A Peptide Against the N-Terminus of Myristoylated Alanine-Rich C Kinase Substrate Inhibits Degranulation of Human LeukocytesIn Vitro

Abstract

Leukocytes synthesize a variety of inflammatory mediators that are packaged and stored in the cytoplasm within membrane-bound granules. Upon stimulation, the cells secrete the granule contents via an exocytotic process whereby the granules translocate to the cell periphery, the granule membranes fuse with the plasma membrane, and the granule contents are released extracellularly. We have reported previously that another exocytotic process, release of mucin by secretory cells of the airway epithelium, is regulated by the myristoylated alanine-rich C kinase substrate ( Keywords: MARCKS protein; leukocytes; degranulationLeukocytes synthesize a number of inflammatory mediators that are packaged and stored in cytoplasmic membrane-bound granules. These mediators include myeloperoxidase (MPO) in neutrophils (1), eosinophil peroxidase (EPO) and major basic pro- (2), lysozyme in monocytes/macrophages (3, 4), and granzyme in natural killer (NK) cells and cytotoxic lymphocytes (5-8). These mediators are released at sites of injury and contribute to inflammation and repair in the lung and elsewhere. Leukocytes release these granules via an exocytotic mechanism (9, 10), but the regulatory molecules and specific pathways involved in the exocytotic process have not been fully described.Several exogenous stimuli can provoke degranulation of leukocytes via a pathway that involves activation of protein kinase C (PKC) and subsequent phosphorylation events (9-13). MARCKS (myristoylated alanine-rich C kinase substrate), a ubiquitous phosphorylation target of PKC, is highly expressed in leukocytes (14-16). We have previously demonstrated that MARCKS protein is involved in exocytotic secretion of mucin by goblet cells that line the respiratory airways (17, 18). In airway epithelial cells, the N-terminus of MARCKS seems to be integral to the secretory process. The mechanism seems to involve the binding of MARCKS to membranes of intracellular mucin granules because a peptide against the N-terminus of MARCKS blocked mucin secretion and binding of MARCKS to mucin granule membranes in these cells (18). Because MARCKS is a prominent protein in leukocytes, we investigated whether or not MARCKS, and specifically its N-terminus, could play a role in leukocyte degranulation.In these studies, we used four different leukocyte types or models that secrete specific granule contents in response to phorbol ester-induced activation of PKC. First, neutrophils were isolated from human blood, and the in vitro release of MPO by these cells was assessed. Due to difficulties in isolating sufficient amounts of other leukocyte types from blood, we investigated the release of membrane-bound inflammatory mediators from commercially available human leukocyte cell lines. The human promyelocytic cell line HL-60 clone 15 was used to assess secretion of , the monocytic leukemia cell line U937 was used to assess secretion of lysozyme (3,4,23), and the lymphocyte NK cell line NK-92 was used to assess the release of granzyme (6-8). In all cases, the cells were preinc...

Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein Regulation of Human Neutrophil Migration

“…A major role for the N-terminal region of MARCKS in other functions, such as mucin secretion and inflammatory cell degranulation, was first described in studies from our laboratory (27,33,36). To our knowledge, these studies were the first to point out a role for the N-terminal region of MARCKS in any physiological functions.…”

“…Since MANS has been shown to inhibit exocytic processes such as myeloperoxidase (MPO) release from neutrophils (36) and mucin release from airway epithelial cells (33), we hypothesized that MANS was disrupting adhesion by reducing the ability of neutrophils to up-regulate surface b2 integrins. PMA-and fMLF-induced up-regulation of surface b2 integrin expression was evaluated using anti-CD18 antibody and flow cytometric analysis as described in MATERIALS AND METHODS.…”

MARCKS Regulation of Mucin Secretion by Airway Epithelium in Vitro

“…This includes secretion of oxytocin (24,25), thyrotropin-releasing hormone (26), catecholamines (27), ACTH (28), insulin (29), pepsinogen (30), noradenaline (31), serotonin (32), neurotransmitters (33,34), and neurotensin (35). We have shown recently that MARCKS plays an important role in leukocyte degranulation (36). These published results implicate PKC activation and subsequent phosphorylation of MARCKS in a variety of secretory functions in numerous cell types.…”

CFTR and lung homeostasis

“…A synthetic amino-terminal peptide from the MARCKS called MANS (MARCKS-N-terminal sequence) inhibits mucin secretion in airway goblet cells, indicating that MARCKS is an important component in the secretory apparatus that is required for granule release in a number of cell types (70). A recent study from Davis and colleagues (32), however, questions this model.…”

“…A recent study from Davis and colleagues (32), however, questions this model. The MANS peptide is believed to compete with the MARCKS protein for membrane binding and therefore acts as a dominant-negative regulator that blocks mucin release (70). In the Davis studies, the investigators tested this hypothesis by using MARCKS-null mice and harvested cells from mouse embryos since the MARCKS-null mutation results in prenatal lethality (69).…”