Increase In Smad2 Phosphorylation Research Articles

Melatonin Protects Human Renal Proximal Tubule Epithelial Cells Against High Glucose-Mediated Fibrosis via the Cellular Prion Protein-TGF-β-Smad Signaling Axis.

Diabetes-mediated hyperglycemia is a major risk factor for renal fibrosis, resulting in the development of chronic kidney diseases. To address this issue, the effect of melatonin, which has an antioxidative potential, on renal fibrosis in human renal proximal tubule epithelial cells under high glucose conditions was investigated. Under high glucose conditions, the generation of reactive oxygen species was drastically increased in human renal proximal tubule epithelial cells, which lead to the inhibition of cell proliferation, enlargement of cell size, reduction of cell survival, and suppression of antioxidant enzyme activities. High glucose also increased the expression of transforming growth factor-β, leading to an increase in Smad2 phosphorylation. These fibrotic phenotype changes increased the expression of fibrosis-mediated extracellular matrix proteins, such as fibronectin, collagen I, and α-smooth muscle actin. In addition, the level of cellular prion protein (PrPC), which is associated with several biological processes, was decreased by exposure to high glucose conditions. Melatonin recovered the expression levels of PrPC under high glucose conditions via phosphorylation of Akt, resulting in the prevention of high glucose-induced fibrosis. In particular, overexpression of PrPC blocked the high glucose-mediated fibrotic phenotype change. These findings indicate that melatonin could be a powerful agent for treating hyperglycemia-induced renal fibrosis.

Connective tissue growth factor contributes to joint homeostasis and osteoarthritis severity by controlling the matrix sequestration and activation of latent TGFβ

ObjectivesOne mechanism by which cartilage responds to mechanical load is by releasing heparin-bound growth factors from the pericellular matrix (PCM). By proteomic analysis of the PCM, we identified connective tissue...

Mechanical stretch inhibits mesenchymal stem cell adipogenic differentiation through TGFβ1/Smad2 signaling

Mesenchymal stem cells (MSCs) are the common precursors of several functionally disparate cell lineages. A plethora of chemical and physical stimuli contribute to lineage decisions and guidance, including mechanical stretch concomitant with physical movement. Here, we examined how stretch regulates MSC differentiation into adipocytes and the intracellular signaling pathways involved. MSCs were cultured under adipogenic conditions and divided into a control and an experimental group. Cultures in the experimental group were subjected to a sinusoidal stretch regimen delivered via flexible culture bottoms (5% magnitude, 10 times per min, 6h/day, 3 or 5 days). Expression levels of the adipocyte markers PPARγ-2, adiponectin, and C/EBPα were measured as indices of differentiation. Compared to controls, MSCs exposed to mechanical stretch exhibited downregulated PPARγ-2, adiponectin, and C/EBPα mRNA expression. Alternatively, stretch upregulated phosphorylation of Smad2. This stretch-induced increase in Smad2 phosphorylation was suppressed by pretreatment with the TGFβ1/Smad2 pathway antagonist SB-431542. Pretreatment with the TGFβ1/Smad2 signaling agonist TGFβ1 facilitated the inhibitory effect of stretch on the expression levels of PPARγ-2, adiponectin, and C/EBPα proteins, while pretreatment with SB-431542 reversed the inhibitory effects of subsequent stretch on the expression levels of these markers. These results strongly suggest that the anti-adipogenic effects of mechanical stretch on MSCs are mediated, at least in part, by activation of the TGFβ1/Smad2 signaling pathway.

Latent transforming growth factor binding protein 4 regulates transforming growth factor beta receptor stability.

Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFβ) signaling. Despite elevated extracellular TGFβ activity, downstream signaling molecules of the TGFβ pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFβ receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFβ1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFβ receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFβ signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFβ receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFβ receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.

