The mosquito melanization response requires hierarchical activation of non-catalytic clip domain serine protease homologs.

Serine protease cascades regulate important insect immune responses namely melanization and Toll pathway activation. An important component of these cascades are clip-domain serine protease homologs (cSPHs), which are non-catalytic, but essential for activating the enzyme prophenoloxidase (PPO) in the melanization response during septic infections. The activation of cSPHs requires their proteolytic cleavage, yet factors that control their activation and the complexity of their interactions within these cascades remain unclear. Here, we report the identification of CLIPA28 as a novel immune-related cSPH in the malaria vector Anopheles gambiae. Functional genetic analysis using RNA interference (RNAi) revealed that CLIPA28 is essential for the melanization of Plasmodium berghei parasites in refractory mosquitoes, and for mosquito resistance to fungal infections. We further show, using combined biochemical and genetic approaches, that CLIPA28 is member of a network of at least four cSPHs, whereby members are activated in a hierarchical manner following septic infections. Depletion of the complement-like protein TEP1 abolished the activation of this network after septic infections, whereas, depletion of the serine protease inhibitor 2 (SRPN2) triggered enhanced network activation, even in naïve mosquitoes, culminating in a dramatic reduction in cSPHs hemolymph levels, which paralleled that of PPO. Our data suggest that cSPHs are engaged in complex and multilayered interactions within serine protease cascades that regulate melanization, and identify TEP1 and SRPN2 as two master regulators of the cSPH network

The epinephrine-induced PGE2 reduces Na+/K+ ATPase activity in Caco-2 cells via PKC, NF-κB and NO.

We showed previously an epinephrine-induced inhibition of the Na+/K+ ATPase in Caco-2 cells mediated via PGE2. This work is an attempt to further elucidate mediators downstream of PGE2 and involved in the observed inhibitory effect. The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme. Changes in the protein expression of the Na+/K+ ATPase were investigated by western blot analysis which revealed a significant decrease in the abundance of the ATPase in plasma membranes. Treating the cells with epinephrine or PGE2 in presence of SC19220, a blocker of EP1 receptors abolished completely the effect of the hormone and the prostaglandin while the effect was maintained unaltered in presence of antagonists to all other receptors. Treatment with calphostin C, PTIO, ODQ or KT5823, respective inhibitors of PKC, NO, soluble guanylate cyclase and PKG, abrogated completely the effect of epinephrine and PGE2, suggesting an involvement of these mediators. A significant inhibition of the ATPase was observed when cells were treated with PMA, an activator of PKC or with 8-Br-cGMP, a cell permeable cGMP analogue. PMA did reduce the protein expression of IκB, as shown by western blot analysis, and its effect on the ATPase was not manifested in presence of an inhibitor of NF-κB while that of SNAP, a nitric oxide donor, was not affected. The results infer that NF-κB is downstream PKC and upstream NO. The data support a pathway in which epinephrine induces the production of PGE2 which binds to EP1 receptors and activates PKC and NF-κB leading to NO synthesis. The latter activates soluble guanylate cyclase resulting in cGMP production and activation of PKG which through direct or indirect phosphorylation inhibits the Na+/K+ ATPase by inducing its internalization

Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2.

Epinephrine, a key stress hormone, is known to affect ion transport in the colon. Stress has been associated with alterations in colonic functions leading to changes in water movements manifested as diarrhea or constipation. Colonic water movement is driven by the Na+-gradient created by the Na+/K+-ATPase. Whether epinephrine acts via an effect on the Na+/K+-ATPase hasn't been studied before. The aim of this work was to investigate the effect of epinephrine on the Na+/K+-ATPase and to elucidate the signaling pathway involved using CaCo-2 cells as a model. The activity of the Na+/K+-ATPase was assayed by measuring the amount of inorganic phosphate released in presence and absence of ouabain, a specific inhibitor of the enzyme. Epinephrine, added for 20 minutes, decreased the activity of the Na+/K+-ATPase by around 50%. This effect was found to be mediated by α2 adrenergic receptors as it was fully abolished in the presence of yohimbine an α2-blocker, but persisted in presence of other adrenergic antagonists. Furthermore, treatment with Rp-cAMP, a PKA inhibitor, mimicked epinephrine's negative effect and didn't result in any additional inhibition when both were added simultaneously. Treatment with indomethacin, PP2, SB202190, and PD98059, respective inhibitors of COX enzymes, Src, p38MAPK, and ERK completely abrogated the effect of epinephrine. The effect of epinephrine did not appear also in presence of inhibitors of all four different types of PGE2 receptors. Western blot analysis revealed an epinephrine-induced increase in the phosphorylation of p38 MAPK and ERK that disappeared in presence of respectively PP2 and SB2020190. In addition, an inhibitory effect, similar to that of epinephrine's, was observed upon incubation with PGE2. It was concluded that epinephrine inhibits the Na+/K+-ATPase by the sequential activation of α2 adrenergic receptors, Src, p38MAPK, and ERK leading to PGE2 release

