How long does it take for TGFβ to activate Smad?
Noteworthy, the peak of a transient TGF-β induced SMAD phosphorylation generally occurs within 30–60 min, turning back to baseline at between 3 and 6 h. On the other hand, the full differentiation of MFB needs between 2–3 days [26,44].
Does TGF-β1 increase phospho-ERK levels after hypoxia treatment?
However, levels of phospho-ERK increased after TGF-β1 treatment and continued to increase after hypoxia co-treatment. The activation of phospho-Smad2/3 and phospho-RhoA induced by TGFβ1 was significantly reduced after hypoxia co-treatment.
Do lipids change with TGF-β2 stimulation for 6'H?
We found a net increase in the extent of neutral lipids including saturated and monounsaturated FA in response to TGF-β2 stimulation for 6 h (Fig. 4a, b and Supplementary Table 2) while the pools of phospholipids and free FA were not altered (Fig. 4a ).
How does TGF-β activate myofibroblasts?
TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression.
What does TGFb pathway do?
The transforming growth factor beta (TGFB) signaling pathway is involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, cell migration, apoptosis, cellular homeostasis and other cellular functions.
How is TGFb activated?
Activation by protease and metalloprotease The TGF-β activation process involves the release of the LLC from the matrix, followed by further proteolysis of the LAP to release TGF-β to its receptors. MMP-9 and MMP-2 are known to cleave latent TGF-β.
Does TGFb promote proliferation?
Promotion of Cell Proliferation through TGF-β Signaling Although TGF-β induces cytostasis in multiple cell types, under certain conditions, it stimulates proliferation of endothelial cells and several types of mesenchymal cells.
How does TGF-beta induced EMT?
TGF-β induces the demethylation of H3K27me3 in Snail1 promoter and overexpresses Snail1, which cause EMT. Referring to these results, it's possible that the mechanism of TGF-β to induce EMT is correlated with Smad, which is the upstream molecule of Snail in the TGF-β signaling cascade21,22.
Is TGFB secreted?
TGF-β is secreted as a latent high-molecular-weight complex consisting of the C-terminal remnant of the TGF-β precursor and a TGF-β-binding protein. The biologically active levels of TGF-β depend on changes in its synthesis and in its activation from its latent form.
What stimulates TGF beta production?
Inflammatory stimuli that activate macrophages enhance the release of active TGF-β by promoting the activation of plasmin. Macrophages can also endocytose IgG-bound latent TGF-β complexes that are secreted by plasma cells and then release active TGF-β into the extracellular fluid.
What is the role of TGF beta in inflammation?
These studies show that TGF-β functions as an anti-inflammatory cytokine in cell types that are also present in atherosclerotic plaques. TGF-β is also known to be an important fibrotic cytokine that plays an important role in matrix remodeling and collagen synthesis.
What does high TGF beta mean?
What does it mean if your TGF-b1 result is too high? - TGF B-1 is often chronically over-expressed in disease states, including cancer, fibrosis and inflammation. - TGF B-1 is moderately to extremely high in Chronic Inflammatory Response Syndrome due to water-damaged buildings (CIRS).
Is TGF beta a tumor suppressor?
TGF-β is a potent proliferation inhibitor of normal colon epithelial cells and acts as a tumor suppressor. However, TGF-β also promotes invasion and metastasis during late-stage CRC, thereby acting as an oncogene.
How will TGF-beta affect F actin?
Addition of TGF beta 1 produced a dramatic reorganization of apical F-actin and development of stress fibers, as well as the loss of normal cell border-associated ZO-1 distribution. The effects of TGF beta 1 were blocked by the neutralizing antibodies to TGF beta 1.
What induces EMT?
EMT has been shown to be induced by androgen deprivation therapy in metastatic prostate cancer. Activation of EMT programs via inhibition of the androgen axis provides a mechanism by which tumor cells can adapt to promote disease recurrence and progression.
How will TGF-beta affect e cadherin expression?
Results: TGF-beta treatment was associated with morphologic and phenotypic changes typical of epithelial-mesenchymal transition (EMT) including increased fibrogenesis in all renal cell types and decreased E-cadherin expression in tubular cells.
