TGF-β1 Induction Research Articles

Oxidative stress and TGF-β1 induction by metformin in MCF-7 and MDA-MB-231 human breast cancer cells are accompanied with the downregulation of genes related to cell proliferation, invasion and metastasis

High doses of metformin induces oxidative stress (OS) and transforming growth factor β1 (TGF-β1) in breast cancer cells, which was associated with increased cancer stem cell population, local invasion, liver metastasis and treatment resistance. Considering the impact of TGF- β1 and OS in breast cancer and the interrelation between these two pathways, the objective of this work was to investigate the effects of consecutive metformin treatments, at a non-cytotoxic dosage, in TGF- β1 targets in MCF-7 and MDA-MB-231 cells. Cells were exposed to 6 μM of metformin for seven consecutive passages. Samples were collected to immunocytochemistry (evaluation of p53, Nf-кB, NRF2 and TGF-β1), biochemical (determination of lipoperoxidation, total thiols and nitric oxide/peroxynitrite levels) and molecular biology analyzes (microarray and Real-time quantitative array PCR). Microarray analysis confirmed alterations in genes related to OS and TGF-β1. Treatment interfered in several TGF-β1 target-genes. Metformin upregulated genes involved in OS generation and apoptosis, and downregulated genes associated with metastasis and epithelial mesenchymal transition in MCF-7 cells. In MDA-MB-231 cells, metformin downregulated genes involved with cell invasion, viability and proliferation. The results shows that even a non-cytotoxic dosage of metformin can promote a less aggressive profile of gene expression in breast cancer cells.

The role of APOE in Microglia regulation in Neurogeneration

Alzheimer’s disease (AD) is the most prevalent senile dementia affecting 4.5 million Americans. Neuroinflammatory changes are prominent and may significantly contribute to the pathologic process. Mononuclear phagocytes (brain resident microglia and recruited peripheral monocytes) accumulate around amyloid plaque in AD brains. However, their exact cellular identity, molecular and functional phenotypes, and their protective or destructive roles in AD are not well understood. This stems in part from the lack of a specific molecular signatures for mononuclear phagocytes, cell type-specific antibodies, and analytic tools for in situ characterization. We recently identified a unique TGFβ-dependent molecular signature of homeostatic (M0)- and APOE-dependent (MGnD)- microglia in mouse models including APP-PS1 mice and human AD. Mechanistically, the TREM2-APOE pathway mediates a switch from M0- to MGnD-microglia phenotype after phagocytosis of apoptotic neurons in a cell-autonomous manner. TREM2 induces APOE signaling which is a negative regulator of the transcription program in M0-microglia.Transcription regulatory network analysis identified direct effect of APOE on suppression of major microglial homeostatic regulators including TGFβ signaling and induction of disease-associated molecules which are essential for pathogenicity in neuroinflammation. Specific genetic ablation of Apoe and/orTrem2 in microglia restored their homeostatic phenotype and genetic ablation of Apoe or Trem2 in TAU (P301S) mice arrested neurodegeneration and brain atrophy. Therefore, APOE plays an important role in microglia phenotype regulation in conditions, and restoration of the homeostatic microglia by targeting the APOE-signaling in microglia represents a novel immunotherapeutic approach. Taken together, our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in diseases and serves as a novel target to restore homeostatic microglia. These advances have major implications not only for understanding normal CNS function, but have opened up new avenues to understand the role of microglia in disease and most importantly have created the opportunity for consideration of ways in which microglial may be imaged and targeted for the treatment of disease. Since APOE e4 is the major risk factor of the disease, we study the role of APOE e4 in microglia regulation by employing novel tools including new mouse models and techniques to specifically target APOE in order to restore microglia-mediated protein clearance and brain function in animal models of tauopathies and AD. I will present recent advances in understanding the new molecular signature of homeostatic microglia, disease associated microglia and how microglia are regulated in health and disease. This impairment triggers a progressive loss of cognitive skills such as memory and decision making. Neurodegeneration is a key aspect of a large number of diseases that come under the umbrella of neurodegenerative diseases. Of these hundreds of different disorders, attention has so far focused mainly on just a handful, with Parkinson's disease, Huntington disease, being the most notable and Alzheimer's disease. A large proportion of the lesser-advertised diseases were essentially ignored. Such disorders can lead to irreversible damage to the brain and neurodegeneration. While all three of the diseases manifest with different clinical characteristics, the mechanisms of disease tend to be identical at the cellular level. Parkinson's disease, for example , affects the basal ganglia in the Mind, it depletes dopamine. It results in swelling, swelling and tremors in the body's main muscles, which are common features of the disease. There are deposits of tiny protein plaques in Alzheimer's disease which damage various parts of the brain and lead to progressive memory loss. Huntington's disease is a progressive genetic disorder which affects the body’s major muscles Severe restriction on the engine, and ultimately death.

