Canonical Smad Signaling Pathway Research Articles

Comparative proteomics analysis of transforming growth factor-beta1-overexpressed human dental pulp stem cell-derived secretome on CD44-mediated fibroblast activation via canonical smad signal pathway

ABSTRACT Purpose The aim of this study investigates whether the secretome collected from human dental pulp stem cells (hDPSCs) transfected with transforming growth factor-beta1 (TGF-β1) is related to CD44 expression of fibroblasts and canonical smad signaling pathway via proteomic analyzes. Materials and Methods In order to obtain secretome, hDPSCs were conditioned with serum-free alpha-MEM in an incubator containing 37°C, 5% CO2, and humidity for 18–24 h. Proteins in control and TGF-β1 secretome were analyzed by tandem mass spectrometry-based shotgun proteomic method. Bioinformatic evaluations were completed via Ingenuity Pathway Analysis (IPA, QIAGEN) software. CD44 expressions in fibroblasts were evaluated by real time-PCR, western blot, and immunofluorescent staining. The relationship of canonical smad pathway and CD44 was analyzed by western blot and LC-MS/MS. Cell cycle, proliferation and wound healing tests were performed in the secretome groups. Results Venn diagram was showed 174 common proteins were identified from each group. In the control secretome 140 unique proteins were identified and 66 entries were exclusive for TGF-β1 secretome. CD44 gene and protein expressions were increased in fibroblasts treated with TGF-β1 secretome. Relationship between targeted protein data showed that activation of the canonical TGF-β1/Smad pathway was up-regulated CD44 expression in fibroblasts. The canonical smad pathway-mediated upregulation of CD44 may increase the mitotic activity, proliferation, and wound healing potential in fibroblasts. Conclusion While TGF-β1-transfected hDPSC secretome may be a potential therapeutic candidate in regenerative connective tissue therapies as it induces fibroblast activation, anti-TGF-β1-based therapies would be considered in histopathological conditions such as pulmonary fibrosis or hepatic fibrosis.

BMP Signaling Pathway in Dentin Development and Diseases.

BMP signaling plays an important role in dentin development. BMPs and antagonists regulate odontoblast differentiation and downstream gene expression via canonical Smad and non-canonical Smad signaling pathways. The interaction of BMPs with their receptors leads to the formation of complexes and the transduction of signals to the canonical Smad signaling pathway (for example, BMP ligands, receptors, and Smads) and the non-canonical Smad signaling pathway (for example, MAPKs, p38, Erk, JNK, and PI3K/Akt) to regulate dental mesenchymal stem cell/progenitor proliferation and differentiation during dentin development and homeostasis. Both the canonical Smad and non-canonical Smad signaling pathways converge at transcription factors, such as Dlx3, Osx, Runx2, and others, to promote the differentiation of dental pulp mesenchymal cells into odontoblasts and downregulated gene expressions, such as those of DSPP and DMP1. Dysregulated BMP signaling causes a number of tooth disorders in humans. Mutation or knockout of BMP signaling-associated genes in mice results in dentin defects which enable a better understanding of the BMP signaling networks underlying odontoblast differentiation and dentin formation. This review summarizes the recent advances in our understanding of BMP signaling in odontoblast differentiation and dentin formation. It includes discussion of the expression of BMPs, their receptors, and the implicated downstream genes during dentinogenesis. In addition, the structures of BMPs, BMP receptors, antagonists, and dysregulation of BMP signaling pathways associated with dentin defects are described.

Pan-Phosphodiesterase Inhibitors Attenuate TGF-β-Induced Pro-Fibrotic Phenotype in Alveolar Epithelial Type II Cells by Downregulating Smad-2 Phosphorylation.

