Activin Receptor-like Kinase-5 Inhibitor Research Articles

Fibrotic remodeling in joint diseases: induction and inhibition of fibrosis in fibroblast-like synoviocytes

BackgroundWe aimed to investigate the development of synovial fibrosis in vitro and how the fibrosis can be halted. Synovial fibrosis causes joint stiffness in arthritic diseases. The pathway of the fibrotic growth factor, transforming growth factor-beta (TGF-β), has been associated with joint pain in osteoarthritis (OA) and with the fibroid phenotype of rheumatoid arthritis (RA). This suggests that synovial fibrosis, thus accumulation of extracellular matrix (ECM) proteins, plays a role in the clinical manifestations of the diseases. Improving our understanding of fibrotic development may aid in selecting appropriate treatments and development of drugs that can target synovial fibrosis.MethodsWe isolated primary fibroblast-like synoviocytes (FLS) from the synovial membrane of patients undergoing total knee replacement surgery. To investigate the development of synovial fibrosis, the FLS were cultured in a crowded in vitro model mimicking the ECM. TGF-β1 was used as the fibrotic initiator, the activin receptor-like kinase 5 inhibitor (ALK5i), the anti-fibrotic drug nintedanib, and the anti-inflammatory drug tofacitinib were used as fibrotic inhibitors. The ECM protein formation was quantified in the conditioned media using specific biomarkers of type I, III, and VI collagen formation and fibronectin turnover.ResultsThe TGF-β stimulation inducted fibrogenesis by increasing the biomarkers of fibronectin turnover, type I, III, and VI collagen formation. ALK5i and nintedanib inhibited the TGF-β response across all biomarkers. Tofacitinib trended towards inhibiting TGF-β response with up to 78% inhibition. All the treatments preserved cell viability.ConclusionWe have established an in vitro model for assessing fibrogenesis in primary FLS, which can be used to assess the anti-fibrotic effect of multiple drug types. Our study implies that synovial fibrosis can be induced by TGF-β, which additionally can be halted by both direct and indirect inhibition with anti-fibrotic substances. The anti-inflammatory drug tofacitinib also halted the fibrogenesis to some extent; thus, it may exert an anti-fibrotic effect.

Design, synthesis and evaluation of a pyrazolo[3,4-d]pyrimidine derivative as a novel and potent TGFβ1R1 inhibitor

The transforming growth factor β1 (TGFβ1)/SMAD signaling pathway regulates many vital physiological processes. The development of potent inhibitors targeting activin receptor-like kinase 5 (ALK5) would provide potential treatment reagents for various diseases. A significant number of ALK5 inhibitors have been discovered, and they are currently undergoing clinical evaluation at various stages. However, the clinical demands were far from being met. In this study, we utilized an alternative conformation-similarity-based virtual screening (CSVS) combined with a fragment-based drug designing (FBDD) strategy to efficiently discover a potent and active hit with a novel chemical scaffold. After structural optimization in the principle of group replacement, compound 57 was identified as the most promising ALK5 inhibitor. Compound 57 demonstrated significant inhibitory effects against the TGF-β1/SMAD signaling pathway. It could markedly attenuate the production of extracellular matrix (ECM) and deposition of collagen. Also, the lead compound showed adequate pharmacokinetic (PK) properties and good in vivo tolerance. Moreover, treatment with compound 57 in two different xerograph models showed significant inhibitory effects on the growth of pancreatic cancer cells. These results suggested that lead compound 57 refers as a promising ALK5 inhibitor both in vitro and in vivo, which merits further validation.

Oocyte-derived growth differentiation factor 9 suppresses the expression of CYP17A1 and androgen production in human theca cells

