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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]

• Neurons express CD109 in the central nervous system. • CD109 is upregulated in the lumbar spinal cord of SOD1-G93A mice. • CD109 upregulation is mainly contributed by proliferated microglia. • CD109 inhibits TGFβ/SMAD pathway and attenuates inflammatory reactions in microglia. CD109 is a multifunctional coreceptor, whose function has been widely studied in tumor progression and metastasis. One of the reported primary roles of CD109 involves down-regulating TGFβ signaling. However, the role of CD109 in central nervous system, especially neurodegenerative disease, is barely known. Here, we examined the expression changes and cellular location of CD109 and TGFβ/SMAD pathway molecules in lumbar spinal cord of SOD1-G93A mice, and explored the role and mechanism of CD109 on LPS-treated BV2 microglia and primary microglia derived from SOD1-G93A mice. Our results showed an increased expression of CD109 and TGFβ/SMAD pathway molecules in lumbar spinal cord of SOD1-G93A mice. Further cellular localization analysis demonstrated that proliferating microglia contributed mainly to the upregulation of CD109 and TGFβ1. Moreover, CD109 intervention in vitro partially reduced inflammatory response and TGFβ/SMAD pathway activation in both LPS-treated BV2 microglia and primary SOD1-G93A microglia. Thus, CD109 was involved in pathogenesis of ALS mice, and interventions targeting on CD109 modulation could be a potential therapeutic strategy for ALS. [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]

Cells compartmentalize biochemical processes using physical barriers in the form of membranes. Eukaryotes have a wide diversity of membrane-based compartments that can be used in this context, with the main ones being the extracellular membrane, which separates the inside from the outside of the cell, and the nuclear membrane, which separates the nucleus from the cytoplasm. The nuclear membrane not only isolates and protects the DNA and the transcription and replication processes from the other processes that are occurring in the cytoplasm but also has an active role in the regulation of cellular signaling. The TGF- β pathway is one of the most important and conserved signaling cascades, and it achieves compartmentalization using a well-tuned balance between the import and export rates of the active and inactive forms of key proteins. Thus, compartmentalization serves as an additional regulatory mechanism, physically isolating transcription factors from their targets, influencing the dynamics and strength of signal transduction. This contribution focuses on this biophysical layer of regulation, using the TGF- β pathway to illustrate the molecular mechanisms underlying this process, as well as the biological consequences of this compartmentalization. We also introduce a simplified mathematical formulation for studying the dynamics of this process using a generalized approach. [ABSTRACT FROM AUTHOR]

Intrauterine adhesion (IUA) resulting from irreversible fibrotic repair of endometrium is the main cause of secondary infertility in women, and current therapeutic approaches to IUA are limited. Increasing evidence has suggested the important role of competitive endogenous RNA (ceRNA) in IUA pathologies. This study aimed to investigate the long noncoding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1)-associated ceRNA in IUA development. We harvested endometrial tissues from patients with or without IUA and extracted endometrial stromal cells (ESCs) from normal endometrial tissues. Transforming growth factor β1 (TGF-β1) was used to induce fibrosis in ESCs. The expression of transforming growth factor β receptor 1 (TGFβR1), α-smooth muscle actin, phosphorylated suppressor of mother against decapentaplegic (p-Smad)2/3, collagen type I alpha 1, MALAT1, and microRNA (miR)-22-3p in endometrial tissues and ESCs was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) or western blotting. Pearson's correlation analysis was conducted to assess the correlation between miR-22-3p expression or TGFβR1 and MALAT1 expression in endometrial tissues. The expression of TGFβR1 in ESCs was also evaluated by immunofluorescence staining. The location of MALAT1 was examined by fluorescence in situ hybridization. Luciferase reporter assays were performed to verify the binding relationship between MALAT1 or TGFβR1 and miR-22-3p. Cell viability was assessed via cell counting kit-8 assays. Our findings revealed that lncRNA MALAT1 and TGFβR1 were upregulated while miR-22-3p was downregulated in IUA endometrial tissues or TGF-β1-stimulated ESCs, and lncRNA MALAT1 expression was negatively correlated with miR-22-3p expression while being positively correlated with TGFβR1 expression in IUA endometrial tissues. Additionally, lncRNA MALAT1 was mainly located in the cytoplasm of ESCs and directly targeted miR-22-3p to regulate TGFβR1 expression. Moreover, knockdown of lncRNA MALAT1 exerted anti-fibrotic effects on ESCs by targeting miR-22-3p, and miR-22-3p overexpression inhibited the fibrosis of ESCs by binding to TGFβR1 3'untranslated region. Collectively, lncRNA MALAT1 promotes endometrial fibrosis by sponging miR-22-3p to regulate TGFβR1 and Smad2/3, and inhibition of MALAT1 may represent a promising therapeutic option for suppressing endometrial fibrosis. [ABSTRACT FROM AUTHOR]

Cells compartmentalize biochemical processes using physical barriers in the form of membranes. Eukaryotes have a wide diversity of membrane-based compartments that can be used in this context, with the main ones being the extracellular membrane, which separates the inside from the outside of the cell, and the nuclear membrane, which separates the nucleus from the cytoplasm. The nuclear membrane not only isolates and protects the DNA and the transcription and replication processes from the other processes that are occurring in the cytoplasm but also has an active role in the regulation of cellular signaling. The TGF-β pathway is one of the most important and conserved signaling cascades, and it achieves compartmentalization using a well-tuned balance between the import and export rates of the active and inactive forms of key proteins. Thus, compartmentalization serves as an additional regulatory mechanism, physically isolating transcription factors from their targets, influencing the dynamics and strength of signal transduction. This contribution focuses on this biophysical layer of regulation, using the TGF-β pathway to illustrate the molecular mechanisms underlying this process, as well as the biological consequences of this compartmentalization. We also introduce a simplified mathematical formulation for studying the dynamics of this process using a generalized approach.

