Calpain and MARCKS protein regulation of airway mucin secretion

Abstract

Hypersecretion of mucin plays an important role in the pathophysiology of many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Myristoylated alanine-rich C-kinase substrate (MARCKS) protein has been shown to play an important role in regulation of airway mucin secretion, as peptides analogous to the amino (N)-terminus of MARCKS attenuate mucin secretion by airway epithelium in vitro and in vivo. Here, we investigated a potential role for the protease Calpain, a calcium-dependent cysteine protease that can cleave MARCKS, in the MARCKS-related secretory mechanism. We theorized that Calpain might cleave MARCKS near the N-terminus, thereby attenuating the ability of MARCKS to bind to membranes and/or creating a small N-terminal peptide that could act as a competitive intracellular inhibitor to remaining endogenous full-length MARCKS molecules. Primary normal human bronchial epithelial (NHBE) cells and the virally-transformed human bronchial epithelial HBE1 cell line were exposed to phorbol-12-myristate-13-acetate (PMA) to stimulate the Protein Kinase C (PKC) pathway, leading to enhanced mucin secretion, and Calpain activity within the cells was measured with a fluorescent cleavage assay. Calpain activity was increased by PMA, and pretreatment of the cells with Calpain inhibitors reduced both Calpain activity and mucin secretion in a concentration-dependent manner. Thus, as opposed to the original hypothesis, inactivating Calpain caused a decrease rather than an increase in secretion. HBE1 cells transfected with DNA constructs encoding a MARCKS-YFP fusion protein showed cleavage at a putative site near the N-terminus in response to PMA. Cleavage of MARCKS by Calpain may have an important role in regulation of the PKC/MARCKS pathway regulating airway mucin secretion.

Figure 1

PMA exposure increases Calpain activity in airway epithelial cells. Relative levels of Calpain activity in HBE1 cells after stimulation with 250nM PMA are shown. Label on X axis (time) refers to time post PMA addition. Calpain activity is significantly increased within 2 min of exposure to PMA. (* p<0.05 relative to 0 time control, all values expressed as mean ± SEM, n=3).

Figure 2

Calpain inhibitors decrease Calpain activity in airway epithelium. HBE1 cells were exposed to 250nM PMA for 3 min after 15 min pretreatment with 20 μM of the Calpain inhibitors Z-LLY-FMK or Z-LL-CHO (* p<0.05 † p<0.005 relative to PMA - stimulated, all values expressed as mean ± SEM, n=3).

Figure 3

Pretreatment of airway epithelial cells with Calpain inhibitors attenuates mucin secretion. Relative levels of mucin secretion in NHBE cells after treatment with 250nM PMA with or without pre-treatment with Calpain inhibitors: 20μM Z-LLY-CHO (CHO) or 20μM Z-LLY-FMK (FMK) for 15 min. Secretion was measured 3 min after PMA addition. († p<0.01, †† p<0.001 relative to PMA-stimulated, all values expressed as mean ± SEM, n=4).

Figure 4

Pretreatment of NHBE cells for 15 min with the Calpain inhibitor Z-LL-CHO (CHO) attenuates mucin secretion in response to 250nM PMA in a concentration-dependant manner. Secretion was measured 3 minutes after PMA addition and is normalized to media control. (* P < 0.05 relative to PMA-stimulated, all values expressed as mean ± SEM, n=3).

Figure 5

Western Blot probing for YFP (using antibody against GFP) in HBE1 cells transfected with YFP fused to the N-terminus of MARCKS. A) the entire blot; B) the area of the blot within the dotted line in “A”; this region was exposed longer to bring out blot details; C) actin used for normalization. Lanes 1,2: MARCKS constructs; Lanes 3,4: empty vector. Treatment of cells for 3 min with 100 μM ATP (B; lane 2) shows apparent induction of MARCKS cleavage products of molecular weight 27 - 32 kDa (indicated by arrows).