Stephane Huet

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 β (TGFβ), which elicits a large variety of cellular responses. Upon TGFβ 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 TGFβ. 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 TGFβ 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 TGFβ responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGFβ. In addition, TGFβ 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 TGFβ‐responsive regions of several other genes suggests that this may be a widely used motif in TGFβ‐regulated transcription.

A short amino-acid sequence in MH1 domain is responsible for functional differences between Smad2 and Smad3

Smad proteins are essential components of the signalling cascade initiated by members of the Transforming Growth Factor-β family. TGFβ binding to heteromeric complexes of transmembrane Ser/Thr kinases induces Smad2 and Smad3 phosphorylation on their C terminus residues. This phosphorylation leads to oligomerization with Smad4, a common mediator of TGF-β, activin and BMP signalling. The Smad complexes then translocate to the nucleus where they play transcription regulator roles. Even if they share 92% identity, the two TGFβ restricted Smad2 and Smad3 are not functionally equivalent. As we have previously shown, Smad3 acts as a transcription factor by binding to a TGFβ-responsive sequence termed CAGA box whereas Smad2 does not. Smad2 differs from Smad3 mainly in the N-terminal MH1 domain where it contains two additional stretches of amino acids that are lacking in Smad3. Here, we show that one of these domains corresponding to exon 3 is responsible for the absence of Smad2 transcriptional activity in CAGA box-containing promoters. Furthermore, in vitro studies indicate that this domain prevents Smad2 from binding to this DNA sequence. This suggests that Smad2 and Smad3 may have different subsets of target genes participating thus in distinct responses among TGFβ pleiotropic effects.

Inhibition of TGF-β signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis

1 Chronic liver disease is characterized by an exacerbated accumulation of matrix, causing progressive fibrosis, which may lead to cirrhosis. Transforming growth factor beta (TGF‐β), a well‐known profibrotic cytokine, transduces its signal through the ALK5 ser/thr kinase receptor, and increases transcription of different genes including PAI‐1 and collagens. The identification of GW6604 (2‐phenyl‐4‐(3‐pyridin‐2‐yl‐1H‐pyrazol‐4‐yl)pyridine), an ALK5 inhibitor, allowed us to evaluate the therapeutic potential of inhibiting TGF‐β pathway in different models of liver disease. 2 A cellular assay was used to identify GW6604 as a TGF‐β signaling pathway inhibitor. This ALK5 inhibitor was then tested in a model of liver hepatectomy in TGF‐β‐overexpressing transgenic mice, in an acute model of liver disease and in a chronic model of dimethylnitrosamine (DMN)‐induced liver fibrosis. 3 In vitro, GW6604 inhibited autophosphorylation of ALK5 with an IC50 of 140 nM and in a cellular assay inhibited TGF‐β‐induced transcription of PAI‐1 (IC50: 500 nM). In vivo, GW6604 (40 mg kg−1 p.o.) increased liver regeneration in TGF‐β‐overexpressing mice, which had undergone partial hepatectomy. In an acute model of liver disease, GW6604 reduced by 80% the expression of collagen IA1. In a chronic model of DMN‐induced fibrosis where DMN was administered for 6 weeks and GW6604 dosed for the last 3 weeks (80 mg kg−1 p.o., b.i.d.), mortality was prevented and DMN‐induced elevations of mRNA encoding for collagen IA1, IA2, III, TIMP‐1 and TGF‐β were reduced by 50–75%. Inhibition of matrix genes overexpression was accompanied by reduced matrix deposition and reduction in liver function deterioration, as assessed by bilirubin and liver enzyme levels. 4 Our results suggest that inhibition of ALK5 could be an attractive new approach to treatment of liver fibrotic diseases by both preventing matrix deposition and promoting hepatocyte regeneration.

Tissue-Specific Liver X Receptor Activation Promotes Macrophage Reverse Cholesterol Transport In Vivo

Objective—We previously reported that a systemic liver X receptor (LXR) agonist promoted macrophage reverse-cholesterol transport (mRCT) in vivo. Because LXR are expressed in multiple tissues involved in RCT (macrophages, liver, intestine), we analyzed the effect of tissue-specific LXR agonism on mRCT. Methods and Results—In initial studies, the systemic LXR agonist GW3965 failed to promote mRCT in a setting in which LXR was expressed in macrophages but not in liver or intestine. To evaluate the effect of LXR activation specifically in small intestine on mRCT, wild-type mice were treated with either intestinal-specific LXR agonist (GW6340) or systemic LXR agonist (GW3965). Both GW3965 and GW6340 significantly promoted excretion of [3H]-sterol in feces by 162% and 52%, respectively. To evaluate the requirement for macrophage LXR activation, we assessed the ability of GW3965 to promote mRCT in wild-type mice using primary macrophages deficient in LXR&agr;/&bgr; vs wild-type macrophages. Whereas GW3965 treatment promoted fecal excretion compared with vehicle, its overall ability to promote mRCT was significantly attenuated using LXR&agr;/&bgr; knockout macrophages. Conclusion—We demonstrate that intestinal-specific LXR agonism promotes macrophage RCT in vivo and that macrophage LXR itself plays an important, but not predominant, role in promoting RCT in response to an LXR agonist.

