LncRNA LITATS1 suppresses TGF-ß-induced EMT and cancer cell plasticity by potentiating TßRI degradation.

Epithelial cells acquire mesenchymal phenotypes through epithelial-mesenchymal transition (EMT) during cancer progression. However, how epithelial cells retain their epithelial traits and prevent malignant transformation is not well understood. Here, we report that the long noncoding RNA LITATS1 (LINC01137, ZC3H12A-DT) is an epithelial gatekeeper in normal epithelial cells and inhibits EMT in breast and non-small cell lung cancer cells. Transcriptome analysis identified LITATS1 as a TGF-ß target gene. LITATS1 expression is reduced in lung adenocarcinoma tissues compared with adjacent normal tissues and correlates with a favorable prognosis in breast and non-small cell lung cancer patients. LITATS1 depletion promotes TGF-ß-induced EMT, migration, and extravasation in cancer cells. Unbiased pathway analysis demonstrated that LITATS1 knockdown potently and selectively potentiates TGF-ß/SMAD signaling. Mechanistically, LITATS1 enhances the polyubiquitination and proteasomal degradation of TGF-ß type I receptor (TßRI). LITATS1 interacts with TßRI and the E3 ligase SMURF2, promoting the cytoplasmic retention of SMURF2. Our findings highlight a protective function of LITATS1 in epithelial integrity maintenance through the attenuation of TGF-ß/SMAD signaling and EMT.

Inhibition of TGF-ß type I receptor by SB505124 down-regulates osteoblast differentiation and mineralization of human mesenchymal stem cells.

Numerous signaling pathways are well-known in osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), including transforming growth factor-beta (TGF-ß) signaling pathway, which sends signals through specific type I and II serine/threonine kinase receptors. However, the key role of TGF-ß signaling during bone formation and remodeling is yet to be studied. A TGF-ß type I receptor inhibitor, SB505124, discovered through a screening of a small molecule library for their effect of osteoblast differentiation of hBMSCs. Alkaline phosphatase quantification and staining were tested as indicators of osteoblastic differentiation and Alizarin red staining was tested as an indicator of in vitro mineralization. Changes in gene expressions were assessed using qRT-PCR. SB505124 showed significant inhibition of the osteoblast differentiation of hBMSCs, as confirmed by reduced alkaline phosphatase, in vitro mineralization, and downregulation of osteoblast-associated gene expression. To further understand the molecular mechanisms involved in the inhibition of the TGF-ß type I receptor, we assessed the effects on signature genes of several signaling pathways identified in the osteoblast differentiation of hBMSCs. SB505124 downregulated gene expression of many genes linked to osteoblast-related signaling pathways including TGF-ß, insulin, focal adhesion, Notch, Vitamin D, interleukin (IL)-6, osteoblast signaling, and cytokines and inflammatory. We report TGF-ß type I receptor inhibitor (SB505124) is a potent inhibitor of osteoblastic differentiation of hBMSCs that could be a valuable innovative therapeutic tool to cure bone disorders with increased bone formation, besides its potential use to treat patients with cancer and fibrosis.

The multifunctional adaptor protein HIP-55 couples Smad7 to accelerate TGF-ß type I receptor degradation.

Transforming growth factor ß (TGF-ß) is a multifunctional polypeptide that plays critical roles in regulating a broad range of cellular functions and physiological processes. TGF-ß signalling dysfunction contributes to many disorders, such as cardiovascular diseases, cancer and immunological diseases. The homoeostasis of negative feedback regulation is critical for signal robustness, duration and specificity, which precisely control physiological and pathophysiological processes. However, the underlying mechanism by which the negative regulation of TGF-ß signalling is integrated and coordinated is still unclear. Here, we reveal that haematopoietic progenitor kinase-interacting protein of 55 kDa (HIP-55) was upregulated upon TGF-ß stimulation, while the loss of HIP-55 caused TGF-ß signalling overactivation and the abnormal accumulation of downstream extracellular matrix (ECM) genes. HIP-55 interacts with Smad7 and competes with Smad7/Axin complex formation to inhibit the Axin-mediated degradation of Smad7. HIP-55 further couples Smad7 to TßRI but not TßRII, driving TßRI degradation. Altogether, our findings demonstrate a new mechanism by which the effector and negative feedback functions of HIP-55 are coupled and may provide novel strategies for the treatment of TGF-ß signalling-related human diseases.

Effects of HMGB1 on Tricellular Tight Junctions via TGF-ß Signaling in Human Nasal Epithelial Cells.

The airway epithelium of the human nasal mucosa acts as a physical barrier that protects against inhaled substances and pathogens via bicellular and tricellular tight junctions (bTJs and tTJs) including claudins, angulin-1/LSR and tricellulin. High mobility group box-1 (HMGB1) increased by TGF-ß1 is involved in the induction of nasal inflammation and injury in patients with allergic rhinitis, chronic rhinosinusitis, and eosinophilic chronic rhinosinusitis. However, the detailed mechanisms by which this occurs remain unknown. In the present study, to investigate how HMGB1 affects the barrier of normal human nasal epithelial cells, 2D and 2.5D Matrigel culture of primary cultured human nasal epithelial cells were pretreated with TGF-ß type I receptor kinase inhibitor EW-7197 before treatment with HMGB1. Knockdown of angulin-1/LSR downregulated the epithelial barrier. Treatment with EW-7197 decreased angulin-1/LSR and concentrated the expression at tTJs from bTJs and increased the epithelial barrier. Treatment with a binder to angulin-1/LSR angubindin-1 decreased angulin-1/LSR and the epithelial barrier. Treatment with HMGB1 decreased angulin-1/LSR and the epithelial barrier. In 2.5D Matrigel culture, treatment with HMGB1 induced permeability of FITC-dextran (FD-4) into the lumen. Pretreatment with EW-7197 prevented the effects of HMGB1. HMGB1 disrupted the angulin-1/LSR-dependent epithelial permeability barriers of HNECs via TGF-ß signaling in HNECs.

TNF-α enhances TGF-ß-induced endothelial-to-mesenchymal transition via TGF-ß signal augmentation.

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer-associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF-ß, which induces endothelial-to-mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor-infiltrating inflammatory cells secrete various cytokines, including TNF-α. However, the role of TNF-α in TGF-ß-induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF-α on TGF-ß-induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF-ß and TNF-α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF-ß and TNF-α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF-ß type I receptor, TGF-ß2, activin A, and integrin αv, suggesting that TNF-α enhanced TGF-ß-induced EndMT by augmenting TGF-ß family signals. Furthermore, oral squamous cell carcinoma-derived cells underwent epithelial-to-mesenchymal transition (EMT) in response to humoral factors produced by TGF-ß and TNF-α-cultured ECs. This EndMT-driven EMT was blocked by inhibiting the action of TGF-ßs. Collectively, our findings suggest that TNF-α enhances TGF-ß-dependent EndMT, which contributes to tumor progression.

Design, synthesis and biological activity evaluation of novel 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as potent transforming growth factor-ß (TGF-ß) type I receptor inhibitors.

TGF-ß type I receptor (also known as activin-like kinase 5 or ALK5) plays a critical role in the progression of fibrotic diseases and tumor invasiveness and metastasis, as well. The development of small inhibitors targeting ALK5 has been validated as a potential therapeutic strategy for fibrotic diseases and cancer. Here, we developed various 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as ALK5 inhibitors. The optimization led to identification of potent and selective ALK5 inhibitors 12r. The compound 12r exhibited strong inhibitory activity both in vitro and in vivo, and pharmacokinetics study showed an oral bioavailability of 57.6%. Thus, compound 12r may provide as new therapeutic option as ALK5 TGF-ßR1 inhibitor.

5-Aza-2'-deoxycytidine induces human Tenon's capsule fibroblasts differentiation and fibrosis by up-regulating TGF-ß type I receptor.

The principle reason of high failure rate of glaucoma filtration surgery is the loss of filtration function caused by postoperative scar formation. We investigated the effects of 5-aza-2'-deoxycytidine (5-Aza-dc), a DNA methyltransferases inhibitor, on human Tenon's capsule fibroblasts (HTFs) differentiation and fibrosis and its mechanism of action, especially in relation to transforming growth factor (TGF)-ß1 signaling. TGF-ß1 was used to induce differentiation of cultured HTFs. 5-Aza-dc suppressed DNA methyltransferases (DNMTs) activity 6 h after treatment with a course corresponding to that of TGF-ß1-induced reduction of DNMT activity without affecting cell viability as measured by Cell Counting Kit-8 assay. 5-Aza-dc also reduced DNMT1 and DNMT3a protein expression from 24 to 48 h. HTFs migration evaluated by scratch-wound assay were significantly increased 24 h after 5-Aza-dc treatment, a time course similar to that of TGF-ß1. Treatment with 5-Aza-dc significantly increased the mRNA and protein levels of α-smooth muscle actin (α-SMA), collagen-1A1 (Col1A1), fibronectin (FN) and TGF-ß type I receptor (TGFßRI). Furthermore, the effects of 5-Aza-dc on DNMT activity suppression, cell migration, and fibrosis were all reversed by a TGFßRI inhibitor- SB-431542. Meanwhile, knockdown of DNMT1 upregulated TGFßRI expression and had the same fibrosis-inducing effect in HTFs, which was also inhibited by SB-431542. Thus, the results indicate that DNA hypomethylation induces HTFs differentiation and fibrosis through up-regulation of TGFßRI. DNA methylation status plays an important role in subconjunctival wound healing.