BACKGROUND: Aerobic exercise can suppress liver fibrosis in diabetic mice. However, the specific mechanism is yet to be elucidated. OBJECTIVE: To investigate the protective effect and mechanism of aerobic exercise on liver fibrosis in db/db mice via the transforming growth factor-β/Smad signaling pathway. METHODS: 8-week-old male db/db mice and age-matched m/m mice were randomly divided into m/m control group, m/m+exercise group, db/db control group, and db/db+exercise group, with 10 mice in each group. Mice in the exercise group were subjected to a 12-week aerobic exercise. After the exercise, fasting blood glucose levels were measured in mice, and glucose tolerance and insulin tolerance tests were conducted. Mouse liver was extracted to calculate liver index and mouse eyeballs were taken to collect blood sample and detect biochemical indicators. Masson, oil red O and Hematoxylin-eosin staining were used to detect and analyze the pathological changes in mouse liver tissues. Immunohistochemistry was used to determine the protein expression levels of transforming growth factor-β1 and p-Smad3. Western blot analysis was applied to determine the protein expression levels of transforming growth factor-β1, Smad3, p-Smad3, α-smooth muscle actin, type I collagen and type III collagen. RESULTS AND CONCLUSION: Compared with the m/m group and m/m+exercise group, body mass, liver mass, liver index, fasting blood glucose, triglycerides, total cholesterol, low-density lipoprotein and creatine kinase levels were significantly increased (P < 0.01), but the high-density lipoprotein level significantly decreased (P < 0.01) in the db/db group; the protein expressions of transforming growth factor-β1, p-Smad3, α-smooth muscle actin, type I collagen and type III collagen significantly increased (P < 0.01) in the db/db group; the area under curve of glucose and insulin tolerance tests significantly increased (P < 0.01) in the db/db group; and pathological staining of the liver in the db/db group showed extensive infiltration of inflammatory cells, increased lipid droplets, and significant fibrosis. In the db/db+exercise group, aerobic exercise could significantly reduce body mass, liver mass, liver index, fasting blood glucose, triglycerides, total cholesterol, low-density lipoprotein and creatine kinase levels (P < 0.05 or P < 0.01), and increase high-density lipoprotein level (P < 0.05). Similarly, a marked decrease was observed in the protein expression levels of transforming growth factor-β1, p-Smad3, α-smooth muscle actin, type I collagen and type III collagen (P < 0.05 or P < 0.01) in the db/db+exercise group. In addition, the area under the curve of glucose tolerance and insulin tolerance tests significantly decreased (P < 0.01, P < 0.05), and pathological changes in liver tissues were significantly improved. In conclusion, aerobic exercise can attenuate liver fibrosis in diabetic mice, which may be related to the regulation of the transforming growth factor-β1/Smad signaling pathway. [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]

Epithelial-to-mesenchymal transitions (EMTs) and extracellular matrix (ECM) remodeling are distinct yet important processes during carcinoma invasion and metastasis. Transforming growth factor β (TGF-β) and RAS, signaling through SMAD and RAS-responsive element-binding protein 1 (RREB1), jointly trigger expression of EMT and fibrogenic factors as two discrete arms of a common transcriptional response in carcinoma cells. Here, we demonstrate that both arms come together to form a program for lung adenocarcinoma metastasis and identify chromatin determinants tying the expression of the constituent genes to TGF-β and RAS inputs. RREB1 localizes to H4K16acK20ac marks in histone H2A.Z-loaded nucleosomes at enhancers in the fibrogenic genes interleukin-11 (IL11), platelet-derived growth factor-B (PDGFB), and hyaluronan synthase 2 (HAS2), as well as the EMT transcription factor SNAI1 , priming these enhancers for activation by a SMAD4-INO80 nucleosome remodeling complex in response to TGF-β. These regulatory properties segregate the fibrogenic EMT program from RAS-independent TGF-β gene responses and illuminate the operation and vulnerabilities of a bifunctional program that promotes metastatic outgrowth. [Display omitted] • TGF-β and RAS drive expression of EMT and fibrogenic genes that jointly fuel metastasis • Epigenetic determinants segregate different programs within a global TGF-β response • RAS/MAPK-regulated RREB1 primes enhancers for activation by SMAD-recruited INO80 • Targeting RREB1 selectively inhibits LUAD metastasis During carcinoma metastasis, malignant progenitors undergo a TGF-β-dependent EMT associated with fibroblast activation and extracellular matrix remodeling in the tumor microenvironment. RAS-activated RREB1 primes enhancers of EMT and fibrogenic genes in lung adenocarcinoma cells for activation by chromatin remodeling complexes that the TGF-β/SMAD pathway recruits to these enhancers. Inhibiting RREB1 disables this pro-metastatic process. [ABSTRACT FROM AUTHOR]

