Transforming growth factor alpha binds to Trypanosoma cruzi amastigotes to induce signaling and cellular proliferation

Abstract

Macrophages secrete transforming growth factor alpha (TGF-alpha) to trigger proliferation of cancer cells. Here, we report a new role for TGF-alpha in modulating the direct cellular proliferation of a parasitic protozoan, Trypanosoma cruzi. Amastigotes present two classes of receptors for TGF-alpha with different binding affinities. (125)I-TGF-alpha binding was competed by an excess of cold epidermal growth factor and TGF-alpha but not by an irrelevant molecule. Upon binding of TGF-alpha to amastigotes, the ligand is internalized, inducing trypanosome tyrosine phosphorylation of 90- and 87-kDa proteins and increasing DNA synthesis and proliferation of amastigotes. Furthermore, exposure of macrophages to TGF-alpha induced increased amastigote proliferation. These results describe a novel mechanism used by amastigotes to regulate their proliferation mediated by a TGF-alpha-dependent signal transduction pathway.

FIG. 1.

¹²⁵I-TGF-α binds to amastigotes, and this binding is inhibited by cold excess TGF-α and EGF. (A) TGF-α binds to amastigotes. (Inset) Scatchard analysis of the binding data. B/F, bound/free (unbound) TGF-α. (B) Competition of ¹²⁵I-TGF-α binding by TGF-α, EGF, and bovine pituitary extract. The same concentration of amastigotes (5 × 10⁶ parasites ml⁻¹) were preincubated with different excess amounts (0- to 100-fold) of cold EGF and TGF-α followed by exposure to ¹²⁵I-TGF-α. Bars show means for triplicate samples of one representative experiment (± 1 standard deviation) selected from three independent experiments with similar results. *, significant difference compared to values for 0 and 1:1 cold/labeled ratios or to bovine pituitary extract values for 0 to 1:100 cold/labeled ratios (P < 0.05). The results are representative of three independent experiments.

FIG. 2.

TGF-α induces tyrosine phosphorylation of amastigote proteins. Amastigotes (5 × 10⁶ organisms) were incubated with TGF-α (2 ng/ml) for 5, 15, and 30 min at 4°C. Lysate samples (10 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, probed with antiphosphotyrosine monoclonal antibody PY20, and developed by chemiluminescence. Control parasites did not receive any growth factor. Blots were stripped, probed with anti-β-actin antibodies, and developed by chemiluminescence as a loading control. Results are from a representative experiment of three performed independently with similar results. Arrows point to enhanced protein tyrosine phosphorylation induced by TGF-α with respect to controls in the absence of growth factor treatment.

FIG. 3.

TGF-α stimulates DNA synthesis and proliferation of T. cruzi amastigotes in cell-free medium and within macrophages. (A) Incorporation of [³H]thymidine into amastigote DNA. Overnight-starved amastigotes (3 × 10⁶ organisms/ml) in RPMI with 1% BSA were incubated at 37°C with different concentrations of TGF-α and 1 μCi of [³H]thymidine. After 24 h, the organisms were harvested and the incorporation of [³H]thymidine was determined by measuring acid-insoluble radioactivity. (B) Amastigote proliferation in cell-free medium. Overnight-starved amastigotes (3 × 10⁶ organisms/ml) were incubated at 37°C in ML-15 medium alone (open bars) or in ML-15 medium containing 2 ng of TGF-α per ml (closed bars). The number of organisms was determined at 24-h intervals. (C) Amastigote proliferation within macrophages. Unelicited CBA/J mouse peritoneal macrophages were cultured with 2 ng of TGF-α per ml and trypomastigotes at a ratio of five parasites per macrophage. Parasite uptake was measured at 2 h and parasite multiplication was measured at 48 h as previously described (13). Bars represent the mean of triplicate samples of one representative experiment (± 1 standard deviation) selected from three independent experiments with similar results. *, significant difference compared to values at 0 and 2 h (P < 0.05).