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(The FASEB Journal. 2004;18:1625-1635.)
© 2004 FASEB

Inhibition of host connective tissue growth factor expression: a novel Trypanosoma cruzi-mediated response

MEERA UNNIKRISHNAN and BARBARA A. BURLEIGH1

Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA

1Correspondence: Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115, USA. E-mail: bburleig{at}hsph.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Connective tissue growth factor (CTGF) is a secreted cytokine that plays a fundamental role in the development of tissue fibrosis by mediating many of the profibrotic effects of TGF-ß. We present the novel finding that the intracellular pathogen Trypanosoma cruzi elicits immediate and sustained repression of basal CTGF expression in dermal fibroblasts, followed by down-regulation of the extracellular matrix proteins, fibronectin, and collagen I {alpha}1. To address mechanisms underlying this response, the major CTGF-regulating pathways were investigated. We report that both T. cruzi trypomastigotes and secreted parasite factor(s) antagonize TGF-ß-dependent induction of CTGF in fibroblasts. Of the TGF-ß-dependent signaling pathways required for CTGF expression, we demonstrate that T. cruzi interferes with cellular Erk1/2 phosphorylation but not Smad3 signaling. While increased stimulation of Erk phosphorylation alone was insufficient to override the parasite-mediated repression of CTGF, stimulation of fibroblasts with increased concentrations of TGF-ß, which activates both Smad3 and Erk1/2, completely abrogated this inhibition. Together with the finding that T. cruzi-mediated down-regulation of CTGF expression requires de novo host cell protein synthesis, our data indicate that the unique ability of T. cruzi to interfere with the host fibrogenic response is a complex process requiring input from multiple host cell signaling pathways.—Unnikrishnan, M., Burleigh, B. A. Inhibition of host connective tissue growth factor expression: a novel Trypanosoma cruzi-mediated response.


Key Words: parasite • extracellular matrix • infection • mitogen-activated kinases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRYPANOSOMA CRUZI is an intracellular protozoan pathogen that causes human Chagas’ disease, the primary contributor to heart disease in Latin America (1) . Infective trypomastigotes are capable of activating a variety of host cell signaling pathways early in the host cell invasion process (2) . T. cruzi-triggered Ca2+ signaling (3) , phosphatidylinositol-3 kinase activation (4) cAMP elevation (5) , and transforming growth factor ß (TGF-ß) receptor signaling (6) are known to facilitate entry of this pathogen into a variety of nonprofessional phagocytic cell types. In a recent study using DNA microarray hybridization to examine the global transcriptional response of human dermal fibroblasts to infection with T. cruzi trypomastigotes, minimal modulation of host cell gene expression was observed (7) . Remarkably, none of the ~27,000 human sequences represented on the arrays were significantly up-regulated in response to T. cruzi at early time points of infection (2–6 h) (7) . Just as striking was the immediate repression of transcripts encoding connective tissue growth factor (CTGF) and related cysteine-rich secreted proteins ADAMTS and Cyr61 observed in dermal fibroblasts infected with T. cruzi. This limited transcriptional response to T. cruzi provides a striking contrast to the rapid and considerable number of changes in host cell gene expression elicited by pathogens in general (8–11). Of the transcripts immediately repressed in response to T. cruzi, CTGF was the only gene for which repression was maintained throughout the time course of infection studied (2–24 h) and for which expression was similarly altered in response to soluble secreted/released T. cruzi factors (7) . Since T. cruzi is known to exploit a cellular wound repair pathway involving a Ca2+-regulated lysosome exocytosis for host cell invasion (12 , 13) , the selective repression of CTGF, a TGF-ß-dependent cytokine normally up-regulated in tissue repair processes (14) , presented an apparent paradox.

TGF-ß plays a fundamental role in extracellular matrix (ECM) synthesis and degradation, tightly regulated processes required for maintenance of tissue homeostasis. In response to injury, TGF-ß induces increased collagen and fibronectin synthesis and promotes fibroblast migration and proliferation required for tissue regeneration (15 , 16) . Aberrant overexpression of TGF-ß leads to fibrosis, as characterized by increased ECM deposition (17 , 18) . Many of the fibrogenic effects of TGF-ß can be attributed to or enhanced by CTGF (14) , a 38 kDa cysteine-rich polypeptide secreted by a variety of cell types. CTGF is a member of the Cyr61/CTGF/NOV (CCN) family with a range of biological activities, including stimulation of DNA synthesis, cell proliferation, ECM production, and angiogenesis (19 20 21) . CTGF is implicated in various normal physiological processes such as embryonic development and differentiation and in the pathogenesis of a range of fibrotic disorders such as scleroderma and renal, hepatic, and cardiac fibrosis (22 23 24 25) .