Airway remodeling is a pathological process that accompanies many chronic lung diseases. One of the important players in this process are epithelial cells, which under the influence of pro-inflammatory and pro-fibrotic factors present in the airway niche, actively participate in the remodeling process by increasing extracellular matrix secretion, acquiring migration properties, and overproducing pro-fibrotic transducers. Here, we investigated the effect of three new 8-arylalkylamino- and 8-alkoxy-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl-N-(5-(tert-butyl)-2-hydroxyphenyl)butanamides (1, 2, and 3), representing prominent pan-phosphodiesterase (pan-PDE) inhibitors on transforming growth factor type β (TGF-β)-induced alveolar epithelial type II cells (A549 cell line) of a pro-fibrotic phenotype. Our results demonstrate for the first time the strong activity of pan-PDE inhibitors in the prevention of TGF-β-induced mesenchymal markers’ expression and A549 cells’ migration. We also showed an increased p-CREB and decreased p-Smad-2 phosphorylation in TGF-β-induced A549 cells treated with 1, 2, and 3 derivatives, thereby confirming a pan-PDE inhibitor mesenchymal phenotype reducing effect in alveolar epithelial type II cells via suppression of the canonical Smad signaling pathway. Our observations confirmed that PDE inhibitors, and especially those active against various isoforms involved in the airway remodeling, constitute an interesting group of compounds modulating the pro-fibrotic response of epithelial cells.

BMP6 increases CD68 expression by up-regulating CTGF expression in human granulosa-lutein cells

Bone morphogenetic protein 6 (BMP6) and connective tissue growth factor (CTGF) are critical growth factors required for normal follicular development and luteal function. Cluster of Differentiation 68 (CD68) is an intraovarian marker of macrophages that plays an important role in modulating the physiological regression of the corpus luteum. The aim of this study was to investigate the effect of BMP6 on the expression of CTGF and the subsequent increase in CD68 expression as well as its underlying mechanisms. Primary and immortalized (SVOG) human granulosa cells obtained from infertile women undergoing in vitro fertilization treatment were used as cell models to conduct the in vitro experiments. Our results showed that BMP6 treatment significantly increased the expression levels of CTGF and CD68. Using BMP type I receptor inhibitors (dorsomorphin, DMH-1 and SB431542), we demonstrated that both activin receptor-like kinase (ALK)2 and ALK3 are involved in BMP6-induced stimulatory effects on the expression of CTGF and CD68. Additionally, SMAD4-knock down reversed the BMP6-induced up-regulation of CTGF and CD68, indicating that the canonical SMAD signaling pathway is required for these effects. Moreover, CTGF-knock down abolished the BMP6-induced up-regulation of CD68 expression. These findings indicate that intrafollicular CTGF mediates BMP6-induced increases in CD68 expression through the ALK2/ALK3-mediated SMAD-dependent signaling pathway.

Growth differentiation factor 5: a neurotrophic factor with neuroprotective potential in Parkinson's disease.

Parkinson’s disease is the most common movement disorder worldwide, affecting over 6 million people. It is an age-related disease, occurring in 1% of people over the age of 60, and 3% of the population over 80 years. The disease is characterized by the progressive loss of midbrain dopaminergic neurons from the substantia nigra, and their axons, which innervate the striatum, resulting in the characteristic motor and non-motor symptoms of Parkinson’s disease. This is paralleled by the intracellular accumulation of α-synuclein in several regions of the nervous system. Current therapies are solely symptomatic and do not stop or slow disease progression. One promising disease-modifying strategy to arrest the loss of dopaminergic neurons is the targeted delivery of neurotrophic factors to the substantia nigra or striatum, to protect the remaining dopaminergic neurons of the nigrostriatal pathway. However, clinical trials of two well-established neurotrophic factors, glial cell line-derived neurotrophic factor and neurturin, have failed to meet their primary end-points. This failure is thought to be at least partly due to the downregulation by α-synuclein of Ret, the common co-receptor of glial cell line-derived neurorophic factor and neurturin. Growth/differentiation factor 5 is a member of the bone morphogenetic protein family of neurotrophic factors, that signals through the Ret-independent canonical Smad signaling pathway. Here, we review the evidence for the neurotrophic potential of growth/differentiation factor 5 in in vitro and in vivo models of Parkinson’s disease. We discuss new work on growth/differentiation factor 5’s mechanisms of action, as well as data showing that viral delivery of growth/differentiation factor 5 to the substantia nigra is neuroprotective in the α-synuclein rat model of Parkinson’s disease. These data highlight the potential for growth/differentiation factor 5 as a disease-modifying therapy for Parkinson’s disease.

