PD98059 inhibited TGF-β1-induced ERK phosphorylation and enhanced TGF-β1-induced <i>NOS</i> expression in the <i>A. stephensi</i> midgut.

<p>(A) PD98059 inhibited ERK activation in mosquito midgut. <i>Anopheles stephensi</i> mosquitoes were fed on artificial bloodmeals supplemented with 2000 pg/ml TGF-β1 with or without 4 or 40 µM PD98059. At 20 min after completion of feeding, 100 midguts from each treatment group were dissected and prepared for immunoblot analysis. Protein lysate concentrations were measured by BCA assay; equal amounts of protein were loaded in each lane. The immunoblot is representative of 3 independent experiments. (B) Inhibition of ERK phosphorylation enhanced TGF-β1-induced <i>NOS</i> expression <i>in vivo</i>. <i>Anopheles stephensi</i> were fed on artificial bloodmeals supplemented with 40 µM PD98059 (PD), 2000 pg/ml TGF-β1, or 40 µM PD98059 and 2000 pg/ml TGF-β1 (TGF-β1+PD). A control group (BM) was provided an identical unsupplemented artificial bloodmeal. Previous studies confirmed that provision of small volumes of PBS and DMSO has no effect on <i>AsNOS</i> expression <i>in vivo</i> (not shown), so these treatments were not included here. <i>AsNOS</i> expression levels were analyzed from midguts (n = 15) collected at 24 h or 48 h after blood feeding from each treatment group. The data are represented as means±standard errors from 5 independent experiments for fold inductions compared with the PBS control. The data were analyzed by ANOVA for overall significance and Student's t-test was used for the pairwise comparison at 24 h.</p

Human TGF-β1 induced ERK phosphorylation in <i>Anopheles</i> cells <i>in vitro</i> and <i>in vivo</i>.

<p>(A) <i>Anopheles stephensi</i> ASE cells were treated with PBS (0) or human TGF-β1 at concentrations from 6–6000 pg/mL. ERK phosphorylation (pERK) was examined by western blotting at 5 min after treatment. Total ERK levels provided an assessment of protein loading and were used to normalize corresponding pERK levels. The fold increases in ERK phosphorylation from densitometry analyses are indicated relative to the PBS control. (B) ASE cells were treated with 6000 pg/ml TGF-β1 for the times indicated; the 0 h timepoint indicates the pre-treatment baseline. The fold increases in ERK phosphorylation from densitometry analyses are indicated relative to 0 h baseline. (C) <i>Anopheles gambiae</i> 4a3B cells were treated with 60 pg/ml TGF-β1 or equivalent volumes of PBS for 5, 15, 30, 60 min. The fold increases in ERK phosphorylation from densitometry analyses are indicated relative to the matched PBS control within each timepoint. (D) Female <i>A. stephensi</i> were allowed to feed on an artificial blood meal supplemented with 2–2000 pg/ml TGF-β1 or with an equivalent volume of PBS as a control (BM). Midguts (n = 100) were collected and processed for protein analysis at 20 min after completion of feeding. ERK phosphorylation levels from densitometry were normalized to total ERK levels and fold inductions relative to the NBM (no blood meal) control are indicated. Figures A–D are representative of immunoblots from 2–3 independent experiments.</p

Treatment effects and trends for mean <i>P. falciparum</i> oocysts per midgut by experiment.

1<p>Lower case letters indicate significant differences among experiments (all data combined) by ANOVA and Student-Neuman-Keuls test (α = 0.05). For Experiments 3 and 5, a total of 20 mosquitoes were dissected per treatment group. For Experiments 1, 2, 4, 6, 7, and 8, a total of 50 mosquitoes were dissected per treatment group.</p>2<p>Upper case letters indicate significant differences among treatment groups within an experiment by ANOVA and Student-Neuman-Keuls test (α = 0.05).</p

Prevalences of mosquitoes without <i>P. falciparum</i> oocysts by experiment after feeding on infected blood supplemented with TGF-β1 or with TGF-β1 and PD98059.

1<p>Lower case letters indicate significant differences among experiments (all data combined) by ANOVA and Student-Neuman-Keuls test (α = 0.05).</p>2<p>Two-tailed p-values calculated by chi-square test (α = 0.05).</p

PD98059 inhibition of ERK phosphorylation increased TGF-β1-induced <i>NOS</i> expression in <i>Anopheles</i> cells.

