A myristoylated alanine-rich C-kinase substrate (MARCKS) inhibitor peptide attenuates neutrophil outside-in ß2-integrin activation and signaling.

MARCKS is an actin and PIP2-binding protein that plays an essential role in neutrophil migration and adhesion; however, the molecular details regarding MARCKS function in these processes remains unclear. Neutrophil adhesion and migration also require the cell surface receptors ß2-integrins. We hypothesized that MARCKS inhibition would alter neutrophil ß2-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in ß2-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out ß2-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn2+-stimulated static adhesion, cell spreading and ß2-integrin clustering, suggesting a role for MARCKS function in outside-in ß2-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in ß2-integrin activation and signaling.

Protective Effects of Astragalus Membranaceus and Ligustrazine on Rat Brain Microvascular Endothelial Cell Injury after Oxygen-Glucose Deprivation/ Reoxygenation by Suppressing the PKCδ/MARCKS Pathway.

AIM AND OBJECTIVE: Cell death is a main pathological change in brain ischemia. Astragalus membranaceus (Ast) and ligustrazine (Lig), as traditional Chinese herbs, have a protective effect against ischemia-reperfusion injury. We aim to find whether the underlying protective mechanism of Astragalus membranaceus and ligustrazine against Oxygen-glucose deprivation/reoxygenation (OGD/R) -induced injury in RBMECs is related to PKCδ/MARCKS pathway. MATERIALS AND METHODS: OGD/R preconditioning was instituted in rat brain microvascular endothelial cells (RBMECs). The survival and apoptosis of RBMECs were detected by a Cell Counting Kit-8 and TUNEL staining; PKCδ/MARCKS and MMP9 expression were examined by immunofluorescence, western blot and quantitative real-time PCR. RESULTS: OGD/R stimulation significantly increased RBMEC apoptosis, whereas Ast+Lig, Rottlerin or Ast+Lig+Rottlerin treatment evidently reduced cellular apoptosis and increased cell viability (P <0.05). Furthermore, Ast+Lig, Rottlerin or Ast+Lig+Rottlerin treatment significantly reduced mRNA expression levels of PKCδ/MARCKS and MMP9 (P <0.05), compared to OGD/R control group. Moreover, Ast+Lig, Rottlerin or Ast+Lig+Rottlerin treatment evidently reduced protein expression levels of PKCδ, MMP9, and MARCKS (P <0.05), compared to OGD/R control group, detected by western blotting or immunofluorescence. CONCLUSION: The administration of Astragalus membranaceus and ligustrazine protected RBMECs against OGD/R-induced apoptosis. PKCδ/MARCKS and MMP9 expression were significantly increased after OGD/R stimulation, while Astragalus membranaceus and ligustrazine treatment evidently suppressed. Collectively, Astragalus membranaceus and ligustrazine play protective effects against OGD/R-induced injury in RBMECs through regulating PKCδ/MARCKS pathway to inhibit MMP9 activation.

MARCKS mediates vascular contractility through regulating interactions between voltage-gated Ca2+ channels and PIP2.

Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP2-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α1-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca2+ currents, which were prevented by depleting PIP2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP2 into the local environment where it increases voltage-gated Ca2+ channel activity.

MARCKS inhibition cooperates with autophagy antagonists to potentiate the effect of standard therapy against drug-resistant multiple myeloma.

Overexpression of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) is implicated in drug resistance and progression of multiple myeloma (MM). The basis for MARCKS induction and impact on MM are not known. Here we show that microRNA-34a (miR-34a), regulates MARCKS translation and is under-expressed in drug-resistant MM cells, leading to increased MARCKS protein level. Over-expression of miR-34a reduces MARCKS expression and sensitizes resistant cells to anti-myeloma drugs. A MARCKS peptide inhibitor (MPS) exerts a dose dependent cytotoxic effect on drug-resistant MM cells with minimal cytotoxicity to normal hematopoietic cells. MPS synergizes with the proteasomal-inhibitor bortezomib to effectively kill drug-resistant MM cells both in vitro and in a xenograft model of MM. While MARCKS inhibition killed MM cells, it also enhanced a pro-survival autophagic pathway that sustained growth following MARCKS inhibition. In accordance, combined treatment with MARCKS antagonists, bortezomib and the autophagy inhibitor, chloroquine, significantly diminished tumor growth in drug-resistant MM cell lines as well as primary MM cells. This study uncovers a mechanism of drug resistance involving miR-34a-MARCKS autoregulatory loop and provides a framework for a potentially new therapeutic strategy to overcome drug resistance in multiple myeloma.