Low-Intensity Pulsed Ultrasound Stimulates Osteogenic Differentiation of Periosteal Cells In Vitro.

Adequate bone volume is required for osseointegrated implants to restore lost teeth and oral function. Several studies have demonstrated potential advantage of stem cells in regenerative medicine using osteoblasts. The periosteum is composed of osteoblasts, fibroblasts, and osteoprogenitor cells. It may be an alternative source for bone tissue engineering because of easy isolation and rapid proliferation in vivo and in vitro. Low-intensity pulsed ultrasound (LIPUS) has proved successful in recoveries from nonunions, delayed unions, and fracture of the bone in both animal experiments and clinical treatments. The study was to investigate the influence of LIPUS on the osteogenic differentiation in murine periosteum-derived cells (PDCs) and the underlying mechanism of LIPUS. PDCs were treated daily with LIPUS for 20 min up to 21 days with 3 MHz frequency, 30 mW/cm2 intensity, and pulse repetition frequency of 1 kHz. The effects of LIPUS on cell proliferation and viability were investigated. Osteogenic differentiation was analyzed by alkaline phosphatase (ALP)-positive cell staining, ALP activity assay, mineralized nodule formation, real-time reverse transcription-polymerase chain reaction, as well as western blotting. The results indicated that ultrasound stimulation did not significantly affect the proliferation of PDCs. But LIPUS significantly increased ALP activity on day 7 and markedly promoted formation of mineralized nodules on day 21. mRNA expression of ALP and osteocalcin was significantly upregulated by stimulation with LIPUS. LIPUS enhanced gene expression of both bone morphogenetic protein-2 (BMP-2) and osterix only in the presence of osteogenic medium. LIPUS stimulation did not affect Smad 1 and Smad 5 protein expression, but significantly upregulated protein levels of BMP-2 and phosphor-Smad 1/5/9 in PDCs. Thus, LIPUS stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of BMP-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition. Therefore, LIPUS might have potential to promote osteogenesis in PDCs. Impact statement There are few studies on periosteum-derived cells (PDCs) because conventional methods of their isolation are relatively difficult to procure abundant cells for cell culture and the total cell numbers are limited. In this study, a modified isolation technique of murine calvarial PDCs using gelatin is described. PDCs were initiated to emerge as early as day 3 and showed increased proliferation, which can be used for further studies. Low-intensity pulsed ultrasound stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of bone morphogenic protein-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition.

Melatonin Inhibits Transforming Growth Factor-β1-Induced Epithelial-Mesenchymal Transition in AML12 Hepatocytes.

Recent studies showed that melatonin, a well-known pineal hormone that modulates the circadian rhythm, exerts beneficial effects against liver fibrosis. However, mechanisms for its protective action against the fibrotic processes remain incompletely understood. Here, we aimed to explore the effects of the hormone on transforming growth factor-β1 (TGF-β1)-stimulated epithelial–mesenchymal transition (EMT) in AML12 hepatocytes. Pretreatment with melatonin dose-dependently reversed downregulation of an epithelial marker and upregulation of mesenchymal markers after TGF-β1 stimulation. Additionally, melatonin dose-dependently suppressed an increased phosphorylation of Smad2/3 after TGF-β1 treatment. Besides the canonical Smad signaling pathway, an increase in phosphorylation of extracellular signal-regulated kinase 1/2 and p38 was also dose-dependently attenuated by melatonin. The suppressive effect of the hormone on EMT stimulated by TGF-β1 was not affected by luzindole, an antagonist of melatonin membrane receptors, suggesting that its membrane receptors are not required for the inhibitory action of melatonin. Moreover, melatonin suppressed elevation of intracellular reactive oxygen species (ROS) levels in TGF-β1-treated cells. Finally, TGF-β1-stimulated EMT was also inhibited by the antioxidant N-acetylcysteine. Collectively, these results suggest that melatonin prevents TGF-β1-stimulated EMT through suppression of Smad and mitogen-activated protein kinase signaling cascades by deactivating ROS-dependent mechanisms in a membrane receptor-independent manner.

