Tackling MARCKS‐PIP3 circuit attenuates fibroblast activation and fibrosis progression

Abstract

Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker a-smooth muscle actin (a-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases a-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.

Targeting the AXL Receptor in Combating Smoking-related Pulmonary Fibrosis

“…The above results demonstrated the importance of AXL signaling in smokeexposed fibroblast cells, so we next interrogated the molecular mechanism of how AXL activity is regulated in lung fibroblasts. We previously identified the signal molecule MARCKS as a druggable target in PF (30), and our current work had confirmed a positive association between phospho-MARCKS and AXL abundance (Figure 4E). Based on these observations, we presume that AXL cooperates with MARCKS to sustain activated phenotypes of fibroblasts in response to cigarette smoke.…”

“…We had previously demonstrated the role of MARCKS, a master regulator of the PI3K/AKT pathway, in modulating fibroblast activities such as invasiveness in IPF fibroblasts (30). Since the PI3K/AKT pathway is the major signaling downstream to AXL activation, we questioned whether the signal molecule MARCKS also played a role in tobacco-smoke mediated fibroblast invasiveness and AXL signaling.…”

“…Given the pro-fibrotic roles of tobacco smoke (23)(24)(25)(26), we first characterized the influence of smoke exposure on the phenotypes of lung fibroblasts. Three normal lung fibroblast cell lines (Normal-1, -2, and -3) derived from non-smoker donors, two normal lung fibroblast cell lines (Smoking Normal-1 and -2) derived from smokers without PF, and three lung fibroblasts (PF-1, -2, and -3) isolated from smokers with PF, as described previously (30), were subjected to colony formation and Matrigel transwell invasion assays, respectively. We observed stronger colony-forming ability (Figure 1A) and higher invasiveness (Figure 1B) in the two smoking normal (Smoking Normal-1 and -2) as well as further elevated activities in the three PF fibroblasts (PF-1, -2, and -3) as compared to normal lung fibroblasts.…”

MARCKS cooperates with NKAP to activate NF-kB signaling in smoke-related lung cancer

“…Notably, treatment with 20% CSE resulted in an approximately 1.9-fold increase of phospho-p65 expression and 2.3-fold decease of IκBα level (Figure 5 B). We previously identified a small peptide, the MPS peptide, which targets the MARCKS PSD and inhibits MARCKS-mediated functions with no cytotoxic effect on normal human epithelial cells 13 , 18 , 26 , 27 . Through treatment with 50 μM MPS peptide in lung cancer cells exposed to 20% CSE, we confirmed that MPS peptide had an inhibitory effect on smoke-enhanced MARCKS phosphorylation in both CL1-0 and H292 cells (Figure 5 C).…”

“…An in vitro cell invasion assay was performed as previously described 13 , 15 , 18 using Transwell chambers (8-μm pore size; Costar, Cambridge, MA). Briefly, 2×10 4 cells were seeded on top of the polycarbonate filters coated with Matrigel (Becton Dickinson, Franklin Lakes, NJ), and 0.5 mL of growth medium was added to both the upper and lower wells.…”