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Biphasic and directed translocation of protein kinase C? inside cultured endothelial cells before migration.

ABSTRACT: Mechanical wounding of an endothelial monolayer induces an immediate Ca²⁺ wave. Several hours later, the denuded area is covered by endothelial cells (ECs) that migrate to the wound. This migration process is closely related to protein kinase C? (PKC?), a Ca²⁺-dependent protein that translocates from the cytosol to the cell membrane. Because the cells adjacent to the wounded area are the first to migrate into the wound, we investigated whether a mechanical wound immediately induces PKC? translocation in adjacent cells. We monitored Ca²⁺ dynamics and PKC? translocation simultaneously using fluorescent microscopy. For this simultaneous observation, we used Fura-2-acetoxymethyl ester to visualize Ca²⁺ and constructed a green fluorescent protein-tagged fusion protein to visualize PKC?. Mechanical wounding of the endothelial monolayer induced an immediate Ca²⁺ wave in cells adjacent to the wounded cells before their migration. Almost concurrently, PKC? in the neighboring cells translocated to the cell membrane, then accumulated at the periphery near the wounded cell. This report is the first description of this biphasic and directed translocation of PKC? in cells before cell migration. Our results may provide new insights into the directed migration of ECs.

SUBMITTER: Arai M

PROVIDER: S-EPMC5613218 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Biphasic and directed translocation of protein kinase Cα inside cultured endothelial cells before migration.

Arai Masataka M Shimada Tomoya T Kora Chihiro C Nakashima Kazuhiro K Sera Toshihiro T Kudo Susumu S

Biochemistry and biophysics reports 20170901

Mechanical wounding of an endothelial monolayer induces an immediate Ca<sup>2+</sup> wave. Several hours later, the denuded area is covered by endothelial cells (ECs) that migrate to the wound. This migration process is closely related to protein kinase Cα (PKCα), a Ca<sup>2+</sup>-dependent protein that translocates from the cytosol to the cell membrane. Because the cells adjacent to the wounded area are the first to migrate into the wound, we investigated whether a mechanical wound immediately ...[more]

PMID: 28955796

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Project description:The use and production of multi-walled carbon nanotubes (MWCNTs) have significantly increased over the last decade due to their versatility in numerous applications. Their unique physical and chemical properties make them desirable for various biomedical applications, but the same properties also raise concerns about their safety to human health, particularly at the cellular level. The vascular endothelium could be exposed to nanomaterials either by direct intravenous administration in nanomedicine or by translocation following inhalational exposure in an occupational setting. We hypothesized that direct exposure to MWCNTs will increase the expression of inflammatory markers in human aortic endothelial cells (HAEC). We also investigated the effect of the route of exposure on activation by changing the suspension medium of the MWCNTs. HAEC were treated in vitro with MWCNTs (1 or 10 ?g/cm(2)) suspended in either cell culture medium [(M)-MWCNTs] or 10% clinical grade pulmonary surfactant [(S)-MWCNTs]. The zeta potential of the (S)-MWCNTs was significantly more negative than the (M)-MWCNTs suggesting a more stable suspension. Treatment of HAEC with (S)-MWCNTs; as compared to (M)-MWCNTs resulted in a significantly higher up-regulation of mRNA transcripts for cell adhesion molecules VCAM1, SELE, ICAM1 and the chemokine CCL2. Time dependent changes in VCAM1 and CCL2 protein levels were confirmed by immunofluorescence, flow cytometry and ELISA. A label free quantitative mass spectrometry proteomic analysis was utilized to compare protein expression patterns between the two suspensions of MWCNTs. We identified significant expression changes in >200 unique proteins in MWCNT treated HAEC. However, the two suspensions of MWCNTs resulted in different protein expression patterns with the eIF2 pathway as the only common pathway identified between the two suspensions. These data suggest that direct exposure to MWCNTs induces acute inflammatory and protein expression changes in HAEC, which is influenced by the type of media used for suspension of MWCNTs and their resulting zeta potential.

Dataset Information

Biphasic and directed translocation of protein kinase C? inside cultured endothelial cells before migration.

Publications

Biphasic and directed translocation of protein kinase Cα inside cultured endothelial cells before migration.

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