Fibrosis is a major medical challenge, as it leads to irreversible tissue remodeling and organ dysfunction. Its progression contributes significantly to morbidity and mortality worldwide, with limited therapeutic options available. Extensive research on the molecular mechanisms of fibrosis has revealed numerous factors and signaling pathways involved. However, the interactions between these pathways remain unclear. A comprehensive understanding of the entire signaling network that drives fibrosis is still missing. The TGF-β and Notch signaling pathways play a key role in fibrogenesis, and this review focuses on their functional interplay and molecular mechanisms. Studies have shown synergy between TGF-β and Notch cascades in fibrosis, but antagonistic interactions can also occur, especially in cardiac fibrosis. The molecular mechanisms of these interactions vary depending on the cell context. Understanding these complex and context-dependent interactions is crucial for developing effective strategies for treating fibrosis. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify miRNAs that could potentially influence the activity of SMAD proteins involved in TGFβ signal transduction in five types of breast cancer in Polish women. Patients with five breast cancer subtypes were included in the study: luminal A (n = 130), luminal B HER2− (n = 100), luminal B HER2+ (n = 96), non-luminal HER2+ (n = 36), and TNBC (n = 43). During surgery, tumor tissue was removed along with a margin of healthy tissue (control). Molecular analysis included determination of the expression of genes related to SMAD protein signal transduction using mRNA microarrays and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Protein expression was determined using an enzyme-linked immunosorbent assay (ELISA). The miRNA profiling was performed using miRNA microarrays and the miRDB database. SMAD3 and SMAD5 were overexpressed in all types of breast cancer, which could be related to the reduced expression of miR-145, and the findings for SMAD4 and miR-155 were similar. Additionally, the level of SMAD7 was reduced, which may be due to the low activity of miR-15b and miR21b. This study determined the gene expression profiles involved in SMAD protein signal transduction across five different types of breast cancer and identified the miRNAs potentially regulating their activity. Overexpression of SMAD3, SMAD4, and SMAD5 suggests excessive activation of the TGFβ pathway, potentially promoting tumor growth and development. Concurrently, a significant reduction in SMAD7 expression removes inhibitory control in the TGFβ pathway, a phenomenon that is particularly evident in more aggressive breast cancer types. [ABSTRACT FROM AUTHOR]

Although several methods are being applied to treat peripheral nerve injury, a perfect treatment that leads to full functional recovery has not yet been developed. SMAD (Suppressor of Mothers Against Decapentaplegic Homolog) plays a crucial role in nerve regeneration by facilitating the survival and growth of nerve cells following peripheral nerve injury. We conducted a systematic literature review on the role of SMAD in this context. Following peripheral nerve injury, there was an increase in the expression of SMAD1, -2, -4, -5, and -8, while SMAD5, -6, and -7 showed no significant changes; SMAD8 expression was decreased. Specifically, SMAD1 and SMAD4 were found to promote nerve regeneration, whereas SMAD2 and SMAD6 inhibited it. SMAD exerts its effects by promoting neuronal survival and growth through BMP/SMAD1, BMP/SMAD4, and BMP/SMAD7 signaling pathways. Furthermore, it activates nerve regeneration programs via the PI3K/GSK3/SMAD1 pathway, facilitating active regeneration of nerve cells and subsequent functional recovery after peripheral nerve damage. By leveraging these mechanisms of SMAD, novel strategies for treating peripheral nerve damage could potentially be developed. We aim to further elucidate the precise mechanisms of nerve regeneration mediated by SMAD and explore the potential for developing targeted nerve treatments based on these findings.

Purpose: To research the role of microRNA (miR)-152 in the pathogenesis of pulpitis using a cell model based on human dental pulp cells (HDPCs) treated with lipopolysaccharides (LPS). Materials and Methods: The biological activity of HDPCs infected by LPS was measured using a cell counting kit (CCK-8), Transwell test, flow cytometry, and fluorescent quantitative PCR. The concentration of superoxide dismutase (SOD) and malondialdehyde (MDA) was evaluated using an assay kit, the levels of interleukin (IL)-1β and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA), and the targeting relationship between SMAD5 and miR-152 was measured by the double-luciferase report test. The expression of cell cycle-related CyclinD1 and BAX was assessed by PCR. By plotting a receiver operating characteristic (ROC) curve, the diagnostic value of miR-152 was shown. Results: The level of miR-152 in HDPCs induced by LPS decreased, while the level of SMAD5 increased. After overexpressing miR-152 in LPS-induced HDPCs, the viability was elevated, the apoptosis rate decreased, CyclinD1 was elevated, BAX diminished, the inflammatory cytokines (IL-6 and IL-1β) were inhibited, the activity of SOD increased, and the MDA content decreased. miR-152 targeted regulation of SMAD5, and SMAD5 modulated the effects of miR-152 on cell viability, apoptosis, inflammation, and the oxidative response of HDPCs. Reduced miR-152 expression was verified in patients with pulpitis, which could be a biomarker for pulpitis. Conclusion: miR-152 was found to be a biomarker correlated with the pathogenesis of pulpitis and the biological behaviour of HDPCs. [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]

