RESUMO
Rationale: Sma mothers against decapentaplegic homologue 4 (Smad4) is a key mediator of the transforming growth factor ß (TGF-ß) pathway and plays complex and contradictory roles in hepatocellular carcinoma (HCC). However, the specific role of Smad4 in hepatocytes in regulating hepatocarcinogenesis remains poorly elucidated. Methods: A diethylnitrosamine/carbon tetrachloride-induced HCC model was established in mice with hepatocyte-specific Smad4 deletion (AlbSmad4-/-) and liver tumorigenesis was monitored. Immune cell infiltration was examined by immunofluorescence and fluorescence activated cell sorting (FACS). Cytokine secretion, glycolysis, signal pathway, and single-cell RNA sequencing were analysed for mechanism. Results: AlbSmad4-/- mice exhibited significantly fewer and smaller liver tumor nodules, less fibrosis, reduced myeloid-derived suppressor cell infiltration and increased CD8+ T cell infiltration. Smad4 deletion in hepatocytes enhanced C-X-C motif ligand 10 (CXCL10) secretion, promoting tumor necrosis factor-α (TNF-α) production in CD8+ T cells. The loss of Smad4 activated the CXCL10/mammalian target of rapamycin (mTOR)/lactate dehydrogenase A (LDHA) pathway, which increased glycolytic activity in CD8+ T cells. HCC patients with high Smad4 expression exhibited decreased CD8+ T cell infiltration and altered glycolysis. Conclusion: Our results demonstrate that Smad4 in hepatocytes promotes hepatocarcinogenesis and is a potential and candidate target for the prevention and therapy of HCC.
Assuntos
Linfócitos T CD8-Positivos , Carcinogênese , Carcinoma Hepatocelular , Quimiocina CXCL10 , Hepatócitos , Neoplasias Hepáticas , Receptores CXCR3 , Proteína Smad4 , Animais , Proteína Smad4/metabolismo , Proteína Smad4/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Hepatócitos/metabolismo , Hepatócitos/imunologia , Camundongos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Receptores CXCR3/metabolismo , Receptores CXCR3/genética , Carcinogênese/imunologia , Carcinogênese/genética , Transdução de Sinais , Camundongos Knockout , Humanos , Camundongos Endogâmicos C57BL , MasculinoRESUMO
TGF-ß-SMAD signaling pathway plays an important role in the progression of various cancers. However, posttranscriptional regulation such as N6-methyladenosine (m6A) of TGF-ß-SMAD signaling axis remains incompletely understood. Here, we reveal that insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) is low expression as well as associated with poor prognosis in clear cell renal cell carcinoma (ccRCC) patients and inhibits proliferation as well as promotes metastasis of ccRCC cells. Mechanistically, IGF2BP2 systematically regulates TGF-ß-SMAD signaling family, including TGF-ß1/2, TGF-ßR1/2 and SMAD2/3/4, through mediating their mRNA stability in an m6A-dependent manner. Furthermore, the functional effects of IGF2BP2 on ccRCC cells is mediated by TGF-ß-SMAD signaling downstream effector SMAD4, which is identified three m6A sites in 5'UTR and CDS. Our study establishes IGF2BP2-TGF-ß-SMAD axis as a new regulatory effector in ccRCC, providing new insights for developing novel therapeutic strategies.
Assuntos
Adenosina , Carcinoma de Células Renais , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais , Proteínas de Ligação a RNA , Transdução de Sinais , Proteínas Smad , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Linhagem Celular Tumoral , Proteínas Smad/metabolismo , Proteínas Smad/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Proteína Smad4/metabolismo , Proteína Smad4/genética , Camundongos , Movimento Celular , Estabilidade de RNA , Metástase NeoplásicaRESUMO
Pancreatic cancer is associated with an oncogenic KRAS mutation in approximately 90% of cases. However, a non-negligible proportion of pancreatic cancer cases harbor wild-type KRAS (KRAS-WT). This study establishes genetically engineered mouse models that develop spontaneous pancreatic cancer in the context of KRAS-WT. The Trp53loxP/loxP;Smad4loxP/loxP;Pdx1-Cre (PPSSC) mouse model harbors KRAS-WT and loss of Trp53/Smad4. The Trp53loxP/loxP;Tgfbr2loxP/loxP;Pdx1-Cre (PPTTC) mouse model harbors KRAS-WT and loss of Trp53/Tgfbr2. We identify that either Trp53/Smad4 loss or Trp53/Tgfbr2 loss can induce spontaneous pancreatic tumor formation in the absence of an oncogenic KRAS mutation. The Trp53/Smad4 loss and Trp53/Tgfbr2 loss mouse models exhibit distinct pancreatic tumor histological features, as compared to oncogenic KRAS-driven mouse models. Furthermore, KRAS-WT pancreatic tumors with Trp53/Smad4 loss reveal unique histological features of pancreatic adenosquamous carcinoma (PASC). Single-cell RNA sequencing (scRNA-seq) analysis reveals the distinct tumor immune microenvironment landscape of KRAS-WT (PPSSC) pancreatic tumors as compared with that of oncogenic KRAS-driven pancreatic tumors.
Assuntos
Mutação , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Proteína Smad4 , Proteína Supressora de Tumor p53 , Proteína Smad4/genética , Proteína Smad4/metabolismo , Animais , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Mutação/genética , Camundongos , Humanos , Carcinoma Adenoescamoso/genética , Carcinoma Adenoescamoso/patologia , Carcinoma Adenoescamoso/metabolismo , Modelos Animais de Doenças , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismoRESUMO
Obesity is a global issue that warrants the identification of more effective therapeutic targets and a better understanding of the pivotal molecular pathogenesis. Annexin A1 (ANXA1) is known to inhibit phospholipase A2, exhibiting anti-inflammatory activity. However, the specific effects of ANXA1 in obesity and the underlying mechanisms of action remain unclear. Our study reveals that ANXA1 levels are elevated in the adipose tissue of individuals with obesity. Whole-body or adipocyte-specific ANXA1 deletion aggravates obesity and metabolic disorders. ANXA1 levels are higher in stromal vascular fractions (SVFs) than in mature adipocytes. Further investigation into the role of ANXA1 in SVFs reveals that ANXA1 overexpression induces lower numbers of mature adipocytes, while ANXA1-knockout SVFs exhibit the opposite effect. This suggests that ANXA1 plays an important role in adipogenesis. Mechanistically, ANXA1 competes with MYC binding protein 2 (MYCBP2) for interaction with PDZ and LIM domain 7 (PDLIM7). This exposes the MYCBP2-binding site, allowing it to bind more readily to the SMAD family member 4 (SMAD4) and promoting its ubiquitination and degradation. SMAD4 degradation downregulates peroxisome proliferator-activated receptor gamma (PPARγ) transcription and reduces adipogenesis. Treatment with Ac2-26, an active peptide derived from ANXA1, inhibits both adipogenesis and obesity through the mechanism. In conclusion, the molecular mechanism of ANXA1 inhibiting adipogenesis was first uncovered in our study, which is a potential target for obesity prevention and treatment.
Assuntos
Adipócitos , Adipogenia , Anexina A1 , Obesidade , PPAR gama , Anexina A1/genética , Anexina A1/metabolismo , Adipogenia/genética , Animais , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Humanos , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Adipócitos/metabolismo , Adipócitos/patologia , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células 3T3-L1 , PeptídeosRESUMO
The progression of pancreatic ductal adenocarcinoma (PDAC) is significantly affected by transforming growth factor (TGF)-ß but targeting TGF-ß can also compromize physiological effects in patients. Our study examined the functions of the ubiquitously expressed protein, PDCD10, as a modulator of TGF-ß signaling in PDAC. Using in silico analyses we found that in patient samples, PDCD10 is significantly higher expressed in PDAC tumor tissue compared with normal pancreas and it is highly correlated with reduced survival. We created stable KO's of PDCD10 in two PDAC lines, PaTu 8902 (SMAD4 +/+) and PaTu 8988t (SMAD4 -/-), and found that KO lines are more sensitive to 5-FU and Gemcitabine treatment than their wild-type counterparts. Performing viability and wound closure assays we further found that PDCD10 promotes cell survival and proliferation by enhancing specifically the mitogenic functions of TGF-ß. The molecular mechanism underlying this effect was further investigated using Western blots and with primary organoid lines derived from patient PDAC tissue samples. The data imply that PDCD10 mediates an increase in p-ERK through a non-SMAD4 pathway, leading to EMT promotion. Furthermore, PDCD10 facilitates deactivation of RB via a SMAD4-dependent pathway, thereby counter-acting the anti-proliferative actions of TGF-ß. By performing proximity ligation assays (PLA) we found that PDCD10 associates with the kinase MST4, translocates it intracellularly and thereby facilitates phosphorylations of RB and ERK1/2. Our study indicates that PDCD10 promotes the proliferative function and EMT induction of TGF-ß in pancreatic cancer cells. Therefore, targeting PDCD10 in PDAC patients could represent a promising new strategy to optimize TGF-ß targeted therapies.
Assuntos
Proteínas Reguladoras de Apoptose , Carcinoma Ductal Pancreático , Proliferação de Células , Desoxicitidina , Neoplasias Pancreáticas , Proteína Smad4 , Fator de Crescimento Transformador beta , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/tratamento farmacológico , Fator de Crescimento Transformador beta/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteína Smad4/metabolismo , Proteína Smad4/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Gencitabina , Transdução de Sinais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Sobrevivência Celular/efeitos dos fármacos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão GênicaRESUMO
BACKGROUND: Integrin-Binding Sialoprotein (IBSP) has been implicated in tumor progression across various cancers. However, the specific role of IBSP in breast cancer remains underexplored. There is a need to investigate the mechanisms by which IBSP influences breast cancer progression and its potential as a therapeutic target. AIMS: This study aims to elucidate the role of IBSP in breast cancer, particularly its impact on tumor progression and its relationship with prognosis. We also seek to understand the underlying mechanisms, including the involvement of the BMP-SMAD signaling pathway, and to explore the potential of targeting IBSP for therapeutic interventions. METHODS AND RESULTS: Overexpression of IBSP in breast cancer cells led to increased migration and invasion, whereas IBSP interference reduced these behaviors, indicating its role in enhancing tumor progression. Differentially expressed genes were significantly enriched in the BMP-SMAD signaling pathway, a critical pathway for osteogenic differentiation. Transcription Factor Binding: Dual luciferase reporter assays demonstrated that SMAD4 specifically binds to the IBSP promoter, establishing a regulatory link between SMAD4 and IBSP expression. Silencing IBSP (si-IBSP) mitigated the effects of SMAD4-induced tumor proliferation, confirming that IBSP acts as a downstream target of SMAD4 in the BMP signaling pathway. CONCLUSION: Our study reveals that IBSP plays a significant role in breast cancer progression through the BMP-SMAD4 signaling pathway. Targeting IBSP could be a promising therapeutic strategy for breast cancer treatment. Further research into IBSP inhibitors may offer new avenues for improving treatment outcomes and managing breast cancer more effectively.
Assuntos
Neoplasias Ósseas , Neoplasias da Mama , Proliferação de Células , Sialoproteína de Ligação à Integrina , Transdução de Sinais , Proteína Smad4 , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Neoplasias Ósseas/secundário , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/genética , Proteína Smad4/metabolismo , Proteína Smad4/genética , Sialoproteína de Ligação à Integrina/metabolismo , Sialoproteína de Ligação à Integrina/genética , Camundongos , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação Neoplásica da Expressão Gênica , Movimento Celular , Linhagem Celular Tumoral , Prognóstico , Camundongos NusRESUMO
Cigarette smoke (CS) is a prevalent chemical indoor air contaminant known to be the primary cause of EMT during airway remodeling in COPD. While some evidence indicates the involvement of SMAD4 in EMT across certain diseases, its specific role in CS-induced EMT in airway remodeling associated with COPD is not established. In our research, we observed a substantial upregulation in SMAD4 expression, O-GlcNAcylation and EMT in patients with COPD, as well as in vitro and in vivo COPD models induced by CS, than those of the controls. Downregulation of SMAD4 resulted in a reduction in CS-induced EMT in vitro and in vivo. As a post-translational modification of proteins, O-GlcNAcylation is dynamically controlled by the duo of enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA). We further discovered the enhancement of O-GlcNAcylation levels induced by CS was due to an elevated OGT expression, as the expression of OGA remained unchanged. Using an OGT inhibitor (OSMI-1) counteracted the effects of SMAD4 on EMT. Whereas, overexpressing OGT increased SMAD4 expression and promoted EMT. OGT-mediated SMAD4 O-GlcNAcylation shielded SMAD4 from proteasomal degradation by reducing its ubiquitination, thereby aiding in SMAD4 stabilization in response to EMT induced by CS. Overall, this research uncovers a fresh pathway for CS-induced EMT in the airway remodeling of COPD and offers valuable insights.
Assuntos
Remodelação das Vias Aéreas , Transição Epitelial-Mesenquimal , N-Acetilglucosaminiltransferases , Doença Pulmonar Obstrutiva Crônica , Proteína Smad4 , Doença Pulmonar Obstrutiva Crônica/patologia , N-Acetilglucosaminiltransferases/metabolismo , Proteína Smad4/metabolismo , Remodelação das Vias Aéreas/efeitos dos fármacos , Humanos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Animais , Fumaça/efeitos adversos , Camundongos , Masculino , FemininoRESUMO
Colorectal Cancer (CRC) is the third most common cancer worldwide, and the occurrence and development of CRC are influenced by the molecular biology characteristics of CRC, especially alterations in key signaling pathways. The transforming growth factor-ß (TGF-ß) plays a crucial role in cellular growth, differentiation, migration, and apoptosis, with SMAD4 protein serving as a key transcription factor in the TGF-ß signaling pathway, thus playing a significant role in the onset and progression of CRC. CRC is one of the malignancies with a high mortality rate worldwide. Despite significant research progress in recent years, especially regarding the role of SMAD4, its dual role in the early and late stages of tumor progression has promoted further discussion on its complexity as a therapeutic target, highlighting the urgent need for a deeper analysis of its role in CRC. This review aims to explore the function of SMAD4 protein in CRC and its potential as a therapeutic target.
Assuntos
Neoplasias Colorretais , Proteína Smad4 , Humanos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/tratamento farmacológico , Proteína Smad4/metabolismo , Proteína Smad4/genética , Transdução de Sinais , Animais , Fator de Crescimento Transformador beta/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
Therapeutic advancements in treatments for cancer, a leading cause of mortality worldwide, have lagged behind the increasing incidence of this disease. There is a growing interest in multifaceted approaches for cancer treatment, such as chemotherapy, targeted therapy, and immunotherapy, but due to their low efficacy and severe side effects, there is a need for the development of new cancer therapies. Recently, the human microbiome, which is comprised of various microorganisms, has emerged as an important research field due to its potential impact on cancer treatment. Among these microorganisms, Bifidobacterium infantis has been shown to significantly improve the efficacy of various anticancer drugs. However, research on the role of B. infantis in cancer treatment remains insufficient. Thus, in this study, we explored the anticancer effect of treatment with B. infantis DS1685 supernatant (BI sup) in colorectal and breast cancer cell lines. Treatment with BI sup induced SMAD4 expression to suppress cell growth in colon and breast cancer cells. Furthermore, a decrease in tumor cohesion was observed through the disruption of the regulation of EMT-related genes by BI sup in 3D spheroid models. Based on these findings, we anticipate that BI sup could play an adjunctive role in cancer therapy, and future cotreatment of BI sup with various anticancer drugs may lead to synergistic effects in cancer treatment.
Assuntos
Bifidobacterium longum subspecies infantis , Neoplasias da Mama , Neoplasias Colorretais , Proteína Smad4 , Fator de Crescimento Transformador beta , Humanos , Proteína Smad4/metabolismo , Proteína Smad4/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Linhagem Celular Tumoral , Fator de Crescimento Transformador beta/metabolismo , Bifidobacterium longum subspecies infantis/metabolismo , Bifidobacterium longum subspecies infantis/genética , Feminino , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Probióticos , Antineoplásicos/farmacologiaRESUMO
BACKGROUND: While many genes linked to colorectal cancer (CRC) contribute to cancer development, a thorough investigation is needed to explore crucial hub genes yet to be fully studied. A pivotal pathway in CRC is transforming growth factor-beta (TGF-ß). This study aimed to assess SMAD2 and SMAD4 gene expression from this pathway. METHODS AND RESULTS: Counted data from the Cancer Genome Atlas (TCGA) were examined, comparing 483 tumor and 41 normal samples. Using clinical data, genes impacting overall survival (OS) were evaluated. GSE39582 was employed to confirmed the levels of genes in CRC compared to the normal samples. Additionally, employing unhealthy samples and the RT-qPCR means our outcomes was validated. Finally, PharmacoGx information were utilized to connect the levels of potential genes to drug tolerance and susceptibility. Our findings showed SMAD2 and SMAD4 levels in TGF-ß signaling were more significant than other pathway genes. Our findings indicated that the protein levels of these genes were lower in malignant tissues than in healthy tissues. Results revealed a significant correlation between low levels of SMAD2 and unfavorable OS in CRC individuals. RT-qPCR results demonstrated decreased expressions of both SMAD2 and SMAD4 in cancer tissues compared to elevated levels in adjacent normal samples. Our results showed significant association between selected genes and immune cell infiltration markers such as CD8+, and B-cells. Our results indicated a potential association among the levels of SMAD2 and SMAD4 genes and tolerance and susceptibility to Nilotinib and Panobinostat drugs. CONCLUSION: Reduced expression of SMAD2 and SMAD4 may be pivotal in CRC progression, impacting downstream genes unrelated to patient OS. These findings suggest a potential role for SMAD2 and SMAD4 as predictive markers for drug response in CRC patients.
Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteína Smad2 , Proteína Smad4 , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Prognóstico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/genética , Transdução de Sinais/genética , Transdução de Sinais/efeitos dos fármacos , Masculino , Regulação para Baixo/genética , Regulação para Baixo/efeitos dos fármacos , FemininoRESUMO
This study investigated the efficacy of using chitosan/alginate nanoparticles loaded with recombinant human bone morphogenetic-2 (rhBMP-2) and SMAD4 encoding plasmid to enhance the chondrogenesis of human bone marrow mesenchymal stem cells (hBM-MSCs) seeded on an extracellular matrix (ECM). The research treatments included the stem cells treated with the biological cocktail (BC), negative control (NC), hBM-MSCs with chondrogenic medium (MCM), hBM-MSCs with naked rhBMP-2 and chondrogenic medium (NB/C), and hBM-MSCs with naked rhBMP-2 and chondrogenic medium plus SMAD4 encoding plasmid transfected with polyethyleneimine (PEI) (NB/C/S/P). The cartilage differentiation was performed with real-time quantitative PCR analysis and alizarin blue staining. The data indicated that the biological cocktail (BC) exhibited significantly higher expression of cartilage-related genes compared to significant differences with MCM and negative control (NC) on chondrogenesis. In the (NB/C/S/P), the expression levels of SOX9 and COLX were lower than those in the BC group. The expression pattern of the ACAN gene was similar to COL2A1 changes suggesting that it holds promising potential for cartilage regeneration.
Assuntos
Alginatos , Proteína Morfogenética Óssea 2 , Cartilagem Articular , Quitosana , Condrogênese , Matriz Extracelular , Células-Tronco Mesenquimais , Nanopartículas , Regeneração , Transdução de Sinais , Proteína Smad4 , Alicerces Teciduais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Quitosana/química , Quitosana/farmacologia , Alginatos/química , Alginatos/farmacologia , Humanos , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Cartilagem Articular/citologia , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/genética , Nanopartículas/química , Condrogênese/efeitos dos fármacos , Alicerces Teciduais/química , Proteína Smad4/metabolismo , Proteína Smad4/genética , Transdução de Sinais/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regeneração/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador betaRESUMO
BACKGROUND: Cyp19a1a is a key enzyme in the pathway that converts androgens into estrogen and is regulated by TGF-ß signaling. Smad4 and FoxH1 are downstream effectors of TGF-ß signaling and may play important roles in ovarian development in M. albus. METHODS: We investigated the expression pattern of the Smad4 and FoxH1 using qRTâPCR and immunofluorescence, then tested the changes of smad4 and foxh1 by qRTâPCR after ovary incubation with FSH in vitro, and analysed the regulation of cyp19a1a transcription by Smad4 and FoxH1 by dual-luciferase reporter assays. RESULTS: We found that Smad4 encoded a putative protein of 449 amino acids and harbored the three conserved domains typical of this protein family. Smad4 and foxh1 exhibited similar expression patterns during ovarian development and after FSH incubation, with Pearson's coefficients of 0.873 and 0.63-0.81, respectively. Furthermore, Smad4, FoxH1 and Cyp19a1a colocalized in the granulosa cells and theca cells of ovaries during the mid-to-late vitellogenic stage. Smad4 repressed cyp19a1a activity via SBE1 (- 1372/-1364) and SBE2 (- 415/-407) in the cyp19a1a promoter, whereas mutating SBE1 or SBE2 restored cyp19a1a promoter activity. Co-overexpression of Smad4 and FoxH1 significantly reduced cyp19a1a promoter activity. CONCLUSIONS: This study provides new insights into the potential functions of transcription factors Smad4 and FoxH1 in ovarian development and the transcriptional regulation mechanism of cyp19a1a in M. albus, which will reveal Smad4/FoxH1-mediated TGF-ß signaling in reproduction and the regulation of the cyp19a1a. Aromatase, encoded by cyp19a1a, is involved in ovarian development and plays an important role in the quality of eggs, as well the sex ratio, of the teleost fish, M. albus. The research on the transcriptional regulation of cyp19a1a has contributed to the understanding of its role in ovarian development. In previous study, it was shown that FoxH1 inhibits cyp19a1a transcription. In the present study, Smad4 was confirmed as a cyp19a1a transcriptional repressor and Smad4 may also coordinate with FoxH1 to repress cyp19a1a transcription. At present, we provide a new perspective for the transcriptional regulation of cyp19a1a by transcription factors Smad4 and FoxH1 in teleost fish ovary. In the future, the regulatory networks of Smad4 and FoxH1 will be further studied and the gene editing technology will be applied to screen specific regulatory factors of cyp191a1a gene, so as to alter the female cycle and modulate the sex ratio of the eggs production.
Assuntos
Aromatase , Enguias , Fatores de Transcrição Forkhead , Ovário , Regiões Promotoras Genéticas , Proteína Smad4 , Animais , Feminino , Ovário/metabolismo , Aromatase/metabolismo , Aromatase/genética , Proteína Smad4/metabolismo , Proteína Smad4/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Enguias/metabolismo , Proteínas de Peixes/metabolismo , Proteínas de Peixes/genética , Hormônio Foliculoestimulante/metabolismoRESUMO
Increasing epidemiological evidence has shown that PM2.5 exposure is significantly associated with the occurrence of osteoporosis. It has been well demonstrated that PM2.5 exposure enhanced the differentiation and function of osteoclasts by indirectly causing chronic inflammation, while the mechanism in osteoblasts remains unclear. In our study, toxic effects were evaluated by direct exposure of 20-80⯵g/ml PM2.5 to MC3T3-E1 cells and BMSCs. The results showed that PM2.5 exposure did not affect cell viability via proliferation and apoptosis, but significantly inhibited osteoblast differentiation in a dose-dependent manner. Osteogenic transcription factors Runx2 and Sp7 and other biomarkers Alp and Ocn decreased after PM2.5 exposure. RNA-seq revealed TGF-ß signaling was involved in PM2.5 exposure inhibited osteoblast differentiation, which led to P-Smad1/5 and P-Smad2 reduction in the nucleus by increasing the ubiquitination and degradation of Smad4. At last, the inflammation response increased in MC3T3-E1 cells with PM2.5 exposure. Moreover, the mRNA levels of Mmp9 increased in bone marrow-derived macrophage cells treated with the conditional medium collected from MC3T3-E1 cells exposed to PM2.5. Overall, these results indicated that PM2.5 exposure inhibits osteoblast differentiation and concurrently increases the maturation of osteoclasts. Our study provides in-depth mechanistic insights into the direct impact of PM2.5 exposure on osteoblast, which would indicate the unrecognized role of PM2.5 on osteoporosis.
Assuntos
Diferenciação Celular , Osteoblastos , Material Particulado , Proteína Smad4 , Ubiquitinação , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proteína Smad4/metabolismo , Proteína Smad4/genética , Camundongos , Material Particulado/toxicidade , Ubiquitinação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Poluentes Atmosféricos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Proteólise/efeitos dos fármacosRESUMO
BACKGROUND: Promoting the balance between bone formation and bone resorption is the main therapeutic goal for postmenopausal osteoporosis (PMOP), and bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation plays an important regulatory role in this process. Recently, several long non-coding RNAs (lncRNAs) have been reported to play an important regulatory role in the occurrence and development of OP and participates in a variety of physiological and pathological processes. However, the role of lncRNA tissue inhibitor of metalloproteinases 3 (lncTIMP3) remains to be investigated. METHODS: The characteristics of BMSCs isolated from the PMOP rat model were verified by flow cytometry assay, alkaline phosphatase (ALP), alizarin red and Oil Red O staining assays. Micro-CT and HE staining assays were performed to examine histological changes of the vertebral trabeculae of the rats. RT-qPCR and western blotting assays were carried out to measure the RNA and protein expression levels. The subcellular location of lncTIMP3 was analyzed by FISH assay. The targeting relationships were verified by luciferase reporter assay and RNA pull-down assay. RESULTS: The trabecular spacing was increased in the PMOP rats, while ALP activity and the expression levels of Runx2, Col1a1 and Ocn were all markedly decreased. Among the RNA sequencing results of the clinical samples, lncTIMP3 was the most downregulated differentially expressed lncRNA, also its level was significantly reduced in the OVX rats. Knockdown of lncTIMP3 inhibited osteogenesis of BMSCs, whereas overexpression of lncTIMP3 exhibited the reverse results. Subsequently, lncTIMP3 was confirmed to be located in the cytoplasm of BMSCs, implying its potential as a competing endogenous RNA for miRNAs. Finally, the negative targeting correlations of miR-214 between lncTIMP3 and Smad4 were elucidated in vitro. CONCLUSION: lncTIMP3 may delay the progress of PMOP by promoting the activity of BMSC, the level of osteogenic differentiation marker gene and the formation of calcium nodules by acting on the miR-214/Smad4 axis. This finding may offer valuable insights into the possible management of PMOP.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Osteoporose Pós-Menopausa , RNA Longo não Codificante , Proteína Smad4 , Animais , Feminino , Humanos , Ratos , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Modelos Animais de Doenças , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/patologia , Ratos Sprague-Dawley , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteína Smad4/metabolismo , Proteína Smad4/genética , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
SMAD4 is a tumor suppressor mutated or silenced in multiple cancers, including oral cavity squamous cell carcinoma (OSCC). Human clinical samples and cell lines, mouse models and organoid culture were used to investigate the role that SMAD4 plays in progression from benign disease to invasive OSCC. Human OSCC lost detectable SMAD4 protein within tumor epithelium in 24% of cases, and this loss correlated with worse progression-free survival independent of other major clinical and pathological features. A mouse model engineered for KrasG12D expression in the adult oral epithelium induced benign papillomas, however the combination of KrasG12D with loss of epithelial Smad4 expression resulted in rapid development of invasive carcinoma with features of human OSCC. Examination of regulatory pathways in 3D organoid cultures of SMAD4+ and SMAD4- mouse tumors with Kras mutation found that either loss of SMAD4 or inhibition of TGFß signaling upregulated the WNT pathway and altered the extracellular matrix. The gene signature of the mouse tumor organoids lacking SMAD4 was highly similar to the gene signature of human head and neck squamous cell carcinoma. In summary, this work has uncovered novel mechanisms by which SMAD4 acts as a tumor suppressor in OSCC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Progressão da Doença , Neoplasias Bucais , Proteína Smad4 , Via de Sinalização Wnt , Proteína Smad4/metabolismo , Proteína Smad4/genética , Humanos , Animais , Via de Sinalização Wnt/fisiologia , Neoplasias Bucais/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/genética , Camundongos , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Mutação , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Masculino , Organoides/metabolismo , Organoides/patologiaRESUMO
Endocrine resistance poses a significant clinical challenge for patients with hormone receptor-positive and human epithelial growth factor receptor 2-negative (HR + HER2-) breast cancer. Dysregulation of estrogen receptor (ER) and ERBB signaling pathways is implicated in resistance development; however, the integration of these pathways remains unclear. While SMAD4 is known to play diverse roles in tumorigenesis, its involvement in endocrine resistance is poorly understood. Here, we investigate the role of SMAD4 in acquired endocrine resistance in HR + HER2- breast cancer. Genome-wide CRISPR screening identifies SMAD4 as a regulator of 4-hydroxytamoxifen (OHT) sensitivity in T47D cells. Clinical data analysis reveals downregulated SMAD4 expression in breast cancer tissues, correlating with poor prognosis. Following endocrine therapy, SMAD4 expression is further suppressed. Functional studies demonstrate that SMAD4 depletion induces endocrine resistance in vitro and in vivo by enhancing ER and ERBB signaling. Concomitant inhibition of ER and ERBB signaling leads to aberrant autophagy activation. Simultaneous inhibition of ER, ERBB, and autophagy pathways synergistically impacts SMAD4-depleted cells. Our findings unveil a mechanism whereby endocrine therapy-induced SMAD4 downregulation drives acquired resistance by integrating ER and ERBB signaling and suggest a rational treatment strategy for endocrine-resistant HR + HER2- breast cancer patients.
Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Receptor ErbB-2 , Receptores de Estrogênio , Transdução de Sinais , Proteína Smad4 , Humanos , Proteína Smad4/metabolismo , Proteína Smad4/genética , Feminino , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Receptores de Estrogênio/metabolismo , Linhagem Celular Tumoral , Animais , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Tamoxifeno/análogos & derivados , Camundongos , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Camundongos Nus , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Receptores ErbB/metabolismo , Receptores ErbB/genéticaRESUMO
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 (TGFß) 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 TGFß 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.
Assuntos
Células da Granulosa , Oócitos , Proteína Smad4 , Animais , Proteína Smad4/metabolismo , Proteína Smad4/genética , Oócitos/metabolismo , Oócitos/crescimento & desenvolvimento , Camundongos , Feminino , Células da Granulosa/metabolismo , Células da Granulosa/fisiologia , Receptor Notch2/metabolismo , Receptor Notch2/genética , Caderinas/metabolismo , Caderinas/genética , Pseudópodes/metabolismo , Pseudópodes/fisiologiaRESUMO
Transforming growth factor-ß (TGF-ß) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion. Targeting TGF-ß signaling is a promising therapeutic approach, but nonspecific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-ß signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-ß signaling, but not the interaction between Smad1 and Smad4 in bone morphogenetic protein (BMP) signaling. Furthermore, VP exhibited inhibitory effects on TGF-ß-induced EMT and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-ß signaling pathway for treatments of related diseases.
Assuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Transdução de Sinais , Proteína Smad2 , Proteína Smad3 , Proteína Smad4 , Fator de Crescimento Transformador beta , Verteporfina , Humanos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Proteína Smad4/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteína Smad3/metabolismo , Movimento Celular/efeitos dos fármacos , Proteína Smad2/metabolismo , Verteporfina/farmacologiaRESUMO
PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.
Assuntos
Fibroblastos , Fibrose , Cirurgia Filtrante , Glaucoma , MicroRNAs , Ratos Sprague-Dawley , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Glaucoma/patologia , Glaucoma/genética , Cirurgia Filtrante/efeitos adversos , Fibroblastos/metabolismo , Masculino , Cápsula de Tenon/metabolismo , Cápsula de Tenon/patologia , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Ratos , Proteína Smad4/metabolismo , Proteína Smad4/genética , NF-kappa B/metabolismo , Mitomicina/farmacologia , Mitomicina/uso terapêutico , Regulação da Expressão GênicaRESUMO
In this study, a novel method for the fabrication of hesperidin/reduced graphene oxide nanocomposite (RGOH) with the assistance of gamma rays is reported. The different RGOHs were obtained by varying hesperidin concentrations (25, 50, 100, and 200 wt.%) in graphene oxide (GO) solution. Hesperidin concentrations (25, 50, 100, and 200 wt.%) in graphene oxide (GO) were varied to produce the various RGOHs. Upon irradiation with 80 kGy from γ-Ray, the successful reduction of GO occurred in the presence of hesperidin. The reduction process was confirmed by different characterization techniques such as FTIR, XRD, HRTEM, and Raman Spectroscopy. A cytotoxicity study using the MTT method was performed to evaluate the cytotoxic-anticancer effects of arbitrary RGOH on Wi38, CaCo2, and HepG2 cell lines. The assessment of RGOH's anti-inflammatory activity, including the monitoring of IL-1B and IL-6 activities as well as NF-kB gene expression was done. In addition, the anti-invasive and antimetastatic properties of RGOH, ICAM, and VCAM were assessed. Additionally, the expression of the MMP2-9 gene was quantified. The assessment of apoptotic activity was conducted by the detection of gene expressions related to BCl2 and P53. The documentation of the JNK/SMAD4/MMP2 signaling pathway was ultimately accomplished. The findings of our study indicate that RGOH therapy has significant inhibitory effects on the JNK/SMAD4/MMP2 pathway. This suggests that it could be a potential therapeutic option for cancer.