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Background and Purpose: Renal interstitial fibrosis is a frequent pathological manifestation of lupus nephritis (LN). tRNA halves (tiRNAs) are acquired from tRNA‐derived small non‐coding RNAs (sncRNAs) and are associated with fibrosis. Our previous study indicated enhanced tiRNA‐Gly‐GCC‐002 (tiRNA002) levels in kidneys were positively related to LN‐related fibrosis. However, the precise molecular mechanism remains unclear. Experimental Approach: The mimic and agomiR of tiRNA002 were introduced into tubular epithelial cells (TECs) and MRL/lpr mice by transfection. The levels of gene and protein expressions were quantified using real‐time quantitative polymerase chain reaction (RT‐qPCR), Western blot and immunofluorescence assays. Key Results: In TECs treated with LN serum, as well as in the kidneys of MRL/lpr mice, high levels of tiRNA002 directly influenced the epithelial‐mesenchymal transition (EMT) and extracellular matrix (ECM) deposition. Furthermore, tiRNA002 overexpression promoted EMT in TECs and accelerated renal interstitial fibrosis in MRL/lpr mice via Smad signalling. The target gene of tiRNA002, FKBP prolyl isomerase 5 (FKBP5), improved Smad signalling by interacting with phosphorylated Smad2/3. Silencing FKBP5 alleviated LN serum‐ or tiRNA002‐mimic‐induced EMT in TECs. In addition, FKBP5 overexpression reversed the tiRNA002 knockdown‐mediated reduction of EMT and ECM accumulation. Conclusions and Implications: These findings indicated that tiRNA002 is markedly increased in LN, which facilitates renal fibrosis by promoting EMT via FKBP5‐mediated Smad signalling. Therefore, targeting tiRNA002 may be an innovative approach to treat renal interstitial fibrosis in LN. [ABSTRACT FROM AUTHOR]

Dye degradation presents a persistent challenge in addressing water pollution. While several methods, including adsorption, biodegradation, and advanced oxidation processes, have been extensively explored, photocatalysis remains one of the most effective techniques. Conventional photocatalytic dye degradation processes often rely on expensive light sources and are time‐intensive. Herein, we synthesized a SnS catalyst by the solvated metal atom dispersion (SMAD) method, using Sn foil and sulfur powder. The catalyst exhibited remarkable performance, achieving complete degradation of methylene blue within 2 minutes under ambient room light, without the need for any external light source. Similar degradation efficiency was achieved for methyl orange. To evaluate the role of light for the degradation, control experiments were conducted in the dark using methylene blue as a model dye. Although the degradation rate was slightly reduced, the catalyst still facilitated dye degradation in the absence of light. Additionally, the catalytic performance was tested with four other dyes under natural light, all of which yielded promising results, demonstrating the versatility and effectiveness of the SnS catalyst in dye degradation. This work highlights the potential of the SnS catalyst for efficient and rapid dye degradation under both light and dark conditions, offering an energy‐efficient solution for wastewater treatment. [ABSTRACT FROM AUTHOR]

Aim: Rutin is a flavonoid glycoside obtained from the plant Ruta graveolens. It was known to have immunosuppressant activities. This study was focused on effect of rutin against immunoglobulin A (IgA) nephropathy. Methods: IgA nephropathy was induced in Sprague–Dawley rats with various inducing agents described in text. During the later part of the induction phase, rutin was administered. Control group rats did not receive any treatment or inducing agent, induced group rats received only the inducing agents, whereas treatment group received the inducing agents as well as rutin. Results: During the study, various biochemical parameters pertaining to kidney function were evaluated and also, the expression of proteins and cytokines responsible for inflammation and fibrosis were assessed. The effect of rutin in IgA nephropathy was promising as treatment with rutin reduced the deposition of IgA in the glomeruli of rats. Along with this we also tried to establish the probable mechanism of action of rutin and based on the summary of the results it was concluded that rutin reduced the inflammation and fibrosis related to IgA nephropathy by inhibiting the TGF‐β/SMAD pathways and ultimately reducing the expression of α‐smooth muscle actin (α‐SMA). Conclusion: Comprehending all the above consideration, it may be safely said that that rutin alleviated inflammation and also fibrosis mediated by IgA, by suppressing the transforming growth factor‐β (TGF‐β) activities through suppressor of mothers against decapentaplegic pathways and reduced the epithelial‐to‐mesenchymal transition by downregulating the α‐SMA which is a marker for fibrosis. [ABSTRACT FROM AUTHOR]

Transforming growth factor‐β (TGF‐β) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor‐activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF‐β regulates the transcription of target genes in a cell context‐dependent manner by screening a double‐stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti‐Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF‐β. The preference of the activated Smad complexes for conventional Smad‐binding motifs such as Smad‐binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor‐binding motifs other than SBE or CAGA, including the Fos/Jun‐binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor‐binding motifs together with Smad‐binding motifs indicated that some of the motifs may be involved in cell type‐dependent transcriptional activation by TGF‐β. The results suggest that the CASTing method is useful for elucidating the molecular basis of context‐dependent Smad signaling. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition characterized by the abnormal regulation of extracellular matrix (ECM) and epithelial‐mesenchymal transition (EMT). In this study, we investigated the potential of rutin, a natural flavonoid, in attenuating transforming growth factor‐β (TGF‐β)‐induced ECM regulation and EMT through the inhibition of the TGF‐β type I receptor (TβRI)‐mediated suppressor of mothers against decapentaplegic (SMAD) signaling pathway. We found that non‐toxic concentrations of rutin attenuated TGF‐β‐induced ECM‐related genes, including fibronectin, elastin, collagen 1 type 1, and TGF‐β, as well as myoblast differentiation from MRC‐5 lung fibroblast cells accompanied by the downregulation of α‐smooth muscle actin. Rutin also inhibited TGF‐β‐induced EMT processes, such as wound healing, migration, and invasion by regulating EMT‐related gene expression. Additionally, rutin attenuated bleomycin‐induced lung fibrosis in mice, thus providing a potential therapeutic option for IPF. The molecular docking analyses in this study predict that rutin occludes the active site of TβRI and inhibits SMAD‐mediated fibrotic signaling pathways in lung fibrosis. These findings highlight the potential of rutin as a promising anti‐fibrotic prodrug for lung fibrosis and other TGF‐β‐induced fibrotic and cancer‐related diseases; however, further studies are required to validate its safety and effectiveness in other experimental models. [ABSTRACT FROM AUTHOR]

At present, there is limited research on the mechanisms underlying moyamoya disease (MMD). Herein, we aimed to determine the role of glutamine in MMD pathogenesis, and 360 adult patients were prospectively enrolled. Human brain microvascular endothelial cells (HBMECs) were subjected to Integrin Subunit Beta 4 (ITGB4) overexpression or knockdown and atorvastatin. We assessed factors associated with various signaling pathways in the context of the endothelial‐to‐mesenchymal transition (EndMT), and the expression level of related proteins was validated in the superficial temporal arteries of patients. We found glutamine levels were positively associated with a greater risk of stroke (OR = 1.599, p = 0.022). After treatment with glutamine, HBMECs exhibited enhanced proliferation, migration, and EndMT, all reversed by ITGB4 knockdown. In ITGB4‐transfected HBMECs, the MAPK–ERK–TGF–β/BMP pathway was activated, with Smad4 knockdown reversing the EndMT. Furthermore, atorvastatin suppressed the EndMT by inhibiting Smad1/5 phosphorylation and promoting Smad4 ubiquitination in ITGB4‐transfected HBMECs. We also found the protein level of ITGB4 was upregulated in the superficial temporal arteries of patients with MMD. In conclusion, our study suggests that glutamine may be an independent risk factor for hemorrhage or infarction in patients with MMD and targeting ITGB4 could potentially be therapeutic approaches for MMD. [ABSTRACT FROM AUTHOR]

Physiological embryogenesis and adult tissue homeostasis are regulated by transforming growth factor‐β (TGF‐β), an evolutionarily conserved family of secreted polypeptide factors, acting in an autocrine and paracrine manner. The role of TGF‐β in inflammation, fibrosis, and cancer is complex and sometimes even contradictory, exhibiting either inhibitory or promoting effects depending on the stage of the disease. Under pathological conditions, especially fibrosis and cancer, overexpressed TGF‐β causes extracellular matrix deposition, epithelial–mesenchymal transition, cancer‐associated fibroblast formation, and/or angiogenesis. In this review article, we have tried to dive deep into the mechanism of action of TGF‐β in inflammation, fibrosis, and carcinogenesis. As TGF‐β and its downstream signaling mechanism are implicated in fibrosis and carcinogenesis blocking this signaling mechanism appears to be a promising avenue. However, targeting TGF‐β carries substantial risk as this pathway is implicated in multiple homeostatic processes and is also known to have tumor‐suppressor functions. There is a need for careful dosing of TGF‐β drugs for therapeutic use and patient selection. [ABSTRACT FROM AUTHOR]