How is TGF- synthesized?
TGF-β is synthesized in the form of a latent precursor that needs to be cleaved by furin-like proteases to become activated. Subsequently, the C-terminus of the TGF-β molecule binds to the N-terminus of the latency associated protein (LAP) to form the latent TGF-β complex (LTC) [21]. The LTC is then released and deposited in the surrounding ECM through binding to latent TGF-β binding protein (LTBP), forming the large latent complex (LLC) [22]. All TGF-β isoforms undergo such process, whereas certain BMPs and activins are not released as latent complexes [13]. Entrapment of TGF-β in the form of LLC ensures focused local effects upon activation. The release of active TGF-β from LLC is actively triggered through variant physical and biochemical factors, such as high or low pH, cleavage by specific proteases, and through interaction with integrins. The integrin interactions are considered the principal activating mechanism for latent TGF-β. Through integrin binding to certain collagen molecules in the ECM, traction forces are applied, which induce a conformational change in the LLC, thereby facilitating the accessibility of proteases that mediate the liberation of the active form of TGF-β [22,23,24,25]. We recently identified extracellular matrix protein 1 (ECM1) as a stabilizer of liver ECM deposited latent TGF-β by interacting with αv integrins. In different animal models of experimental liver fibrosis and in patients with chronic liver diseases (CLD), ECM1 expression decreased along with disease severity. Ecm1-KOmice spontaneously developed severe liver fibrosis with tremendous TGF-β/Smad3 and subsequent HSC activation. The animals die between 8 and 12 weeks of age. This phenotype could be rescued by adenoassociated virus (AAV) mediated expression of ECM1 or by interfering with TGF-β signaling using AAV expressing soluble TβRII. Moreover, carbon tetrachloride (CCl4)-induced liver damage was blunted by ECM1 overexpression [25].
What is the difference between SMAD2 and SMAD3?
Although SMAD2 and SMAD3 share 92% of their amino acid sequence, they show distinct biological effects. As already mentioned above, SMAD2, in contrast to SMAD3, has no DNA binding capacity, as it has extra sequences inserted in its Mad homology (MH1) domain, which prevents DNA binding [27]. This difference in DNA binding ability provides one explanation for the variant functions of the two SMAD molecules in general, in the different liver cell types and during liver fibrosis [27]. SMAD3 is considered the crucial inducer of the fibrogenic program in HSC, whereas SMAD2 was described as having antifibrotic effects [41,42]. A mechanism mediating such antifibrotic effects of SMAD2 was recently deciphered by Xu et al. and was linked to its ability to increase ligand (TRAIL) mediated HSC apoptosis through downregulation of X-linked inhibitor of apoptosis protein (XIAP). This then leads to enhanced caspase-3 activity and cell death [42]. In addition, deletion of SMAD3, but not SMAD2, in fibroblasts efficiently reduced pressure overload-induced cardiac fibrosis [43], which again underlines the differential roles of SMAD2 and SMAD3 in mediating organ fibrosis.
What is GP73 in hepatocytes?
GP73 is a type II transmembrane glycoprotein, which is highly expressed in hepatocytes during acute and CLD [68]. GP73 was recently described as a novel target and regulator of the TGF-β pathway. Its promoter contains a SMAD binding element (SBE) which is responsible for enhanced transcription upon stimulation with TGF-β [69]. Subsequently, GP73 feeds back on decision making between canonical and non-canonical arms of TGF-β signaling, thereby downregulating SMAD signaling and facilitating ERK/AKT signaling [69]. The modulatory effect of GP73 on TGF-β signaling was attributed to GP73 mediated upregulation of caveolin that subsequently modulates TGF-β signaling as discussed above [69]. However, the in vivo relevance of GP73 in HSC activation and liver fibrosis awaits further studies.
How many members are there in the TGF family?
