NEK2 promotes colorectal cancer progression by activating the TGF-ß/Smad2 signaling pathway.

Colorectal cancer (CRC) is a prevalent malignancy with poor patient survival, and NIMA-associated kinase 2 (NEK2) has been implicated in the pathogenesis and progression of various cancers, including CRC. This study aimed to investigate the impact of NEK2 on CRC cell functionality and its interaction with the TGF-ß/Smad signaling pathway. NEK2 expression in CRC tissues and cell lines was assessed, and its association with patient survival was analyzed. Functional assays, including NEK2 knockdown via lentiviral infection, RT-qPCR, Western blotting, CCK-8 assay, Transwell migration, invasion assays, and goblet cell formation assays, were employed to evaluate NEK2's effects on CRC cell proliferation, migration, invasion, and stemness. Mechanistic studies explored the TGF-ß/Smad2 signaling pathway, utilizing co-immunoprecipitation (Co-IP) and protein interaction analyses. In vivo experiments further evaluated NEK2's role in tumor initiation, metastasis, and chemoresistance. NEK2 was found to be upregulated in CRC tissues and correlated with poor survival. NEK2 knockdown inhibited CRC cell behaviors, while NEK2 activated the TGF-ß/Smad2 signaling pathway through Smad2/3 phosphorylation. Overexpression of Smad2/3 reversed NEK2 knockdown effects, confirming the importance of this pathway in CRC. In vivo, NEK2 promoted tumor initiation, metastasis, and chemoresistance, effects partially reversed by Smad2/3 overexpression. These findings reveal the critical role of NEK2 in CRC progression and underscore its potential as a therapeutic target, offering new insights into the molecular mechanisms driving CRC and informing targeted therapy development.

TGF-ß/Smad signaling pathway in fatty liver disease: a case-control study.

BACKGROUND: Fatty liver disease is a metabolic disorder that recently has been classified into two categories: metabolic dysfunction-associated fatty liver disease (MAFLD) and non-MAFLD. TGF-ß signaling pathway is likely a significant factor in the pathogenesis of this condition, exerting its effects through its downstream signaling proteins, Smad2/3. Accordingly, this study aimed to investigate the TGF-ß signaling pathway in the white blood cells (WBCs) of patients with MAFLD compared to those with non-MAFLD and control groups. METHODS AND RESULTS: In this study, 41 patients with fatty liver were evaluated, comprising 22 patients with MAFLD and 19 patients with non-MAFLD, and compared to 22 healthy controls. Gene expression of TGF-ß1, TGF-ß3, and CTGF were quantified using qRT-PCR, and the protein expressions of Smad2/3 and P-Smad2/3 were analyzed using western blotting. Gene expression analysis revealed a significant decrease in the gene expressions of the TGF-ß1 and TGF-ß3 and an increase in CTGF gene expression in patients with MAFLD and non-MAFLD compared to the control group. Notably, the Smad2/3 protein expression was significantly higher in the non-MAFLD group compared to the control group (P < 0.05). On the other hand, the P-smad2/3 protein expression was significantly elevated in the MAFLD group compared to the control group (P < 0.001). CONCLUSIONS: TGF-ß signaling pathway in WBCs of patients with fatty liver are affected by a complex signaling pathway. However, metabolic factors most probably affect TGF-ß1 gene expression and its downstream signaling proteins more than TGF-ß3.

TGF-ß and TNF-α interaction promotes the expression of MMP-9 through H3K36 dimethylation: implications in breast cancer metastasis.

Increased MMP-9 expression in the tumor microenvironment (TME) plays a crucial role in the extracellular matrix remodeling to facilitate cancer invasion and metastasis. However, the mechanism of MMP-9 upregulation in TME remains elusive. Since TGF-ß and TNF-α levels are elevated in TME, we asked whether these two agents interacted to induce/augment MMP-9 expression. Using a well-established MDA-MB-231 breast cancer model, we found that the synergy between TGF-ß and TNF-α led to MMP-9 upregulation at the transcriptional and translational levels, compared to treatments with each agent alone. Our in vitro findings are corroborated by co-expression of elevated MMP-9 with TGF-ß and TNF-α in human breast cancer tissues. Mechanistically, we found that the MMP-9 upregulation driven by TGF-ß/TNF-α cooperativity was attenuated by selective inhibition of the TGF-ßRI/Smad3 pathway. Comparable outcomes were observed upon inhibition of TGF-ß-induced phosphorylation of Smad2/3 and p38. As expected, the cells defective in Smad2/3 or p38-mediated signaling did not exhibit this synergistic induction of MMP-9. Importantly, the inhibition of histone methylation but not acetylation dampened the synergistic MMP-9 expression. Histone modification profiling further identified the H3K36me2 as an epigenetic regulatory mark of this synergy. Moreover, TGF-ß/TNF-α co-stimulation led to increased levels of the transcriptionally permissive dimethylation mark at H3K36 in the MMP-9 promoter. Comparable outcomes were noted in cells deficient in NSD2 histone methyltransferase. In conclusion, our findings support a cooperativity model in which TGF-ß could amplify the TNF-α-mediated MMP-9 production via chromatin remodeling and facilitate breast cancer invasion and metastasis.

