Extracellular TGF-beta Research Articles

Abstract MP39: FHL5 , A Novel Cofactor Associated With Coronary Artery Disease, Regulates Smooth Muscle Cell Function Through A Transcriptional Network Linking Multiple Risk Loci

Introduction: Coronary artery disease (CAD) is the leading cause of death worldwide with an estimated heritability of ~50%. The most recent genome wide association study (GWAS) for CAD and myocardial infarction (MI) identified over 200 loci. One such novel CAD/MI locus, UFL1-FHL5 (P = 1.1E-8), is associated with additional vascular pathologies, including hypertension, migraines, and coronary calcification. We previously showed through statistical fine-mapping that FHL5 is the top candidate causal gene underlying each of these vascular trait associations and highly enriched in the contractile mural cell populations in the artery. These preliminary studies motivate our hypothesis that FHL5 functions as a transcriptional regulator of SMC contractility to affect vascular disease risk. Methods and Results: Given the reported role of FHL5 as a cofactor, we mapped 17,201 candidate FHL5 binding sites in coronary artery SMCs using the Cleavage Under Targets and Release Using Nuclease (CUT&RUN) method. Binding sites were enriched for AP-1 family motifs and strongly overlapped CREB binding sites and H3K27ac enhancer marks. FHL5 target genes were functionally enriched in well characterized CAD pathways, such as extracellular matrix organization (P=5.2E-15, OR=2.1) and TGF-beta signaling (P=8.0E-5, OR=2.0). Interestingly, FHL5 binding sites were also enriched for CAD (P=1.8E-7, OR=2.3) and blood pressure (BP) risk variants (P=0.04, OR=1.2), thereby linking FHL5 with the regulation of multiple downstream CAD/BP loci. Weighted gene co-expression network and key driver analyses of human coronary artery transcriptomic data further support this regulatory role. Lastly, we validated these findings by performing confocal based calcium imaging and collagen gel contraction assays. Consistent with our functional genomic analyses, overexpression of FHL5 elevated intracellular calcium levels 2.5X and increased SMC contractility by ~45%. Conclusion: Taken together, our results provide evidence that FHL5 may impact CAD risk by regulating a network of disease-associated genes mediating SMC functions. These findings further contribute to our understanding of the heritable risk for multiple vascular diseases.

Abstract A192: Proteomic analysis of epithelial-to-mesenchymal transition induced by EGF in breast cancer cells

Abstract Metastasis is responsible for most of the deaths related to cancer. Therefore, the most effective methods to decrease cancer morbidity and mortality consist of early detection, prevention, and treatment of metastasis. Epithelial-to-mesenchymal transition (EMT) is one of the processes involved in cancer progression and metastasis. EMT induces cellular and microenvironmental changes that result in loss of the epithelial phenotype and acquisition of mesenchymal properties, thus promoting cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including EGF and TGF-beta. Overexpression of transcription factors such as SNAIL also induce EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was treated with the growth factor EGF (10 ng/mL) to identify proteomic alterations and specific mechanisms involved in the induction of EMT and metastasis. EGF treatment led to EMT, which was validated by molecular, morphologic, and functional experiments. Subcellular proteome enrichment followed by target proteomics was performed to identify changes in protein expression between the subcellular compartments cytoplasm, nucleus, and membrane. Thirty micrograms of each subcellular compartment of cells induced (or not) to EMT were processed to obtain tryptic peptide digest for subsequent MRM analysis. Proteotypic peptides representing relevant proteins for EMT and cancer progression, including proteins identified in our previous studies, were selected to compose panels for MRM analysis. EGF treatment activated the ERK/MAPK signaling pathway, which is involved in cancer progression by deregulating cell growth. Changes in expression of important proteins related to the EMT process were observed after EGF treatment, such as CDH1, CDH2, VIME, SOD2, and TGFB1. Subcellular compartment analysis allowed the identification of the main location of the proteins selected as well as differential regulation between subcellular compartments and potential mechanisms of subcellular translocation resulting from the EGF treatment and EMT induction, such as the regulation of PRDX3. Changes in the cellular protein localization may lead to alterations in the protein function and may thus be correlated with tumor progression. Other proteins related to cancer progression, including cytoskeleton rearrangement and cell cycle control, were also regulated after EGF treatment in specific compartments, such as CDK1, ARPC2, CKAP5, CNN3, and ARHGDIB. Besides that, we could identify the sumoylation as a possible post-translational modification regulated during EMT, which was verified by the expression of SUMO1/2 proteins, mainly in the cytoplasmic compartment. This detailed analysis allowed a better comprehension of the mechanisms of the EMT induction by EGF in a breast cancer cell line and can help to identify the biologic process and proteins relevant to cancer progression and that can potentially serve as targets for metastasis inhibition and/or diagnosis. (Financial support: FAPESP 2015/08693-0, 2011-0947-1, 2016/03809-3; CISBi NAP/USP grant 12.1.17598.1.3 and CTC-CEPID 2013/08135-2.) Citation Format: Camila S. Palma, Mariana L. Grassi, Carolina H. Thomé, Aline Poersch, Guilherme P. Lanfredi, Vitor M. Faça. Proteomic analysis of epithelial-to-mesenchymal transition induced by EGF in breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A192.