Role of MMP-9 in epithelial-mesenchymal transition of thyroid cancer

BackgroundThe purpose of this study is to explore the role and mechanism of MMP-9 in the EMT process of thyroid cancer (TC), so as to provide a basis for clinical exploration of invasion and metastasis process of TC, looking for biological markers of tumor metastasis and molecular intervention therapy.MethodsWestern blot and RT-PCR were employed to detect the expression of MMP-9 in human normal thyroid cell line HT-ori3 and human TC cell lines IHH-4 (PTC), FTC-133, and 8505C. Expression levels of EMT-related markers: epithelial cell marker E-cadherin and stromal cell marker Vimentin in TGF-1-induced TC cell lines were detected by Western blot and RT-PCR, respectively. The effects of MMP-9 downregulation on cell invasion and metastasis were investigated by wound-healing assay and cell invasion experiment.ResultsThe protein and mRNA expression levels of MMP-9 in TC cell lines were increased compared with the human normal thyroid cell line HT-ori3. When TGF-β1 was added, the expression of EMT and Vimentin increased while the expression of E-cadherin decreased. Compared with the control group, the TC cells stably transfected with MMP-9 shRNA showed inhibited EMT, decreased Vimentin expression, and increased E-cadherin expression. The induction of TGF-β1 did not promote the occurrence of EMT in TC cells which were stably transformed with MMP-9 shRNA. The addition of TGF-β1 to TC cells increased the ability of the cells to migrate and invade. Compared with the control group, the migration and invasion ability of TC cells stably transfected with MMP-9 shRNA was significantly reduced, and the induction of TGF-β1 could not restore the migration and invasion ability of cells without MMP-9.ConclusionsIn conclusion, we found that MMP-9 can be used as a biomarker for TC, which can promote the EMT process of TGF-β1 induced TC, and thus affecting the cell migration and invasion ability.

Small molecule inhibition of non-canonical (TAK1-mediated) BMP signaling results in reduced chondrogenic ossification and heterotopic ossification in a rat model of blast-associated combat-related lower limb trauma.