ObjectiveTo investigate the direct effect of growth differentiation factor 9 (GDF9) on androgen production in human theca cells. DesignExperimental study SettingTertiary hospital-based research laboratory Patient(s)Women underwent IVF/intracytoplasmic sperm injection (ICSI) at our clinic were included in this study. Intervention(s)Primary cultured human theca cells from women undergoing IVF/ICSI treatment were treated with GDF9, Activin receptor-like kinase 5 (ALK5) inhibitor, and SMAD4 agonist. Main Outcome Measure(s)The expression of androgen synthesis-related gene StAR, CYP17A1, and LHCGR, levels of androstenedione and testosterone, phosphorylation of SMAD2/3 and the interaction between bone morphogenetic protein receptor II (BMPRII) and ALK5 were evaluated by RT-qPCR, Western blot, enzyme-linked immunosorbent assays, and co-immunoprecipitation assays respectively. ResultsGDF9 decreased StAR, CYP17A1, and LHCGR expression levels in human theca cells, which was prevented by the treatment with the ALK5 inhibitor, and suppressed production of androgen in human theca cells. GDF9 increased SMAD2/3 phosphorylation, and the ALK5 inhibitor also suppressed this effect. BMPRII and ALK5 bound to each other following GDF9 stimulation. The SMAD4 agonist kartogenin also decreased mRNA levels of StAR and CYP17A1, and protein levels of StAR in human theca cells. ConclusionGDF9 can activate the BMPRII-ALK5-SMAD2/3 signaling pathway, suppress CYP17A1 expression and androgen production in human theca cells.

The effects of ALK5 inhibition and simultaneous inhibition or activation of HIF-1α in melanoma tumor growth and angiogenesis.

Hypoxia is the most common signature of the tumor microenvironment that drives tumorigenesis through the complex crosstalk of a family of transcription factors called hypoxia-inducible factors (HIFs), with other intercellular signaling networks. Hypoxia increases transforming growth factor-beta (TGF-β) expression. TGF-β and HIF-1α play critical roles in several malignancies and their interactions in melanoma progression remain unknown. Therefore, the aim of this study was to assess the impact of inhibiting activin receptor-like kinase-5 (ALK5), a TGF-β receptor, on the response to HIF-1α activation or inhibition in melanoma tumor progression. Tumors were induced in C57BL/6J mice by subcutaneous inoculation with B16F10 melanoma cells. Mice were divided into HIF-1α inhibitor, ALK5 inhibitor (1 mg/kg) and HIF-1α inhibitor (100 mg/kg), ALK5 inhibitor, HIF-1α activator (1000 mg/kg), HIF-1α activator and ALK5 inhibitor, and control groups to receive inhibitors and activators through intraperitoneal injection. The expression of E-cadherin was evaluated by RT-qPCR. Vessel density and platelet-derived growth factor receptor beta (PDGFR)-β+ cells around the vessels were investigated using immunohistochemistry. The groups receiving HIF-1α inhibitor and activator showed lower and higher tumor growth compared to the control group, respectively. E-cadherin expression decreased in all groups compared to the control group, illustrating the dual function of E-cadherin in the tumor microenvironment. Vascular density was reduced in the groups given HIF-1α inhibitor, ALK5 inhibitor, and ALK5 and HIF-1α inhibitor simultaneously. The percentage of PDGFR-β+ cells was reduced in the presence of HIF-1α inhibitor, ALK5 inhibitor, HIF-1α and ALK5 inhibitors, and upon simultaneous treatment with HIF-1α activator and ALK5 inhibitor. Despite increased expression and interaction between TGF-β and HIF-1α pathways in some cancers, in melanoma, inhibition of either pathway alone may have a stronger effect on tumor inhibition than simultaneous inhibition of both pathways. The synergistic effects may be context-dependent and should be further evaluated in different cancer types.

Synthesis of and anti-fibrotic effect of pyrazole derivative J-1048: Inhibition of ALK5 as a novel approach to liver fibrosis targeting inflammation

Liver fibrosis is a worldwide challenge of health issue. Developing effective new drugs for treating liver fibrosis is of great importance. In recent years, chemically synthesized drugs have significant advantages in treating liver fibrosis. Small molecule pyrazole derivatives as activin receptor-like kinase 5 (ALK5) inhibitors have also shown anti-fibrotic and tumor growth inhibitory effects. To develop the candidate with anti-fibrotic effect, we synthesized a novel pyrazole derivative, J-1048. The inhibitory effect of J-1048 on ALK5 and p38α mitogen-activated protein (MAP) kinase activity was assessed by enzymatic assays. We established an in vivo liver fibrosis model by injecting thioacetamide (TAA) into mice and in vitro model of TGF-β stimulated hepatic stellated cells to explore the inhibition mechanisms and therapeutic potential of J-1048 as an ALK5 inhibitor in liver fibrosis. Our data showed that J-1048 inhibited TAA-induced liver fibrosis in mice by explicitly blocking the TGF-β/Smad signaling pathway. Additionally, J-1048 inhibited the production of inflammatory cytokine Interleukin-1β (IL-1β) by inhibiting the purinergic ligand-gated ion channel 7 receptor (P2X7r) -Nucleotide-binding domain-(NOD-)like receptor protein 3 (NLRP3) axis, thereby alleviating liver fibrosis. Our findings demonstrated that a novel small molecule ALK5 inhibitor, J-1048, exhibited strong potential as a clinical therapeutic candidate for liver fibrosis.