Expression of α‐smooth muscle actin (αSMA) is constitutive in vascular smooth muscle cells, but is induced in nonmuscle cells such as hepatic stellate cells (HSCs). HSCs play important roles in both physiological homeostasis and pathological response. HSC activation is characterized by αSMA expression, which is regulated by the TGFβ‐induced Smad pathway. Recently, protein kinase C (PKC) was identified to regulate αSMA expression. Diacylglycerol kinase (DGK) metabolizes a second‐messenger DG, thereby controlling components of DG‐mediated signaling, such as PKC. In the present study we aimed to investigate the putative role of DGKα in αSMA expression. Use of a cellular model indicated that the DGK inhibitor R59949 promotes αSMA expression and PKCδ phosphorylation. It also facilitates Smad2 phosphorylation after 30 min of TGFβ stimulation. Furthermore, immunocytochemical analysis revealed that DGK inhibitor pretreatment without TGFβ stimulation engenders αSMA expression in a granular pattern, whereas DGK inhibitor pretreatment plus TGFβ stimulation significantly induces αSMA incorporation in stress fibers. Through animal model experiments, we observed that DGKα‐knockout mice exhibit increased expression of αSMA in the liver after 48 h of carbon tetrachloride injection, together with enhanced phosphorylation levels of Smad2 and PKCδ. Together, these findings suggest that DGKα negatively regulates αSMA expression by acting on the Smad and PKCδ signaling pathways, which differentially regulate stress fiber incorporation and protein expression of αSMA, respectively. [ABSTRACT FROM AUTHOR]

Objectives: Effective treatments for vocal fold fibrosis remain elusive. Tamoxifen (TAM) is a selective estrogen receptor modulator and was recently reported to have antifibrotic actions. We hypothesized that TAM inhibits vocal fold fibrosis via altered transforming growth factor beta 1 (TGF‐β1) signaling. Both in vitro and in vivo approaches were employed to address this hypothesis. Methods: In vitro, vocal fold fibroblasts were treated with TAM (10−8 or 10−9 M) ± TGF‐β1 (10 ng/ml) to quantify cell proliferation. The effects of TAM on genes related to fibrosis were quantified via quantitative real‐time polymerase chain reaction. In vivo, rat vocal folds were unilaterally injured, and TAM was administered by oral gavage from pre‐injury day 5 to post‐injury day 7. The rats were randomized into two groups: 0 mg/kg/day (sham) and 50 mg/kg/day (TAM). Histological changes were examined on day 56 to assess tissue architecture. Results: TAM (10−8 M) did not affect Smad3, Smad7, Acta2, or genes related to extracellular matrix metabolism. TAM (10−8 or 10−9 M) + TGF‐β1, however, significantly increased Smad7 and Has3 expression and decreased Col1a1 and Acta2 expression compared to TGF‐β1 alone. In vivo, TAM significantly increased lamina propria area, hyaluronic acid concentration, and reduced collagen deposition compared to sham treatment. Conclusions: TAM has antifibrotic potential via the regulation of TGF‐β1/Smad signaling in vocal fold injury. These findings provide foundational data to develop innovative therapeutic options for vocal fold fibrosis. Level of Evidence: NA Laryngoscope, 133:2248–2254, 2023 [ABSTRACT FROM AUTHOR]

Autocrine stimulation of tumor cells is an important mechanism for the growth of skeletal tumors. In tumors that are sensitive, growth factor inhibitors can dramatically reduce tumor growth. In this study, our aim was to investigate the effects of Secreted phosphoprotein 24 kD (Spp24) on the growth of osteosarcoma (OS) cells in the presence and absence of exogenous BMP‐2 both in vitro and in vivo. Our study demonstrated that Spp24 inhibited proliferation and promoted apoptosis of OS cells as confirmed by 3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide assay and immunohistochemical staining. We found that BMP‐2 increased the mobility and invasiveness of tumor cells in vitro whereas Spp24 inhibited both of these processes alone and in the presence of exogenous BMP‐2. Phosphorylation of Smad1/5/8 and Smad8 gene expression was enhanced by treatment with BMP‐2 but inhibited by treatment with Spp24. Subcutaneous and intratibial tumor models in nude mice demonstrated that BMP‐2 promoted OS growth in vivo, while Spp24 significantly inhibited tumor growth. We conclude that the BMP‐2/Smad signaling pathway is involved in the pathogenesis of OS growth and that Spp24 inhibits the growth of human OS induced by BMP‐2 both in vitro and in vivo. Interruption of Smad signaling and increased apoptosis appear to be the primary mechanisms involved. These results confirm the potential of Spp24 as a therapeutic agent for the treatment of OS and other skeletal tumors. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis (PF) is a progressive and fatal interstitial lung disease with limited therapeutic options at present, and epithelial–mesenchymal transition (EMT) is recognized as a major cause of lung fibrosis. Our previous work has confirmed that total extract of Anemarrhena asphodeloides Bunge [Asparagaceae] exerted the effect of anti‐PF. As a main constituent of Anemarrhena asphodeloides Bunge [Asparagaceae], the effect of timosaponin BII (TS BII) on drug‐induced EMT process in PF animals and alveolar epithelial cells remains unknown. In this study, we evaluated the effect of TS BII on bleomycin (BLM)‐induced PF. The results showed that TS BII could restore the structure of lung architecture and MMP‐9/TIMP‐1 balance in fibrotic rat lung and inhibit collagen deposition. Moreover, we found that TS BII could reverse the abnormal expression of TGF‐β1 and EMT‐related marker proteins including E‐cadherin, vimentin, and α‐SMA. Besides, aberrant TGF‐β1 expression and phosphorylation of Smad2 and Smad3 in BLM‐induced animal model and TGF‐β1‐induced cell model were downregulated by TS BII treatment, indicating that EMT in fibrosis was suppressed by inhibition of TGF‐β/Smad pathway both in vivo and in vitro. In summary, our study suggested that TS BII could be a promising candidate for PF treatment. [ABSTRACT FROM AUTHOR]

Background: Endometrial dysfunction is closely correlated with the development of multiple severe gynecological disorders including intrauterine adhesion. Accumulating evidence supports that some long non‐coding RNAs (lncRNAs) have peptide‐coding potential. In this text, the peptide‐coding ability of lncRNA SNHG6 was examined. Also, the effects of an SNHG6‐encoded peptide on the viability and migration of human endometrial stromal cells (hESCs) and human endometrial epithelial cells (hEECs) and related molecular mechanisms were explored. Methods: The peptide‐encoding potential of SNHG6 was predicted by FuncPEP and getorf databases and validated by western blot assay. Cell viability was tested by cell counting kit‐8 assay. Cell migratory ability was examined by wound healing and transwell migration assays. Protein levels of genes were measured by western blot assay. Results: Prediction analysis suggested that SNHG6 had the potential peptide‐coding ability and multiple open‐reading frames (ORFs). Western blot validated that SNHG6 ORF#1 and ORF#2 could translate into short peptides. SNHG6 ORF#2 overexpression facilitated cell migration and epithelial–mesenchymal transition (EMT) in hESCs and hEECs, while these effects were abrogated by transforming growth factor‐beta (TGF‐β)/SMAD signaling inhibitor GW788388. Moreover, GW788388 inhibited the increase of p‐SMAD2 and p‐SMAD3 levels induced by SNHG6 ORF#2 in hESCs. SNHG6 ORF#2‐encoded peptide did not influence endometrial stromal and epithelial cell viability. Conclusions: LncRNA SNHG6 ORF#1 and ORF#2 could translate into small peptides and SNHG6 ORF#2 overexpression promoted cell migration and EMT by activating the TGF‐β/SMAD pathway in hESCs and hEECs, suggesting the potential roles of SNHG6‐encoded peptides in the development of endometrial stromal and epithelial cells and related gynecological diseases. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) is one of the most prevalent malignancies of the digestive tract. Ginsenoside Rh1 was reported to exert effects on GC. The current study set out to explore the mechanism underlying Ginsenoside Rh1 effects on GC. With oxaliplatin (OXA) serving as the positive control, human GC cells AGS were treated with 0, 10, 25, 50, 74, or 100 μM of ginsenoside Rh1 for 48 h. Proliferation, migration, invasion, and apoptosis were subsequently assessed by means of MTT, scratch test, Transwell, and TUNEL, respectively. AGS cells were further jointly treated with Rh1 and the TGF‐β/Smad pathway activator Kartogenin, followed by detection of TGF‐β/Smad pathway effects on AGS biological behaviours. Moreover, TGF‐β/Smad pathway activation was detected with a Western blot assay. Furthermore, xenograft tumour models were established and tumour growth was recorded. Ki‐67 expression patterns and apoptosis were detected with immunohistochemistry and TUNEL, respectively. In vitro, Ginsenoside Rh1 repressed AGS cell proliferation, migration, and invasion, and further promoted apoptosis, with a concentration of 50 μM Rh1 exerting the equivalent effects as OXA. In vivo, Ginsenoside Rh1 inhibited GC proliferation and induced tumour cell apoptosis. Mechanistically, Ginsenoside Rh1 reduced TGF‐β1 and TGF‐β2 levels and Smad2 and Smad3 phosphorylation levels. Collectively, our findings highlighted that ginsenoside Rh1 inhibited GC cell growth and tumour growth in xenograft tumour models via inhibition of the TGF‐β/Smad pathway. [ABSTRACT FROM AUTHOR]