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues.ResultsWe isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four.ConclusionsOur results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

The face in Marfan syndrome: a 3D morphometric study

Marfan syndrome (MFS) is a rare congenital disorder of the connective tissue mainly caused by mutations in the FBN1 gene, resulting in an altered assembly of extracellular matrix microfibrils and TGF-beta signalling dysregulation. Major clinical manifestations of MFS involve the skeletal, ocular, and cardiovascular systems, with a high risk of life-threatening aortic dissection and rupture. An early recognition of the disorder is essential, but it could be difficult, due to the variable phenotypic expression of the disease and the current incomplete sensitivity of molecular genetic testing of FBN1. It has been suggested that craniofacial dysmorphism associated with MFS could facilitate obstructive sleep apnea, which in turn may promote aortic dilation. The study aimed to investigate the face in MFS through a 3D not invasive approach [1], identifying new morphometric features which could facilitate the early diagnosis of the disease. The 3D coordinates of 50 anatomical facial landmarks were obtained using a stereophotogrammetric system in 68 Italian subjects diagnosed with MFS, aged 4-64 years (27 males, mean ± SD age 29.6 ± 18.2 years; 41 females, mean ± SD age 37.2 ± 15.5 years). Subjects were divided in 11 non-overlapping age groups. Facial linear distances and angles were measured; z score values were calculated comparing patients with healthy Italian reference subjects (347 males, 388 females), matched for gender and age. Subjects with MFS showed a shorter mandibular ramus than controls (mean z score = -1.9), a greater facial divergence (mean z score = +2.0), a reduced ratio between posterior and anterior facial height (mean z score = -1.9), and a reduced ratio between facial width and facial height (mean z score = -1.5), together with an expected but overall mild increase of facial height (mean z score = +1.3). Noteworthy gender differences or age trends were not observed. Facial abnormalities pointed out in the current study could represent phenotypic traits of MFS; since they were observed also in young patients, their detection could facilitate the early recognition, management, and follow up of the disease. These promising findings need to be confirmed extending the study on more patients.