TGFβ1 signaling protects chondrocytes against oxidative stress via FOXO1–autophagy axis

The forkhead box O1 (FOXO1) transcription factor is a key regulator of autophagy. In chondrocytes, reduced FOXO1 expression with aging causes osteoarthritis due to dysfunction of autophagy, but the mechanisms underlying regulation of FOXO1 expression and the reduction in expression with aging remain unclear. We investigated the mechanism by which transforming growth factor β1 (TGFβ1) signaling regulates the FOXO1-autophagy axis. Expression of FOXO1 was measured in chondrocytes after TGFβ1 treatment. Immunohistochemistry was performed to estimate the levels of activin receptor-like kinase 5 (ALK5) and FOXO1 in the knee joints of young, middle-aged and old mice. The effects of the ALK5 inhibitor and SMAD3 or SMAD2 knockdown on FOXO1 expression were evaluated. The role of TGFβ1 in autophagy after hydrogen peroxide (H2O2) treatment was analyzed. The protective effect of TGFβ1 against H2O2 treatment was assessed by cell viability assay and TUNEL assay. TGFβ1 promoted the expression of FOXO1 mRNA and protein. Both ALK5 and FOXO1 expression decreased with aging. ALK5 inhibition and SMAD3 knockdown suppressed induction of FOXO1 expression by TGFβ1, whereas SMAD2 knockdown increased it. TGFβ1 promoted the expression of microtubule-associated proteins 1A/1B light chain 3B (LC3)-I protein via the SMAD3-FOXO1 pathway. Furthermore, under H2O2 treatment, TGFβ1 promoted expression of LC3-II. TGFβ1 pretreatment suppressed cell death of chondrocytes following H2O2 treatment, but this protective effect was abolished by FOXO1 knockdown. TGFβ1 protects chondrocytes against oxidative stress via the FOXO1-autophagy axis, and a reduction in ALK5 expression might cause reduced FOXO1 expression with aging.

Modulatory effects of TGF-β1 and BMP6 on thecal angiogenesis and steroidogenesis in the bovine ovary.

Angiogenesis plays an integral role in follicular and luteal development and is positively regulated by several intra-ovarian factors including vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2). Various transforming growth factor-β (TGF-β) superfamily members function as intra-ovarian regulators of follicle and luteal function, but their potential roles in modulating ovarian angiogenesis have received little attention. In this study, we used a bovine theca interna culture model (exhibiting characteristics of luteinization) to examine the effects of TGF-β1 and bone morphogenetic protein 6 (BMP6) on angiogenesis and steroidogenesis. VEGFA/FGF2 treatment promoted endothelial cell network formation but had little or no effect on progesterone and androstenedione secretion or expression of key steroidogenesis-related genes. TGF-β1 suppressed basal and VEGFA/FGF2-induced endothelial cell network formation and progesterone secretion, effects that were reversed by an activin receptor-like kinase 5 (ALK5) inhibitor (SB-431542). The ALK5 inhibitor alone raised androstenedione secretion and expression of several transcripts including CYP17A1. BMP6 also suppressed endothelial cell network formation under VEGFA/FGF2-stimulated conditions and inhibited progesterone secretion and expression of several steroidogenesis-related genes under basal and VEGFA/FGF2-stimulated conditions. These effects were reversed by an ALK1/2 inhibitor (K02288). Moreover, the ALK1/2 inhibitor alone augmented endothelial network formation, progesterone secretion, androstenedione secretion and expression of several steroidogenesis-related genes. The results indicate dual suppressive actions of both TGF-β1 and BMP6 on follicular angiogenesis and steroidogenesis. Further experiments are needed to unravel the complex interactions between TGF-β superfamily signalling and other regulatory factors controlling ovarian angiogenesis and steroidogenesis.

