MARCKS and Lung Disease.

MARCKS (myristoylated alanine-rich C kinase substrate) is a prominent PKC substrate expressed in all eukaryotic cells. It is known to bind to and cross-link actin filaments, to serve as a bridge between Ca2+/calmodulin and PKC signaling, and to sequester the signaling molecule phosphatidylinositol 4,5-bisphosphate in the plasma membrane. Since the mid-1980s, this evolutionarily conserved and ubiquitously expressed protein has been associated with regulating cellular events that require dynamic actin reorganization, including cellular adhesion, migration, and exocytosis. More recently, translational studies have implicated MARCKS in the pathophysiology of a number of airway diseases, including chronic obstructive pulmonary disease, asthma, lung cancer, and acute lung injury/acute respiratory distress syndrome. This article summarizes the structure and cellular function of MARCKS (also including MARCKS family proteins and MARCKSL1 [MARCKS-like protein 1]). Evidence for MARCKS's role in several lung diseases is discussed, as are the technological innovations that took MARCKS-targeting strategies from theoretical to therapeutic. Descriptions and updates derived from ongoing clinical trials that are investigating inhalation of a MARCKS-targeting peptide as therapy for patients with chronic bronchitis, lung cancer, and ARDS are provided.

An Inhaled Inhibitor of Myristoylated Alanine-Rich C Kinase Substrate Reverses LPS-Induced Acute Lung Injury in Mice.

Intratracheal instillation of bacterial LPS is a well-established model of acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). Because the myristoylated alanine-rich C kinase substrate (MARCKS) protein is involved in neutrophil migration and proinflammatory cytokine production, we examined whether an aerosolized peptide that inhibits MARCKS function could attenuate LPS-induced lung injury in mice. The peptide, BIO-11006, was delivered at 50 µM via inhalation either just before intratracheal instillation of 5 µg of LPS into Balb/C mice, or 4, 12, 24, or 36 hours after LPS instillation. Effects of BIO-11006 were evaluated via analysis of mouse disease-related behavior, lung histology, bronchoalveolar lavage fluid total protein, neutrophil counts and percentages, cytokine (KC [CXCl1, mouse IL-8 equivalent] and TNF-α) expression, and activation of NF-κB in lung tissue. Treatment with aerosolized BIO-11006 at 0, 4, 12, 24, and even 36 hours after LPS instillation reversed the disease process: mouse behavior returned to normal after two treatments 12 hours apart with the inhaled peptide after LPS injury, whereas control LPS-instilled animals treated with PBS only remained moribund. Histological appearance of inflammation, bronchoalveolar lavage fluid protein levels, leukocyte and neutrophil numbers, KC and TNF-α gene and protein expression, and NF-κB activation were all significantly attenuated by inhaled BIO-11006 at all time points. These results implicate MARCKS protein in the pathogenesis of ALI/ARDS and suggest that MARCKS-inhibitory peptide(s), delivered by inhalation, could represent a new and potent therapeutic treatment for ALI/ARDS, even if administered well after the disease process has begun.

MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro.

Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

Calpain and MARCKS protein regulation of airway mucin secretion.

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.

Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein.

A role for MARCKS protein in directed migration of macrophages toward a chemoattractant was investigated. A peptide identical to the N-terminus of MARCKS (the MANS peptide), shown previously to inhibit the function of MARCKS in various cell types, was used. We investigated whether this MARCKS-related peptide could affect migration of macrophages, using the mouse macrophage-like J774A.1 cell line and primary murine macrophages. Both of these cell types migrated in response to the chemoattractants macrophage/MCPs, MCP-1 (25-100 ng/ml) or C5a (5-20 ng/ml). Cells were preincubated (15 min) with MANS or a mis-sense control peptide (RNS), both at 50 µM, and effects on migration determined 3 h after addition of chemoattractants. The movement and interactions of MARCKS and actin also were followed visually via confocal microscopy using a fluorescently labeled antibody to MARCKS and fluorescently tagged phalloidin to identify actin. MANS, but not RNS, attenuated migration of J774A.1 cells and primary macrophages in response to MCP-1 or C5a, implicating MARCKS in the cellular mechanism of directed migration. Exposure of cells to MCP-1 resulted in rapid phosphorylation and translocation of MARCKS from plasma membrane to cytosol, whereas actin appeared to spread through the cell and into cell protrusions; there was visual and biochemical evidence of a transient interaction between MARCKS and actin during the process of migration. These results suggest that MARCKS is involved in directed migration of macrophages via a process involving its phosphorylation, cytoplasmic translocation, and interaction with actin.

