Inhibitory effects of Scutellaria barbata D. Don. and Euonymus alatus Sieb. on aromatase activity of human leiomyomal cells

It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase, we have examined the aromatase-inhibiting potency of the Scutellaria barbata D. Don. (SB) and Euonymus alatus Sieb. (EA) in myometrial and leiomyomal cells which contain aromatase. We have also used human placental tissues. Although SB and EA are approximately equipotent in a cell-free aromatase system (human placental microsomes), EA is consistently 10-30 times more potent than SB in inhibiting intracellular aromatase in myometrial and leiomyomal cells. To provide insights into the effect of SB and EA on aromatase activity in leiomyomal cells, we examined the cell lines, which is induced to differentiate toward the more transformed cell phenotype by 12-tetradecanoylphorbal-13-acetate (TPA) as a protein kinase C activator and transforming growth factor-beta1 (TGF-beta1). Enzyme activity was inhibited in a time-and dose-dependent fashion by SB and EA and by either 1-50 nM TPA or 0.01-0.5 ng/ml TGF-beta1, with maximal responses after 2-3 h exposure.     

Regulation of PTC phenotype and function by TGF-β1: implications for transdifferentiation

Introduction There is now increasing evidence that proximal tubular cells (PTCs) contribute to renal interstitial fibrosis by alteration of matrix turnover and by the generation of pro‐fibrotic cytokines such as TGF‐β1. Recent studies suggest that, through a process of transdifferentiation, the PTCs are one source of the interstitial myofibroblasts that directly drive the fibrotic process. The aim of this work was to examine the role and mechanism by which TGF‐β1 may regulate PTC phenotype and function. Methods Experiments were performed using both primary‐cultures of PTC and the human PTC cell line HK2. All experiments were performed on growth‐arrested cells in the absence of serum. Results TGF‐β1 altered cell phenotype, assessed by light microscopy, with cells appearing elongated and spindle‐shaped. This was associated with loss of cell–cell contact and rearrangement of the actin cytoskeleton, increased formation of stress fibres and focal adhesions. Disruption of the actin cytoskeleton with cytochalasin‐D prevented phenotypic alterations following addition of TGF‐β1. Transient transfection with Smad‐2/‐4 or Smad‐3/‐4 expression vectors did not alter cell phenotype. Previously, we have demonstrated β‐catenin translocation to PTC nuclei and its association with Smad proteins following addition of TGF‐β1, suggesting the possibility that TGF‐β1 may modulate Wnt signalling. Wnt‐responsive Xtwn‐reporter construct was, however, silent in response to TGF‐β1. Similarly, a second Wnt‐/LEF‐1‐regulated element Toplflash, which does not contain Smad‐binding sites, was insensitive to TGF‐β1 signalling. In contrast, phenotypic changes in response to TGF‐β1 were abrogated by inhibitors of the RhoA downstream target ROCK, which also prevented loss of cell–cell contact and adherens junction disassembly. Removal of TGF‐β1 and addition of 1% FCS, however, reverted cell phenotype to a typical cobblestone epitheliod appearance, suggesting that TGF‐β1 did not result in terminal PTC transdifferentiation. Cells grown on tissue culture dishes coated with either type‐I or type‐III collagen also acquired an elongated fibroblastic phenotype; this effect was exaggerated by the addition of TGF‐β1. In contrast to the cells stimulated with TGF‐β1 alone, following stimulation by both TGF‐β1 and exposure to interstitial collagens, cell phenotype was stable in that it was not reversed upon removal of TGF‐β1 and addition of FCS. Addition of TGF‐β1 to cells grown on type‐IV collagen had no greater effect than TGF‐β1 alone. Addition of TGF‐β1 alone had little effect on the expression of α‐SMA. In contrast, cells grown on either type‐I or type‐III collagen, following addition of TGF‐β1, demonstrated marked increased expression of α‐SMA, which appeared to be incorporated into the cell cytoskeleton. Similarly, the combination of interstitial collagen (either type‐I or type‐III) and TGF‐β1 had synergistic effect on the relocation and down‐regulation of the epithelial markers E‐cadherin and cytokeratin. Finally, the results demonstrated synergistic effects of coating with interstitial collagen (either type‐I or type‐III), on cell ‘fibroblastic’ cell function as assessed by cell migration and by the synthesis of type‐III and type‐IV collagen. Conclusion The results of these in vitro experiments suggest that terminal transdifferentiation of proximal tubular epithelial cells is the result of a combination of the effects of the pro‐fibrotic cytokine TGF‐β1 and exposure of the cells to components of the interstitial extra‐cellular matrix to which the cells are not exposed in the absence of damage to the tubular basement membrane.     

