a molecular determinant of tumor growth, invasion, and angiogenesis. The regulation of HYAL-1 expression is unknown. Real time reverse transcription-PCR using 11 bladder and prostate cancer cells and 69 bladder tissues showed that HYAL-1 mRNA levels are elevated 10-30-fold in cells/tissues that express high hyaluronidase activity. Although multiple transcription start sites (TSS) for HYAL-1 mRNA were detected in various tissues, the major TSS in many tissues, including bladder and prostate, was at nucleotide 27274 in the cosmid clone LUCA13 (AC002455). By analyzing the 1532 base sequence 5' to this TSS, using cloning and luciferase reporter assays, we identified a TACAAA sequence at position -31 and the minimal promoter region between nucleotides -93 and -38. Mutational analysis identified that nucleotides -73 to -50 (which include overlapping binding consensus sites for SP1, Egr-1, and AP-2), bases C(-71) and C(-59), and an NFkappaB-binding site (at position -15) are necessary for promoter activity. The chromatin immunoprecipitation assay identified that Egr-1, AP-2, and NFkappaB bind to the promoter in HYAL-1-expressing cells, whereas SP1 binds to the promoter in non-HYAL-1-expressing cells. 5-Aza-2'-deoxycytidine treatment, bisulfite DNA sequencing, and methylation-specific PCR revealed that HYAL-1 expression is regulated by methylation at C(-71) and C(-59); both Cs are part of the SP1/Egr-1-binding sites. Thus, HYAL-1 expression is epigenetically regulated by the binding of different transcription factors to the methylated and unmethylated HYAL-1 promoter.

Atypical protein kinase C (iota) activates ezrin in the apical domain of intestinal epithelial cells.

Atypical protein kinase iota (PKCiota) is a key organizer of the apical domain in epithelial cells. Ezrin is a cytosolic protein that, upon activation by phosphorylation of T567, is localized under the apical membrane where it connects actin filaments to membrane proteins and recruits protein kinase A (PKA). To identify the kinase that phosphorylates ezrin T567 in simple epithelia, we analyzed the expression of active PKC and the appearance of T567-P during enterocyte differentiation in vivo. PKCiota phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human ezrin. In CACO-2 human intestinal cells in culture, PKCiota co-immunoprecipitated with ezrin and was knocked down by shRNA expression. The resulting phenotype showed a modest decrease in total ezrin, but a steep decrease in T567 phosphorylation. The PKCiota-depleted cells showed fewer and shorter microvilli and redistribution of the PKA regulatory subunit. Expression of a dominant-negative form of PKCiota also decreased T567-P signal, and expression of a constitutively active PKCiota mutant showed depolarized distribution of T567-P. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKCiota is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.

Calcium-mediated, purinergic stimulation and polarized localization of calcium-sensitive adenylyl cyclase isoforms in human airway epithelia.

Purinergic stimulation of human airway epithelia results in a prolonged increase in ciliary beat frequency that depends on calcium-mediated cAMP production [Lieb, T., Wijkstrom Frei, C., Frohock, J.I., Bookman, R.J. and Salathe, M. (2002) Prolonged increase in ciliary beat frequency after short-term purinergic stimulation in human airway epithelial cells. J. Physiol. (Lond.) 538, 633-646]. Here, fully differentiated human airway epithelial cells in culture are shown to express calcium-stimulated transmembrane adenylyl cyclase (tmAC) isoforms (types 1, 3, and 8) by reverse transcription polymerase chain reaction. Immunohistochemistry of tracheal sections and fully differentiated airway epithelial cell cultures revealed polarized expression of these tmACs, with types 1 and 8 localized to the apical membrane and thus at the position required for ciliary regulation. Real-time, ciliated-cell specific cAMP production by tmACs upon apical, purinergic stimulation with UTP was confirmed using fluorescent energy resonance transfer between fluorescently tagged PKA subunits.

Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP.

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO(2)/HCO(3)(-) during exacerbations of airway diseases, but the role of CO(2)/HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO(2)/HCO(3)(-)-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO(2)/HCO(3)(-)-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO(2)/HCO(3)(-) increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.

Control of core 2 beta1,6 N-acetylglucosaminyltransferase-I transcription by Sp1 in lymphocytes and epithelial cells.

Core 2 beta1,6 N-acetylglucosaminyltransferase-I (C2GnT-I) catalyzes the synthesis of one of the major core structures in GalNAc alpha-Ser/Thr O-linked oligosaccharides, the core 2 branch. The production of the core 2 branch is required for the synthesis of glycoforms that are important for the cellular functions of lymphocytes, mucin-producing epithelial cells and other cell types. Therefore, proper molecular control of C2GnT-I expression is very important for different types of cells. C2GnT-I is transcribed from 4 promoters, with promoter 2 being the major promoter. C2GnT-I promoter 2 lacks a TATA box and is very GC rich. In this study, the analysis of this promoter finds that the transcription factor Sp1 is essential for transcription of C2GnT-I in both mesodermally derived T-cells (Jurkat) and in endodermal mucin producing epithelial cells (NCI H292). In Jurkat cells, all nine of the Sp1 binding sites within the minimal promoter region contribute to transcription, and there is a linear relationship between the number of Sp1 sites and the transcriptional activity of the promoter. In NCI H292 cells, only three of these Sp1 binding sites are required for transcription from promoter 2. Chromatin immunoprecipitation confirms that Sp1 binds to promoter 2 in NCI H292 cells in vivo.