Mucin granule-associated proteins in human bronchial epithelial cells: the airway goblet cell "granulome".

BACKGROUND: Excess mucus in the airways leads to obstruction in diseases such as chronic bronchitis, asthma, and cystic fibrosis. Mucins, the highly glycosolated protein components of mucus, are stored in membrane-bound granules housed in the cytoplasm of airway epithelial "goblet" cells until they are secreted into the airway lumen via an exocytotic process. Precise mechanism(s) of mucin secretion, including the specific proteins involved in the process, have yet to be elucidated. Previously, we have shown that the Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein regulates mucin secretion by orchestrating translocation of mucin granules from the cytosol to the plasma membrane, where the granules dock, fuse and release their contents into the airway lumen. Associated with MARCKS in this process are chaperone (Heat Shock Protein 70 [HSP70], Cysteine string protein [CSP]) and cytoskeletal (actin, myosin) proteins. However, additional granule-associated proteins that may be involved in secretion have not yet been elucidated. METHODS: Here, we isolated mucin granules and granule membranes from primary cultures of well differentiated human bronchial epithelial cells utilizing a novel technique of immuno-isolation, based on the presence of the calcium activated chloride channel hCLCA1 (the human ortholog of murine Gob-5) on the granule membranes, and verified via Western blotting and co-immunoprecipitation that MARCKS, HSP70, CSP and hCLCA1 were present on the granule membranes and associated with each other. We then subjected the isolated granules/membranes to liquid chromatography mass spectrometry (LC-MS/MS) to identify other granule associated proteins. RESULTS: A number of additional cytoskeletal (e.g. Myosin Vc) and regulatory proteins (e.g. Protein phosphatase 4) associated with the granules and could play a role in secretion were discovered. This is the first description of the airway goblet cell "granulome."

A common MUC5B promoter polymorphism and pulmonary fibrosis.

BACKGROUND: The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk. METHODS: Using a genomewide linkage scan, we detected linkage between idiopathic interstitial pneumonia and a 3.4-Mb region of chromosome 11p15 in 82 families. We then evaluated genetic variation in this region in gel-forming mucin genes expressed in the lung among 83 subjects with familial interstitial pneumonia, 492 subjects with idiopathic pulmonary fibrosis, and 322 controls. MUC5B expression was assessed in lung tissue. RESULTS: Linkage and fine mapping were used to identify a region of interest on the p-terminus of chromosome 11 that included gel-forming mucin genes. The minor-allele of the single-nucleotide polymorphism (SNP) rs35705950, located 3 kb upstream of the MUC5B transcription start site, was present at a frequency of 34% among subjects with familial interstitial pneumonia, 38% among subjects with idiopathic pulmonary fibrosis, and 9% among controls (allelic association with familial interstitial pneumonia, P=1.2×10(-15); allelic association with idiopathic pulmonary fibrosis, P=2.5×10(-37)). The odds ratios for disease among subjects who were heterozygous and those who were homozygous for the minor allele of this SNP were 6.8 (95% confidence interval [CI], 3.9 to 12.0) and 20.8 (95% CI, 3.8 to 113.7), respectively, for familial interstitial pneumonia and 9.0 (95% CI, 6.2 to 13.1) and 21.8 (95% CI, 5.1 to 93.5), respectively, for idiopathic pulmonary fibrosis. MUC5B expression in the lung was 14.1 times as high in subjects who had idiopathic pulmonary fibrosis as in those who did not (P<0.001). The variant allele of rs35705950 was associated with up-regulation in MUC5B expression in the lung in unaffected subjects (expression was 37.4 times as high as in unaffected subjects homozygous for the wild-type allele, P<0.001). MUC5B protein was expressed in lesions of idiopathic pulmonary fibrosis. CONCLUSIONS: A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis. Our findings suggest that dysregulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis. (Funded by the National Heart, Lung, and Blood Institute and others.).