Background: Human multipotent progenitor cells (hiMPCs) created from induced pluripotent stem cells (iPSCs) represent a new cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) are needed to enhance transforming growth factor‐β (TGFβ)‐induced hiMPC chondrogenesis. In contrast, TGFβ alone is sufficient to result in robust chondrogenesis of human primary mesenchymal stromal cells (hMSCs). Currently, the mechanism underlying this difference between hiMPCs and hMSCs has not been fully understood. Methods: In this study, we first tested different growth factors alone or in combination in stimulating hiMPC chondrogenesis, with a special focus on chondrocytic hypertrophy. The reparative capacity of hiMPCs‐derived cartilage was assessed in an osteochondral defect model created in rats. hMSCs isolated from bone marrow were included in all studies as the control. Lastly, a mechanistic study was conducted to understand why hiMPCs and hMSCs behave differently in responding to TGFβ. Results: Chondrogenic medium supplemented with TGFβ3 and BMP6 led to robust in vitro cartilage formation from hiMPCs with minimal hypertrophy. Cartilage tissue generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and resulted in a hyaline cartilage‐like regeneration in osteochondral defects in rats. Interestingly, TGFβ3 induced phosphorylation of both Smad2/3 and Smad1/5 in hMSCs, but only activated Smad2/3 in hiMPCs. Supplementing BMP6 activated Smad1/5 and significantly enhanced TGFβ's compacity in inducing hiMPC chondrogenesis. The chondro‐promoting function of BMP6 was abolished by the treatment of a BMP pathway inhibitor. Conclusions: This study describes a robust method to generate chondrocytes from hiMPCs with low hypertrophy for hyaline cartilage repair, as well as elucidates the difference between hMSCs and hiMPCs in response to TGFβ. Our results also indicated the importance of activating both Smad2/3 and Smad1/5 in the initiation of chondrogenesis. [ABSTRACT FROM AUTHOR]

Aging is a complex and highly regulated process of interwoven signaling mechanisms. As an ancient transcriptional regulator of thermal adaptation and protein homeostasis, the Heat Shock Factor, HSF‐1, has evolved functions within the nervous system to control age progression; however, the molecular details and signaling dynamics by which HSF‐1 modulates age across tissues remain unclear. Herein, we report a nonautonomous mode of age regulation by HSF‐1 in the Caenorhabditis elegans nervous system that works through the bone morphogenic protein, BMP, signaling pathway to modulate membrane trafficking in peripheral tissues. In particular, HSF‐1 represses the expression of the neuron‐specific BMP ligand, DBL‐1, and initiates a complementary negative feedback loop within the intestine. By reducing receipt of DBL‐1 in the periphery, the SMAD transcriptional coactivator, SMA‐3, represses the expression of critical membrane trafficking regulators including Rab GTPases involved in early (RAB‐5), late (RAB‐7), and recycling (RAB‐11.1) endosomal dynamics and the BMP receptor binding protein, SMA‐10. This reduces cell surface residency and steady‐state levels of the type I BMP receptor, SMA‐6, in the intestine and further dampens signal transmission to the periphery. Thus, the ability of HSF‐1 to coordinate BMP signaling along the gut–brain axis is an important determinate in age progression. [ABSTRACT FROM AUTHOR]

Background: Asthma is a heterogeneous chronic inflammatory disease of the bronchi, the course of which is significantly influenced by extrinsic factors (specific and non‐specific). Methods: The aim of this study was to evaluate the effect of these factors represented by nasal allergen challenge (specific factors) and methacholine challenge test (non‐specific) on changes in mRNA expression of genes encoding the TGF‐β (TGF‐β1 and TGF‐β3)‒Smad (mitogen‐activated protein kinase 1/3 [MPK1/3], Smad1/3/6/7) signaling pathway in asthmatic patients. Results: Seventy‐five subjects were included in the study, of whom 27 were applied an intranasal allergen provocation and 48 a methacholine provocation. There were 9 men and 18 women in the intranasal provocation group, and 17 men and 31 women in the methacholine test group. We found that both examined the types of challenges contributed to changes in the relative expression of genes of the TGF‐β (TGF‐β1 and TGF‐β3)‒Smad (MPK1/3, Smad1/3/6/7) signaling pathway in asthmatic patients. A decrease was noted for MAPK1, MAPK3, Smad3, Smad6, and Smad7 genes and an increase of up to 2.5 times for TGF‐β1 gene. Conclusions: Our experiment allows us to conclude that the change in the mRNA expression of the TGF‐β1–MPK1/3 and Smad3/6/7 genes occurs after an intranasal allergen and bronchial methacholine challenge. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) are essential regulators of skeletal homeostasis, and BMP9 is the most potently osteogenic among them. Here, we found that BMP9 and BMP2 rapidly induced early growth response 1 (EGR1) protein expression in osteoblasts through MEK/ERK pathway-dependent transcriptional activation. Knock-down of EGR1 using siRNA significantly inhibited BMP9-induced matrix mineralization and osteogenic marker gene expression in osteoblasts. Knock-down of EGR1 significantly reduced SMAD1/5 phosphorylation and inhibited the expression of their transcriptional targets in osteoblasts stimulated by BMP9. In contrast, forced EGR1 overexpression in osteoblasts enhanced BMP9-mediated osteoblast differentiation and SMAD1/5 phosphorylation. An intracellular association between EGR1 and SMAD1/5 was identified using immunoprecipitation assays. These results indicated that EGR1 plays an important role in BMP9-stimulated osteoblast differentiation by enhancing SMAD1/5 phosphorylation. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)‐β signalling commences with the engagement of TGF‐β ligand to cell surface TGF‐β receptors (TGFBR) stimulating Smad2 carboxyl‐terminal phosphorylation (phospho‐Smad2C) and downstream biological responses. In several cell models, G protein‐coupled receptors (GPCRs) transactivate the TGF‐β receptors type‐1 (TGFBR1) leading to phospho‐Smad2C, however, we have recently published that in keratinocytes thrombin did not transactivate the TGFBR1. The bulk of TGFBRs reside in the cytosol and in response to protein kinase B (Akt phosphorylation) can translocate to the cell surface increasing the cell's responsiveness to TGF‐β. In this study, we investigate the role of Akt in GPCR transactivation of the TGFBR1. We demonstrate that angiotensin II and thrombin do not phosphorylate Smad2C in human vascular smooth muscle cells and in keratinocytes respectively. We used Akt agonist, SC79 to sensitise the cells to Akt and observed that Ang II and thrombin phosphorylate Smad2C via Akt/AS160‐dependent pathways. We show that SC79 rapidly translocates TGFBRs to the cell surface thus increasing the cell's response to the GPCR agonist. These findings highlight novel mechanistic insight for the role of Akt in GPCR transactivation of the TGFBR1. [ABSTRACT FROM AUTHOR]

Atherosclerosis, the major underlying pathology of cardiovascular disease, commences with the binding and trapping of lipids on modified proteoglycans, with hyperelongated glycosaminoglycan chains. Transforming growth factor (TGF)‐β stimulates glycosaminoglycan elongation in vascular smooth muscle cells. We have recently shown that this TGF‐β signaling pathway involves reactive oxygen species (ROS). YY‐11 is a dodecapeptide derived from camel milk and it has antioxidant activity. We have investigated the role of YY‐11 in blocking ROS signaling and downstream atherogenic responses. YY‐11 inhibited TGF‐β stimulated ROS production and inhibited the expression of genes for glycosaminoglycan chain elongation as a component of an in vitro model of atherosclerosis. This study provides a biochemical mechanism for the role of camel milk as a potential nutritional product to contribute to the worldwide amelioration of cardiovascular disease. Practical applications: The identification of readily accessible foods with antioxidant properties would provide a convenient and cost‐effective approach community wide reducing oxidative stress induced pathologies such as atherosclerosis. We demonstrate that camel milk‐derived peptide is an antioxidant that can inhibit growth factor‐mediated proteoglycan modification in vitro. As proteoglycan modification is being recognized as one of the earliest atherogenic responses, these data support the notion of camel milk as a suitable nutritional product to contribute to the prevention of early stage of atherosclerosis development. [ABSTRACT FROM AUTHOR]

