Suppression of TGF-β1/Smad Signaling Pathway by Sesamin Contributes to the Attenuation of Myocardial Fibrosis in Spontaneously Hypertensive Rats

This study investigated the effect of sesamin on myocardial fibrosis in spontaneously hypertensive rats (SHRs) and the possible mechanisms involved. Twenty-eight male SHRs were randomly allocated to SHR group, Ses160 group (sesamin 160 mg/kg), Ses80 group (sesamin 80 mg/kg) and Cap30 group (captopril 30 mg/kg). Seven male WKY rats were used as control. Sesamin and captopril were administered intragastrically for 12 weeks. Captopril significantly reduced systolic blood pressure and angiotensin II (Ang II) levels in SHRs, accompanied by a marked attenuation of left ventricular hypertrophy (LVH) and collagen deposition (P <0.05 or P <0.01). Though sesamin had no significant influence on Ang II levels, and the hypotensive effect was also significantly inferior to that of captopril (P <0.05 or P <0.01), however, the improvement of LVH and collagen deposition was similar to that in captopril group. Sesamin markedly reduced transforming growth factor-β₁ (TGF-β₁) content in cardiac tissues, with Smad3 phosphorylation decreased and Smad7 protein expression increased notably (P <0.05 or P <0.01). Protein expression of type I collagen and type III collagen, target genes of Smad3, was down-regulated markedly by sesamin (P <0.05 or P <0.01). In addition, sesamin significantly increased total antioxidant capacity and superoxide dismutase protein in cardiac tissues (P <0.05 or P <0.01), while the expression of NADPH oxidase subunit p47phox and malondialdehyde content were reduced markedly (P <0.05 or P <0.01). In vitro studies also demonstrated that sesamin was able to suppress Ang II induced phosphorylation of Smad3 and secretion of TGF-β₁ and type I and type III collagen in cultured rat cardiac fibroblasts. These data suggest that sesamin is capable of attenuating hypertensive myocardial fibrosis through, at least partly, suppression of TGF-β₁/Smad signaling pathway.     

TGF-β2/Smad3 Signaling Pathway Activation Through Enhancing VEGF and CD34 Ameliorates Cerebral Ischemia/Reperfusion Injury After Isoflurane Post-conditioning in Rats

Neurochemical Research - Evidence has shown the therapeutic potential of isoflurane (ISO) in cerebral stroke. The present study investigated the mechanism of ISO on vascular endothelial growth...     

FTO Promotes Adipogenesis through Inhibition of the Wnt/β-catenin Signaling Pathway in Porcine Intramuscular Preadipocytes

Numerous studies have demonstrated that FTO plays an important role in adipogenesis. Herein, we designed a small interfering RNA targeting FTO to knock down its endogenous expression and investigated its effects on the proliferation and differentiation of porcine intramuscular preadipocytes. Its possible mechanism was also investigated. We showed that FTO silencing significantly decreased the level of phospho-Histone H3 protein and inhibited the proliferation of porcine intramuscular preadipocytes. In addition, the expressions of peroxisome proliferators-activated receptor γ (PPARγ) and CAAT/enhancer binding protein (C/EBPα) were down-regulated, but the expression of β-catenin was up-regulated, by FTO silencing. Of specific interest here was that LiCl, a Wnt/β-catenin signaling specific activator, attenuated the FTO-induced upregulation of PPARγ and downregulation of β-catenin. Collectively, our data demonstrated that FTO silence decreased the proliferation and differentiation of porcine intramuscular preadipocytes, and FTO affects the porcine intramuscular preadipocytes differentiation might be via Wnt/β-catenin signaling pathway.     

Effects of TRAP-1-Like Protein (TLP) Gene on Collagen Synthesis Induced by TGF-β/Smad Signaling in Human Dermal Fibroblasts

Background

Hypertrophic scars are pathologic proliferations of the dermal skin layer resulting from excessive collagen deposition during the healing process of cutaneous wounds. Current research suggests that the TGF-β/Smad signaling pathway is closely associated with normal scar and hypertrophic scar formation. TRAP-1-like protein (TLP), a cytoplasmic protein, has been reported to efficiently regulate Smad2- and Smad3-dependent signal expression in the TGF-β pathway. The relationship between TLP and Type I/III collagen (Col I/III) synthesis explored in the present study provides an effective target for wound healing and gene therapy of hypertrophic scarring.

Objective

To investigate the effects of TLP on collagen synthesis in human dermal fibroblasts.

Methods

Lentiviral vectors encoding TLP was constructed to transfect fibroblasts derived from normal human skin. The expression of Col I/III and phosphorylation of Smad2 and Smad3 in fibroblasts were examined after TLP treatment. In addition, the comparison of TLP expression in normal skin tissues and in hypertrophic scar tissues was performed, and the effect of TLP on cell viability was analyzed by MTT assay.