The TGF-β family comprises 33 members including TGF-βs, activins, and bone morphogenetic proteins (BMPs) [13]. TGF-β proteins exist in three isoforms, TGF-β1, 2, and 3, which share overlapping but non-redundant functions, as most importantly shown by knockout (KO) mice of the respective TGF-β isoforms [14,15,16]. Generally, TGF-β1 is the most widely and most intensely investigated isoform in liver fibrogenesis [10,17]. Therefore, when using the term TGF-β, the knowledge is based on experimental data from TGF-β1 throughout this review. In this regard, we previously reported a predominant role for TGF-β2 in the pathogenesis of biliary fibrosis. In liver tissue of bile-duct ligated (BDL) and Mdr2-/-mice, TGF-β2 was elevated to a higher level than TGF-β1 [18]. Moreover, a TGF-β2 antisense oligonucleotide efficiently attenuated biliary fibrosis, as shown by reduced sirius red and α-SMA staining (data under revision). Abd El-Mequid et al. describe a correlation between TGF-β2 expression in peripheral leucocytes and protein levels in serum with hepatic fibrogenesis in a cohort of 89 HCV patients and 21 healthy controls. The authors thus suggest TGF-β2 as a promising biomarker for liver fibrosis in HCV [19]. Mechanistically, the increased expression of TGF-β2 in HCV-induced liver fibrosis is mediated via a cAMP-responsive element-binding protein (CREBH) site in the promoter of the TGF-β2 gene in hepatocytes that is activated from HCV infection [20].
What are BMPs in liver?
BMPs belong to the TGF-β superfamily and signal through SMAD1/5/8 complexes [13,89]. BMPs have diverse effects on liver fibrosis development and progression. For example, we recently reported upregulation of BMP9 expression in HSC and determined its fibrogenic role in the chronic CCl4mouse liver fibrosis model as well as in human patients with CLD, whereas BMP2 was proven to have antifibrotic effects in CCl4and BDL models of liver fibrosis [90,91]. Further, BMP-2 suppressed expression of TGF-β, TβRI and TβRII in HSC, whereas exogenous TGF-β and TGF-β signaling decreased BMP-2 expression, suggesting the existence of mutual regulation between the pathways of these two cytokines [91]. In addition, gremlin1, a TGF-β target gene, acts as antagonist of BMP7. It was upregulated in the porcine serum-induced hepatic fibrosis model [92]. Taking into consideration the antagonistic functions of BMP7 and TGF-β in liver fibrosis, gremlin1 could act as a bridge connecting these two signaling pathways [10].
What is the pathway that drives HSC activation?
In cooperation with other signaling pathways, triggered by e.g., reactive oxygen species (ROS), platelet-derived growth factor (PDGF), and connective tissue growth factor (CTGF), TGF-β signaling is considered the key fibrogenic pathway that drives HSC activation and induces ECM production [8,10]. In normal liver, quiescent HSC express a minute amount of TGF-β, which is upregulated shortly after liver injury. Besides HSC, there are additional cellular sources for TGF-β in the liver such as LSECs, macrophages, and hepatocytes [11]. Very recently, platelets were also described as an important source of TGF-β during liver fibrosis [12].
What are the cells that activate hepatic stellate cells?
Activation of hepatic stellate cells (HSCs) and origin of myofibroblasts (MFBs) in chronic liver diseases. During activation, HSCs lose intracellular lipid droplets, acquire a fibroblast-like shape, and express a large amount of alpha-smooth muscle actin (α-SMA) and extracellular matrix proteins (ECM). Beside HSCs, which represent a major source of MFBs, other cells such as pericytes, portal fibroblasts can differentiate into MFBs. Also, endothelial cells (ECs) and epithelial cells, i.e., hepatocytes and cholangiocytes, might contribute to liver MFBs pool through an endothelial-mesenchymal transition (EndMT) and epithelial-mesenchymal transition (EMT), respectively. However, unequivocal in vivo evidence of EMT during liver fibrosis is still missing.
How does acidosis affect TGF-2?