Activin A receptor type 1–mediated BMP signaling regulates RANKL-induced osteoclastogenesis via canonical SMAD-signaling pathway

Bone morphogenetic proteins (BMPs) are important mediators of osteoclast differentiation. Although accumulating evidence has implicated BMPs in osteoblastogenesis, the mechanisms by which BMPs regulate osteoclastogenesis remain unclear. Activin A receptor type 1 (ACVR1) is a BMP type 1 receptor essential for skeletal development. Here, we observed that BMP-7, which preferentially binds to ACVR1, promotes osteoclast differentiation, suggesting ACVR1 is involved in osteoclastogenesis. To investigate this further, we isolated osteoclasts from either Acvr1-floxed mice or mice with constitutively-activated Acvr1 (caAcvr1) carrying tamoxifen-inducible Cre driven by a ubiquitin promotor and induced Cre activity in culture. Osteoclasts from the Acvr1-floxed mice had reduced osteoclast numbers and demineralization activity, whereas those from the caAcvr1-mutant mice formed large osteoclasts and demineralized pits, suggesting that BMP signaling through ACVR1 regulates osteoclast fusion and activity. It is reported that BMP-2 binds to BMPR1A, another BMP type 1 receptor, whereas BMP-7 binds to ACVR1 to activate SMAD1/5/9 signaling. Here, Bmpr1a-disrupted osteoclasts displayed reduced phospho-SMAD1/5/9 (pSMAD1/5/9) levels when induced by BMP-2, whereas no impacts on pSMAD1/5/9 were observed when induced by BMP-7. In contract, Acvr1-disrupted osteoclasts displayed reduced pSMAD1/5/9 levels when induced either by BMP-2 or BMP-7, suggesting that ACVR1 is the major receptor for transducing BMP-7 signals in osteoclasts. Indeed, LDN-193189 and LDN-212854, which specifically block SMAD1/5/9 phosphorylation, inhibited osteoclastogenesis of caAcvr1-mutant cells. Moreover, increased BMP signaling promoted nuclear translocation of nuclear factor-activated T-cells 1 (NFATc1), which was inhibited by LDN treatments. Taken together, ACVR1-mediated BMP-SMAD signaling activates NFATc1, a regulatory protein crucial for receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis.

BMP6 increases TGF-β1 production by up-regulating furin expression in human granulosa-lutein cells

Bone morphogenetic protein 6 (BMP6) and transforming growth factor-β1 (TGF-β1) are key intraovarian regulators that play essential roles in regulating mammalian follicular function and promoting oocyte maturation. Furin, a member of the subtilisin-like proprotein convertase family, promotes the activation of diverse functional proteins by cleaving protein precursors in the secretory pathway. The aim of this study was to investigate the effect and underlying molecular mechanisms by which BMP6 regulates the expression of furin to increase TGF-β1 production. Primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used as study models. Our results show that BMP6 significantly up-regulated the expression of furin and increased the production of TGF-β1 in hGL cells. Using dual inhibition approaches (kinase receptor inhibitors and small interfering RNA-targeted knockdown), we demonstrate that both activin receptor-like (ALK)2 and ALK3 are involved in the BMP6-induced up-regulation of furin. Additionally, knockdown of furin abolished BMP6-induced increases in TGF-β1 production. Moreover, knockdown of endogenous SMAD4 reversed the BMP6-induced increase in furin expression. These results indicate that the ALK2/3-mediated canonical SMAD signaling pathway is required for the stimulatory effect of BMP6 on furin expression, which in turn increases the production of TGF-β1 in hGL cells. Our findings provide insights into the molecular interactions and mechanisms of two intrafollicular growth factors in hGL cells.