Inflammation and fibrosis are two pathological features of chronic kidney disease (CKD). Renal fibrosis is considered to be one of the most important conditions, as it may be the result of excessive extracellular matrix protein production and deposition, or prolonged exposure to nephrotoxic substances or drugs. Unfortunately, no suitable therapies or medications are currently available to prevent renal fibrosis. We conducted this study for the evaluation of the protective potential of vanillin by reversing TAA (250 mg/kg TAA for 6 weeks) induced renal injury in rats. The concentrations of the proteins tumour necrosis factor alpha (TNFα), interleukin‐6 (IL‐6), extracellular signal regulated kinase 1/2 (Erk1/2), and transforming growth factor beta‐1 (TGF‐β1) in kidney tissues were assessed using ELISA. Kidney Injury Molecule‐1 (KIM‐1) and mothers against decapentaplegic homologue 2, 3 (SMAD 2, 3) expressions were evaluated using real time PCR. We also estimated the expression of α‐smooth muscle actin (α‐SMA) using immunohistochemistry. Treatment with vanillin (100 mg/kg) significantly ameliorated kidney Injury and improved the kidney function. Vanillin treatment also significantly decreased the malondialdehyde (MDA) content, and elevated glutathione peroxidase (GPx) and catalase (CAT) activities in kidney tissues. Vanillin also reduced α‐SMA renal expression and TNFα, IL‐6, TGF‐β1, and Erk1/2 renal levels. Vanillin significantly decreased the expression of the genes encoding KIM‐1 and SMAD 2, 3 and ameliorated histological abnormalities in kidney architecture. Our molecular docking findings showed that vanillin has a good binding mode inside TGF‐β type I receptors (ALK5) biding site. [ABSTRACT FROM AUTHOR]

This commentary focuses on the emerging intersection between BMP/TGF‐β signaling roles in nervous system function and the amyotrophic lateral sclerosis (ALS) disease state. Future research is critical to elucidate the molecular underpinnings of this intersection of the cellular processes disrupted in ALS and those influenced by BMP/TGF‐β signaling, including synapse structure, neurotransmission, plasticity, and neuroinflammation. Such knowledge promises to inform us of ideal entry points for the targeted modulation of dysfunctional cellular processes in an effort to abrogate ALS pathologies. It is likely that different interventions are required, either at discrete points in disease progression, or across multiple dysfunctional processes which together lead to motor neuron degeneration and death. We discuss the challenging, but intriguing idea that modulation of the pleiotropic nature of BMP/TGF‐β signaling could be advantageous, as a way to simultaneously treat defects in more than one cell process across different forms of ALS. Key Findings: Gene mutations and environmental triggers of ALS primarily perturb protein homeostasis, RNA function, and cytoskeletal dynamics.Emerging data reveal specific molecular links between BMP/TGF‐β signaling and dysfunctional cellular processes in ALS.The pleiotropic nature of BMP/TGF‐β signaling may prove advantageous as a pathway whose modulation will impact multiple processes simultaneously versus the need to develop multiple interventions to slow or prevent motor decline in ALS. [ABSTRACT FROM AUTHOR]

High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease‐related mutations of SLC39A5 dysregulate the TGF‐β pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA‐seq and qRT‐PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGF‐β signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R‐Smads) via phosphorylation was greatly blocked. SLC39A5 re‐expression reversed the phenotype of TGF‐β signalling and ECM synthesis in the KO cells. The fact that TGF‐β signalling was zinc‐regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGF‐β signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGF‐β signalling. Overall, the SLC39A5 depletion–induced zinc deficiency destabilized Smad proteins, which inhibited the TGF‐β signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development. [ABSTRACT FROM AUTHOR]

The 2021 Annual Review Issue of The Journal of Pathology contains 14 invited reviews on current research areas of particular importance in pathology. The subjects included here reflect the broad range of interests covered by the journal, including both basic and applied research fields but always with the aim of improving our understanding of human disease. This year, our reviews encompass the huge impact of the COVID‐19 pandemic, the development and application of biomarkers for immune checkpoint inhibitors, recent advances in multiplexing antigen/nucleic acid detection in situ, the use of genomics to aid drug discovery, organoid methodologies in research, the microbiome in cancer, the role of macrophage‐stroma interactions in fibrosis, and TGF‐β as a driver of fibrosis in multiple pathologies. Other reviews revisit the p53 field and its lack of clinical impact to date, dissect the genetics of mitochondrial diseases, summarise the cells of origin and genetics of sarcomagenesis, provide new data on the role of TRIM28 in tumour predisposition, review our current understanding of cancer stem cell niches, and the function and regulation of p63. The reviews are authored by experts in their field from academia and industry, and provide comprehensive updates of the chosen areas, in which there has been considerable recent progress. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Growing tumors are dynamic and nonlinear ecosystems, wherein cancer cells adapt to their local microenvironment, and these adaptations further modify the environment, inducing more changes. From nascent intraductal neoplasms to disseminated metastatic disease, several levels of evolutionary adaptations and selections occur. Here, we focus on one example of such an adaptation mechanism, namely, "niche construction" promoted by adaptation to acidosis, which is a metabolic adaptation to the early harsh environment in intraductal neoplasms. The avascular characteristics of ductal carcinoma in situ (DCIS) make the periluminal volume profoundly acidic, and cancer cells must adapt to this to survive. Based on discovery proteomics, we hypothesized that a component of acid adaptation involves production of collagen by pre‐cancer cells that remodels the extracellular matrix (ECM) and stabilizes cells under acid stress. The proteomic data were surprising as collagen production and deposition are commonly believed to be the responsibility of mesenchymally derived fibroblasts, and not cells of epithelial origin. Subsequent experiments in 3D culture, spinning disk and second harmonic generation microscopy of DCIS lesions in patients' samples are concordant. Collagen production assay by acid‐adapted cells in vitro demonstrated that the mechanism of induction involves the RAS and SMAD pathways. Secretome analyses show upregulation of ECM remodeling enzymes such as TGM2 and LOXL2 that are collagen crosslinkers. These data strongly indicate that acidosis in incipient cancers induces collagen production by cancer cells and support the hypothesis that this adaptation initiates a tumor‐permissive microenvironment promoting survival and growth of nascent cancers. [ABSTRACT FROM AUTHOR]

Skin grafting is a surgical method of cutaneous reconstruction, which provides volumetric replacement in wounds unable to heal by primary intention. Clinically, full‐thickness skin grafts (FTSGs) are placed in aesthetically sensitive and mechanically demanding areas such as the hands, face, and neck. Complete or partial graft failure is the primary complication associated with this surgical procedure. Strategies aimed at improving the rate of skin graft integration will reduce the incidence of graft failure. Cold atmospheric plasma (CAP) is an emerging technology offering innovative clinical applications. The aim of this study was to test the therapeutic potential of CAP to improve wound healing and skin graft integration into the recipient site. In vitro models that mimic wound healing were used to investigate the ability of CAP to enhance cellular migration, a key factor in cutaneous tissue repair. We demonstrated that CAP enhanced the migration of epidermal keratinocytes and dermal fibroblasts. This increased cellular migration was possibly induced by the low dose of reactive oxygen and nitrogen species produced by CAP. Using a mouse model of burn wound reconstructed with a full‐thickness skin graft, we showed that wounds treated with CAP healed faster than did control wounds. Immunohistochemical wound analysis showed that CAP treatment enhanced the expression of the dermal–epidermal junction components, which are vital for successful skin graft integration. CAP treatment was characterised by increased levels of Tgfbr1 mRNA and collagen I protein in vivo, suggesting enhanced wound maturity and extracellular matrix deposition. Mechanistically, we show that CAP induced the activation of the canonical SMAD‐dependent TGF‐β1 pathway in primary human dermal fibroblasts, which may explain the increased collagen I synthesis in vitro. These studies revealed that CAP improved wound repair and skin graft integration via mechanisms involving extracellular matrix formation. CAP offers a novel approach for treating cutaneous wounds and skin grafts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A disintegrin and metalloproteinase 8 (ADAM8) protein is a multi‐domain transmembrane glycoprotein which involves in extracellular matrix remodelling, cell adhesion, invasion and migration. ADAM8 and epithelial‐mesenchymal transition (EMT) play an important role in tumour invasion has been well established. However, the interaction between ADAM8 and EMT has remained unclear. The data of colon cancer patients obtained from TCGA (The Cancer Genome Atlas) and GTEx (Genotype‐Tissue Expression Project) were analysed by the bioinformatics research method. The expression of ADAM8 in colon cancer cells was up‐regulated and down‐regulated by transfecting with the expression plasmid and small interfering RNA, respectively. Transwell invasion assay, immunohistochemistry, immunocytochemistry, Western blotting and qRT‐PCR were utilized to study the effect of ADAM8 on colon cancer cell's EMT and its related mechanisms. Analysis of TCGA and GTEx data revealed that ADAM8 was linked to poor overall survival in colon cancer patients. Besides, ADAM8 was correlated with multiple EMT biomarkers (E‐cadherin, N‐cadherin, Vimentin, Snail2 and ZEB2). In vitro, we also proved that the up‐regulation of ADAM8 could promote EMT effect and enhance the invasive ability of colon cancer cells. On the contrary, the down‐regulation of ADAM8 in colon cancer cells attenuated these effects above. Further studies suggested that ADAM8 modulated EMT on colon cancer cells through TGF‐β/Smad2/3 signalling pathway. Our research suggested that ADAM8 could be a potential biomarker for the prognosis of colon cancer and induced EMT to promote the invasion of colon cancer cells via activating TGF‐β/Smad2/3 signalling pathway. [ABSTRACT FROM AUTHOR]