TGF-ß is the main positive regulator of CTGF expression. TGF-ß-dependent expression of CTGF requires input from Smad3 and MAP kinase pathways (26 27 28 29) . Activation of the type I, II TGF-ß receptor Ser/Thr kinase complex by TGF-ß promotes phosphorylation of the TGF-ß I receptor kinase, followed by binding and subsequent phosphorylation of receptor-activated Smad, Smad3. Activated Smad3 is then escorted into the nucleus by cytosolic Smad4, where it binds and activates TGF-ß-responsive promoters, including CTGF, which contains a functional Smad3 binding site essential for TGF-ß-induced expression (28 , 30) . Inhibitory Smads, Smad6, and Smad7 block phosphorylation of Smad3 (31 , 32) , and consequently repress TGF-ß-induced CTGF transcription (28) . In addition to Smad activation, TGF-ß-dependent CTGF expression requires input from the Ras/MEK/Erk pathway (27 , 29 , 33) . It was recently demonstrated that Iloprost, a prostacyclin derivative used to treat scleroderma, inhibits CTGF expression by inducing elevated cAMP levels and activating protein kinase A (PKA), which in turn inhibits Ras/MEK/Erk signaling (27 , 34) . Other negative regulators of CTGF expression include protein kinase C (PKC), tyrosine kinases, and cytokines such as TNF-{alpha}, IL-1{alpha}, and IL-4 (35 36 37 38) . Given that CTGF is rapidly down-regulated in T. cruzi-infected fibroblasts (7) , it is predicted that one or more of the early signaling pathways activated by T. cruzi (2) mediate repression of CTGF in response to this pathogen.

The immediate down-regulation of CTGF expression in response to T. cruzi appears to be a unique feature of the early interaction of this pathogen with host cells (7 , 10) . The role of this response in the T. cruzi infective process is not understood, but specific repression of fibrogenic genes may represent an important mechanism by which this parasite establishes a successful infection in the host. Thus, characterization of the mechanisms involved in T. cruzi-mediated repression of this important fibrogenic cytokine is critical for understanding its role in the parasite infective process. Studies of the fibrogenic response in pathogen infection present a unique opportunity to elucidate novel regulators of this pathway with potential implications for novel therapeutic approaches.

In this study, we characterize the T. cruzi-mediated repression of CTGF expression in dermal fibroblasts and investigate the mechanisms underlying this unique response. We present the novel finding that the intracellular pathogen T. cruzi causes a rapid and sustained down-regulation of the key fibrogenic cytokine CTGF, followed by decreased expression of the TGF-ß/CTGF-regulated extracellular matrix proteins, fibronectin, and collagen I {alpha}1. In addition to exerting repressive effects on basal expression of host fibrogenic genes, T. cruzi infection as well as a secreted/released parasite activity was found to antagonize TGF-ß-mediated induction of CTGF expression. Our findings indicate that the mechanism of T. cruzi-mediated repression of CTGF is complex involving targeted inhibition of the TGF-ß-induced host signaling pathways and de novo host cell protein synthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Parasites and parasite-conditioned medium (PCM)
Tissue culture-derived T. cruzi trypomastigotes (Y strain) were generated by weekly passage in confluent monolayers of LLcMK2 cells in DMEM containing 2% fetal bovine serum (FBS) as described (39) . Trypomastigotes harvested from culture supernatants were washed twice in DMEM containing 2% FBS (DMEM-2). For PCM preparation, trypomastigotes were washed twice in cold DMEM-2, resuspended in warm DMEM-2 at a concentration of 3–5 x 107 trypomastigotes/mL, and incubated at 37°C with 5% CO2 for 14–18 h. The supernatant was clarified by centrifugation and filter sterilized through a 0.2 µm membrane.

Cell infections
Human foreskin fibroblasts (HFF, passages 10–16) were cultured in DMEM supplemented with 10% FBS. HFF were infected with freshly harvested and washed trypomastigotes at a multiplicity of infection of 100 to achieve an infection efficiency of 1–2 parasites/cell in 2 h. Remaining extracellular parasites were removed by extensive washing and cells were further incubated for 4, 18, 24, 48, or 72 h. To quantitate infection, 12 mm2 glass coverslips with infected cells were removed from tissue culture dishes prior to cell lysis, fixed with 2% paraformaldehyde, and the number of intracellular parasites/100 cells was quantitated using an immunofluorescence assay as described previously (40) . Drug pretreatments of mammalian cells were as follows: bisindolymaleimide (0.1 µM) or genistein (10 µM) for 2 h or H-89 (10 µM) or MDL-12,330A (10 µM) for 30 min (all inhibitors were purchased from Calbiochem, San Diego, CA, USA).

Northern hybridizations
HFF RNA (12 µg) extracted using an RNeasy kit (Qiagen, Chatsworth, CA, USA) was size-fractionated on a 1.0 or 1.5% formaldehyde-agarose gel and blotted onto Hybond N+ (Amersham) membranes. Membranes were hybridized using ExpressHyb (Clontech, Palo Alto, CA, USA) using the manufacturer’s protocols with the appropriate 32P-labeled probe. Specific DNA probes for CTGF and fibronectin were generated by PCR amplification of cDNA generated from total HFF RNA using the following primers: CTGF (forward 5'-GAGGAAAACATTAAGAAGGGCAAA-3', reverse-5'-CGGCACAGGTCTTGATGA-3') (25) and fibronectin (forward 5'-AGAAGGGCGACAGGACGGACAT-3', reverse-5'-GCCACGGCCATAGCAGTAGCAC-3'). Human cDNA clones for collagen I {alpha}1 and TGF-ß were obtained from ATCC (Manassas, VA, USA). Probes were sequenced for verification before use. To quantitate mRNA, band intensities were measured using a PhosphorImager and ImageQuant Software (Molecular Dynamics, Sunnyvale, CA, USA).

