Am J Physiol Cell Physiol AJP: Lung Cellular and Molecular Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 293: C1445-C1454, 2007. First published August 29, 2007; doi:10.1152/ajpcell.00051.2007
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/C1445    most recent
00051.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ehre, C.
Right arrow Articles by Davis, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ehre, C.
Right arrow Articles by Davis, C. W.

RECEPTORS AND SIGNAL TRANSDUCTION

nPKC{varepsilon}, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Camille Ehre,1 Yunxiang Zhu,1 Lubna H. Abdullah,1 John Olsen,1 Keiichi I. Nakayama,3 Keiko Nakayama,3 Robert O. Messing,4 and C. William Davis1,2

1Cystic Fibrosis/Pulmonary Research and Treatment Center, 2Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina; 3Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoda, Japan; and 4Ernest Gallo Clinic & Research Center, University of California-San Franciso, San Francisco, California

Submitted 3 February 2007 ; accepted in final form 20 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Airway goblet cell mucin secretion is controlled by agonist activation of P2Y2 purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP3), diacylglycerol, Ca2+ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC{alpha}, nPKC{delta}, nPKC{varepsilon}, and nPKC{eta}; of these, only nPKC{delta} translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149–L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC{alpha}, nPKC{delta}, and nPKC{eta} had the same levels of ATP{gamma}S- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC{varepsilon} (14.6 and 23.5%, for ATP{gamma}S and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC{varepsilon} exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATP{gamma}S-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y2-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC{delta} knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC{varepsilon} KO mice relative to its WT littermates. We conclude that nPKC{varepsilon} is the effector isoform downstream of P2Y2-R activation in the goblet cell secretory response. The translocation of nPKC{delta} observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology.

protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung

OLIGOMERIC MUCINS provide the molecular scaffolding of mucus (58), which in the lung is crucial for defense against inhaled particulates and pathogens by the mucociliary clearance system (34). In healthy subjects, oligomeric mucins are produced and secreted from goblet cells in the superficial epithelium of the airways and from submucosal glands (20), in quantities sufficient for normal mucociliary clearance. The conditions of infection and inflammation in obstructive lung disease (chronic bronchitis, emphysema, cystic fibrosis, asthma), however, drive metaplastic, hyperplasic, and hypertrophic processes, resulting in mucus hyperproduction and plugged airways. In humans and possibly other primates, innervation of the superficial epithelium is sparse and regulation of goblet cell secretion is effected primarily through local mediators (see Ref. 15). Two such mediators, the nucleotides ATP and UTP, appear to be major agonists in goblet cell mucin secretion acting via P2Y2 purinoceptors (P2Y2-Rs) (15, 17, 31, 62). P2Y2-R activation by agonist elicits a coupling with PLC via Gq, production of the primary intracellular messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3) from phosphatidylinositol-4,5-bisphosphate (PIP2), followed by activation of protein kinase C (PKC) and mobilization of Ca2+ (62). Pharmacological studies in goblet cells have shown that mucin secretion can be stimulated via intracellular messenger pathways by the DAG mimic phorbol-1,2-myristate-13-acetate (PMA) (15, 17, 30, 31), by IP3 (52), and by the Ca2+-mobilizing agents ionomycin and thapsigargin (15, 30, 51). Each of these signaling molecules is thought to activate or modulate a series of protein-mediated events, ultimately to elicit exocytosis of mucin secretory granules. Extrapolating from other secretory cells, the likely sequence regulated by DAG, PKC, and Ca2+ are the disassembly of actin filaments to allow granule access to the plasma membrane (18, 60), docking and priming of the granule (1, 7), and following a final Ca2+ trigger exocytosis (8, 10, 52). Little specific information is available regarding effector molecules regulating this pathway downstream of messenger production in goblet cells. In this paper, we focus on identification of the PKC isoform(s) participating in agonist-induced mucin release.

The members of the PKC family have been implicated in signaling pathways of a wide variety of cellular processes, including regulated mucin secretion (15, 17, 30, 31). Previously, we have shown that membrane-associated PKC activity is enhanced by P2Y2 agonists and PMA in the mucin-secreting SPOC1 cell line (1), indicating a likely PKC participation in regulated mucin secretion. The PKC family of serine-threonine kinases is divided into three subfamilies (see Refs. 44 and 45): conventional (cPKC{alpha}, cPKCβI, cPKCβII, and cPKC{gamma}), novel (nPKC{delta}, nPKC{varepsilon}, nPKC{eta}, and nPKC{theta}), and atypical (aPKC{zeta} and aPKC{iota}/{lambda}). These subdivisions are based primarily on the characteristics of two conserved NH2-terminal domains. C1 domains in cPKCs and nPKCs (as they are in most other C1 domain-containing proteins) are responsible for binding DAG (and phorbol esters). C2 domains in cPKCs bind phospholipids in a Ca2+-dependent manner, but in nPKCs they are Ca2+-independent and have diverse binding functions. Thus cPKCs are activated by both DAG and Ca2+ and nPKCs by DAG but not Ca2+. aPKCs are both DAG and Ca2+ insensitive: they lack C2 domains and their C1 domain is unique among all protein C1 domains studied in being DAG insensitive (44, 45). For these reasons, aPKCs are not considered in this study.

In a previous study (1), cPKC{alpha}, nPKC{delta}, nPKC{varepsilon}, nPKC{eta}, and aPKC{zeta} were identified in SPOC1 cell extracts at both the mRNA and protein levels. Of these, cPKC{alpha} and nPKC{delta} translocated to the membrane fraction under PMA stimulation. Only nPKC{delta} translocated to the membrane in response to agonist, and because the response was concentration dependent, we concluded tentatively that it was the PKC effector isoform in regulated mucin secretion. Even though such a correlation is consistent with causality, alone it offers an insufficient level of proof. Additionally, only cPKCβI and nPKC{varepsilon} have been implicated strongly in the regulation of exocytotic secretion (5, 9, 25, 26, 28, 37, 42), whereas nPKC{delta} has been associated primarily with cell differentiation and apoptosis (27, 57). Hence, in the present study we have pursued the identity of the PKC isoform active in regulated mucin secretion, using pharmacological, molecular, and genetic manipulations to alter PKC isoform activity or expression levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DMEM/F12 culture medium was obtained from GIBCO-BRL (Gaithersburg, MD), and the supplements were from Collaborative Research (Bedford, MD). PMA, Gö6976, and Gö6983 were obtained from Calbiochem (LaJolla, CA), and ATP{gamma}S was from Roche Applied Science (Indianapolis, IN). Unless otherwise noted, all other chemicals were purchased from Sigma Chemical (St. Louis, MO).

SPOC1 cell culture, experimental procedure, and mucin enzyme-linked lectin assay. SPOC1 cells, a mucin-secreting cell line derived from rat trachea, were grown as described previously in a fully defined medium (50) for 2–3 wk in tissue culture plastic plates appropriate to the particular experiment. For mucin secretion experiments, differentiated cultures were washed 3 x 30 min with warm DMEM/F12. For PKC inhibitor experiments, cells were pretreated with the appropriate concentration of inhibitor for 15 min as the last wash. Cultures were then exposed or not (control) to ATP{gamma}S (100 µM) to stimulate mucin secretion for 40 min, following which samples were collected from and assessed for mucin content using an enzyme-linked lectin assay (ELLA) described previously (3). Standard curves were generated from purified SPOC1 mucins applied to each plate, and the results were expressed as nanograms mucin released per culture. Importantly, in Western blots analyses of SPOC1 cell extracts, soybean agglutinin stains only high-molecular-weight glycoconjugates (3).