Regulation of mucin secretion and inflammation in asthma: a role for MARCKS protein?

BACKGROUND: A major characteristic of asthmatic airways is an increase in mucin (the glycoprotein component of mucus) producing and secreting cells, which leads to increased mucin release that further clogs constricted airways and contributes markedly to airway obstruction and, in the most severe cases, to status asthmaticus. Asthmatic airways show both a hyperplasia and metaplasia of goblet cells, mucin-producing cells in the epithelium; hyperplasia refers to enhanced numbers of goblet cells in larger airways, while metaplasia refers to the appearance of these cells in smaller airways where they normally are not seen. With the number of mucin-producing and secreting cells increased, there is a coincident hypersecretion of mucin which characterizes asthma. On a cellular level, a major regulator of airway mucin secretion in both in vitro and in vivo studies has been shown to be MARCKS (myristoylated alanine-rich C kinase substrate) protein, a ubiquitous substrate of protein kinase C (PKC). GENERAL SIGNIFICANCE: In this review, properties of MARCKS and how the protein may regulate mucin secretion at a cellular level will be discussed. In addition, the roles of MARCKS in airway inflammation related to both influx of inflammatory cells into the lung and release of granules containing inflammatory mediators by these cells will be explored. This article is part of a Special Issue entitled: Biochemistry of Asthma.

MARCKS-related peptide modulates in vivo the secretion of airway Muc5ac.

In a mouse model of neutrophil elastase-induced bronchitis that exhibits goblet cell metaplasia and inflammation, we investigated the effects of intratracheal instillation of the MANS peptide, a peptide identical to the NH(2) terminus of the myristoylated alanine-rich C kinase substrate (MARCKS) on mucin protein airway secretion, inflammation, and airway reactivity. To induce mucus cell metaplasia in the airways, male BALB/c mice were treated repetitively with the serine protease, neutrophil elastase, on days 1, 4, and 7. On day 11, when goblet cell metaplasia was fully developed and profiles of proinflammatory cytokines were maximal, the animals were exposed to aerosolized methacholine after intratracheal instillation of MANS or a missense control peptide (RNS). MANS, but not RNS, attenuated the methacholine-stimulated secretion of the major respiratory mucin protein, Muc5ac (50% reduction). Concurrently, elastase-induced proinflammatory cytokines typically recovered in bronchoalveolar lavage (BAL), including KC, IL-1beta, IL-6, MCP-1, and TNFalpha, were reduced by the MANS peptide (mean levels decreased 50-60%). Secondary to the effects of MANS on mucin secretion and inflammation, mechanical lung function by forced oscillation technique was characterized with respect to airway reactivity in response to cumulative aerosol stimulation with serotonin. The MANS peptide was also found to effectively attenuate airway hyperresponsiveness to serotonin in this airway hypersecretory model. Collectively, these findings support the concept that even in airway epithelia remodeled with goblet cell metaplasia and in a state of mucin hypersecretion, exogenous attenuation of function of MARCKS protein via the MANS peptide decreases airway mucin secretion, inflammation, and hyperreactivity.

MARCKS and related chaperones bind to unconventional myosin V isoforms in airway epithelial cells.