15-Deoxy-Δ-12, 14-prostaglandin J2 acts cooperatively with prednisolone to reduce TGF-β-induced pro-fibrotic pathways in human osteoarthritis fibroblasts

Background/AimsSynovial fibrosis is a pathological process that is observed in several musculoskeletal disorders and characterized by the excessive deposition of extracellular matrix, as well as cell migration and proliferation. Despite the fact that glucocorticoids are widely employed in the treatment of rheumatic pathologies such as osteoarthritis (OA) and rheumatoid arthritis, the mechanisms by which glucocorticoids act in the joint and their impacts on pro-fibrotic pathways are still unclear. MaterialsHuman OA synovial fibroblasts were obtained from knee and hip joints. Cells were treated with prednisolone (1 mM) or transforming growth factor-beta 1 (TGF-β1) (10 ng/ml) for 1 and 7 days for quantification of RNA and protein expression (by real-time quantitative reverse transcription-PCR and western blot, respectively), 72 h for immunocytochemistry analysis, and 48 h for proliferation (by BrdU assay) and migration (by wound assay) studies. In addition, cells were preincubated with prednisolone and/or the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) for 6 h before adding TGF-β1. pSmad1/5, pSmad2 and β-catenin levels were analyzed by Western blot. The activin receptor-like kinase-5 (ALK-5) inhibitor (SB-431542) was employed for the mechanistic assays. ResultsPrednisolone showed a predominant anti-fibrotic impact on fibroblast-like synoviocytes as it attenuated the spontaneous and TGF-β-induced gene expression of pro-fibrotic markers. Prednisolone also reduced α-sma protein and type III collagen levels, as well as cell proliferation and migration after TGF-β stimulation. However, prednisolone did not downregulate the gene expression of all the pro-fibrotic markers tested and did not restore the reduced PPAR-γ levels after TGF-β stimulation. Interestingly, anti-fibrotic actions of the glucocorticoid were reinforced in the presence of the PPAR-γ agonist 15d-PGJ2. Combined pretreatment modulated Smad2/3 levels and, similar to the ALK-5 inhibitor, blocked β-catenin accumulation elicited by TGF-β. ConclusionsPrednisolone, along with 15d-PGJ2, modulates pro-fibrotic pathways activated by TGF-β in synovial fibroblasts at least partially through the inhibition of ALK5/Smad2 signaling and subsequent β-catenin accumulation. These findings shed light on the potential therapeutic effects of glucocorticoids treatment combined with a PPAR-γ agonist against synovial fibrosis, although future studies are warranted to further evaluate this concern.

Overexpression of ALK5 Induces Human Tenon’s Capsule Fibroblasts Transdifferentiation and Fibrosis In Vitro

ABSTRACTPurpose: To investigate the involvement of activin receptor-like kinase 5 (ALK5) in human Tenon’s capsule fibroblasts (HTFs) transdifferentiation and fibrosis.Methods: (1) Cultured HTFs were treated with transforming growth factor beta 1 (TGF-β1) at different concentrations for different durations, mRNA expression of ALK5 and plasminogen activator inhibitor-1 (PAI-1) was measured by quantitative polymerase chain reaction (PCR) while protein expression of ALK5, α-smooth muscle actin (α-SMA), and extracellular matrix deposition including fibronectin (FN) and collagen I (Col1) was assessed by western blot. HTFs with or without TGF-β1 were also treated with an ALK5 activity inhibitor, SB-431542, and fibrosis-related genes were assessed.(2) HTFs were transduced with ALK5 lentivirus (ALK5-OE group) or empty lentivirus (NC-OE) with or without the treatment of SB-431542. Protein expression of ALK5, α-SMA, FN, and Col1 was evaluated.(3) HTFs in the ALK5-OE group and NC-OE group were subjected to a scratch-wound assay and their migratory activities assessed.Results: (1) TGF-β1, in a concentration-dependent manner, upregulated ALK5 and PAI-1 expressions in the HTFs, which peaked between 24 and 36 h. These changes were associated with increases in protein levels of FN, Col1, and α-SMA. These TGF-β1 effects were blocked by the ALK5 inhibitor SB-431542.(2) Similarly, overexpression of ALK5 by lentiviral vector significantly increased protein expression of α-SMA, FN, and Col1. Addition of TGF-β1 to the ALK5-OE cells did not produce additional expression of any of the marker proteins. The upregulation of extracellular matrix and α-SMA can be reduced by SB-431542.(3) In ALK5-OE group, HTFs migration was significantly increased compared with normal control and TGF-β1 could still promote ALK5-OE cells migration.Conclusions: Our findings suggest that ALK5 is an important mediator of HTFs fibrosis. ALK5 is a potential therapeutic target to suppress scar formation after filtration surgery.