Heterotopic ossification (HO) is defined as ectopic bone formation around joints and in soft tissues following trauma, particularly blast-related extremity injuries, thermal injuries, central nerve injuries, or orthopaedic surgeries, leading to increased pain and diminished quality of life. Current treatment options include pharmacotherapy with non-steroidal anti-inflammatory drugs, radiotherapy, and surgical excision, but these treatments have limited efficacy and have associated complication profiles. In contrast, small molecule inhibitors have been shown to have higher specificity and less systemic cytotoxicity. Previous studies have shown that bone morphogenetic protein (BMP) signaling and downstream non-canonical (SMAD-independent) BMP signaling mediated induction of TGF-β activated kinase-1 (TAK1) contributes to HO. In the current study, small molecule inhibition of TAK1, NG-25, was evaluated for its efficacy in limiting ectopic bone formation following a rat blast-associated lower limb trauma and a murine burn tenotomy injury model. A significant decrease in total HO volume in the rat blast injury model was observed by microCT imaging with no systemic complications following NG-25 therapy. Furthermore, tissue-resident mesenchymal progenitor cells (MPCs) harvested from rats treated with NG-25 demonstrated decreased proliferation, limited osteogenic differentiation capacity, and reduced gene expression of Tac1, Col10a1, Ibsp, Smad3, and Sox2 (P<0.05). Single cell RNA-sequencing of murine cells harvested from the injury site in a burn tenotomy injury model showed increased expression of these genes in MPCs during stages of chondrogenic differentiation. Additional in vitro cell cultures of murine tissue-resident MPCs and osteochondrogenic progenitors (OCPs) treated with NG-25 demonstrated reduced chondrogenic differentiation by 10.2-fold (P<0.001) and 133.3-fold (P<0.001), respectively, as well as associated reduction in chondrogenic gene expression. Induction of HO in Tak1 knockout mice demonstrated a 7.1-fold (P<0.001) and 2.7-fold reduction (P<0.001) in chondrogenic differentiation of murine MPCs and OCPs, respectively, with reduced chondrogenic gene expression. Together, our in vivo models and in vitro cell culture studies demonstrate the importance of TAK1 signaling in chondrogenic differentiation and HO formation and suggest that small molecule inhibition of TAK1 is a promising therapy to limit the formation and progression of HO.

WITHDRAWN: Immunomodulatory effects of sCD83 via induction of TGF-β1 and IDO: an in vitro and rat orthotopic liver transplantation study

WITHDRAWN: Immunomodulatory effects of sCD83 via induction of TGF-β1 and IDO: an in vitro and rat orthotopic liver transplantation study

The oncogenic role of MUC12 in RCC progression depends on c-Jun/TGF-β signalling.

Renal cell carcinoma (RCC) is a common kidney cancer worldwide. Even though current treatments show promising therapeutic effectiveness, metastatic RCC still has limited therapeutic options so that novel treatments were urgently needed. Here, we identified that MUC12 was overexpressed in RCC patients and served as poor prognostic factor for RCC progression. Overexpression of MUC12 increased RCC cell growth and cell invasion while deficiency of MUC12 exerted opposite effects on RCC cells. Mechanistic dissection demonstrated that MUC12‐mediated RCC cell growth and cell invasion were dependent of TGF‐β1 signalling because they could be blocked in the presence of TGF‐β1 inhibitor. Moreover, the regulation of TGF‐β1 by MUC12 relied on the transactivation of c‐Jun. MUC12 promoted the recruitment of c‐Jun on the promoter of TGF‐β1, leading to its transcription. Importantly, knockdown of c‐Jun also attenuated MUC12‐mediated TGF‐β1 induction and RCC cell invasion. In summary, our study defines the role of MUC12 in RCC progression and provides rational to develop novel targeted therapy to battle against RCC.

Epigallocatechin-3-gallate (EGCG) inhibits myofibroblast transformation of human Tenon's fibroblasts

Myofibroblast transformation of human Tenon's fibroblasts severely challenges the outcome of glaucoma filtration surgery. epigallocatechin-3-gallate (EGCG) is considered as a potential reagent to overcome this issue for its anti-fibrosis effect on various human diseases, but it is unclear on the fibrosis of Tenon's fibroblasts. This study was conducted to investigate the effect of EGCG on TGF-β1-induced myofibroblast transformation of human Tenon's fibroblasts. The human Tenon's fibroblasts were incubated in the medium containing 10 ng/mL TGF-β1, and subsequently treated with EGCG or mitomycin C (MMC). The cell proliferation and migration were analyzed. The expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col-I), and p-Smad2/3 were also evaluated. It showed that EGCG and MMC strongly inhibited the elevation in cell number in tissue explants compared to the tissues treated with TGF-β1 alone. Scratch-Wound assay showed that 48 h after TGF-β1 induction, only 10% of the wound width remained. But cells treated with EGCG still showed over 93% wound width. Further, EGCG effectively inhibited TGF-β1-induced expression of α-SMA and Col-I as well as phosphorylation of Smad2/3 in Tenon's fibroblasts. Altogether, we concluded that EGCG suppressed the myofibroblast transformation in Tenon's fibroblasts through inactivating TGF-β1/Smad signaling. These findings demonstrate that EGCG can be considered as one of the possible antifibrotic reagents for preventing postoperative scarring in glaucoma filtration surgery.