Subcellular fractionation and Western blot analysis. SPOC1 cell lysates were prepared for Western blot analysis following a quick wash with ice-cold phosphate-buffered saline. The cells were lysed, on ice, in a hypotonic buffer (in mM: 20 Tris·HCl, 2 EGTA, 2 EDTA, 6 β-mercaptoethanol, 0.1 protein cocktail inhibitor). The lysed cells were homogenized (~15 strokes with Potter-Elvehjem tissue grinder) at 4°C and centrifuged for 1 h at 100,000 g, and the supernatant was taken as the cytosolic fraction. The pellet was solubilized in buffer supplemented with Nonidet P-40 (1:100) detergent, incubated 30 to 60 min on ice, and centrifuged (100,000 g); this supernatant was taken as the particulate fraction. Protein concentrations were determined for all samples (BCA Protein Assay Kit, Pierce Biotechnology, Rockford, IL), following which they were frozen and stored at –70°C. Note that in this preparation, the membrane fraction includes nuclear membranes, as well as the cortical actin cytoskeleton.

Equivalent amounts of protein (15 µg/lane) were resolved by 10% SDS-PAGE and electrophoretically transferred to polyvinyldifluoride membranes. The blots were probed with PKC isoform-specific antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) diluted appropriately (1:20,000 to 1:500). Immunoreactive proteins were detected using enhanced chemiluminescence (SuperSignal West Pico Stable Kit, Pierce Biotechnology, Rockford, IL; secondary antibody purchased from Jackson ImmunoResearch, West Grove, PA).

RT-PCR. Total RNA was extracted from SPOC1 cells using the RNeasy extraction kit (cat. no. 74104, Qiagen) and stored at –80°C. Reverse transcription was achieved from 2 µg total RNA using the First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) applying oligo (dT) for mRNA probing and SuperScript II Reverse Transcriptase for polymerization, stored at –20°C. cDNA (1 µl) was amplified by PCR in a 25-µl reaction volume containing 10 mM dNTP (0.5 µl) and Taq (1 µl). For novel and conventional PKCs, the forward (5'-AARGSAGYTTTGGSAAGGT-3') and reverse (5'-TAGTCWGGRGTSCCRCAGAA-3') degenerate primers correspond to conserved regions between the isoforms of both subgroups. The 476-bp PCR product was digested for 1 h at 37°C by restriction enzymes specific for each isoform within the amplified piece of cDNA. To confirm novel and conventional PKC isoform expression, the common forward primer and the following isoform-specific reverse primers were used: for cPKC{alpha}, 5'-CGAAGTACAGCCGATCCACT-3'; for cPKCβ, 5'-GCTCCTTAGACCGGCCGACT-3'; for cPKC{gamma}, 5'-CGGGTTGTCGTCCCGGGGAA-3'; for nPKC{varepsilon}, 5'-CATGAGGGCCGATGTGACCT-3'; for nPKC{eta}, 5'-TCATCAAAAAGACGAGACTT-3', and for PKC{theta}, 5'-TATTCGTCTTGGCATCTCCT-3'.

PKC cloning, mutagenesis, and retroviral construction. CPKC{alpha}, nPKC{delta}, nPKC{varepsilon}, and nPKC{eta} cDNAs were 3'-tagged with a HA construct composed of three HA tag sequences in frame behind a Not1 site and cloned into LXPIN retrovirus expression vectors. CPKC{alpha} and nPKC{delta} cDNAs, obtained from Yoshitaka Ono (Biosignal Research Center, Kobe University, Japan), were mutated to replace the stop codon with a Not1 site and extracted by digestion with EcoRI and NotI, and the HA tag was extracted from its construct after digestion by NotI and XhoI. After pLXPIN was opened by EcoRI and XhoI, cPKC{alpha} and nPKC{delta} (~2 kb) were cloned into the vector, along with the HA tag (~0.2 kb). Full-length nPKC{varepsilon} and nPKC{eta} were obtained by RT-PCR from SPOC1 cells cDNA using Pfu polymerase (GIBCO-BRL) and by the following forward and reverse primers: for nPKC{varepsilon}, 5'-GAATTCCGGAATCCGGCGAGGAAATA-3' and 5'-GCGGCCGCCAAAGTAGGAGAAGCCTTTAAATT-3'; for nPKC{eta}, 5'-GAATTCTATCGGGGCTCCGGCAGCA-3', and 5'-GCGGCCGCGTTGCAATTCTGGTGACACATA-3'. The products were cloned using the ZeroBlunt TOPO PCR cloning kit (Invitrogen), and their nucleotide sequences were confirmed by direct DNA sequencing. NPKC{varepsilon} and nPKC{eta} were then transferred into pLXPIN with an HA tag in the same manner as for cPKC{alpha} and nPKC{delta}.

Kinase-deficient mutants of HA-tagged cPKC{alpha}, nPKC{delta}, and nPKC{varepsilon} were created the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the following primers: for cPKC{alpha} (K368R), 5'-GGAACTGTACGCCATCAGAATCCTGAAGAAGG-3'; for nPKC{delta} (K376R), 5'-GGTACTTTGCAATCAGGTACCTGAAGAAGGACGTGG-3'; and for nPKC{varepsilon} (K436R), 5'-GTCTATGCTGTGAGGGTCTTAAAGAAGGACGTC-3'. In each primer, the mutated base pair is underlined. Retroviral constructs for the mutants followed the procedure specified above for the wild-type (WT) constructs.

The PKC cDNAs encoding for HA-tagged PKCs were inserted into the multiple cloning site of the retroviral pLXPIN vector, and retroviral particles were generated as previously described (47); titers were determined to be between 5 x 105 and 5 x 106 virus particles/ml. Retroviral infection of SPOC1 cells (passage 6) was performed by exposure to 1 ml of virus-containing solution for 4 h in the presence of 8 µg/ml polybrene (Aldrich Chemical, St. Louis, MO). Neo selection for successfully infected cells started the following day and continued for about a week using G418 at a concentration of 125 µg/ml with daily media changes. PKC-overexpressing cells were used for experiments between cell passages 8 and 13.

Mice. P2Y2-R–/– and P2Y2-R+/+ 129S6/SvEv inbred mice were kindly provided by University of North Carolina colleague, Dr. Beverly Koller (Department of Genetics; 13). nPKC{delta}+/– C57/B6 mice, developed at Kobe University, Japan (43), were obtained from Dr. Mary Reyland (Department of Craniofacial Biology, University of Colorado, Denver, CO). nPKC{varepsilon}+/– C57/B6 mice, developed at the University of California, San Francisco (24), were obtained from University of North Carolina colleague Dr. Clyde Hodge (Department of Psychiatry). All animals were bred and raised at the University of North Carolina and were allowed food and water ad libitum until euthanized. All experimental procedures using mice were conducted under protocols approved by the University of North Carolina Institutional Animal Use and Care Committee.