Epinephrine modulates Na⁺/K⁺ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2 - Fig 7

<p>(A) Effect of epinephrine (0.5 mM, 20min) and (B) PGE2 (1nM, 20min) on the activity of the Na⁺/K⁺ ATPase when ERK was inhibited with PD98059 (50μM, added 15 min before epinephrine or PGE2). Values are means ± SEM of 3 observations. Bars not sharing the same letter are considered significantly different from each other at p<0.01. (C) Epinephrine but not PGE2 increased significantly the phosphorylation of ERK. Values of the normalized data (p-ERK/ERK) are means ± SEM of 3 observations. Densitometry bars that do not share the same letter are considered significantly different from each other at p<0.01. The blots are representative of an experiment repeated 3 times.</p

Proposed signaling pathway of epinephrine’s action on the Na+/K+ ATPase.

<p>Proposed signaling pathway of epinephrine’s action on the Na+/K+ ATPase.</p

Epinephrine modulates Na⁺/K⁺ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2 - Fig 5

<p>(A) Effect of epinephrine (0.5 mM, 20min) and (B) PGE2 (1nM,20min) on the activity of the Na⁺/K⁺ ATPase in presence of the Src inhibitor PP2 (20μM, added 15 min before epinephrine or PGE2). Values are means ± SEM of 3 observations. Bars not sharing the same letter are considered significantly different from each other at p<0.01. (C) Epinephrine but not PGE2 increased Src phosphorylation. Values of the normalized data (p-Src/Src) are means ± SEM of 3 observations. Densitometry bars that do not share the same letter are considered significantly different from each other at p<0.01. The blots are representative of an experiment repeated 3 times.</p

(A)RpcAMP (30 μM, 20min), an inhibitor of PKA, reduced the Na⁺/K⁺ ATPase activity while (B) dbcAMP (10μM, 20min), had no effect.

<p>Values are means ±SEM of 3 observations. Bars not sharing a common letter are significantly different from each other at P<0.01.</p

Epinephrine inhibits the Na⁺/K⁺ ATPase by activating α2 adrenergic receptors.

<p>Effect of epinephrine (0.5 mM, 20min) on the Na⁺/K⁺ ATPase activity in presence of (A) prazosin (50 μM), an α1 blocker (B) propranolol (0.03M), a β adrenergic blocker, and (C) yohimbine (0.1mM), an α2 blocker. Blockers were added 20 min prior to the addition of epinephrine. Values are means ±SEM of a minimum of 3 observations. Bars not sharing a common letter are significantly different from each other at P<0.01.</p

Involvement of PGE2 in epinephrine signaling.

<p>(A) Effect of epinephrine (0.5 mM, 20min) on the activity of the Na⁺/K⁺ ATPase in presence of indomethacin (100μM, a COX inhibitor added 20 min before epinephrine; (B) Effect of exogenous PGE2 (1nM,20min) on pump’s activity. (C) Effect of epinephrine in presence of inhibitors of all PGE2 receptors. Values are means ± SEM of 4 observations. Bars not sharing the same letter are considered significantly different from each other at p<0.01.</p

Epinephrine modulates Na⁺/K⁺ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2 - Fig 6

<p>(A) Effect of epinephrine (0.5 mM, 20min) and (B) PGE2 (1nM,20min) on the activity of the Na⁺/K⁺ ATPase when p38MAPK was inhibited with SB202190 (50μM, added 15 min before epinephrine or PGE2). Values are means ± SEM of 3 observations. Bars not sharing the same letter are considered significantly different from each other at p<0.01. (C)Epinephrine increased significantly the phosphorylation of p38MAPK but PGE2 exerted no effect. Values of the normalized data (p38MAPK / p38MAPK) are means ± SEM of 3 observations. Densitometry bars that do not share the same letter are considered significantly different from each other at p<0.01. The blots are representative of an experiment repeated 4 times.</p