Effective interplay between the uterus and the embryo is essential for pregnancy establishment; however, convenient methods to screen embryo implantation success and maternal uterine response in experimental mouse models are currently lacking. Here, we report 3DMOUSEneST, a groundbreaking method for analyzing mouse implantation sites based on label-free higher harmonic generation microscopy, providing unprecedented insights into the embryo--uterine dynamics during early pregnancy. The 3DMOUSEneST method incorporates second-harmonic generation microscopy to image the three-dimensional structure formed by decidual fibrillar collagen, named 'decidual nest', and third-harmonic generation microscopy to evaluate early conceptus (defined as the embryo and extra-embryonic tissues) growth. We demonstrate that decidual nest volume is a measurable indicator of decidualization efficacy and correlates with the probability of early pregnancy progression based on a logistic regression analysis using Smad1/5 and Smad2/3 conditional knockout mice with known implantation defects. 3DMOUSEneST has great potential to become a principal method for studying decidual fibrillar collagen and characterizing mouse models associated with early embryonic lethality and fertility issues. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the use of 5,7,3′,4′-tetramethoxyflavone (TMF) to treat pulmonary fibrosis (PF), a chronic and fatal lung disease. In vitro and in vivo models were used to examine the impact of TMF on PF. NIH-3T3 (Mouse Embryonic Fibroblast) were exposed to transforming growth factor‑β1 (TGF-β1) and treated with or without TMF. Cell growth was assessed using the MTT method, and cell migration was evaluated with the scratch wound assay. Protein and messenger ribonucleic acid (mRNA) levels of extracellular matrix (ECM) genes were analyzed by western blotting and quantitative reverse transcription-polymerase chain reaction (RT–PCR), respectively. Downstream molecules affected by TGF-β1 were examined by western blotting. In vivo, mice with bleomycin-induced PF were treated with TMF, and lung tissues were analyzed with staining techniques. The in vitro results showed that TMF had no significant impact on cell growth or migration. However, it effectively inhibited myofibroblast activation and ECM production induced by TGF-β1 in NIH-3T3 cells. This inhibition was achieved by suppressing various signaling pathways, including Smad, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/AKT (PI3K/AKT), and WNT/β-catenin. The in vivo experiments demonstrated the therapeutic potential of TMF in reducing PF induced by bleomycin in mice, and there was no significant liver or kidney toxicity observed. These findings suggest that TMF has the potential to effectively inhibit myofibroblast activation and could be a promising treatment for PF. TMF achieves this inhibitory effect by targeting TGF-β1/Smad and non-Smad pathways. [ABSTRACT FROM AUTHOR]

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Hundreds of millions of people worldwide are expected to suffer from diabetes mellitus. Diabetes is characterized as a dynamic and heterogeneous disease that requires deeper understanding of the pathophysiology, genetics, and metabolic shaping of this disease and its macro/microvascular complications. Macrophages play an essential role in regulating local immune responses, tissue homeostasis, and disease pathogenesis. Here, we have analyzed transforming growth factor beta 1 (TGFβ1)/Smad signaling in primary human macrophages grown in normal (NG) and high-glucose (HG; +25 mM glucose) conditions. Cell culture lactate concentration and cellular phosphofructokinase (PFK) activity were increased in HG concentrations. High glucose levels in the growth media led to increased macrophage mRNA expression of TGFβ1, and TGFβ-regulated HAMP and PLAUR mRNA levels, while the expression of TGFβ receptor II remained unchanged. Stimulation of cells with TGFβ1 protein lead to Smad2 phosphorylation in both NG and HG conditions, while the phosphorylation of Smad1/5 was detected only in response to TGFβ1 stimulation in HG conditions. The use of the specific Alk1/2 inhibitor dorsomorphin and the Alk5 inhibitor SB431542, respectively, revealed that HG conditions led TGFβ1 to activation of Smad1/5 signaling and its downstream target genes. Thus, high-glucose activates TGFβ1 signaling to the Smad1/5 pathway in primary human macrophages, which may contribute to cellular homeostasis in a harmful manner, priming the tissues for diabetic complications. [ABSTRACT FROM AUTHOR]

The liver plays a crucial role in maintaining whole-body homeostasis in both health and disease, engaging in important communication with other organs. The coordination of multiple signaling pathways is essential for facilitating such interorgan communication. Among these pathways, the transforming growth factor-ß (TGF-ß) and HIPPO signaling pathways serve critical functions as tumor suppressors, exerting pivotal control over liver development, size, and tissue regeneration. In the normal hepatic context, these pathways exhibit significant crosstalk through various molecular mechanisms. This interaction is context-dependent within the hepatic microenvironment, regulating diverse cellular processes from development to adulthood. Disruptions in the regulation of these pathways and their crosstalk contribute to the onset of liver diseases. This review delves into the intricate interplay between the canonical pathways of TGF-ß and HIPPO, exploring their involvement in liver development and various pathologies such as fibrosis, cirrhosis, and tumorigenesis. Special attention is given to their impact on the epithelial-to-mesenchymal transition process, a crucial element associated with liver wound healing and cancer metastasis. By addressing these molecular interactions, the review aimed to provide insights into the underlying mechanisms that influence liver physiology and pathology, offering potential avenues for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-β) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-β and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-β1 (β1), 1 μM sphingosine-1-phosphate (S1P), and 5 μM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) β1/S1P; (3) β1/I2; prevention groups; (4) S1P/β1; and (5) I2/β1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-β signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-β binding proteins (LTBPs), TGF-β receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-β receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis. [ABSTRACT FROM AUTHOR]