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux.

Peroxisome proliferator-activated receptor delta (PPAR&dgr;) is involved in regulation of energy homeostasis. Activation of PPAR&dgr; markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPAR&dgr; activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPAR&dgr; agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion (∼200%) and TICE (∼100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPAR&dgr; activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPAR&dgr; stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis.

Design of novel quinazoline derivatives and related analogues as potent and selective ALK5 inhibitors

Starting from quinazoline 3a, we designed potent and selective ALK5 inhibitors over p38MAP kinase from a rational drug design approach based on co-crystal structures in the human ALK5 kinase domain. The quinazoline 3d exhibited also in vivo activity in an acute rat model of DMN-induced liver fibrosis when administered orally at 5mg/kg (bid).

Inhibition of ALK5 Signaling Induces Physeal Dysplasia in Rats

TGF-|β|, and its type 1 (ALK5) receptor, are critical to the pathogenesis of fibrosis. In toxicologic studies of 4 or more days in 10-week-old Sprague–Dawley rats, using an ALK5 inhibitor (GW788388), expansion of hypertrophic and proliferation zones of femoral physes were noted. Subphyseal hyperostosis, chondrocyte hypertrophy/hyperplasia, and increased matrix were present. Physeal zones were laser microdissected from ALK5 inhibitor-treated and control rats sacrificed after 3 days of treatment. Transcripts for TGF-|β|1, TGF-|β|2, ALK5, IHH, VEGF, BMP-7, IGF-1, bFGF, and PTHrP were amplified by real-time PCR. IGF and IHH increased in all physis zones with treatment, but were most prominent in prehypertrophic zones. TGF-|β|2, bFGF and BMP7 expression increased in proliferative, pre- and hypertrophic zones. PTHrP expression was elevated in proliferative zones but decreased in hypertrophic zones. VEGF expression was increased after treatment in pre- and hypertrophic zones. ALK5 expression was elevated in prehypertrophic zones. Zymography demonstrated gelatinolytic activity was reduced after treatment. Apoptotic markers (TUNEL and caspase-3) were decreased in hypertrophic zones. Proliferation assessed by Topoisomerase II and Ki67 was increased in multiple zones. Movat stains demonstrated that proteoglycan deposition was altered. Physeal changes occurred at doses well above those resulting in fibrosis. Interactions of factors is important in producing the physeal dysplasia phenotype.

Intestine-Specific Regulation of PPARα Gene Transcription by Liver X Receptors

Liver X receptor-alpha (LXRalpha) and LXRbeta are ligand-activated transcription factors belonging to the nuclear receptor superfamily. They have been identified as key players in cholesterol homeostasis and lipid and glucose metabolism as well as immune and inflammatory responses. In the small intestine, LXRs have been shown not only to regulate cholesterol absorption and excretion but also to promote high-density lipoprotein biogenesis via the ATP-binding cassette A1 signaling pathway. Here, using gene expression assays, we identified PPARalpha as an intestine-specific LXR target gene. Chronic administration of LXR synthetic agonists led to a significant increase of PPARalpha mRNA levels in the small intestine but not in the liver. In addition, this specific PPARalpha gene up-regulation occurred in the duodenum, jejunum, and ileum in a dose-dependent manner and translated at the protein level as demonstrated by Western blot analysis. Furthermore, PPARalpha gene induction was completely abolished in LXR-deficient mice. Finally, the physiological relevance of LXR-mediated PPARalpha up-regulation in the small intestine was assessed in PPARalpha-deficient mice. Administration of a synthetic LXR agonist to wild-type mice led to the induction of several PPARalpha target genes including PDK4 and CPT1. Those effects were completely abolished in PPARalpha-deficient mice, demonstrating the biological relevance of this LXR-PPARalpha transcriptional cascade. Taken together, these results demonstrate that PPARalpha is an intestine-specific LXR target gene and suggest the existence of a transcriptional cross talk between those members of the nuclear receptor superfamily.

Discovery of a novel class of PPARδ partial agonists

Anthranilic acid GW9371 was identified as a novel class of PPARdelta partial agonist through high-throughput screening. The design and synthesis of SAR analogues is described. GSK1115 and GSK7227 show potent partial agonism of the PPARdelta target genes CPT1a and PDK4 in skeletal muscle cells.

TGF-β Receptor Kinase Inhibitors for Treatment of Fibrosis

Because TGF-β is central to the progression of fibrosis, selective inhibition of this signaling pathway could provide a novel treatment for many fibrotic diseases. Small molecule inhibitors of the kinase activity of the TGF-β type I receptor (ALK5) have been developed by several companies and institutions. These inhibitors prevent the phosphorylation of the Smad proteins and many if not most of the sequelae following TGF-β release and activation. Thus, it has been demonstrated that these inhibitors prevent the extra-cellular matrix accumulation and organ destruction associated with fibrosis. The effectiveness of these inhibitors in several animal models suggests broad application in many fibrotic diseases. However, due to the pleiotropic activity of TGF-β there may be unwanted side effects of TGF-β type I receptor kinase inhibition, especially when considering chronic therapy