Airway mucus metaplasia was induced in all mice used in this study through ovalbumin (OVA) sensitization and challenge (see Refs. 11 and 19). OVA, 40 µg in 0.2 ml aluminum hydroxide adjuvant (Alhydrogel), was injected intraperitoneally into each mouse on days 0, 7, and 14. On days 21 and 24, 50 µl of 2.0% OVA in PBS was instilled by aspiration into the tracheas of isoflurane-anesthetized mice. The mice were anesthetized in a closed, 4'' x 4'' cylindrical glass jar using 150 µl isoflurane. Individual mice were exposed to the vapors just sufficient to enter deep anesthesia (2–3 min, ~60 breaths/min). The mice were then removed and laid supine on an inclined platform, the tongue grasped very gently and extended forward using a pair of forceps with polyethylene tubing-covered tips, and 50 µl of OVA solution was gently pipetted into the oropharyx. With the tongue extended, the solution is aspirated quickly into the airways. This entire procedure was completed in <20 s, well within the ~1-min period of time it takes the mouse to recover from the isoflurane anesthesia; recovery from the procedure was 100%. Experimental procedures were performed 3 to 14 days following the second OVA instillation.

Tracheas were harvested from CO2-euthanized mice, cannulated at the proximal end, mounted vertically inside a short cylinder with an inner diameter just sufficient to allow the trachea to be inserted (~4 mm), connected to a peristaltic pump, and perfused at 50 µl/min with warmed 5% CO2-95% O2-equilibrated DMEM/F12. A pair of preparations were mounted on a custom bracket attached to the arm of a Gilson FC204 microtiter plate fraction collector (Middleton, WI) such that the effluent dripped from the open ends of the tracheas directly into the wells of 96-well, nonbinding, polystyrene microtiter storage plates, with collections timed at 5 min. The entire setup (pump, solutions, tissues, fraction collector) was housed in a humidified Nuaire DH Autoflow CO2 incubator (Plymouth, MN), factory customized to lie horizontally. Mucins in the collected fractions were assessed by an ELISA, using a mucin "subunit antibody" that recognizes all vertebrate oligomeric mucins (received from Dr. John Sheehan, University of North Carolina; Ref. 54, and see RESULTS). As for the mucin ELLA (see SPOC1 cell culture, experimental procedure, and mucin enzyme-linked lectin assay), standard curves were generated from purified SPOC1 mucins applied to each plate, and the results were expressed as the equivalent ng mucin released per trachea.

Statistical analysis. Data are presented as means ± SE for the specified number of SPOC1 cell cultures, each culture in a given experiment originating from a different passage, or mouse tracheas. Student's t-test was used, where appropriate, to test for differences between experimental means, with P < 0.05 taken as indicating statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of selective PKC inhibitors on agonist-induced mucin secretion. As an initial functional test of whether conventional or novel PKC isoforms modulate regulated mucin secretion, we tested the efficacy of the semispecific PKC indolocarbazole inhibitors Gö6983 and Gö6976. These inhibitors, like their archetypal PKC inhibitor parent compound staurosporine, act competitively with ATP against the active site of the enzyme (40). Gö6976 selectively inhibits the cPKCs, whereas Gö6983 inhibits most c-PKCs and nPKCs. SPOC1 cells were grown for 3 wk and preincubated for 15 min with a series of increasing inhibitor concentrations, ranging from 0.3 to 30 µM. The preincubation was followed by 40 min stimulation with ATP{gamma}S (100 µM) plus inhibitor. The results were normalized first to baseline secretion and then to an ATP{gamma}S-stimulated control (no inhibitor). Measured baseline control secretion rates for the two sets of SPOC1 cell cultures used in the experiment were 1.1 ± 0.17 and 1.0 ± 0.22 mg/ml mucin per culture, and these were elevated approximately fivefold in the two agonist controls to 5.7 ± 0.09 and 5.7 ± 0.13, respectively (n = 4 each). As with our previous work with PKC inhibitors (2), there were no measured effects of the inhibitors on baseline secretion.

As expected (2), treatment of SPOC1 cells with Gö6983 diminished agonist-induced mucin secretion in a concentration-dependent manner, with a maximum inhibition of 50.1 ± 1.2% at 30 µM (Fig. 1). These results confirmed that at lease one DAG-responsive PKC isoform is involved in the purinergic signaling pathway leading to mucin secretion in SPOC1 cells. Treatment of SPOC1 cells by the cPKC inhibitor Go6976, however, did not affect stimulated mucin secretion, even at the maximal 30 µM dose (Fig. 1). These results are consistent with our previous data (1) regarding the selective involvement of one or more nPKCs in agonist-induced mucin secretion.


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 1. Effects of semispecific protein kinase C (PKC) inhibitors on ATP{gamma}S-stimulated mucin secretion from SPOC1 cells. SPOC1 cells were pretreated (15 min) with increasing doses of Gö6983, which inhibits most cPKCs and nPKCs, or Gö6976, which selectively inhibits cPKCs, then challenged with the purinergic agonist ATP{gamma}S for 40 min in the continued presence of inhibitor. The mucin secretion data (means of triplicate determinations) were normalized first to baseline secretion and then to agonist-stimulated cells (no inhibitor) for each experiment. See RESULTS for more details. Values are means ± SE (n = 4) *P < 0.05.

 
PKC isoform expression and translocation in SPOC1 cells. Conventional RT-PCR with isoform-specific PCR primers was used to confirm our original identification of PKC isoforms expressed in SPOC1 cells using a combination of RT-PCR and restriction enzyme digestion. After total mRNA extraction from differentiated SPOC1 cells, PKC cDNAs were probed using a common forward primer recognizing all c-PKCs and nPKCs, and isoform-specific reverse primers. Analysis of the RT-PCR products revealed the expression of cPKC{alpha}, nPKC{delta}, nPKC{varepsilon}, and nPKC{eta} (Fig. 2A). Products for cPKCβ, cPKC{gamma}, nPKC{theta} mRNAs were not detected, consistent with the apparent absence of these isoforms by Western blot analysis (1).


Figure 2
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 2. PKC isoform expression and translocation in SPOC1 cells. A: PKC isoforms. Sequence-specific primers were used in the detection of PKC isoforms by RT-PCR. B: PKC translocation. SPOC1 cells were extracted at the indicated times following exposure to agonist (ATP{gamma}S, 100 µM). After 10% SDS-PAGE of the membrane fractions and transfer to nitrocellulose, the Western blot analyses were probed with PKC isoform-specific antibodies.

 
Given the uncertainties with nPKC{varepsilon} involvement in regulated mucin secretion in our previous study, we tested whether it translocated to the membrane fraction following stimulation with the purinergic agonist ATP{gamma}S comparing it with cPKC{alpha} and nPKC{delta}. Membrane fractions of SPOC1 cells collected at intervals following ATP{gamma}S stimulation were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with PKC isoform-specific antibodies (Fig. 2B). As previously described, there was no agonist-induced translocation to the membrane fraction by cPKC{alpha}, and nPKC{delta} exhibited a clear, steady increase over time. The behavior of nPKC{varepsilon} was not easy to decipher: there was no clear increase in membrane signal strength for the first 20 min, but its mobility showed a slight shift between 10 and 20 min in a manner suggestive of a phosphorylation event. Additionally, there may have been an increase in membrane-associated nPKC{varepsilon} at 40 and 60 min, long after the major effects of agonist on mucin secretion would have occurred. Hence, the data suggest that the purinergic signaling pathway activates nPKC{delta} but are equivocal regarding nPKC{varepsilon}. In independent experiments the behavior of nPKC{eta} was also reexamined, and as described previously (1), there was no apparent change in its distribution following agonist stimulation (data not shown).