The fruit of Rosa odorata sweet var. gigantea (Coll. et Hemsl.) Rehd. et Wils attenuates chronic atrophic gastritis induced by MNNG and its potential mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosa odorata Sweet var. gigantea (Coll. et Hemsl.) Rehd. et Wils is a commonly utilized traditional medicine among the Yi nationality, also known as "Gugongguo", for the treatment of gastrointestinal disorders. Previous studies have indicated that the extract of Rosa odorata sweet var. gigantea (FOE) fruit has demonstrated a protective effect on the stomach; however, its impact on chronic atrophic gastritis (CAG) with severe disease remains unknown. AIM OF THE STUDY: This study aimed to investigate the impact of FOE on CAG and its underlying mechanisms both in vitro and in vivo. MATERIALS AND METHODS: By employing Ultra Performance Liquid Chromatography/Quadrupole-Time of Flight Mass Spectrometry (UPLC-QTOF-MS/MS) and network pharmacology, the primary active compounds and action targets of FOE were identified. In vitro, the impact of FOE on CAG was investigated through scratch, migration, and invasion assays. Subsequently, guided by network pharmacology, EMT and TGF-ß signaling pathway-related proteins were assessed using Western blot and immunofluorescence experiments. Additionally, an in vivo CAG rat model was established to validate the effects of FOE and confirm its mechanism of action through hematoxylin-eosin (H&E), immunohistochemistry, Western blot, as well as untargeted metabolomics analysis of rat serum. It was observed that FOE inhibited scratch healing abilities, migration, invasion capabilities, as well as the expression of EMT-related proteins (E-cadherin, N-cadherin, Snail, Vimentin) in CAG model cells (MC cells), providing initial evidence for its efficacy. RESULTS: Through the analysis of UPLC-QTOF-MS/MS, a total of 51 major compounds were identified in the FOE. Subsequent network pharmacological analysis suggested that FOE may regulate Epithelial mesenchymal transition (EMT) through the transforming growth factor ß (TGF-ß) pathway. Furthermore, experimental verification demonstrated that FOE inhibited the protein expression of TGF-ß1 and its downstream protein Smad2/3 in vitro. In vivo findings also indicated similar mechanisms in MC cells, suggesting a reversal of the CAG process and significant inhibition of EMT and TGF-ß signaling pathways. Additionally, untargeted metabolomics of rat serum confirmed the therapeutic effect of FOE on CAG and predicted its potential involvement in the arachidonic acid metabolic pathway. CONCLUSION: This study initially demonstrated that FOE effectively reverses the process of EMT through the TGF-ß1/Smad2/3 signaling pathway, thereby providing a therapeutic benefit for CAG.

SOX17 Prevents Endothelial-Mesenchymal Transition of Pulmonary Arterial Endothelial Cells in Pulmonary Hypertension through Mediating TGF-ß/Smad2/3 Signaling.

Endothelial to mesenchymal transition (EndMT) has been reported to cause pulmonary vascular remodeling of pulmonary hypertension (PH). We have demonstrated that SOX17, a member of the SRY-Box (SOX) transcription factor family, affects pulmonary artery vascular homeostasis through exosomes in an autocrine and paracrine manner. However, the role of SOX17 in mediating EndMT of pulmonary arterial endothelial cells (PAECs) in PH and its underlying intracellular mechanisms are not yet clarified. Here, we show that in the remodeling pulmonary vascular of idiopathic PH patients and Sugen 5416/hypoxia (Sugen/hypoxia)-induced PH rats, the downregulation of SOX17 expression was accompanied by a significant pulmonary arterial EndMT and TGF-ß/Smad2/3 signaling activation. In primary HPAECs, the expression of SOX17 was inhibited by canonical TGF-ß signaling. SOX17 overexpression reversed TGF-ß- and hypoxia-induced EndMT. It is suggested that SOX17 is required for HPAECs to acquire TGF-ß-mediated EndMT. Mechanistically, SOX17 prevented TGF-ß-induced EndMT of PAECs through trans-suppressing ROCK1 expression by binding to the specific promoter region of ROCK1, thereby inhibiting the phosphorylation of MYPT1 and MLC. Further, we found that Tie2-Cre rats with endothelial cell-specific SOX17 overexpression were prevented from Sugen/hypoxia-induced EndMT and pulmonary vascular remodeling. In keeping with the in vitro data, compared with the Tie2-Cre rats treated by Sugen/hypoxia, the rats with SOX17 overexpression showed decreased expression of ROCK1 as well as the MYPT1 and MLC phosphorylation. Overall, our studies demonstrate a novel TGF-ß/SOX17/ROCK1 pathway regulating EndMT of PAECs and propose SOX17 as a potential target for exploring therapeutics to alleviate pulmonary vascular remodeling in PH.

