Continuous culture of limbal epithelial stem cells (LSCs) slows down proliferation, which inevitably results in differentiation. Transforming growth factor-beta (TGFβ)-assisted epithelial–mesenchymal transition (EMT) is often found in the late stage of LSC culture. Thus, EMT is proposed to be part of the mechanism responsible for the loss of LSCs in culture. To explore the regulation mechanism of EMT, we investigated the early stage culture for factor(s) that may potentially prevent EMT.
LSCs from the corneal limbus region of rabbits were isolated and expanded to confluence in culture (P0), and then serial passage of these LSCs (P1 to P3) was performed. EMT in LSCs was induced with TGFβ1, and the corresponding EMT signaling was confirmed with Smad2/3 phosphorylation. The expression of mesenchymal markers, including alpha-smooth muscle actin (α-SMA) and vimentin, was determined with western blot analysis. Proteins extracted from different passaged cells were also subjected to western blot analysis of TGFβ signaling components, including TGFβ1, TGFβ receptor I/II, and Smad2/3 as well as Smad7, the main negative regulator of TGFβ signaling. The mitogenic response was measured with the bromodeoxyuridine (BrdU) labeling index and real-time PCR using primers for Ki67. N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor, and Jagged-1 Notch ligand were used to block and activate Notch signaling, respectively, and their efficacy was evaluated by determining the expression of Hes1, a Notch signaling target.
Mesenchymal marker induction and growth arrest were found in the TGFβ1-treated P1 cells, and the changes were less significant in the TGFβ1-treated P0 cells. Western blot analysis confirmed that the expressed levels of TGFβ signaling components, including TGFβ1, TGFβ receptor I/II, and Smad2/3, were relatively stable with passages. In contrast, the expression of Hes1 and Smad7 markedly decreased after the first passage, and with each passage, the levels diminished even further. Hes1 and Smad7 were expressed only in the limbal epithelium and not in the corneal epithelium. DAPT effectively blocked the expression of Hes1. DAPT also dose-dependently suppressed Smad7 expression in P0 cells, which was associated with the susceptibility of P0 cells to TGFβ1-induced Smad2/3 phosphorylation, EMT formation, and growth arrest. Reciprocally, Jagged-1 upregulated Smad7 expression in LSCs against TGFβ signaling.
These findings indicate that Smad7 plays a crucial role in antagonizing EMT induced by TGFβ signaling and support our proposition that Smad7 is a Notch signaling target in LSCs, and may mediate the Notch function in preventing the occurrence of EMT.
Coronary vessel development depends on a subpopulation of epicardial cells that undergo epithelial to mesenchymal transformation (EMT) and invade the subepicardial space and myocardium. These cells form the smooth muscle of the vessels and fibroblasts, but the mechanisms that regulate these processes are poorly understood. Mice lacking the Type III Transforming Growth Factor β Receptor (TGFβR3) die by E14.5 due to failed coronary vessel development accompanied by reduced epicardial cell invasion. BMP2 signals via TGFβR3 emphasizing the importance of determining the relative contributions of the canonical BMP signaling pathway and TGFβR3-dependent signaling to BMP2 responsiveness. Here we examined the role of TGFβR3 in BMP2 signaling in epicardial cells. Whereas TGFβ induced loss of epithelial character and smooth muscle differentiation, BMP2 induced an ALK3-dependent loss of epithelial character and modestly inhibited TGFβ-stimulated differentiation. Tgfbr3−/− cells respond to BMP2 indicating that TGFβR3 is not required. However, Tgfbr3−/− cells show decreased invasion in response to BMP2 and overexpression of TGFβR3 in Tgfbr3−/− cells rescued invasion. Invasion was dependent on ALK5, ALK2, ALK3, and Smad4. Expression of TGFβR3 lacking the 3 C-terminal amino acids required to interact with the scaffolding protein GIPC (GAIP-interacting protein, C terminus) did not rescue. Knockdown of GIPC in Tgfbr3+/+ or Tgfbr3−/− cells rescued with TGFβR3 decreased BMP2-stimulated invasion confirming a requirement for TGFβR3/GIPC interaction. Our results reveal the relative roles of TGFβR3-dependent and TGFβR3-independent signaling in the actions of BMP2 on epicardial cell behavior and demonstrate the critical role of TGFβR3 in mediating BMP2-stimulated invasion.