Effects of overexpression PKC isoforms in SPOC1 cells. The effect of PKC isoform overexpression on regulated mucin release was tested in SPOC1 cells. Since these cells require 2–3 wk to differentiate to a full mucin secretory phenotype, we used retroviral expression vectors to overexpress cPKC{alpha}, nPKC{delta}, nPKC{varepsilon}, and nPKC{eta}, using PKC cDNAs labeled with an HA tag at the COOH-terminal of each kinase gene. Transgene expression was driven by a long-term repeat (LTR) promoter, leading to a moderate approximate twofold overexpression, relative to endogenous PKC expression (Fig. 3A). To ensure that the results were not due to an inopportune insertion of the provirus, each retroviral infection for each isoform was repeated a minimum of four times on passage 6 SPOC1 cells. SPOC1 cell secretagogue-induced mucin release from each infection was measured, after selection, on six consecutive SPOC1 cell passages for each infected line. Mucin secretion was normalized to basal mucin release for each infected cell line, and these values were reduced to a group mean. SPOC1 cells infected by an empty retroviral vector were selected and served as secretagogue-induced controls. Basal mucin release in all of the retrovirally infected SPOC1 cells was unchanged from uninfected control cells (data not shown).


Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 3. Overexpression of PKC in SPOC1 cells. A: Western blot analyses for PKC expression. Whole cell extracts from SPOC1 cells infected with retroviruses carrying cPKC{alpha}-HA, nPKC{delta}-HA, nPKC{epsilon} or an empty vector were resolved by SDS-PAGE, blotted, and probed with PKC-specific antibodies. B: effects of PKC isoform overexpression on regulated mucin secretion. SPOC1 cells stably expressing the indicated COOH-terminal HA-tagged PKC isoforms, or empty vector (Control), were exposed to ATP{gamma}S (100 µM) or phorbol-1,2-myristate-13-acetate (PMA, 300 nM) for 40 min, and the mucins secreted were assessed. Data are means ± SE for 4–7 retroviral infections and are normalized to their respective, nonstimulated controls (e.g., cPKC{alpha} cells ± secretagogue). There were no differences in baseline secretion rates between the different treatments and controls. *P < 0.05.

 
Overexpression of cPKC{alpha}-HA or nPKC{eta}-HA had no effect on stimulated mucin secretion, whether cells were challenged by ATP{gamma}S (100 µM) or by PMA (300 nM) (Fig. 3B), i.e., there was no difference in normalized, secretagogue-induced mucin release, relative to the empty-vector controls. An apparent increase in stimulated mucin secretion was observed when cells expressed nPKC{delta}-HA for both ATP{gamma}S and PMA stimulation, but in neither case were the results significant. Only the overexpression of nPKC{varepsilon}-HA increased secretagogue-induced mucin secretion significantly: these cells exhibited a mean increase of 14.6% over empty vector control cells with 100 µM ATP{gamma}S stimulation and of 23.5% with 300 nM PMA (P < 0.05). Interestingly, even at the supramaximal dose of PMA used in these studies (300 nM), no other PKC isoform overexpression induced a significant change in stimulated mucin release (Fig. 3B, right), suggesting that the role played by nPKC{varepsilon} in the regulation of mucin secretion cannot be substituted by the generalized activation of other isoforms.

Overexpression of kinase-defective PKCs in SPOC1 cells. Dominant negative mutations have long been used to indicate protein function (22), and early studies of PKC demonstrated the dominant negative effect of disabling PKC phosphorylation by mutating an invariant lysine in the catalytic domain to an arginine (5, 36, 46). In the experiments described below, retroviral infection of SPOC1 cells with cPKC{alpha}, nPKC{delta}, and nPKC{varepsilon} constructs mutated with the respective substitutions, K368R, K376R, and K436R, failed to show a dominant negative effect, likely because of the relatively low levels of overexpression afforded by our use of retroviral vectors. These kinase-deficient (KD) mutants, however, offered a powerful control, illustrating the specificity of effect of WT PKC overexpression. Procedurally, these studies were similar to those in the previous section; hence, we did not repeat the noninfected or empty vector-infected controls of Fig. 3B, and we used younger SPOC1 cell cultures, which resulted in mucin secretory responses that were less robust.

SPOC1 cells were infected with retroviruses carrying either the WT or KD mutant constructs of cPKC{alpha}, nPKC{delta}, or nPKC{varepsilon}. For cells overexpressing cPKC{alpha} and nPKC{delta} constructs, the same relative mucin secretory response was recorded for the cells expressing the WT or KD variant of each gene when stimulated with ATP{gamma}S or PMA (Fig. 4). Cells expressing the WT and KD variants of cPKC{alpha} or nPKC{delta} and stimulated with ATP{gamma}S exhibited about a 1.5-fold increase over nonstimulated controls, whereas PMA-stimulated cells exhibited a 2.5- to 3-fold response. We interpret the lack of difference between the WT and KD cPKC{alpha} and nPKC{delta} overexpressing cells to indicate that the cellular response elicited by ATP{gamma}S and PMA is most likely attributable to the endogenous PKC isoform, not that expressed heterologously (c.f., Fig. 3B). In contrast, cells overexpressing KD-nPKC{varepsilon} had a reduced mucin secretory response of 42% with ATP{gamma}S (100 µM) stimulation, and of 49% with PMA (100 nM) stimulation, relative to cells overexpressing the WT-nPKC{varepsilon} construct. Also note that cells overexpressing WT-nPKC{varepsilon} had more than twice the response to ATP{gamma}S, relative to cells overexpressing cPKC{alpha} or nPKC{delta}. The response of WT-nPKC{varepsilon} overexpressing cells to PMA was also higher, relative to cells expressing cPKC{alpha} or nPKC{delta} constructs, but the differences were somewhat less than with ATP{gamma}S. Western blot analysis (Fig. 4B) indicated that the total levels of nPKC{varepsilon} in cells overexpressing the WT and KD constructs were similar, and that both levels were greater than cellular content of endogenous nPKC{varepsilon}. This experiment verifies the kinase specificity of nPKC{varepsilon} and confirms that the phosphorylation of a nPKC{varepsilon} substrate is necessary for the increase in mucin secretion observed in cells overexpressing WT-nPKC{varepsilon}.


Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 4. Overexpression of kinase-deficient (KD) PKCs. A: effects of KD-PKC mutant overexpression on SPOC1 cell mucin secretion. SPOC1 cells were infected with wild-type (WT) or KD-PKC mutants, the latter being generated by a point mutation in the respective ATP-binding sites. WT and KD PKC isoform overexpressing SPOC1 cells were exposed to ATP{gamma}S (100 µM) or PMA (100 nM) for 40 min and mucins assessed. Data are means ± SE; n = 3–4 infections, normalized to unstimulated WT cells for each culture. *P < 0.05. B: WT and KD nPKC{varepsilon} overexpression. Western blot analysis, prepared as for Fig. 3, was probed with the anti-nPKC{varepsilon} antibody on whole cell lysates from uninfected (Control) and WT and KD nPKC{varepsilon}-overexpressing cells.

