The complexity of TGFβ/activin signaling in regeneration

The role of transforming growth factor β TGFβ/activin signaling in wound repair and regeneration is highly conserved in the animal kingdom. Various studies have shown that TGF-β/activin signaling can either promote or inhibit different aspects of the regeneration process (i.e., proliferation, differentiation, and re-epithelialization). It has been demonstrated in several biological systems that some of the different cellular responses promoted by TGFβ/activin signaling depend on the activation of Smad-dependent or Smad-independent signal transduction pathways. In the context of regeneration and wound healing, it has been shown that the type of R-Smad stimulated determines the different effects that can be obtained. However, neither the possible roles of Smad-independent pathways nor the interaction of the TGFβ/activin pathway with other complex signaling networks involved in the regenerative process has been studied extensively. Here, we review the important aspects concerning the TGFβ/activin signaling pathway in the regeneration process. We discuss data regarding the role of TGF-β/activin in the most common animal regenerative models to demonstrate how this signaling promotes or inhibits regeneration, depending on the cellular context.     

Naa10p and IKKα interaction regulates EMT in oral squamous cell carcinoma via TGF‐β1/Smad pathway

Epithelial‐mesenchymal transition (EMT) has been contributed to increase migration and invasion of cancer cells. However, the correlate of Naa10p and IKKα with EMT in oral squamous cell carcinoma (OSCC) is not yet fully understood. In our present study, we found N‐α‐acetyltransferase 10 protein (Naa10p) and IκB kinase α (IKKα) were abnormally abundant in oral squamous cell carcinoma (OSCC). Bioinformatic results indicate that the expression of Naa10p and IKKα is correlated with TGF‐β1/Smad and EMT‐related molecules. The Transwell migration, invasion, qRT‐PCR and Western blot assay indicated that Naa10p repressed OSCC cell migration, invasion and EMT, whereas IKKα promoted TGF‐β1–mediated OSCC cell migration, invasion and EMT. Mechanistically, Naa10p inhibited IKKα activation of Smad3 through the interaction with IKKα directly in OSCC cells after TGF‐β1 stimulation. Notably, knockdown of Naa10p reversed the IKKα‐induced change in the migration, invasion and EMT‐related molecules in OSCC cells after TGF‐β1 stimulation. These findings suggest that Naa10p interacted with IKKα mediates EMT in OSCC cells through TGF‐β1/Smad, a novel pathway for preventing OSCC.     

TGFβ Governs the Pleiotropic Activity of NDRG1 in Triple-Negative Breast Cancer Progression

In triple-negative breast cancer (TNBC), the pleiotropic NDRG1 (N-Myc downstream regulated gene 1) promotes progression and worse survival, yet contradictory results were documented, and the mechanisms remain unknown. Phosphorylation and localization could drive NDRG1 pleiotropy, nonetheless, their role in TNBC progression and clinical outcome was not investigated. We found enhanced p-NDRG1 (Thr346) by TGFβ1 and explored whether it drives NDRG1 pleiotropy and TNBC progression. In tissue microarrays of 81 TNBC patients, we identified that staining and localization of NDRG1 and p-NDRG1 (Thr346) are biomarkers and risk factors associated with shorter overall survival. We found that TGFβ1 leads NDRG1, downstream of GSK3β, and upstream of NF-κB, to differentially regulate migration, invasion, epithelial-mesenchymal transition, tumor initiation, and maintenance of different populations of cancer stem cells (CSCs), depending on the progression stage of tumor cells, and the combination of TGFβ and GSK3β inhibitors impaired CSCs. The present study revealed the striking importance to assess both total NDRG1 and p-NDRG1 (Thr346) positiveness and subcellular localization to evaluate patient prognosis and their stratification. NDRG1 pleiotropy is driven by TGFβ to differentially promote metastasis and/or maintenance of CSCs at different stages of tumor progression, which could be abrogated by the inhibition of TGFβ and GSK3β.     