Cancer is driven by genetic mutations in oncogenes and tumor suppressor genes and by cellular events that develop a misregulated molecular microenvironment in the growing tumor tissue. The tumor microenvironment is guided by the excessive action of specific cytokines including transforming growth factor‐β (TGF‐β), which normally controls embryonic development and the homeostasis of young or adult tissues. As a consequence of the genetic alterations generating a given tumor, TGF‐β can preserve its homeostatic function and attempt to limit neoplastic expansion, whereas, once the tumor has progressed to an aggressive stage, TGF‐β can synergize with various oncogenic stimuli to facilitate tumor invasiveness and metastasis. TGF‐β signaling mechanisms via Smad proteins, various ubiquitin ligases, and protein kinases are relatively well understood. Such mechanisms regulate the expression of genes encoding proteins or non‐coding RNAs. Among non‐coding RNAs, much has been understood regarding the regulation and function of microRNAs, whereas the role of long non‐coding RNAs is still emerging. This article emphasizes TGF‐β signaling mechanisms leading to the regulation of non‐coding genes, the function of such non‐coding RNAs as regulators of TGF‐β signaling, and the contribution of these mechanisms in specific hallmarks of cancer. [ABSTRACT FROM AUTHOR]

Following peripheral nerve injury, remnant Schwann cells adopt a migratory phenotype and remodel the extracellular matrix allowing axonal regrowth. Although much evidence has demonstrated that TGF‐β1 promotes glioma cell motility and induces the expression of extracellular matrix proteins, the effects of TGF‐β1 on Schwann cell migration has not yet been studied. We therefore investigated the cellular effects and the signal transduction pathways evoked by TGF‐β1 in rattus norvegicus neuronal Schwann RSC96 cell. TGF‐β1 significantly increased migration and invasion of Schwann cells assessed by the wound‐healing assay and by cell invasion assay. TGF‐β1‐enhanced migration/invasion was blocked by inhibition of MMP‐2 and MMP‐9. Consistently, by real‐time and western blot analyses, we demonstrated that TGF‐β1 increased MMP‐2 and MMP‐9 mRNA and protein levels. TGF‐β1 also increased MMPs activities in cell growth medium, as shown by gelatin zymography. The selective TGF‐β Type I receptor inhibitor SB431542 completely abrogated any effects by TGF‐β1. Indeed, TGF‐β1 Type I receptor activation provoked the cytosol‐to‐nucleus translocation of SMAD2 and SMAD3. SMAD2 knockdown by siRNA blocked MMP‐2 induction and cell migration/invasion due to TGF‐β1. TGF‐β1 also provoked phosphorylation of MAPKs extracellular regulated kinase 1/2 and JNK1/2. Both MAPKs were upstream to p65/NF‐kB inasmuch as both MAPKs' inhibitors PD98059 and SP600125 or their down‐regulation by siRNA significantly blocked the TGF‐β1‐induced nuclear translocation of p65/NF‐kB. In addition, p65/NF‐κB siRNA knockdown inhibited the effects of TGF‐β1 on both MMP‐9 and cell migration/invasion. We conclude that TGF‐β1 controls RSC96 Schwann cell migration and invasion through MMP‐2 and MMP‐9 activities. MMP‐2 is controlled by SMAD2 whilst MMP‐9 is controlled via an ERK1/2‐JNK1/2‐NF‐κB dependent pathway. [ABSTRACT FROM AUTHOR]

Background: Transforming growth factor‐β (TGF‐β) is a major regulator of extracellular matrix (ECM) events, particularly collagen production. Aim: We explored whether the expression of matrix metalloproteinases (MMPs) and collagen are transcriptionally regulated by the TGF‐β and Smad signaling pathways, and the roles played by NF‐κB and mitogen‐activated protein kinase (MAPK) signaling in normal, aged, human dermal fibroblasts. Methods: We quantified mRNA and protein expression using real‐time PCR and immunoblotting of proteins from cells in passage 5‐15. Results: The levels of mRNAs encoding TGF‐β1, TGF‐β3, and TGF‐β receptor type I (TGFβ RI) decreased with increasing passage number. The levels of mRNAs encoding TGF‐β2, TGFβ RII, and TGFβ RIII increased to passage 10 but decreased by passage 15. The levels of mRNAs encoding Smad‐2, ‐3, ‐4, and ‐7 decreased with increasing passage number. The level of mRNA encoding MMP‐1 increased with increasing passage number, and the levels of mRNAs encoding MMP‐2, TIMP‐1, and TIMP‐2 increased to passage 10 but decreased by passage 15. The levels of mRNAs encoding collagen types I and II decreased with increasing passage number. At the protein level, NF‐κB, IκBα, p38, ERK, Akt, and JNK became increasingly phosphorylated at higher passage numbers. Conclusion: Our results suggest that reductions in the expression levels of MMPs and collagen types I and III in aging human dermal fibroblasts reflect reduced expression of TGF‐β/Smad and TGF‐β receptors, thus compromising the TGF‐β receptor‐binding capacity of fibroblasts; the NF‐κB and Akt‐JNK/MAPK signaling pathways may play active roles in this process. [ABSTRACT FROM AUTHOR]

Regeneration remains a major challenge in skeletal muscle repair after injury. Recently, transforming growth factor‐β (TGF‐β)/Smad pathway was found to play an important role in inhibiting myogenesis, a crucial stage in skeletal muscle regeneration. In our previous study, microRNA‐122‐5p (miR‐122) was proved to have the function of downregulating TGF‐β/Smad pathway. Theoretically, miR‐122 might also be involved in the process of skeletal muscle myogenesis through the regulation of TGF‐β/Smad pathway. In this study, we aimed to investigate the impact of miR‐122 on skeletal muscle myogenesis and explore its underlying mechanism. Results showed that miR‐122 and myogenic markers were downregulated in C2C12 cells after TGF‐β stimulation, and miR‐122 overexpression could restore the myogenesis inhibited by TGF‐β. We then located TGFBR2 as the direct target of miR‐122 and discovered the effect of miR‐122 overexpression could be rescued by TGFBR2 overexpression. Further, the downstream molecules of TGFBR2 in the TGF‐β/Smad pathway were found to be suppressed by miR‐122. In conclusion, miR‐122 could suppress the TGF‐β/Smad signalling pathway by directly targeting TGFBR2 and, consequently, restore myogenesis. Significance of the study: Regeneration remains a major challenge in skeletal muscle repair after injury. In this study, it was found that miR‐122 could suppress the TGF‐β/Smad signalling pathway by directly targeting TGFBR2 and, consequently, restore myogenesis. Our findings could inspire future experiments on the role of miRs in skeletal muscle diseases and future translational studies on potential novel gene therapy for skeletal muscle injury. [ABSTRACT FROM AUTHOR]

Receptor‐interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor‐β 1 (TGF‐β1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF‐β signaling during epidermal homeostasis‐related events and suppression of RIPK4 expression by TGF‐β1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor‐κB (NF‐κB), protein kinase C (PKC), wingless‐type MMTV integration site family (Wnt), and (mitogen‐activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad‐mediated TGF‐β1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad‐mediated TGF‐β1 signaling events through suppression of TGF‐β1‐induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3‐Smad4 interaction, nuclear localization, and TGF‐β1‐induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound‐scratch assay demonstrated that RIPK4 suppressed TGF‐β1‐mediated wound healing through blocking TGF‐β1‐induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF‐β1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF‐β1 signaling. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC), a leading cause of cancer death, has recently been known as the most prevalent malignancy worldwide. Although chemotherapy is an important therapeutic option for CRC patients, multidrug resistance (MDR) still remains a major cause of chemotherapy failure. Transmembrane protein 45A (TMEM45A) has been found highly expressed in various cancers, and is also proposed as an interesting biomarker for chemoresistance. However, the association between TMEM45A and MDR in CRC remains unclear. This study aimed to investigate the key role of TMEM45A in CRC by knockdown of its expression in 5‐FU‐resistant CRC cells (HCT‐8/5‐FU and SW480/5‐FU) and their parental cells (HCT‐8 and SW480). Data showed that TMEM45A was significantly up‐regulated in HCT‐8/5‐FU and SW480/5‐FU cells in comparison with their parental HCT‐8 and SW480 cells. Knockdown of TMEM45A enhanced 5‐FU sensitivity and 5‐FU‐induced apoptosis in HCT‐8/5‐FU and SW480/5‐FU cells. It was also found that inhibition of TMEM45A increased the intracellular accumulation of Rhodamine‐123 and down‐regulated the expression of MDR1 in HCT‐8/5‐FU and SW480/5‐FU cells. In addition, knockdown of TMEM45A suppressed migration and invasion of HCT‐8/5‐FU and SW480/5‐FU cells. Furthermore, knockdown of TMEM45A not only attenuated MDR‐enhanced epithelial–mesenchymal transition (EMT), but also suppressed MDR‐enhanced activation of the TGF‐β signalling pathway in HCT‐8/5‐FU and SW480/5‐FU cells. Taken together, our study suggests that knockdown of TMEM45A can effectively overcome MDR and inhibit EMT via suppression of the TGF‐β signalling pathway in human CRC cells, and that targeting TMEM45A will be a potential strategy in the treatment of MDR in CRC. [ABSTRACT FROM AUTHOR]

