Activation of extracellular signal-regulated kinase by TGF-β1 via TβRII and Smad7 dependent mechanisms in human bronchial epithelial BEP2D cells

Transforming growth factor-β1 (TGF-β1) can activate mitogen-activated protein kinases (MAPKs) in many types of cells. The mechanism of this activation is not well elucidated. Here, we explore the role of TGF-β/Smads signaling compounds in TGF-β1-mediated activation of extracellular signal-regulated kinase (ERK) MAPK in human papillomavirus (HPV)-18 immortalized human bronchial epithelial cell line BEP2D and the role of TGF-β1-induced phosphorylation of ERK in proliferation and apoptosis of BEP2D. The cell models of siRNA-mediated silencing of TGF-β receptor type II (TβRII), Smad2, Smad3, Smad4, and Smad7 were employed in this study. Our results demonstrate that TGF-β1 activates ERK in a time-dependent manner with a maximum effect at 60 min; overexpression of Smad7 increased this TGF-β1-mediated phosphorylation of the ERK; and siRNA-mediated silencing of TβRII, Smad3, Smad4, and Smad7 abrogated this effect. Moreover, we observed that overexpression of Smad7 restored TGF-β1-mediated ERK phosphorylation in Smad4 knockdown cells but not in TβRII knockdown cells. In BEP2D cells, TGF-β1 treatment effectively inhibited cells’ proliferation and induced their apoptosis. Pretreatment with U0126, an inhibitor of ERK1/2, significantly enhanced the TGF-β1-mediated antiproliferative and apoptosis induction effects in BEP2D cells. These data revealed that TβRII and Smad7 play the critical roles in TGF-β1-mediated activation of ERK; Smad3 and Smad4 can play an indirect role through up-regulating Smad7 expression; and TGF-β1-induced phosphorylation of ERK may participate in BEP2D cell proliferation and apoptosis regulation.     

CD8 Co-receptor promotes susceptibility of CD8<Superscript>+</Superscript> T cells to transforming growth factor-β (TGF-β)-mediated suppression

CD8⁺ T cell function depends on a finely orchestrated balance of activation/suppression signals. While the stimulatory role of the CD8 co-receptor and pleiotropic capabilities of TGF-β have been studied individually, the influence of CD8 co-receptor on TGF-β function in CD8⁺ T cells is unknown. Here, we show that while CD8 enhances T cell activation, it also enhances susceptibility to TGF-β-mediated immune suppression. Using Jurkat cells expressing a full-length, truncated or no αβCD8 molecule, we demonstrate that cells expressing full-length αβCD8 were highly susceptible, αβCD8-truncated cells were partially susceptible, and CD8-deficient cells were completely resistant to suppression by TGF-β. Additionally, we determined that inhibition of Lck rendered mouse CD8⁺ T cells highly resistant to TGF-β suppression. Resistance was not associated with TGF-β receptor expression but did correlate with decreased Smad3 and increased Smad7 levels. These findings highlight a previously unrecognized third role for CD8 co-receptor which appears to prepare activated CD8⁺ T cells for response to TGF-β. Based on the important role which TGF-β-mediated suppression plays in tumor immunology, these findings unveil necessary considerations in formulation of CD8⁺ T cell-related cancer immunotherapy strategies.     

