Shijing Fang

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.

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.

A Myristoylated Alanine-Rich C Kinase Substrate–Related Peptide Suppresses Cytokine mRNA and Protein Expression in LPS-Activated Canine Neutrophils

Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C substrate that has emerged as a potential therapeutic target for the amelioration of mucin secretion and inflammation in patients with chronic obstructive pulmonary disease. MARCKS also plays a key role in regulating the adhesion, migration, and degranulation of neutrophils. Moreover, given its biological role in epithelial and immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion by neutrophils. Because the amino terminus of MARCKS is highly conserved across vertebrate species, we successfully applied the well-characterized human MARCKS inhibitory peptide, myristoylated N-terminal sequence (MANS), to attenuate the function of MARCKS in isolated canine neutrophils. Pretreatment of canine neutrophils with MANS peptide significantly reduced both mRNA and protein expression in a broad range of LPS-induced cytokines, including IL-8, a chemokine (C-X-C motif) ligand-1 orthologue, and TNF-α, in comparison with untreated cells or those treated with a control peptide. This reduction in cytokine expression was observed even when neutrophils were treated with MANS 2 hours after LPS exposure. The observed reduction in cytokine secretion was not attributable to protein retention or cell death, but was associated with reduced cytokine transcript synthesis. These observations identify MARCKS protein as a promising therapeutic target in the treatment of inflammatory diseases or syndromes attributed to neutrophil influx and inflammatory cytokine production, such as sepsis, acute lung injury, and acute respiratory distress syndrome.

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.

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.

Abstract 4042: MARCKS protein inhibitors attenuate cancer cell migration/metastasis

Background: Metastasis causes most deaths from cancer. In a number of studies, high expressions of the MARCKS protein (Myristoylated Alanine-Rich C Kinase Substrate, a 332-amino acid protein known to play a key role in normal cell motion) in cancer cells have been associated with higher rates of metastasis in breast, lung, prostate, brain, skin and other neoplasias. Recently, Chen et al. showed that the 24-amino acid N-terminal sequence of MARCKS, called the MANS peptide, blocked metastasis of lung cancer cells orthotopically injected into mouse lungs (http://www.ncbi.nlm.nih.gov/pubmed/23955080). Unfortunately, for various reasons, the MANS peptide is not practical as an anti-cancer drug candidate. We have identified shorter and far more soluble N-terminal peptide inhibitors of MARCKS that appear to block migration of cancer cells in vitro and metastasis in mice in-vivo at lower concentrations than the MANS peptide, and when administered either IP or as an inhaled aerosol. Methods: For the in vitro cancer cell migration assays, 1 X 105 of three separate human lung cancer cell lines (A549, PC-9, and CL1-5) were cultured in Transwell ® plates (24-well, 8-um pore size). Either PBS (control), MANS, or 3 different N-terminal peptide inhibitor analogs of MANS; BIO-11006, BIO-11002, and BIO-10901 were incubated with the cells for 12 hrs, and cells that migrated from the upper to lower surface of the chambers were stained with hematoxylin and counted. For the in vivo metastasis evaluations, PC-9 cells were orthotopically-injected into the lungs of NOD-SCID mice with or without preincubation with either MANS or BIO-11006, and animals were given MANS or BIO-11006 IP after 7 days and every 3 days up until day 25, when they were harvested and examined. Another group of orthotopically injected animals received treatment with BIO-11006 as an inhaled aerosol starting at day 4 and every day thereafter until day 25. Animals were euthanized at day 26; locations and magnitudes of systemic metastases were assessed macroscopically and microscopically. Results: All of the N-terminal peptide inhibitors showed concentration - dependent attenuation of in vitro migration of each of the cancer cell lines, with an inhibition of close to 75% at the highest concentration (100μM). The smaller MANS analogs appeared to be the most effective at the lower concentrations. IP injection of MANS or BIO-11006 dramatically inhibited metastasis of the primary tumor to other sites in the lung, heart and diaphragm, with BIO-11006 showing potent inhibition of metastasis even at a concentration an order of magnitude lower than MANS. Administration of 50 uM BIO-11006daily via inhaled aerosol had the same inhibitory effect on metastasis as IP injection of 50 uM every three days. Conclusion: N-terminal peptide inhibitors of MARCKS suitable for development as potential new anti-cancer drugs show substantial promise in inhibiting metastasis of lung cancer cells both in vitro and in vivo. Citation Format: Walker A. Long, Indu Parikh, Qi Yen, Shijing Fang, Anne L. Crews, Kenneth B. Adler. MARCKS protein inhibitors attenuate cancer cell migration/metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2014-4042