EGF-mediated EGFR/ERK signaling pathway promotes germinative cell proliferation in Echinococcus multilocularis that contributes to larval growth and development

Abstract

Background: Larvae of the tapeworm E. multilocularis cause alveolar echinococcosis (AE), one of the most lethal helminthic infections in humans. A population of stem cell-like cells, the germinative cells, is considered to drive the larval growth and development within the host. The molecular mechanisms controlling the behavior of germinative cells are largely unknown.

Methodology/principal findings: Using in vitro cultivation systems we show here that the EGFR/ERK signaling in the parasite can promote germinative cell proliferation in response to addition of human EGF, resulting in stimulated growth and development of the metacestode larvae. Inhibition of the signaling by either the EGFR inhibitors CI-1033 and BIBW2992 or the MEK/ERK inhibitor U0126 impairs germinative cell proliferation and larval growth.

Conclusions/significance: These data demonstrate the contribution of EGF-mediated EGFR/ERK signaling to the regulation of germinative cells in E. multilocularis, and suggest the EGFR/ERK signaling as a potential therapeutic target for AE and perhaps other human cestodiasis.

Fig 1. EGF stimulates E. multilocularis larval growth and development.

(A-B) Metacestode vesicles were cultivated in conditioned medium (C-medium) (A) or serum-containing DMEM (B) supplemented with 100 ng/mL recombinant human EGF or not. Vesicle growth is shown as the increase of vesicle diameter as compared to day 0. Comparison between the EGF group and the control group at the same timepoint was performed using two-tailed Student’s t-test. (C) Vesicle formation from protoscolex in conditioned medium (control) with addition of EGF. Control was set to 1 and results were normalized against the control (right). Representative images of the formation process are shown (left). Bar = 50 μm. (D) Survival of the vesicles in serum-free DMEM supplemented with EGF. Data in (A-D) are shown as mean ± SD of at least three replicates, representative of 2–3 independent experiments.*P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 2. EGF promotes E. multilocularis germinative cell proliferation.

(A) Vesicles were incubated with BrdU for two days and chromosomal DNA was isolated for BrdU incorporation assay. Control was set to 1 and results were normalized against the control. Data are shown as mean ± SD of triplicates. ** P < 0.01. (B) Vesicles were in vitro cultivated under normal conditions and germinative cell proliferation was visualized by EdU-BrdU dual labeling. Insert shows the magnified view. Arrows indicate EdU⁺BrdU⁺ cells. Bar = 20 μm. (C) Vesicles were pretreated with 40 mM of hydroxyurea for three days and allowed for recovery in conditioned medium (control) supplemented with EGF. Germinative cell proliferation was analyzed after 4 days of removal of hydroxyurea and representative images are shown. Quantification of EdU⁺ and EdU⁺BrdU⁺ cells is shown in the right panel. Data are shown as mean ± SD of 3 separate labeling experiments. * P < 0.05; ** P < 0.01. Bar = 20 μm.

Fig 3. EGF activates E. multilocularis EGF receptor EmER in Xenopus oocyte expression system.

(A) Membrane extracts were prepared from Xenopus oocytes expressing EmER or not (NI, noninjected) and immunoprecipitated and analyzed by western blot using the anti-flag antibody. The results of two independent injection experiments are presented (I-1 and I-2). The bands exhibited a molecular mass larger than expected (178 kDa), which could be attributable to glycosylation. (B) Oocytes expressing EmER were incubated with CI-1033 or DMSO for 4 h followed by 20 minutes of EGF stimulation. Membrane extracts were immunoprecipitated by the anti-flag antibody and analyzed by western blot using the anti-flag or anti-phospho-tyrosine antibodies. (C) Induction of GVBD (germinal vesicle breakdown) in EmER-expressing Xenopus oocytes. Oocytes that had been expressing EmER for 48 h were pretreated with CI-1033 or DMSO for 4 h and then incubated with EGF. GVBD was monitored after 16 h of EGF incubation and the mean percentages of oocytes exhibiting GVBD for three separate experiments are shown. Noninjected oocytes were used as controls. DMSO (final concentration 0.25%) was found no effects on GVBD. PG: progesterone; “/”: not tested.