Introduction: Colorectal cancer (CRC) is the second common cancer and the fourth major reason of cancer death worldwide. Dysregulation of intracellular pathways, such as TGF-β/SMAD signaling, contributes to CRC development. MicroRNAs (miRNAs) are post-transcriptional regulators that are involved in CRC pathogenesis. Here, we aimed to investigate the effect of miR-3613-3p on the TGF-β /SMAD signaling pathway in CRC. Methods & results: Bioinformatics analysis suggested that miR-3613-3p is a regulator of TGF-Β signaling downstream genes. Then, miR-3613-3p overexpression was followed by downregulation of TGF-βR1, TGF-βR2, and SMAD2 expression levels, detected by RT-qPCR. Additionally, dual luciferase assay supported the direct interaction of miR-3613-3p with 3'UTR sequences of TGF-βR1 and TGF-βR2 genes. Furthermore, reduced SMAD3 protein level following the miR-3613-3p overexpression verified its suppressive effect against TGF-β signaling in HCT-116 cells, detected by western blot analysis. Finally, miR-3613-3p overexpression induced sub-G1 arrest in HCT116 cells, detected by flow cytometry, and promoted downregulation of cyclin D1 protein expression, which was detected by western blotting analysis. Conclusion: Our findings indicated that miR-3613-3p plays an important role in CRC by targeting the TGF-β/SMAD signaling pathway and could be considered as a new candidate for further therapy investigations. [ABSTRACT FROM AUTHOR]

Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness (AHR), inflammation, and remodeling. Epithelial-mesenchymal transition (EMT) is an essential player in these alterations. Scutellarin is isolated from Erigeron breviscapus. Its vascular relaxative, myocardial protective, and anti-inflammatory effects have been well established. This study was designed to detect the biological roles of scutellarin in asthma and its related mechanisms. The asthma-like conditions were induced by ovalbumin challenges. The airway resistance and dynamic compliance were recorded as the results of AHR. Bronchoalveolar lavage fluid (BALF) was collected and processed for differential cell counting. Hematoxylin and eosin staining, periodic acid-Schiff staining, and Masson staining were conducted to examine histopathological changes. The levels of asthma-related cytokines were measured by enzyme-linked immunosorbent assay. For in vitro analysis, the 16HBE cells were stimulated with 10 ng/mL transforming growth beta-1 (TGF-β1). Cell migration was estimated by Transwell assays and wound healing assays. E-cadherin, N-cadherin, and α-smooth muscle actin (α-SMA) were analyzed by western blotting, real-time quantitative polymerase chain reaction, immunofluorescence staining, and immunohistochemistry staining. The underlying mechanisms of the mitogen-activated protein kinase (MAPK) and Smad pathways were investigated by western blotting. In an ovalbumin-induced asthmatic mouse model, scutellarin suppressed inflammation and inflammatory cell infiltration into the lungs and attenuated AHR and airway remodeling. Additionally, scutellarin inhibited airway EMT (upregulated E-cadherin level and downregulated N-cadherin and α-SMA) in ovalbumin-challenged asthmatic mice. For in vitro analysis, scutellarin prevented the TGF-β1-induced migration and EMT in 16HBE cells. Mechanistically, scutellarin inhibits the phosphorylation of Smad2, Smad3, ERK, JNK, and p38 in vitro and in vivo. In conclusion, scutellarin can inactivate the Smad/MAPK pathways to suppress the TGF-β1-stimulated epithelial fibrosis and EMT and relieve airway inflammation and remodeling in asthma. This study provides a potential therapeutic strategy for asthma. [ABSTRACT FROM AUTHOR]

Abstact: With advancing age, the incidence of sarcopenia increases, eventually leading to a cascade of adverse events. However, there is currently a lack of effective pharmacological treatment for sarcopenia. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) empagliflozin demonstrates anti-fibrotic capabilities in various organs. This study aims to determine whether empagliflozin can improve skeletal muscle fibrosis induced by sarcopenia in naturally aging mice. A natural aging model was established by feeding male mice from 13 months of age to 19 months of age. A fibrosis model was created by stimulating skeletal muscle fibroblasts with TGF-β1. The Forelimb grip strength test assessed skeletal muscle function, and expression levels of COL1A1, COL3A1, and α-SMA were analyzed by western blot, qPCR, and immunohistochemistry. Additionally, levels of AMPKα/MMP9/TGFβ1/Smad signaling pathways were examined. In naturally aging mice, skeletal muscle function declines, expression of muscle fibrosis markers increases, AMPKα expression is downregulated, and MMP9/TGFβ1/Smad signaling pathways are upregulated. However, treatment with empagliflozin reverses this phenomenon. At the cellular level, empagliflozin exhibits similar anti-fibrotic effects, and these effects are attenuated by Compound C and siAMPKα. Empagliflozin exhibits anti-fibrotic effects, possibly associated with the AMPK/MMP9/TGFβ1/Smad signaling pathways. [ABSTRACT FROM AUTHOR]