VprBP mitigates TGF-ß and Activin signaling by promoting Smurf1-mediated type I receptor degradation.

The transforming growth factor-ß (TGF-ß) family controls embryogenesis, stem cell differentiation, and tissue homeostasis. However, how post-translation modifications contribute to fine-tuning of TGF-ß family signaling responses is not well understood. Inhibitory (I)-Smads can antagonize TGF-ß/Smad signaling by recruiting Smurf E3 ubiquitin ligases to target the active TGF-ß receptor for proteasomal degradation. A proteomic interaction screen identified Vpr binding protein (VprBP) as novel binding partner of Smad7. Mis-expression studies revealed that VprBP negatively controls Smad2 phosphorylation, Smad2-Smad4 interaction, as well as TGF-ß target gene expression. VprBP was found to promote Smad7-Smurf1-TßRI complex formation and induce proteasomal degradation of TGF-ß type I receptor (TßRI). Moreover, VprBP appears to stabilize Smurf1 by suppressing Smurf1 poly-ubiquitination. In multiple adult and mouse embryonic stem cells, depletion of VprBP promotes TGF-ß or Activin-induced responses. In the mouse embryo VprBP expression negatively correlates with mesoderm marker expression, and VprBP attenuated mesoderm induction during zebrafish embryogenesis. Our findings thereby uncover a novel regulatory mechanism by which Smurf1 controls the TGF-ß and Activin cascade and identify VprBP as a critical determinant of embryonic mesoderm induction.

A TGF-ß type I receptor-like molecule with a key functional role in Haemonchus contortus development.

Here we investigated the gene of a transforming growth factor (TGF)-ß type I receptor-like molecule in Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of small ruminants. Designated Hc-tgfbr1, this gene is transcribed in all developmental stages of H. contortus, and the encoded protein has glycine-serine rich and kinase domains characteristic of a TGF-ß family type I receptor. Expression of a GFP reporter driven by the putative Hc-tgfbr1 promoter localised to two intestinal rings, the anterior-most intestinal ring (int ring I) and the posterior-most intestinal ring (int ring IX) in Caenorhabditis elegans in vivo. Heterologous genetic complementation using a plasmid construct containing Hc-tgfbr1 genomic DNA failed to rescue the function of Ce-daf-1 (a known TGF-ß type I receptor gene) in a daf-1-deficient mutant strain of C. elegans. In addition, a TGF-ß type I receptor inhibitor, galunisertib, and double-stranded RNA interference (RNAi) were employed to assess the function of Hc-tgfbr1 in the transition from exsheathed L3 (xL3) to the L4 of H. contortus in vitro, revealing that both galunisertib and Hc-tgfbr1-specific double-stranded RNA could retard L4 development. Taken together, these results provide evidence that Hc-tgfbr1 is involved in developmental processes in H. contortus in the transition from the free-living to the parasitic stage.

Smad2 is involved in Aggregatibacter actinomycetemcomitans-induced apoptosis.