NLRP3 deficiency improves bone healing of tooth extraction sockets through SMAD2/3-RUNX2 mediated osteoblast differentiation.

Impaired bone healing following tooth extraction poses a significant challenge for implantation. As a crucial component of the natural immune system, the NLRP3 inflammasome is one of the most extensively studied Pattern-Recognition Receptors (PRRs), and is involved in multiple diseases. Yet, the role of NLRP3 in bone healing remains to be clarified. Here, to investigate the effect of NLRP3 on bone healing, we established a maxillary first molar extraction model in wild-type (WT) and NLRP3KO mice using minimally invasive techniques. We observed that NLRP3 was activated during the bone repair phase, and its depletion enhanced socket bone formation and osteoblast differentiation. Moreover, NLRP3 inflammasome activation was found to inhibit osteogenic differentiation in alveolar bone-derived mesenchymal stem cells (aBMSCs), an effect mitigated by NLRP3 deficiency. Mechanistically, we established that SMAD2/3-RUNX2 signaling pathway is a downstream target of NLRP3 inflammasome activation, and SMAD2/3 knockdown partially reversed the significant decrease in expression of RUNX2, OSX, and ALP induced by NLRP3. Thus, our findings demonstrate that NLRP3 negatively modulates alveolar socket bone healing and contribute to the understanding of the NLRP3-induced signaling pathways involved in osteogenesis regulation.

LMK235 ameliorates inflammation and fibrosis after myocardial infarction by inhibiting LSD1-related pathway.

BACKGROUND: Histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5) are two isoforms of class IIa HDACs, and LMK235 is an HDAC inhibitor with higher selectivity for HDAC4/5. This study aimed to explore the expression and subcellular localization of HDAC4/5 and determine the mechanisms underlying the impact of LMK235 on ventricular remodelling post-MI. METHODS: The MI model was established by left anterior descending branch (LAD) ligation, and LMK235 or vehicle was intraperitoneally injected daily for 21 days. Cardiac function was determined by echocardiography. Inflammation was evaluated by HE staining and measuring inflammatory cytokine expression, and fibrosis was evaluated by Masson staining and measuring fibrotic biomarker expression. RESULTS: We found that LMK235 ameliorated cardiac dysfunction post-MI by suppressing inflammation and fibrosis, and LMK235 inhibited upregulation of lysine-specific demethylase 1 (LSD1) expression post-MI. In macrophages, LMK235 attenuated lipopolysaccharide (LPS) - induced inflammatory cytokine expression and inhibited LSD1 expression, while overexpression of LSD1 abrogated the anti-inflammatory effect of LMK235. In cardiac fibroblasts, LMK235 attenuated transforming growth factor-ß1 (TGF-ß1) - induced fibrotic biomarker expression and inhibited LSD1 expression, while overexpression of LSD1 abrogated the antifibrotic effect of LMK235. CONCLUSION: LMK235 attenuates chronic inflammation and fibrosis post-MI, leading to improved cardiac function. The anti-inflammatory effect of LMK235 may result from inhibition of the LSD1-NF-κB pathway in macrophages. The antifibrotic effect of LMK235 may result from inhibition of the LSD1-Smad2/3 pathway in cardiac fibroblasts.

ß-Mangostin Alleviates Renal Tubulointerstitial Fibrosis via the TGF-ß1/JNK Signaling Pathway.