<p>(A) The MEK1/2 inhibitor PD98059 dose-dependently inhibited TGF-β1-dependent ERK phosphorylation in 4a3B cells. Cells were pre-treated with PD98059 for 40 min then treated with 60 pg/ml human TGF-β1 for 15 min. Cell lysates were analyzed by western blotting using anti-phospho-ERK or anti-ERK antibody. (B) PD98059 inhibited TGF-β1-dependent ERK phosphorylation in ASE cells. Cells were pre-treated with PD98059 at 4 or 10 µM for 40 min then treated with 6000 pg/ml TGF-β1 for 5 min. Cell lysates were subjected to western blot analysis as in (A). (C) Inhibition of ERK phosphorylation in 4a3B cells is persistent. 4a3B cells were pre-treated with 4 µM PD98059 or with an equivalent volume of dimethyl sulfoxide (DMSO; PD98059 diluent) then treated with 60 pg/ml TGF-β1 for 5 min to 6 h. Cell lysates were subjected to western blotting analysis as in (A). Immunoblots in A–C are representative of 2–3 independent experiments. (D) PD98059 reversed the inhibitory effect of ERK activation on TGF-β1 induced <i>NOS</i> expression. 4a3B cells were pre-treated with 4 µM PD98059 or an equivalent volume of DMSO, then treated with 60 pg/ml TGF-β1. An additional control group was treated with PBS (TGF-β1 diluent) at a volume equivalent to the TGF-β1 treatment. The data are represented as means±standard errors from three independent experiments. The data were analyzed using ANOVA for overall significance and by Student-Neuman-Keuls for multiple pairwise comparisons. Legend: * = p<0.05 (TGF-β1+PD98059 versus all other groups).</p

U0126 inhibition of ERK phosphorylation increased TGF-β1-induced <i>NOS</i> expression in <i>Anopheles</i> cells.

<p>(A) U0126 inhibited basal ERK phosphorylation and TGF-β1-induced ERK phosphorylation. ASE cells were pre-treated with 10 µM U0126 for 40 min then treated with 6 or 6000 pg/ml TGF-β1 or an equivalent volume of PBS. ERK phosphorylation (pERK) was examined by western blotting at 5 min after treatment; total ERK levels provided an assessment of protein loading. The immunoblot is representative of 3 independent experiments. (B) U0126 enhanced hTGF-β1-induced <i>AsNOS</i> expression. ASE cells were pre-treated with 10 µM U0126 then treated with 6 pg/ml TGF-β1 for 6, 24 and 48 h. DMSO and U0126 alone had no impact on <i>AsNOS</i> expression compared to the PBS control (not shown). <i>AsNOS</i> expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay. The data are represented as means±standard errors from three independent experiments for fold inductions compared with the PBS control. The data were analyzed by ANOVA for overall significance and Student's t-test was used for the pairwise comparison at 48 h.</p

A model of MEK-ERK signaling in TGF-β1-dependent control of <i>P. falciparum</i> development.

<p>Human TGF-β1 ingested during the blood meal activates MEK-ERK signaling in mosquito cells. Activation of ERK inhibits <i>NOS</i> gene expression which reduces nitric oxide synthase levels and the synthesis of reactive oxygen and nitrogen species. MEK-ERK signaling may inhibit the expression of other anti-parasite genes as well that function together with <i>NOS</i> to limit parasite development. Inhibition of the expression of <i>NOS</i> and other anti-parasite gene products would favor <i>P. falciparum</i> development in the mosquito midgut. In contrast, inhibition of MEK-ERK signaling by the MEK inhibitors PD98059 or U0126 increases anti-parasite activity, including TGF-β1-dependent <i>NOS</i> gene expression. Increased <i>NOS</i> expression results in higher nitric oxide synthase enzyme levels and the generation of inflammatory levels of reactive oxygen and nitrogen species <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000366#ppat.1000366-Peterson1" target="_blank">[7]</a> that are toxic to the parasite.</p

Effect of treatments on growth of asexual stage <i>Plasmodium falciparum</i>.

<p>Replicated cultures of <i>P. falciparum</i> NF54 were divided into treatment and control groups with 4 replicates per group and incubated for 50 h with the indicated treatments. The volume of PBS was equivalent to that used for TGF-β1 treatments. Parasite growth was analyzed as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000366#s2" target="_blank">Materials and Methods</a>. Relative growth is compared to the PBS control which is indicated as 100%. The data are represented as means and were analyzed using ANOVA for overall significance and by Student-Neuman-Keuls for multiple pairwise comparisons. Different lower case letters indicate differences (p<0.05) among treatment groups.</p

Inhibitor Competition

<p>The indicated amounts of falstatin and anti-falstatin antibody were incubated with lysates from asynchronous parasite cultures before addition of [¹²⁵I] DCG04, electrophoresis, and analysis by autoradiography. Results with increasing concentrations of falstatin (A), increasing concentrations of antibody (B), and increasing falstatin in the presence of antibody (C) are shown. Labels above the gels represent concentrations of falstatin and antibody (μg/ml). Proteins are labeled based on known migration patterns that were previously confirmed by mass spectrometry. FP, falcipain; DPAP1, dipeptidyl aminopeptidase1[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020117#ppat-0020117-b028" target="_blank">28</a>].</p

Immunolocalization of Falstatin

<div><p>(A) Immunofluorescence microscopy. Erythrocytes infected with synchronized 3D7 or W2 parasites were collected every 8 h, stained with DAPI and anti-falstatin antibodies and FITC-second antibody, and then evaluated by immunofluorescence microscopy.</p><p>(B) Immunoelectron microscopy. Late-schizont stage parasites were incubated with anti-falstatin antibodies and gold-conjugated second antibody and then evaluated by electron microscopy. Labels show individual merozoites (M) and erythrocyte cytosol (EC).</p></div