 
Mucin secretory response to agonist by mouse trachea. The data above implicate nPKC{varepsilon} as the active PKC isoform in regulated mucin secretion, despite its translocation behavior in the traditional Western blot anaysis experiments. With the use of RT-PCR, the same PKC isoform mRNAs were expressed in whole tracheal extracts (data not shown) as in SPOC1 cells (Fig. 2) plus cPKCβ. Mucin secretion was studied in tracheas harvested from mice that had been sensitized and challenged with OVA to induce goblet cell metaplasia (19, 35; Fig. 5A). All the mice used in these studies exhibited similar degrees of mucous metaplasia (data not shown). Tracheas harvested from euthanized mice were cannulated at the proximal end, mounted vertically on the arm of a fraction collector, and perfused with DMEM/F12, at 37°C and 5% CO2, with the distal end positioned over a 96-well collection plate. The 5-min, 50-µl fractions collected were assessed for mucins by ELISA (see Fig. 5C, inset), using a mucin subunit antibody that recognizes vertebrate oligomeric mucins (54; Fig. 5B). Figure 5D shows the time course of mucin release in a mounting control experiment with WT mice in which the tracheas were not exposed to agonist. Not unexpectedly, mucin secretion was stimulated by the handling of the tissue during its harvest and mounting, but it waned over a period of ~40 min to a stable steady state that lasted for the duration of the experiment. From these data, a 2-h equilibration period was selected for the subsequent experiments, following which a 40-min baseline collection period (marked by the bar labeled Baseline in Fig. 5D) preceded introduction of ATP{gamma}S into the perfusate. To test whether changes in agonist-induced, tracheal mucin secretory responses could be detected with our experimental system in a control, gene-targeted mouse model, we took advantage of the major role the P2Y2-R plays in regulated mucin secretion from airway goblet cells (13). Figure 5E shows the time course and integrated mucin secretory responses of ATP{gamma}S-induced release of mucins from tracheas of P2Y2-R+/+ (WT) and P2Y2-R–/– (KO) mice. Agonist-stimulated mucin release was decreased substantially in the P2Y2-R KO mouse tracheas: the integrated secretory response of the KO tracheas was reduced by 82%, relative to WT. Presumably, the small response of the KO tracheas to ATP{gamma}S was mediated by the P2Y4 or other P2Y receptor (12).


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. Purinergic stimulation of mucin secretion in ovalbumin (OVA)-challenged mice. A: periodic acid-Schiffs (PAs)-stained tracheal epithelium. Tracheas were harvested and sections stained with PAS from control mice (OVA–) or from mice (OVA+) that were sensitized to OVA, 7 days after OVA challenge by tracheal instillation. B: agarose Western blot analyses of human and mouse mucins. Mucins from human bronchial epithelial cells (HBE: lanes 1 and 2) and whole mouse lung extract (mLung: lanes 3–5) were resolved on a 1% agarose gel, and the blot was probed with a human MUC5AC (see Ref. 33) or mucin subunit antibody. C: mucin ELISA. Standard curve using purified SPOC1 cell mucin standards and the mucin subunit antibody (means ± SE, n = 6). The line represents a standard 4-parameter logistic fit to the data. D: equilibration of perfused tracheas from OVA+ mice. Tracheas harvested from WT mice 7 days after OVA challenge were mounted and perfused for 3 h at 37°C, 5% CO2, and the perfusate was collected and assessed for mucins by ELISA. (Baseline, stable period selected for determination of baseline mucin secretion in subsequent experiments). E: mucin secretory responses to agonist by WT and P2Y2-R KO mice. Left: time course of mucin secretory responses of isolated, perfused tracheas to ATP{gamma}S (100 µM). After a 2-h equilibration, perfusate was collected during 40 min baseline and agonist (ATP{gamma}S, 100 µM) exposure periods; data were normalized to the mean of the baseline period. Right: normalized integrated mucin secretory responses calculated as the suprabasal area under each curve during the ATP{gamma}S exposure in left-hand graph. Data are means ± SE; n = 4. *P < 0.05.

 
Since our previous (1) and present results (Fig. 2) indicated that purinergic stimulation of goblet cells activates nPKC{delta}, we tested the mucin secretory response of the nPKC{delta} KO mouse to ATP{gamma}S. As shown in Fig. 6, the secretory responses of littermate, WT, and KO tracheas were nearly identical. Integrated mucin secretion was slightly diminished (–28%) in the KO tracheas, but statistically it was indistinguishable from WT. Consistent with the SPOC1 cell nPKC{delta} overexpression data of Fig. 3, this isoform does not appear to function in agonist-induced mucin secretion from goblet cells.


Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 6. Mucin secretory responses to agonist in WT and nPKC{delta} KO mice. Left: time course of mucin secretory responses to ATP{gamma}S (100 µM) during the period indicated by the horizontal line bar; data normalized to baseline period (Note: the 1st two points were excluded from the calculation of the mean baseline for each set of mice). Right: normalized, integrated mucin secretory responses (area under each curve during ATP{gamma}S exposure; left). Data are means ± SE; n = 4.

 
Finally, because nPKC{varepsilon} overexpression in SPOC1 cells caused an increased mucin secretory response to agonist (Fig. 3), we tested the secretory responses of tracheas removed from nPKC{varepsilon} KO mice. Figure 7 shows that the nPKC{varepsilon} KO tracheas exhibited a vastly reduced ATP{gamma}S-induced mucin secretory response, relative to WT littermate controls. In fact, the mean integrated mucin secretory response was nearly zero, relative to baseline, but the individual responses were variable, with some tracheas exhibiting small, suprabasal mucin release following exposure to agonist. Consistent with the nPKC{varepsilon} SPOC1 cell overexpression data of Figs. 3 and 4, ablation of the nPKC{varepsilon} gene in mice results in a diminished ability of airway goblet cells to mount an effective mucin secretory response to purinergic agonist.


Figure 7
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 7. Mucin secretory responses to agonist in WT and nPKC{varepsilon} KO mice. Left: time course of mucin secretory responses to ATP{gamma}S (100 µM) during the period indicated by bar; data normalized to baseline period. Right: normalized integrated mucin secretory responses (area under each curve during ATP{gamma}S exposure; left). Data are means ± SE; n = 4 *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The regulation of mucin secretion from airway epithelium has been intensively studied at the receptor level for the past decade and a half. Studies using apical purinergic agonists to stimulate mucin release in a wide variety of human (12, 30) and animal (16, 31) experimental models have demonstrated that ATP and UTP stimulate mucin secretion, most likely via an apically localized purinoceptor P2Y2-R. In addition to these pharmacological indications, P2Y2-R mRNA has been identified in SPOC1 cell (3) and in hamster (32) airway goblet cell models. Though it was intended primarily as a control in the present study, our results showing a major loss of ATP{gamma}S-stimulated mucin secretion in P2Y2-R KO mouse tracheas (Fig. 5) clearly substantiates the hypothesis that this receptor is a major factor in luminal regulation of mucin secretion in the airways by purinergic agonists.

Activated P2Y-Rs couple via G{alpha}q to phospholipase C-β to cause the production of the cellular messengers IP3 and DAG from PIP2, leading to Ca2+ mobilization and PKC activation (62). Pharmacological studies conducted over many years with ionomycin and PMA in goblet cell models have suggested consistently that Ca2+ and PKC are important in regulated mucin secretion (16, 31), but mechanistic details remain elusive. Previously, we showed that nPKC{delta} activation in SPOC1 cells correlates positively with regulated mucin secretion, a result that identified this isoform as a potential P2Y2-R downstream effector (1). In this study we sought a more rigorous level of scrutiny. Studies of PKC can be problematic in at least two ways. First, the use of PMA as a generalized activator of PKC has come under question in recent years as other proteins with C1 domains have been identified, several of which may subserve important functions in the secretory pathway (6, 29). Indeed, we have shown that PKC activation in SPOC1 cells is maximal at 30 nM PMA, but an order of magnitude higher concentration (300 nM) is required to achieve a maximal mucin secretory response (1, 2). The PKC-independent effects of PMA on mucin release may relate to the exocytic priming protein MUNC13-2 (61) expressed in SPOC1 cells (1). This notion was supported by the recent finding that the stimulatory effects of PMA at high concentrations on mucin secretion is resistant to inhibition by BAPTA, possibly due to the close molecular proximity of MUNC13–2 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex (52). The second problematic aspect of PKC research is the identification of specific isoforms using inhibitors, reagents generally not as specific as their reputation and common use might suggest. There is no better illustration of this problem than the use of the widely popular inhibitor rottlerin to identify nPKC{delta} function (21). Not only is this inhibitor known to inhibit other kinases (14) and other enzymes (41), it also uncouples mitochondria (56). Consequently, after we verified that a novel isoform mediates the purinergic-related actions of PKC in SPOC1 cell mucin secretion (Fig. 1), along with the expression of nPKC{delta} and nPKC{varepsilon} protein (Fig. 2), we turned to retroviral gene expression tools and mouse molecular genetics to pursue the PKC isoform identity question in airway goblet cells.