We have shown previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule in the process of mucin secretion by airway epithelial cells, and that part of the secretory mechanism involves intracellular associations of MARCKS with specific chaperones: heat shock protein 70 (Hsp70) and cysteine string protein (CSP). Here, we report that MARCKS also interacts with unconventional myosin isoforms within these cells, and further molecular interactions between MARCKS and these chaperones/cytoskeletal proteins are elucidated. Primary human bronchial epithelial cells and the HBE1 cell line both expressed myosin V and VI proteins, and both MARCKS and CSP were shown to bind to myosin V, specifically Va and Vc. This binding was enhanced by exposing the cells to phorbol-12-myristate-13-acetate, an activator of protein kinase C and stimulator of mucin secretion. Binding of MARCKS, Hsp70, and CSP was further investigated by His-tagged pull down assays of purified recombinant proteins and multiple transfections of HBE1 cells with fusion proteins (MARCKS-HA; Flag-Hsp70; c-Myc-CSP) and immunoprecipitation. The results showed that MARCKS binds directly to Hsp70, and that Hsp70 binds directly to CSP, but that MARCKS binding to CSP appears to require the presence of Hsp70. Interrelated binding(s) of MARCKS, chaperones, and unconventional myosin isoforms may be integral to the mucin secretion process.

MARCKS regulation of mucin secretion by airway epithelium in vitro: interaction with chaperones.

We have reported previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. The results of those studies supported a mechanism whereby MARCKS, upon phosphorylation by protein kinase C (PKC), translocates from plasma membrane to cytoplasm, where its binding to membranes of intracellular mucin granules is a key component of the secretory pathway. It remains unknown how MARCKS is targeted to and/or preferentially attaches to mucin granule membranes. We hypothesized that the chaperone cysteine string protein (CSP) may play an important role in this process. CSP was shown to associate with membranes of intracellular mucin granules in well-differentiated normal human bronchial epithelial (NHBE) cells in vitro, as determined by ultrastructural immunohistochemistry and Western blotting of isolated granule membranes. CSP in these cells complexed with MARCKS, as shown by co-immunoprecipitation. Given reported associations between CSP and a second chaperone, heat shock protein 70 (HSP70), a role for HSP70 in the MARCKS-dependent secretory mechanism also was investigated. HSP70 appeared to form a trimeric complex with MARCKS and CSP associated with mucin granule membranes within airway epithelial cells. Transfection of the HBE1 human bronchial epithelial cell line with siRNAs targeting sequences of MARCKS, CSP, or HSP70 resulted, in each case, in significant knockdown of expression of these proteins and subsequent attenuation of mucin secretion. The results provide the first evidence that CSP and HSP70, and their interactions with MARCKS, are involved in mucin secretion.

Protease-activated receptor-2 (PAR-2) is a weak enhancer of mucin secretion by human bronchial epithelial cells in vitro.

PAR-2, a member of a family of G-protein-coupled receptors, can be activated by serine proteases via proteolytic cleavage. PAR-2 expression is known to be upregulated in respiratory epithelium subsequent to inflammation in asthma and chronic obstructive pulmonary disease (COPD). Since these diseases also are characterized by excessive mucus production and secretion, we investigated whether PAR-2 could be linked to mucin hypersecretion by airway epithelium. Normal human bronchial epithelial (NHBE) cells in primary culture or the human bronchial epithelial cell lines, NCI-H292 and HBE-1, were used. NHBE, NCI-H292, and HBE-1 cells expressed prominent levels of PAR-2 protein. Short-term (30min) exposure of cells to the synthetic PAR-2 agonist peptide (SLIGKV-NH2) elicited a small but statistically significant increase in mucin secretion at high concentrations (100microM and 1000microM), compared to a control peptide with reversed amino acid sequence (VKGILS-NH2). Neither human lung tryptase nor bovine pancreatic trypsin, both PAR-2 agonists, affected NHBE cell mucin secretion when added over a range of concentrations. Knockdown of PAR-2 expression by siRNA blocked the stimulatory effect of the AP. The results suggest that, since PAR-2 activation only weakly increases mucin secretion by human airway epithelial cells in vitro, PAR-2 probably is not a significant contributor to mucin hypersecretion in inflamed airways.