Results

TLP expression in hypertrophic scar tissue was markedly higher than in normal skin tissue. The Real Time PCR and Western blot test results both revealed that the synthesis of Col I/III was positively correlated with the expression of TLP. TLP also facilitate Smad2 phosphorylation while, conversely, inhibiting Smad3 phosphorylation. TLP may play a cooperative role, along with the cytokine TGF-β1, in improving the overall cell viability of skin fibroblasts.

Conclusions

TLP likely acts as a molecular modulator capable of altering the balance of Smad3- and Smad2-dependent signaling through regulation of phosphorylation, thus facilitating collagen synthesis in fibroblasts. Based on genetic variation in TLP levels in different tissues, these results suggest that TLP plays a key role in the process of TGF-β1/Smad3 signaling that contributes to wound healing and genesis of pathologic scars.     

Developmental Changes of TGF-β1 and Smads Signaling Pathway in Intestinal Adaption of Weaned Pigs

Weaning stress caused marked changes in intestinal structure and function. Transforming growth factor-β1 (TGF-β1) and canonical Smads signaling pathway are suspected to play an important regulatory role in post-weaning adaptation of the small intestine. In the present study, the intestinal morphology and permeability, developmental expressions of tight junction proteins and TGF-β1 in the intestine of piglets during the 2 weeks after weaning were assessed. The expressions of TGF-β receptor I/II (TβRI, TβRII), smad2/3, smad4 and smad7 were determined to investigate whether canonical smads signaling pathways were involved in early weaning adaption process. The results showed that a shorter villus and deeper crypt were observed on d 3 and d 7 postweaning and intestinal morphology recovered to preweaning values on d 14 postweaning. Early weaning increased (P<0.05) plasma level of diamine oxidase (DAO) and decreased DAO activities (P<0.05) in intestinal mucosa on d 3 and d 7 post-weaning. Compared with the pre-weaning stage (d 0), tight junction proteins level of occludin and claudin-1 were reduced (P<0.05) on d 3, 7 and 14 post-weaning, and ZO-1 protein was reduced (P<0.05) on d 3 and d 7 post-weaning. An increase (P<0.05) of TGF-β1 in intestinal mucosa was observed on d 3 and d 7 and then level down on d 14 post-weaning. Although there was an increase (P<0.05) of TβR II protein expression in the intestinal mucosa on d3 and d 7, no significant increase of mRNA of TβRI, TβRII, smad2/3, smad4 and smad7 was observed during postweaning. The results indicated that TGF-β1 was associated with the restoration of intestinal morphology and barrier function following weaning stress. The increased intestinal endogenous TGF-β1 didn''t activate the canonical Smads signaling pathway.     

Aberrant Activation of the WNT/β-Catenin Signaling Pathway in Lupus Nephritis

Objective

The canonical WNT pathway has been implicated as playing important roles in the pathogenesis of a variety of kidney diseases. Recently, WNT pathway activity was reported to be elevated in the renal tissue of a lupus mouse model. This study aimed to evaluate the potential role of the WNT pathway in the pathogenesis of human lupus nephritis.

Methods

The expression of β-catenin was evaluated in renal biopsy specimens from lupus nephritis patients and control kidney tissues by immunohistochemistry and western blotting. Real-time polymerase chain reaction (RT-PCR) was used to detect RNA expression of β-catenin, Dkk-1 and Axin2. Plasma concentrations of Dkk-1 were measured by ELISA.

Results

Immunohistochemistry and western blotting revealed increased expression of β-catenin in the kidneys of patients with lupus nephritis compared with control kidney tissues (p<0.05), accompanied by an increase in mRNA expression of β-catenin (p<0.01) and axin2 (p<0.05).β-catenin was significantly greater in LN patients without renal interstitial fibrosis compared with those with renal interstitial fibrosis (p<0.01) at the mRNA expression level; the increase in β-catenin mRNA positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury. Greater plasma Dkk-1 concentrations were found in LN patients compared with controls (p<0.05). Plasma Dkk-1 concentrations also correlated negatively with anti-dsDNA antibody levels and positively with serum C3 levels.

Conclusions

The canonical WNT/β-catenin signaling pathway was activated in lupus nephritis patients, accompanied by an increase in plasma levels of Dkk-1. Altered WNT/β-catenin signaling was related to the pathogenesis of lupus nephritis and might play a role in renal fibrosis.     

Targeted BRAF Inhibition Impacts Survival in Melanoma Patients with High Levels of Wnt/β-Catenin Signaling

Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of β-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.     