With the use of retroviral, LTR promoter-driven constructs, overexpression of all the conventional and novel isoforms expressed in SPOC1 cells (cPKC{alpha}, nPKC{delta}, nPKC{varepsilon}, and nPKC{eta}) led to increases in ATP{gamma}S- and PMA-stimulated mucin secretion in only a single instance, namely, by overexpression of nPKC{varepsilon} (Fig. 3B). The increase in stimulated mucin release by nPKC{varepsilon} overexpression was modest (~14–23%), likely because the degree of PKC overexpression was also modest (Fig. 3A). Importantly, cells overexpressing cPKC{alpha}, nPKC{delta}, or nPKC{eta} exhibited the same levels of PMA-stimulated mucin secretion as control, empty vector-expressing cells, indicating that the stimulation observed was due to an endogenous PKC isoform. That this isoform is likely nPKC{varepsilon} was also indicated by the observation that overexpression of a kinase-deficient nPKC{varepsilon} mutant was ineffective in stimulating mucin secretion from secretagogue-exposed cells, relative to the levels of stimulation observed in cells overexpressing WT nPKC{varepsilon} (Fig. 4). Cells overexpressing kinase-deficient mutants of cPKC{alpha} and nPKC{delta}, in contrast, had the same levels of PMA-stimulated mucin release as their WT kinase-overexpressing cells, as well as empty vector control-expressing cells, again indicating a lack of participation by these isoforms in regulated mucin secretion.

Interestingly, the PKC translocation data in Fig. 2 verified our earlier observation that purinergic stimulation of SPOC1 cells leads to a classical activation of nPKC{delta} (1); however, nPKC{varepsilon} did not appear to be similarly activated (Fig. 2B). The changes observed in membrane-associated nPKC{varepsilon}, a mobility shift between 10 and 20 min and an apparent increase in quantity after 40 min, occurred well after the known effects of agonist on mucin secretion (Fig. 2B and Refs. 1 and 3). The apparent absence of nPKC{varepsilon} translocation may be explained by its unique actin-binding domain (49), because nPKC{varepsilon} bound to cortical actin would appear in the membrane fraction under the conditions of our experiments. It is also possible that nPKC{varepsilon} does translocate to the membrane in a classical manner, but the fraction of total nPKC{varepsilon} that translocates is low. Because of the apparent lack of translocation and the modest increases in stimulated mucin secretion from nPKC{varepsilon} overexpressing SPOC1 cells, we turned to the use of gene-targeted mouse models to test independently whether this isoform, not nPKC{delta}, is the primary PKC effector active in regulated mucin secretion downstream of purinergic activation. Consistent with our findings of nPKC{delta} and nPKC{varepsilon} mRNA expression in mouse tracheal extracts, both isoforms have been shown by immunohistochemistry to be expressed in airway epithelium (38, 59).

After validating a perfusion model for regulated mucin secretion using tracheas from OVA-sensitized and challenged control and P2Y2-R KO mice (Fig. 5), we tested the effects of agonist (ATP{gamma}S) on tracheas from nPKC{delta} KO mice and their WT littermate controls. Consistent with the lack of effect of overexpressing nPKC{delta} in SPOC1 cells, agonist-stimulated mucin secretion was unchanged in the tracheas of nPKC{delta} KO mice, relative to WT controls (Fig. 6). In contrast to the lack of effect of nPKC{delta} deficiency, the release of mucins from nPKC{varepsilon} KO mouse tracheas following ATP{gamma}S challenge was largely abolished (Fig. 7). Hence, the data from the different models used in this study are consistent: overexpression of nPKC{delta} in SPOC1 cells or ablation of the gene in mice has no observable effect on agonist-induced mucin secretion, whereas overexpression or ablation of nPKC{varepsilon} leads to increases or decreases in regulated mucin secretion, respectively. Consequently, we conclude that nPKC{varepsilon} is a downstream PKC effector for P2Y2-R signaling in airway goblet cells. The activation of nPKC{delta} that also occurs (Fig. 2) likely relates not to regulated mucin granule exocytosis but to some other cellular function in purinergically stimulated cells, e.g., increased mucin gene expression (23, 53, 63). Importantly, the data are also consistent with the notion that PKC isoforms have well defined, nonredundant cellular functions, and suggest that the selective targeting of the different isoforms plays a crucial role in determining the specificity of cellular effects (see Ref. 44).

nPKC{varepsilon} appears to be an important downstream effector for agonist-regulated exocytosis in many secretory cells. For instance, overexpression of nPKC{varepsilon} caused increased secretion of prolactin from GH4 cells (5) and of insulin from pancreatic β cells (26). Conversely, overexpression of a dominant negative nPKC{varepsilon} mutant or downregulation of nPKC{varepsilon} using siRNAs diminished or abolished stimulated secretion from HT29 cells (25), pancreatic β cells (42), lacrimal gland cells (28), and chromaffin cells (26). Other PKC isoforms have been implicated in regulated secretion from other cells [e.g., cPKCβ (9, 37)], but the reports generally are neither as rigorous in approach, as numerous, nor as consistently positive in their identification as for nPKC{varepsilon}.

PKC activation of myristoylated alanine-rich C kinase substrate (MARCKS) and the subsequent remodeling of cortical actin are events postulated to precede secretory granule-plasma membrane docking before exocytosis in activated secretory cells (39, 60), including airway goblet cells (18, 55). Interestingly, nPKC{varepsilon} has been shown to phosphorylate MARCKS in GH4 and chromaffin cells (4, 48), and the isoform is essential for cortical actin remodeling in lacrimal gland cells (28). Since nPKC{varepsilon} appears to be the effector isoform downstream of purinoceptor-stimulated mucin secretion, it is likely performing a similar function in airway goblet cells. Formally demonstrating the relationship between nPKC{varepsilon} activation and actin remodeling, as well as the docking, priming, and exocytosis of mucin secretory granules, however, remain major challenges for the future.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by grants from La Foundation Vaincre la Mucoviscidose, Paris (to C. Ehre) and from the National Heart, Lung, and Blood Institute Grant HL-063756 and by the North American Cystic Fibrosis Foundation (to C. W. Davis).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. W. Davis, CF/Pulmonary Research & Treatment Center, 6009 Thurston Bowles, CB 7248, Univ. of North Carolina, Chapel Hill, NC 27599-7248 (e-mail: cwdavis{at}med.unc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Abdullah LH, Bundy JT, Ehre C, Davis CW. Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA. Am J Physiol Lung Cell Mol Physiol 285: L149–L160, 2003.[Abstract/Free Full Text]

