Vascular Smooth Muscle Cell-Derived Exosomal MicroRNAs Regulate Endothelial Cell Migration Under PDGF Stimulation

Intercellular communication between vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is essential for the maintenance of vascular homeostasis. The presence of exosomes, a recently discovered player in vascular cell communication, has been associated with vascular disease progression. However, the detailed mechanism of how the signal mediated by exosomes affects the function of vascular cells during vascular pathogenesis is yet to be further understood. In this study, we investigated the expression of exosomal microRNAs (miRNAs) secreted by VSMCs and their functional relevance to ECs in pathogenesis, including their role in processes such as platelet-derived growth factor (PDGF) stimulation. We observed that PDGF stimulation contributes to a change in exosomal miRNA release from VSMCs; specifically, miR-1246, miR-182, and miR-486 were deficient in exosomes derived from PDGF-stimulated VSMCs. The reduced miRNA expression in these exosomes is associated with an increase in EC migration. These findings increase our understanding of exosome-mediated crosstalk between vascular cells under a pathological condition.

Gab1 mediates PDGF signaling and is essential to oligodendrocyte differentiation and CNS myelination

Oligodendrocytes (OLs) myelinate axons and provide electrical insulation and trophic support for neurons in the central nervous system (CNS). Platelet-derived growth factor (PDGF) is critical for steady-state number and differentiation of oligodendrocyte precursor cells (OPCs), but its downstream targets are unclear. Here, we show for the first time that Gab1, an adaptor protein of receptor tyrosine kinase, is specifically expressed in OL lineage cells and is an essential effector of PDGF signaling in OPCs in mice. Gab1 is downregulated by PDGF stimulation and upregulated during OPC differentiation. Conditional deletions of Gab1 in OLs cause CNS hypomyelination by affecting OPC differentiation. Moreover, Gab1 binds to downstream GSK3β and regulated its activity, and thereby affects the nuclear accumulation of β-catenin and the expression of a number of transcription factors critical to myelination. Our work uncovers a novel downstream target of PDGF signaling, which is essential to OPC differentiation and CNS myelination.

Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors

Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5–6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP3 and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing “excess p85,” p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.

CRISPR/Cas9-mediated modification of the extreme C-terminus impairs PDGF-stimulated activity of Duox2

Duox2 belongs to the large family of NADPH-oxidase enzymes that are implicated in immune response, vasoregulation, hormone synthesis, cell growth and differentiation via the regulated synthesis of H2O2 and reactive oxygen species. We and others have shown that Duox2 and H2O2 are involved in platelet-derived growth factor (PDGF) induced migration of fibroblasts. Now, using the CRISPR/Cas9-mediated genome editing we demonstrate that the extreme C-terminal region of Duox2 is required for PDGF-stimulated activity of Duox2 and H2O2 production. We generated the fibroblast cells that stably co-express the wild-type or C-terminally modified Duox2 and fluorescent H2O2 probe Hyper. We found that nonsense substitution of the last 23 amino acids in Duox2 results in complete loss of PDGF stimulation of intracellular H2O2 and fibroblast migration, yet these mutations have no effects on the expression of Duox2 and other NADPH-oxidases in cells. These findings illustrate for the first time that the extreme C-terminus of Duox2 is required for the functional activity of the enzyme. Furthermore, the conservative nature of the C-terminus suggests its role for activity in other NADPH-oxidases.

Cell signaling promoting protein carbonylation does not cause sulfhydryl oxidation: Implications to the mechanism of redox signaling