Quantitative RT-PCR
The SUPERSCRIPTTM II RT kit (Invitrogen, San Diego, CA, USA) was used for cDNA synthesis from total RNA extracted from cells using RNeasy kits (Qiagen). Quantitative multiplex real-time PCR was performed using an ABI 7000 PCR machine (Applied Biosystems, Foster City, CA, USA). Specific primers and probes were designed using the Primer Express program (ABI): 20 nM each CTGF primer (forward 5'-CTGCCCTCGCGGCTTA-3' and reverse 5'GGACCAGGCAGTTGGCTCTA-3') and 10 nM FAM-labeled CTGF probe (6FAM-ACACGTTTGGCCCAGACCCAACTATG-TAMRA) were used per reaction, along with the pre-made VIC-labeled human GAPDH (ABI). {Delta}Ct values were calculated to account for GAPDH.

Preparation of nuclear extracts
Cells were washed twice with cold Dulbecco’s phosphate-buffered saline, then incubated with ice-cold DIGNAM lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM ß-mercaptoethanol, 0.5 mM PMSF, 1x proteinase inhibitor cocktail, 0.1% NP-40, 1 mM Na3VO4, 1 mM NaF) for 20 min. The cytosolic fraction was removed by centrifugation at 1200 g for 4 min and the nuclear pellet was resuspended in cold nuclear lysis buffer (20 mM HEPES pH 7.8, 25% glycerol, 520 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM ß-mercaptoethanol, 0.5 mM PMSF, 1x proteinase inhibitor cocktail, 0.2% NP-40, 1 mM Na3VO4, 1 mM NaF), incubated for 1 h on ice, then centrifuged at 10,000 g for 20 min. The supernatant or nuclear extract was removed and stored at –20°C. Protein concentrations in the extracts were measured using a BCA protein assay kit (Pierce, Rockford, IL, USA).

Western blot analysis
Total cell lysates were prepared from cells stimulated with medium, TGF-ß, EGF, trypomastigotes, or PCM in RIPA buffer (50 mM Tris Cl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1x proteinase inhibitor cocktail, 1 mM Na3VO4, 1 mM NaF). SDS-PAGE and Western blot were performed with samples (total lysates or nuclear lysates) as described (41) . Anti-hFibronectin antibodies were a kind gift from K. Huang, Princeton University, and anti-collagen antibodies were purchased from Biodesign International (Kennebunkport, ME, USA). Anti-Smad 2/3 and anti-CTGF antibodies were purchased from Santa Cruz Technologies (Santa Cruz, CA, USA); anti-Erk1/2 was from Upstate Biotech (Lake Placid, NY, USA). The ECL PlusTM chemiluminescent kit (Amersham Pharmacia, Arlington Heights, IL, USA) was used for detection. Blots were scanned in a UMAX Powerlook III scanner; the software program UN-SCAN-IT gel (Silk Scientific Corporation Inc., Orem, UT, USA) was used to quantitate relative band intensity.

Statistics
Samples were compared using the 2-tailed, one-sample t test; P values <0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T. cruzi elicits immediate and sustained repression of host cell CTGF expression
In a recent study exploiting a DNA microarray hybridization approach, we demonstrated that dermal fibroblasts undergo minimal changes in gene expression in response to the intracellular pathogen T. cruzi (7) . An exception to this trend is the rapid repression of the key TGF-ß-regulated fibrogenic cytokine, CTGF (7) . To further characterize this novel T. cruzi-mediated response, we first analyzed relative CTGF transcript levels in control and parasite-infected human fibroblasts by Northern hybridization (Fig. 1 B) and quantitative RT-PCR (Fig. 1A, C ). A 3- to 5-fold reduction in CTGF mRNA levels was consistently observed in T. cruzi-infected fibroblasts and cells treated with parasite-conditioned medium (PCM), which contains soluble, secreted, or released parasite factors. Down-regulation of CTGF in response to infection or PCM treatment was observed within 2 h (Fig. 1A ) and was sustained for the duration of the intracellular replicative cycle (72 h) (Fig. 1B, C ).



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  		Figure 1. T. cruzi-mediated down-regulation of CTGF in human foreskin fibroblasts (HFF). Quantitative real-time RT-PCR (multiplex) analysis with total RNA extracted from HFF incubated with medium (C), PCM or infected with T. cruzi (Tc) for 2 h (A) or infected with T. cruzi for 24, 48, 72 h (C) using FAM-labeled CTGF and VIC-labeled human GAPDH probes. CTGF mRNA levels relative to GAPDH levels are shown (2-{Delta}Ct values are plotted); n = 3 (±SD), P < 0.003. B) Northern blot analysis of total RNA extracted from HFF incubated with medium (C), TGF-ß, parasite-conditioned medium (PCM) or infected with T. cruzi (Tc) for 18 h with 32P-labeled hCTGF 3' UTR or ß-actin probes. Densitometric analysis of the Northern blot is shown in the graph below.

