Protein kinase C delta regulates airway mucin secretion via phosphorylation of MARCKS protein

Abstract

Mucin hypersecretion is a major pathological feature of many respiratory diseases, yet cellular mechanisms regulating secretion of mucin have not been fully elucidated. Previously, we reported that mucin hypersecretion induced by human neutrophil elastase involves activation of protein kinase C (PKC), specifically the delta-isoform (PKC delta). Here, we further investigated the role of PKC delta in mucin hypersecretion using both primary human bronchial epithelial cells and the human bronchial epithelial 1 cell line as in vitro model systems. Phorbol-12-myristate-13-acetate (PMA)-induced mucin hypersecretion was significantly attenuated by rottlerin, a PKC delta-selective inhibitor. Rottlerin also reduced PMA- or human neutrophil elastase-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in these cells. Both secretion and MARCKS phosphorylation were significantly enhanced by the PKC delta activator bryostatin 1. A dominant-negative PKC delta construct (pEGFP-N1/PKC delta K376R) transfected into human bronchial epithelial 1 cells significantly attenuated both PMA-induced mucin secretion and phosphorylation of MARCKS, whereas transfection of a wild-type construct increased PKC delta and enhanced mucin secretion and MARCKS phosphorylation. Similar transfections of a dominant-negative or wild-type PKC epsilon construct did not affect either mucin secretion or MARCKS phosphorylation. The results suggest that PKC delta plays an important role in mucin secretion by airway epithelium via regulation of MARCKS phosphorylation.

Figure 1

Rottlerin, a selective PKCδ inhibitor, attenuates PMA-induced mucin secretion and phosphorylation of MARCKS in well differentiated NHBE cells. Cells were preincubated with 15 μmol/L rottlerin (or dimethyl sulfoxide solvent control) for 20 minutes before addition of 100 nmol/L PMA (B and C) or 500 nmol/L HNE (C). A: PKCδ translocates from cytosol to membrane in response to PMA (100 nmol/L) at 15 minutes. After detecting PKCδ, the polyvinylidene difluoride membrane was stripped and reprobed with antibodies against α-tubulin and E-cadherin, which were used as controls to detect the cytosolic and the membrane fraction, respectively. B: Rottlerin significantly attenuates PMA-induced mucin secretion in a concentration-dependent manner. *Significantly different from vehicle control (P < 0.05); ^†significantly different from PMA alone (P < 0.05). Data are presented as mean ± SEM (n = 4). C: Rottlerin attenuates phosphorylation of MARCKS in NHBE cells exposed to two different mucin secretagogues, HNE (500 nmol/L) or PMA (100 nmol/L). Phosphorylated MARCKS was detected by Western blot using a specific antibody against MARCKS phosphorylated at serine 152/156. After detecting phosphorylated MARCKS, the polyvinylidene difluoride membrane was stripped and reprobed with an antibody against total MARCKS protein as a loading control for each lane. Blots are representative of two replicate experiments. Densitometry measurements of the band intensities were quantified using LabWorks (BioImaging Systems, UVP, Inc., Cambridge, UK) and are shown below the blots.

Figure 2

Effect of bryostatin 1, a PKCδ activator, on mucin secretion in well differentiated NHBE cells. NHBE cells were exposed to bryostatin 1 over a range of concentrations from 1 to 1000 nmol/L for 15 minutes. A: PKCδ translocates from cytosol to membrane in response to bryostatin 1. α-Tubulin and E-cadherin were used as controls for the cytosolic and membrane fractions, respectively. Blots are representative of three replicate experiments. B: Bryostatin 1 provokes mucin secretion by NHBE cells in a concentration-dependent manner. Significantly different from vehicle control: *P < 0.05; ^†P < 0.001; ^‡P < 0.005. Data are presented as mean ± SEM (n = 4). C: Phosphorylation of MARCKS in NHBE cells is induced by bryostatin 1 in a concentration-dependent manner. Blots are representative of three replicate experiments.

Figure 3

HBE-1 cells secrete mucin in response to PKC activation. HBE-1 cells maintained in air/liquid interface were exposed to 100 or 500 nmol/L PMA for 15 minutes. Mucin secretion and phosphorylation of MARCKS were assessed by enzyme-linked immunosorbent assay and Western blot analysis, respectively. A: Mucin secretion is significantly enhanced by PMA at 500 nmol/L (but not 100 nmol/L) in HBE1 cells. *Significantly different from vehicle control (P < 0.05). Data are presented as mean ± SEM (n = 4). B: Phosphorylation of MARCKS is increased by exposure of HBE1 cells to 500 nmol/L PMA. Blots are representative of three replicate experiments.

Figure 4

Transient transfection of HBE1 cells with a dominant-negative PKCδ construct results in reduction of mucin hypersecretion. HBE1 cells were transiently transfected with empty vector (pEGFP-N1), a wild-type PKCδ construct (pEGFP-N1/PKCδ), or a dominant-negative construct (pEGFP-N1/PKCδ^K376R) using the FuGene 6 transfection reagent as described in Materials and Methods. As an additional control, a wild-type (pHACE/PKCε) and a dominant-negative PKCε construct (pHACE/PKCε^K437R) were also transfected. A: After 48 hours transfection, cells were exposed to 500 nmol/L PMA (lanes 2 to 8) or vehicle control (lane 1) for 15 minutes, at which time media were collected and mucin secretion assessed by enzyme-linked immunosorbent assay. Data are significantly different from media control (*P < 0.05; **P < 0.001); significantly different from cells transfected with no DNA or empty vector and exposed to PMA (^†P < 0.05, ^‡P < 0.001); and significantly different from cells transfected with the pEGFP-N1/PKCδ^K376R and exposed to PMA (^§P < 0.05). Data are presented as mean ± SEM (n = 3∼4). After collecting media from transfected HBE1 cells exposed to PMA, cell lysates were analyzed by Western blot for MARCKS phosphorylated at Serine 152/156 residues (B and C). Blots are representative of three replicate experiments. B: Transfection of the dnPKCδ construct results in decreased phosphorylation of MARCKS in response to PMA, whereas overexpression of PKCδ via transfection of the wild-type construct enhances MARCKS phosphorylation after PMA exposure. Lane 1, control medium; lane 2, PMA plus control medium; lane 3, PMA plus mock transfection; lane 4, PMA plus empty vector (pEGFP-N1); lane 5, PMA plus wild-type PKCδ (pEGFP-N1/PKCδ); and lane 6, PMA plus dnPKCδ (pEGFP-N1/PKCδ^K376R). C: In a separate experiment, phosphorylation of MARCKS is attenuated by transfection of the dnPKCδ (pEGFP-N1/PKCδ^K376R; lane 2), whereas similar transfection of the dnPKCε (pHACE/PKCε^K437R; lane 3) does not appear to decrease PMA-induced phosphorylation of MARCKS.