Endoglin in liver fibrosis

Liver fibrosis occurs in most types of chronic liver diseases and is characterized by excessive accumulation of extracellular matrix proteins, leading to disruption of tissue function and eventually organ failure. Transforming growth factor (TGF)-β represents an important pro-fibrogenic factor and aberrant TGF-β action has been implicated in many disease processes of the liver. Endoglin is a TGF-β co-receptor expressed mainly in endothelial cells that has been shown to differentially regulates TGF-β signal transduction by inhibiting ALK5-Smad2/3 signalling and augmenting ALK1-Smad1/5 signalling. Recent reports demonstrating upregulation of endoglin expression in pro-fibrogenic cell types such as scleroderma fibroblasts and hepatic stellate cells have led to studies exploring the potential involvement of this TGF-β co-receptor in organ fibrosis. A recent article by Meurer and colleagues now shows that endoglin expression is increased in transdifferentiating hepatic stellate cells in vitro and in two different models (carbon tetrachloride intoxication and bile duct ligation) of liver fibrosis in vivo. Moreover, they show that endoglin overexpression in hepatic stellate cells is associated with enhanced TGF-β-driven Smad1/5 phosphorylation and α-smooth muscle actin production without altering Smad2/3 signaling. These findings suggest that endoglin may play an important role in hepatic fibrosis by altering the balance of TGF-β signaling via the ALK1-Smad1/5 and ALK-Smad2/3 pathways and raise the possibility that targeting endoglin expression in transdifferentiating hepatic stellate cells may represent a novel therapeutic strategy for the treatment of liver fibrosis.     

A milk growth factor extract reduces chemotherapeutic drug toxicity in epithelial cells in vitro

Summary Transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF-I) can attenuate drug-induced cell death in epithelial cells. Since milk whey contains a mixture of these and other growth factors, we evaluated mitogenic bovine whey extract (MBWE) for protective activity against chemotherapy drug damage in cultured epithelial cells (mink lung, Mu1.Lu). Etoposide and vinblastine reduced cell survival by up to 90%. This was attenuated by the addition of MBWE before and during drug exposure, but not following drug removal. MBWE was compared with individual growth factors known to be present in the mixture. IGF-I and platelet-derived growth factor were ineffective, whereas TGF-β2 induced growth inhibition and cell survival, with a maximum response at 3 ng/ml. TGF-β2 bioactivity was also demonstrated by showing that acidification of MBWE (A-MBWE), to activate TGF-β2, enhanced its growth inhibitory and chemoprotective activities 60- and 12-fold, respectively. However, MBWE contained additional protective factors. When TGF-β2 and the MBWE preparations were compared, on the basis of growth inhibition equivalents, MBWE protected cells against drug toxicity at concentrations an order of magnitude lower than with TGF-β2 or A-MBWE. Immunoneutralization of the TGF-β present in MBWE and A-MBWE eliminated all growth inhibitory activity but not all cell survival activity. We conclude that the MBWE preparations are cytoprotective against two chemotherapy drugs when added before and during drug exposure. TGF-β contributes to this activity, but the extracts contain other factors that promote the survival of epithelial cells after chemotherapy drug exposure.     

Chymase induces profibrotic response via transforming growth factor-β1/Smad activation in rat cardiac fibroblasts

Mast cell-derived chymase is implicated in myocardial fibrosis (MF), but the underlying mechanism of intracellular signaling remains unclear. Transforming growth factor-β1 (TGF-β1) is identified as the most important profibrotic cytokine, and Smad proteins are essential, but not exclusive downstream components of TGF-β1 signaling. Moreover, novel evidence indicates that there is a cross talk between Smad and mitogen-activated protein kinase (MAPK) signaling cascade. We investigated whether chymase activated TGF-β1/Smad pathway and its potential role in MF by evaluating cardiac fibroblasts (CFs) proliferation and collagen synthesis in neonatal rats. MTT assay and ³H-Proline incorporation revealed that chymase induced CFs proliferation and collagen synthesis in a dose-dependent manner. RT-PCR and Western blot assay demonstrated that chymase not only increased TGF-β1 expression but also upregulated phosphorylated-Smad2/3 protein. Furthermore, pretreatment with TGF-β1 neutralizing antibody suppressed chymase-induced cell growth, collagen production, and Smad activation. In contrast, the blockade of angiotensin II receptor had no effects on chymase-induced production of TGF-β1 and profibrotic action. Additionally, the inhibition of MAPK signaling had no effect on Smad activation elicited by chymase. These results suggest that chymase can promote CFs proliferation and collagen synthesis via TGF-β1/Smad pathway rather than angiotensin II, which is implicated in the process of MF.     