T. cruzi down-regulates expression of host cell fibrogenic genes
As T. cruzi and T. cruzi - shed/secreted products promote immediate and sustained repression of basal CTGF gene expression, we predicted that genes encoding major fibrogenic proteins downstream of CTGF, fibronectin, and collagen I {alpha}1 would also be negatively regulated in response to this pathogen. Examination of the relative abundance of fibronectin and collagen I {alpha}1 transcripts in dermal fibroblasts by Northern blot analysis demonstrated that these genes are down-regulated at 24 h in response to T. cruzi and PCM, contrasting with the up-regulation of these genes in response to TGF-ß (Fig. 2 A, B). Unlike CTGF, for which repression is first observed at 2 h (Fig. 1A ), significant decreases in fibronectin and collagen I {alpha}1 levels in T. cruzi or PCM-stimulated cells is not observed until 24 h post-treatment. Since TGF-ß1 mRNA levels remain unaltered in T. cruzi-infected cells (Fig. 2A ), this suggests that the block imposed on the host cell fibrogenic pathway does not involve inhibition of TGF-ß transcription.



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  		Figure 2. T. cruzi-mediated down-regulation of fibronectin and collagen in HFF. A) Northern blot analysis of total RNA extracted from HFF incubated with medium (C), TGF-ß 1, parasite-conditioned medium (PCM) or infected with T. cruzi (Tc) for 24 h and probed with 32P-labeled hCTGF, hFibronectin, hCollagen, hTGF-ß, or ß-actin DNA. B) Densitometric analysis of Northern blots for CTGF (upper panel), fibronectin (middle panel), and collagen (lower panel); n = 3 (±SD), P < 0.03.

The decrease observed in transcript abundance for host cell fibrogenic genes is reflected in decreased protein expression for CTGF at 48 and 72 h postinfection (Fig. 3 A). The effects of PCM treatment on HFF were generally comparable to that observed after T. cruzi infection, however this response is not consistently sustained at later time points (Fig. 3A ). This is likely to be due to depletion of the active factor(s) present in PCM. We observed a modest but reproducible decrease in fibronectin and collagen proteins 48 h after treatment with T. cruzi or PCM with a more pronounced decrease 72 h after treatment (Fig. 3B, C ). A less prominent effect on these fibrogenic proteins may be due to a possible role for additional regulatory pathways in the control of these proteins. Together, the results demonstrate that T. cruzi infection of dermal fibroblasts, as well as stimulation of cells with secreted/released T. cruzi factors, result in down-regulation of critical components of the host fibrogenic pathway, at both mRNA and protein levels.



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  		Figure 3. T. cruzi-mediated down-regulation of CTGF, fibronectin, and collagen proteins in HFF. Western blot analysis of total cell lysates from HFF incubated with medium (C), TGF-ß, parasite-conditioned medium (PCM) or infected with T. cruzi (Tc) for 48 or 72 h probed with anti-CTGF (A), anti-human fibronectin (B), or anti-mouse collagen type I (C) antibodies. Lower panels of A–C represent densitometric analysis of the respective Western blots. Data are representative of at least 2 experiments.

Known CTGF inhibitory pathways do not contribute to T. cruzi-mediated CTGF repression
Negative regulators of CTGF expression include intracellular signaling pathways involving cAMP, PKA, tyrosine kinases, or PKC (38 , 42 , 43) . Since T. cruzi trypomastigotes are able to trigger a variety of host cell signaling pathways early in the infective process (2) , we examined the possibility that parasite-dependent CTGF repression could be mediated by activation of one or more of the signaling pathways known to be inhibitory for CTGF expression. Membrane-permeant inhibitors of adenyl cyclase (MDL-12,330A), protein kinase A (H-89), protein kinase C (bisindolymaleimide), and tyrosine kinases (genestein) were used to pretreat fibroblasts before infection with T. cruzi. As demonstrated by Northern hybridization and quantitative RT-PCR, none of the inhibitors tested prevented parasite-mediated repression of CTGF (Fig. 4 A, B). To exclude potential deleterious effects of inhibitor pretreatments on parasite invasion, similar experiments were carried out with PCM added in the continued presence of this inhibitor (Fig. 4C ). Comparable to the results with live trypomastigotes, inhibition of these signaling pathways failed to block PCM-mediated CTGF repression, although bisindolymaleimide and MDL-12,330A alone caused a slight decrease in basal CTGF mRNA levels. Thus, the CTGF regulatory pathways involving host cell cAMP, PKA, PKC, or tyrosine kinases do not appear to play a role in the mechanism of T. cruzi-mediated repression of the fibrogenic pathway.



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  		Figure 4. Effect of signaling pathway inhibitors on T cruzi-induced CTGF repression. A) Northern blot analysis of total RNA extracted from HFF preincubated with medium, 0.1 µM bisindolymaleimide (B) or 10 µM genistein (Gen) for 2 h, washed, and infected with T. cruzi for 2 h with 32P-labeled CTGF probe. B) Real-time RT-PCR with total RNA extracted from HFF preincubated with either medium, 1 µM bisindolymaleimide (Bis) for 2 h or 10 µM H-89 for 30 min, washed, and infected with T. cruzi (Tc) for 2 h. Results are representative of at least 3 separate experiments. C) Real-time RT-PCR with total RNA extracted from HFF incubated with either medium, 1 µM bisindolymaleimide (Bis), 10 µM genistein (Gen) for 2 h or 10 µM MDL-12,330A (MDL) for 30 min, followed by treatment with PCM in the presence of the respective drug for 2 h.

