Peptide Inhibitors of MARCKS Suppress Endotoxin Induced Uveitis in Rats.

Purpose: To determine if inhibition of Myristoylated Alanine Rich C Kinase Substrate (MARCKS) protein, using novel MARCKS inhibitor peptides, will reduce the severity of endotoxin-induced uveitis (EIU) in rats. Methods: EIU was induced in Lewis rats using subcutaneous administration of lipopolysaccharide. In the first phase of the study, 3 different novel MARCKS inhibitor peptides that mimic the N-terminal region of MARCKS (BIO-11006, or lower molecular weight analogs BIO-91201 or BIO-91202; Biomarck Pharmaceuticals, Ltd., Newtown, PA) were administered intravitreally (IVT) at 50 and 100 µM. In the second phase, BIO-91201 was administered IVT at 10, 50, and 100 µM and topically at the 100 µM concentration. The efficacy of MARCKS inhibitor peptides was assessed by clinical examination using slit lamp biomicroscopy, optical coherence tomography (OCT) anterior chamber cell counts, histopathology, and aqueous humor cytokine analysis. Results: Clinical scores were significantly reduced 24 h following uveitis induction in the first phase of the study in the following treatment groups: BIO-11006 50 µM IVT and 100 µM IVT, BIO-91201 50 µM IVT, and BIO-91202 100 µM IVT (P < 0.05). OCT anterior chamber cell counts were significantly reduced in the first phase of the study in all treatment groups (P < 0.001). OCT anterior chamber cell counts and histopathology scores were significantly reduced in the second phase of the study in the BIO-91201 50 µM IVT group (P < 0.05). No effect was seen with topical administration. Conclusion: MARCKS inhibitor peptides were effective in reducing the severity of ocular inflammation and cellular influx in EIU.

Phosphorylation-dependent proteome of Marcks in ependyma during aging and behavioral homeostasis in the mouse forebrain.

Ependymal cells (ECs) line the ventricular surfaces of the mammalian central nervous system (CNS) and their development is indispensable to structural integrity and functions of the CNS. We previously reported that EC-specific genetic deletion of the myristoylated alanine-rich protein kinase C substrate (Marcks) disrupts barrier functions and elevates oxidative stress and lipid droplet accumulation in ECs causing precocious cellular aging. However, little is known regarding the mechanisms that mediate these changes in ECs. To gain insight into Marcks-mediated mechanisms, we performed mass spectrometric analyses on Marcks-associated proteins in young and aged ECs in the mouse forebrain using an integrated approach. Network analysis on annotated proteins revealed that the identified Marcks-associated complexes are in part involved in protein transport mechanisms in young ECs. In fact, we found perturbed intracellular vesicular trafficking in cultured ECs with selective deletion of Marcks (Marcks-cKO mice), or upon pharmacological alteration to phosphorylation status of Marcks. In comparison, Marcks-associated protein complexes in aged ECs appear to be involved in regulation of lipid metabolism and responses to oxidative stress. Confirming this, we found elevated signatures of inflammation in the cerebral cortices and the hippocampi of young Marcks-cKO mice. Interestingly, behavioral testing using a water maze task indicated that spatial learning and memory is diminished in young Marcks-cKO mice similar to aged wildtype mice. Taken together, our study provides first line of evidence for potential mechanisms that may mediate differential Marcks functions in young and old ECs, and their effect on forebrain homeostasis during aging.

A pre-investigational new drug study of lung spheroid cell therapy for treating pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a lethal interstitial lung disease with unknown etiology, no cure, and few treatment options. Herein, a therapy option is presented that makes use of a heterogeneous population of lung cells, including progenitor cells and supporting cells lines, cultured in adherent and suspension conditions, the latter of which induces spontaneous spheroid formation. Within these spheroids, progenitor marker expression is augmented. The cells, called lung spheroid cells, are isolated from fibrotic lungs, expanded, and delivered in single cell suspensions into rat models of pulmonary fibrosis via tail-vein injections. Two bleomycin-induced fibrotic rat models are used; a syngeneic Wistar-Kyoto rat model, treated with syngeneic cells, and a xenogeneic nude rat model, treated with human cells. The first objective was to study the differences in fibrotic progression in the two rat models after bleomycin injury. Nude rat fibrosis formed quickly and extended for 30 days with no self-resolution. Wistar-Kyoto rat fibrosis was more gradual and began to decrease in severity between days 14 and 30. The second goal was to find the minimum effective dose of cells that demonstrated safe and effective therapeutic value. The resultant minimum effective therapeutic dose, acquired from the nude rat model, was 3 × 106 human cells. Histological analysis revealed no evidence of tumorigenicity, increased local immunological activity in the lungs, or an increase in liver enzyme production. These data demonstrate the safety and efficacy of lung spheroid cells in their application as therapeutic agents for pulmonary fibrosis, as well as their potential for clinical translation.

Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal and incurable form of interstitial lung disease in which persistent injury results in scar tissue formation. As fibrosis thickens, the lung tissue loses the ability to facilitate gas exchange and provide cells with needed oxygen. Currently, IPF has few treatment options and no effective therapies, aside from lung transplant. Here we present a series of studies utilizing lung spheroid cell-secretome (LSC-Sec) and exosomes (LSC-Exo) by inhalation to treat different models of lung injury and fibrosis. Analysis reveals that LSC-Sec and LSC-Exo treatments could attenuate and resolve bleomycin- and silica-induced fibrosis by reestablishing normal alveolar structure and decreasing both collagen accumulation and myofibroblast proliferation. Additionally, LSC-Sec and LSC-Exo exhibit superior therapeutic benefits than their counterparts derived from mesenchymal stem cells in some measures. We showed that an inhalation treatment of secretome and exosome exhibited therapeutic potential for lung regeneration in two experimental models of pulmonary fibrosis.

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.

Derivation of therapeutic lung spheroid cells from minimally invasive transbronchial pulmonary biopsies.

BACKGROUND: Resident stem and progenitor cells have been identified in the lung over the last decade, but isolation and culture of these cells remains a challenge. Thus, although these lung stem and progenitor cells provide an ideal source for stem-cell based therapy, mesenchymal stem cells (MSCs) remain the most popular cell therapy product for the treatment of lung diseases. Surgical lung biopsies can be the tissue source but such procedures carry a high risk of mortality. METHODS: In this study we demonstrate that therapeutic lung cells, termed "lung spheroid cells" (LSCs) can be generated from minimally invasive transbronchial lung biopsies using a three-dimensional culture technique. The cells were then characterized by flow cytometry and immunohistochemistry. Angiogenic potential was tested by in-vitro HUVEC tube formation assay. In-vivo bio- distribution of LSCs was examined in athymic nude mice after intravenous delivery. RESULTS: From one lung biopsy, we are able to derive >50 million LSC cells at Passage 2. These cells were characterized by flow cytometry and immunohistochemistry and were shown to represent a mixture of lung stem cells and supporting cells. When introduced systemically into nude mice, LSCs were retained primarily in the lungs for up to 21 days. CONCLUSION: Here, for the first time, we demonstrated that direct culture and expansion of human lung progenitor cells from pulmonary tissues, acquired through a minimally invasive biopsy, is possible and straightforward with a three-dimensional culture technique. These cells could be utilized in long-term expansion of lung progenitor cells and as part of the development of cell-based therapies for the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

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.

In Vitro Neutrophil Migration Requires Protein Kinase C-Delta (δ-PKC)-Mediated Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Phosphorylation.

Dysregulated release of neutrophil reactive oxygen species and proteolytic enzymes contributes to both acute and chronic inflammatory diseases. Therefore, molecular regulators of these processes are potential targets for new anti-inflammatory therapies. We have shown previously that myristoylated alanine-rich C-kinase substrate (MARCKS), a well-known actin binding protein and protein kinase C (PKC) substrate, is a key regulator of neutrophil functions. In the current study, we investigate the role of PKC-mediated MARCKS phosphorylation in neutrophil migration and adhesion in vitro. We report that treatment of human neutrophils with the δ-PKC inhibitor rottlerin significantly attenuates f-Met-Leu-Phe (fMLF)-induced MARCKS phosphorylation (IC50=5.709 µM), adhesion (IC50=8.4 µM), and migration (IC50=6.7 µM), while α-, ß-, and ζ-PKC inhibitors had no significant effect. We conclude that δ-PKC-mediated MARCKS phosphorylation is essential for human neutrophil migration and adhesion in vitro. These results implicate δ-PKC-mediated MARCKS phosphorylation as a key step in the inflammatory response of neutrophils.

Targeting myristoylated alanine-rich C kinase substrate phosphorylation site domain in lung cancer. Mechanisms and therapeutic implications.