Acidosis triggers TGF-β2 upregulation/activation in a TSP1-dependent manner ( 1) and promotes an autocrine signaling pathway through TGF-βRI receptor and subsequent Smad2/3 phosphorylation ( 2) in cancer cells. TGFβ2-mediated signaling pathways induce a partial EMT gene programme ( 3) as well as a PKCζ-dependent translocation of CD36 fatty acid translocase to the plasma membrane ( 4 ). TGF-β2 autocrine signaling, in acidosis-adapted cancer cells, favors the uptake of long-chain fatty acids ( 5 ), the latter being oxidized in mitochondria for energy production ( 6) but also stored, as neutral lipids (triacylglycerols) in lipid droplets, in a DGAT1-dependent manner ( 7 ); the resulting increase in the cellular acetyl-CoA pool accounts for an overall increase in the acetylation of proteins, including Smad2 (thereby increasing its transcriptional activity). Both TGFβ2-induced partial EMT and lipid storage participate to increase survival (i.e., anoikis resistance) and invasion capacities that support in vivo metastatic spreading, ATP needs being fulfilled by FAO upon preferred hydrolysis of TG stored into LD ( 8 ). Inhibitors able to block different steps of the acidosis-induced TGF-β2/DGAT1 axis are indicated in red. ACSL1: acyl-CoA synthetase long chain family member 1; ATGL: adipose triglyceride lipase; CPT1: carnitine palmitoyltransferase 1; DAG: diacylglycerol; endo: endosomes; FA: fatty acids; PKCζ: protein kinase C ζ; PLIN2: perilipin 2; TAG: triacylglycerol; TSP-1: thrombospondin-1.
What is the main entry path for FA in the cytosol?
Expressions of both CD36 and DGAT1 , the main entry path for FA in the cytosol and the final actor of their accumulation as neutral lipids, respectively, are regulated by TGF-β2 produced by acidosis-adapted cancer cells. We identified the upregulation of TSP-1 in response to extracellular acidosis as a critical actor of integrin-independent TGF-β2 activation 37. We further documented a positive feedback loop wherein TGF-β2 transcription is stimulated by TGF-β2 itself, thereby reinforcing TGF-β2 signaling under acidosis. Remarkably, this mode of autocrine, isoform-specific TGF-β2 activation in cancer cells differs from that described in models where a reactive stroma leads to TGF-β secretion 58, 59, 60 or where excess free FA uptake itself promotes TGF-β signaling to initiate EMT 56. In our study, we provide evidence that addition of exogenous TGF-β2 on native cancer cells led to LD formation and that inhibition of TGF-β2 signaling in 3D spheroids undergoing spontaneous acidification dramatically prevented LD accumulation (Fig. 6f–h ). Of note, TGF-β1 was reported to shift cell metabolism from FA synthesis to enhanced oxidative phosphorylation 54. Whether, in the latter study, FA uptake was increased leading to LD formation was, however, not addressed. Although we cannot exclude this possibility, TGF-β2 was in our study the only TGF-β isoform induced in response to ambient acidosis. Thus, collectively, our results allow to propose a model wherein acidosis, by promoting autocrine TSP1-dependent TGF-β2 activation, triggers signaling pathways driving the shift toward a mesenchymal-like invasive phenotype from one hand, and supporting fatty acid uptake, oxidation but also storage in LD from the other hand (Fig. 7 ). Each arm reinforces each other since FA oxidation favors Smad2 acetylation/activity and thereby supports EMT while the bona fide EMT transcription factor ZEB1 is necessary for LD formation; ZEB-1-driven TGF-β2 expression 61 may actually account for the capacity of TGF-β2 to promote its own gene expression.
How does acidosis affect cancer cells?
We also documented that fatty acid (FA) metabolism is profoundly altered in response to ambient acidosis with FA oxidation (FAO) acting as a main source of acetyl-CoA to support TCA cycle 15. Moreover, acidosis-mediated metabolic rewiring is closely associated with sirtuin-mediated deacetylation of hypoxia-inducible factors HIF-1α and HIF-2α as well as histones H3 and H4 in the nucleus, leading to the differential expression of several metabolism-related enzymes 14, 15. In addition, acidosis can abolish hypoxia-induced gene reprogramming, in particular by inhibiting HIF-1α protein stabilization 17, 18. Altogether, these data indicate that acidosis, on its own, may strongly influence cancer cell metabolic phenotype.