Abstract The transforming growth factor (TGF)-β3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-β3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-β3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-β3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-β3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-β3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

Abstract Background Osteoporosis is a genetic disease caused by the imbalance between osteoblast-led bone formation and osteoclast-induced bone resorption. However, further gene-related pathogenesis remains to be elucidated. Methods The aberrant expressed genes in osteoporosis was identified by analyzing the microarray profile GSE100609. Serum samples of patients with osteoporosis and normal group were collected, and the mRNA expression of candidate genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mouse cranial osteoblast MC3T3-E1 cells were treated with dexamethasone (DEX) to mimic osteoporosis in vitro. Alizarin Red staining and alkaline phosphatase (ALP) staining methods were combined to measure matrix mineralization deposition of MC3T3-E1 cells. Meanwhile, the expression of osteogenesis related genes including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Osterix, and bone morphogenetic protein 2 (BMP2) were evaluated by qRT-PCR and western blotting methods. Then the effects of candidate genes on regulating impede bone loss caused by ovariectomy (OVX) in mice were studied. Results Cyclin A1 (CCNA1) was found to be significantly upregulated in serum of osteoporosis patients and the osteoporosis model cells, which was in line with the bioinformatic analysis. The osteogenic differentiation ability of MC3T3-E1 cells was inhibited by DEX treatment, which was manifested by decreased Alizarin Red staining intensity, ALP staining intensity, and expression levels of ALP, OCN, OPN, Osterix, and BMP2. The effects of CCNA1 inhibition on regulating osteogenesis were opposite to that of DEX. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that genes negatively associated with CCNA1 were enriched in the TGF-beta signaling pathway. Inhibitor of TGF-beta signaling pathway partly reversed osteogenesis induced by suppressed CCNA1. Furthermore, suppressed CCNA1 relieved bone mass of OVX mice in vivo. Conclusion Downregulation of CCNA1 could activate TGF-beta signaling pathway and promote bone formation, thus playing a role in treatment of osteoporosis.

Abstract Osteoporosis is caused by the imbalance of osteoblasts and osteoclasts. The regulatory mechanisms of differentially expressed genes (DEGs) in pathogenesis of osteoporosis are of significant and needed to be further investigated. GSE100609 dataset downloaded from Gene Expression Omnibus (GEO) database was used to identified DEGs in osteoporosis patients. KEGG analysis was conducted to demonstrate signaling pathways related to enriched genes. Osteoporosis patients and the human mesenchymal stem cells (hMSCs) were obtained for in vivo and in vitro resaerch. Lentivirus construction and viral infection was used to knockdown genes. mRNA expression and protein expression were detected via qRT-PCR and western blot assay separately. Alkaline phosphatase (ALP) activity detection, alizarin Red S (ARS) staining, and expression of bone morphogenetic protein 2 (BMP2), osteocalcin (OCN) and Osterix were evaluated to determine osteoblast differentiation capacity. UL-16 binding protein 1 (ULBP1) gene was upregulated in osteoporosis and downregulated in differentiated hMSCs. Knockdown of ULBP1 increased ALP activity, mineralization ability evaluated by ARS staining, expression of BMP2, OCN and Osterix in differentiated hMSCs. Furthermore, rescue experiment demonstrated that suppressed ULBP1 boosted osteoblast differentiation by activating TNF-β signaling pathway. Knockdown of ULBP1 gene could promoted osteoblast differentiation by activating TNF-β signaling pathway in differentiated hMSCs. ULBP1 may be a the Achilles’ heel of osteoporosis, and suppression of ULBP1 could be a promising treatment for osteoporosis.

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]

Purpose: The aim of the study was to verify hypotheses: Are transforming growth factors TGFβ1-3, their receptors TGFβI-III, and intracellular messenger proteins Smad1-7 involved in the pathogenesis of kidney cancer? What is the expression of genes of the TGFβ/Smads pathway in renal cell carcinoma (RCC) tissues, peritumoral tissues (TME; tumor microenvironment), and in normal kidney (NK) tissue?. Methods: Twenty patients with RCC who underwent total nephrectomy were included into the molecular analysis. The mRNA expression of the genes was quantified by RT-qPCR. Results: The study showed that the expression of the genes of TGFβ/Smads pathway is dysregulated in both RCC and the TME: TGFβ1, TGFβ3 expression is increased in the TME in comparison to the NK tissues; TGFβ2, TGFβ3, TGFβRI, TGFβRIII, Smad1, Smad2, Smad3, and Smad6 are underexpressed in RCC comparing to the TME tissues; TGFβRI, TGFβRIII, and Smad2 are underexpressed in RCC in comparison to the NK tissues. Conclusion: On the one hand, the underexpression of the TGFβ signaling pathway genes within the malignant tumor may result in the loss of the antiproliferative and pro-apoptotic activity of this cytokine. On the other hand, the overexpression of the TGFβ/Smads pathway genes in the TME than in tumor or NK tissues most probably results in an immunosuppressive effect in the space surrounding the tumor and may have an antiproliferative and pro-apoptotic effect on non-neoplastic cells present in the TME. The functional and morphological consistency of this area may determine the aggressiveness of the tumor and the time in which the neoplastic process will spread. [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]