The epithelial-to-mesenchymal transition (EMT) plays a key role in the pathogenesis of kidney fibrosis, and kidney fibrosis is associated with an adverse renal prognosis. Beta-mangostin (ß-Mag) is a xanthone derivative obtained from mangosteens that is involved in the generation of antifibrotic and anti-oxidation effects. The purpose of this study was to examine the effects of ß-Mag on renal tubulointerstitial fibrosis both in vivo and in vitro and the corresponding mechanisms involved. As shown through an in vivo study conducted on a unilateral ureteral obstruction mouse model, oral ß-Mag administration, in a dose-dependent manner, caused a lesser degree of tubulointerstitial damage, diminished collagen I fiber deposition, and the depressed expression of fibrotic markers (collagen I, α-SMA) and EMT markers (N-cadherin, Vimentin, Snail, and Slug) in the UUO kidney tissues. The in vitro part of this research revealed that ß-Mag, when co-treated with transforming growth factor-ß1 (TGF-ß1), decreased cell motility and downregulated the EMT (in relation to Vimentin, Snail, and N-cadherin) and phosphoryl-JNK1/2/Smad2/Smad3 expression. Furthermore, ß-Mag co-treated with SB (Smad2/3 kinase inhibitor) or SP600125 (JNK kinase inhibitor) significantly inhibited the TGF-ß1-associated downstream phosphorylation and activation of JNK1/2-mediated Smad2 targeting the Snail/Vimentin axis. To conclude, ß-Mag protects against EMT and kidney fibrotic processes by mediating the TGF-ß1/JNK/Smad2 targeting Snail-mediated Vimentin expression and may have therapeutic implications for renal tubulointerstitial fibrosis.

SIRT7 protects against liver fibrosis by suppressing stellate cell activation via TGF-ß/SMAD2/3 pathway.

BACKGROUND: SIRT7 is a class III HDACs deacetylase which plays critical roles in various biological processes. Aberrant SIRT7 expression is associated with tumorigenesis and disease progression while role of SIRT7 in hepatic fibrosis remain elusive. METHODS: SIRT7 expression was examined in fibrotic liver sample via WB and IHC. Myeloid cell-specific knockout (SIRT7MKO) mice were generated by crossing SIRT7flox/flox mice with LysM-Cre mice. Primary hepatic stellate cells (HSCs) was isolated to examine stellate cells activation. SIRT7 and SMAD2/3 interaction were analyzed by immunoprecipitation. SB525334 was used to prevent SMAD2/3 phosphorylation. RESULTS: SIRT7 expression was decreased during chronic liver disease progression but was increased in liver cancer. IHC staining indicated that SIRT7 was primarily expressed in non-parenchymal cells in both fibrotic and cirrhotic liver. Knockout SIRT7 in myeloid cells resulted in significant elevation of serum ALT and liver fibrosis, but mildly affected hepatic inflammation after CCl4 treatment. We further observed significant elevation of elevation of stellate cell activation and SMAD2/3 activation in SIRT7MKO mice. By using primary HSCs and stellate cell line, we confirmed that SIRT7 interacted with SMAD2/3, induced its deacetylation and was critical in regulation of SMAD2/3 activation and stellate cell activation upon TGF-ß stimulation. Pharmacological inhibition of SMAD2/3 reversed the hyperactivation of SIRT7MKO HSCs after TGF-ß stimulation, and abolished stellate cell activation and liver fibrosis in SIRT7MKO mice. CONCLUSION: Our findings revealed previously unidentified role of SIRT7 in regulating HSCs activation via modulating TGF-ß/SMAD2/3 signaling pathway. Targeting SIRT7 might offer novel therapeutic option against liver fibrosis.

TIPE2 inhibits the migration and invasion of epithelial ovarian cancer cells by targeting Smad2 to reverse TGF-ß1-induced EMT.

Epithelial ovarian cancer is the deadliest gynecologic malignancy, characterized by high metastasis. Transforming growth factor-ß1 (TGF-ß1) drives epithelial- mesenchymal transformation (EMT), a key process in tumor metastasis. Tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TIPE2) acts as a negative regulator of innate and adaptive immunity and involves in various cancers. However, its relationship with TGF-ß1 in ovarian cancer and its role in reversing TGF-ß1-induced EMT remain unclear. This study examined TIPE2 mRNA and protein expression using quantitative RT-PCR (qRT-PCR), western blot and immunohistochemistry. The effects of TIPE2 overexpression and knockdown on the proliferation, migration and invasion of epithelial ovarian cancer cells were assessed through 5-ethynyl-2-deoxyuridine, colony-forming, transwell migration and invasion assays. The relationship between TIPE2 and TGF-ß1 was investigated using qRT-PCR and enzyme-linked immunosorbent assay, while the interaction between TIPE2 and Smad2 was identified via co-immunoprecipitation. The results revealed that TIPE2 protein was significantly down-regulated in epithelial ovarian cancer tissues and correlated with the pathological type of tumor, patients' age, tumor differentiation degree and FIGO stage. TIPE2 and TGF-ß1 appeared to play an opposite role to each other during the progression of human ovarian cancer cells. Furthermore, TIPE2 inhibited the metastasis and EMT of ovarian cancer cells by combining with Smad2 in vitro or in an intraperitoneal metastasis model. Consequently, these findings suggest that TIPE2 plays a crucial inhibitory role in ovarian cancer metastasis by modulating the TGF-ß1/Smad2/EMT signaling pathway and may serve as a potential target for ovarian cancer, providing important direction for future diagnostic and therapeutic strategies.