Cross-Talk and Information Transfer in Mammalian and Bacterial Signaling

In mammalian and bacterial cells simple phosphorylation circuits play an important role in signaling. Bacteria have hundreds of two-component signaling systems that involve phosphotransfer between a receptor and a response regulator. In mammalian cells a similar pathway is the TGF-beta pathway, where extracellular TGF-beta ligands activate cell surface receptors that phosphorylate Smad proteins, which in turn activate many genes. In TGF-beta signaling the multiplicity of ligands begs the question as to whether cells can distinguish signals coming from different ligands, but transduced through a small set of Smads. Here we use information theory with stochastic simulations of networks to address this question. We find that when signals are transduced through only one Smad, the cell cannot distinguish between different levels of the external ligands. Increasing the number of Smads from one to two significantly improves information transmission as well as the ability to discriminate between ligands. Surprisingly, both total information transmitted and the capacity to discriminate between ligands are quite insensitive to high levels of cross-talk between the two Smads. Robustness against cross-talk requires that the average amplitude of the signals are large. We find that smaller systems, as exemplified by some two-component systems in bacteria, are significantly much less robust against cross-talk. For such system sizes phosphotransfer is also less robust against cross-talk than phosphorylation. This suggests that mammalian signal transduction can tolerate a high amount of cross-talk without degrading information content. This may have played a role in the evolution of new functionalities from small mutations in signaling pathways, allowed for the development of cross-regulation and led to increased overall robustness due to redundancy in signaling pathways. On the other hand the lack of cross-regulation observed in many bacterial two-component systems may partly be due to the loss of information content due to cross-talk.

Syndromic and non‐syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta1 mRNA level. The increase in stored TGF-beta1 was associated with enhanced LTBP-1 protein and mRNA levels. These dysregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta1 and LTBP-1 staining and between their respective mRNA expressions. In parallel, decorin was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta.

Extracellular control of TGFβ signalling in vascular development and disease

The intracellular mechanism of transforming growth factor-beta (TGFbeta) signalling via kinase receptors and SMAD effectors is firmly established, but recent studies of human cardiovascular syndromes such as Marfan syndrome and pre-eclampsia have refocused attention on the importance of regulating the availability of active extracellular TGFbeta. It seems that elastic extracellular matrix (ECM) components have a crucial role in controlling TGFbeta signalling, while soluble and membrane bound forms of TGFbeta co-receptors add further layers of regulation. Together, these extracellular interactions determine the final bioavailability of TGFbeta to vascular cells, and dysregulation is associated with an increasing number of vascular pathologies.

Pioglitazone Induces Apoptosis in Human Vascular Smooth Muscle Cells from Diabetic Patients Involving the Transforming Growth Factor-β/Activin Receptor-Like Kinase-4/5/7/Smad2 Signaling Pathway

Alterations in vascular wall remodeling are a typical complication in type 2 diabetes mellitus due to an imbalance between cell proliferation and apoptosis. In this context, we have previously shown that vascular smooth muscle cells (VSMC) from diabetic patients were resistant to induced apoptosis. Thiazolidinediones, such as pioglitazone, seem to exert direct antiatherosclerotic effects on type 2 diabetes. Here, we aimed to study whether pioglitazone was able to induce apoptosis in VSMC from diabetic patients (DP) and, if so, whether the transforming growth factor (TGF)-beta1/Smad-2 pathway was involved. We isolated human internal mammary artery VSMC from patients who had undergone coronary-artery bypass graft. Pioglitazone (100 microM) induced apoptosis in human VSMC from diabetic and nondiabetic patients (NDP), analyzed by DNA fragmentation and by degradation of Bcl-2, in high-glucose-containing medium (15 and 25 mM). This apoptotic effect was inhibited by the activin receptor-like kinase-4/5/7/Smad2 inhibitor 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide (SB-431542), denoting that the TGF-beta1/Smad-2 pathway was involved. Pioglitazone rapidly increased the extracellular TGF-beta1 levels and concomitantly induced phosphorylation of Smad2 in VSMC from DP and NDP. Thus, we demonstrated that pioglitazone induced apoptosis in human VSMC from DP, which are strongly resistant to the induced apoptosis. This effect of pioglitazone might contribute in the treatment of alterations of vascular remodeling in type 2 diabetes mellitus.

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.

Overexpression of latent transforming growth factor-beta binding protein 1 (LTBP-1) in dioxin receptor-null mouse embryo fibroblasts.