Ginsenoside Rd Attenuates Myocardial Ischemia/Reperfusion Injury via Akt/GSK-3β Signaling and Inhibition of the Mitochondria-Dependent Apoptotic Pathway

Evidence suggests Ginsenoside Rd (GSRd), a biologically active extract from the medical plant Panax Ginseng, exerts antioxidant effect, decreasing reactive oxygen species (ROS) formation. Current study determined the effect of GSRd on myocardial ischemia/reperfusion (MI/R) injury (a pathological condition where ROS production is significantly increased) and investigated the underlying mechanisms. The current study utilized an in vivo rat model of MI/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of simulated ischemia/reperfusion (SI/R) injury. Infarct size was measured by Evans blue/TTC double staining. NRC injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. ROS accumulation and apoptosis were assessed by flow cytometry. Mitochondrial membrane potential (MMP) was determined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetrathylbenzimidazol carbocyanine iodide (JC-1). Cytosolic translocation of mitochondrial cytochrome c and expression of caspase-9, caspase-3, Bcl-2 family proteins, and phosphorylated Akt and GSK-3β were determined by western blot. Pretreatment with GSRd (50 mg/kg) significantly augmented rat cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF) and ±dP/dt. GSRd reduced myocardial infarct size, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels after MI/R. In NRCs, GSRd (10 µM) inhibited SI/R-induced ROS generation (P<0.01), decreased cellular apoptosis, stabilized the mitochondrial membrane potential (MMP), and attenuated cytosolic translocation of mitochondrial cytochrome c. GSRd inhibited activation of caspase-9 and caspase-3, increased the phosphorylated Akt and GSK-3β, and increased the Bcl-2/Bax ratio. Together, these data demonstrate GSRd mediated cardioprotective effect against MI/R–induced apoptosis via a mitochondrial-dependent apoptotic pathway.     

Essential Role of TGF-β/Smad Pathway on Statin Dependent Vascular Smooth Muscle Cell Regulation

Background

The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-β (TGF-β) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-β/Smad pathway in atherosclerosis and vascular cells.

Methodology

In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-β. In addition, statins upregulated TGF-β receptor type II (TRII), and increased TGF-β synthesis and TGF-β/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-β induced apoptosis and increased TGF-β-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-β/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected.

Conclusions

Statins enhance TGF-β/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-β/Smad pathway is essential for statins-dependent actions in VSMCs.     

Pentoxifylline Alleviates Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats: Possibly via Inhibiting TLR 4/NF-κB Signaling Pathway

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) generally causes significant and lasting damage. Pentoxifylline (PTX), a nonselective phosphodiesterase inhibitor, has shown anti-inflammatory and neuroprotective properties in several brain injury models, but the role of PTX with respect to EBI following SAH remains uncertain. The purpose of this study was to investigate the effects of PTX on EBI after SAH in rats. Adult male Sprauge–Dawley rats were randomly assigned to the sham and SAH groups. PTX (30 or 60 mg/kg) or an equal volume of the administration vehicle (normal saline) was administrated at 30 min intervals following SAH. Neurological scores, brain edema, and neural cell apoptosis were evaluated. In order to explore other mechanisms, changes in the toll-like receptor 4 (TLR4) and the nuclear factor-κB (NF-κB) signaling pathway, in terms of the levels of apoptosis-associated proteins, were also investigated. We found that administration of PTX (60 mg/kg) notably improved neurological function and decreased brain edema at both 24 and 72 h following SAH. Treatment with PTX (60 mg/kg) significantly inhibited the protein expressions of TLR4, NF-κB, MyD88 and the downstream pro-inflammatory cytokines, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). PTX also significantly reduced neural cell death and BBB permeability. Our observations may be the first time that PTX has been shown to play a neuroprotective role in EBI after SAH, potentially by suppressing the TLR4/NF-κB inflammation-related pathway in the rat brain.     

In Vivo Disruption of TGF-β Signaling by Smad7 in Airway Epithelium Alleviates Allergic Asthma but Aggravates Lung Carcinogenesis in Mouse

Background

TGF-β has been postulated to play an important role in the maintenance of epithelial homeostasis and the development of epithelium-derived cancers. However, most of previous studies are mainly focused on the function of TGF-β in immune cells to the development of allergic asthma and how TGF-β signaling in airway epithelium itself in allergic inflammation is largely unknown. Furthermore, the in vivo TGF-β function specifically in the airway epithelium during lung cancer development has been largely elusive.

Methodology/Principal Findings

To evaluate the in vivo contribution of TGF-β signaling in lung epithelium to the development of allergic disease and lung cancer, we generated a transgenic mouse model with Smad7, an intracellular inhibitor of TGF-β signaling, constitutively expressed in mouse airway Clara cells using a mouse CC10 promoter. The mice were subjected to the development of OVA-induced allergic asthma and urethane-induced lung cancer. The Smad7 transgenic animals significantly protected from OVA-induced asthma, with reduced airway inflammation, airway mucus production, extracellular matrix deposition, and production of OVA-specific IgE. Further analysis of cytokine profiles in lung homogenates revealed that the Th2 cytokines including IL-4, IL-5 and IL-13, as well as other cytokines including IL-17, IL-1, IL-6, IP10, G-CSF, and GM-CSF were significantly reduced in the transgenic mice upon OVA induction. In contrast, the Smad7 transgenic animals had an increased incidence of lung carcinogenesis when subjected to urethane treatment.