[Retracted] Jumonji AT­rich interactive domain 1B overexpression is associated with the development and progression of glioma.

Following the publication of this paper, and subsequently to the publication of a corrigendum (DOI: 10.3892/ijmm.2016.2682) that was intended to address the issue of misassembled data in Figs. 3, 5 and 8, it was drawn to the Editor's attention by a concerned reader that certain of the scratch­wound assay data shown in Fig. 5B were strikingly similar to data appearing in different form in an article written by different authors at different research institutes that had already been published in the journal Cancer Research. In view of the fact that the abovementioned data had already apparently been published prior to its submission to International Journal of Molecular Medicine, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Journal of Molecular Medicine 38: 172­182, 2016; DOI: 10.3892/ijmm.2016.2614].

Evaluation of CNPase and TGFß1/Smad Signalling Pathway Molecule Expression in Sinus Epithelial Tissues of Patients with Chronic Rhinosinusitis with (CRSwNP) and without Nasal Polyps (CRSsNP).

Chronic rhinosinusitis with and without nasal polyps (CRSwNP and CRSsNP, respectively) is a chronic inflammatory disease affecting almost 5 to 12% of the population and exhibiting high recurrence rates after functional endoscopic sinus surgery (FESS). TGFß1-related pathways contribute to tissue remodelling, which is one of the key aspects of CRS pathogenesis. Additionally, adenosine signalling participates in inflammatory processes, and CNPase was shown to elevate adenosine levels by metabolizing cyclic monophosphates. Thus, the aim of this study was to assess the expression levels of Smad2, pSmad3, TGFß1, and CNPase protein via immunohistochemistry in sinus epithelial tissues from patients with CRSwNP (n = 20), CRSsNP (n = 23), and non-CRS patients (n = 8). The expression of Smad2, pSmad3, TGFß1, and CNPase was observed in the sinus epithelium and subepithelial area of all three groups of patients, and their expression correlated with several clinical symptoms of CRS. Smad2 expression was increased in CRSsNP patients compared to CRSwNP patients and controls (p = 0.001 and p < 0.001, respectively), pSmad3 expression was elevated in CRSwNP patients compared to controls (p = 0.007), TGFß1 expression was elevated in CRSwNP patients compared to controls (p = 0.009), and CNPase was decreased in CRSsNP patients compared to controls (p = 0.03). To the best of our knowledge, we are the first to demonstrate CNPase expression in the upper airway epithelium of CRSwNP, CRSsNP, and non-CRS patients and point out a putative synergy between CNPase and TGFß1/Smad signalling in CRS pathogenesis that emerges as a novel still undiscovered aspect of CRS pathogenesis; further studies are needed to explore its function in the course of the chronic inflammation of the upper airways.

GAMG ameliorates silica-induced pulmonary inflammation and fibrosis via the regulation of EMT and NLRP3/TGF-ß1/Smad signaling pathway.

Silicosis is an occupational disease caused by exposure to silica characterized by pulmonary inflammation and fibrosis, for which there is a lack of effective drugs. Glycyrrhetinic acid 3-O-ß-D-glucuronide (GAMG) can treat silicosis due to its anti-inflammatory and anti-fibrotic properties. Here, the effect of therapeutic interventions of GAMG was evaluated in early-stage and advanced silicosis mouse models. GAMG significantly improved fibrotic pathological changes and collagen deposition in the lungs, alleviated lung inflammation in the BALF, reduced the expression of TNF-α, IL-6, NLRP3, TGF-ß1, vimentin, Col-Ⅰ, N-cadherin, and inhibited epithelial-mesenchymal transition (EMT), thereby ameliorating pulmonary fibrosis. Moreover, the dose of 100 mg/kg GAMG can effectively prevent early-stage silicosis, while that of 200 mg/kg was recommended for advanced silicosis. In vitro and in vivo study verified that GAMG can suppress EMT through the NLRP3/TGF-ß1/Smad2/3 signaling pathway. Therefore, GAMG could be a promising preventive (early-stage silicosis) and therapeutic (advanced silicosis) strategy, which provides a new idea for formulating prevention and treatment strategies.

Cannabidiol suppresses silica-induced pulmonary inflammation and fibrosis through regulating NLRP3/TGF-ß1/Smad2/3 pathway.