The calpain inhibitor calpeptin prevents bleomycin-induced pulmonary fibrosis in mice

Pulmonary fibrosis is characterized by progressive worsening of pulmonary function leading to a high incidence of death. Currently, however, there has been little progress in therapeutic strategies for pulmonary fibrosis. There have been several reports on cytokines being associated with lung fibrosis, including interleukin (IL)‐6 and transforming growth factor (TGF)‐β1. We reported recently that two substances (ATRA and thalidomide) have preventive effects on pulmonary fibrosis by inhibiting IL‐6‐dependent proliferation and TGF‐β1‐dependent transdifferentiation of lung fibroblasts. Rheumatoid arthritis is a chronic autoimmune disorder, and its pathogenesis is also characterized by an association with several cytokines. It has been reported that calpain, a calcium‐dependent intracellular cysteine protease, plays an important role in the progression of rheumatoid arthritis. In this study, we examined the preventive effect of Calpeptin, a calpain inhibitor, on bleomycin‐induced pulmonary fibrosis. We performed histological examinations and quantitative measurements of IL‐6, TGF‐β1, collagen type Iα1 and angiopoietin‐1 in bleomycin‐treated mouse lung tissues with or without the administration of Calpeptin. Calpeptin histologically ameliorated bleomycin‐induced pulmonary fibrosis in mice. Calpeptin decreased the expression of IL‐6, TGF‐β1, angiopoietin‐1 and collagen type Iα1 mRNA in mouse lung tissues. In vitro studies disclosed that Calpeptin reduced (i) production of IL‐6, TGF‐β1, angiopoietin‐1 and collagen synthesis from lung fibroblasts; and (ii) both IL‐6‐dependent proliferation and angiopoietin‐1‐dependent migration of the cells, which could be the mechanism underlying the preventive effect of Calpeptin on pulmonary fibrosis. These data suggest the clinical use of Calpeptin for the prevention of pulmonary fibrosis.     

Polymorphism in codons 10 and 25 of the transforming growth factor‐beta 1 (TGF‐β1) gene in patients with invasive squamous cell carcinoma of the uterine cervix

Transforming growth factor‐beta 1 (TGF‐β1) has a multifactorial role in the development of cervical cancer. It potently inhibits the growth of epithelial cells that harbour oncogenic human papilloma viruses (HPVs). TGF‐β1 also inhibits the expression of the early viral transforming regions E6 and E7, which appear to be the key oncoproteins. It has been suggested that squamous cell carcinomas are devoid of TGF‐β1, raising the possibility that elevated levels of this growth factor could protect against cervical cancer. It is also recognized that the production and levels of TGF‐β1 are genetically predetermined and individually variable. Two genetic polymorphisms in the DNA encoding the leader sequence of the TGF‐β1 gene have been described and shown to be associated with the production of high or low TGF‐β1 levels in vivo and in vitro. We hypothesized that the inheritance of these polymorphisms could influence the development of invasive cervical cancer. This hypothesis was investigated by studying polymorphism in codons 10 and 25 of the TGF‐β1 gene. We studied 97 patients with invasive cervical cancer and 73 healthy controls and found that the distributions of alleles T (Leu) and/or C (Pro) and alleles G (Arg) and/or C (Pro) in codons 10 and 25, respectively, were similar. There was no significant association between the alleles and the histological degree of cancer differentiation. It appears that the role of this growth factor in cervical oncogenesis is not related to the point mutations that we examined in codons 10 and 25 of the TGF‐β1 gene. We speculate that other factors, including additional polymorphisms of the TGF‐β1 gene, the status of TGF‐β1 receptors, the complex cytokine network, differential responsiveness of cells to the stimuli, and the status of the precancer/cancer genome, may play a role in development of invasive cervical cancer.     