The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobiotic metabolism. Increasingly clear is also the role of the AhR in the control of cell growth and proliferation. By analyzing differential patterns of gene expression between wild-type (AhR+/+) and null (AhR-/-) mouse embryo fibroblasts (MEF), we have identified latent transforming growth factor-beta binding protein 1 (LTBP-1) as a negatively AhR-regulated gene in the absence of xenobiotics. Ltbp-1 mRNA and protein expression were markedly increased in AhR-/- MEF. Furthermore, secreted LTBP-1 was elevated in the culture medium and the extracellular matrix of AhR-null MEF. Actinomycin D inhibited Ltbp-1 mRNA overexpression, suggesting regulation at the transcriptional level. AhR activation by dioxin (TCDD) downregulated Ltbp-1, again suggesting an AhR-regulated mechanism. Treatment of AhR+/+ MEF with transforming growth factor-beta(TGF-beta) downregulated AhR and, simultaneously, increased Ltbp-1, further supporting the role of this receptor in LTBP-1 expression. AhR-/- conditioned medium had higher levels of active and total TGF-beta activity, suggesting a role for LTBP-1 in maintaining extracellular TGF-beta concentrations. TGF-beta did not appear to directly regulate Ltbp-1 given that addition of TGFbeta neutralizing antibody or TGFbeta protein to AhR-/- MEF had no effect on Ltbp-1 expression. AhR-/- MEF had lower levels of matrix metalloproteinase 2 (MMP-2) activity, which could not be attributable to MMP-2 mRNA downregulation or MMP-inhibitors Timp-1 and Timp-2 overexpression. These data identify LTBP-1 as one of the few AhR-regulated genes not involved in xenobiotic metabolism and also support the implication of the AhR in controlling TGFbeta activity and cell proliferation.

Extracellular regulation of TGF-beta activity in wound repair: growth factor latency as a sensor mechanism for injury.

The transforming growth factor-beta (TGFbeta) family of growth factors are major regulators of wound repair, scar formation, and fibrosis. One of the prominent features of TGFbeta biology is the fact that this growth factor is secreted as a latent precursor, which may be directed to and stored at specific sites in the cellular microenvironment. Targeting and mobilization, and particularly extracellular activation of latent TGF-beta control the biological action of this growth factor. This review will focus on mechanisms of extracellular TGF-beta regulation relevant to and potentially operating in wound repair and scarring.

Transforming growth factor-beta1 expression in early biopsy specimen predicts long-term graft function following pediatric renal transplantation.

The main cause of late graft loss or declining long-term graft function is chronic allograft nephropathy (CAN), characterized by progressive interstitial fibrosis. Transforming growth factor (TGF)-beta1 plays a key role in fibrogenesis. We immunohistochemically investigated whether the degree of TGF-beta1 expression in early biopsy specimens routinely obtained from stable allografts at 100 d could predict fibrosis and graft dysfunction in the late phase. Patients were children with grafts from related donors. We immunohistochemically determined intracellular and extracellular expression of TGF-beta1 in the graft using LC antibody (LC) for intracellular TGF-beta1 and CC antibody (CC) for extracellular TGF-beta1. The change in creatinine clearance between 100 d and 3 yr after transplantation (DeltaCcr) was used as an index of long-term graft function. We also used image analysis to calculate the relative area involved by interstitial fibrosis in the trichrome-stained section of graft biopsy specimens at 100 d and 3 yr, designating the change as DeltaFI. DeltaCcr was -4.2+/-9.4 mL/min in subjects with minimal early immunoreactivity for CC and -20.5+/-15.9 mL/min in subjects with strong reactivity (p<0.05). DeltaCcr was -14.5+/-18.6 mL/min in subjects with minimal early immunoreactivity for LC and -11.7+/-12.8 mL/min in those with strong reactivity. DeltaFI in subjects with minimal CC reactivity (1.28+/-4.11%) tended to be lower than that in subjects with strong reactivity (8.45+/-15.47%). Neither fibrosis at 100 d nor DeltaFI differed between subjects with minimal and strong LC reactivity. Thus, strong extracellular TGF-beta1 expression in grafts at 100 d after transplantation is associated with a long-term decline in graft function and tends to be associated with increased graft fibrosis at 3 yr.