P0734THE DELETION OF AKT1 PROMOTES THE RENAL FIBROSIS IN THE MURINE MODEL OF UNILATERAL URETERAL OBSTRUCTION

Abstract Background and Aims Previous studies have found the increased Akt activity in experimental renal fibrosis. We investigated the role of Akt1, one of the three Akt isoforms, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). Method In vivo, we subjected the wild type and Akt1−/− mice to UUO. In vitro, gene silencing of Akt1 was achieved using the short hairpin RNA delivered by the lentiviral vector in immortalized human proximal tubular cells (HK2 cells) and rat kidney fibroblasts (NRK-49F cells). Results In immunohistochemical stain, the expression of Akt1 was significantly higher in obstructed kidneys of wild type mice compared with control sham kidneys and increased gradually as UUO progressed. The fibronectin, type I collagen, and heat shock protein 47 (HSP47) were markedly more expressed in obstructed kidneys of Akt1−/− mice than in those of the wild type mice. Transforming growth factor β1 (TGFβ1) was highly induced within 1 day of UUO in obstructed kidneys of Akt1−/− mice and the expression of TGFβ1 was significantly higher in the Akt1−/− mice than in the wild type mice as UUO progressed. Western blot showed that silencing of Akt1 increased the expression of TGFβ1, which was enhanced by angiotensin II stimulation in HK2 cells, but not in NRK-49F cells. Immunohistochemical stain demonstrated that the expression of cleaved caspase-3 in renal tubules was significantly higher in the Akt1−/− mice than in the wild type mice. Western blot showed that silencing of Akt1 increased the expression of cleaved caspase-3 in HK2 cells, but not in NRK-49F cells. Conclusion Our findings suggest that the deletion of Akt1 might contribute to tubulointerstitial fibrosis and tubular apoptosis via TGFβ1 induction. We also showed that TGFβ1 upregulation by genetic deletion of Akt1 is associated with activation of STAT3 independently of the TGFβ1/Smad signaling pathway.

Cholesterol 25-hydroxylase promotes efferocytosis and resolution of lung inflammation.

Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol 25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds the expression of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the antiinflammatory nuclear receptor, liver X receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural oxysterols or sterols, 25HC is induced in the inflamed lung of mice and humans. Ch25h-/- mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia, which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGF-β. CH25H/25HC/LXR is, thus, an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.

Tγδ17 cells build up the nerve.

γδ T cells are critical contributors to tissue homeostasis. Recent research identifies an unexpected role for γδ T cell–derived IL-17F in promoting sympathetic innervation and tissue thermogenesis through the induction of the cytokine TGF-β in adipose cells.

The effect of adrenocorticotropic hormone on alpha-2-macroglobulin in osteoblasts derived from human mesenchymal stem cells.

Nowadays, alpha‐2‐macroglobulin (A2M) gene has allocated escalating interest among several genes involved in the pathogenesis of avascular necrosis of the femoral head (ANFH). This molecule could interact with several osteogenic‐related proteins. It was reported that adrenocorticotropic hormone (ACTH) affects bones through its receptor located on osteoblasts, suggesting it as a potential target in ANFH treatment. In this study, the effect of ACTH on A2M expression was investigated in osteoblasts as well as during the differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. In this study, MSCs derived from bone marrow were isolated and purified using Ficoll gradient and several passaging. MSCs were characterized by induction with osteogenic and adipogenic medium followed by Oil Red O, Alizarin Red and alkaline phosphatase staining. Besides, MSCs were exposed to various concentrations of ACTH to evaluate the cell variability by MTT assay. MSCs and differentiated osteoblasts were treated with 10−8 molar ACTH for 16 and 26 days, respectively. Then, the total RNA was extracted and A2M expression was quantified by real‐time qPCR. The protein expression levels of osteoblast markers including alkaline phosphatase (ALPL) and bone gamma‐carboxyglutamate protein (BGLAP) were also measured. The results showed that A2M expression in cells treated with ACTH was up‐regulated significantly compared to the control group. Similarly, the expression of osteoblast gene markers including ALPL and BGLAP was significantly increased. ACTH, as an osteoblastic differentiation enhancer, up‐regulates A2M, which promotes osteoblastic differentiation probably through TGF‐β induction.

Involvement of TGF-β and ROS in G1 Cell Cycle Arrest Induced by Titanium Dioxide Nanoparticles Under UVA Irradiation in a 3D Spheroid Model.

BackgroundAs one of the most widely produced engineered nanomaterials, titanium dioxide nanoparticles (nano-TiO2) are used in biomedicine and healthcare products, and as implant scaffolds; therefore, the toxic mechanism of nano-TiO2 has been extensively investigated with a view to guiding application. Three-dimensional (3D) spheroid models can simplify the complex physiological environment and mimic the in vivo architecture of tissues, which is optimal for the assessment of nano-TiO2 toxicity under ultraviolet A (UVA) irradiation.Methods and ResultsIn the present study, the toxicity of nano-TiO2 under UVA irradiation was investigated in 3D H22 spheroids cultured in fibrin gels. A significant reduction of approximately 25% in spheroid diameter was observed following treatment with 100 μg/mL nano-TiO2 under UVA irradiation after seven days of culture. Nano-TiO2 under UVA irradiation triggered the initiation of the TGF-β/Smad signaling pathway, increasing the expression levels of TGF-β1, Smad3, Cdkn1a, and Cdkn2b at both the mRNA and protein level, which resulted in cell cycle arrest in the G1 phase. In addition, nano-TiO2 under UVA irradiation also triggered the production of reactive oxygen species (ROS), which were shown to be involved in cell cycle regulation and the induction of TGF-β1 expression.ConclusionNano-TiO2 under UVA irradiation induced cell cycle arrest in the G1 phase and the formation of smaller spheroids, which were associated with TGF-β/Smad signaling pathway activation and ROS generation. These results reveal the toxic mechanism of nano-TiO2 under UVA irradiation, providing the possibility for 3D spheroid models to be used in nanotoxicology studies.

SAT-159 MAD2B Promotes Podocyte Injury through Regulating Numb-Dependent Notch1 Pathway in Diabetic Nephropathy

The pathological features of diabetic nephropathy（DN）include glomerulosclerosis, thickening of the glomerular basement membrane, glomerular hypertrophy, mesangial cell expansion, podocyte loss, renal-cell hypertrophy and tubulointerstitial fibrosis. Recent studies have demonstrated the loss of podocyte is a critical event in DN. Many studies suggested that DN podocyte injury was induced by the association of multiple factors, including mechanical stress, inflammatory reaction, oxidative stress, TGF-β1 induction, renin angiotensin aldosterone system (RAAS) activation, and AGEs accumulation. Previously, our group have found the mitotic arrest deficient protein MAD2B is implicated in high glucose (HG)-induced podocyte injury by regulating cell cycle. However, the exact mechanism of MAD2B in podocyte injury is lacking. Numb was originally discovered as an intrinsic cell fate determinant in Drosophila by antagonizing Notch signaling which may play a pivotal pathogenic role in podocyte injury. In vivo study, DN model was constructed on C57BL/6 mice by a single intra-peritoneal injection of STZ. Our group also successfully constructed gene knockout mice with podocyte-specific depletion of MAD2B (NMKO). In vitro study, immortalized human podocyte cell line was employed, and exposed to different treatments after differentiation. The expression of MAD2B and Numb was suppressed by recombinant lentivirus infection，while Numb was elevated by adenovirus infection. Our latest finding revealed that HG exposure downregulated Numb expression in podocytes and DN mice resulting in increase of NICD and Hes-1 and mitotic catastrophe of podocyte. In addition, the overexpression of Numb could alleviate podocyte injury through decreasing the expression of Desmin and increasing autophagy. Previously we have demonstrated the enhancement of MAD2B in DN mice and HG treated podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes when exposed to HG. Accordingly, the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased. Afterwards we identified Numb as a novel MAD2B binding protein in human kidney through screens using a yeast two-hybrid interaction trap. Through confocal laser scanning microscopy we confirmed the co-localization of MAD2B and Numb in podocyte in vivo and in vitro. Subsequent endogenous co-immunoprecipitation established the direct interaction between MAD2B and Numb. The mechanisms of interaction between them still remains to be established. Upregulated MAD2B expression contributes to Numb depletion and its downstream protein accumulation such as NICD and Hes-1 in HG condition，which ultimately leads to podocyte mitosis abnormality and cell polarity disorder. However, inhibiting MAD2B expression could reverse podocyte injury by maintaining normal cell cycle and increasing autophagy.

Dead Cells Induce Innate Anergy via Mertk after Acute Viral Infection

Infections can result in a temporarily restricted unresponsiveness of the innate immune response, thereby limiting pathogen control. Mechanisms of such unresponsiveness are well studied in lipopolysaccharide tolerance; however, whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here, we find that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days. Innate anergy is associated with induction of apoptotic cells, which activates the Tyro3, Axl, and Mertk (TAM) receptor Mertk and induces high levels of interleukin-10 (IL-10) and transforming growth factorβ (TGF-β). Lack of Mertk in Mertk-/- mice prevents induction of IL-10 and TGF-β, resulting in abrogation of innate anergy. Innate anergy is associated with enhanced VSV replication and poor survival after infection. Mechanistically, Mertk signaling upregulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Dexamethasone treatment upregulates Mertk and enhances innate anergy in a Mertk-dependent manner. In conclusion, we identify Mertk as one major regulator of innate tolerance during infection with VSV.

IPF pathogenesis is dependent upon TGFβ induction of IGF-1.

Pathogenic fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), have some of the worst prognoses and affect millions of people worldwide. With unclear etiology and minimally effective therapies, two-thirds of IPF patients die within 2-5years from this progressive interstitial lung disease. Transforming Growth Factor Beta (TGFβ) and insulin-like growth factor-1 (IGF-1) are known to promote fibrosis; however, myofibroblast specific upregulation of IGF-1 in the initiation and progression of TGFβ-induced fibrogenesis and IPF have remained unexplored. To address this, the current study (1) documents the upregulation of IGF-1 via TGFβ in myofibroblasts and fibrotic lung tissue, as well as its correlation with decreased pulmonary function in advanced IPF; (2) identifies IGF-1's C1 promoter as mediating the increase in IGF-1 transcription by TGFβ in pulmonary fibroblasts; (3) determines that SMAD2 and mTOR signaling are required for TGFβ-dependent Igf-1 expression in myofibroblasts; (4) demonstrates IGF-1R activation is essential to support TGFβ-driven profibrotic myofibroblast functions and excessive wound healing; and (5) establishes the effectiveness of slowing the progression of murine lung fibrosis with the IGF-1R inhibitor OSI-906. These findings expand our knowledge of IGF-1's role as a novel fibrotic-switch, bringing us one step closer to understanding the complex biological mechanisms responsible for fibrotic diseases and developing effective therapies.