Transforming Growth Factor‐β Suppressed Id‐1 Expression in a smad3‐Dependent Manner in LoVo Cells

TGF‐β plays an important role in regulating cell differentiation and proliferation in human cancers such as colorectal cancer. Id‐1 has been identified as a marker in colorectal cancer progression. The aim of this study was to investigate the role of TGF‐β in regulating Id‐1 in LoVo cells. siRNA was used to silence smad2, smad3, and p38 MAPK gene expression in Lovo cells. Interference efficiency and the role of TGF‐β on Id‐1 expression were analyzed using a luciferase reporter assay, RT‐PCR, and Western blotting. Cell viability was determined using the MTT assay. In this study, we demonstrated that TGF‐β1 downregulated Id‐1 protein expression in LoVo cells. Smad2 and smad3 siRNA inhibited TGF‐β1‐induced 4×SBE luciferase reporter activity. p38 MAPK siRNA inhibited TGF‐β1‐induced 3×AP‐1 luciferase reporter activity. However, the suppression of Id‐1 by TGF‐β1 was recovered by smad3 siRNA but not smad2 or p38 MAPK siRNA. Moreover, TGF‐β1 stimulated cellular proliferation and p21^Waf1 protein expression, which might be mediated by suppressing Id‐1 expression. In conclusion, this study demonstrated that TGF‐β1 suppressed Id‐1 expression in a smad3‐dependent manner in LoVo cells using RNAi technology. These results provide new insight into the mechanisms of TGF‐β function in colorectal cancer cells. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Titration of non-replicating adenovirus as a vector for transducing active TGF-β1 gene expression causing inflammation and fibrogenesis in the lungs of C57BL/6 mice

Investigators have shown that interstitial pulmonary fibrosis (IPF) can be induced in rats by overexpressing transforming growth factor beta₁ (TGF‐β₁) through a replication‐deficient recombinant adenovirus vector instilled into the lungs ( Sime et al. 1997 ). We have shown that this vector induces IPF in fibrogenic‐resistant tumour necrosis factor alpha‐receptor knockout (TNF‐αRKO) mice ( Liu et al. 2001 ). The object of our studies is to understand how peptide growth factors, such as TGF‐β₁, mediate interstitial lung disease (ILD). To do so, we must be able to manipulate the dose of the factor and sort out its effects on multiple other mediators in the lung parenchyma. As a step in this complex process, in the studies reported here, we have determined the concentrations of the recombinant adenovirus vector carrying the gene for porcine active TGF‐β₁ (AVTGFβ₁) that have little apparent effect, cause clear induction of disease, or severe disease. The disease largely resolves by 28 days in all cases, thus providing a valuable model to understand the mechanisms of the IPF that is mediated, at least in part, by TGF‐β₁. The findings here show that 10⁶ plaque‐forming units (pfu) of AVTGFβ₁, provide essentially a ‘no‐effect’ dose, but even this amount of TGF‐β₁ causes a significant increase in whole‐lung collagen by day 28 after treatment. In contrast, 10⁸ and 10⁹ pfu cause severe IPF in 4 days, whereas 10⁷ and 5 × 10⁷ are intermediate for all parameters studied, i.e. TGF‐β protein, inflammatory cells, cell proliferation, pro‐α 1(I) collagen gene expression and whole‐lung collagen accumulation, and expression of growth factors such as TGF‐β₁, TNF‐α and PDGF‐A and ‐B. Interestingly enough, TGF‐β₁, as a potent blocker of epithelial cell proliferation, appears to suppress airway epithelial cell growth that would be expected during the inflammatory phase of IPF. Thus, this model system helps us to understand some quantitative aspects of TGF‐β₁ biological activity and allows us to manipulate this potent factor as a mediator of interstitial fibrogenesis.     

Transforming growth factor‐β1 in asthmatic airway smooth muscle enlargement: is fibroblast growth factor‐2 required?

Enlargement of airway smooth muscle (ASM) tissue around the bronchi/bronchioles is a histopathological signature of asthmatic airway remodelling and has been suggested to play a critical role in the increased lung resistance and airway hyperresponsiveness seen in asthmatic patients. The pleiotropic cytokine, TGF‐β1, is believed to contribute to several aspects of asthmatic airway remodelling and is known to influence the growth of many cell types. Increased TGF‐β1 expression/signalling and ASM growth have been shown to occur concurrently in animal models of asthma. Abundant studies further substantiate this association by showing that therapeutic strategies that reduce or prevent TGF‐β1 overexpression/signalling lead to a parallel decrease or prevention of ASM enlargement. Finally, recent findings have supported a direct link of causality between TGF‐β1 overexpression/signalling and the overgrowth of ASM tissue. To follow‐up on these in vivo studies, many investigators have pursued detailed investigation of ASM in cell culture conditions, assessing the direct role of TGF‐β1 on cellular proliferation and/or hypertrophy. Inconsistencies among the in vitro studies suggest that the effect of TGF‐β1 on ASM cell proliferation/hypertrophy is contextual. A hypothesis focusing on fibroblast growth factor‐2 is presented at the end of this review, which could potentially reconcile the apparent discrepancy between the conflicting in vitro findings with the consistent in vivo finding that TGF‐β1 is required for ASM enlargement in asthma. Cite this as: Y. Bossé, J. Stankova and M. Rola‐Pleszczynski, Clinical & Experimental Allergy, 2010 (40) 710–724.     