Conclusion/Significance

These studies, therefore, demonstrate for the first time the in vivo function of TGF-β signaling specifically in airway epithelium during the development of allergic asthma and lung cancer.     

Estrogen Exerts Neuroprotective Effects in Vascular Dementia Rats by Suppressing Autophagy and Activating the Wnt/β-Catenin Signaling Pathway

Vascular dementia (VD) is a clinical syndrome of acquired cognitive dysfunction caused by various cerebrovascular factors. Estrogen is a steroid hormone involved in promoting neuronal survival and in regulating many signaling pathways. However, the mechanism by which it confers neuroprotective effects in VD remains unclear. Here, we aimed to investigate the effect of estrogen on neuronal injury and cognitive impairment in VD rats. Adult female rats were randomly divided into four groups (sham, model, estrogen early and estrogen later treatment) and received sham surgery or bilateral ovariectomy and permanent occlusion of bilateral common carotid arteries (BCCAO). The early treatment group received daily intraperitoneal injections of 17β-estradiol (100 µg/kg/day) for 8 weeks starting the day after BCCAO. The later treatment group was administered the same starting 1 week after BCCAO. Learning and memory functions were assessed using the Morris water maze. Morphological changes within the hippocampal CA1 region were observed by hematoxylin/eosin staining and electron microscopy. Expression of proteins associated with autophagy and signaling were detected by immunohistochemical staining and Western blot. We found that estrogen significantly alleviated cognitive damage and neuronal injury and reduced the expression of Beclin1 and LC3B, indicating a suppression of autophagy. Moreover, estrogen enhanced expression of β-catenin and Cyclin D1, while reducing glycogen synthase kinase 3β, suggesting activation of Wnt/β-catenin signaling. These results indicate that estrogen ameliorates learning and memory deficiencies in VD rats, and that this neuroprotective effect may be explained by the suppression of autophagy and activation of Wnt/β-catenin signaling.

     

Triptolide Improves Cognitive Dysfunction in Rats with Vascular Dementia by Activating the SIRT1/PGC-1α Signaling Pathway

Neurochemical Research - Tripterygium Wilfordii Hook F has been exploited as a treatment for several diseases due to its neuroprotective, anti-tumor, and anti-inflammatory effects. Triptolide is...     

SNPs in the TGF-β Signaling Pathway Are Associated with Increased Risk of Brain Metastasis in Patients with Non–Small-Cell Lung Cancer

Purpose

Brain metastasis (BM) from non-small cell lung cancer (NSCLC) is relatively common, but identifying which patients will develop brain metastasis has been problematic. We hypothesized that genotype variants in the TGF-β signaling pathway could be a predictive biomarker of brain metastasis.

Patients and Methods

We genotyped 33 SNPs from 13 genes in the TGF-β signaling pathway and evaluated their associations with brain metastasis risk by using DNA from blood samples from 161 patients with NSCLC. Kaplan-Meier analysis was used to assess brain metastasis risk; Cox hazard analyses were used to evaluate the effects of various patient and disease characteristics on the risk of brain metastasis.

Results

The median age of the 116 men and 45 women in the study was 58 years; 62 (39%) had stage IIIB or IV disease. Within 24 months after initial diagnosis of lung cancer, brain metastasis was found in 60 patients (37%). Of these 60 patients, 16 had presented with BM at diagnosis. Multivariate analysis showed the GG genotype of SMAD6: rs12913975 and TT genotype of INHBC: rs4760259 to be associated with a significantly higher risk of brain metastasis at 24 months follow-up (hazard ratio [HR] 2.540, 95% confidence interval [CI] 1.204–5.359, P = 0.014; and HR 1.885, 95% CI 1.086–3.273, P = 0.024), compared with the GA or CT/CC genotypes, respectively. When we analyzed combined subgroups, these rates showed higher for those having both the GG genotype of SMAD6: rs12913975 and the TT genotype of INHBC: rs4760259 (HR 2.353, 95% CI 1.390–3.985, P = 0.001).

Conclusions

We found the GG genotype of SMAD6: rs12913975 and TT genotype of INHBC: rs4760259 to be associated with risk of brain metastasis in patients with NSCLC. This finding, if confirmed, can help to identify patients at high risk of brain metastasis.     

Involvement of the Wnt/β-Catenin Signaling Pathway in the Cellular and Molecular Mechanisms of Fibrosis in Endometriosis

Background

During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified.

Objectives

The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice.

Methods

Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice.

Results

Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis.

Conclusion

Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis.