The regulation of connective tissue growth factor in fibroblasts by a soluble Trypanosoma cruzi factor

Trypanosoma cruzi infection of human foreskin fibroblasts results in repression of a group of genes involved in extracellular matrix remodeling, including the fibrogenic cytokine connective tissue growth factor (CTGF; CCN2). The same repression is observed after treatment with parasite-conditioned medium (PCM), which contains a complex mixture of secreted/shed trypomastigote molecules. Treatment with PCM results in repression of basal CCN2 levels and antagonizes several CCN2 inducing agents including TGF-β LPA, serum and endothelin-1. We have determined a variety of physical and biochemical characteristics of the parasite protein responsible for the repression of CCN2. Through a combination of purification methods, we have produced an enriched fraction for which mass spectrometry data was obtained. We identified a group of candidate proteins that may be responsible for the CCN2 repression and have investigated a subset of that group. While we were unable to attain purity, efforts to date indicate that T. cruzi trypomastigotes release a proteinaceous complex capable of repressing mammalian CCN2 expression. An investigation of the host cell signaling pathways affected by PCM treatment showed a profound inhibition of the three arms of MAP kinase signaling. We demonstrate that both the CArG-like box and ETS sequence in the CCN2 promoter are involved in PCM-mediated repression of expression. We demonstrate an inhibition of SRF binding to the CArG box, which is concomitant with a reduction in Elk-1 activation. This is the first evidence that PCM treatment results in an inhibition of MAP kinase signaling, which is upstream of ETS family transcription factors that are involved in the gene expression inhibition at multiple sites of the CCN2 promoter. As an outgrowth of the signaling study we also measured the effects of PCM treatment and parasite infection on cytoskeletal mechanical properties and remodeling dynamics in the host cell. HFF incubated with PCM stiffened, while infected fibroblasts displayed a time-dependant softening. In addition only those HFF treated with PCM displayed accelerated cytoskeletal remodeling. This is the first direct mechanical evidence that the initial stages of infection favour an intact, stiff, and rapidly remodeling cytoskeleton, followed by cytoskeletal softening and disassembly during subsequent parasite replication.