Role of T cell TGF‐β signaling in intestinal cytokine responses and helminthic immune modulation

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL‐10 or TGF‐β, that are important in suppressing colitis. Helminths induce mucosal T cell IL‐10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL‐10‐dependent manner in WT mice. Helminths also stimulate mucosal TGF‐β release. As TGF‐β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF‐β signaling in helminthic modulation of intestinal immunity. T cell TGF‐β signaling is interrupted in TGF‐β receptor II dominant negative (TGF‐βRII DN) mice by T‐cell‐specific over‐expression of a TGF‐βRII DN. We studied LPMC responses in WT and TGF‐βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF‐β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL‐10 secretion requires intact T cell TGF‐β‐signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF‐βRII DN mice. Thus, T cell TGF‐β signaling is essential for helminthic stimulation of mucosal IL‐10 production, helminthic modulation of intestinal IFN‐γ generation and H. polygyrus‐mediated suppression of chronic colitis.     

The GARP/Latent TGF‐β1 complex on Treg cells modulates the induction of peripherally derived Treg cells during oral tolerance

Treg cells can secrete latent TGF‐β1 (LTGF‐β1), but can also utilize an alternative pathway for transport and expression of LTGF‐β1 on the cell surface in which LTGF‐β1 is coupled to a distinct LTGF‐β binding protein termed glycoprotein A repetitions predominant (GARP)/LRRC32. The function of the GARP/LTGF‐β1 complex has remained elusive. Here, we examine in vivo the roles of GARP and TGF‐β1 in the induction of oral tolerance. When Foxp3^? OT‐II T cells were transferred to wild‐type recipient mice followed by OVA feeding, the conversion of Foxp3^? to Foxp3⁺ OT‐II cells was dependent on recipient Treg cells. Neutralization of IL‐2 in the recipient mice also abrogated this conversion. The GARP/LTGF‐β1 complex on recipient Treg cells, but not dendritic cell‐derived TGF‐β1, was required for efficient induction of Foxp3⁺ T cells and for the suppression of delayed hypersensitivity. Expression of the integrin αvβ8 by Treg cells (or T cells) in the recipients was dispensable for induction of Foxp3 expression. Transient depletion of the bacterial flora enhanced the development of oral tolerance by expanding Treg cells with enhanced expression of the GARP/LTGF‐β1 complex.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

Leukotriene D4-induced, epithelial cell-derived transforming growth factor β1 in human bronchial smooth muscle cell proliferation

Background Cysteinyl‐leukotrienes (cys‐LTs) orchestrate many pathognomonic features of asthma in animal models of allergic airway inflammation, including bronchial smooth muscle cell (BSMC) hyperplasia. However, because cys‐LTs alone do not induce mitogenesis in monocultures of human BSMC, the effect observed in vivo seemingly involves indirect mechanisms, which are still undefined. Objective This study aims to investigate the regulatory role of leukotriene (LT)D₄ on TGF‐β1 expression in airway epithelial cells and the consequence of this interplay on BSMC proliferation. Methods HEK293 cells stably transfected with cys‐LT receptor 1 (CysLT1) (293LT1) were stimulated with LTD₄ and TGF‐β1 mRNA and protein expression was measured using Northern blot and ELISA, respectively. Conditioned medium (CM) harvested from LTD₄‐treated cells was then assayed for its proliferative effect on primary human BSMC. TGF‐β1 mRNA expression was also determined in tumoural type II pneumocytes A549 and in normal human bronchial epithelial cells (NHBE) following LTD₄ stimulation. Results The results demonstrated that LTD₄‐induced TGF‐β1 mRNA production in a time‐ and concentration‐dependent manner in 293LT1. TGF‐β1 secretion was also up‐regulated and CM from LTD₄‐treated 293LT1 was shown to increase BSMC proliferation in a TGF‐β1‐dependent manner. The increased expression of TGF‐β1 mRNA by LTD₄ also occured in A549 and NHBE cells via a CysLT1‐dependent mechanism. Conclusion In conclusion, elevated expression of cys‐LTs in asthmatic airways might contribute to BSMC hyperplasia and concomitant clinical features of asthma such as airway hyperresponsiveness via a paracrine loop involving TGF‐β1 production by airway epithelial cells.     