Fig 4. Inhibition of E. multilocularis EGFR impairs germinative cell proliferation.

(A) Metacestode vesicles were treated with 10 μM CI-1033 and the representative images for day 0, 3 and 6 are shown on the left (red: EdU; blue: DAPI). Quantifications of the EdU⁺ germinative cells in the vesicles treated with 10 μM CI-1033 for indicated time (middle) and 5–10 μM CI-1033 or DMSO control (0) for 6 days (right) are shown. Values represent the mean ± SD of 5 separate labeling experiments. * P < 0.05; *** P < 0.001. (B) Effects of CI-1033 on germinative cell proliferation during the recovery from hydroxyurea treatment. Vesicles were allowed for recovery in conditioned medium supplemented with EGF, and CI-1033 was added into the medium to a final concentration of 10 μM immediately after the initial EdU pulse. Germinative cell proliferation was analyzed by EdU-BrdU dual labeling after 4 days of recovery. Images show rare EdU⁺ and EdU⁺BrdU⁺ cells following CI-1033 treatment (see the text). (C) Representative images of the accumulations of EdU⁺ germinative cells in some cell aggregates (indicated by the dashed-line boxes) in the vesicles treated with DMSO (control) or 10 μM CI-1033 for 6 days (red: EdU; blue: DAPI). (D) Effects of CI-1033 on the larval growth and development. Vesicles or protoscoleces were cultivated in DMSO-containing conditioned medium (control) supplemented with the ingredients as indicated. Vesicle growth (left) and vesicle formation from protoscoleces (right) were analyzed after 28 days and 18 days of cultivation, respectively. Data are shown as mean ± SD of triplicates, representative of 3 independent experiments. ** P < 0.01 and *** P < 0.001. Bar = 20 μm in (A), (B) and (C).

Fig 5. EGFR/ERK signaling contributes to E. multilocularis germinative cell proliferation.

(A) CI-1033 impairs E. multilocularis ERK phosphorylation. Vesicles were treated with 10 μM CI-1033 for indicated time. (B) CI-1033 inhibits EGF-stimulated E. multilocularis ERK phosphorylation. Vesicles were treated with 10 μM CI-1033 (+) or DMSO (-) for 6 h, followed by stimulation with EGF (+) or not (-) for 20 minutes. (C) U0126 impairs E. multilocularis ERK phosphorylation. Vesicles were treated with indicated concentrations of U0126 for 6 h. (D) U0126 inhibits EGF-stimulated E. multilocularis ERK phosphorylation. Vesicles were treated with 20 μM U0126 (+) or DMSO (-) for 6 h, followed by stimulation with EGF (+) or not (-) for 20 minutes. (E) U0126 reduces the number of EdU⁺ germinative cells. Vesicles were treated with 20 μM U0126 for 6 days. Representative images are shown on the left and the quantification is shown on the right. Values represent the mean ± SD of 4 separate labeling experiments. *** P < 0.001. Bar = 20 μm. (F) Effects of U0126 on germinative cell proliferation during the recovery from hydroxyurea treatment. Vesicles were allowed for recovery in conditioned medium supplemented with EGF and U0126 was added into the medium to a final concentration of 20 μM immediately after the initial EdU pulse. Germinative cell proliferation was analyzed after 4 days of recovery. Images show rare EdU⁺ and EdU⁺BrdU⁺ cells following U0126 treatment (see the text). Bar = 20 μm. (G) Effects of U0126 on the larval growth. Vesicles were incubated with 10 μM U0126 and parasite growth was analyzed after 28 days of cultivation. Data are shown as mean ± SD of triplicates. ** P < 0.01.