RATIONALE: Phosphorylation of myristoylated alanine-rich C kinase substrate (phospho-MARCKS) at the phosphorylation site domain (PSD) is crucial for mucus granule secretion and cell motility, but little is known concerning its function in lung cancer. OBJECTIVES: We aimed to determine if MARCKS PSD activity can serve as a therapeutic target and to elucidate the molecular basis of this potential. METHODS: The clinical relevance of phospho-MARCKS was first confirmed. Next, we used genetic approaches to verify the functionality and molecular mechanism of phospho-MARCKS. Finally, cancer cells were pharmacologically inhibited for MARCKS activity and subjected to functional bioassays. MEASUREMENTS AND MAIN RESULTS: We demonstrated that higher phospho-MARCKS levels were correlated with shorter overall survival of lung cancer patients. Using shRNA silencing and ectopic expression of wild-type and PSD-mutated (S159/163A) MARCKS, we showed that elevated phospho-MARCKS promoted cancer growth and erlotinib resistance. Further studies demonstrated an interaction of phosphoinositide 3-kinase with MARCKS, but not with phospho-MARCKS. Interestingly, phospho-MARCKS acted in parallel with increased phosphatidylinositol (3,4,5)-triphosphate pools and AKT activation in cells. Through treatment with a 25-mer peptide targeting the MARCKS PSD motif (MPS peptide), we were able to suppress tumor growth and metastasis in vivo, and reduced levels of phospho-MARCKS, phosphatidylinositol (3,4,5)-triphosphate, and AKT activity. This peptide also enhanced the sensitivity of lung cancer cells to erlotinib treatment, especially those with sustained activation of phosphoinositide 3-kinase/AKT signaling. CONCLUSIONS: These results suggest a key role for MARCKS PSD in cancer disease and provide a unique strategy for inhibiting the activity of MARCKS PSD as a treatment for lung cancer.

Myristoylated Alanine Rich C Kinase Substrate (MARCKS) is essential to ß2-integrin dependent responses of equine neutrophils.

Neutrophil infiltration is a prominent feature in a number of pathologic conditions affecting horses including recurrent airway obstruction, ischemia-reperfusion injury, and laminitis. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. In order to migrate into tissue, neutrophils must respond to chemoattractant signals in their external environment through activation of adhesion receptors (i.e. integrins) and reorganization of the actin cytoskeleton. Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a highly conserved actin-binding protein, has a well demonstrated role in cytoskeletal dependent cellular functions (i.e. adhesion, spreading, and migration), but the details of MARCKS involvement in these processes remain vague. We hypothesized that MARCKS serves as a link between the actin cytoskeleton and integrin function in neutrophils. Using a MARCKS-specific inhibitor peptide known as MANS on equine neutrophils in vitro, we demonstrate that inhibition of MARCKS function significantly attenuates ß2-integrin-dependent neutrophil functions including migration, adhesion, and immune complex-mediated respiratory burst. The MANS peptide did not, however, inhibit the ß2-integrin-independent PMA mediated respiratory burst. These results attest to the essential role of MARCKS function in regulating neutrophil responses, and strongly implicate MARCKS as a potential regulator of ß2-integrins in neutrophils.

Regulated mucin secretion from airway epithelial cells.

Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3 × 10(6) Da per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ∼1 µm in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the generation of DAG and of IP3 that releases calcium from apical ER. Stimulated secretion requires activation of the low affinity calcium sensor Synaptotagmin-2, while a corresponding high affinity calcium sensor in basal secretion is not known. The core exocytic machinery is comprised of the SNARE proteins VAMP8, SNAP23, and an unknown Syntaxin protein, together with the scaffolding protein Munc18b. Common and distinct features of this exocytic system in comparison to neuroendocrine cells and neurons are highlighted.

Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.

IL-17A induces signal transducers and activators of transcription-6-independent airway mucous cell metaplasia.

Mucous cell metaplasia is a hallmark of asthma, and may be mediated by signal transducers and activators of transcription (STAT)-6 signaling. IL-17A is increased in the bronchoalveolar lavage fluid of patients with severe asthma, and IL-17A also increases mucus production in airway epithelial cells. Asthma therapeutics are being developed that inhibit STAT6 signaling, but the role of IL-17A in inducing mucus production in the absence of STAT6 remains unknown. We hypothesized that IL-17A induces mucous cell metaplasia independent of STAT6, and we tested this hypothesis in two murine models in which increased IL-17A protein expression is evident. In the first model, ovalbumin (OVA)-specific D011.10 Th17 cells were adoptively transferred into wild-type (WT) or STAT6 knockout (KO) mice, and the mice were challenged with OVA or PBS. WT-OVA and STAT6 KO-OVA mice demonstrated increased airway IL-17A and IL-13 protein expression and mucous cell metaplasia, compared with WT-PBS or STAT6 KO-PBS mice. In the second model, WT, STAT1 KO, STAT1/STAT6 double KO (DKO), or STAT1/STAT6/IL-17 receptor A (RA) triple KO (TKO) mice were challenged with respiratory syncytial virus (RSV) or mock viral preparation, and the mucous cells were assessed. STAT1 KO-RSV mice demonstrated increased airway mucous cell metaplasia compared with WT-RSV mice. STAT1 KO-RSV and STAT1/STAT6 DKO-RSV mice also demonstrated increased mucous cell metaplasia, compared with STAT1/STAT6/IL17RA TKO-RSV mice. We also treated primary murine tracheal epithelial cells (mTECs) from WT and STAT6 KO mice. STAT6 KO mTECs showed increased periodic acid-Schiff staining with IL-17A but not with IL-13. Thus, asthma therapies targeting STAT6 may increase IL-17A protein expression, without preventing IL-17A-induced mucus production.

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.

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.

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.