Submandibular gland morphogenesis: Stage‐specific expression of TGF‐α/EGF,IGF, TGF‐β, TNF,and IL‐6 signal transduction in normal embryonic mice and the phenotypic effects of TGF‐β2, TGF‐β3, and EGF‐r null mutations

Branching morphogenesis of the mouse submandibular gland (SMG) is dependent on cell‐cell conversations between and within epithelium and mesenchyme. Such conversations are typically mediated in other branching organs (lung, mammary glands, etc.) by hormones, growth factors, cytokines, and the like in such a way as to translate endocrine, autocrine, and paracrine signals into specific gene responses regulating cell division, apoptosis, and histodifferentiation. We report here the protein expression in embryonic SMGs of four signal transduction pathways: TGF‐α/EGF/EGF‐R; IGF‐II/IGF‐IR/IGF‐IIR; TGF‐βs and cognate receptors; TNF, IL‐6, and cognate receptors. Their in vivo spatiotemporal expression is correlated with specific stages of progressive SMG development and particular patterns of cell proliferation, apoptosis, and mucin expression. Functional necessity regarding several of these pathways was assessed in mice with relevant null mutations (TGF‐β2, TGF‐β₃, EGF‐R). Among many observations, the following seem of particular importance: (1) TGF‐α and EGF‐R, but not EGF, are found in the Initial and Pseudoglandular Stages of SMG development; (2) ductal and presumptive acini lumena formation was associated with apoptosis and TNF/TNF‐R1 signalling; (3) TGF‐β2 and TGF‐β3 null mice have normal SMG phenotypes, suggesting the presence of other pathways of mitostasis; (4) EGF‐R null mice displayed an abnormal SMG phenotype consisting of decreased branching. These and other findings provide insight into the design of future functional studies. Anat Rec 256:252–268, 1999. © 1999 Wiley‐Liss, Inc.     

Decorin in renal inflammation and fibrosis – more than an inhibitor of TGF-β

Introduction Decorin, a small leucine‐rich proteoglycan, participates in extracellular matrix assembly and influences cell behaviour by interacting with signalling membrane receptors and TGF‐β. Treatment with decorin has been shown to have beneficial effects in an acute model of mesangioproliferative glomerulonephritis due to interaction with TGF‐β. The underlying mechanisms, however, remain unclear because upon complex formation with TGF‐β, the cytokine's activity may become increased, decreased or not influenced at all. Hence, the objectives of the present study were twofold: firstly, to provide evidence that decorin influences the course and final outcome of renal inflammation and secondly, to find anti‐fibrotic mechanisms of decorin both related and unrelated to the regulation of TGF‐β activity. Results Based on our studies in human diabetic nephropathy, we postulate two regulatory mechanisms by which decorin modulates TGF‐β‐mediated fibrosis: (1) increased quantities of glomerular decorin are synthesized and form complexes with TGF‐β, which then are removed via glomerular capillaries or the urinary tract, and (2) in the presence of type‐I collagen, decorin is able to sequester TGF‐β in the extracellular matrix, thereby withdrawing the cytokine from its cell‐surface receptors. Furthermore, we have compared the evolution of tubulointerstitial fibrosis in wild‐type (WT) and decorin^–/– mice in a model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin^–/– mice did not differ in any aspect from their WT counterparts. However, already 12 h after obstruction, decorin^–/– animals showed lower levels of p27^KIP1 and soon thereafter a more pronounced up‐regulation and activation of initiator and effector caspases, followed by enhanced apoptosis of tubular epithelial cells. At later stages, a higher increase of TGF‐β1 became apparent. After 7 days, there was a 15‐fold transient up‐regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan‐expressing mononuclear cells. Other small proteoglycans showed no similar response. Owing to enhanced degradation of type‐I collagen and increased tubular epithelial cell apoptosis, end‐stage kidneys from decorin^–/– animals were more atrophic than WT kidneys. Conclusion These data suggest that decorin exerts beneficial effects on renal inflammation, primarily by influencing the expression of a key cyclin‐dependent kinase inhibitor, thereby limiting the degree of apoptosis and tubular atrophy. In later stages, anti‐fibrotic effects of decorin are based on the regulation of TGF‐β1 expression, mononuclear cell infiltration and collagen turnover.