epithelial to mesenchymal transformation; transforming growth factor beta; bone morphogenic protein; epicardium; invasion; differentiation; coronary vessels
Transforming growth factor β1 (TGFβ1) is a potent stimulator of epithelial-to-mesenchymal transition (EMT) and has been associated with chronic kidney diseases by activating profibrotic gene expression. In this study, we investigated the role of the KEAP1-NRF2 pathway, which is a master regulator of the cellular antioxidant system, in TGFβ1-stimulated EMT gene changes using human renal tubular epithelial HK2. Treatment with TGFβ1 enhanced the levels of reactive oxygen species (ROS) and TGFβ1-stimulated EMT gene changes, including an increase in profibrotic fibronectin-1 and collagen 1A1, were diminished by the antioxidant N-acetylcysteine. In HK2, TGFβ1 suppressed NRF2 activity and thereby reduced the expression of GSH synthesizing enzyme through the elevation of ATF3 level. Therefore, the activation of NRF2 signaling with sulforaphane effectively attenuated the TGFβ1-stimulated increase in fibronectin-1 and collagen 1A1. Conversely, the TGFβ1-EMT gene changes were further enhanced by NRF2 knockdown compared to the control cells. The relationship of NRF2 signaling and TGFβ1-EMT changes was further confirmed in a stable KEAP1-knockdown HK2, which is a model of pure activation of NRF2. The TGFβ1-mediated increase of collagen 1A1 and fibronectin-1 in KEAP1 knockdown HK2 was suppressed. In particular, TGFβ1-SMAD signaling was modulated in KEAP1 knockdown HK2: the TGFβ1-stimulated SMAD2/3 phosphorylation and SMAD transcriptional activity were repressed. Additionally, the protein level of SMAD7, an inhibitor of SMAD signaling, was elevated and the level of SMURF1, an E3 ubiquitin ligase for SMAD7 protein, was diminished in KEAP1 knockdown HK2. Finally, the inhibition of SMAD7 expression in KEAP1 knockdown HK2 restored TGFβ1 response, indicating that SMURF1-SMAD7 may be a molecular signaling linking the NRF2-GSH pathway to TGFβ1-EMT changes. Collectively, these results indicate that the KEAP1-NRF2 antioxidant system can be an effective modulator of TGFβ1-stimulated renal epithelial transition to fibroblastic cells through the SMUR1-SMAD7 signaling, and further implies the beneficial role of NRF2 in chronic renal diseases.
Transforming growth factor β (TGFβ) derived from the tumor microenvironment induces malignant phenotypes such as epithelial-mesenchymal transition (EMT) and aberrant cell motility in lung cancers. TGFβ-induced translocation of β-catenin from E-cadherin complexes into the cytoplasm is involved in the transcription of EMT target genes. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is known to exert phosphatase activity by binding to E-cadherin complexes via β-catenin, and recent studies suggest that phosphorylation of the PTEN C-terminus tail might cause loss of this PTEN phosphatase activity. However, whether TGFβ can modulate both β-catenin translocation and PTEN phosphatase activity via phosphorylation of the PTEN C-terminus remains elusive. Furthermore, the role of phosphorylation of the PTEN C-terminus in TGFβ-induced malignant phenotypes has not been evaluated. To investigate whether modulation of phosphorylation of the PTEN C-terminus can regulate malignant phenotypes, here we established lung cancer cells expressing PTEN protein with mutation of phosphorylation sites in the PTEN C-terminus (PTEN4A). We found that TGFβ stimulation yielded a two-fold increase in the phosphorylated -PTEN/PTEN ratio. Expression of PTEN4A repressed TGFβ-induced EMT and cell motility even after snail expression. Our data showed that PTEN4A might repress EMT through complete blockade of β-catenin translocation into the cytoplasm, besides the inhibitory effect of PTEN4A on TGFβ-induced activation of smad-independent signaling pathways. In a xenograft model, the tumor growth ratio was repressed in cells expressing PTEN4A. Taken together, these data suggest that phosphorylation sites in the PTEN C-terminus might be a therapeutic target for TGFβ-induced malignant phenotypes in lung cancer cells.
TGFβ has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT). Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21Cip1 and pRB, three critical regulators of the G1/S transition are variably involved in TGFβ-induced cell cycle arrest in hepatocytes. As these proteins are also involved in the regulation of apoptosis in many circumstances, we investigated their contribution to other relevant TGFβ-induced effects, namely apoptosis and EMT, and examined how the various processes were interrelated.
Primary mouse hepatocytes deficient in p53, p21 and/or Rb, singly or in combination were treated with TGFβ for 24 to 96 hours. Apoptosis was quantified according to morphology and by immunostaining for cleaved-capsase 3. Epithelial and mesenchymal marker expression was studied using immunocytochemistry and real time PCR.
We found that TGFβ similarly induced morphological changes regardless of genotype and independently of proliferation index or sensitivity to inhibition of proliferation by TGFβ. Morphological changes were accompanied by decrease in E-cadherin and increased Snail expression but the mesenchymal markers (N-cadherin, SMAα and Vimentin) studied remained unchanged. TGFβ induced high levels of apoptosis in p53-/-, Rb-/-, p21cip1-/- and control hepatocytes although with slight differences in kinetics. This was unrelated to proliferation or changes in morphology and loss of cell-cell adhesion. However, hepatocytes deficient in both p53 and p21cip1were less sensitive to TGFβ-induced apoptosis.
Although p53, p21Cip1 and pRb are well known regulators of both proliferation and apoptosis in response to a multitude of stresses, we conclude that they are critical for TGFβ-driven inhibition of hepatocytes proliferation, but only slightly modulate TGFβ-induced apoptosis. This effect may depend on other parameters such as proliferation and the presence of other regulatory proteins as suggested by the consequences of p53, p21Cip1 double deficiency. Similarly, p53, p21Cip1 and pRB deficiency had no effect on the morphological changes and loss of cell adhesion which is thought to be critical for metastasis. This indicates that possible association of these genes with metastasis potential would be unlikely to involve TGFβ-induced EMT.