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Osteoporosis is caused by the imbalance of osteoblasts and osteoclasts. The regulatory mechanisms of differentially expressed genes (DEGs) in pathogenesis of osteoporosis are of significant and needed to be further investigated. GSE100609 dataset downloaded from Gene Expression Omnibus (GEO) database was used to identified DEGs in osteoporosis patients. KEGG analysis was conducted to demonstrate signaling pathways related to enriched genes. Osteoporosis patients and the human mesenchymal stem cells (hMSCs) were obtained for in vivo and in vitro resaerch. Lentivirus construction and viral infection was used to knockdown genes. mRNA expression and protein expression were detected via qRT-PCR and western blot assay separately. Alkaline phosphatase (ALP) activity detection, alizarin Red S (ARS) staining, and expression of bone morphogenetic protein 2 (BMP2), osteocalcin (OCN) and Osterix were evaluated to determine osteoblast differentiation capacity. UL-16 binding protein 1 (ULBP1) gene was upregulated in osteoporosis and downregulated in differentiated hMSCs. Knockdown of ULBP1 increased ALP activity, mineralization ability evaluated by ARS staining, expression of BMP2, OCN and Osterix in differentiated hMSCs. Furthermore, rescue experiment demonstrated that suppressed ULBP1 boosted osteoblast differentiation by activating TNF-β signaling pathway. Knockdown of ULBP1 gene could promoted osteoblast differentiation by activating TNF-β signaling pathway in differentiated hMSCs. ULBP1 may be a the Achilles' heel of osteoporosis, and suppression of ULBP1 could be a promising treatment for osteoporosis. [ABSTRACT FROM AUTHOR]

Background: Osteoporosis is a genetic disease caused by the imbalance between osteoblast-led bone formation and osteoclast-induced bone resorption. However, further gene-related pathogenesis remains to be elucidated. Methods: The aberrant expressed genes in osteoporosis was identified by analyzing the microarray profile GSE100609. Serum samples of patients with osteoporosis and normal group were collected, and the mRNA expression of candidate genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mouse cranial osteoblast MC3T3-E1 cells were treated with dexamethasone (DEX) to mimic osteoporosis in vitro. Alizarin Red staining and alkaline phosphatase (ALP) staining methods were combined to measure matrix mineralization deposition of MC3T3-E1 cells. Meanwhile, the expression of osteogenesis related genes including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Osterix, and bone morphogenetic protein 2 (BMP2) were evaluated by qRT-PCR and western blotting methods. Then the effects of candidate genes on regulating impede bone loss caused by ovariectomy (OVX) in mice were studied. Results: Cyclin A1 (CCNA1) was found to be significantly upregulated in serum of osteoporosis patients and the osteoporosis model cells, which was in line with the bioinformatic analysis. The osteogenic differentiation ability of MC3T3-E1 cells was inhibited by DEX treatment, which was manifested by decreased Alizarin Red staining intensity, ALP staining intensity, and expression levels of ALP, OCN, OPN, Osterix, and BMP2. The effects of CCNA1 inhibition on regulating osteogenesis were opposite to that of DEX. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that genes negatively associated with CCNA1 were enriched in the TGF-beta signaling pathway. Inhibitor of TGF-beta signaling pathway partly reversed osteogenesis induced by suppressed CCNA1. Furthermore, suppressed CCNA1 relieved bone mass of OVX mice in vivo. Conclusion: Downregulation of CCNA1 could activate TGF-beta signaling pathway and promote bone formation, thus playing a role in treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

The Wnt/β-catenin signaling pathway plays important roles in several cancer cells, including cell proliferation and development. We previously succeeded in synthesizing a small molecule compound inhibiting the Wnt/β-catenin signaling pathway, named LPD-01 (1), and 1 inhibited the growth of human colorectal cancer (HT-29) cells. In this study, we revealed that 1 inhibits the growth of HT-29 cells stronger than that of another human colorectal cancer (SW480) cells. Therefore, we have attempted to identify the target proteins of 1 in HT-29 cells. Firstly, we investigated the effect on the expression levels of the Wnt/β-catenin signaling pathway-related proteins. As a result, 1 inhibited the expression of target proteins of Wnt/β-catenin signaling pathway (c-Myc and Survivin) and their genes, whereas the amount of transcriptional co-activator (β-catenin) was not decreased, suggesting that 1 inhibited the Wnt/β-catenin signaling pathway without affecting β-catenin. Next, we investigated the target proteins of 1 using magnetic FG beads. Chemical pull-down assay combined with mass spectrometry suggested that 1 directly binds to importin7. As expected, 1 inhibited the nuclear translocation of importin7 cargoes such as Smad2 and Smad3 in TGF-β-stimulated HT-29 cells. In addition, the knockdown of importin7 by siRNA reduced the expression of target genes of Wnt/β-catenin signaling pathway. These results suggest that importin7 is one of the target proteins of 1 for inhibition of the Wnt/β-catenin signaling pathway. [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.