Abstract 5359: TGFβ promotes the DNA damage response and repair of DNA damage induced by benzo[a]pyrene

Abstract Transforming growth factor beta (TGFβ) is involved in coordinating a number of cellular processes including proliferation, differentiation and apoptosis, and as such, dysregulation of TGFβ signalling has significant implications for cancer development. TGFβ is rapidly and persistently activated in response to irradiation-induced DNA damage (Barcellos-Hoff et al, J Clin Invest 1994; 93:892-9) and has been implicated in regulation of the irradiation-induced DNA damage response (Kirshner et al, Cancer Res 2006; 66:10861-9). We have recently published two studies investigating the activation of DNA damage signalling in cells after exposure to two highly potent polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) (Niziolek-Kierecka et al, Chem Res Toxicol 2012; 25:862-72 and Jarvis et al, Toxicol Appl Pharmacol 2013; 266: 408-18). The aim of this study was therefore to investigate a potential role for TGFβ in the response to PAH-induced DNA damage. Using human hepatocellular carcinoma cells (HepG2) we studied activation of DNA damage signalling by Western blotting. Formation of γH2AX and ATM foci was assessed by immunofluorescence and DNA damage levels were quantified by Comet assay. HepG2 cells were exposed to non-cytotoxic concentrations of BP and DBP (confirmed by MTT assay). Exposure to BP or DBP caused a concentration- and time-dependent increase in DNA damage signalling assessed by phosphorylation of Chk1, H2AX and p53. DBP was approximately 60-fold more potent at inducing prolonged DNA damage signalling than BP. Prolonged γH2AX and ATM foci were observed after exposure to 1 µM BP or 50 nM DBP for 48 hours consistent with persistent DNA damage whereas exposure to 0.1 µM BP or 5 nM DBP caused a transient increase in foci up to 24h and lower levels at 48h suggesting repair. To investigate the role of TGFβ signalling in repair of PAH-induced DNA damage, exogenous TGFβ (1 ng/ml) was added during incubation with BP and DBP. Addition of TGFβ significantly decreased the number of γH2AX and ATM foci in cells exposed to either BP or DBP and this was consistent with a decrease in DNA damage. Levels of both damage foci and Comet tail moments were similar to control levels suggesting activation of DNA repair in response to addition of TGFβ. Exposure to BP or DBP with or without TGFβ caused an increase in Smad2 phosphorylation confirming activation of TGFβ signalling. Smad2 activation was more pronounced after exposure to BP compared to DBP and RNA interference experiments are ongoing to further elucidate the mechanism of PAH induction of TGFβ signalling. Taken together our data suggest an important role for TGFβ signalling in repair of PAH-induced DNA damage. Furthermore, our data suggest a possible link between compromised TGFβ signalling and persistent DNA damage that might have important implications in understanding PAH-induced carcinogenesis. Citation Format: Ian WH Jarvis, Kristian Dreij, Ulla Stenius. TGFβ promotes the DNA damage response and repair of DNA damage induced by benzo[a]pyrene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5359. doi:10.1158/1538-7445.AM2014-5359

Transgenic overexpression of the adenine nucleotide translocase 1 protects cardiomyocytes against TGFβ1-induced apoptosis by stabilization of the mitochondrial permeability transition pore

Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin-angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor β (TGFβ) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFβ/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFβ(1)-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected. Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFβ(1) (1 ng/ml). TGFβ(1) treatment of WT cells enhanced the number of apoptotic cells by 31.8 ± 11.7% (p<0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1μM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFβ(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFβ(1) decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0 ± 3.7% in WT cardiomyocytes, but only by 7.2 ± 2.8% (p<0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFβ(1) in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFβ signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFβ receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFβ(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFβ(1) treatment, SMAD signaling was still responsive to TGFβ(1) indicated by an upregulation of SMAD7, a TGFβ(1) target protein. Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFβ(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFβ signaling.

TGFβ Inhibition Radiosensitizes Murine Glioblastoma Cells and Decreases Neurosphere-forming Capacity

