The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells

Diacylglycerol kinase alpha produced phosphatidic acid controls myosin light chain kinase localization and activity in HGF stimulated epithelial cells

Diacylglycerol kinases (DGKs) regulate lipid signaling by phosphorylating diacylglycerol to phosphatidic acid (PA). Membrane recruitment and activation of DGK-alpha (DGKA) is required for cell proliferation, migration and matrix invasion induced by growth factor, chemokines and oncogenes in epithelial and endothelial cells. Through a proteomic approach we identified 76 proteins associated to tyrosine phosphorylated DGKA in Kaposi sarcoma cells. Among these proteins we selected non-muscle myosin light chain kinase (MLCK), for further characterization. MLCK is a regulator of acto-myosin driven contraction, which phosphorylates myosin light chain (MLC). DGKA co-immunoprecipitates with both long (non-muscle) and short (smooth muscle) MLCK isoforms, consistently with the interaction observed with the proteomic strategy. Inhere we show that in epithelial cells: (i) HGF-induced activation of DGKA produces PA at the nascent ruffle; (ii) DGKA-mediated PA generation is necessary and sufficient to recruit MLCK at ruffling site were MLCK promotes ruffle extension; (iii) expression of a constitutively active and membrane-bound DGKA mutant induces ruffles formation by recruiting MLCK at plasma membrane and promoting MLCK-mediated MLC phosphorylation; (iv) expression of constitutively active MLCK is sufficient to lead to ruffle assembly independently from DGKA activity. Altogether those data indicate that PA produced by HGFinduced activation of DGKA, is a novel regulator of the localization and the activity of MLCK required for ruffle formation in epithelial cells

Mouse Satellite Cell Isolation and Transplantation

: Satellite cell (SC) transplantation represents a powerful strategy to investigate SC biology during muscle regeneration. We described here a protocol for SC isolation from green fluorescent protein (GFP)-expressing mice and their transplantation into murine muscles. This procedure was originally used to assess the effects of the hormone unacylated ghrelin on muscle regeneration, in particular evaluating how the increase of unacylated ghrelin in the recipient muscle affected the engraftment of donor SCs ( Reano et al., 2017 )

Unacylated Ghrelin Enhances Satellite Cell Function and Relieves the Dystrophic Phenotype in Duchenne Muscular Dystrophy mdx Model

Muscle regeneration depends on satellite cells, quiescent precursors that, in consequence of injury or in pathological states such as muscular dystrophies, activate, proliferate, and differentiate to repair the damaged tissue. A subset of satellite cells undergoes self-renewal, thus preserving the satellite cell pool and its regenerative potential. Unacylated ghrelin (UnAG) is a circulating hormone that protects muscle from atrophy, promotes myoblast differentiation, and enhances ischemia-induced muscle regeneration. Here we show that UnAG increases satellite cell activity and stimulates Par polarity complex/p38-mediated asymmetric division, fostering both satellite cell self-renewal and myoblast differentiation. Because of those activities on different steps of muscle regeneration, we hypothesized a beneficial effect of UnAG in mdx dystrophic mice, in which the absence of dystrophin leads to chronic muscle degeneration, defective muscle regeneration, fibrosis, and, at later stages of the pathology, satellite cell pool exhaustion. Upregulation of UnAG levels in mdx mice reduces muscle degeneration, improves muscle function, and increases dystrophin-null satellite cell self-renewal, maintaining the satellite cell pool. Our results suggest that UnAG has significant therapeutic potential for preserving the muscles in dystrophies. This article is protected by copyright. All rights reserved

Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc

Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data)