Role of TGF-ß-induced pathways in T. cruzi-elicited CTGF repression
TGF-ß-dependent signaling events play an important role in regulating gene expression of CTGF and downstream fibrogenic genes. Since our data indicate that many of the predicted CTGF inhibitory pathways do not appear to play a role in the mechanism of T. cruzi-mediated CTGF repression, we examined the possibility that T. cruzi could interfere with TGF-ß-dependent regulation of CTGF gene expression.

T. cruzi antagonizes TGF-ß-induced CTGF expression
Stimulation of dermal fibroblasts with TGF-ß results in significantly increased levels of CTGF mRNA. However, addition of T. cruzi or PCM to TGF-ß-treated cells prevents induction of CTGF gene expression (Fig. 5 A, B). Infective T. cruzi trypomastigotes blocked TGF-ß-mediated up-regulation of CTGF expression when TGF-ß was added in the range of 0.01–0.1 ng/mL (Fig. 5A, C ), whereas PCM was able to inhibit CTGF expression in cells treated with TGF-ß at a much higher concentration (10 ng/mL TGF-ß) (Fig. 5B ). TGF-ß-dependent stimulation of CTGF expression was also blocked in cells that had been preinfected with T. cruzi for 24 h (Fig. 5C ). These data clearly demonstrate the ability of both extracellular and intracellular forms of T. cruzi, as well as parasite soluble factors, to antagonize TGF-ß-mediated CTGF gene expression. Interference with TGF-ß-dependent host cell signaling pathways regulating ECM synthesis suggests this is an important mechanism for T. cruzi-mediated repression of basal CTGF expression.



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  		Figure 5. Repression of TGF-ß-induced CTGF by T. cruzi/PCM. A) Real-time RT-PCR with total RNA extracted from HFF incubated with medium (C), TGF-ß1 (0.01 ng/mL), for 2 h, followed by treatment with medium or T. cruzi (in continued presence of 0.01 ng/mL TGF-ß) for 2 h. n = 3 (±SD), P < 0.005. B) Real-time RT-PCR with total RNA extracted from HFF incubated with medium (C), TGF-ß2 (10 ng/mL), followed by addition of medium or PCM (in presence of 10 ng/mL TGF-ß2) for 2 h. n = 3 (±SD), P < 0.03. C) Real-time RT-PCR with total RNA extracted from HFF incubated with medium or infected with T. cruzi for 24 h, followed by treatment with medium (C) or TGF-ß1 (0.1 ng/mL) for 2 h. n = 3 (±SD), P < 0.01.

TGF-ß signaling pathways in the T. cruzi-mediated repression
In dermal fibroblasts, TGF-ß-regulated CTGF gene expression requires input from both Smad and Ras/MEK/Erk signaling pathways (27 28 29 , 33) , where inhibition of either pathway inhibits CTGF expression. To further explore the mechanisms of T. cruzi-mediated repression of CTGF, we tested the ability of this parasite to modulate signaling through Smad3 and the Ras/MEK/Erk pathways in HFF.

Phosphorylation of receptor Smads and their translocation to the nucleus are key steps initiating a cellular response to TGF-ß following binding to the TGF-ß receptor. Activation of Smad3 has been specifically linked to CTGF expression in response to TGF-ß (28) . To determine whether T. cruzi stimulation of fibroblasts leads to inhibition of Smad signaling, Smad3 phosphorylation and nuclear translocation were examined. Under conditions where T. cruzi or PCM treatment cause decreased CTGF transcript levels, no decrease in basal Smad3 phosphorylation (Fig. 6 A) or nuclear localization (Fig. 6B ) was observed. Furthermore, no impairment of TGF-ß-induced Smad3 phosphorylation was detected after incubation of cells with T. cruzi in the presence of TGF-ß (Fig. 6C ), even though the parasite clearly blocks TGF-ß-induced CTGF expression at lower concentrations of TGF-ß (Fig. 5A ). This is not surprising since T. cruzi alone caused an increase in Smad3 phosphorylation similar to that observed after TGF-ß stimulation (Fig. 6A, C ). Increased expression of inhibitory Smad7 was not observed in T. cruzi-infected/PCM-treated fibroblasts, indicating that this mechanism of dampening TGF-ß-dependent Smad signaling does not contribute to parasite-mediated antagonism of the TGF-ß signaling pathway (data not shown). Combined, these data indicate that interference with TGF-ß signaling at the level of receptor Smad3 signaling does not contribute to the mechanism of T. cruzi-mediated CTGF repression.