Transforming growth factor-β (TGFβ) is a pleotropic cytokine in the tumor microenvironment that can promote malignant behaviors, including invasion and motility. Glioblastomas produce abundant TGFβ, are routinely treated with radiation, and have a very poor prognosis. A role for TGFβ in the DNA damage response has recently been discovered in which TGFβ inhibition in vitro and in vivo compromises ATM-kinase activity induced by ionizing radiation (Cancer Research 66:10861 - 68; 62:5627 - 31). These data suggest that TGFβ could protect cancer cells from the cytotoxic effects of radiation by promoting ATM dependent responses; if so, TGFβ inhibitors, which are in clinical trials, might increase therapeutic response to radiation. We used the murine glioblastoma cell line, GL261, to test the effects of TGFβ inhibition by LY364947 (a small molecule inhibitor of the TGFβ type I receptor kinase) on proliferation, radiosensitivity, and neurosphere-forming capacity. Experiments were performed in triplicate and differences tested by ANOVA. GL261 cells produce 0.9 ng/mL per 106 cells of total TGFβ in media conditioned for 24 hr, the majority of which is latent TGFβ2. They also respond to exogenous TGFβ1 with an increase in Smad2 phosphorylation. Despite intact TGFβ receptor kinase activity, GL261 cells displayed no growth modulation response to exogenous TGFβ1 (0.5 - 2ng/mL) treatment or to inhibition by LY364947 (400nM). Nonetheless, inhibition of TGFβ with LY364947 for 24 hours prior to radiation treatment significantly increased GL261 radiosensitivity in the clonogenic assay (p<0.001), with a 1.25 dose enhancement ratio at 10% surviving fraction. This correlated with a significant 55% decrease in γH2AX foci following radiation treatment with 2Gy (p<0.0001). Irradiation of GL261 cells with 2Gy also decreased primary neurosphere-forming capacity by 28% (p<0.001, ANOVA), but had no effect on secondary neurosphere formation. Treatment with LY364947 alone had no effect on neurosphere formation. In contrast, treatment for 24 hours prior to irradiation decreased the primary neurosphere-forming capacity of irradiated GL261 cells by an additional 47% (p<0.001) and decreased secondary neurosphere formation by 68% (p<0.001). Given the radiosensitization and specifically the response of the neurosphere assay, which is thought to measure the glioma initiating cell population, our results suggest that inhibition of TGFβ in combination with radiation represents a promising therapeutic strategy. By targeting the putative stem cells long term benefit of TGFβ inhibition in glioblastoma may be achieved compared to poor response rates seen with the standard regimen of chemotherapy and radiotherapy.

Abstract LB-361: Radiosensitization and decreased neurosphere-forming capacity with TGFβ inhibition in glioma cells

Abstract Transforming growth factor-β (TGFβ) is a pleotropic cytokine in the tumor microenvironment that can promote malignant behaviors, including invasion and motility, at late stages of tumorigenesis. Glioblastomas produce abundant TGFβ, are routinely treated with radiation, and have a very poor prognosis (median survival of only 14.6 months). An unexpected role for TGFβ in the DNA damage response has recently been discovered in which TGFβ inhibition in vitro and in vivo compromises ATM-kinase activity induced by ionizing radiation (Cancer Research 66:10861–68; 62:5627–31). These data suggest that TGFβ could actually protect cancer cells from the cytotoxic effects of radiation by promoting ATM dependent responses; if so, TGFβ inhibitors, which are in clinical trials, might increase therapeutic response to radiation. To test this idea, we asked whether TGFβ inhibition could radiosensitize glioblastoma cells. We used the murine glioblastoma cell line, GL261, to test the effects of TGFβ inhibition by LY364947 (a small molecule inhibitor of the TGFβ type I receptor kinase) on proliferation, radiosensitivity, and neurosphere-forming capacity. GL261 cells were found to produce 0.9 ng/mL per 106 cells of total TGFβ in media conditioned for 24 hr, the majority of which is latent TGFβ2. They also respond to exogenous TGFβ1 with an increase in Smad2 phosphorylation by Western blot. Despite intact TGFβ receptor kinase activity, GL261 cells displayed no growth modulation response to exogenous TGFβ1 (0.5–2ng/mL) treatment or to inhibition by LY364947 (400nM). Nonetheless, inhibition of TGFβ with LY364947 for 24 hours prior to radiation treatment significantly increased GL261 radiosensitivity in the clonogenic assay, with a 1.25 (p&amp;lt;0.001, ANOVA) dose enhancement ratio at 10% surviving fraction; in other words, an increase of 25% in clonogenic cell death. The increase in radiosensitivity correlated with a significant 55% decrease in γH2AX foci, which is a substrate of ATM, following radiation treatment with 2Gy (p&amp;lt;0.0001, ANOVA). We then assessed the response of glioma initiating cells (GIC) in a neurosphere assay. Irradiation of GL261 cells with 2Gy decreased neurosphere-forming capacity by 28% (p&amp;lt;0.001, ANOVA). Treatment with LY364947 alone had no effect on neurosphere formation, but treatment for 24 hours prior to irradiation decreased the neurosphere-forming capacity of irradiated GL261 cells by an additional 47% (p&amp;lt;0.001, ANOVA). Further studies using an orthotopic GL261 intracranial glioblastoma model are underway to test how these radiation effects mediated by TGFβ affect tumor growth in vivo. Given the radiosensitization and specifically the response of the GIC population, our results suggest that inhibition of TGFβ in combination with radiation represents promising therapeutic strategy in glioblastoma to improve poor response rates seen with the standard regimen of chemotherapy and radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-361. doi:10.1158/1538-7445.AM2011-LB-361