Silica-induced pulmonary fibrosis is an irreversible and progressive lung disease with limited treatments available. In this work, FDA-approved cannabidiol (CBD) was studied for its potential medical use in silicosis. In silicosis female C57BL/6 mice model, oral CBD or pirfenidone (PFD) on day 1 after intratracheal drip silica (150 mg/mL) and continued for 42 days. Lung inflammatory and fibrotic changes were studied using ELISA kits, H&E staining and Masson staining. Osteopontion (OPN) and α-smooth muscle actin (α-SMA) expression in lung tissues was determined using immunohistochemical staining. The results indicated that CBD attenuated silica-induced pulmonary inflammation and fibrosis. Human myeloid leukemia mononuclear cells (THP-1) were treated with silica (200 µg/mL) to induce cell damage, then CBD (10 µM, 20 µM) and PFD (100 µM) were incubated. In vitro experiments showed that CBD can effectively reduce the expression of NLRP3 inflammasome in THP-1 cells and subsequently block silica-stimulated transformation of fibromuscular-myofibroblast transition (FMT) by culturing human embryonic lung fibroblasts (MRC-5) in conditioned medium of THP-1 cells. Therefore, CBD exhibited the potential therapy for silicosis through inhibiting the silica-induced pulmonary inflammation and fibrosis via the NLRP3/TGF-ß1/Smad2/3 signaling pathway.

Low-dose SAHA enhances CD8+ T cell-mediated antitumor immunity by boosting MHC I expression in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a highly aggressive type of lung cancer with poor responses to traditional therapies such as surgery, radiotherapy, and chemotherapy. While immunotherapy has become an effective approach for treating multiple types of cancer, solid tumors frequently exhibit immune escape through various mechanisms, including downregulation of MHC I expression. However, whether the upregulation of MHC I expression can improve the immunotherapeutic effect on NSCLC remains unexplored. Suberoylanilide hydroxamic acid (SAHA) is a potent histone deacetylase (HDAC) inhibitor that has been applied clinically to treat lymphoma, but a high dose of SAHA kills tumor cells and normal cells without preference. Here, we report that low-dose SAHA enhances CD8+ T cell-mediated antitumor immunity by upregulating MHC I expression in NSCLC cells. METHODS: Flow cytometric analysis, quantitative real-time PCR and western blot were used to analyze the expression of MHC I, STAT1 and Smad2/3 in both human and mouse NSCLC cell lines after SAHA treatment. The nuclear translocation of phosphorylated STAT1 and Smad2/3 was investigated by western blot and immunofluorescence staining. The mechanisms underlying STAT1 and Smad2/3 upregulation were analyzed through database searches and chromatin immunoprecipitation-qPCR. Finally, we assessed the antitumor effect of specific CD8+ T cells with SAHA treatment in vivo and in vitro. RESULTS: We showed that low-dose SAHA upregulated the expression of MHC I in NSCLC cell lines without affecting cell viability. We also provided evidence that high levels of MHC I induced by SAHA promoted the activation, proliferation, and cytotoxicity of specific CD8+ T cells in mouse models. Mechanistically, low-dose SAHA increased the levels of H3K9ac and H3K27ac in the promoters of the STAT1, Smad2 and Smad3 genes in NSCLC cells by inhibiting HDAC activity, resulting in elevated expression levels of STAT1, Smad2 and Smad3. The nuclear translocation of phosphorylated STAT1 and Smad2/3 markedly upregulated the expression of MHC I in NSCLC cells. CONCLUSIONS: Low-dose SAHA enhances CD8+ T cell-mediated antitumor immunity by boosting MHC I expression in NSCLC cells. Thus, we revealed a key mechanism of SAHA-mediated enhanced antitumor immunity, providing insights into a novel immunotherapy strategy for NSCLC.

LMTK2 switches on canonical TGF-ß1 signaling in human bronchial epithelial cells.

Transforming growth factor (TGF-ß1) is a critical profibrotic mediator in chronic lung disease, and there are no specific strategies to mitigate its adverse effects. Activation of TGF-ß1 signaling is a multipart process involving ligands, transmembrane receptors, and transcription factors. In addition, an intricate network of adaptor proteins fine-tunes the signaling strength, duration, and activity. Namely, Smad7 recruits growth arrest and DNA damage (GADD34) protein that then interacts with the catalytic subunit of phosphoprotein phosphatase 1 (PP1c) to inactivate TGF-ß receptor (TßR)-I and downregulate TGF-ß1 signaling. Little is known about how TGF-ß1 releases TßR-I from the GADD34-PP1c inhibition to activate its signaling. Transmembrane lemur tyrosine kinase 2 (LMTK2) is a PP1c inhibitor, and our published data showed that TGF-ß1 recruits LMTK2 to the cell surface. Here, we tested the hypothesis that TGF-ß1 recruits LMTK2 to inhibit PP1c, allowing activation of TßR-I. First, LMTK2 interacted with the TGF-ß1 pathway in the human bronchial epithelium at multiple checkpoints. Second, TGF-ß1 inhibited PP1c by an LMTK2-dependent mechanism. Third, TGF-ß1 used LMTK2 to activate canonical Smad3-mediated signaling. We propose a model whereby the LMTK2-PP1c and Smad7-GADD34-PP1c complexes serve as on-and-off switches in the TGF-ß1 signaling in human bronchial epithelium.NEW & NOTEWORTHY Activation of the transforming growth factor (TGF)-ß1 signaling pathway is complex, involving many ligands, transmembrane receptors, transcription factors, and modulating proteins. The mechanisms of TGF-ß1 signaling activation/inactivation are not fully understood. We propose for the first time a model by which transmembrane lemur tyrosine kinase 2 (LMTK2) forms a complex with phosphoprotein phosphatase 1 (PP1c) to activate TGF-ß1 signaling and Smad7, growth arrest and DNA damage (GADD34), and PP1C form a complex to inactivate TGF-ß1 signaling in human bronchial epithelium.