The crosstalk between RNA m6A epitranscriptome and TGFβ signaling pathway contributes to the arrest of cell cycle.

TGFβ signaling pathway is critical for the cell division, differentiation and apoptosis, the aberrant regulation of which will result in severe diseases including cancer. N6-methyl-adenosine (m6A) is one of the most abundant modifications on mRNA, it is unclear yet how m6A epitranscriptome response to stimulation of TGFβ. Here, we found that cellular m6A level of RNA was elevated after TGFβ treatment, which might be regulated by upregulation of WTAP and METTL3. MeRIP-Seq of mRNAs of MCF7 with or without treated by TGFβ showed that mRNA with upregulated m6A modification level after TGFβ treatment were enriched in TGFβ signaling pathway. Phosphorylated level of SMAD2 or SMAD3 induced by TGFβ was impaired when WTAP was silenced. Moreover, the m6A modification and mRNA level of JunB, which is known as a cell cycle inhibitor, both were increased after induction of TGFβ and decreased after knockdown of WTAP. Intriguingly, growth inhibition caused by TGFβ was rescued in WTAP-knockdown cells. Collectively, these results reveal the key role that m6A pathway playing in the cell cycle arrest induced by TGFβ signaling, providing new mechanisms explanation for growth inhibition mediated by TGFβ.

Homeobox D3, A Novel Link Between Bone Morphogenetic Protein 9 and Transforming Growth Factor Beta 1 Signaling

AimsSeveral signaling pathways contribute to endothelial-mesenchymal transitions and vascular calcification, including bone morphogenetic protein (BMP) and transforming growth factor (TGF) β signaling. The transcription factor homeobox D3 (Hoxd3) is known to regulate an invasive endothelial phenotype, and the aim of the study is to determine if HOXD3 modulates BMP and TGFβ signaling in the endothelium. Methods and ResearchWe report that the endothelium with high BMP activity due to the loss of BMP inhibitor matrix Gla protein (MGP) shows induction of Hoxd3. HOXD3 is part of a BMP-triggered cascade. When activated by BMP9, activin receptor–like kinase (ALK) 1 induces HOXD3 expression. Hoxd3 promoter is a direct target of phosphorylated (p) SMAD1, a mediator of BMP signaling. High BMP activity further results in enhanced TGFβ signaling due to induction of TGFβ1 and its receptor, ALK5. This is mediated by HOXD3, which directly targets the Tgfb1 promoter. Finally, TGFβ1 and BMP9 stimulate the expression of MGP, which limits the enhanced ALK1 induction by counteracting BMP4. The cascade of BMP9-HOXD3-TGFβ also affects Notch signaling and angiogenesis through induction of Notch ligand Jagged 2 and suppression of Notch ligand delta-like 4 (Dll4). ConclusionThe results suggest that HOXD3 is a novel link between BMP9/ALK1 and TGFβ1/ALK5 signaling. Translational perspectiveBMP and TGFβ signaling are instrumental in vascular disease such as vascular calcification and atherosclerosis. This study demonstrated a novel type of cross talk between endothelial BMP and TGFβ signaling as mediated by HOXD3. The results provide a possible therapeutic approach to control dysfunctional BMP and TGFβ signaling by regulating HOXD3.

Protein phosphatase Mg2+ /Mn2+ dependent-1A and PTEN deregulation in renal fibrosis: Novel mechanisms and co-dependency of expression.