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  		Figure 6. Effect of T cruzi infection on Smad3 activation.A) Western blot analysis of immunoprecipitates from total cell lysates from HFF incubated with medium (C), TGF-ß, parasite-conditioned medium (PCM) or infected with T cruzi (Tc) for 2 h. Total cell lysates were immunoprecipitated with anti-Smad2/3, then probed with an anti-phosphoserine antibody. Blots were stripped and reprobed with anti-Smad2/3. B) Western blot analysis of 250 µg nuclear lysates from HFF cells incubated with medium (C), TGF-ß, PCM or infected with T. cruzi (Tc) for 2 h with anti-Smad2/3. Results are representative of at least 3 separate experiments. C) Western blot analysis of immunoprecipitates from HFF. Cells were incubated with medium (C) or TGF-ß (0.01–1 ng/mL) for 2 h, followed by treatment with the respective concentration of TGF-ß (0.01–1 ng/mL) or T. cruzi (Tc) (in the presence of corresponding pretreatment concentration of TGF-ß), for 2 h. Total cell lysates were immunoprecipitated with anti-Smad2/3 and blots probed with an anti-phosphoserine antibody. Blots were stripped and reprobed with anti-Smad2/3. Levels of Smad3P relative to Smad3 were quantitated using densitometry.

Upon examination of the Ras/MEK/Erk pathway, we noted a marked and reproducible decrease in the level of phosphorylated Erk1/2 in fibroblasts incubated with T. cruzi or PCM (Fig. 7 A), suggesting that the ability of T. cruzi to inhibit Erk phosphorylation and CTGF expression were correlated. In fibroblasts stimulated with low levels of TGF-ß (0.01 ng/mL), expression of both Erk phosphorylation and CTGF expression was impaired by T. cruzi (Fig. 7B, C ) 2 h postinfection. In contrast, treatment of HFF with higher concentrations of TGF-ß (0.1–1 ng/mL) resulted in complete abrogation of parasite-mediated inhibition of Erk phosphorylation and CTGF expression (Fig. 7B, C ). Since signaling through the Ras/MEK/Erk pathway is required for TGF-ß-regulated CTGF gene expression, these observations suggested that dephosphorylation of Erk in response to T. cruzi may cause down-regulation of CTGF expression. We attempted to address this more specifically by hyperactivating the Ras/MEK/Erk pathway with EGF (Fig. 7D ). Stimulation of cells with high concentrations of EGF (5 ng/mL) overcame T. cruzi-dependent Erk dephosphorylation but failed to block down-regulation of CTGF in response to the parasite (Fig. 7D ). T. cruzi-mediated CTGF inhibition was still observed in cells transiently overexpressing constitutively active MEK (data not shown). Thus, activation of the Ras/MEK/Erk pathway alone, i.e., in the absence of additional pathways triggered by TGF-ß, is insufficient to override T. cruzi-mediated down-regulation of CTGF expression. These data therefore indicate a requirement for additional, and perhaps balanced, inputs from other signaling pathways.



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  		Figure 7. Effects of T. cruzi/PCM on Erk phosphorylation. A) Western blot analysis of total cell lysates from HFF incubated with medium (C), TGF-ß (10 ng/mL), TGF-ß, EGF (1 ng/mL), PCM or infected with T. cruzi (Tc) for 30 min or 2 h, with anti-phospho-Erk. Membranes were stripped and reprobed with anti-Erk. The graph represents densitometric analysis of the Western blots. n = 5 (±SD), P < 0.004. B) Western blot analysis of total cell lysates from HFF incubated with medium (C) or TGF-ß (0.01–1 ng/mL) for 2 h, followed by treatment with respective concentration of TGF-ß (0.01–1 ng/mL) or T. cruzi (Tc) (in the presence of corresponding pretreatment concentration of TGF-ß) for 2 h with anti-phospho-Erk. Membranes were stripped and reprobed with anti-Erk; n = 3 (±SD), P < 0.05. C) Relative CTGF mRNA levels measured by real-time PCR from similar experiments; total RNA was extracted from HFF incubated with medium (C) or TGF-ß (0.01–1 ng/mL) for 2 h, followed by treatment with respective concentration of TGF-ß (0.01–1 ng/mL) or T. cruzi (Tc) for 2 h; n = 3 (±SD), P < 0.05. D) Western blot analysis of total cell lysates from HFF incubated with medium (C) or EGF (0.1 or 5 ng/mL) for 30 min, followed by treatment with EGF (0.1 or 5 ng/mL) or T. cruzi (Tc) (in the continued presence of EGF) for 2 h with anti-phospho-Erk. Membranes were stripped and reprobed with anti-Erk. Blots are representative of at least 4 independent experiments. The graph represents relative CTGF mRNA levels measured by real-time PCR in similar experiments performed in parallel with 0.1 and 5 ng/mL EGF.

T. cruzi requires de novo host cell protein synthesis to elicit CTGF repression
To investigate the possibility that T. cruzi interferes with multiple cellular processes to bring about CTGF repression, we studied other possible points of intervention. Experiments with actinomycin D indicated that the decrease observed in CTGF mRNA on T. cruzi infection was not the result of reduced mRNA stability and does not appear to require new host cell transcription (data not shown). To examine whether de novo protein synthesis is required for T. cruzi-mediated CTGF repression, T. cruzi infection or PCM treatment was performed in the presence of the protein synthesis inhibitor cycloheximide (CHX). CTGF transcript abundance was not decreased in CHX-treated fibroblasts incubated with T. cruzi or PCM compared with the CHX-treated, mock-infected controls (Fig. 8 ). In contrast, transcript levels were slightly elevated in CHX-treated cells, possibly due to stabilization of the transcript. Thus, these data strongly suggest that in addition to host signaling pathways such as the TGF-ß-induced MAP kinase pathway, de novo host cell protein synthesis plays a role in the mechanism of T. cruzi-mediated CTGF repression.