MiR-17-92 cluster and its paralogues have emerged as crucial regulators of many oncogenes and tumor suppressors. Transforming growth factor-β receptor II (TGFβR2), as an important tumor suppressor, is involved in various cancer types. However, it is in cancer that only two miRNAs of this cluster and its paralogues have been reported so far to regulate TGFβR2. MiR-93 is oncogenic, but its targetome in cancer has not been fully defined. The role of miR-93 in nasopharyngeal carcinoma (NPC) still remains largely unknown.
We firstly evaluated the clinical signature of TGFβR2 down-regulation in clinical samples, and next used a miRNA expression profiling analysis followed by multi-validations, including Luciferase reporter assay, to identify miRNAs targeting TGFβR2 in NPC. In vitro and in vivo studies were performed to further investigate the effects of miRNA-mediated TGFβR2 down-regulation on NPC aggressiveness. Finally, mechanism studies were conducted to explore the associated pathway and genes influenced by this miRNA-mediated TGFβR2 down-regulation.
TGFβR2 was down-regulated in more than 50% of NPC patients. It is an unfavorable prognosis factor contributing to clinical NPC aggressiveness. A cluster set of 4 TGFβR2-associated miRNAs was identified; they are all from miR-17-92 cluster and its paralogues, of which miR-93 was one of the most significant miRNAs, directly targeting TGFβR2, promoting cell proliferation, invasion and metastasis in vitro and in vivo. Moreover, miR-93 resulted in the attenuation of Smad-dependent TGF-β signaling and the activation of PI3K/Akt pathway by suppressing TGFβR2, further promoting NPC cell uncontrolled growth, invasion, metastasis and EMT-like process. Impressively, the knockdown of TGFβR2 by siRNA displayed a consentaneous phenocopy with the effect of miR-93 in NPC cells, supporting TGFβR2 is a major target of miR-93. Our findings were also substantiated by investigation of the clinical signatures of miR-93 and TGFβR2 in NPC.
The present study reports an involvement of miR-93-mediated TGFβR2 down-regulation in NPC aggressiveness, thus giving extended insights into molecular mechanisms underlying cancer aggressiveness. Approaches aimed at blocking miR-93 may serve as a promising therapeutic strategy for treating NPC patients.
miR-93; TGFβR2; Aggressiveness; PI3K/Akt; Nasopharyngeal carcinoma
The TGFβ1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFβ1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation.
The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFβ1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes.
Our results reveal potential new signaling pathways and several new genes affected by TGFβ in epithelial cell differentiation. The differentiation induced by TGFβ1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells.
Transforming growth factor beta (TGFβ) is transiently increased in the mammary gland during involution and by radiation. While TGFβ normally has a tumour suppressor role, prolonged exposure to TGFβ can induce an oncogenic epithelial to mesenchymal transition (EMT) program in permissive cells and initiate the generation of cancer stem cells. Our objective is to mimic the transient exposure to TGFβ during involution to determine the persistent effects on premalignant mammary epithelium.
CDβGeo cells, a transplantable mouse mammary epithelial cell line, were treated in vitro for 14 days with TGFβ (5 ng/ml). The cells were passaged for an additional 14 days in media without TGFβ and then assessed for markers of EMT and transformation.
The 14-day exposure to TGFβ induced EMT and transdifferentiation in vitro that persists after withdrawal of TGFβ. TGFβ-treated cells are highly tumorigenic in vivo, producing invasive solid de-differentiated tumours (100%; latency 6.7 weeks) compared to control (43%; latency 32.7 weeks). Although the TGFβ-treated cells have initiated a persistent EMT program, the stem cell population was unchanged relative to the controls. The gene expression profiles of TGFβ-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells. Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFβ2 and changes in extra cellular matrix.
This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.
Transforming growth factor beta; TGFβ; Epithelial to mesenchymal transition; EMT; Transdifferentiation
Valvular heart disease is a major cause of mortality and morbidity. Revealing the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. A key step in valve formation during heart development is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endocardial cells in the atrioventricular cushion (AVC). The type III transforming growth factor-β receptor (TGFβR3) regulates AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Little is known concerning the signaling mechanisms downstream of TGFβR3. Here we use endocardial cell EMT in vitro to determine the role of 2 well-characterized downstream TGFβ signaling pathways in TGFβR3-dependent endocardial cell EMT. Targeting of Smad4, the common mediator Smad, demonstrated that Smad signaling is required for EMT in the AVC and TGFβR3-dependent EMT stimulated by TGFβ2 or BMP-2. Although we show that Smads 1, 2, 3, and 5 are required for AVC EMT, overexpression of Smad1 or Smad3 is not sufficient to induce EMT. Consistent with the activation of the Par6/Smurf1 pathway downstream of TGFβR3, targeting ALK5, Par6, or Smurf1 significantly inhibited EMT in response to either TGFβ2 or BMP-2. The requirement for ALK5 activity, Par6, and Smurf1 for TGFβR3-dependent endocardial cell EMT is consistent with the documented role of this pathway in the dissolution of tight junctions. Taken together, our data demonstrate that TGFβR3-dependent endocardial cell EMT stimulated by either TGFβ2 or BMP-2 requires Smad4 and the activation of the Par6/Smurf1 pathway.
Transforming growth factor-β; Epithelial mesenchymal transformation; Atrioventricular cushion; Heart valve; Receptors
Background: SHP2 regulates cancer development and progression.