BMP6 Increases TGF-Beta1 Production by Up-Regulating Furin Expression in Human Granulosa-Lutein Cells

Background: Bone morphogenetic protein 6 (BMP6) and transforming growth factor--β1 (TGF--β1) are key intraovarian regulators that play essential roles in regulating mammalian follicular function and promoting oocyte maturation. Dysregulated BMP6 or TGF-β1 signaling is associated with polycystic ovary syndrome. Furin is a proprotein convertase that promotes the activation of diverse functional proteins by cleaving protein precursors. Methods: Primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used as study models. RT-qPCR, western blot analyses, and enzyme immunoassay were used to examine the effects of recombinant human BMP6 on the expression of furin and production of TGF-β1. Dual inhibition approaches (kinase receptor inhibitors and small interfering RNAs targeted knockdown) were used to investigate the underlying molecular mechanisms by which BMP6 regulated the expression of furin. Findings: BMP6 significantly up-regulated the expression of furin and increased the production of TGF-β1. Additionally, both activin receptor-like (ALK)2 and ALK3 were involved in BMP6-induced up-regulation of furin. Furthermore, knockdown of furin abolished BMP6-induced increases in TGF--β1 production, and knockdown of endogenous SMAD4 reversed the increase in furin expression induced by BMP6. Interpretation: The ALK2/3-mediated canonical SMAD signaling pathway is required for the stimulatory effect of BMP6 on furin expression, which in turn increases the production of TGF-β1 in hGL cells. Our findings shed insights into the molecular interactions and mechanisms of two intrafollicular growth factors in hGL cells, which could be potentially applied as therapeutic targets for patients with polycystic ovary syndrome. Funding Statement: Canadian Institutes of Health Research Foundation Scheme Grant #143317. Declaration of Interests: The authors have no conflict of interests to declare. Ethics Approval Statement: All participants signed a written informed consent. The study was approved by Research Ethics Board from the University of British Columbia.

ERK1/2-mediated EGFR-signaling is required for TGFβ-induced lens epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) plays a critical role in the pathogenesis of fibrotic cataract. Transforming growth factor-beta (TGFβ) is a potent inducer of this fibrotic process in lens. Recent studies in cancer progression have shown that in addition to activating the canonical Smad signaling pathway, TGFβ can also transactivate the epidermal growth factor receptor (EGFR) to enhance invasive cell migration. The present study aims to elucidate the involvement of EGFR-signaling in TGFβ-induced EMT in LECs. Treatment with TGFβ2 induced transdifferentiation of LECs into myofibroblastic cells, typical of an EMT. TGFβ2 induced the phosphorylation of the EGFR and upregulation of Egfr and Hb-egf gene expression. Pharmacologic inhibition of EGFR-signaling using PD153035 inhibited TGFβ-induced EMT, including the upregulation of mesenchymal markers and downregulation of epithelial markers. Crosstalk between TGFβ2-induced EGFR and ERK1/2 was evident, with both pathways impacting on Smad2/3-signaling. Our finding that TGFβ2 transactivates downstream EGFR-signaling reveals a previously unknown mechanism in the pathogenesis of cataract. Understanding the complex interplay between divergent canonical and non-canonical signaling pathways, as well as downstream target genes involved in TGFβ-induced EMT, will enable the development of more effective targeted therapies in the pharmacological treatment of cataract.

Targeting TAZ and YAP as a New Therapeutic Approach to Diabetic Nephropathy

Introduction
 Fibrosis is a major contributor to chronic kidney disease (CKD), for which no effective clinical treatment exists. The primary source of fibrosis is the activation of fibroblasts to the myofibroblast state. Fibroblast-myofibroblast transition requires transforming growth factor-β (TGF-β) and its canonical Smad signaling pathway.
 
 Purpose
 Recent findings suggest that mechanical stimuli affect fibroblast behavior. Nuclear localization of YAP/TAZ, closely associated mechanosensitive transcriptional co-factors, are regulated by substrate stiffness. As YAP/TAZ are Smad nuclear retention factors promoting TGF- β signaling, we hypothesized that YAP/TAZ inhibition could attenuate stiffness-mediated, TGF- β induced pro-fibrotic responses.
 
 Methods
 Immunostaining and immunoblotting were used to analyze localization and activity of YAP/TAZ and Smad levels, respectively.
 
