Title

Protein Kinase C and D Isoform Signaling at the Blood-Brain Barrier in Response to Interleukin-1β

Poster Number

24

Lead Author Major

Biological Sciences and Biochemistry

Format

Poster Presentation

Faculty Mentor Name

Robert Rigor

Faculty Mentor Department

Biological Sciences

Abstract/Artist Statement

Interleukin (IL)-1β is a pro-inflammatory cytokine that participates in inflammation in the brain and other tissues. In the brain, IL-1β causes microvascular leakage and brain edema. Specifically, brain microvascular endothelial cells that protect the brain (the blood-brain barrier (BBB)) become leaky at intercellular tight junctions in response to IL-1β exposure. Previously, we found that IL-1β exposure decreases electrical resistance (TER) across BBB endothelial cell monolayers, which is preceded by a transient increase in resistance (tightening). We showed that the IL-1β dependent decrease in resistance occurs following activation of protein kinase isoforms PKC-Ө and PKD1, and that PKC-Ө is required for this response. In addition, PKD activity is required for increased barrier resistance in response to IL-1β, as well as physiological maintenance of BBB integrity in brain capillaries. Based on published reports of direct interactions between novel PKC isoforms and PKD1, we hypothesized that PKC-Ө and PKD1 interactions may account for the bimodal response to IL-1β. In the present study, we examined phosphorylation states of PKC-Ө and PKD1 using phosphorylation epitope specific antibodies and Western blotting. We found that the IL-1β induced phosphorylation of PKC-Ө at T538 (kinase activity site) is decreased by pretreatment with CID755673 (PKD inhibitor) indicating that PKD activity is required for PKC- Ө activation. On the other hand, sotrastaurin (PKC-Ө specific inhibitor) failed to prevent PKD phosphorylation at S916 (kinase activity site). Therefore PKC-Ө activity is not required for PKD activation. In contrast, sotrastaurin prevented PKD phosphorylation at S744/S748 (translocation site) in response to IL-1β, indicating that PKC-Ө activity is required for PKD1 translocation away from the plasma membrane. This suggests that PKD1 tightens the BBB during the early transient rise in resistance,

Location

DeRosa University Center, Ballroom

Start Date

30-4-2016 10:00 AM

End Date

30-4-2016 12:00 PM

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Apr 30th, 10:00 AM Apr 30th, 12:00 PM

Protein Kinase C and D Isoform Signaling at the Blood-Brain Barrier in Response to Interleukin-1β

DeRosa University Center, Ballroom

Interleukin (IL)-1β is a pro-inflammatory cytokine that participates in inflammation in the brain and other tissues. In the brain, IL-1β causes microvascular leakage and brain edema. Specifically, brain microvascular endothelial cells that protect the brain (the blood-brain barrier (BBB)) become leaky at intercellular tight junctions in response to IL-1β exposure. Previously, we found that IL-1β exposure decreases electrical resistance (TER) across BBB endothelial cell monolayers, which is preceded by a transient increase in resistance (tightening). We showed that the IL-1β dependent decrease in resistance occurs following activation of protein kinase isoforms PKC-Ө and PKD1, and that PKC-Ө is required for this response. In addition, PKD activity is required for increased barrier resistance in response to IL-1β, as well as physiological maintenance of BBB integrity in brain capillaries. Based on published reports of direct interactions between novel PKC isoforms and PKD1, we hypothesized that PKC-Ө and PKD1 interactions may account for the bimodal response to IL-1β. In the present study, we examined phosphorylation states of PKC-Ө and PKD1 using phosphorylation epitope specific antibodies and Western blotting. We found that the IL-1β induced phosphorylation of PKC-Ө at T538 (kinase activity site) is decreased by pretreatment with CID755673 (PKD inhibitor) indicating that PKD activity is required for PKC- Ө activation. On the other hand, sotrastaurin (PKC-Ө specific inhibitor) failed to prevent PKD phosphorylation at S916 (kinase activity site). Therefore PKC-Ө activity is not required for PKD activation. In contrast, sotrastaurin prevented PKD phosphorylation at S744/S748 (translocation site) in response to IL-1β, indicating that PKC-Ө activity is required for PKD1 translocation away from the plasma membrane. This suggests that PKD1 tightens the BBB during the early transient rise in resistance,