Identification of TGF-β receptor-1 as a key regulator of carbon nanotube-induced fibrogenesis.

Carbon nanotubes (CNTs) induce rapid interstitial lung fibrosis, but the underlying mechanisms are unclear. Previous studies indicated that the ability of CNTs to penetrate lung epithelium, enter interstitial tissue, and stimulate fibroblasts to produce collagen matrix is important to lung fibrosis. In this study, we investigated the activation of transforming growth factor-β receptor-1 [TGF-β R1; i.e., activin receptor-like kinase 5 (ALK5) receptor] and TGF-β/Smad signaling pathway in CNT-induced collagen production in human lung fibroblasts. Human lung fibroblasts and epithelial cells were exposed to low, physiologically relevant concentrations (0.02-0.6 μg/cm(2)) of single-walled CNTs (SWCNT) and multiwalled CNTs (MWCNT) in culture and analyzed for collagen, TGF-β1, TGF-β R1, and SMAD proteins by Western blotting and immunofluorescence. Chemical inhibition of ALK5 and short-hairpin (sh) RNA targeting of TGF-β R1 and Smad2 were used to probe the fibrogenic mechanism of CNTs. Both SWCNT and MWCNT induced an overexpression of TGF-β1, TGF-β R1 and Smad2/3 proteins in lung fibroblasts compared with vehicle or ultrafine carbon black-exposed controls. SWCNT- and MWCNT-induced collagen production was blocked by ALK5 inhibitor or shRNA knockdown of TGF-β R1 and Smad2. Our results indicate the critical role of TGF-β R1/Smad2/3 signaling in CNT-induced fibrogenesis by upregulating collagen production in lung fibroblasts. This novel finding may aid in the design of mechanism-based risk assessment and development of rapid screening tests for nanomaterial fibrogenicity.

Activin receptor‐like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8+ T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation.

Transforming Growth Factor-β Type I Receptor Inhibitor Induces Functional and Morphologic Recovery in a Rat Model of Erectile Dysfunction and Cavernous Fibrosis

Purpose: To examine the effectiveness of small-molecule inhibitor of transforming growth factor-β (TGF-β) type I receptor, an activin receptor-like kinase 5 (ALK5), on erectile dysfunction (ED) in a rat model of cavernous fibrosis, in which fibrosis was induced by intracavernous injection of adenovirus expressing TGF-β1 (Ad-TGF-β1). Materials and Methods: Four-month-old Sprague-Dawley rats were divided into four groups (n=10 per group): age-matched controls without treatment, age-matched controls receiving intracavernous injection of LacZ adenovirus, and cavernous fibrosis rats receiving an intracavernous injection of saline or ALK5 inhibitor (5 mg/kg). ALK5 inhibitor or saline was administered on day 5 after injection of Ad-TGF-β1. On day 30, erectile function was assessed by electrical stimulation of the cavernous nerve and the penis was then harvested for histologic studies (n=6 per group) and for the measurement of the hydroxyproline level (n=4 per group). Results: Ad-TGF-β1-induced cavernous fibrosis rats treated with saline showed a significant decrease in cavernous smooth muscle and endothelial content, and an increase in collagen deposition, which resulted in profound deterioration of all erectile function parameters, such as the ratios of maximal intracavernous pressure (ICP), total ICP, and slope to mean arterial pressure. ALK5 inhibitor significantly restored erectile function in a rat model of cavernous fibrosis by increasing cavernous smooth muscle and endothelial content, and by blocking cavernous fibrosis. Conclusions: The results suggest that inhibition of the TGF-β pathway is a promising therapeutic strategy for the treatment of ED related to cavernous fibrosis from various cause