Glial-Cell-Line-Derived Neurotrophic Factor Promotes Glioblastoma Cell Migration and Invasion via the SMAD2/3-SERPINE1-Signaling Axis.

Glial-cell-line-derived neurotrophic factor (GDNF) is highly expressed and is involved in the malignant phenotype in glioblastomas (GBMs). However, uncovering its underlying mechanism for promoting GBM progression is still a challenging work. In this study, we found that serine protease inhibitor family E member 1 (SERPINE1) was a potential downstream gene of GDNF. Further experiments confirmed that SERPINE1 was highly expressed in GBM tissues and cells, and its levels of expression and secretion were enhanced by exogenous GDNF. SERPINE1 knockdown inhibited the migration and invasion of GBM cells promoted by GDNF. Mechanistically, GDNF increased SERPINE1 by promoting the phosphorylation of SMAD2/3. In vivo experiments demonstrated that GDNF facilitated GBM growth and the expressions of proteins related to migration and invasion via SERPINE1. Collectively, our findings revealed that GDNF upregulated SERPINE1 via the SMAD2/3-signaling pathway, thereby accelerating GBM cell migration and invasion. The present work presents a new mechanism of GDNF, supporting GBM development.

SIRT3 sulfhydration using hydrogen sulfide inhibited angiotensin II-induced atrial fibrosis and vulnerability to atrial fibrillation via suppression of the TGF-ß1/smad2/3 signalling pathway.

Atrial fibrosis is associated with the occurrence of atrial fibrillation (AF) and regulated by the transforming growth factor-ß1 (TGF-ß1)/Smad2/3 signalling pathway. Unfortunately, the mechanisms of regulation of TGF-ß1/Smad2/3-induced atrial fibrosis and vulnerability to AF remain still unknown. Previous studies have shown that sirtuin3 (SIRT3) sulfhydration has strong anti-fibrotic effects. We hypothesised that SIRT3 sulfhydration inhibits angiotensin II (Ang-II)-induced atrial fibrosis via blocking the TGF-ß1/Smad2/3 signalling pathway. In this study, we found that SIRT3 expression was decreased in the left atrium of patients with AF compared to that in those with sinus rhythm (SR). In vitro, SIRT3 knockdown by small interfering RNA significantly expanded Ang-II-induced atrial fibrosis and TGF-ß1/Smad2/3 signalling pathway activation, whereas supplementation with Sodium Hydrosulfide (NaHS, exogenous hydrogen sulfide donor and sulfhydration agonist) and SIRT3 overexpression using adenovirus ameliorated Ang-II-induced atrial fibrosis. Moreover, we observed suppression of the TGF-ß1/Smad2/3 pathway when Ang-II was combined with NaHS treatment, and the effect of this co-treatment was consistent with that of Ang-II combined with LY3200882 (Smad pathway inhibitor) on reducing atrial fibroblast proliferation and cell migration in vitro. Supplementation with dithiothreitol (DTT, a sulfhydration inhibitor) and adenovirus SIRT3 shRNA blocked the ameliorating effect of NaHS and AngII co-treatment on atrial fibrosis in vitro. Finally, continued treatment with NaHS in rats ameliorated atrial fibrosis and remodelling, and further improved AF vulnerability induced by Ang-II, which was reversed by DTT and adenovirus SIRT3 shRNA, suggesting that SIRT3 sulfhydration might be a potential therapeutic target in atrial fibrosis and AF.

Effects of vitamin D status on cutaneous wound healing through modulation of EMT and ECM.