Reactive oxygen species (ROS) have been recognized as second messengers, however, targeting mechanisms for ROS in cell signaling have not been defined. While ROS oxidizing protein cysteine thiols has been the most popular proposed mechanism, our laboratory proposed that ligand/receptor-mediated cell signaling involves protein carbonylation. Peroxiredoxin-6 (Prx6) is one protein that is carbonylated at 10 min after the platelet-derived growth factor (PDGF) stimulation of human pulmonary artery smooth muscle cells. In the present study, the SulfoBiotics Protein Redox State Monitoring Kit Plus (Dojindo Molecular Technologies) was used to test if cysteine residues of Prx6 are oxidized in response to the PDGF stimulation. Human Prx6 has a molecular weight of 25 kDa and contains two cysteine residues. The Dojindo system adds the 15 kDa Protein-SHifter if these cysteine residues are reduced in the cells. Results showed that, in untreated cells, the Prx6 molecule predominantly exhibited the 55 kDa band, indicating that both cysteine residues are reduced in the cells. Treatment of cells with 1 mM H2O2 caused the disappearance of the 55 kDa band and the appearance of a 40 kDa band, suggesting that the high concentration of H2O2 oxidized one of the two cysteine residues in the Prx6 molecule. By contrast, PDGF stimulation had no effects on the thiol status of the Prx6 molecule. We concluded that protein carbonylation is a more sensitive target of ROS during ligand/receptor-mediated cell signaling than sulfhydryl oxidation.

A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

Although cell migration plays a central role in development and disease, the underlying molecular mechanism is not fully understood. Here we report that a phosphorylation-mediated molecular switch comprising deleted in liver cancer 1 (DLC1), tensin-3 (TNS3), phosphatase and tensin homologue (PTEN) and phosphoinositide-3-kinase (PI3K) controls the spatiotemporal activation of the small GTPases, Rac1 and RhoA, thereby initiating directional cell migration induced by growth factors. On epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) stimulation, TNS3 and PTEN are phosphorylated at specific Thr residues, which trigger the rearrangement of the TNS3–DLC1 and PTEN–PI3K complexes into the TNS3–PI3K and PTEN–DLC1 complexes. Subsequently, the TNS3–PI3K complex translocates to the leading edge of a migrating cell to promote Rac1 activation, whereas PTEN–DLC1 translocates to the posterior for localized RhoA activation. Our work identifies a core signalling mechanism by which an external motility stimulus is coupled to the spatiotemporal activation of Rac1 and RhoA to drive directional cell migration.

Abstract 645: Heparin’s Effects on Vascular Cells Require Transmembrane Receptor 184A

Introduction: Though heparin has been used in the clinic for decades, molecular mechanism(s) underlying heparin’s functions independent of anti-coagulation are still unclear. Transmembrane protein 184A (TMEM184A) is conserved, yet its physiological and molecular functions remain unknown. There are few studies reporting its expression and potential role in membrane trafficking in exocrine cells, and involvement in signaling during male germ cell differentiation. Sequence analysis reveals that the C terminal domain includes a putative binding site for heparin. We have previously shown that heparin decreases proliferation in vascular smooth muscle cells (VSMCs) by inducing expression of MKP-1 that decreases Elk-1 and ERK activation. Hypothesis: We hypothesized that TMEM184A plays a role in mediating heparin effects in vascular cells. Methods and Results: We observed TMEM184A expression in vascular cells through immunofluorescence and western blotting using three primary antibodies against different regions in TMEM184A, and visualized cells with confocal microscopy. To investigate whether heparin effects were dependent on TMEM184A, VSMCs were electroporated with 20 μg/ml control or TMEM184A shRNA. Control and knockdown VSMCs were treated with 200 μg/ml heparin for 20 min followed by 2 μg/ml platelet-derived growth factor (PDGF). Activated ERK or Elk-1 in the nucleus was compared to untreated controls or cells treated with PDGF alone. Quantitative immunofluorescence of over 100 cells for each treatment from at least three independent experiments showed that heparin treatment prior to 20 min PDGF stimulation significantly decreased active ERK by nearly 50% in control shRNA cells compared to cells treated with PDGF alone (20 min PDGF = 100.0% ± 3.76%, heparin pre-treatment = 55.5% ± 2.20%; p<0.001). In TMEM184A knockdown cells, heparin pre-treatment did not decrease ERK activation (20 min PDGF = 100.0% ± 3.27%, heparin pre-treatment = 109.8% ± 3.06%). Similar results were observed for Elk-1. Heparin also did not decrease proliferation in response to PDGF in knockdown VSMCs as shown with BrdU incorporation assays. Conclusions: Our results provide functional evidence that heparin signaling in VSMCs is mediated at least in part by TMEM184A.