CD151 promotes Colorectal Cancer progression by a crosstalk involving CEACAM6, LGR5 and Wnt signaling via TGFβ1

CD151 impacts various signaling pathways in different cancers, and promotes colorectal cancer (CRC) cell malignancy by yet undefined mechanisms. This study aimed to comprehensively assess CD151''s function in CRC. CD151 levels were significantly higher in CRC tissues and cells compared with controls in the tissue microarray. Cell viability, migration and invasion were suppressed by CD151 downregulation in CRC cells. Consistently, mouse xenografts were inhibited by CD151 silencing. RNA-seq revealed that multiple genes were significantly altered by CD151 knockdown in cultured CRC cells and xenografts. Particularly, transforming growth factor β1 (TGFβ1), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) alongside CD151 were downregulated both in vitro and in vivo. Co-immunoprecipitation and mass spectrometry results were validated by qRT-PCR and immunoblot. Moreover, pull-down assay and immunofluorescence confirmed the associations of TGFβ1, CEACAM6 and LGR5 with CD151. This study demonstrated CEACAM6, LGR5 and Wnt pathway suppression by CD151 silencing might occur through TGFβ1 regulation, offering a comprehensive view of CD151''s roles in colorectal carcinogenesis. Our findings provide an insight into the CD151-involved signaling network in CRC oncogenesis, which could be utilized to design novel targeted therapies against CD151-based signaling in treatment for CRC.     

A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins

Rationale: The αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFβ complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called “5a”), which selectively recognizes the LAP/TGFβ complex-binding site of αvβ6 and αvβ8.Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with ¹⁸F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvβ6/αvβ8 integrins and various αvβ6/αvβ8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvβ8-positive prostate tumors.Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFβ activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvβ6/αvβ8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvβ8-positive prostate tumors.Conclusions: The results indicate that 5a can home to αvβ6- and/or αvβ8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFβ activators.     

Simvastatin inhibits stem cell proliferation in human leiomyoma via TGF‐β3 and Wnt/β‐Catenin pathways

Uterine leiomyoma (UL) is the most common gynaecologic tumour, affecting an estimated 70 to 80% of women. Leiomyomas develop from the transformation of myometrial stem cells into leiomyoma stem (or tumour‐initiating) cells. These cells undergo self‐renewal and differentiation to mature cells, both are necessary for the maintenance of tumour stem cell niche and tumour growth, respectively. Wnt/β‐catenin and TGF‐β/SMAD pathways, both overactive in UL, promote stem cell self‐renewal, crosstalk between stem and mature cells, cellular proliferation, extracellular matrix (ECM) accumulation and drive overall UL growth. Recent evidence suggests that simvastatin, an antihyperlipidemic drug, may have anti‐leiomyoma properties. Herein, we investigated the effects of simvastatin on UL stem cells. We isolated leiomyoma stem cells by flow cytometry using DyeCycle Violet staining and Stro‐1/CD44 surface markers. We found that simvastatin inhibits proliferation and induces apoptosis in UL stem cells. In addition, it also suppressed the expression of the stemness markers Nanog, Oct4 and Sox2. Simvastatin significantly decreased the production of the key ECM proteins, collagen 1 and fibronectin. Finally, it inhibited genes and/or proteins expression of TGF‐β1, 2 and 3, SMAD2, SMAD4, Wnt4, β‐Catenin, LRP6, AXIN2 and Cyclin D1 in UL stem cells, all are key drivers of the TGF‐β3/SMAD2 and Wnt4/β‐Catenin pathways. Thus, we have identified a novel stem cell‐targeting anti‐leiomyoma simvastatin effect. Further studies are needed to replicate these findings in vivo.     

A disintegrin and metalloproteinase 8 induced epithelial‐mesenchymal transition to promote the invasion of colon cancer cells via TGF‐β/Smad2/3 signalling pathway