Results: SHP2 promotes TGFβ1-induced EMT requiring its phosphatase activity. PTP and N-SH2 domains of SHP2 interact with Hook1, which negatively regulates TGFβ1-induced EMT.
Conclusion: SHP2-Hook1 complex regulates TGFβ1-induced EMT.
Significance: SHP2 plays important roles in tumor metastases due to promoting EMT in cancer cells.
The epithelial-mesenchymal transition (EMT) is an essential process for embryogenesis. It also plays a critical role in the initiation of tumor metastasis. Src homology 2 (SH2)-domain containing protein-tyrosine phosphatase-2 (SHP2) is a ubiquitously expressed protein-tyrosine phosphatase and is mutated in many tumors. However, its functional role in tumor metastasis remains largely unknown. We found that TGFβ1-induced EMT in lung epithelial A549 cells was partially blocked when SHP2 was decreased by transfected siRNA. The constitutively active form (E76V) promoted EMT while the phosphatase-dead mutation (C459S) and the SHP2 inhibitor PHPS1 blocked EMT, which further demonstrated that the phosphatase activity of SHP2 was required for promoting TGFβ1-induced EMT. Using the protein-tyrosine phosphatase domain of SHP2 as bait, we identified a novel SHP2-interacting protein Hook1. Hook1 was down-regulated during EMT in A549 cells. Overexpression of Hook1 inhibited EMT while knockdown of Hook1 promoted EMT. Moreover, both the protein-tyrosine phosphatase domain and N-terminal SH2 domain of SHP2 directly interacted with Hook1. Down-regulation of Hook1 increased SHP2 activity. These results suggested that Hook1 was an endogenous negative regulator of SHP2 phosphatase activity. Our data showed that the protein-tyrosine phosphatase SHP2 was involved in the process of EMT and Hook1 repressed EMT by regulating the activation of SHP2. SHP2-Hook1 complex may play important roles in tumor metastases by regulating EMT in cancer cells.
Epithelial to Mesenchymal Transition; Epithelial-Mesenchymal Transition (EMT); Metastasis; Protein Phosphatase; Protein-Protein Interaction; Hook1; Mesenchymal; Protein-tyrosine Phosphatase; shp2
Epithelial–mesenchymal transition (EMT) is a crucial step in tumor progression, and the TGFβ–SMAD signaling pathway as an inductor of EMT in many tumor types is well recognized. However, the role of non-canonical TGFβ–TAK1 signaling in EMT remains unclear. Herein, we show that TAK1 deficiency drives metastatic skin squamous cell carcinoma earlier into EMT that is conditional on the elevated cellular ROS level. The expression of TAK1 is consistently reduced in invasive squamous cell carcinoma biopsies. Tumors derived from TAK1-deficient cells also exhibited pronounced invasive morphology. TAK1-deficient cancer cells adopt a more mesenchymal morphology characterized by higher number of focal adhesions, increase surface expression of integrin α5β1 and active Rac1. Notably, these mutant cells exert an increased cell traction force, an early cellular response during TGFβ1-induced EMT. The mRNA level of ZEB1 and SNAIL, transcription factors associated with mesenchymal phenotype is also upregulated in TAK1-deficient cancer cells compared with control cancer cells. We further show that TAK1 modulates Rac1 and RhoA GTPases activities via a redox-dependent downregulation of RhoA by Rac1, which involves the oxidative modification of low-molecular weight protein tyrosine phosphatase. Importantly, the treatment of TAK1-deficient cancer cells with Y27632, a selective inhibitor of Rho-associated protein kinase and antioxidant N-acetylcysteine augment and hinders EMT, respectively. Our findings suggest that a dysregulated balance in the activation of TGFβ–TAK1 and TGFβ–SMAD pathways is pivotal for TGFβ1-induced EMT. Thus, TAK1 deficiency in metastatic cancer cells increases integrin:Rac-induced ROS, which negatively regulated Rho by LMW-PTP to accelerate EMT.
reactive oxygen species; cell traction force; epithelial–mesenchymal transition
By interfering with the TGFβ/Smad pathway in the human HaCaT keratinocytes, this study provides novel insights into the role of Smad signaling for regular tissue homeostasis and demonstrates its crucial role in terminal epidermal differentiation and in the decision between alternative epithelial differentiation programs.
The mechanism by which transforming growth factor-β (TGFβ) regulates differentiation in human epidermal keratinocytes is still poorly understood. To assess the role of Smad signaling, we engineered human HaCaT keratinocytes either expressing small interfering RNA against Smads2, 3, and 4 or overexpressing Smad7 and verified impaired Smad signaling as decreased Smad phosphorylation, aberrant nuclear translocation, and altered target gene expression. Besides abrogation of TGFβ-dependent growth inhibition in conventional cultures, epidermal morphogenesis and differentiation in organotypic cultures were disturbed, resulting in altered tissue homeostasis with suprabasal proliferation and hyperplasia upon TGFβ treatment. Neutralizing antibodies against TGFβ, similar to blocking the actions of EGF-receptor or keratinocyte growth factor, caused significant growth reduction of Smad7-overexpressing cells, thereby demonstrating that epithelial hyperplasia was attributed to TGFβ-induced “dermis”-derived growth promoting factors. Furthermore impaired Smad signaling not only blocked the epidermal differentiation process or caused epidermal-to-mesenchymal transition but induced a switch to a complex alternative differentiation program, best characterized as mucous/intestinal-type epithelial differentiation. As the same alternative phenotype evolved from both modes of Smad-pathway interference, and reduction of Smad7-overexpression caused reversion to epidermal differentiation, our data suggest that functional TGFβ/Smad signaling, besides regulating epidermal tissue homeostasis, is not only essential for terminal epidermal differentiation but crucial in programming different epithelial differentiation routes.
A substantial body of evidence implicates TGFβ as a tumor promoter in epithelial cells that have become resistant to its tumor suppressor activity. To better understand early, genome-wide TGFβ responses in cells resistant to growth inhibition by TGFβ, we used microarray analysis in a well-defined cell culture system of sensitive and resistant intestinal epithelial cells. TGFβ-regulated gene expression in TGFβ-growth-sensitive, nontransformed rat intestinal epithelial cells (RIE-1) was compared to expression in TGFβ-growth-resistant RIE cells stably transformed by oncogenic Ras(12V). Treatment of RIE-1 cells with 2 ng/ml TGFβ1 for 1 hour increased the expression of eight gene sequences by 2.6-fold or more, whereas eight were down regulated 2.6-fold. In RIE-Ras(12V) cells, 42 gene sequences were upregulated and only 3 were down-regulated. Comparison of RIE and RIE-Ras(12V) identified 37 gene sequences as unique, Ras-dependent genomic targets of TGFβ1. TGFβ-regulation of connective tissue growth factor and vascular endothelial growth factor, two genes up-regulated in RIE-Ras cells and previously implicated in tumor promotion, was independently confirmed and further characterized by Northern analysis. Our data indicate that overexpression of oncogenic Ras in intestinal epithelial cells confers a significantly expanded repertoire of robust, early transcriptional responses to TGFβ via signaling pathways yet to be fully elucidated but including the canonical Raf-1/MAPK/Erk pathway. Loss of sensitivity to growth inhibition by TGFβ does not abrogate TGFβ signaling and actually expands the early transcriptional response to TGFβ1. Expression of some of these genes may confer to Ras-transformed cells characteristics favorable for tumor promotion.
Transforming growth factor beta (TGFβ) plays a central role in morphogenesis, growth, and cell differentiation. This cytokine is particularly important in cartilage where it regulates cell proliferation and extracellular matrix synthesis. While the action of TGFβ on chondrocyte metabolism has been extensively catalogued, the modulation of specific genes that function as mediators of TGFβ signalling is poorly defined. In the current study, elements of the Smad component of the TGFβ intracellular signalling system and TGFβ receptors were characterised in human chondrocytes upon TGFβ1 treatment.
Human articular chondrocytes were incubated with TGFβ1. Then, mRNA and protein levels of TGFβ receptors and Smads were analysed by RT-PCR and western blot analysis. The role of specific protein 1 (Sp1) was investigated by gain and loss of function (inhibitor, siRNA, expression vector).
We showed that TGFβ1 regulates mRNA levels of its own receptors, and of Smad3 and Smad7. It modulates TGFβ receptors post-transcriptionally by affecting their mRNA stability, but does not change the Smad-3 and Smad-7 mRNA half-life span, suggesting a potential transcriptional effect on these genes. Moreover, the transcriptional factor Sp1, which is downregulated by TGFβ1, is involved in the repression of both TGFβ receptors but not in the modulation of Smad3 and Smad7. Interestingly, Sp1 ectopic expression permitted also to maintain a similar expression pattern to early response to TGFβ at 24 hours of treatment. It restored the induction of Sox9 and COL2A1 and blocked the late response (repression of aggrecan, induction of COL1A1 and COL10A1).
These data help to better understand the negative feedback loop in the TGFβ signalling system, and enlighten an interesting role of Sp1 to regulate TGFβ response.
Transforming growth factor beta 1 (TGFβ1) plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFβ1 activates a signal transduction cascade that results in the transcriptional regulation of genes in the nucleus, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify genome-wide scale map of SMAD3 binding targets and the molecular pathways and networks affected by the TGFβ1/SMAD3 signaling in lung epithelial cells.
We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip) along with gene expression microarrays to study global transcriptional regulation of the TGFβ1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFβ1/SMAD3 signaling were identified using computational approaches. Validation of selected target gene expression and direct binding of SMAD3 to promoters were performed by quantitative real time RT-PCR and electrophoretic mobility shift assay on A549 and human primary lung epithelial cells.
Results and Conclusions
Known TGFβ1 target genes such as SERPINE1, SMAD6, SMAD7, TGFB1 and LTBP3, were found in both ChIP-on-chip and gene expression analyses as well as some previously unrecognized targets such as FOXA2. SMAD3 binding of FOXA2 promoter and changed expression were confirmed. Computational approaches combining ChIP-on-chip and gene expression microarray revealed multiple target molecular pathways affected by the TGFβ1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFβ1/SMAD3 signaling allow for a better understanding of the mechanisms that determine epithelial cell phenotypes in fibrogenesis and carcinogenesis as does the discovery of the direct effect of TGFβ1 on FOXA2.
Transforming growth factor β1 (TGFβ1) is a potent inhibitor of epithelial cell growth, thus playing an important role in tissue homeostasis. Most carcinoma cells exhibit a reduced sensitivity for TGFβ1 mediated growth inhibition, suggesting TGFβ1 participation in the development of these cancers. The tumor suppresor gene DPC4/SMAD4, which is frequently inactivated in carcinoma cells, has been described as a key player in TGFβ1 mediated growth inhibition. However, some carcinoma cells lacking functional SMAD4 are sensitive to TGFβ1 induced growth inhibition, thus requiring a SMAD4 independent TGFβ1 pathway.
Here we report that mature TGFβ1 is a ligand for the integrin αVβ6, independent of the common integrin binding sequence motif RGD. After TGFβ1 binds to αVβ6 integrin, different signaling proteins are activated in TGFβ1-sensitive carcinoma cells, but not in cells that are insensitive to TGFβ1. Among others, interaction of TGFβ1 with the αVβ6 integrin resulted in an upregulation of the cell cycle inhibitors p21/WAF1 and p27 leading to growth inhibition in SMAD4 deleted as well as in SMAD4 wildtype carcinoma cells.
Our data provide support for the existence of an alternate TGFβ1 signaling pathway that is independent of the known SMAD pathway. This alternate pathway involves αVβ6 integrin and the Ras/MAP kinase pathway and does not employ an RGD motif in TGFβ1-sensitive tumor cells. The combined action of these two pathways seems to be necessary to elicit a complete TGFβ1 signal.
TGFβ1; signaling; cytoskeleton; growth inhibition; integrin.
Immaculate and complete palatal seam disintegration, which takes place at the last phase of palate development, is essential for normal palate development. And in absence of palatal medial edge seam (MES) disintegration, cleft palate may arise. It has been established that Transforming Growth Factor (TGF) β induces both Epithelial Mesenchymal Transition (EMT) and/or apoptosis during MES disintegration. It is likely that MES might cease cell cycle to facilitate cellular changes prior to undergoing transformation or apoptosis, which has never been studied before. This study was designed to explore whether TGFβ, which is crucial for palatal MES disintegration, is capable of inducing cell cycle arrest. We studied the effects of TGFβ1 and TGFβ3, potent negative regulators of the cell cycle, on p15ink4b activity in MES cells. We surprisingly found that TGFβ1, but not TGFβ3, plays a major role in activation of the p15ink4b gene. In contrast, following successful cell cycle arrest by TGFβ1, it is TGFβ3 but not TGFβ1 that causes later cellular morphogenesis, such as EMT and apoptosis. Since TGFβ signaling activates Smads, we analyzed the roles of three Smad binding elements (SBEs) on the p15ink4b mouse promoter by site specific mutagenesis and found that these binding sites are functional. The ChIP assay demonstrated that TGFβ1, not TGFβ3, promotes Smad4 binding to two 5’ terminal SBEs but not the 3’ terminal site. Thus, TGFβ1 and TGFβ3 play separate yet complimentary roles in achieving cell cycle arrest and EMT/apoptosis and cell cycle arrest is a prerequisite for later cellular changes.
cell cycle; palate; TGFβ; apoptosis; EMT
Hepatocyte Nuclear Factor 1α (HNF1α) is an atypical homeodomain-containing transcription factor that transactivates liver-specific genes including albumin, α-1-antitrypsin and α- and β-fibrinogen. Biallelic inactivating mutations of HNF1A have been frequently identified in hepatocellular adenomas (HCA), rare benign liver tumors usually developed in women under oral contraceptives, and in rare cases of hepatocellular carcinomas developed in non-cirrhotic liver. HNF1α-mutated HCA (H-HCA) are characterized by a marked steatosis and show activation of glycolysis, lipogenesis, translational machinery and mTOR pathway. We studied the consequences of HNF1α silencing in hepatic cell lines, HepG2 and Hep3B and we reproduced most of the deregulations identified in H-HCA.
We transfected hepatoma cell lines HepG2 and Hep3B with siRNA targeting HNF1α and obtained a strong inhibition of HNF1α expression. We then looked at the phenotypic changes by microscopy and studied changes in gene expression using qRT-PCR and Western Blot.
Hepatocytes transfected with HNF1α siRNA underwent severe phenotypic changes with loss of cell-cell contacts and development of migration structures. In HNF1α-inhibited cells, hepatocyte and epithelial markers were diminished and mesenchymal markers were over-expressed. This epithelial-mesenchymal transition (EMT) was related to the up regulation of several EMT transcription factors, in particular SNAIL and SLUG. We also found an overexpression of TGFβ1, an EMT initiator, in both cells transfected with HNF1α siRNA and H-HCA. Moreover, TGFβ1 expression is strongly correlated to HNF1α expression in cell models, suggesting regulation of TGFβ1 expression by HNF1α.
Our results suggest that HNF1α is not only important for hepatocyte differentiation, but has also a role in the maintenance of epithelial phenotype in hepatocytes.