Activin Receptor-Like Kinase 5 Inhibitor Attenuates Fibrosis in Fibroblasts Derived from Peyronie's Plaque

PurposeTransforming growth factor-β1 (TGF-β1) is the key fibrogenic cytokine associated with Peyronie's disease (PD). The aim of this study was to determine the antifibrotic effect of 3-((5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl) methyl)benzamide (IN-1130), a small-molecule inhibitor of the TGF-β type I receptor activin receptor-like kinase 5 (ALK5), in fibroblasts isolated from human PD plaque.Materials and MethodsPlaque tissue from a patient with PD was used for primary fibroblast culture, and we then characterized primary cultured cells. Fibroblasts were pretreated with IN-1130 (10 µM) and then stimulated with TGF-β1 protein (10 ng/ml). We determined the inhibitory effect of IN-1130 on TGF-β1-induced phosphorylation of Smad2 and Smad3 or the nuclear translocation of Smad proteins in fibroblasts. Western blot analyses for plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV were performed to evaluate effect of IN-1130 on the production of extracellular matrix proteins.ResultsThe treatment of fibroblasts with TGF-β1 significantly increased phosphorylation of Smad2 and Smad3 and induced translocation of Smad proteins from the cytoplasm to the nucleus. Pretreatment with IN-1130 substantially inhibited TGF-β1-induced phosphorylation of Smad2 and Smad3 and nuclear accumulation of Smad proteins. The TGF-β1-induced production of extracellular matrix proteins was also significantly inhibited by treatment with IN-1130 and returned to basal levels.ConclusionsOverexpression of TGF-β and activation of Smad transcriptional factors are known to play a crucial role in the pathogenesis of PD. Thus, inhibition of the TGF-β signaling pathway by ALK5 inhibitor may represent a promising therapeutic strategy for treating PD.

EW‐7203, a novel small molecule inhibitor of transforming growth factor‐β (TGF‐β) type I receptor/activin receptor‐like kinase‐5, blocks TGF‐β1‐mediated epithelial‐to‐mesenchymal transition in mammary epithelial cells

Recently, small molecule inhibitors of transforming growth factorβ (TGF-β) type I receptor kinase ⁄ activin receptor-like kinase-5 (ALK5) have been developed to target TGF-β signalling as a therapeutic strategy for combating cancer. In the present study, the authors examined a novel small molecule inhibitor of ALK5, 3-((5- ([1,2,4]triazolo[1,5-a]pyridin-6-yl)-4-(6-methylpyridin-2-yl)thiazol-2-ylamino)methyl)benzonitrile (EW-7203) in breast cancer cells to determine if it has potential for cancer treatment. The inhibitory effects of EW-7203 on TGF-β-induced Smad signalling and epithelial- to-mesenchymal transition (EMT) were investigated in mammary epithelial cells using luciferase reporter assays, immunoblotting, confocal microscopy and wound healing assays. In addition, the suppressive effects of EW-7203 on mammary cancer metastasis to the lung were examined using a Balb ⁄ c xenograft model system. The novel ALK5 inhibitor, EW-7203, inhibited the TGF-β1-stimulated transcriptional activation of p3TP-Lux and pCA-GA₁₂- Luc. In addition, EW-7203 decreased phosphorylated Smad2 levels and the nuclear translocation of Smad2 was increased by TGF-β1. In addition, EW-7203 inhibited TGF-β1-induced EMT and wound healing of NMuMG cells. Furthermore, in xenografted Balb ⁄ c mice, EW-7203 inhibited metastasis to the lung from breast tumors. The novel ALK5 inhibitor, EW-7203, efficiently inhibited TGF-β1-induced Smad signalling, EMT and breast tumor metastasis to the lung in vivo, demonstrating that EW-7203 has therapeutic potential for breast cancer metastasis to the lung.

