J.W. Kim

Vimentin filaments regulate integrin-ligand interactions by binding to the cytoplasmic tail of integrin β3.

ABSTRACT Vimentin, an intermediate filament protein induced during epithelial-to-mesenchymal transition, is known to regulate cell migration and invasion. However, it is still unclear how vimentin controls such behaviors. In this study, we aimed to find a new integrin regulator by investigating the H-Ras-mediated integrin suppression mechanism. Through a proteomic screen using the integrin β3 cytoplasmic tail protein, we found that vimentin might work as an effector of H-Ras signaling. H-Ras converted filamentous vimentin into aggregates near the nucleus, where no integrin binding can occur. In addition, an increase in the amount of vimentin filaments accessible to the integrin β3 tail enhanced talin-induced integrin binding to its ligands by inducing integrin clustering. In contrast, the vimentin head domain, which was found to bind directly to the integrin β3 tail and compete with endogenous vimentin filaments for integrin binding, induced nuclear accumulation of vimentin filaments and reduced the amount of integrin–ligand binding. Finally, we found that expression of the vimentin head domain can reduce cell migration and metastasis. From these data, we suggest that filamentous vimentin underneath the plasma membrane is involved in increasing integrin adhesiveness, and thus regulation of the vimentin–integrin interaction might control cell adhesion. Summary: Vimentin filaments underneath the plasma membrane might provide integrin-binding sites to mediate integrin clustering, which in turn can enhance integrin–ligand interaction.

Vimentin filament controls integrin α5β1‐mediated cell adhesion by binding to integrin through its Ser38 residue

Regulation of integrin affinity for its ligand is essential for cell adhesion and migration. Here, we found that direct interaction of vimentin with integrin β1 can enhance binding of integrin α5β1 to its ligand, fibronectin. Conversely, blocking the interaction reduced fibronectin binding, cell migration on a fibronectin‐coated surface, and neural tube closure during Xenopus embryogenesis. We also found that withaferin A (WFA), a natural compound known to inhibit vimentin function, can suppress the vimentin–integrin interaction and abolish fibronectin binding. Finally, we identified Ser38 of vimentin as a critical residue for integrin binding. Our results suggest that phosphorylation of vimentin at Ser38 may regulate the integrin–ligand interaction, thus providing a molecular basis for antivimentin therapeutic strategies.

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin β3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-β TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

Calmodulin Mediates Ca2+-Dependent Inhibition of Tie2 Signaling and Acts as a Developmental Brake During Embryonic Angiogenesis

Objective— Angiogenesis, the process of building complex vascular structures, begins with sprout formation on preexisting blood vessels, followed by extension of the vessels through proliferation and migration of endothelial cells. Based on the potential therapeutic benefits of preventing angiogenesis in pathological conditions, many studies have focused on the mechanisms of its initiation as well as control. However, how the extension of vessels is terminated remains obscure. Thus, we investigated the negative regulation mechanism. Approach and Results— We report that increased intracellular calcium can induce dephosphorylation of the endothelial receptor tyrosine kinase Tie2. The calcium-mediated dephosphorylation was found to be dependent on Tie2–calmodulin interaction. The Tyr1113 residue in the C-terminal end loop of the Tie2 kinase domain was mapped and found to be required for this interaction. Moreover, mutation of this residue into Phe impaired both the Tie2-calmodulin interaction and calcium-mediated Tie2 dephosphorylation. Furthermore, expressing a mutant Tie2 incapable of binding to calmodulin or inhibiting calmodulin function in vivo causes unchecked growth of the vasculature in Xenopus. Specifically, knockdown of Tie2 in Xenopus embryo retarded the sprouting and extension of intersomitic veins. Although human Tie2 expression in the Tie2-deficient animals almost completely rescued the retardation, the Tie2(Y1113F) mutant caused overgrowth of intersomitic veins with strikingly complex and excessive branching patterns. Conclusions— We propose that the calcium/calmodulin-dependent negative regulation of Tie2 can be used as an inhibitory signal for vessel growth and branching to build proper vessel architecture during embryonic development.

Linoleic Acid Attenuates the Toxic Dose of Bupivacaine-Mediated Reduction of Vasodilation Evoked by the Activation of Adenosine Triphosphate-Sensitive Potassium Channels

The goal of this study was to investigate the effect of lipid emulsion on a toxic dose of local anesthetic-mediated reduction of vasodilation evoked by the ATP-sensitive potassium (KATP) channel agonist levcromakalim. The effect of lipid emulsion (LE) and linoleic acid on the local anesthetic-mediated reduction of vasodilation and membrane hyperpolarization evoked by levcromakalim was assessed in isolated endothelium-denuded vessels (rat aorta and mesenteric artery) and aortic vascular smooth muscle cells. The effect of LE and linoleic acid on KATP channel activity in transfected HEK-293 cells was investigated, as was the effect of LE on bupivacaine concentration. The efficacy of LE in attenuating the local anesthetic-mediated reduction of vasodilation evoked by levcromakalim was correlated with the lipid solubility of the local anesthetic. Linoleic acid attenuated the bupivacaine-mediated reduction of vasodilation evoked by levcromakalim. LE decreased the bupivacaine-mediated reduction of membrane hyperpolarization evoked by levcromakalim but did not significantly alter the mepivacaine-mediated reduction. LE and linoleic acid both reversed the bupivacaine-mediated decrease of KATP activity and enhanced KATP activity. LE decreased the bupivacaine concentration. Linoleic acid may be the major contributor to LE-induced attenuation of bupivacaine-mediated reduction of vasodilation evoked by levcromakalim via the direct activation of KATP channels and indirect effects.

Lipid Emulsion Inhibits the Late Apoptosis/Cardiotoxicity Induced by Doxorubicin in Rat Cardiomyoblasts

This study aimed to examine the effect of lipid emulsion on the cardiotoxicity induced by doxorubicin in H9c2 rat cardiomyoblasts and elucidates the associated cellular mechanism. The effects of lipid emulsion on cell viability, Bax, cleaved caspase-8, cleaved capase-3, Bcl-XL, apoptosis, reactive oxygen species (ROS), malondialdehyde, superoxide dismutase (SOD), catalase and mitochondrial membrane potential induced by doxorubicin were examined. Treatment with doxorubicin decreased cell viability, whereas pretreatment with lipid emulsion reduced the effect of doxorubicin by increasing cell viability. Lipid emulsion also suppressed the increased expression of cleaved caspase-3, cleaved caspase-8, and Bax induced by doxorubicin. Moreover, pretreatment with lipid emulsion decreased the increased Bax/Bcl-XL ratio induced by doxorubicin. Doxorubicin-induced late apoptosis was reduced by treatment with lipid emulsion. In addition, pretreatment with lipid emulsion prior to doxorubicin enhanced glycogen synthase kinase-3β phosphorylation. The increased malondialdehyde and ROS levels by doxorubicin were reduced by lipid emulsion pretreatment. Furthermore, lipid emulsion attenuated the reduced SOD and catalase activity and the decreased mitochondrial membrane potential induced by doxorubicin. Taken together, these results suggest that lipid emulsion attenuates doxorubicin-induced late apoptosis, which appears to be associated with the inhibition of oxidative stress induced by doxorubicin.