Altered expression of transforming growth factor betas during urethral and bulbourethral gland tumor progression in transgenic mice carrying the androgen-responsive C3(1) 5' flanking region fused to SV40 large T antigen.

We demonstrate that targeted expression of SV40 large T antigen (TAg) to the urethral (periurethral) and bulbourethral gland epithelium leads to adenocarcinoma formation in these tissues after 7 months of age, which are extremely rare sites for spontaneous tumor formation in humans. The development of proliferative lesions in the urethral gland predictably follows a temporal course of progression with approximately one third of male animals developing urethral tumors by 1 year of age. Tumor progression in these organs correlates to the level of TAg and p53 expression. Immunoprecipitation confirmed that SV40 TAg protein was bound to p53 and Rb p110 in vivo. Expression of transforming growth factor beta (TGFbetas) was evaluated during tumor progression of urethral gland carcinomas. Elevations of intracellular and extracellular TGFbeta1 and extracellular TGFbeta3 were found in preneoplastic and neoplastic lesions, suggesting that increased TGFbetas may augment tumor growth. c-Met expression showed a tendency for increased expression in the urethral gland carcinomas. We speculate that the directed expression of SV40 TAg by the hormone responsive C3(1) gene and subsequent tumor formation in these organs is influenced by androgens, since these tissues and carcinomas express androgen receptor (AR) and arise only in male transgenic mice. Several cell lines established from the urethral carcinomas were also shown to express AR, but are not androgen dependent in culture. To our knowledge, this is the first transgenic animal model for urethral and bulbourethral carcinomas. This transgenic mouse model and the cell lines derived from it may provide a unique opportunity for dissecting molecular mechanisms involved in the tumorigenesis of these organs which otherwise rarely develop cancer.

Renal TGF-β in HIV-associated kidney diseases

Human immunodeficiency virus (HIV)-1 infection may be complicated by progressive renal glomerular disease, including focal segmental glomerulosclerosis (FSGS) and proliferative glomerulonephritis. We examined renal tissue from 71 patients, including biopsies and autopsies from patients in the presence and absence of HIV-1 infection. We assessed the extent of TGF-beta, interstitial fibrosis, and interstitial CD45-positive cellular infiltrate using immunohistochemistry. Extracellular TGF-beta 1/beta 3 was largely confined to the renal interstitium, with the highest scores in HIV-seropositive renal disease and crescentic nephritis. Among all biopsies, the TGF-beta 1/beta 3 score correlated with the fibrosis score (r = 0.79, P < 0.0001) and with the CD45 score (r = 0.60, P < 0.0001). Biopsies from HIV-infected patients, taken together, showed marginally more TGF-beta 1/beta 3 compared to biopsies from HIV-uninfected patients (P = 0.05); similarly, HIV-associated FSGS showed marginally more TGF-beta 1/beta 3 compared to FSGS biopsies obtained from HIV-uninfected patients (P = 0.05). Intracellular TGF-beta 1 and TGF-beta 3 were both expressed by renal tubular epithelial cells and in extraglomerular crescents, whereas TGF-beta 3 was also present within interstitial mononuclear cells and eosinophils, and, exclusively in HIV-infected patients, within glomerular cells. In conclusion, TGF-beta expression was increased in several progressive glomerular diseases, and was particularly but not uniquely elevated in HIV-associated renal diseases.

TGF-beta-mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes.