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  		Figure 8. Effect of cycloheximide treatments on T. cruzi-mediated CTGF repression. Densitometric analysis of Northern blots with total RNA extracted from HFF, pretreated for 30 min with 1 µg/mL cycloheximide (CHX), followed by treatment with medium (C), TGF-ß (10 ng/mL), PCM or infected with T. cruzi (Tc) in the presence of 1 µg/mL CHX for 2 h with 32P-labeled CTGF or ß-actin probes. Data are representative of 2 independent experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have characterized the novel repression of CTGF, a key regulator of the TGF-ß-induced fibrogenic response, by the intracellular protozoan pathogen T. cruzi. The rapid T. cruzi-elicited inhibition of CTGF is followed by down-regulation of genes encoding CTGF-regulated extracellular matrix proteins fibronectin and collagen I {alpha}1. Despite our initial prediction that this early host cell response may be mediated by signaling pathways involving cAMP, PKC, or tyrosine kinases activated by infective T. cruzi trypomastigotes (38 , 42 , 43) , our data demonstrate that these common negative regulators of CTGF expression do not contribute to the repressive effects of T. cruzi, but instead strongly argue that parasite-mediated inhibition of CTGF expression occurs at the level of TGF-ß-dependent signaling. Strikingly, we find that T. cruzi trypomastigotes, as well as soluble factors secreted or released from infective parasites, antagonize TGF-ß-induced CTGF gene expression and inhibit the TGF-ß-dependent MAP kinase signaling pathway. The demonstration that de novo host cell protein synthesis is required for down-regulation of CTGF in response to T. cruzi indicates the complexity of mechanisms underlying this unique pathogen-mediated host response.

It was recently shown that TGF-ß-dependent CTGF expression involves Smad3 and Ras/MEK/Erk signaling (27 28 29 , 33) . Here we demonstrate that T. cruzi elicits a rapid inhibition of Erk1/2 phosphorylation in fibroblasts exposed to infective trypomastigotes or soluble secreted/released parasite factors whereas Smad3 phosphorylation and nuclear localization were not compromised. T. cruzi was also able to block Erk phosphorylation and CTGF expression in fibroblasts stimulated with low TGF-ß concentrations 0.01 ng/mL (2 h postinfection) or 0.1 ng/mL (24 h postinfection). When TGF-ß was applied at a higher concentration (1–10 ng/mL), the parasite-mediated down-regulation of Erk signaling and CTGF expression was completely abrogated. Given that input from the Ras/MEK/Erk pathway is required for CTGF expression, these findings suggest that Erk dephosphorylation may be linked to down-regulation of CTGF expression in T. cruzi-infected cells. To examine the Ras/MEK/Erk pathway without affecting Smad signaling pathways, we attempted to override CTGF repression by stimulating cells with EGF before infection with T. cruzi. While parasite-mediated Erk dephosphorylation was overcome with high concentrations of EGF (5 ng/mL), a concomitant increase in CTGF expression was not observed in EGF-stimulated cells. Similarly, transient overexpression of active MEK failed to override the repression of CTGF in two different cell lines (HFF, NIH 3T3; M. Unnikrishnan, unpublished data). Together, these data indicate that activation of the MAP kinase pathway without input from other TGF-ß-dependent signaling pathways is insufficient to rescue the T. cruzi-mediated repression of CTGF gene expression. As our data do not directly implicate Smad-dependent signaling in the mechanism of T. cruzi-elicited CTGF repression, it is possible that additional TGF-ß-dependent modulators of Smad3 activity in the nucleus may be involved in parasite-mediated down-regulation of CTGF gene transcription.

The complex regulation of CTGF expression is reflected in the diversity of signaling events involved in the suppression of CTGF promoter activity (28 , 29 , 33 , 35) . Although infective T. cruzi trypomastigotes are capable of initiating a variety of signaling pathways in mammalian host cells (2) , including elevation of cAMP/PKA levels (12) , an event known to negatively regulate CTGF expression (42 , 43) , our data suggest that T. cruzi-mediated repression of CTGF does not involve signaling through these pathways. Moreover, our results indicate that the T. cruzi-dependent modulation of CTGF expression is distinct from the effects of the prostacyclin derivative Iloprost, an effective inhibitor of the fibrotic process used for treatment of scleroderma (27 , 34) . In cells treated with Iloprost, CTGF levels decrease considerably due to targeted inhibition of the Ras/MEK/ERK pathway after PKA activation (27) . While we demonstrate that Ras/MEK/Erk signaling is compromised in dermal fibroblasts by infective T. cruzi trypomastigotes and released soluble parasite factors, the mechanism CTGF repression elicited by T. cruzi appears to be distinct from that of Iloprost since it is not dependent on cAMP/PKA and requires de novo host cell protein synthesis.