Transforming growth factor‐beta type 1 receptor (ALK5) and Smad proteins mediate TIMP‐1 and collagen synthesis in experimental intestinal fibrosis

Transforming growth factor β (TGF-β) is known to play a key role in intestinal fibrosis; however, the underlying mechanisms are not well understood. TGF-β signal transduction is through TGF-β receptors, including the TGF-β type 1 receptor. Most cell types contain a TGF-β type 1 receptor form known as activin receptor-like kinase 5 (ALK5), which propagates the signal to the nucleus through the phosphorylation of Smad2 and Smad3 proteins. Therefore, we assessed the effect of the disruption of TGF-β/ALK5/Smad signalling by an ALK5 inhibitor (SD-208) in two experimental animal models of intestinal fibrosis: anaerobic bacteria- and trinitrobenzensulphonic acid-induced colitis. In addition, isolated myofibroblasts were pretreated with SD-208 and exposed to recombinant TGF-β1. Finally, myofibroblasts were transfected with ALK5, Smad2, and Smad3-specific siRNA. Up-regulation of ALK5 and TIMP-1, phosphorylation of Smad2 and Smad3 proteins, and increased intestinal wall collagen deposition were found in both experimental animal models. These effects were decreased by SD-208. TGF-β1 treatment also induced phosphorylation of Smad2 and Smad3 and up-regulation of ALK5 protein, TIMP-1, and α2 type 1 collagen gene expression in isolated myofibroblasts. Again these effects were inhibited by SD-208. Also, ALK5, Smad2, and Smad3 siRNA abolished the induction of TIMP-1 and α2 type 1 collagen. Our findings provide evidence that the TGF-β/ALK5/Smad pathway participates in the pathogenesis of experimental intestinal fibrosis. These data show promise for the development of an effective therapeutic intervention in this condition.

TGF-β Inhibits CYP17 Transcription in H295R Cells Acting via Activin Receptor-Like Kinase 5

Objective. Transforming growth factor β (TGF-β) is a potent inhibitor of 17α-hydroxylase/17,20 lyase activity and CYP17 gene expression. We investigated the mechanism how CYP17 is inhibited by TGF-β in adrenocortical cells. Methods. H295R cells were culture and incubated with TGF-β, transcription inhibitor (DRB), activin receptor-like kinase 5 ALK5 (TβRII) inhibitor (SB431542), mitogen activated kinases inhibitors (PD98059 and SB203580), subsequently using reverse transcription and quantitative PCR (RT-qPCR) we determined CYP17 expression. Results. TGF-β significantly decreased the level of cytochrome P450c17 mRNA and this inhibitory effect of TGF-β on CYP17 expression required activin receptor-like kinase 5 (ALK5) and on-going transcription. Mitogen activated kinases MEK1 and p38 MAPK are not involved it the inhibitory effect of TGF-β on CYP17 expression. Conclusion. We concluded that the TGF-β-dependent decrease of 17α-hydroxylase/17,20 lyase activity in the H295R cells is caused by inhibition of CYP17 transcription and is mediated by the ALK5 receptor.

Inhibition of activin receptor-like kinase 5 attenuates Bleomycin-induced pulmonary fibrosis

Activin receptor-like kinase 5 (ALK5) is a type I receptor of transforming growth factor (TGF)-beta. ALK5 inhibition has been reported to attenuate the tissue fibrosis including pulmonary fibrosis, renal fibrosis and liver fibrosis. To elucidate the inhibitory mechanism of ALK5 inhibitor on pulmonary fibrosis in vivo, we performed the histopathological assessment, gene expression analysis of extracellular matrix (ECM) genes and immunohistochemistry including receptor-activated Smads (R-Smads; Smad2/3), CTGF, myofibroblast marker (alpha-smooth muscle actin; aSMA) and type I collagen deposition in the lung using Bleomycin (BLM)-induced pulmonary fibrosis model. ALK5 inhibitor, SB-525334 (10 mg/kg or 30 mg/kg) was orally administered at twice a day. Lungs were isolated 5, 7, 9 and 14 days after BLM treatment. BLM treatment led to significant pulmonary fibrotic changes accompanied by significant upregulation of ECM mRNA expressions, Smad2/3 nuclear translocation, CTGF expression, myofibroblast proliferation and type I collagen deposition. SB-525334 treatment attenuated the histopathological alterations in the lung, and significantly decreased the type I and III procollagen and fibronectin mRNA expression. Immunohistochemistry revealed that SB-525334 treatment showed significant attenuation in Smad2/3 nuclear translocation, decrease in CTGF-expressing cells, myofibroblast proliferation and type I collagen deposition. These results suggest that ALK5 inhibition attenuates R-Smads activation thereby attenuates pulmonary fibrosis.