Abnormal tumor microenvironment and the epithelial‐mesenchymal transition (EMT) are important features of tumor metastasis. However, it remains unknown how signals can form complicated networks to regulate the sustainability of the EMT process. The aim of our study is to explore the possible interaction between tumor‐associated macrophages and tumor cells in the EMT process mediated by microRNA (miR)‐362‐3p. In this study, we found that by releasing TGF‐β, M2 macrophages mediate binding of Smad2/3 to miR‐362‐3p promoter, leading to overexpression of miR‐362‐3p. MicroRNA‐362‐3p maintains EMT by regulating CD82, one of the most important members of the family of tetraspanins. Our finding suggests that miR‐362‐3p can serve as a core factor mediating cross‐talk between the TGF‐β pathway in tumor‐associated macrophages and tetraspanins in tumor cells, and thus facilitates the EMT process. [ABSTRACT FROM AUTHOR]

Liver cirrhosis is associated with increased morbidity and mortality with important health and social consequences; however, an effective treatment has not been found yet. Previous reports have shown some beneficial effects of stevioside (SVT) in different diseases, but the ability of SVT to inhibit liver cirrhosis has not been reported. Therefore, we studied the potential of this diterpenoid to inhibit liver cirrhosis induced by thioacetamide, a model that shares many similarities with the human disease, and investigated the possible underlying molecular mechanism using in vivo and in vitro approaches. Cirrhosis was induced in male Wistar rats by chronic thioacetamide administration (200 mg/kg) intraperitoneally three times per week. Rats received saline or SVT (20 mg/kg) two times daily intraperitoneally. In addition, co‐cultures were incubated with either lipopolysaccharide or ethanol. Liver fibrosis, hepatic stellate cells activation, metalloproteinases activity, canonical and non‐canonical Smads pathway and expression of several profibrogenic genes were evaluated. Thioacetamide activated hepatic stellate cells and distorted the liver parenchyma with the presence of abundant thick bands of collagen. In addition, thioacetamide up‐regulated the protein expression of α‐smooth muscle actin, transforming growth factor‐β1, metalloproteinases‐9,‐2 and ‐13 and overstimulate the canonical and non‐canonical Smad pathways. SVT administration inhibited all of these changes. In vitro, SVT inhibited the up‐regulation of several genes implicated in cirrhosis when cells were exposed to lipopolysaccharides or ethanol. We conclude that SVT inhibited liver damage by blocking hepatic stellate cells activation, down‐regulating canonical and non‐canonical profibrotic Smad pathways. [ABSTRACT FROM AUTHOR]

Herein, we report a simple solid state synthetic route to prepare carbon‐Fe based magnetic nanoparticles with different compositions and morphologies through annealing of amorphous Fe nanoparticles under appropriate conditions. Tri‐n‐octylphosphine (TOP) capped amorphous Fe nanoparticles with a mean diameter of 3.2 nm were synthesized using solvated metal atom dispersion (SMAD) method. Annealing of as‐prepared Fe nanoparticles at 300 °C produced carbon encapsulated crystalline bcc‐Fe nanoparticles, whereas at higher temperatures i.e., 400 °C and 500 °C, spherical Fe3C/C core‐shell nanoparticles were obtained. Annealing of as‐prepared Fe nanoparticles in the presence of tri‐n‐octylphosphine oxide (TOPO) ligand under optimized conditions yielded rod shaped Fe3C/C core‐shell morphology. The size, composition and particle morphology of these magnetic nanoparticles could be controlled by changing the reaction time, temperature and the concentration of the TOPO ligand. Magnetic measurements show that rod shaped Fe3C nanoparticles exhibit enhanced coercivity (Hc) values compared with spherical Fe3C nanoparticles, which is due to shape anisotropy. Spherical and rod‐shaped Fe3C nanoparticles encapsulated in carbon have been prepared via annealing under different reaction conditions. [ABSTRACT FROM AUTHOR]

Aim: The aims of the present study were to investigate the capacity of stevia leaves to prevent experimental cirrhosis induced by chronic administration of carbon tetrachloride (CCl4) in rats and to explore the action mechanism involved. Methods: Liver cirrhosis was established by CCl4 treatment (400 mg/kg i.p. three times a week for 12 weeks); stevia powder was administered (100 mg/kg by gavage daily) during the CCl4 treatment. Serum markers of liver damage and hydroxyproline were evaluated and histopathological analyses were carried out. The profibrotic pathways were analyzed by western blot and immunohistochemistry. Results: We found for the first time that stevia cotreatment prevented the elevation of serum markers of necrosis and cholestasis and the occurrence of liver fibrosis. It is worth noting that stevia downregulated several profibrogenic pathways, including the reduction of hepatic myofibroblasts and decreased matrix metalloproteinase (MMP)2 and MMP13 expression, thereby blocking the liberation of transforming growth factor‐β from the extracellular matrix. Notably, stevia reduced the phosphorylation of pSmad3L, the most profibrogenic and mitogenic Smad, by inhibiting the activation of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase. Interestingly, Smad7, an important antifibrotic molecule, was upregulated by stevia treatment in cirrhotic rats. These multitarget mechanisms led to the prevention of experimental cirrhosis. Conclusions: Because stevia possesses a reasonable safety profile, our results indicate that it could be useful in the clinical setting to treat chronic liver diseases. [ABSTRACT FROM AUTHOR]

Cyclin‐dependent kinase (CDK) 4 and CDK6 inhibitors are effective therapeutic options for hormone receptor (HR)‐positive, human epidermal growth factor receptor 2 (HER2)‐negative advanced breast cancer. Although CDK4/6 inhibitors mainly target the cyclin D‐CDK4/6‐retinoblastoma tumor suppressor protein (RB) axis, little is known about the clinical impact of inhibiting phosphorylation of other CDK4/6 target proteins. Here, we focused on other CDK4/6 targets, SMAD proteins. We showed that a CDK4/6 inhibitor palbociclib and activin‐SMAD2 signaling cooperatively inhibited cell cycle progression of a luminal‐type breast cancer cell line T47D. Palbociclib enhanced SMAD2 binding to the genome by inhibiting CDK4/6‐mediated linker phosphorylation of the SMAD2 protein. We also showed that cyclin G2 plays essential roles in SMAD2‐dependent cytostatic response. Moreover, comparison of the SMAD2 ChIP‐seq data of T47D cells with those of Hs578T (triple‐negative breast cancer cells) indicated that palbociclib augmented different SMAD2‐mediated functions based on cell type, and enhanced SMAD2 binding to the target regions on the genome without affecting its binding pattern. In summary, palbociclib enhances the cytostatic effects of the activin‐SMAD2 signaling pathway, whereas it possibly strengthens the tumor‐promoting aspect in aggressive breast cancer. A CDK4/6 inhibitor palbociclib and activin‐SMAD2 signaling cooperatively inhibited cell cycle progression of a luminal‐type breast cancer cell line T47D. Palbociclib enhanced SMAD2 binding to the genome by inhibiting CDK4/6‐mediated linker phosphorylation of the SMAD2 protein. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the hypothesis that emodin inhibits extracellular matrix (ECM)-related gene expression in activated hepatic stellate cells (HSCs) by blocking canonical or/and noncanonical components of transforming growth factor β1 (TGFβ1) intracellular signaling. Here, we demonstrate that emodin suppressed the gene expression of HSCs activation markers type I collagen, fibronectin, and α-smooth muscle actin, as well as HSCs proliferation. Mechanistically, emodin suppresses TGFβ1, TGFβ receptor II, TGFβ receptor I, and Smad4 gene expression, as well as Smad luciferase activity. Simultaneously, emodin reduced p38 mitogen-activated protein kinase (p38MAPK ) activity but not c-Jun N-terminal kinases and extracellular signal-regulated kinases 1 and 2 phosphorylation in HSC-T6 cells. Interestingly, deprivation of TGFβ using a neutralizing antibody abolished emodin-mediated inhibitions of the both Smad transcriptional activity and p38MAPK phosphorylation. Furthermore, emodin-mediated inhibition of HSCs activation could be partially blocked by PD98059 inhibition of p38MAPK or short hairpin RNA-imposed knockdown of Smad4. Conversely, simultaneous inhibition of Smad4 and p38MAPK pathways completely reverses the effects of emodin, suggesting that Smad and p38MAPK locate downstream of TGFβ1 and regulate collagen genes expression in HSCs. Collectively, these data suggest that emodin is a promising candidate for the treatment of hepatic fibrosis. [ABSTRACT FROM AUTHOR]