The activation of hepatic stellate cells (HSCs) is regarded as the primary driving factor of liver fibrosis. miR-192, a miRNA associated with hepatocellular carcinoma and enriched in HSCs, has an undisclosed role in HSC activation and liver fibrosis. In this study, a CCl4-induced rat liver fibrosis model and transforming growth factor-beta 1 (TGF-β1)-treated HSC lines (LX-2 and HSC-T6) were used to detect miR-192 and Rictor levels in vivo and in vitro. Bioinformatic analysis and a dual luciferase assay were used to predict and confirm the interaction of Rictor with miR-192. Gain- and/or loss-of-function methods evaluated molecular changes and HSC activation phenotypes, detected by quantitative real-time PCR, western blotting, and immunofluorescence. We observed a gradual downregulation of miR-192 and upregulation of Rictor during CCl4-induced liver fibrosis/cirrhosis in rats. Enriched miR-192 was downregulated, while Rictor was upregulated in TGF-β1-activated HSCs. miR-192 inhibited the activation of HSCs by directly targeting Rictor. High miR-192/low Rictor expression attenuated the fibrotic-related gene expression by AKT/mTORC2 signaling. In conclusion, miR-192 could inhibit the activation of HSCs by directly targeting Rictor in the AKT/mTORC2 signaling pathway. This study provides insights into potential therapeutic targets for liver fibrosis and cirrhosis.

AbstractContext Fibroblast senescence was reported to contribute to the pathological development of idiopathic pulmonary fibrosis (IPF), and baicalein is reported to attenuate IPF.Objective This study explores whether baicalein attenuates lung fibrosis by regulating lung fibroblast senescence.Materials and methods Institute of Cancer Research (ICR) mice were randomly assigned to control, bleomycin (BLM), baicalein and BLM + baicalein groups. Lung fibrosis was established by a single intratracheal dose of BLM (3 mg/kg). The baicalein group received baicalein orally (100 mg/kg/day). Sirtuin 3 (Sirt3) siRNA (50 μg) was injected through the tail vein once a week for 2 weeks to explore its effect on the anti-pulmonary fibrosis of baicalein.Results BLM-treated mice exhibited obvious lung fibrosis and fibroblast senescence by showing increased levels of collagen deposition (27.29% vs. 4.14%), hydroxyproline (208.05 vs. 40.16 ng/mg), collagen I (25.18 vs. 9.15 μg/mg), p53, p21, p16, MCP-1, PAI-1, TNF-α, MMP-10 and MMP-12 in lung tissues, which were attenuated by baicalein. Baicalein also mitigated BLM-mediated activation of TGF-β1/Smad signalling pathway. Baicalein restored the BLM-induced downregulation of Sirt3 expression in lung tissues and silencing of Sirt3 abolished the inhibitory role of baicalein against BLM-induced lung fibrosis, fibroblast senescence and activation of TGF-β1/Smad signalling pathway.Conclusions Baicalein preserved the BLM-induced downregulation of lung Sirt3 expression, and thus the suppression of TGF-β1/Smad signalling pathway and lung fibrosis, which might provide an experimental basis for treatment of IPF.

Abstract Methyltransferase-like 3 (METTL3) is the key subunit of methyltransferase complex responsible for catalyzing N6-methyladenosine (m6A) modification on mRNA, which is the most prevalent post-transcriptional modification in eukaryotes. In this study, we utilized online databases to analyze the association between METTL3 expression and various aspects of tumorigenesis, including gene methylation, immunity, and prognosis. Our investigation revealed that METTL3 serves as a prognostic marker and therapeutic target for liver hepatocellular carcinoma (LIHC). Through experimental studies, we observed frequent upregulation of METTL3 in LIHC tumor tissue and cells. Subsequent inhibition of METTL3 using a novel small molecule inhibitor, STM2457, significantly impeded tumor growth in LIHC cell lines, spheroids, and xenograft tumor model. Further, transcriptome and m6A sequencing of xenograft bodies unveiled that inhibition of METTL3-m6A altered genes enriched in SMAD and MAPK signaling pathways that are critical for tumorigenesis. These findings suggest that targeting METTL3 represents a promising therapeutic strategy for LIHC.

Development of the mammalian oocyte requires physical contact with the surrounding granulosa cells of the follicle, which provide it with essential nutrients and regulatory signals. This contact is achieved through specialized filopodia, termed transzonal projections (TZPs), that extend from the granulosa cells to the oocyte surface. Transforming growth factor (TGFβ) family ligands produced by the oocyte increase the number of TZPs, but how they do so is unknown. Using an inducible Cre recombinase strategy together with expression of green fluorescent protein to verify Cre activity in individual cells, we examined the effect of depleting the canonical TGFβ mediator, SMAD4, in mouse granulosa cells. We observed a 20–50% decrease in the total number of TZPs in SMAD4-depleted granulosa cell-oocyte complexes, and a 50% decrease in the number of newly generated TZPs when the granulosa cells were reaggregated with wild-type oocytes. Three-dimensional image analysis revealed that TZPs of SMAD4-depleted cells were longer than controls and more frequently oriented towards the oocyte. Strikingly, the transmembrane proteins, N-cadherin and Notch2, were reduced by 50% in SMAD4-depleted cells. SMAD4 may thus modulate a network of cell adhesion proteins that stabilize the attachment of TZPs to the oocyte, thereby amplifying signalling between the two cell types.

Abstract 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.

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]