Primary cultures of rat hepatocytes, rat (FAO) and human (HepG2) hepatoma cells were studied by immunocytochemistry for expression of transforming growth factor (TGF)-beta, for the release of TGF-beta into the medium, and generation of hepatocellular apoptosis by the respective cell-conditioned media. Using the alkaline-phosphatase anti-alkaline-phosphatase technique, intense TGF-beta immunostaining was shown in all cell types. The cytokine is released almost entirely in the latent form into the culture medium; only the FAO-cells had a substantial fraction of bioactive TGF-beta in the native (unacidified) culture fluid. Exposure of hepatocytes with the respective cell-conditioned media in the activated, but not in the native form (except for FAO-cell media), induced severe detrimental effects as evidenced by: (i) gross morphological alterations, (ii) functional impairment (reduction of WST-1 test, detachment of cells, lactate dehydrogenase increase in the medium), and (iii) generation of apoptosis. The latter phenomenon was confirmed by an increase of internucleosomal DNA fragments, positive TUNEL reaction, and intense binding of the fluorochrome Hoechst 33342 to fragmented nuclei. All these effects, which were mimicked by addition of recombinant human TGF-beta 1, were almost entirely antagonized by pre-incubation of the conditioned media with latency associated peptide. In contrast to hepatocytes, both types of hepatoma cells were completely resistant to the multiple actions of TGF-beta and activated conditioned media. It is concluded that hepatocytes might have the ability to induce autocrine, TGF-beta-mediated apoptosis, whereas hepatoma cells, because of their TGF-beta resistance, might generate TGF-beta-mediated peritumorous apoptosis of hepatocytes in a paracrine way, which could facilitate their expansion in situ. Both mechanisms, however, are critically dependent on extracellular TGF-beta activation.

Hepatic silicosis, cirrhosis, and liver tumors in mice and hamsters: Studies of transforming growth factor beta expression

Hepatic silicosis, cirrhosis, liver cell adenoma, and carcinomas developed in nude mice (NCr-Nu) given quartz by the subcutaneous and intraperitoneal routes. Syrian golden hamsters (15:16 EHS:cr) given quartz by both routes developed extensive fibrosis and cirrhosis and had higher morbidity and mortality rates after 3 months. Crystalline silica (quartz) induces fibrosis, adenomas, and carcinomas in the lungs of Fisher 344 rats, but certain strains of mice and hamsters are resistant to quartz-induced pulmonary carcinogenesis. Pulmonary fibrosis, however, is minimal in mice and absent in hamsters who received quartz intratracheally. To determine whether species differences are due to organ-specific rather than species-specific factors, susceptibility of the liver to quartz toxicity was investigated in nude mice and hamsters. The present study shows that the differential manifestations of quartz toxicity by these rodent species are dependent on factors that are organ-specific rather than host-specific. At 3 months, hepatocytes in mice were immunostained with intracellular transforming growth factor (TGF) beta 1 (LC 1-30) but not with TGF-beta 1 latency-associated peptide (LAP) protein (266-278); at 12 months, hepatocytes were immunostained with TGF-beta 1 LAP (266-278) but not with TGF-beta 1 (LC1-30). The hepatocytes of hamsters at 3 months showed immunoreactivities to TGF-beta 1 LAP (266-278) and TGF-beta 1 (LC1-30); immunostaining to TGF-beta 1 (LC1-30) was detected in nonparenchymal cells. Extracellular TGF-beta 1 (CC1-30) was detected in the silicotic granulomas and fibrous tissue in livers of both species. Quartz-induced liver carcinoma did not express TGF-beta 1 LAP (266-278) and LC (1-30) proteins, but these were detected in the cells of the adenoma in the same liver. Control animals showed no hepatic lesions nor immunoreactivity to TGF-beta 1. The spatial and temporal patterns of expression of TGF-beta 1, TGF-beta 2, TGF-beta receptor type II messenger RNAs (mRNAs), and TGF-beta 1 proteins in the different hepatic lesions suggests that TGF-beta isoforms may play a role in the pathogenesis of quartz-induced fibrosis, cirrhosis, liver cell adenoma, and carcinoma. (Hepatology 1996 May;23(5):1268-75)

Hepatic silicosis, cirrhosis, and liver tumors in mice and hamsters: studies of transforming growth factor beta expression.

Hepatic silicosis, cirrhosis, liver cell adenoma, and carcinomas developed in nude mice (NCr-Nu) given quartz by the subcutaneous and intraperitoneal routes. Syrian golden hamsters (15:16 EHS:cr) given quartz by both routes developed extensive fibrosis and cirrhosis and had higher morbidity and mortality rates after 3 months. Crystalline silica (quartz) induces fibrosis, adenomas, and carcinomas in the lungs of Fisher 344 rats, but certain strains of mice and hamsters are resistant to quartz-induced pulmonary carcinogenesis. Pulmonary fibrosis, however, is minimal in mice and absent in hamsters who received quartz intratracheally. To determine whether species differences are due to organ-specific rather than species-specific factors, susceptibility of the liver to quartz toxicity was investigated in nude mice and hamsters. The present study shows that the differential manifestations of quartz toxicity by these rodent species are dependent on factors that are organ-specific rather than host-specific. At 3 months, hepatocytes in mice were immunostained with intracellular transforming growth factor (TGF) beta 1 (LC 1-30) but not with TGF-beta 1 latency-associated peptide (LAP) protein (266-278); at 12 months, hepatocytes were immunostained with TGF-beta 1 LAP (266-278) but not with TGF-beta 1 (LC1-30). The hepatocytes of hamsters at 3 months showed immunoreactivities to TGF-beta 1 LAP (266-278) and TGF-beta 1 (LC1-30); immunostaining to TGF-beta 1 (LC1-30) was detected in nonparenchymal cells. Extracellular TGF-beta 1 (CC1-30) was detected in the silicotic granulomas and fibrous tissue in livers of both species. Quartz-induced liver carcinoma did not express TGF-beta 1 LAP (266-278) and LC (1-30) proteins, but these were detected in the cells of the adenoma in the same liver. Control animals showed no hepatic lesions nor immunoreactivity to TGF-beta 1. The spatial and temporal patterns of expression of TGF-beta 1, TGF-beta 2, TGF-beta receptor type II messenger RNAs (mRNAs), and TGF-beta 1 proteins in the different hepatic lesions suggests that TGF-beta isoforms may play a role in the pathogenesis of quartz-induced fibrosis, cirrhosis, liver cell adenoma, and carcinoma.

Loss of high affinity transforming growth factor-beta 1 (TGF-beta 1) binding to a nephritogenic T cell results in absence of growth inhibition by TGF-beta 1 and augmented nephritogenicity.

Transforming growth factor-beta (TGF-beta) affects cellular proliferation and differentiation and has been linked to the development of pathologic extracellular matrix production characteristic of progressive renal disease. TGF-beta 1 is conventionally regarded as having growth-inhibitory activity on T lymphocytes. The growth-regulatory activity of TGF-beta 1 depends on the interaction of TGF-beta 1 with its receptors, especially the type I and II receptors. In this study, we describe a CD4+ nephritogenic T cell clone that, unlike a sister clone with an identical TCR Ag receptor, is not growth inhibited by TGF-beta 1, nor is its nephritogenicity affected by exogenous TGF-beta 1. Although this TGF-beta-resistant T cell clone expresses both type I and II TGF-beta receptors on the cell surface, the affinity of this interaction is markedly diminished compared with that of the sister T cell clone, which does undergo growth arrest in response to extracellular TGF-beta 1. The resistant T cell clone expresses one mutant allele for the type I TGF-beta receptor. This mutation consists of a leucine to glutamine substitution at codon 122, a conserved amino acid in the transmembrane domain. We speculate that this mutation alters the interaction between the type I and II TGF-beta receptors following recognition of ligand by the type II receptor.

Expression of transforming growth factor-beta 1 in prostate cancer.