How many genes are upregulated in acidosis?
This led us to identify a list of 349 genes upregulated in each 6.5/cell line while 306 transcripts were significantly downregulated in the same three cell types (FDR < 0.01 and FPKM > 0.5) (Supplementary Fig. 3a and Supplementary Data 1 ). Interestingly, in the short list of the 20 genes the most upregulated in each of the three screened 6.5/cancer cells, we found TGFB2 that encodes TGF-β2, which belongs to the family of transforming growth factors often associated with cancer cell invasiveness and the epithelial-mesenchymal transition (EMT) 35, and FST (follistatin), a gene under the control of TGF-β2 36 (Fig. 3a ). A net increase in TGF-β2 mRNA transcript was confirmed by qPCR in various acidosis-adapted cancer cells (Fig. 3b ). ELISA confirmed a significant increase of active TGF-β2 protein levels in acidosis-adapted cancer cell extracts (Fig. 3c) and corresponding extracellular media (Supplementary Fig. 3b ). Of note, in the same conditions, active TGF-β1 protein levels remained unchanged (Supplementary Fig. 3c, d ). Increases in TGF-β2 mRNA and protein could be recapitulated by 12–24 h exposure of native cancer cells to acidic pH 6.5 (Fig. 3d and Supplementary Fig. 3e) and further evidence of the activation of TGF-β2 signaling pathway in acidosis-adapted cancer cells was obtained by documenting an increase in the extent of phospho-Smad2/3 (Fig. 3e and Supplementary Fig. 3f ). Our search for the mechanism driving the preferential increase in TGF-β2 activity under acidosis then led us to document an autocrine TGF-β2-induced TGF-β2 transcription loop (Fig. 3f and Supplementary Fig. 3g) and more importantly, the upregulation of thrombospondin-1 (TSP-1), a critical actor of TGF-β2 maturation (Fig. 3g ). Contrary to other TGF-β isoforms that contain a RGD motif in their latency-associated peptide to promote integrin-dependent activation, TGF-β2 activation requires TSP-1 to facilitate maturation in its active form 37. To further prove a role of TSP-1, we showed that TSP1 silencing reduced the extent of mature (active) TGF-β2 (Fig. 3h) and associated phospho-Smad signaling (Supplementary Fig. 3h ).
What is acidosis in the tumor microenvironment?
Acidosis, like hypoxia, is a hallmark of the tumor microenvironment (TME) 1, 2, 3, 4, 5, 6. It largely arises from the conjunction of the exacerbated metabolism of cancer cells (and cancer-associated cells) with a spatio-temporal disorganization of the tumor vasculature which limits the access to O 2 and prevents the rapid elimination of waste products including protons (H +) and CO 2 7. The most common source of H + is the one associated with lactate production from glucose metabolism 8 but hydration of CO 2 molecules generated upon decarboxylation of metabolic intermediates also leads to the production of H + 9, 10.
How long to fix cells on coverslip?
Cells grown on coverslips were fixed with 4% PFA for 10 min. After blocking with 5% BSA for 1 h, cells were incubated overnight at 4 °C with anti-E-cadherin and anti-vimentin antibodies (#14472 and #5741, respectively; Cell Signaling Technology). Cells were then incubated with Alexa Fluor 488- and 568-conjugated anti-rabbit secondary antibodies (#A11034 and #A11036, respectively; Thermo Fisher Scientific) for 1 h and nuclei were counterstained with DAPI. For neutral lipid staining, PFA-fixed cells were incubated with 0.5 µg/ml BODIPY 493/503 (#D3922; Thermo Fisher Scientific) for 30 min at room temperature. Slides were prepared with a fluorescence mounting medium (Dako) and staining was visualized with a Zeiss Imager 1.0 Apotome microscope.
What is the accession number for RNA sequencing data?
The source data underlying all figures are provided as a Source Data file. The accession number for the RNA sequencing data reported in this paper is GEO: GSE116035. Any further information about resources and reagents should be directed to, and will be fulfilled by the corresponding authors upon reasonable request.