To investigate the effects of vitamin D status on cutaneous wound healing, C57BL/6J mice were fed diets with different vitamin D levels or injected intraperitoneally with 1α,25(OH)2D3. Dorsal skin wounds were created and wound edge tissues were collected on days 4, 7, 11, and 14 postwounding. The proliferation and migration of HaCaT cells treated with shVDR or 1α,25(OH)2D3 were assessed. Vitamin D deficiency (VDD) decreased wound closure and might delay inflammatory response, shown by slower inflammatory cell infiltration, decreased IL6 and TNF expression in early phase followed by an increase later. VDD might postpone epithelial-mesenchymal transition (EMT), initially characterized by higher epithelial markers and lower mesenchymal markers, followed by opposite appearance later. Dietary vitamin D supplementation and 1α,25(OH)2D3 intervention tended to accelerate EMT. Regarding extracellular matrix (ECM), VDD appeared to reduce collagen deposition on day 4 and downregulated fibronectin, COL3A1, and MMP9 expression early, followed by an increase later, together with an initial increase and subsequent decrease in Timp1 mRNA expression. Dietary vitamin D intervention promoted fibronectin and MMP9 expression on day 4 and then downregulated their expression on day 14. TGFb1/SMAD2/3 signaling seemed to be downregulated by VDD and upregulated by 1α,25(OH)2D3. In vitro, partial inhibition of VDR by shVDR tended to inhibit HaCaT cell proliferation and migration, EMT, and TGFb1/SMAD2/3 signaling, whereas 1α,25(OH)2D3 appeared to generate opposite effects. In conclusion, VDD hindered cutaneous wound healing, potentially due to impaired inflammatory response, delayed EMT, decreased ECM, and inhibited TGFb1/SMAD2/3 pathway. Vitamin D and 1α,25(OH)2D3 tended to enhance EMT and ECM.

Asiaticoside modulates human NK cell functional fate by mediating metabolic flexibility in the tumor microenvironment.

BACKGROUND: Transforming growth factor-beta (TGF-ß), an immunosuppressive cytokine, is often elevated in various tumors and inhibits the immune system's ability to combat tumor cells. Despite promising results from TGF-ß inhibitor therapies, their clinical efficacy remains limited. PURPOSE: This study aimed to enhance the antitumor capabilities of natural killer (NK) cells in the presence of TGF-ß by exploring the potential of asiaticoside, a natural compound with established clinical safety. STUDY DESIGN: The effects of asiaticoside on NK cells were investigated to determine its potential to counteract TGF-ß-induced immunosuppression and elucidate the underlying mechanisms. METHODS: Natural compounds were screened using a Luminex assay to identify those promoting Interferon-γ (IFN-γ) secretion from NK cells. Asiaticoside-pretreated NK cells' cytotoxicity was assessed against K562, OVCAR8, and A2780 cells using organoids from ascites-derived ovarian cancer (OC) cells. In vivo efficacy was evaluated with B16 melanoma lung metastasis and subcutaneous tumor models in C57BL/6 mice, using asiaticoside as a 50 mg/kg injection. The compound's ability to enhance NK cell-driven anti-neoplastic responses was further assessed in an OC murine model. Effects on TGF-ß/SMAD pathways and mitochondrial functions were examined through various microscopy and metabolomic techniques. The involvement of the mTOR/DRP1 axis in asiaticoside-mediated restoration of mitochondrial oxidation in NK cells after TGF-ß suppression was determined using the mTOR inhibitor rapamycin and the DRP1 inhibitor Mdivi-1. RESULTS: Asiaticoside-treated NK cells retained their ability to suppress tumor growth and metastasis despite TGF-ß presence. Asiaticoside downregulated TGF-ß receptors 1 (TGFBR1) expression, impaired the protein stability of TGFBR1 and TGF-ß receptors 2 (TGFBR2), and reduced SMAD2 phosphorylation, preventing SMAD2 translocation from the mitochondria. This preserved mitochondrial respiration and maintained NK cell antitumor activity. CONCLUSION: The study concludes that asiaticoside has significant potential as a strategy for "priming" NK cells in cellular immunotherapy. By demonstrating that asiaticoside degrades the TGF-ß receptor, leading to reduced phosphorylation of SMAD2 and preventing its mitochondrial translocation, thereby maintaining mitochondrial integrity. Meantime, asiaticoside counteracts TGF-ß-induced suppression of mitochondrial oxidative and aerobic respiration through the mTOR/DRP1 pathways. The research uncovers a previously unreported pathway for preserving mitochondrial respiration and NK cell functionality. A detailed mechanistic insight into how asiaticoside functions at the molecular level was explored. Its ability to counteract the immunosuppressive effects of TGF-ß makes it a valuable candidate for enhancing the effectiveness of immunotherapies in treating a variety of tumors with elevated TGF-ß levels.