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      Pentoxifylline Attenuates Tubulointerstitial Fibrosis by Blocking Smad3/4-Activated Transcription and Profibrogenic Effects of Connective Tissue Growth Factor

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          Abstract

          Pentoxifylline (PTX) is a potent inhibitor of connective tissue growth factor (CTGF), but its underlying mechanism is poorly understood. Here, it was demonstrated that PTX inhibited not only TGF-beta1-induced CTGF expression but also CTGF-induced collagen I (alpha1) [Col I (alpha1)] expression in normal rat kidney fibroblasts (NRK-49F) and alpha-smooth muscle actin expression in normal rat kidney proximal tubular epithelial cells (NRK-52E). Furthermore, PTX attenuated tubulointerstitial fibrosis, myofibroblasts accumulation, and expression of CTGF and Col I (alpha1) in unilateral ureteral obstruction kidneys. The mechanism by which PTX reduced CTGF in NRK-49F and NRK-52E was investigated. Activation of Smad3/4 was essential for TGF-beta1-induced CTGF transcription, but PTX did not interfere with TGF-beta1 signaling to Smad2/3 activation and association with Smad4 and their nuclear translocation. However, PTX was capable of blocking activation of TGF-beta1-induced Smad3/4-dependent reporter as well as CTGF promoter, suggesting that PTX affects a factor that acts cooperatively with Smad3/4 to execute transcriptional activation. It was found that PTX increased intracellular cAMP and caused cAMP response element binding protein phosphorylation. The protein kinase A antagonist H89 abolished the inhibitory effect of PTX on Smad3/4-dependent CTGF transcription, whereas dibutyryl cAMP and forskolin recapitulated the inhibitory effect. In conclusion, these results indicate that PTX inhibits CTGF expression by interfering with Smad3/4-dependent CTGF transcription through protein kinase A and blocks the profibrogenic effects of CTGF on renal cells. Because of the dual blockade, PTX potently attenuates the tubulointerstitial fibrosis in unilateral ureteral obstruction kidneys.

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          Most cited references 41

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          Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.

          Smad proteins play a key role in the intracellular signalling of transforming growth factor beta (TGF beta), which elicits a large variety of cellular responses. Upon TGF beta receptor activation, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transcriptional regulation is largely unknown. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGF beta. Here we report the identification of Smad3/Smad4 binding sequences, termed CAGA boxes, within the promoter of the human PAI-1 gene. The CAGA boxes confer TGF beta and activin, but not bone morphogenetic protein (BMP) stimulation to a heterologous promoter reporter construct. Importantly, mutation of the three CAGA boxes present in the PAI-1 promoter was found to abolish TGF beta responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGF beta. In addition, TGFbeta induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Furthermore, bacterially expressed Smad3 and Smad4 proteins, but not Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGF beta-responsive regions of several other genes suggests that this may be a widely used motif in TGF beta-regulated transcription.
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            Receptor-associated Mad homologues synergize as effectors of the TGF-beta response.

            Transforming growth factor-beta TGF-beta is the prototype for a family of extracellular proteins that affect cell proliferation and tissue differentiation. TGF-beta-related factors, including BMP-2/4, Dpp and activin, act through two types of serine/threonine kinase receptors which can form a heteromeric complex. However, the mechanism of signal transduction by these receptors is largely unknown. In Drosophila, Mad is required for signalling by Dpp. We have isolated complementary DNAs for four human Mad homologues, one of which, hMAD-4, is identical to DPC-4, a candidate tumour suppressor. hMAD-3 and -4 synergized to induce strong ligand-independent TGF-beta-like responses. When truncated at their carboxy termini, hMAD-3 and -4 act as dominant-negative inhibitors of the normal TGF-beta response. The activity of hMAD-3 and -4 was regulated by the TGF-beta receptors, and hMAD-3 but not hMAD-4 was phosphorylated and associated with the ligand-bound receptor complex. These results define hMAD-3 and -4 as effectors of the TGF-beta response and demonstrate a function for DPCA-4/hMAD-4 as a tumour suppressor.
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              Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.

              TGF-beta is a key mediator in renal fibrosis. Kidney-targeted gene therapy with anti-TGF-beta strategies is expected to have therapeutic potential, but this has been hampered by concerns over the safety and practicability of viral vectors and the inefficiency of nonviral transfection techniques. The present study explored the potential role of TGF-beta/Smad signaling in renal fibrosis in vivo and developed a safe and effective gene therapy to specifically block TGF-beta signaling and renal fibrosis in a rat unilateral ureteral obstruction (UUO) model by transferring a doxycycline-regulated Smad7 gene or control empty vectors using an ultrasound-microbubble (Optison)-mediated system. The Smad7 transgene expression was tightly controlled by addition of doxycycline in the daily drinking water. Groups of six rats were sacrificed at day 7, and the transfection rate, Smad7 transgene expression, and tubulointerstitial fibrosis including alpha-smooth muscle actin and collagen matrix mRNA and protein expression were determined. Compared with the non-ultrasound treatment, the combination of ultrasound with Optison largely increased the transfection rate of FITC-ODN and Smad7 transgene expression up to a 1000-fold, and this was found in all kidney tissues. Compared with normal rats, Smad7 expression within the UUO kidney was significantly reduced, and this was associated with up to a sixfold increase in Smad2 and Smad3 activation and severe tubulointerstitial fibrosis. In contrast, treatment with inducible Smad7 resulted in a fivefold increase in Smad7 expression with complete inhibition of Smad2 and Smad3 activation and tubulointerstitial fibrosis in terms of tubulointerstitial myofibroblast accumulation (85% downward arrow ) and collagen I and III mRNA and protein expression (60 to 70% downward arrow ). In conclusion, the ultrasound-mediated inducible Smad7 gene transfer is a safe, effective, and controllable gene therapy. TGF-beta-mediated renal fibrosis is regulated positively by Smad2/3, but negatively by Smad7. Target blockade of TGF-beta/Smad signaling by expression of Smad7 may provide a new therapeutic potential for renal fibrosis.
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                Author and article information

                Journal
                Journal of the American Society of Nephrology
                JASN
                American Society of Nephrology (ASN)
                1046-6673
                1533-3450
                August 25 2005
                September 2005
                September 2005
                June 29 2005
                : 16
                : 9
                : 2702-2713
                Article
                10.1681/ASN.2005040435
                15987746
                © 2005

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