Summary: T helper 17 (Th17) cells play critical roles in inflammatory and autoimmune diseases. The lineage‐specific transcription factor RORγt is the key regulator for Th17 cell fate commitment. A substantial number of studies have established the importance of transforming growth factor β (TGF‐β) ‐dependent pathways in inducing RORγt expression and Th17 differentiation. TGF‐β superfamily members TGF‐β1, TGF‐β3 or activin A, in concert with interleukin‐6 or interleukin‐21, differentiate naive T cells into Th17 cells. Alternatively, Th17 differentiation can occur through TGF‐β‐independent pathways. However, the mechanism of how TGF‐β‐dependent and TGF‐β‐independent pathways control Th17 differentiation remains controversial. This review focuses on the perplexing role of TGF‐β in Th17 differentiation, depicts the requirement of TGF‐β for Th17 development, and underscores the multiple mechanisms underlying TGF‐β‐promoted Th17 generation, pathogenicity and plasticity. With new insights and comprehension from recent findings, this review specifically tackles the involvement of the canonical TGF‐β signalling components, SMAD2, SMAD3 and SMAD4, summarizes diverse SMAD‐independent mechanisms, and highlights the importance of TGF‐β signalling in balancing the reciprocal conversion of Th17 and regulatory T cells. Finally, this review includes discussions and perspectives and raises important mechanistic questions about the role of TGF‐β in Th17 generation and function. [ABSTRACT FROM AUTHOR]

Abstract: Background: Melatonin regulates circadian and seasonal rhythms and the activities of hormones and cytokines that are expressed in various tissues, including the ovary, in which melatonin receptors are expressed. In the ovary, follicular growth occurs as a result of complex interactions between pituitary gonadotropins and autocrine and paracrine factors, including bone morphogenetic proteins (BMPs) that are expressed in the ovary. Methods: The effects of melatonin and BMPs on steroidogenesis were examined by using the primary cultures of rat granulosa cells. Main findings (Results): It was shown that melatonin has antagonistic effects on BMP‐6 actions in the granulosa cells, suggesting that melatonin is likely to contribute to balancing the biological activity of endogenous BMPs that maintain progesterone production and luteinization in the growing follicles. Similar interactions between melatonin and BMP–smad signaling also were shown in the mechanism of controlling ovarian steroidogenesis by other ligands. Conclusion: A new role of melatonin in the regulation of endocrine homeostasis in relation to BMP activity is introduced in this review. [ABSTRACT FROM AUTHOR]

Abstract: Background & Aims: We explored whether growth differentiation factor 15 (GDF15) affects the histological severity of non‐alcoholic fatty liver disease (NAFLD) independent of insulin resistance. Methods: In a biopsy‐proven NAFLD cohort, we measured serum GDF15 levels using enzyme‐linked immunosorbent assays. Results: Among 190 subjects (mean age, 53 ± 14 years; men, 52.1%), 72 (men, 65.3%) and 78 (men, 44.9%) were diagnosed with biopsy‐proven non‐alcoholic fatty liver (NAFL) and non‐alcoholic steatohepatitis (NASH) respectively. GDF15 levels were significantly higher in NASH patients than in controls (P = .010) or NAFL patients (P = .001). Subjects with advanced fibrosis (≥F3) also showed higher GDF15 levels compared to the others (F0‐2; P < .001). Among NAFLD patients, the highest quartile of GDF15 levels was significantly associated with a risk of advanced fibrosis even after adjustment for age, gender, body mass index, smoking status, hypertension, diabetes, aspartate aminotransferase, platelet, albumin, insulin resistance and low skeletal muscle mass (odds ratio, 4.27; 95% confidence interval, 1.04‐17.63), but not with NASH risk. GDF15 levels showed a significant positive correlation with liver stiffness (Spearman's ρ, .525; P < .001). Palmitate treatment increased the GDF15 mRNA expression level significantly in Kupffer cells, but not in hepatocytes. In LX‐2 cells, GDF15 treatment resulted in enhanced expression of α‐smooth muscle actin and collagen I, as well as phosphorylation of SMAD2 and SMAD3. Conclusions: Our findings suggest that GDF15 may serve as a novel biomarker of advanced fibrosis in NAFLD, thereby indicating the need for urgent anti‐fibrotic pharmacotherapy. [ABSTRACT FROM AUTHOR]

Objective To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination. Specifically, we sought to examine the ECF differentiation after (a) silencing of Smad2, 3, and 4 profibrotic genes individually and (b) overexpression of antifibrotic Smad7 gene and in a combination with pro- and antifibrotic Smad genes. Methods Equine corneal fibroblast primary cultures were generated as previously described. ECFs were transfected with individual plasmids which silenced gene expression of either Smad2, 3, or 4 or in combination with a plasmid overexpressing Smad7 using Lipofectamine 2000™ or Lipofectamine BLOCK-iTTM. Smad-transfected clones were then exposed to TGF-β1 to induce differentiation to myofibroblasts. Immunofluorescence and qRT-PCR techniques quantified levels of ECF differentiation to ECM by measuring alpha smooth muscle actin, a known marker of ECM transdifferentiation. Results Silencing of individual Smad2, 3, or 4 genes or overexpression of Smad7 showed significant inhibition of ECF transdifferentiation (73-83% reduction). Silencing of Smad2 showed the greatest inhibition of ECF transdifferentiation in (a) and was therefore utilized for the combination gene transfer testing. The combination gene transfer consisting of Smad7 overexpression and Smad2 silencing attenuated ECF differentiation significantly; however, the level was not significant compared to the overexpression of Smad7 individually. Conclusions Using gene transfer technology involving profibrotic Smad silencing, antifibrotic Smad overexpression or its combination is a novel strategy to control TGF-β1-mediated fibrosis in equine fibroblasts. Combination gene therapy was not better than single gene therapy in this study. [ABSTRACT FROM AUTHOR]

Latent transforming growth factor binding proteins (LTBP) are a family of extracellular matrix glycoproteins that play an important role in the regulation of transforming growth factor beta (TGF‐ß) activation. Dysregulation of the TGF‐ß pathway has been implicated in the pathogenesis of inherited disorders predisposing to thoracic aortic aneurysms syndromes (TAAS) including Marfan syndrome (MFS; FBN1) and Loeys–Dietz syndrome (LDS; TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, SMAD3). While these syndromes have distinct clinical criteria, they share clinical features including aortic root dilation and musculoskeletal findings. LTBP1 is a component of the TGF‐ß pathway that binds to fibrillin‐1 in the extracellular matrix rendering TGF‐ß inactive. We describe a three‐generation family case series with a heterozygous ∼5.1 Mb novel contiguous gene deletion of chromosome 2p22.3‐p22.2 involving 11 genes, including LTBP1. The deletion has been identified in the proband, father and grandfather, who all have a phenotype consistent with a TAAS. Findings include thoracic aortic dilation, ptosis, malar hypoplasia, high arched palate, retrognathia, pes planus, hindfoot deformity, obstructive sleep apnea, and low truncal tone during childhood with joint laxity that progressed to reduced joint mobility over time. While the three affected individuals did not meet criteria for either MFS or LDS, they shared features of both. Although the deletion includes 11 genes, given the relationship between LTBP1, TGF‐ß, and fibrillin‐1, LTBP1 stands out as one of the possible candidate genes for the clinical syndrome observed in this family. More studies are necessary to evaluate the potential role of LTBP1 in the pathophysiology of TAAS. [ABSTRACT FROM AUTHOR]

Chronic allograft dysfunction (CAD) induced by kidney interstitial fibrosis is the main cause of allograft failure in kidney transplantation. Endothelial-to-mesenchymal transition (EndMT) may play an important role in kidney fibrosis. We, therefore, undertook this study to characterize the functions and potential mechanism of EndMT in transplant kidney interstitial fibrosis. Proteins and mRNAs associated with EndMT were examined in human umbilical vein endothelial cells (HUVECs) treated with transforming growth factor-beta1 (TGF-β1) at different doses or at different intervals with western blotting, qRT-PCR and ELISA assays. Cell motility and migration were evaluated with motility and migration assays. The mechanism of EndMT induced by TGF-β1 was determined by western blotting analysis of factors involved in various canonical and non-canonical pathways. In addition, human kidney tissues from control and CAD group were also examined for these proteins by HE, Masson's trichrome, immunohistochemical, indirect immunofluorescence double staining and western blotting assays. TGF-β1 significantly promoted the development of EndMT in a time-dependent and dose-dependent manner and promoted the motility and migration ability of HUVECs. The TGF-β/Smad and Akt/mTOR/p70S6K signalling pathways were found to be associated with the pathogenesis of EndMT induced by TGF-β1, which was also proven in vivo by the analysis of specimens from the control and CAD groups. EndMT may promote transplant kidney interstitial fibrosis by targetting the TGF-β/Smad and Akt/mTOR/p70S6K signalling pathways, and hence, result in the development of CAD in kidney transplant recipients. [ABSTRACT FROM AUTHOR]