 Results
 YAP/TAZ are in an active nuclear location in fibroblasts grown on stiff, fibrotic-like substrates (100kPa). In fibroblasts grown on soft substrates (2kPa), YAP/TAZ are primarily in an inactive cytosolic position. Cells grown on soft surfaces demonstrated strongly attenuated nuclear Smad 2/3 translocation and Smad-3 dependent transcription upon TGF-β stimulation, indicating impaired pro-fibrotic signaling. Verteporfin, a clinically approved drug with YAP inhibitory properties, was used to test the role of YAP/TAZ in reduced TGF-β signaling. Verteporfin reduced TGF-β-induced nuclear Smad2/3 accumulation and Smad3-mediated 
 transcription in fibroblasts grown on stiff surfaces. In vivo, Verteporfin significantly reduces markers of renal fibrosis.
 
 Conclusions
 Soft, healthy kidney-like substrates inhibit, while stiff fibrotic-like substrates promote, pro-fibrotic TGF-β Smad signalling. Verteporfin inactivates the YAP/TAZ fibroblast mechanosensor, reduces stiffness-augmented fibroblast responses to TGF-β through blockade of Smad signalling, and may be a novel anti-fibrotic agent for CKD.

Chinese medicine Bu-Fei decoction attenuates epithelial-mesenchymal transition of non-small cell lung cancer via inhibition of transforming growth factor β1 signaling pathway in vitro and in vivo

Ethnopharmacological relevanceTraditional Chinese medicine Bu-Fei decoction (BFD) has been utilized to treat patients with Qi deficiency for decades, with the advantages of invigorating vital energy, clearing heat-toxin and moistening lung, etc. According to previous clinical experience and trials, BFD has been found to indeed improve life quality of lung cancer patients and prolong survival time. Nevertheless, little is known on its potential mechanisms so far. Being regarded as a pivotal cytokine in the tumor microenvironment, transforming growth factor β (TGF-β) stands out as a robust regulator of epithelial-mesenchymal transition (EMT), which is closely linked to tumor progression. Aim of the studyThe present study was designed to explore whether BFD antagonized EMT via blocking TGF-β1-induced signaling pathway, and then help contribute to create a relatively steady microenvironment for confining lung cancer. Materials and methodsThis experiment was performed in lung adenocarcinoma A549 cells both in vitro and in vivo. In detail, the influences mediated by TGF-β1 alone or in combination with different concentrations of BFD on migration were detected by wound healing and transwell assays, and the effects of BFD on cell viability were determined by cell counting kit-8 (CCK-8) assay. TGF-β1, EMT relevant proteins and genes were evaluated by western blotting, confocal microscopy, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and enzyme-linked immuno sorbent assay (ELISA). Female BALB/C nude mice were subcutaneously implanted A549 cells and given BFD by gavage twice daily for 28 days. The tumor volume was monitored every 4 days to draw growth curve. The tumor weight, expression levels of EMT-related protein in tumor tissues and TGF-β1 serum level were evaluated, respectively. ResultsBFD only exerted minor effects on A549 cell proliferation and this was in accordance with the in vivo result, which showed that the tumor growth and weight were not be restrained by BFD administration. However, the data elucidated that BFD could dose-dependently suppress EMT induced by TGF-β1 in vitro via attenuating canonical Smad signaling pathway. In the A549 xenograft mouse model, BFD also inhibited protein markers that are associated with EMT and TGF-β1 secretion into serum. ConclusionsBased on these above data, the conclusion could be put forward that BFD probably attenuated TGF-β1 mediated EMT in A549 cells via decreasing canonical Smad signaling pathway both in vitro and in vivo, which may help restrain the malignant phenotype induced by TGF-β1 in A549 cells to some extent.

