A soluble factor from Trypanosoma cruzi inhibits transforming growth factor-ß-induced MAP kinase activation and gene expression in dermal fibroblasts

Abstract

The protozoan parasite Trypanosoma cruzi, which causes human Chagas' disease, exerts a variety of effects on host extracellular matrix (ECM) including proteolytic degradation of collagens and dampening of ECM gene expression. Exposure of primary human dermal fibroblasts to live infective T. cruzi trypomastigotes or their shed/secreted products results in a rapid down-regulation of the fibrogenic genes collagenIα1, fibronectin and connective tissue growth factor (CTGF/CCN2). Here we demonstrate the ability of a secreted/released T. cruzi factor to antagonize ctgf/ccn2 expression in dermal fibroblasts in response to TGF-ß, lysophosphatidic acid or serum, where agonist-induced phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2, p38 and JNK was also inhibited. Global analysis of gene expression in dermal fibroblasts identified a discrete subset of TGF-ß-inducible genes involved in cell proliferation, wound repair, and immune regulation that are inhibited by T. cruzi secreted/released factors, where the genes exhibiting the highest sensitivity to T. cruzi are known to be regulated by MAP kinase-activated transcription factors. Consistent with this observation, the Ets-family transcription factor binding site in the proximal promoter region of the ctgf/ccn2 gene (-91 bp to -84 bp) was shown to be required for T. cruzi-mediated down-regulation of ctgf/ccn2 reporter expression. The cumulative data suggest a model in which T. cruzi-derived molecules secreted/released early in the infective process dampen MAP kinase signaling and the activation of transcription factors that regulate expression of fibroblast genes involved in wound repair and tissue remodelling, including ctgf/ccn2. These findings have broader implications for local modulation of ECM synthesis/remodelling by T. cruzi during the early establishment of infection in the mammalian host and highlight the potential for pathogen-derived molecules to be exploited as tools to modulate the fibrogenic response.

Figure 1. T. cruzi PCM inhibits agonist-induced ctgf/ccn2 expression.

HFF were incubated with medium or T. cruzi PCM, alone or in combination with 5 ng/ml TGF-ß, 2 hours prior to mRNA harvest for quantitative real-time PCR analysis (A) or 24 hours prior to analysis of CTGF/CCN2 protein levels by western blot (B). ctgf/ccn2 mRNA levels were analyzed by quantitative real-time PCR following stimulation of HFF with serum (2% v/v) (C) or 10 µM LPA (D) for 2 hours in the presence or absence of T. cruzi PCM. Data is represented as the mean ± S.E. from 5 independent experiments carried out in duplicate. Statistical significance was assessed using the Student's t-test, (** p<0.01).

Figure 2. T. cruzi PCM abrogates MAP kinase activation and decreased MAP kinase signaling results in inhibition of ctgf/ccn2 expression.

(A) HFF were stimulated with 5 ng/ml TGF-ß1 in the presence of medium, T. cruzi PCM or with 10 µM SP600125, 20 µM SB203580 or 50 µM PD98059 as indicated for 2 hours prior to mRNA harvest for quantitative real-time PCR analysis. MAP kinase inhibitor treatments were carried out following a 30-minute pre-incubation step. Data is represented as the mean ± s.e. from triplicate experiments (n = 4). Statistical significance was assessed using the Student's t-test (** p<0.01, * p<0.05). (B) Western blot of phospho-Erk, phospho-JNK and phospho-p38 normalized to total Erk, JNK and p38 respectively in lysates from HFF stimulated with 5 ng/ml TGF-ß1 or 10 µM LPA for 5 or 15 minutes in serum-free media or parasite-conditioned medium. Results for densitometric analysis are shown as numerical values below each panel and represented as phosphorylation relative to mock-treated controls (arbitrarily set to a value of 1.0).

Figure 3. The Ets transcription factor binding site in the ctgf/ccn2 promoter is required for T. cruzi PCM-mediated repression of reporter expression.

SEAP reporter constructs driven by nucleotides −805 to +17 (−805), −244 to +17 (−244), −86 to +17 (−86) or −805 to +17 with a mutated ETS sequence (−805 ETS) representing the proximal ctgf/ccn2 promoter region (A) along with a ß-galactosidase containing control plasmid were transfected into HFF. (B) Serum-starved transfected cells were stimulated with medium alone or 5 ng/ml TGF-ß1 in the presence or absence of T. cruzi PCM for 6 hours. Relative reporter expression in treated cells relative to mock-treated controls is shown as the mean ± s.e. (n = 3). (*p<0.05, p<0.01).

Figure 4. T. cruzi PCM inhibits the expression of a broader range of TGF-ß-inducible genes.

Quantitative real-time PCR analysis of the relative expression of representative PCM-sensitive and PCM-insensitive TGF-ß-inducible genes in HFF after 2 hours of treatment with 5 ng/ml TGF-ß in media or PCM. Genes analyzed: HBEGF, heparin-binding EGF-like growth factor; PTSG2, prostaglandin-endoperoxide synthase 2; CTGF, connective tissue growth factor; Cyr61, cysteine-rich angiogenic factor; SERPINE2, plasminogen activator inhibitor type 1, member 2; CDKN2B; cyclin-dependent kinase 4 inhibitor B; SMAD7, MAD, mothers against decapentaplegic homolog 7; TSPAN2, tetraspanin 2.

Figure 5. Top biological network relevant to the subset of PCM-sensitive TGF-ß-inducible genes.

Biological network was generated through the use of Ingenuity Pathway Analysis software (Ingenuity® Systems, www.ingenuity.com) by uploading the group of TGF-ß-inducible genes that were highly sensitive to T. cruzi PCM. The analysis reports direct (solid lines) and indirect (dotted lines) relationships between genes as established in the literature. Thirteen (shaded symbols) of 15 genes entered into the analysis were included as focus genes in this network.