Differential Regulation of Cysteinyl Leukotriene Receptor Signaling by Protein Kinase C in Human Mast Cells

Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT1R and CysLT2R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT1R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD4 and LTE4-induced calcium influx through CysLT1R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1β generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1β generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT1R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology

TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation

Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells (epithelial) themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation in vivo. Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation

Effect of siRNA-mediated knockdown of PKCα and PKCε on cys-LT-mediated calcium, c-fos and MIP1β generation by LAD2 cells.

<p>PKCα and PKCε isoforms were Knocked down using specific siRNAs against PKCα and PKCε (10 nM). Non-specific (NS) siRNA was used as a control. The siRNA treated cells were analyzed for (A) the expression of PKCα, PKCε and GAPDH, (B, C) LTD₄ (500 nM)-induced calcium influx, and quantitative analysis, (D, E) cys-LT-induced c-fos expression, (F) MIP1β production. Cells were treated with 500 nM of LTD₄ and LTE₄ for 5 minutes (calcium flux), 1 h (c-fos expression) and 6 h (MIP1β). Data shown are±SD of three separate experiments. *P<0.05, **P<0.001.</p

A model depicting PKC regulated cys-LT signaling in MCs.

<p>Hypothetical mechanism(s) depicting cys-LT-mediated signaling in MCs by PKCs. CysLT₁R activation by LTD₄ or LTE₄ activates PKCα and PKCε. PKCα desensitize CysLT₁R by phosphorylating the receptor and negatively regulating the calcium flux. On the other hand, PKCε activation by CysLT₁R activates c-fos expression, MIP1β production. Cys-LTs also activate Erk and CREB independent of PKCs.</p

LTD₄ and LTE₄-induced phosphorylation and expression of c-fos in LAD2 cells and the effect of PKC inhibition.

<p>Relative levels of c-fos transcript (A) upon treatment with 500 nM of LTD₄ and LTE₄, c-fos phosphorylation and expression with LTD₄ or LTE₄ (500 nM) for indicated period (B), Dose response (C), Pre-treated with GFX (2 µM) or MK571 (1 µM) (D), stimulated with 500 nM of cys-LTs and analyzed by western blotting. Blots were stripped and blotted for GAPDH. The data shown are representative of three separate experiments.</p

Phosphorylation of Erk and CREB by LAD2 cells in response to LTD₄ and LTE₄ and the role of PKC in mediating these effects.

<p>(A) phosphorylation as well as total expression of Erk and CREB proteins by Western blotting in cell lysates of LAD2 cells pre-treated with GFX (2 µM) and stimulated with 500 nM of LTD₄ and LTE₄ respectively for 30 minutes (B) Quantitative analysis. The shown data represents±SD of three separate experiments. *P<0.05. NS = non-significant.</p

Involvement of PKC in LTD₄ and LTE₄-induced MIP1β secretion in LAD2 cells.

<p>LAD2 cells were stimulated with 500 nM of LTD₄ or LTE₄ for 6 h in presence or absence of GFX (2 µM). The generation of MIP1β was analyzed from the culture supernatant using MIP1β-specific ELISA. Data shown are±SD of three independent experiments. ** P<0.001. NS = non-significant.</p

Effect of PKC inhibition on calcium signaling by LTD₄ and LTE₄ in LAD2 cells.

<p>Calcium transients from hMC (A) and LAD2 cells (B) with cys-LTs (500 nM) in presence or absence of PKC inhibitor, GFX (2 µM) (C) Quantitative analysis of calcium influx from A and B and the effect of MK571 (1 µM) on the enhanced calcium flux with GFX pre-treatment. The data shown are±SD of three experiments. The significance was tested using Student’s t-test as well as one-way ANOVA followed by Tukey post-hoc analysis. P<0.05. NS = non-significant.</p