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

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.

Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro.

BACKGROUND: Mucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis. Expression of two mucin genes, MUC2 and MUC5AC, and their protein products (mucins), is modulated in certain disease states. Understanding the signaling mechanisms that regulate the production and secretion of these major mucus components may contribute significantly to development of effective therapies to modify their expression in inflamed airways. METHODS: To study the differential expression of Muc2 and Muc5ac, a novel monoclonal antibody recognizing guinea pig Muc2 and a commercially-available antibody against human MUC5AC were optimized for recognition of specific guinea pig mucins by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). These antibodies were then used to analyze expression of Muc2 and another mucin subtype (likely Muc5ac) in guinea pig tracheal epithelial (GPTE) cells stimulated with a mixture of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interferon- gamma (IFN-gamma)]. RESULTS: The anti-Muc2 (C4) and anti-MUC5AC (45M1) monoclonal antibodies specifically recognized proteins located in Muc2-dominant small intestinal and Muc5ac-dominant stomach mucosae, respectively, in both Western and ELISA experimental protocols. IHC protocols confirmed that C4 recognizes murine small intestine mucosal proteins while 45M1 does not react. C4 and 45M1 also stained specific epithelial cells in guinea pig lung sections. In the resting state, Muc2 was recognized as a highly expressed intracellular mucin in GPTE cells in vitro. Following cytokine exposure, secretion of Muc2, but not the mucin recognized by the 45M1 antibody (likely Muc5ac), was increased from the GPTE cells, with a concomitant increase in intracellular expression of both mucins. CONCLUSION: Given the tissue specificity in IHC and the differential hybridization to high molecular weight proteins by Western blot, we conclude that the antibodies used in this study can recognize specific mucin subtypes in guinea pig airway epithelium and in proteins from GPTE cells. In addition, Muc2 is highly expressed constitutively, modulated by inflammation, and secreted differentially (as compared to Muc5ac) in GPTE cells. This finding contrasts with expression patterns in the airway epithelium of a variety of mammalian species in which only Muc5ac predominates.

Modeling the airway epithelium in allergic asthma: interleukin-13- induced effects in differentiated murine tracheal epithelial cells.

Mucous cells of the airway epithelium play a crucial role in the pathogenesis of human inflammatory airway diseases. Therefore, it is of importance to complement in vivo studies that use murine models of allergic asthma with in vitro mechanistic studies that use murine airway epithelial cells, including mucus-containing cells. In this study, we report the development and characterization of an in vitro culture system for primary murine tracheal epithelial (MTE) cells comprising ciliated cells and a substantial number of mucous cells. The increase in mucous cell number over that observed in the native murine airway, or in previously described murine cultures, creates a culture intermediate between the in vivo murine airway epithelium and in vitro cultures of human airway epithelial cells. To establish the usefulness of this culture system for the study of epithelial effects during inflammatory airway diseases, the cells were exposed to interleukin (IL)-13, a central inflammatory mediator in allergic asthma. The IL-13 induced two characteristic epithelial effects, proliferation and modulation of MUC5AC gene expression. There was a concentration dependence of these events, wherein high concentrations of IL-13 (10 ng/ml) induced proliferation, whereas lower concentrations (1 ng/ml) increased MUC5AC mRNA (where mRNA is messenger RNA). Interestingly, these effects occurred in an inverse manner, with the high concentration of IL-13 also provoking a significant decrease in MUC5AC gene expression. Thus, MTE cells cultured in this manner may provide an important link between experimental findings from animal models of allergic asthma and their application to human disease.