Hepatocyte Nuclear Factor 1α; hepatocellular adenoma; tumor suppressor gene; benign tumor; siRNA; EMT; TGFβ1
Development of the palate comprises sequential stages of growth, elevation, and fusion of the palatal shelves. The mesenchymal component of palates plays a major role in early phases of palatogenesis, such as growth and elevation. Failure in these steps may result in cleft palate, the second most common birth defect in the world. These early stages of palatogenesis require precise and chronological orchestration of key physiological processes, such as growth, proliferation, differentiation, migration, and apoptosis. There is compelling evidence for the vital role of TGFβ-mediated regulation of palate development. We hypothesized that the isoforms of TGFβ regulate different cellular biofunctions of the palatal mesenchyme to various extents. Human embryonic palatal mesenchyme (HEPM) cells were treated with TGFβ1, β2, and β3 for microarray-based gene expression studies in order to identify the roles of TGFβ in the transcriptome of the palatal mesenchyme. Following normalization and modeling of 28,869 human genes, 566 transcripts were detected as differentially expressed in TGFβ-treated HEPM cells. Out of these altered transcripts, 234 of them were clustered in cellular biofunctions, including growth and proliferation, development, morphology, movement, cell cycle, and apoptosis. Biological interpretation and network analysis of the genes active in cellular biofunctions were performed using IPA. Among the differentially expressed genes, 11 of them are known to be crucial for palatogenesis (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGFβ1, TGFβ2, and TGFβR1). These genes were used for a merged interaction network with cellular behaviors. Overall, we have determined that more than 2% of human transcripts were differentially expressed in response to TGFβ treatment in HEPM cells. Our results suggest that both TGFβ1 and TGFβ2 orchestrate major cellular biofunctions within the palatal mesenchyme in vitro by regulating expression of 234 genes.
TGFβ; microarray; transcriptome; palatogenesis; mesenchyme; HEPM; craniofacial; palate
TGFβ induces epithelial-mesenchymal transdifferentiation (EMT) accompanied by cellular differentiation and migration. Despite extensive transcriptomic profiling, identification of TGFβ-inducible, EMT-specific genes has met with limited success. Here, we identify a post-transcriptional pathway by which TGFβ modulates expression of EMT-specific proteins, and EMT itself. We show that heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) binds a structural, 33 nucleotides (nt) TGF beta-activated translation (BAT) element in the 3’-UTR of disabled-2 (Dab2) and interleukin-like EMT inducer (ILEI) transcripts, and repress their translation. TGFβ activation leads to phosphorylation at Ser43 of hnRNP E1 by protein kinase Bβ/Akt2, inducing its release from the BAT element and translational activation of Dab2 and ILEI mRNAs. Modulation of hnRNP E1 expression or its post-translational modification alters TGFβ-mediated reversal of translational silencing of the target transcripts and EMT. These results suggest the existence of a TGFβ-inducible post-transcriptional regulon that controls EMT during development and metastatic progression of tumors.
Enhanced TGFβ activity contributes to the accumulation of matrix proteins including collagen I (α2) by proximal tubular epithelial cells in progressive kidney disease. Although TGFβ rapidly activates its canonical Smad signaling pathway, it also recruits noncanonical pathway involving mTOR kinase to regulate renal matrix expansion. The mechanism by which chronic TGFβ treatment maintains increased mTOR activity to induce the matrix protein collagen I (α2) expression is not known. Deptor is an mTOR interacting protein that suppresses mTOR activity in both mTORC1 and mTORC2. In proximal tubular epithelial cells, TGFβ reduced deptor levels in a time-dependent manner with concomitant increase in both mTORC1 and mTORC2 activities. Expression of deptor abrogated activity of mTORC1 and mTORC2, resulting in inhibition of collagen I (α2) mRNA and protein expression via transcriptional mechanism. In contrast, neutralization of endogenous deptor by shRNAs increased activity of both mTOR complexes and expression of collagen I (α2) similar to TGFβ treatment. Importantly, downregulation of deptor by TGFβ increased the expression of Hif1α by increasing translation of its mRNA. TGFβ-induced deptor downregulation promotes Hif1α binding to its cognate hypoxia responsive element in the collagen I (α2) gene to control its protein expression via direct transcriptional mechanism. Interestingly, knockdown of raptor to specifically block mTORC1 activity significantly inhibited expression of collagen I (α2) and Hif1α while inhibition of rictor to prevent selectively mTORC2 activation did not have any effect. Critically, our data provide evidence for the requirement of TGFβ-activated mTORC1 only by deptor downregulation, which dominates upon the bystander mTORC2 activity for enhanced expression of collagen I (α2). Our results also suggest the presence of a safeguard mechanism involving deptor-mediated suppression of mTORC1 activity against developing TGFβ-induced renal fibrosis.