A disintegrin and metalloproteinase 8 (ADAM8) protein is a multi‐domain transmembrane glycoprotein which involves in extracellular matrix remodelling, cell adhesion, invasion and migration. ADAM8 and epithelial‐mesenchymal transition (EMT) play an important role in tumour invasion has been well established. However, the interaction between ADAM8 and EMT has remained unclear. The data of colon cancer patients obtained from TCGA (The Cancer Genome Atlas) and GTEx (Genotype‐Tissue Expression Project) were analysed by the bioinformatics research method. The expression of ADAM8 in colon cancer cells was up‐regulated and down‐regulated by transfecting with the expression plasmid and small interfering RNA, respectively. Transwell invasion assay, immunohistochemistry, immunocytochemistry, Western blotting and qRT‐PCR were utilized to study the effect of ADAM8 on colon cancer cell''s EMT and its related mechanisms. Analysis of TCGA and GTEx data revealed that ADAM8 was linked to poor overall survival in colon cancer patients. Besides, ADAM8 was correlated with multiple EMT biomarkers (E‐cadherin, N‐cadherin, Vimentin, Snail2 and ZEB2). In vitro, we also proved that the up‐regulation of ADAM8 could promote EMT effect and enhance the invasive ability of colon cancer cells. On the contrary, the down‐regulation of ADAM8 in colon cancer cells attenuated these effects above. Further studies suggested that ADAM8 modulated EMT on colon cancer cells through TGF‐β/Smad2/3 signalling pathway. Our research suggested that ADAM8 could be a potential biomarker for the prognosis of colon cancer and induced EMT to promote the invasion of colon cancer cells via activating TGF‐β/Smad2/3 signalling pathway.     

Dok5 regulates proliferation and differentiation of osteoblast via canonical Wnt/β-catenin signaling

Objective:In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.Methods:A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.Results:Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.Conclusion:Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.     

TGFβ signaling in cartilage development and maintenance

Members of the transforming growth factor beta (TGFβ) superfamily of secreted factors play essential roles in nearly every aspect of cartilage formation and maintenance. However, the mechanisms by which TGFβs transduce their effects in cartilage in vivo remain poorly understood. Mutations in several TGFβ family members, their receptors, extracellular modulators, and intracellular transducers have been described, and these usually impact the development of the cartilaginous skeleton. Furthermore, genome‐wide association studies have linked components of the (TGFβ) superfamily to susceptibility to osteoarthritis. This review focuses on recent discoveries from genetic studies in the mouse regarding the regulation of TGFβ signaling in developing growth plate and articular cartilage, as well as the different modes of crosstalk between canonical and noncanonical TGFβ signaling. These new insights into TGFβ signaling in cartilage may open new prospects for therapies that maintain healthy articular cartilage. Birth Defects Research (Part C) 102:37–51, 2014. © 2014 Wiley Periodicals, Inc.     

TNFα activation and TGFβ blockage act synergistically for smooth muscle cell calcification in patients with venous thrombosis via TGFβ/ERK pathway

Venous calcification has been observed in post‐thrombotic syndrome (PTS) patients; yet, the cell types and possible mechanisms regulating this process are still unclear. We evaluated the calcium deposition within the venous wall, the cell type involved in the calcified remodelling of the venous wall after thrombosis and explored possible mechanisms in vitro. Calcium deposition was found in human specimens of superficial thrombotic veins and was co‐localized with VSMCs markers αSMA and TAGLN (also known as SM22α). Besides, the expression of osteogenesis‐related genes was dramatically changed in superficial thrombotic veins. Moreover, the inhibition of the TGFβ signalling pathway after TNFα treatment effectively induced the expression of osteogenic phenotype markers, the calcium salt deposits and the obvious phosphorylation of ERK1/2 and JNK2 in the VSMCs calcification model. Supplementing TGFβ2 or blocking the activation of the ERK/MAPK signalling pathway prevented the transformation of VSMCs into osteoblast‐like cells in vitro. Taken together, VSMCs have an important role in venous calcification after thrombosis. Supplementing TGFβ2 or inhibiting the ERK/MAPK signalling pathway can reduce the appearance of VSMCs osteogenic phenotype. Our findings may present a novel therapeutic approach to prevent of vascular calcification after venous thrombosis.     

The interactions between integrin α5β1 of liver cancer cells and fibronectin of fibroblasts promote tumor growth and angiogenesis