Transforming growth factor-beta 1 (TGF beta 1) is a potent modulator of cell proliferation, differentiation, angiogenesis, and the immune system. TGF beta 1 messenger RNA (mRNA) levels were much higher in several rat prostate adenocarcinomas (Dunning R3327 MATLyLu, AT2, G, HI, and H sublines) than in normal prostate. Normal prostate and the well differentiated H and HI tumors produced two TGF beta 1 mRNA transcripts, 2.4 kilobases (major) and 1.6 kilobases (minor). The poorly differentiated MATLyLu and AT2 sublines produced these plus additional TGF beta 1 mRNA transcripts that were present in the primary tumors, metastases, and cultured cell lines. TGF beta 1 mRNA levels were unchanged 2 weeks after castration. Immunohistochemical staining of TGF beta 1 protein was more prominent and more extensive in prostate cancer than in normal prostate. Only extracellular TGF beta 1 staining was detected. In normal prostate and in well differentiated tumors (H and HI), extracellular TGF beta 1 staining was located in the interacinar stroma, suggesting that it may be produced there. In contrast, in the poorly differentiated tumors (MATLyLu, AT2, and G) that contain sheets of epithelial cells, extracellular TGF beta 1 staining was present throughout the tumor, suggesting that TGF beta 1 may be made and secreted by the tumor epithelial cells. MATLyLu, AT2, and G tumor cells were cultured in vitro, and the conditioned medium was analyzed for the presence of TGF beta using a bioassay. TGF beta 1 is produced and secreted as an inactive latent precursor and must be activated to release bioactive TGF beta 1. Cells secreted about 100-500 pg TGF beta/10(6) cells.24 h and were able to activate about 50% of the total TGF beta secreted. Because TGF beta 1 mRNA and protein expression are higher in cancerous than normal tissue and because prostate cancer cells themselves can activate latent TGF beta 1 to a bioactive form, TGF beta 1 produced endogenously by prostate cancer has the potential to affect tumor behavior in vivo. Therefore, TGF beta 1 may represent a new therapeutic target in prostate cancer.

Molecular characterization and in situ localization of murine endoglin reveal that it is a transforming growth factor-beta binding protein of endothelial and stromal cells

Endoglin is an integral membrane glycoprotein predominantly expressed on human endothelial cells and recently shown to bind transforming growth factor-beta 1 (TGF beta 1) with high affinity. We now report the cloning and sequencing of a full-length murine endoglin complementary DNA of 2902 base pairs which hybridizes specifically with a single messenger RNA (mRNA) species. The polypeptide of 653 amino acids has an overall identity of 72% with human and porcine endoglin. The transmembrane and cytoplasmic domains of all three proteins differ by two to four amino acids and are 70% identical to the corresponding regions of the TGF beta binding protein, betaglycan. Relative levels of murine endoglin mRNA were estimated by polymerase chain reaction and found to be high in ovary and uterus, intermediate in heart and muscle, and low in placenta and spleen. In situ hybridization and immunofluorescence confirmed that murine endoglin, like its human counterpart, is present in blood vessels and capillaries in all tissues examined. In addition, the stromal cells in the connective tissue of intestine, stomach, heart, muscle, uterus, ovary, and testis were strongly and specifically reactive with complementary RNA probes and with a polyclonal antibody to endoglin; epithelial cell layers were distinctly unreactive. This distribution is similar to that of extracellular TGF beta 1, particularly in heart and uterus, and suggests that endoglin on stromal fibroblast-like cells might be regulating access of TGF beta 1 to the signaling receptor complex. NCTC-2071 fibroblasts in culture were shown to express high levels of endoglin mRNA by polymerase chain reaction. After chemical cross-linking with [125I]TGF beta 1 and immunoprecipitation with the polyclonal antihuman endoglin serum, a radiolabeled band of mol wt 180,000 corresponding to dimeric endoglin was observed under nonreducing conditions, whereas a single band of mol wt 90,000 was seen under reducing conditions. Thus murine fibroblast endoglin is capable of binding TGF beta 1. Future studies should establish the specialized role of endoglin in the TGF beta receptor complex of endothelial and stromal cells.