Transforming growth factor-β (TGFβ) promotes glomerular hypertrophy and matrix expansion, leading to glomerulosclerosis. MicroRNAs are well suited to promote fibrosis because they can repress gene expression, which negatively regulate the fibrotic process. Recent cellular and animal studies have revealed enhanced expression of microRNA, miR-21, in renal cells in response to TGFβ. Specific miR-21 targets downstream of TGFβ receptor activation that control cell hypertrophy and matrix protein expression have not been studied. Using 3′UTR-driven luciferase reporter, we identified the tumor suppressor protein PTEN as a target of TGFβ-stimulated miR-21 in glomerular mesangial cells. Expression of miR-21 Sponge, which quenches endogenous miR-21 levels, reversed TGFβ-induced suppression of PTEN. Additionally, miR-21 Sponge inhibited TGFβ-stimulated phosphorylation of Akt kinase, resulting in attenuation of phosphorylation of its substrate GSK3β. Tuberin and PRAS40, two other Akt substrates, and endogenous inhibitors of mTORC1, regulate mesangial cell hypertrophy. Neutralization of endogenous miR-21 abrogated TGFβ-stimulated phosphorylation of tuberin and PRAS40, leading to inhibition of phosphorylation of S6 kinase, mTOR and 4EBP-1. Moreover, downregulation of miR-21 significantly suppressed TGFβ-induced protein synthesis and hypertrophy, which were reversed by siRNA-targeted inhibition of PTEN expression. Similarly, expression of constitutively active Akt kinase reversed the miR-21 Sponge-mediated inhibition of TGFβ-induced protein synthesis and hypertrophy. Furthermore, expression of constitutively active mTORC1 prevented the miR-21 Sponge-induced suppression of mesangial cell protein synthesis and hypertrophy by TGFβ. Finally, we show that miR-21 Sponge inhibited TGFβ-stimulated fibronectin and collagen expression. Suppression of PTEN expression and expression of both constitutively active Akt kinase and mTORC1 independently reversed this miR-21-mediated inhibition of TGFβ-induced fibronectin and collagen expression. Our results uncover an essential role of TGFβ-induced expression of miR-21, which targets PTEN to initiate a non-canonical signaling circuit involving Akt/mTORC1 axis for mesangial cell hypertrophy and matrix protein synthesis.
Microglia are the resident immune cells of the central nervous system and are accepted to be involved in a variety of neurodegenerative diseases. Several studies have demonstrated that microglia, like peripheral macrophages, exhibit two entirely different functional activation states, referred to as classical (M1) and alternative (M2) activation. TGFβ is one of the most important anti-inflammatory cytokines and its effect on inhibiting microglia or macrophage classical activation has been extensively studied. However, the role of TGFβ during alternative activation of microglia has not been described yet.
To investigate the role of TGFβ in IL4-induced microglia alternative activation, both, BV2 as well as primary microglia from new born C57BL/6 mice were used. Quantitative RT-PCR and western blots were performed to detect mRNA and protein levels of the alternative activation markers Arginase1 (Arg1) and Chitinase 3-like 3 (Ym1) after treatment with IL4, TGFβ or both. Endogenous TGFβ release after IL4 treatment was evaluated using the mink lung epithelial cell (MLEC) assay and a direct TGFβ2 ELISA. TGFβ receptor type I inhibitor and MAPK inhibitor were applied to address the involvement of TGFβ signalling and MAPK signalling in IL4-induced alternative activation of microglia.
TGFβ enhances IL4-induced microglia alternative activation by strongly increasing the expression of Arg1 and Ym1. This synergistic effect on Arg1 induction is almost completely blocked by the application of the MAPK inhibitor, PD98059. Further, treatment of primary microglia with IL4 increased the expression and secretion of TGFβ2, suggesting an involvement of endogenous TGFβ in IL4-mediated microglia activation process. Moreover, IL4-mediated induction of Arg1 and Ym1 is impaired after blocking the TGFβ receptor I indicating that IL4-induced microglia alternative activation is dependent on active TGFβ signalling. Interestingly, treatment of primary microglia with TGFβ alone results in up regulation of the IL4 receptor alpha, indicating that TGFβ increases the sensitivity of microglia for IL4 signals.
Taken together, our data reveal a new role for TGFβ during IL4-induced alternative activation of microglia and consolidate the essential functions of TGFβ as an anti-inflammatory molecule and immunoregulatory factor for microglia.
Microglia; TGFβ; IL4; Alternative activation; Arg1; Ym1
Transforming growth factor beta (TGFβ) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGFβ receptor (TβRII) for signaling. Previously, we have shown that deletion of TβRII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGFβ signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGFβ signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression.
All studies were performed on MMTV-Neu mice with and without dominant-negative TβRII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer.
Attenuated TGFβ signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients.
Our studies provide insights into a novel mechanism by which epithelial TGFβ signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGFβ pathway in vivo during tumor progression remain controversial. The targeting of TGFβ signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGFβ signaling.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0425-7) contains supplementary material, which is available to authorized users.
Transforming Growth Factor β is a potent modifier of the malignant phenotype in ErbB2-expressing breast cancers. We demonstrate that epithelial-derived breast cancer cells, which undergo a TGFβ-induced EMT, engage signaling molecules that normally facilitate cellular migration and invasion of mesenchymal cells. We identify Lipoma Preferred Partner (LPP) as an indispensable regulator of TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. We show that LPP re-localizes to focal adhesion complexes upon TGFβ stimulation and is a critical determinant in TGFβ-mediated focal adhesion turnover. Finally, we have determined that the interaction between LPP and α-Actinin, an actin cross-linking protein, is necessary for TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. Thus, our data reveals that LPP, which is normally operative in cells of mesenchymal origin, can be co-opted by breast cancer cells during an EMT to promote their migration and invasion.
breast cancer; ErbB2; TGFβ; EMT; LPP; migration; invasion