PPM1A and PTEN emerged as novel suppressors of chronic kidney disease (CKD). Since loss of PPM1A and PTEN in the tubulointerstitium promotes fibrogenesis, defining molecular events underlying PPM1A/PTEN deregulation is necessary to develop expression rescue as novel therapeutic strategies. Here we identify TGF-β1 as a principle repressor of PPM1A, as conditional renal tubular-specific induction of TGF-β1 in mice dramatically downregulates kidney PPM1A expression. TGF-β1 similarly attenuates PPM1A and PTEN expression in human renal epithelial cells and fibroblasts, via a protein degradation mechanism by promoting their ubiquitination. A proteasome inhibitor MG132 rescues PPM1A and PTEN expression, even in the presence of TGF-β1, along with decreased fibrogenesis. Restoration of PPM1A or PTEN similarly limits SMAD3 phosphorylation and the activation of TGF-β1-induced fibrotic genes. Concurrent loss of PPM1A and PTEN levels in aristolochic acid nephropathy further suggests crosstalk between these repressors. PPM1A silencing in renal fibroblasts, moreover, results in PTEN loss, while PTEN stable depletion decreases PPM1A expression with acquisition of a fibroproliferative phenotype in each case. Transient PPM1A expression, conversely, elevates cellular PTEN levels while lentiviral PTEN introduction increases PPM1A expression. PPM1A and PTEN, therefore, co-regulate each other's relative abundance, identifying a previously unknown pathological link between TGF-β1 repressors, contributing to CKD.

Moxibustion Promotes Formation of Granulation in Wound Healing Process through Induction of Transforming Growth Factor-β in Rats.

To examine the effect of moxibustion on the wound healing process and its mechanism using a rat wound model. Sixty male Sprague-Dawley rats were randomly divided into a sham-treated group (n=30, wound surgery only) and a moxibustion group (n=30, wound treated with moxibustion). Circular full-thickness skin wounds were produced in rats. Moxibustion was applied to the edge of wound and was continued on alternating days till 14 days after surgery, followed by measurement of wound size. Expression of collagens, prolyl-4-hydroxylase (P4H) and transforming growth factor-β (TGF-β) were evaluated by histochemical study and real-time polymerase chain reaction. The size of the wound lesion was significantly reduced in rats treated with moxibustion as compared to that in sham-treated rats at 4-10 days after wounding (P<0.01). Moxibustion stimulated mRNA expression of collagens at 4 days (P<0.01), but not at 7 days, accompanied by enhanced proliferation of P4H-positive fibroblasts. Of importance, expression of TGF-β in tissue from the wound lesion treated with moxibustion was significantly increased as compared to that in sham-treated rats at 4 days (P<0.01 or P<0.05), but not at 7 days. The treatment with moxibustion promoted the wound healing process in the early phase through proliferation of fibroblasts and rapid formation of granulation, possibly mediated by induction of TGF-β which is a key molecule in the physiological process of wound healing. Moxibustion can be expected to be effective as complementary treatment for intractable ulcers.

TOPK promotes epithelial-mesenchymal transition and invasion of breast cancer cells through upregulation of TBX3 in TGF-β1/Smad signaling

TOPK has been suggested to contribute to invasion of lung, prostate, gastric, pancreatic or breast cancer cells. However, how TOPK mediates TGF-β1/Smad signaling leading to epithelial-mesenchymal transition (EMT) and invasion of breast cancer cells remains unknown. Here we report that TOPK upregulates T-box transcription factor TBX3 to enhance TGF-β1-induced EMT and invasion of MDA-MB-231 breast cancer cells. Expression of endogenous TOPK was promoted by TGF-β1 treatment of MDA-MB-231 cells time-dependently. In addition, knockdown of TOPK attenuated TGF-β1-induced phosphorylation or transcriptional activity of Smad3. Meanwhile, levels of both mRNA and protein of TBX3 induced by TGF-β1 were abolished by TOPK depletion. Also, knockdown of TBX3 inhibited TGF-β1 induction of EMT-related genes Snail, Slug or Fibronectin. Furthermore, ablation of TOPK or TBX3 suppressed TGF-β1-induced MDA-MB-231 cell invasion. Collectively, we conclude that TOPK positively regulates TBX3 in TGF-β1/Smad signaling pathway, thereby enhancing EMT and invasion of breast cancer cells, implying a mechanistic role of TOPK in TGF-β1/Smad signaling.