Apoptosis is thought to contribute to the progression of periodontitis. It has been suggested that the apoptosis of epithelial cells may contribute to the loss of epithelial barrier function. Smad2, a downstream signaling molecule of TGF-ß receptors (TGF-ßRs), is critically involved in apoptosis in several cell types. However, the relationship between smad2 and bacteria-induced apoptosis has not yet been elucidated. It is possible that the regulation of apoptosis induced by periodontopathic bacteria may lead to novel preventive therapies for periodontitis. Therefore, in the present study, we investigated the involvement of smad2 phosphorylation in apoptosis of human gingival epithelial cells induced by Aggregatibacter actinomycetemcomitans (Aa). Aa apparently induced the phosphorylation of smad2 in primary human gingival epithelial cells (HGECs) or the human gingival epithelial cell line, OBA9 cells. In addition, Aa induced phosphorylation of the serine residue of the TGF-ß type I receptor (TGF-ßRI) in OBA9 cells. SB431542 (a TGF-ßRI inhibitor) and siRNA transfection for TGF-ßRI, which reduced both TGF-ßRI mRNA and protein levels, markedly attenuated the Aa-induced phosphorylation of smad2. Furthermore, the disruption of TGF-ßRI signaling cascade by SB431542 and siRNA transfection for TGF-ßRI abrogated the activation of cleaved caspase-3 expression and repressed apoptosis in OBA9 cells treated with Aa. Thus, Aa induced apoptosis in gingival epithelial cells by activating the TGF-ßRI-smad2-caspase-3 signaling pathway. The results of the present study may suggest that the periodontopathic bacteria, Aa, activates the TGF-ßR/smad2 signaling pathway in human gingival epithelial cells and induces apoptosis in epithelial cells, which may lead to new therapeutic strategies that modulate the initiation of periodontitis.

RanBPM interacts with TßRI, TRAF6 and curbs TGF induced nuclear accumulation of TßRI.

Transforming growth factor ß (TGF-ß), a cytokine, and its receptors play a vital role during normal embryogenesis, cell proliferation, differentiation, apoptosis and migration. Ran-binding protein in the microtubule-organizing center (RanBPM) serves as a scaffold protein that has been shown to interact with many other proteins, such as MET, Axl/Sky, TRAF6, IFNR, TrKA and TrkB in addition to p75NTR. In the current study, we have identified RanBPM as a novel binding partner of TßRI by yeast two-hybrid assay. The TßRI and RanBPM association was confirmed by co-immunoprecipitation and GST pull-down experiments. Additionally, expression of RanBPM abrogated the interaction between TßRI and TRAF6. Furthermore, RanBPM could depress TGF-ß induced TRAF6 ubiquitination, subsequent NF-κB signaling pathway, and block TGF-ß induced TßRI nuclear accumulation. Taken together, our results reveal that RanBPM may modulate TGF-ß-mediated downstream signaling and biological functions.

Role of caveolin-1 in fibrotic diseases.

Fibrosis underlies the pathogenesis of numerous diseases and leads to severe damage of vital body organs and, frequently, to death. Better understanding of the mechanisms resulting in fibrosis is essential for developing appropriate treatment solutions and is therefore of upmost importance. Recent evidence suggests a significant antifibrotic potential of an integral membrane protein, caveolin-1. While caveolin-1 has been widely studied for its role in the regulation of cell signaling and endocytosis, its possible implication in fibrosis remains largely unclear. In this review we survey involvement of caveolin-1 in various cellular processes and highlight different aspects of its antifibrotic activity. We hypothesize that caveolin-1 conveys a homeostatic function in the process of fibrosis by (a) regulating TGF-ß1 and its downstream signaling; (b) regulating critical cellular processes involved in tissue repair, such as migration, adhesion and cellular response to mechanical stress; and (c) antagonizing profibrotic processes, such as proliferation. Finally, we consider this homeostatic function of caveolin-1 as a possible novel approach in treatment of fibroproliferative diseases.

TGF-ß-induced expression of IGFBP-3 regulates IGF1R signaling in human osteosarcoma cells.

Signaling pathways initiated by transforming growth factor-ß (TGF-ß) and insulin-like growth factors (IGFs) are important in osteosarcoma cell growth. We have investigated a role for endogenous IGF binding protein-3 (IGFBP-3) in mediating cross-talk between TGF-ß receptor and type I IGF receptor (IGF1R) signaling pathways in MG-63 osteosarcoma cells. TGF-ß1 indirectly activated the Ras/Raf/MAPK pathway and induced the expression of IGFBP-3, an important regulator of IGF1R activity. IGFBP-3 attenuated TGF-ß1 activation of ERK1/2 and Akt in MG-63 cells, and inhibited TGF-ß1-induced cell cycle progression and proliferation. This effect of IGFBP-3 was blocked by inhibiting IGF1R signaling. TGF-ß1 phosphorylated Smad2 on the non-receptor substrate sites (Ser245/250/255). Blocking the TGF-ß1-induced expression of IGFBP-3 enhanced pSmad2(Ser245/250/255) and increased its nuclear accumulation. These results suggest an important role for TGF-ß1 in osteosarcoma cell growth, with the induction of IGFBP-3 by TGF-ß1 serving in a negative-feedback loop to control cell growth by preventing activation of the IGF1R.