Date Published: September 8, 2011
Publisher: Public Library of Science
Author(s): G. Adam Mott, Jaime A. Costales, Barbara A. Burleigh, Najib M. El-Sayed. http://doi.org/10.1371/journal.pone.0023482
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.
Partial Text: The kinetoplastid protozoan parasite Trypanosoma cruzi causes Chagas’ disease in humans, a chronic and debilitating condition affecting several million individuals in Latin America. T. cruzi is transmitted by an insect vector which gains access to the host via breaches in the skin or through mucosal membranes, mainly conjunctival or gastric mucosa , . As an obligate intracellular parasite that disseminates from initial infection sites to tissues such as heart and smooth muscle, T. cruzi undergoes multiple rounds of invasion, growth and egress from infected cells during the acute stage of infection. Very little is currently known regarding the early interactions between T. cruzi and its host that facilitate establishment of infection in vivo. Cellular models of T. cruzi infection have been very useful for defining the molecular and cellular events that regulate the early parasite-host cell interactions and host cell invasion. During its early interaction with mammalian host cells, trypomastigotes, the invasive forms of T. cruzi, trigger rapid changes in a number of cellular signaling pathways to facilitate the process of parasite entry into non-professional phagocytic cells (reviewed in , ). While these early signaling events have been relatively well-studied in the context of T. cruzi invasion, little is known regarding the impact of these parasite-induced signaling cascades downstream of the invasion process. Transcriptional profiling of T. cruzi-infected fibroblasts revealed that the earliest detectable changes triggered by infective T. cruzi trypomastigotes involve down-regulation of a small subset of genes including members of the CCN family (cyr61 and ctgf/ccn2) , which play important roles in angiogenesis and extracellular matrix (ECM) homeostasis . T. cruzi-dependent dampening of ctgf/ccn2 expression occurs at both the mRNA and protein levels and is mediated by a secreted/released parasite factor that is capable of antagonizing TGF-ß-mediated induction of ctgf/ccn2.
This study demonstrates that mammalian-infective forms of Trypanosoma cruzi release a factor (or factors) that significantly impacts host cell signaling cascades, thereby altering the expression of a subset of genes involved in cell proliferation, wound repair and inflammation. Focusing on the pro-fibrogenic cytokine, CTGF/CCN2, a multifunctional secreted protein with a central role in wound repair and fibrosis , we demonstrate that a shed/secreted T. cruzi factor inhibits both basal and agonist-induced up-regulation of ctgf/ccn2 gene expression. In line with previous reports demonstrating a critical role for MAP kinases in regulating ctgf/ccn2 gene expression in response to pro-fibrogenic agonists , , , , , our data demonstrate a role for Erk1/2, JNK and p38 in TGF-ß-mediated ctgf/ccn2 expression in low passage primary HFF. Exposure of HFF to the shed/secreted T. cruzi fraction inhibits basal and agonist-dependent phosphorylation of Erk1/2, JNK and p38 and the induction of ctgf/ccn2 expression, a downstream target of MAP kinase activation . Analysis of the global transcriptional response to T. cruzi PCM in HFF revealed significant repression of a subset of TGF-ß-inducible genes involved in cell proliferation, wound healing and inflammatory responses, where the genes most sensitive to T. cruzi-mediated inhibition (EGR1, HBEGF, PTGS2, LIF, EDN1, IL6, and CYR61) are known to be regulated by MAP kinase-activated transcription factors such as the ETS-family members and AP-1 , , , , , , . In addition, genes encoding components of the AP-1 complex, c-jun and c-fos, as well as egr-1 were also rapidly down-regulated in T. cruzi PCM treated cells, suggesting the disruption of an important regulatory network centered around MAP kinase signaling, which is required for efficient expression of ctgf/ccn2 and a host of other TGF-ß-inducible genes. Consistent with the notion that T. cruzi might negatively regulate MAP kinase-activated transcription factors, we demonstrated that a functional Ets-binding site (spanning −91 and −84 bp) in the upstream ctgf/ccn2 promoter region, which is critical for TGF-ß stimulated expression of ctgf/ccn2, , , is required for T. cruzi PCM repression of both basal and TGF-ß-stimulated reporter expression. Thus, we propose that the observed inhibition of agonist-induced phosphorylation of the MAP kinases, Erk, p38 and JNK, would impact the activation of MAP kinase-activated transcription factors such as Ets-1. Failure to assemble Ets transcription factor(s) at the Ets binding site, possibly in conjunction with other accessory proteins such as AP-1 , would prevent activation of the ctgf/ccn2 promoter in response to signals mediated by exogenous agonists such as TGF-ß . Overall, our findings reinforce recent literature regarding the critical role for MAP kinase signaling in regulation of ctgf/ccn2 expression , ,  as well as for the role of the Ets-binding site on the ctgf/ccn2 promoter ,  in driving ctgf/ccn2 gene expression. Given that CTGF/CCN2 is a therapeutic target for the prevention and treatment of a number of disease states in which the expression of this critical cytokine is dysregulated , , , , our finding that the mammalian-infective forms of T. cruzi release a factor that interferes with agonist-dependent up-regulation of CTGF/CCN2 warrants further study.