p38 Mitogen-Activated Protein Kinase Affects Transforming Growth Factor-β/Smad Signaling in Human Dental Pulp Cells

Transforming Growth Factor-β (TGF-β) plays an essential role in differentiation of dental pulp cells into odontoblasts during reparative dentine formation. However, the mechanism by which TGF-β stimulates dental repair remains rather obscure. Human dental pulp cells were used as an in vitro model in the present work. We showed that TGF-β signaled through mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38, along with Smad pathway. Distinct pathways exerted different time response. SB203580, a specific p38 MAPK inhibitor, reduced phosphorylation of Smad3, while it slightly enhanced phosphorylation of Smad2. Increased phosphorylation of ERK1/2 and p38 confirmed that SB203580 did not block activation of TGF-β receptors. In addition, the inhibition of ERK1/2 activity with MEK1/2 inhibitor U0126 increased TGF-β mediated phosphorylation of Smad3. Our results suggest that p38 affects the phosphorylation of Smad2 and Smad3 differentially during TGF-β signaling in human dental pulp cells and ERK1/2 might be involved in the process.     

Transforming growth factor-β1 blocks the enhancement of tumor necrosis factor cytotoxicity by hyaluronidase Hyal-2 in L929 fibroblasts

Background  

Functional antagonism between transforming growth factor beta (TGF-β) and hyaluronidase has been demonstrated. For example, testicular hyaluronidase PH-20 counteracts TGF-β1-mediated growth inhibition of epithelial cells. PH-20 sensitizes various cancer cells to tumor necrosis factor (TNF) cytotoxicity by upregulating proapoptotic p53 and WW domain-containing oxidoreductase (WOX1). TGF-β1 blocks PH-20-increased TNF cytotoxicity. In the present study, the functional antagonism between TGF-β1 and lysosomal hyaluronidases Hyal-1 and Hyal-2 was examined.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.     

The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process     

Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins

Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. Our data suggest that 14-3-3 bound to B-Raf protects the enzyme from protein kinase A-mediated inhibition; the amount of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity.     

支架蛋白GIT2通过与Smad3相互作用调节其转录活性

G蛋白偶联受体激酶相互作用蛋白2(G protein-coupled receptor kinase interacting proteins 2,GIT2)是一种信号支架蛋白,可募集多种信号通路的关键分子,参与肌动蛋白细胞骨架组装、整合素介导的细胞粘附、G蛋白偶联受体的内化及胞内信号传递等生物学过程. 采用酵母双杂交实验证明,TGF-β1信号通路的转录因子Smad3是GIT2的相互作用蛋白质,内、外源免疫共沉淀实验均证实,GIT2与Smad3存在蛋白质相互作用. 报告基因实验及免疫印迹结果表明,GIT2增加Smad3的转录活性并增强TGF-β1诱导的Smad3的磷酸化.研究还发现,Git2-/-小鼠骨髓间充质干细胞(MSC)的Smad3磷酸化受到抑制,其骨形成相关靶基因的表达水平也低于Git2+/+小鼠. 本研究表明,GIT2通过与Smad3的相互作用调节其转录活性并活化TGF-β1信号通路,可能参与调节骨髓间充质干细胞的分化.     

Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells

Hepatocellular carcinoma (HCC) usually arises from hepatic fibrosis caused by chronic inflammation. In chronic liver damage, hepatic stellate cells undergo progressive activation to myofibroblasts (MFB), which are important extracellular-matrix-producing mesenchymal cells. Concomitantly, perturbation of transforming growth factor (TGF)-β signaling by pro-inflammatory cytokines in the epithelial cells of the liver (hepatocytes) promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into fibro-carcinogenic effects on chronically damaged hepatocytes have come from recent detailed analyses of the TGF-β signaling process. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create phosphoisoforms phosphorylated at the COOH-terminal, linker, or both (L/C) regions. After acute liver injury, TGF-β-mediated pSmad3C signaling terminates hepatocytic proliferation induced by the pro-inflammatory cytokine-mediated mitogenic pSmad3L pathway; TGF-β and pro-inflammatory cytokines synergistically enhance collagen synthesis by activated hepatic stellate cells via pSmad2L/C and pSmad3L/C pathways. During chronic liver disease progression, pre-neoplastic hepatocytes persistently affected by TGF-β together with pro-inflammatory cytokines come to exhibit the same carcinogenic (mitogenic) pSmad3L and fibrogenic pSmad2L/C signaling as do MFB, thereby accelerating liver fibrosis while increasing risk of HCC. This review of Smad phosphoisoform-mediated signals examines similarities and differences between epithelial and mesenchymal cells in acute and chronic liver injuries and considers Smad linker phosphorylation as a potential target for the chemoprevention of fibro-carcinogenesis.     

Dimethylfumarate Attenuates Renal Fibrosis via NF-E2-Related Factor 2-Mediated Inhibition of Transforming Growth Factor-β/Smad Signaling

TGF-β plays a key role in the development of renal fibrosis. Suppressing the TGF-β signaling pathway is a possible therapeutic approach for preventing this disease, and reports have suggested that Nrf2 protects against renal fibrosis by inhibiting TGF-β signaling. This study examines whether dimethylfumarate (DMF), which stimulates Nrf2, prevents renal fibrosis via the Nrf2-mediated suppression of TGF-β signaling. Results showed that DMF increased nuclear levels of Nrf2, and both DMF and adenovirus-mediated overexpression of Nrf2 (Ad-Nrf2) decreased PAI-1, alpha-smooth muscle actin (α-SMA), fibronectin and type 1 collagen expression in TGF-β-treated rat mesangial cells (RMCs) and renal fibroblast cells (NRK-49F). Additionally, DMF and Ad-Nrf2 repressed TGF-β-stimulated Smad3 activity by inhibiting Smad3 phosphorylation, which was restored by siRNA-mediated knockdown of Nrf2 expression. However, downregulation of the antioxidant response element (ARE)-driven Nrf2 target genes such as NQO1, HO-1 and glutathione S-transferase (GST) did not reverse the inhibitory effect of DMF on TGF-β-induced upregulation of profibrotic genes or extracellular matrix proteins, suggesting an ARE-independent anti-fibrotic activity of DMF. Finally, DMF suppressed unilateral ureteral obstruction (UUO)-induced renal fibrosis and α-SMA, fibronectin and type 1 collagen expression in the obstructed kidneys from UUO mice, along with increased and decreased expression of Nrf2 and phospho-Smad3, respectively. In summary, DMF attenuated renal fibrosis via the Nrf2-mediated inhibition of TGF-β/Smad3 signaling in an ARE-independent manner, suggesting that DMF could be used to treat renal fibrosis.     

Smad7 and protein phosphatase 1α are critical determinants in the duration of TGF-β/ALK1 signaling in endothelial cells

Background  

In endothelial cells (EC), transforming growth factor-β (TGF-β) can bind to and transduce signals through ALK1 and ALK5. The TGF-β/ALK5 and TGF-β/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-β signaling is an important specificity determinant for signaling responses. TGF-β/ALK1-induced Smad1/5 phosphorylation in ECs occurs transiently.     