Our data indicate that a newly synthesized host cell factor(s) contributes to the mechanism of CTGF expression triggered by infective T. cruzi trypomastigotes. Since our previous DNA microarray hybridization experiments (7) revealed a striking absence of detectable increases in host cell transcript abundance at early time points of T. cruzi infection (2–6 h), this suggests that host cell transcription is not required for expression of this host protein. This is supported by our data that actinomycin D pretreatment of HFF failed to block T. cruzi-mediated repression of CTGF (M. Unnikrishnan, unpublished data). Moreover, changes in CTGF mRNA stability do not appear to contribute to the mechanism of repression elicited by T. cruzi. It is unclear what role this putative host protein(s) might play in inhibiting CTGF expression. Since cytokines TNF{alpha}, IL-1{alpha}, and IL-4 are known to negatively regulate CTGF expression (35 36 37) , it is possible that a host cell secreted factor plays a role in the T. cruzi-mediated process. However, no induction of TNF{alpha}, IL-1{alpha}, and IL-4 or other proinflammatory cytokines was observed in HFF during the first 24 h of infection (7) . Collectively, the data indicate that post-transcriptional regulation of a host protein or set of proteins in response to T. cruzi contributes to the down-regulation of CTGF gene expression. Although the precise mechanisms underlying the anti-fibrogenic effects of T. cruzi are unclear at present, our findings demonstrate the complexity of CTGF regulation and suggest a role for novel regulatory mechanisms in the control of CTGF expression. Studies of CTGF gene regulation using T. cruzi and purified T. cruzi soluble factors are likely to yield important new insights into the complex regulation of this important cellular process.

To our knowledge, such a potent inhibition of TGF-ß-regulated host cell fibrogenic genes by an intracellular pathogen has not been reported in the literature (7 8 9 10 11) . The selectivity and apparent specificity of this unique host cell response to T. cruzi suggests it may play an important role in the infective process. Certainly, targeted modulation of extracellular matrix proteins by microbial pathogens is a common strategy exploited to colonize and to disseminate within the mammalian host. For example, the bacterial pathogens Streptococcus pyogenes, Pseudomonas aeruginosa, and Porphyromonas gingivalis produce a range of matrix-degrading proteases that aid in the initial colonization process (44 45 46) . Dissemination from the initial site of infection often is required for the establishment of chronic infection and development of disease. This is particularly true for many vector-borne pathogens (47 , 48) such as the Lyme disease spirochete Borrelia burgdorferi, which facilitates its dissemination in the host by induction of host matrix metalloproteases (collagenases and gelatinases) and degradation of ECM (49) . Similarly, critical steps in the development of Chagas’ disease include invasion at the primary site of infection (skin, mucosal membranes) and dissemination of infective T. cruzi trypomastigotes to internal organs (heart, gastrointestinal tract), the major sites of chronic infection and pathology. Migration from tissue to the peripheral blood is also required for successful transmission of T. cruzi by the hematophagous triatomine vector. A recent report suggests that an 80 kDa collagen degrading prolyl endopeptidase produced by T. cruzi may play a role in invasion and dissemination of this parasite (50 , 51) . Thus, the ability of T. cruzi to degrade matrix proteins during invasion and to block expression of components of the ECM at the transcriptional level may be critical for establishment of T. cruzi infection in the host. Although the mechanisms by which T. cruzi accomplish migration in the host have not been established, our novel findings strongly suggest that down-regulation of the host fibrogenic response by T. cruzi, coupled with the unique ability of this pathogen to block TGF-ß-dependent up-regulation of this pathway, may ensure a permissive environment for parasite release into interstitial spaces, reinfection of neighboring cells, and access to the circulation after initial colonization. Since CTGF can trigger an apoptotic response in some cell types (52 , 53) , down-regulation of this cytokine may be vital to the survival of the host cell and parasite after infection. While these hypotheses clearly require further investigation, it would not be surprising if T. cruzi-elicited anti-fibrogenic effects were found to play a role early in the acute stages of infection when parasite proliferation in tissue and migration occur.

In summary, we report the novel ability of the human pathogen T. cruzi to down-regulate the host cell fibrogenic response at the level of mRNA and protein expression. We have demonstrated that the anti-fibrogenic effects of T. cruzi are, at least in part, mediated by a soluble parasite factor that blocks the induction of this pathway in response to TGF-ß. Identification of a parasite-derived factor with anti-fibrogenic properties has exciting potential implications for the development of therapeutic strategies against a wide range of fibrotic disorders. Biochemical approaches to isolate this soluble factor are currently underway (G. A. Mott and B. Burleigh, unpublished data). Characterization of the host fibrogenic response in the context of T. cruzi infection and its role in Chagas’ disease pathogenesis promises to have broader implications for elucidating the molecular mechanisms underlying fibrosis in disease states with noninfectious etiology.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. D. F. Wirth and A. Sinai for critical reading of the manuscript and we thank K. Krumholz and B. K. Ghods for excellent technical assistance. This research has been supported by an American Heart Association postdoctoral fellowship to M.U. and by National Institutes of Health grants AI47960 and HL073227 and the Burroughs Wellcome Fund Investigators in Pathogenesis of Infectious Diseases Award to B.A.B.

Received for publication February 10, 2004. Accepted for publication July 12, 2004.


   REFERENCES
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 RESULTS
 DISCUSSION
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