2. Abdullah LH, Conway JD, Cohn JA, Davis CW. Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells. Am J Physiol Lung Cell Mol Physiol 273: L201–L210, 1997.[Abstract/Free Full Text]

3. Abdullah LH, Davis SW, Burch L, Yamauchi M, Randell SH, Nettesheim P, Davis CW. P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways. Biochem J 316: 943–951, 1996.[Web of Science][Medline]

4. Akita Y, Kawasaki H, Ohno S, Suzuki K, Kawashima S. Involvement of protein kinase C epsilon in thyrotropin-releasing hormone-stimulated phosphorylation of the myristoylated alanine-rich C kinase substrate in rat pituitary clonal cells. Electrophoresis 21: 452–459, 2000.[CrossRef][Web of Science][Medline]

5. Akita Y, Ohno S, Yajima Y, Konno Y, Saido TC, Mizuno K, Chida K, Osada S, Kuroki T, Kawashima S. Overproduction of a Ca(2+)-independent protein kinase C isozyme, nPKC epsilon, increases the secretion of prolactin from thyrotropin-releasing hormone-stimulated rat pituitary GH4C1 cells. J Biol Chem 269: 4653–4660, 1994.[Abstract/Free Full Text]

6. Brose N, Rosenmund C. Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters. J Cell Sci 115: 4399–4411, 2002.[Abstract/Free Full Text]

7. Brose N, Rosenmund C, Rettig J. Regulation of transmitter release by Unc-13 and its homologues. Curr Opin Neurobiol 10: 303–311, 2000.[CrossRef][Web of Science][Medline]

8. Brunger AT. Structure and function of SNARE and SNARE-interacting proteins. Q Rev Biophys 38: 1–47, 2005.[Web of Science][Medline]

9. Chang EY, Szallasi Z, Acs P, Raizada V, Wolfe PC, Fewtrell C, Blumberg PM, Rivera J. Functional effects of overexpression of protein kinase C-alpha, -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3. J Immunol 159: 2624–2632, 1997.[Abstract]

10. Chapman ER. Synaptotagmin: a Ca(2+) sensor that triggers exocytosis? Nat Rev Mol Cell Biol 3: 498–508, 2002.[CrossRef][Web of Science][Medline]

11. Cohn L, Whittaker L, Niu N, Homer RJ. Cytokine regulation of mucus production in a model of allergic asthma. Novartis Found Symp 248: 201–213, 2002.[Web of Science][Medline]

12. Conway JD, Bartolotta T, Abdullah LH, Davis CW. Regulation of mucin secretion from human bronchial epithelial cells grown in murine hosted xenografts. Am J Physiol Lung Cell Mol Physiol 284: L945–L954, 2003.[Abstract/Free Full Text]

13. Cressman VL, Lazarowski E, Homolya L, Boucher RC, Koller BH, Grubb BR. Effect of loss of P2Y(2) receptor gene expression on nucleotide regulation of murine epithelial Cl(–) transport. J Biol Chem 274: 26461–26468, 1999.[Abstract/Free Full Text]

14. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][Web of Science][Medline]

15. Davis CW. Goblet cells: physiology and pharmacology. In: Airway Mucus: Basic Mechanisms and Clinical Perspectives, edited by Rogers DF and Lethem MI. Basel: Berkhauser, 1997, p. 150–177.

16. Davis CW. Regulation of mucin secretion from in vitro cellular models. Novartis Found Symp 248: 113–125, 2002.[Web of Science][Medline]

17. Davis CW, Abdullah LH. In vitro models for airways mucin secretion. Pulm Pharmacol Ther 10: 145–155, 1997.[CrossRef][Web of Science][Medline]

18. Ehre C, Rossi AH, Abdullah LH, De Pestel K, Hill S, Olsen JC, Davis CW. Barrier role of actin filaments in regulated mucin secretion from airway goblet cells. Am J Physiol Cell Physiol 288: C46–C56, 2005.[Abstract/Free Full Text]

19. Evans CM, Williams OW, Tuvim MJ, Nigam R, Mixides GP, Blackburn MR, DeMayo FJ, Burns AR, Smith C, Reynolds SD, Stripp BR, Dickey BF. Mucin is produced by clara cells in the proximal airways of antigen-challenged mice. Am J Respir Cell Mol Biol 31: 382–394, 2004.[Abstract/Free Full Text]

20. Groneberg DA, Peiser C, Dinh QT, Matthias J, Eynott PR, Heppt W, Carlstedt I, Witt C, Fischer A, Chung KF. Distribution of respiratory mucin proteins in human nasal mucosa. Laryngoscope 113: 520–524, 2003.[CrossRef][Web of Science][Medline]

21. Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G, Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun 199: 93–98, 1994.[CrossRef][Web of Science][Medline]

22. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature 329: 219–222, 1987.[CrossRef][Medline]

23. Hewson CA, Edbrooke MR, Johnston SL. PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms. J Mol Biol 344: 683–695, 2004.[CrossRef][Web of Science][Medline]

24. Hodge CW, Raber J, McMahon T, Walter H, Sanchez-Perez AM, Olive MF, Mehmert K, Morrow AL, Messing RO. Decreased anxiety-like behavior, reduced stress hormones, and neurosteroid supersensitivity in mice lacking protein kinase Cepsilon. J Clin Invest 110: 1003–1010, 2002.[CrossRef][Web of Science][Medline]

25. Hong DH, Petrovics G, Anderson WB, Forstner J, Forstner G. Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-epsilon. Am J Physiol Gastrointest Liver Physiol 277: G1041–G1047, 1999.[Abstract/Free Full Text]

26. Hoy M, Berggren PO, Gromada J. Involvement of protein kinase C-epsilon in inositol hexakisphosphate-induced exocytosis in mouse pancreatic beta-cells. J Biol Chem 278: 35168–35171, 2003.[Abstract/Free Full Text]

27. Jackson DN, Foster DA. The enigmatic protein kinase Cdelta: complex roles in cell proliferation and survival. FASEB J 18: 627–636, 2004.[Abstract/Free Full Text]

28. Jerdeva GV, Yarber FA, Trousdale MD, Rhodes CJ, Okamoto CT, Dartt DA, and Hamm-Alvarez SF. Dominant-negative PKC-{varepsilon} impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini. Am J Physiol Cell Physiol 289: C1052–C1068, 2005.[Abstract/Free Full Text]

29. Kazanietz MG. Novel "nonkinase" phorbol ester receptors: the C1 domain connection. Mol Pharmacol 61: 759–767, 2002.[Abstract/Free Full Text]

30. Kemp PA, Sugar RA, Jackson AD. Nucleotide-mediated mucin secretion from differentiated human bronchial epithelial cells. Am J Respir Cell Mol Biol 31: 446–455, 2004.[Abstract/Free Full Text]

31. Kim KC, Hisatsune A, Kim dJ, Miyata T. Pharmacology of airway goblet cell mucin release. J Pharm Sci 92: 301–307, 2003.[CrossRef][Web of Science]

32. Kim KC, Park HR, Shin CY, Akiyama T, Ko KH. Nucleotide-induced mucin release from primary hamster tracheal surface epithelial cells involves the P2u purinoceptor. Eur Respir J 9: 542–548, 1996.[Abstract]

33. Kirkham S, Sheehan JK, Knight D, Richardson PS, Thornton DJ. Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochem J 361: 537–546, 2002.[CrossRef][Web of Science][Medline]

34. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 109: 571–577, 2002.[CrossRef][Web of Science][Medline]

35. Kung TT, Jones H, Adams GK, III, Umland SP, Kreutner W, Egan RW, Chapman RW, Watnick AS. Characterization of a murine model of allergic pulmonary inflammation. Int Arch Allergy Immunol 105: 83–90, 1994.[CrossRef][Web of Science][Medline]

36. Li W, Yu JC, Shin DY, Pierce JH. Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity. J Biol Chem 270: 8311–8318, 1995.[Abstract/Free Full Text]

37. Long A, Kelleher D, Lynch S, Volkov Y. Cutting edge: protein kinase C beta expression is critical for export of Il-2 from T cells. J Immunol 167: 636–640, 2001.[Abstract/Free Full Text]

38. Lounsbury KM, Stern M, Taatjes D, Jaken S, Mossman BT. Increased localization and substrate activation of protein kinase C delta in lung epithelial cells following exposure to asbestos. Am J Pathol 160: 1991–2000, 2002.[Abstract/Free Full Text]

39. Malacombe M, Bader MF, Gasman S. Exocytosis in neuroendocrine cells: new tasks for actin. Biochim Biophys Acta 1763: 1175–1183, 2006.[Medline]

40. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J Biol Chem 268: 9194–9197, 1993.[Abstract/Free Full Text]

41. McGovern SL, Shoichet BK. Kinase inhibitors: not just for kinases anymore. J Med Chem 46: 1478–1483, 2003.[CrossRef][Web of Science][Medline]

42. Mendez CF, Leibiger IB, Leibiger B, Hoy M, Gromada J, Berggren PO, Bertorello AM. Rapid association of protein kinase C-epsilon with insulin granules is essential for insulin exocytosis. J Biol Chem 278: 44753–44757, 2003.[Abstract/Free Full Text]

43. Miyamoto A, Nakayama K, Imaki H, Hirose S, Jiang Y, Abe M, Tsukiyama T, Nagahama H, Ohno S, Hatakeyama S, Nakayama KI. Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cdelta. Nature 416: 865–869, 2002.[CrossRef][Medline]

44. Newton AC. Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev 101: 2353–2364, 2001.[CrossRef][Web of Science][Medline]

45. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9: 484–496, 1995.[Abstract]

46. Ohno S, Konno Y, Akita Y, Yano A, Suzuki K. A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation. J Biol Chem 265: 6296–6300, 1990.[Abstract/Free Full Text]

47. Olsen JC, Sechelski J. Use of sodium butyrate to enhance production of retroviral vectors expressing CFTR cDNA. Hum Gene Ther 6: 1195–1202, 1995.[Web of Science][Medline]

48. Park YS, Hur EM, Choi BH, Kwak E, Jun DJ, Park SJ, Kim KT. Involvement of protein kinase C-epsilon in activity-dependent potentiation of large dense-core vesicle exocytosis in chromaffin cells. J Neurosci 26: 8999–9005, 2006.[Abstract/Free Full Text]

49. Prekeris R, Hernandez RM, Mayhew MW, White MK, Terrian DM. Molecular analysis of the interactions between protein kinase C-epsilon and filamentous actin. J Biol Chem 273: 26790–26798, 1998.[Abstract/Free Full Text]

50. Randell SH, Liu JY, Ferriola PC, Kaartinen L, Doherty MM, Davis CW, Nettesheim P. Mucin production by SPOC1 cells–an immortalized rat tracheal epithelial cell line. Am J Respir Cell Mol Biol 14: 146–154, 1996.[Abstract]

51. Rossi AH, Salmon WC, Chua M, Davis CW. Calcium signaling in human airway goblet cells following purinergic activation. Am J Physiol Lung Cell Mol Physiol 292: L92–L98, 2007.[Abstract/Free Full Text]

52. Rossi AH, Sears PR, Davis CW. Ca2+ dependency of "Ca2+-independent" exocytosis in SPOC1 airway goblet cells. J Physiol 559: 555–565, 2004.[Abstract/Free Full Text]

53. Shao MX, Nadel JA. Dual oxidase 1-dependent MUC5AC mucin expression in cultured human airway epithelial cells. Proc Natl Acad Sci USA 102: 767–772, 2005.[Abstract/Free Full Text]

54. Sheehan JK, Boot-Handford RP, Chantler E, Carlstedt I, Thornton DJ. Evidence for shared epitopes within the 'naked' protein domains of human mucus glycoproteins. A study performed by using polyclonal antibodies and electron microscopy. Biochem J 274: 293–296, 1991.[Web of Science][Medline]

55. Singer M, Martin LD, Vargaftig BB, Park J, Gruber AD, Li Y, Adler KB. A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma. Nat Med 10: 193–196, 2004.[CrossRef][Web of Science][Medline]

56. Soltoff SP. Rottlerin is a mitochondrial uncoupler that decreases cellular ATP levels and indirectly blocks protein kinase Cdelta tyrosine phosphorylation. J Biol Chem 276: 37986–37992, 2001.[Abstract/Free Full Text]

57. Steinberg SF. Distinctive activation mechanisms and functions for protein kinase Cdelta. Biochem J 384: 449–459, 2004.[CrossRef][Web of Science][Medline]

58. Thornton DJ, Sheehan JK. From mucins to mucus: toward a more coherent understanding of this essential barrier. Proc Am Thorac Soc 1: 54–61, 2004.[Abstract/Free Full Text]

59. Tourkina E, Gooz P, Oates JC, Ludwicka-Bradley A, Silver RM, Hoffman S. Curcumin-induced apoptosis in scleroderma lung fibroblasts: role of protein kinase cepsilon. Am J Respir Cell Mol Biol 31: 28–35, 2004.[Abstract/Free Full Text]

60. Trifaro JM, Lejen T, Rose SD, Pene TD, Barkar ND, Seward EP. Pathways that control cortical F-actin dynamics during secretion. Neurochem Res 27: 1371–1385, 2002.[CrossRef][Web of Science][Medline]

61. Varoqueaux F, Sigler A, Rhee JS, Brose N, Enk C, Reim K, Rosenmund C. Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming. Proc Natl Acad Sci USA 99: 9037–9042, 2002.[Abstract/Free Full Text]

62. von Kugelgen I. Pharmacological profiles of cloned mammalian P2Y-receptor subtypes. Pharmacol Ther 110: 415–432, 2006.[CrossRef][Web of Science][Medline]

63. Yuan-Chen WD, Wu R, Reddy SP, Lee YC, Chang MM. Distinctive epidermal growth factor receptor/extracellular regulated kinase-independent and -dependent signaling pathways in the induction of airway mucin 5b and mucin 5ac expression by phorbol 12-myristate 13-acetate. Am J Pathol 170: 20–32, 2007.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
O. R. Farah, D. Li, B. A. S. McIntyre, J. Pan, and J. Belik
Airway epithelial-derived factor relaxes pulmonary vascular smooth muscle
Am J Physiol Lung Cell Mol Physiol, January 1, 2009; 296(1): L115 - L120.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
Y. Zhu, C. Ehre, L. H. Abdullah, J. K. Sheehan, M. Roy, C. M. Evans, B. F. Dickey, and C. W. Davis
Munc13-2-/- baseline secretion defect reveals source of oligomeric mucins in mouse airways
J. Physiol., April 1, 2008; 586(7): 1977 - 1992.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/C1445    most recent
00051.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ehre, C.
Right arrow Articles by Davis, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ehre, C.
Right arrow Articles by Davis, C. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.