Bone morphogenetic protein 4 is overexpressed in and promotes migration and invasion of drug-resistant cancer cells

Drug resistance and metastasis significantly hinder chemotherapy and worsen prognoses in cancer. Bone morphogenetic protein 4 (BMP4) belongs to the TGF-β superfamily, has broad biological activities in cell proliferation and cartilage differentiation and is also able to induce migration and invasion. Herein, we investigated the role of BMP4 in the regulation of metastasis in paclitaxel-resistant human esophageal carcinoma EC109 cells (EC109/Taxol) and docetaxel-resistant human gastric cancer MGC803 cells (MGC/Doc). In these drug-resistant cell lines, we found the cell motility was enhanced and BMP4 was up-regulated relative to their respective parental cell lines. Consistent with in vitro assays, migration potential and BMP4 expression were increased in EC109/Taxol nude mice. Furthermore, to address whether BMP4 was required to enhance the metastatic in EC109/Taxol cells, the pharmacological inhibitor of BMP signaling dorsomorphin was used; meanwhile, we found that the migration and invasion abilities were inhibited. Moreover, the canonical Smad signaling pathway was investigated. Overall, our studies demonstrated that BMP4 participates in the regulation of invasion and migration by EC109/Taxol cells, and inhibition of BMP4 may be a novel strategy to interfere with metastasis in cancer therapy.

TGF-β1 protects intestinal integrity and influences Smads and MAPK signal pathways in IPEC-J2 after TNF-α challenge.

The aim of this study was to investigate the protective effects of TGF-β1 on intestinal epithelial barrier, as well as canonical Smad and MAPK signal pathways involved in these protection processes by a IPEC-J2 model stimulated with TNF-α. IPEC-J2 monolayers were treated without or with TNF-α in the absence or presence of TGF-β1. The results showed that TGF-β1 pretreatment ameliorated TNF-α-induced intestinal epithelial barrier disturbances as indicated by decrease of transepithelial electrical resistance (TER) and increase of paracellular permeability. TGF-β1 also dramatically alleviated TNF-α-induced alteration of TJ proteins ZO-1 and occludin. Moreover, TGF-β1 pretreatment increased TβRII protein expression in IPEC-J2 monolayers challenged with TNF-α. In addition, a significant increase of Smad4 and Smad7 mRNA was also observed in the TGF-β1 pretreatment after TNF-α challenge compared with the control group. Furthermore, TGF-β1 pretreatment enhanced smad2 protein activation. These results indicated that the canonical Smad signaling pathway was activated by TGF-β1 pretreatment. Finally, TGF-β1 pretreatment decreased the ratios of the phosphorylated to total JNK and p38 (p-JNK/JNK and p-p38/p38) and increased the ratio of ERK (p-ERK/ERK). Anti-TGF-β1 Abs reduced these TGF-β1 effects. These results indicated that TGF-β1 protects intestinal integrity and influences Smad and MAPK signal pathways in IPEC-J2 after TNF-α challenge.

A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

SMAD-independent down-regulation of caveolin-1 by TGF-β: effects on proliferation and survival of myofibroblasts.

Transforming growth factor-β (TGF-β) mediates growth-inhibitory effects on most target cells via activation of the canonical SMAD signaling pathway. This growth-inhibitory activity may be coupled with cellular differentiation. Our studies demonstrate that TGF-β1 inhibits proliferation of primary, non-transformed human lung fibroblasts in association with the induction of myofibroblast differentiation. Differentiated myofibroblasts maintain the capacity to proliferate in response to exogenous mitogenic stimuli and are resistant to serum deprivation-induced apoptosis. These proliferative and anti-apoptotic properties of myofibroblasts are related, in part, to the down-regulation of caveolin-1 (Cav-1) by TGF-β1. Cav-1 down-regulation is mediated by early activation of p38 MAPK and does not require SMAD signaling. In contrast, myofibroblast differentiation is dependent on activation of the SMAD pathway, but not on p38 MAPK. Thus, combinatorial signaling by TGF-β1 of myofibroblast differentiation and down-regulation of Cav-1 by SMAD and p38 MAPK pathways, respectively, confer proliferative and apoptosis-resistant properties to myofibroblasts. Selective targeting of this SMAD-independent, p38-MAPK/Cav-1-dependent pathway is likely to be effective in the treatment of pathological conditions characterized by TGF-β signaling and myofibroblast activation.