It been shown that IL-6 modulates TGF-β1 expression in fibroblasts, however, what role IL-6 plays concerning TGF-βR expression and function in skin is unknown. Therefore, the aim of this study was to investigate the mechanism by which IL-6 might modulates TGF-β receptors in skin. Skin from WT, IL-6 over-expressing mice and IL-6 treated keratinocyte cultures was analysed for TGF-β RI and TGF-β RII expression via histology, PCR and flow cytometry. Receptor function was assessed by cell migration, bromodeoxyuridine (BrdU) proliferation assays, and Smad7 expression and Smad2/3 phosphorylation. Receptor localization within the membrane was determined by co-immunoprecipitation. IL-6 overexpression and treatment increased TGF-β RII expression in the epidermis. IL-6 treatment of keratinocytes induced TGF-β RI and II expression and augmented TGF-β1-induced function as demonstrated through increased migration and decreased proliferation. Additionally, IL-6 treatment of keratinocytes altered receptor activity as indicated by altered Smad2/3 phosphorylation and increased Smad7 and membrane localization. These results suggest that IL-6 regulates keratinocyte function by modulating TGF-β RI and II expression and signal transduction via trafficking of the receptor to lipid raft pools. [ABSTRACT FROM AUTHOR]

As the most prevalent and lethal type of brain tumours, gliomas, especially malignant ones, are relatively resistant to conventional therapies. Gremlin 1 (GREM1) is a secreted glycoprotein that is implicated in the maintenance of cancer stem cells in tumour hierarchy. In the current study, the role of GREM1 in the carcinogenesis of glioma was studied using a knockdown approach. We first examined the expression level of GREM1 in the clinical samples, and then evaluated the effect of GREM1 knockdown on the viability and colony formation capacity of U87-MG cells. Moreover, the migration ability, invasiveness, cell cycle, and apoptosis of GREM1-silenced cells were assessed. Furthermore, the involvement of functional GREM1 in the epithelial-mesenchymal transition (EMT) process of glioma was investigated by detecting the expression levels of glioma-associated oncogene homologue 3 (GLI3) and EMT-related molecules. Our results demonstrated that knockdown of GREM1 reduced cell viability, suppressed migration and invasion, and inhibited GLI3 expression and the EMT process in U87-MG cells. Meanwhile, GREM1 silencing promoted apoptosis in U87-MG cells through the accumulation of Bax, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) as well as the downregulation of Bcl-2. In addition, GREM1 knockdown abolished transforming growth factor (TGF)-β1-mediated activation of the Smad pathway, which may underlie the mechanism of GREM1-regulated EMT. In conclusion, GREM1 plays an important role in the development of glioma, and it may serve as a potential target in glioma therapy. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP-ribose) polymerases-1 ( PARP-1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP-1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP-1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor-β ( TGF-β) expression and TGF-β/ SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF-β/ SMAD signalling pathway with alpha-smooth muscle actin (α SMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor-α, interleukin-1β, iNOS and COX-2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF-β levels, pSMAD3 expression, α SMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above-mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF-β expression and the TGF-β/ SMAD transduction pathway. [ABSTRACT FROM AUTHOR]

Background IL-22- and IL-17-producing T cells have important roles in allergic diseases. Micro RNAs (mi RNAs) are posttranscriptional regulators of gene expression and modulate numerous biological processes. Little is known about the functions of mi RNAs in IL-22/ IL-17-producing T cells. Material and Methods IL-22- and IL-17-positive T cells were sorted from human peripheral blood mononuclear cells ( PBMCs) by intracellular staining and dual-secretion assay. mi RNA expression profiles were detected with TaqMan array microfluidic cards. T cells were transfected with mi RNA mimics. Gene expression was analyzed using RT- qPCR and/or enzyme-linked immunosorbent assay in T-cell subsets and PBMCs from patients with asthma and atopic dermatitis. Results The increased expression of miR-323-3p and noncoding RNA nc886 and reduced expression of miR-93, miR-181a, miR-26a, and miR-874 were detected in IL-22-producing T cells. The pathway analysis of the putative targets suggested that these differentially expressed mi RNAs could impact the proliferation, differentiation, and effector functions of T cells. Further analyses showed the highest expression for miR-323-3p in IL-22- and IL-17-double-positive T cells and its capacity to suppress multiple genes from the transforming growth factor-β pathway and the production of IL-22 in T cells. An increased expression of miR-323-3p in PBMCs from patients with asthma and reverse correlation between miR-323-3p levels and IL-22 production in PBMCs cultured in T-cell growth conditions was observed. Conclusions Our data suggest that miR-323-3p acts in a negative feedback loop to control the production of IL-22 in IL-22/ IL-17-producing T cells and might thus impact the T-cell responses in asthma. [ABSTRACT FROM AUTHOR]

Objective To investigate molecular mechanisms mediating anti-fibrotic effect of SAHA in the canine cornea using an in vitro model. We hypothesized that SAHA attenuates corneal fibrosis by modulating Smad-dependent and, to a lesser extent, Smad-independent signaling pathways activated by TGF-β1, as well as matrix metalloproteinase ( MMP) activity. Methods Cultured canine corneal fibroblasts ( CCF) were incubated in the presence/absence of TGF-β1 (5 ng/mL) and SAHA (2.5 μ m) for 24 h. Western blot analysis was used to quantify non-phosphorylated and phosphorylated isoforms of Smad2/3, p38 MAP kinase ( MAPK), ERK1/2, and JNK1. Real-time PCR and zymography were utilized to quantify MMP1, MMP2, MMP8, and MMP9 mRNA expressions and MMP2 and MMP9 protein activities, respectively. Results TGF-β1 treatment caused a significant increase in phospho-Smad2/3 and phospho-p38 MAPK. SAHA treatment reduced TGF-β1-induced phosphorylation of Smad2/3 but not of p38 MAPK. TGF-β1 did not modulate the phosphorylation of ERK1/2 or JNK1. SAHA caused a significant reduction in phospho- ERK1/2 expression regardless of concurrent TGF-β1 treatment. Neither SAHA alone nor in combination with TGF-β1 altered phospho- JNK1 expression. TGF-β1 significantly increased MMP1 and MMP9 mRNA expressions but did not alter MMP2 mRNA. SAHA treatment attenuated TGF-β1-induced MMP9 mRNA expression while significantly enhancing TGF-β1-induced MMP1 mRNA expression. Zymography detected reduced expression of MMP2 and MMP9 proteins in untreated control CCF. TGF-β1 treatment did not alter their expression, but SAHA treatment +/− TGF-β1 significantly increased MMP2 and MMP9 protein expressions. Conclusions The corneal anti-fibrotic effects of SAHA involve multiple mechanisms including modulation of canonical and non-canonical components of TGF-β1 intracellular signaling and MMP activity. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the role of transforming growth factor β1 ( TGF-β1) in transcriptional regulation and function of the guanylyl cyclase A/natriuretic peptide receptor A gene ( Npr1) and whether cross-talk exists between these two hormonal systems in target cells. After treatment of primary cultured rat thoracic aortic vascular smooth muscle cells and mouse mesangial cells with TGF-β1, the Npr1 promoter construct containing a δ-crystallin enhancer binding factor 1 (δ EF1) site showed 85% reduction in luciferase activity in a time- and dose-dependent manner. TGF-β1 also significantly attenuated luciferase activity of the Npr1 promoter by 62%, and decreased atrial natriuretic peptide-mediated relaxation of mouse denuded aortic rings ex vivo. Treatment of cells with TGF-β1 increased the protein levels of δ EF1 by 2.4-2.8-fold, and also significantly enhanced the phosphorylation of Smad 2/3, but markedly reduced Npr1 mRNA and receptor protein levels. Over-expression of δ EF1 showed a reduction in Npr1 promoter activity by 75%, while deletion or site-directed mutagenesis of δ EF1 sites in the Npr1 promoter eliminated the TGF-β1-mediated repression of Npr1 transcription. TGF-β1 significantly increased the expression of α-smooth muscle actin and collagen type I α2 in rat thoracic aortic vascular smooth muscle cells, which was markedly attenuated by atrial natriuretic peptide in cells over-expressing natriuretic peptide receptor A. Together, the present results suggest that an antagonistic cascade exists between the TGF-β1/Smad/δ EF1 pathways and Npr1 expression and receptor signaling that is relevant to renal and vascular remodeling, and may be critical in the regulation of blood pressure and cardiovascular homeostasis. [ABSTRACT FROM AUTHOR]