Hepatocellular carcinoma (HCC) progression is closely related to pathological fibrosis, which involves heterotypic intercellular interactions (HIIs) between liver cancer cells and fibroblasts. Here, we studied them in a direct coculture model, and identified fibronectin from fibroblasts and integrin-α₅β₁ from liver cancer cells as the primary responsible molecules utilizing CRISPR/Cas9 gene-editing technology. Coculture led to the formation of 3D multilayer microstructures, and obvious fibronectin remodeling was caused by upregulated integrin-α₅β₁, which greatly promoted cell growth in 3D microstructures. Integrin-α₅ was more sensitive and specific than integrin-β₁ in this process. Subsequent mechanistic exploration revealed the activation of integrin-Src-FAK, AKT and ERK signaling pathways. Importantly, the growth-promoting effect of HIIs was verified in a xenograft tumor model, in which more blood vessels were observed in bigger tumors derived from the coculture group than that derived from monocultured groups. Hence, we conducted triculture by introducing human umbilical vein endothelial cells, which aligned to and differentiated along multilayer microstructures in an integrin-α₅β₁ dependent manner. Furthermore, fibronectin, integrin-α₅, and integrin-β₁ were upregulated in 52 HCC tumors, and fibronectin was related to microvascular invasion. Our findings identify fibronectin, integrin-α₅, and integrin-β₁ as tumor microenvironment-related targets and provide a basis for combination targeted therapeutic strategies for future HCC treatment.     

Nobiletin suppresses cholangiocarcinoma proliferation via inhibiting GSK3β

Background: Cholangiocarcinoma (CCA) is a type of hepatobiliary cancer characterized by uncontrolled cell proliferation, with a poor prognosis and high mortality. Nobiletin (NBT) is a promising anti-tumor compound derived from the peels of oranges and other citrus plants, citrus plant. But the effect of NBT on CCA remains unknown.Results: Our data showed that NBT suppressed CCA cell proliferation in vitro and in vivo. Colony formation and Edu assay indicated that NBT inhibited cell proliferation. Cell cycle analysis showed that NBT arrested the cell cycle in G0/G1 phase. Target prediction showed that GSK3β was a direct target. Western blot and immunofluorescence confirmed that NBT reduced the phosphorylation of GSK3β. The antiproliferative effect of NBT was intercepted in GSK3β knockdown CCA cells. The cellular thermal shift assay (CETSA) showed NBT directly bound to GSK3β. Finally, NBT showed an anti-proliferative effect in tumor-bearing mice with no hepatotoxicity.Conclusion: NBT could inhibit CCA proliferation, and the pharmacological activity of NBT in CCA was attributed to its direct binding to GSK3β. We suggested that NBT might be a potential natural medicine in CCA treatment.     

Kips off to Myc: Implications for TGFβ signaling

Loss of sensitivity to the negative growth regulator transforming growth factor β (TGFβ) is a feature of many different tumor types and is likely involved in tumor progression. In some cases this loss of sensitivity to TGFβ has been shown to be manifest in the absence of membrane-associated TGFβ receptor complexes, thus preventing initiation of antiproliferative signals from the cell surface. In others, loss of sensitivity to TGFβ-induced inhibitory signals has been attributed to loss of function of intracellular effectors of TGFβ-induced inhibitory signals due to mutation or allelic loss of effector genes and their products. The intracellular effectors of TGFβ inhibitory signals have been shown to be involved in the normal regulation of progression through the cell cycle, specifically during G₁ phase. In this manner, elucidation of the mechanisms by which TGFβ inhibits cell growth not only helps us identify steps involved in tumor progression, but also allows us to better understand how cells regulate progression through the cell cycle. J. Cell. Biochem. 66:427–432, 1997. © 1997 Wiley-Liss, Inc.     

TGFβ regulation of membrane mucin Muc4 via proteosome degradation

Muc4 is a heterodimeric membrane mucin implicated in epithelial differentiation and tumor progression. It is expressed from a single gene as a 300 kDa precursor protein which is cleaved in the endoplasmic reticulum to its two subunits. Our previous work has shown that Muc4 is regulated by TGFβ, which represses the precursor cleavage. Working with Muc4‐transfected A375 tumor cells, we now show that Muc4 undergoes proteosomal degradation. Proteosome inhibitors prolong the life of the precursor, shunt the Muc4 into cytoplasmic aggresomes, increase the level of Muc4 associated with the endoplasmic reticulum chaperones calnexin and calreticulin and increase the levels of ubiquitinated Muc4. Most importantly, proteosome inhibitors repress the TGFβ inhibition of Muc4 expression. These results suggest a model in which TGFβ inhibits precursor cleavage, shunting the precursor into the proteosomal degradation pathway. Thus, the cells have evolved a mechanism to use the quality control pathway for glycoproteins to control the quantity of the protein produced. J. Cell. Biochem. 107: 797–802, 2009. © 2009 Wiley‐Liss, Inc.