TGF-β Induces Up-Regulation of Chondroitin Sulfate Synthase 1 (CHSY1) in Nucleus Pulposus Cells Through MAPK Signaling

Background/Aims: Chondroitin sulfate synthase 1 (CHSY1) is a glycosyltransferases involved in the biosynthesis of chondroitin and dermatan sulfate glycosaminoglycan (GAG). TGF-β can stimulate sulfated GAG production in nucleus pulposus cells; however, the underlying mechanisms are poorly understood. Methods: CHSY1 expression was examined in rat nucleus pulposus treated with TGF-β using real-time PCR and Western blot analysis. Lentiviral knockdown was performed to determine the downstream effectors of TGF-β and to measure the effect of c-Jun and Sp1 on TGF-β mediated CHSY1 promoter activity and CHSY1 expression. Results: TGF-β increased CHSY1 expression and promoter activity in the nucleus pulposus partially through activation of canonical Smad signaling pathway. Knockdown of c-Jun and Sp1 decreased CHSY1 promoter activity, CHSY1 expression and sGAG accumulation induced by TGF-β. Furthermore, we found that TGF-β-induced expression of CHSY1 was mediated through the activation of MAPK signaling. Moreover, we showed that silencing CHSY1 decreased sGAG accumulation in nucleus pulposus cells induced by TGF-β. Conclusion: Our results suggest that TGF-β induced CHSY1 expression in the nucleus pulposus through the activation of MAPK signaling.

TGFβ-induced deptor suppression recruits mTORC1 and not mTORC2 to enhance collagen I (α2) gene expression.

Enhanced TGFβ activity contributes to the accumulation of matrix proteins including collagen I (α2) by proximal tubular epithelial cells in progressive kidney disease. Although TGFβ rapidly activates its canonical Smad signaling pathway, it also recruits noncanonical pathway involving mTOR kinase to regulate renal matrix expansion. The mechanism by which chronic TGFβ treatment maintains increased mTOR activity to induce the matrix protein collagen I (α2) expression is not known. Deptor is an mTOR interacting protein that suppresses mTOR activity in both mTORC1 and mTORC2. In proximal tubular epithelial cells, TGFβ reduced deptor levels in a time-dependent manner with concomitant increase in both mTORC1 and mTORC2 activities. Expression of deptor abrogated activity of mTORC1 and mTORC2, resulting in inhibition of collagen I (α2) mRNA and protein expression via transcriptional mechanism. In contrast, neutralization of endogenous deptor by shRNAs increased activity of both mTOR complexes and expression of collagen I (α2) similar to TGFβ treatment. Importantly, downregulation of deptor by TGFβ increased the expression of Hif1α by increasing translation of its mRNA. TGFβ-induced deptor downregulation promotes Hif1α binding to its cognate hypoxia responsive element in the collagen I (α2) gene to control its protein expression via direct transcriptional mechanism. Interestingly, knockdown of raptor to specifically block mTORC1 activity significantly inhibited expression of collagen I (α2) and Hif1α while inhibition of rictor to prevent selectively mTORC2 activation did not have any effect. Critically, our data provide evidence for the requirement of TGFβ-activated mTORC1 only by deptor downregulation, which dominates upon the bystander mTORC2 activity for enhanced expression of collagen I (α2). Our results also suggest the presence of a safeguard mechanism involving deptor-mediated suppression of mTORC1 activity against developing TGFβ-induced renal fibrosis.

Regulation of TGF-β1-driven Differentiation of Human Lung Fibroblasts: EMERGING ROLES OF CATHEPSIN B AND CYSTATIN C

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-β1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation, and led to an accumulation of intracellular 50-kDa TGF-β1. Moreover, the addition of Cat B generated a 25-kDa mature form of TGF-β1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation but had no effect on p38 MAPK and JNK phosphorylation, indicating that Cat B mostly disturbs TGF-β1-driven canonical Smad signaling pathway. Although mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-β1-induced differentiation of idiopathic pulmonary fibrosis and CCD-19Lu fibroblasts. In addition, cystatin C participated in the control of extracellular Cats, because its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-β1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.