Antagonism of Transforming Growth Factor-Β Signaling Inhibits Fibrosis-Related Genes

In the fibrotic process, the transforming growth factor-β1 (TGF-β1)/Smad3 (Sma- and Mad-related protein␣3) signaling plays a central role. To screen for antagonists of TGF-β1/Smad3 signaling and to investigate their effects on the genes related to fibrosis, we construct a molecular model with a luciferase reporter gene. Results showed that both SB-431542 [4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide] and small interference RNA (siRNA) against Smad3 could dose-dependently suppress the reporter gene. More importantly, they both significantly inhibited the expression of plasminogen activator inhibitor-type 1 (PAI-1) and type I collagenα1 (Col Iα1) genes in rat hepatic stellate cells. Thus, SB-431542 and Smad3/siRNA may be potential therapeutics for fibrosis.     

Regulation of melanin synthesis by the TGF-β family in B16 melanoma cells

Effects of representative members of the transforming growth factor-β (TGF-β) family, TGF-β1, activin A and BMP-2, on melanin content and expression of pigment-producing enzymes were examined in B16 melanoma cells. Treatment with TGF-β1 or activin A but not with BMP-2 significantly decreased melanin content and expression of Tyrosinase and Tyrp-1, suggesting an inhibitory effect of TGF-β1 and activin A on melanin synthesis. TGF-β1 completely inhibited melanin synthesis induced by α-melanin stimulating hormone (α-MSH), whereas activin A only slightly did. As compared with parental B16 cells, the inhibitory effects of TGF-β1 and activin A on melanin content were relative smaller in B16 F10 cells, a subline of B16 cells that contain more pigment. The present study indicates that in addition to TGF-β, activin negatively regulates melanogenesis in the absence of α-MSH, but that the activity in the presence of α-MSH was slightly different between TGF-β and activin.     

Evaluation of the role of extracellular matrix proteins,polyunsaturated fatty acids and C-MYC expression in the inhibition of the serum-free growth of epithelial cells by TGF-β1

Summary Novel or modified serum-free media were developed for the anchorage-dependent growth of nontransformed murine mammary epithelial cells (MMEC) and Balb/MK murine keratinocytes respectively. Growth rates for both cell lines were similar in serum-containing and serum-free media. The serum-free media were used to evaluate potential mechanisms of epithelial cell growth regulation by type 1 transforming growth factor β(TGF-β1). The growth of MMEC and Balb/ MK cells was reversibly inhibited 40–65% in a time- and dose-dependent fashion by TGF-β1 under both serum-containing and serum-free conditions. Constitutive over-expression of a stranfected c-myc oncogene inMMEC did not result in loss of sensitivity to growth inhibition by TGF-β1. In addition, Balb/MK and MMEC growth inhibition by TGF-β1 was not potentiated by polynsaturated fatty acids or reversed by vitamin E. Expgenous type V collagen was able to mimic the inhibitory effects of TGF-β1 on the serum-free growth of Balb/MK and MMEC. In contrast, collagen type I and IV, fibronectin and laminin did not inhibit the growth of these cells. The type V collagen used was not contaminated with TGF-β, and subsaturating, but not saturating concentrations of type V collagen and TGF-β1 were additive with respect to Balb/MK and MMEC growth inhibition. These results demonstrate that nontransformed epithelial cell growth inhibition by TGF-β1 is mediated by mechanisms distinct from those observed with certain carcinoma and melanoma cells. Our results also suggest the possible involvement of type V collagen in Balb/MK and MMEC growth inhibition by TGF-β1. This work was supported, in part, by grant #R29 CA 44741 from the National Institutes of Health, Bethesda, MD to NTT.     

<Emphasis Type="Italic">Scutellaria</Emphasis> extract and wogonin inhibit tumor-mediated induction of T<Subscript>reg</Subscript> cells via inhibition of TGF-β1 activity

A number of studies have implicated tumor-induced T_reg cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T_reg cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-β1-induced T_reg activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-β1 and T_reg cell frequency as well as peripheral blood TGF-β1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-β1-mediated T_reg activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T_reg culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3β and ERK1/2 signaling by SocL and wogonin in T_reg cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells’ response to TGF-β1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-β1 secretion as well as via inhibition of T cells’ response to TGF-β1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.