Cardiac Myocyte-specific Ablation of Follistatin-like 3 Attenuates Stress-induced Myocardial Hypertrophy

Transforming growth factor-β family cytokines have diverse actions in the maintenance of cardiac homeostasis. Follistatin-like 3 (Fstl3) is an extracellular regulator of certain TGF-β family members, including activin A. The aim of this study was to examine the role of Fstl3 in cardiac hypertrophy. Cardiac myocyte-specific Fstl3 knock-out (KO) mice and control mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Cardiac hypertrophy was assessed by echocardiography and histological and biochemical methods. KO mice showed reduced cardiac hypertrophy, pulmonary congestion, concentric LV wall thickness, LV dilatation, and LV systolic dysfunction after TAC compared with control mice. KO mice displayed attenuated increases in cardiomyocyte cell surface area and interstitial fibrosis following pressure overload. Although activin A was similarly up-regulated in KO and control mice after TAC, a significant increase in Smad2 phosphorylation only occurred in KO mice. Knockdown of Fstl3 in cultured cardiomyocytes inhibited PE-induced cardiac hypertrophy. Conversely, adenovirus-mediated Fstl3 overexpression blocked the inhibitory action of activin A on hypertrophy and Smad2 activation. Transduction with Smad7, a negative regulator of Smad2 signaling, blocked the antihypertrophic actions of activin A stimulation or Fstl3 ablation. These findings identify Fstl3 as a stress-induced regulator of hypertrophy that controls myocyte size via regulation of Smad signaling.

Gfi-1B controls human erythroid and megakaryocytic differentiation by regulating TGF-β signaling at the bipotent erythro-megakaryocytic progenitor stage

AbstractGrowth factor independence-1B (Gfi-1B) is a transcriptional repressor essential for erythropoiesis and megakaryopoiesis. Targeted gene disruption of GFI1B in mice leads to embryonic lethality resulting from failure to produce definitive erythrocytes, hindering the study of Gfi-1B function in adult hematopoiesis. We here show that, in humans, Gfi-1B controls the development of erythrocytes and megakaryocytes by regulating the proliferation and differentiation of bipotent erythro-megakaryocytic progenitors. We further identify in this cell population the type III transforming growth factor-β receptor gene, TGFBR3, as a direct target of Gfi-1B. Knockdown of Gfi-1B results in altered transforming growth factor-β (TGF-β) signaling as shown by the increase in Smad2 phosphorylation and its inability to associate to the transcription intermediary factor 1-γ (TIF1-γ). Because the Smad2/TIF1-γ complex is known to specifically regulate erythroid differentiation, we propose that, by repressing TGF-β type III receptor (TβRΙII) expression, Gfi-1B favors the Smad2/TIF1-γ interaction downstream of TGF-β signaling, allowing immature progenitors to differentiate toward the erythroid lineage.

Nitric Oxide Induces TIMP-1 Expression by Activating the Transforming Growth Factor β-Smad Signaling Pathway

Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.

Transforming Growth Factor-β Regulates Stearoyl Coenzyme A Desaturase Expression through a Smad Signaling Pathway

The regulation of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is physiologically important because the ratio of saturated to unsaturated fatty acids is thought to control cellular functions by modulating the structural integrity and fluidity of cell membranes. Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, increased SCD mRNA expression in cultured human retinal pigment epithelial cells. This response was elicited by all three TGF-beta isoforms, beta1, beta2, and beta3. However, SCD mRNA expression was not increased either by other members of the TGF-beta family or by other growth factors or cytokines. TGF-beta also increased SCD mRNA expression in several other cell lines tested. The increase in SCD mRNA expression was preceded by a marked increase in Smad2 phosphorylation in TGF-beta-treated human retinal pigment epithelial cells. TGF-beta did not induce SCD mRNA expression in a Smad4-deficient cell line. However, Smad4 overexpression restored the TGF-beta effect in this cell line. Moreover, TGF-beta-induced SCD mRNA expression was effectively blocked by the overexpression of Smad7, an inhibitory Smad. Thus, a TGF-beta signal transduction pathway involving Smad proteins appears to regulate the cellular expression of the SCD gene, and this regulation may play an important role in lipid metabolism.