Rapamycin Inhibits the Growth and Collagen Production of Fibroblasts Derived from Human Urethral Scar Tissue

Rapamycin can inhibit fibroblast proliferation, collagen accumulation, and urethral stricture in rabbits. Transforming growth factor-beta-1 (TGF-β1) signaling, with downstream recruitment of Smad2, is known to promote fibrosis. This in vitro study examined the effects of rapamycin on fibroblasts derived from human urethral scar tissue (FHUS) and investigated the possible mechanism with respect to regulation of TGF-β1 signaling. FHUS were cultured from urethral scar tissues collected from four patients with urethral stricture. The cells were exposed to different concentrations of rapamycin (0, 10, 20, 40, 80, or 160 ng/ml) for 24 or 48 hours. Cell growth was assessed by the MTT assay. Collagen content was measured based on hydroxyproline levels. The mRNA expressions of Smad2, eIF-4E, and alpha-1 chains of collagen types I and III (Col1α1 and Col3α1) were determined by semiquantitative reverse-transcription PCR. The protein expressions of Smad2, phospho-Smad2, and eIF-4E were evaluated by western blot. Rapamycin caused a concentration-dependent inhibition of FHUS growth at 24 and 48 hours (P<0.01). Rapamycin decreased total collagen content (P<0.01), collagen content per 105 cells (P<0.05), and mRNA expressions of Col1α1 and Col3α1 (P<0.05) in a concentration-dependent manner. Rapamycin elicited concentration-dependent reductions in the mRNA (P<0.05) and protein (P<0.01) expressions of Smad2 and eIF-4E. The two highest concentrations of rapamycin also enhanced phospho-Smad2 levels (P<0.01). In conclusion, the present study confirmed that rapamycin may reduce the growth and collagen production of FHUS, possibly through inhibition of TGF-β1 signaling.

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Expression and secretion of transforming growth factor-beta by bleomycin-stimulated rat alveolar macrophages

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.1.l36 ◽

1993 ◽

Vol 264 (1) ◽

pp. L36-L42 ◽

Cited By ~ 2

Author(s):

E. M. Denholm ◽

S. M. Rollins

Keyword(s):

Growth Factor ◽

Alveolar Macrophages ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Chemotactic Activity ◽

Beta Activity ◽

Dependent Manner ◽

Tgf Beta ◽

Growth Factor Beta

Bleomycin-induced fibrosis in rodents has been used extensively as a model of human pulmonary fibrosis. The influx of monocytes observed during the early stages of fibrosis is at least partially regulated by the elaboration of chemotactic factors in the lung. Exposure of alveolar macrophages (AM phi) to bleomycin either in vivo or in vitro stimulated secretion of monocyte chemotactic activity (MCA). This MCA has been previously characterized as being primarily due to fibronectin fragments. The present experiments revealed that bleomycin also induced AM phi to secrete a second chemotactic factor, transforming growth factor-beta (TGF-beta). However, the TGF-beta secreted by macrophages was in latent form, since no TGF-beta activity was detected unless AM phi conditioned medium (CM) was acid-activated. After acidification, chemotactic activity in CM from AM phi stimulated with bleomycin in vitro was increased by 3.6, whereas activity in AM phi CM from fibrotic rats increased by 2 and that of a bleomycin-stimulated AM phi cell line increased by 1.6. This acid-activatable chemotactic activity was inhibited by antibody to TGF-beta. Bleomycin-stimulated AM phi s secreted significantly more TGF-beta than did unstimulated controls. Further, in vitro exposure of AM phi to bleomycin induced TGF-beta mRNA expression in a time- and concentration-dependent manner, with maximal mRNA being detected following a 16-h incubation with 1 microgram/ml bleomycin.

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TGF-β Promotes the Proliferation of Microglia In Vitro

Brain Sciences ◽

10.3390/brainsci10010020 ◽

2019 ◽

Vol 10 (1) ◽

pp. 20 ◽

Cited By ~ 1

Author(s):

Costansia Bureta ◽

Takao Setoguchi ◽

Yoshinobu Saitoh ◽

Hiroyuki Tominaga ◽

Shingo Maeda ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Transforming Growth Factor Beta ◽

Microglial Cell ◽

Transforming Growth Factor ◽

Dependent Manner ◽

In Vitro Proliferation ◽

Inhibition Of Proliferation ◽

Significant Difference

The activation and proliferation of microglia is characteristic of the early stages of brain pathologies. In this study, we aimed to identify a factor that promotes microglial activation and proliferation and examined the in vitro effects on these processes. We cultured microglial cell lines, EOC 2 and SIM-A9, with various growth factors and evaluated cell proliferation, death, and viability. The results showed that only transforming growth factor beta (TGF-β) caused an increase in the in vitro proliferation of both microglial cell lines. It has been reported that colony-stimulating factor 1 promotes the proliferation of microglia, while TGF-β promotes both proliferation and inhibition of cell death of microglia. However, upon comparing the most effective doses of both (assessed from the proliferation assay), we identified no statistically significant difference between the two factors in terms of cell death; thus, both have a proliferative effect on microglial cells. In addition, a TGF-β receptor 1 inhibitor, galunisertib, caused marked inhibition of proliferation in a dose-dependent manner, indicating that inhibition of TGF-β signalling reduces the proliferation of microglia. Therefore, galunisertib may represent a promising therapeutic agent for the treatment of neurodegenerative diseases via inhibition of nerve injury-induced microglial proliferation, which may result in reduced inflammatory and neuropathic and cancer pain.

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Transforming growth factor beta directly regulates primitive murine hematopoietic cell proliferation

Blood ◽

10.1182/blood.v75.3.596.596 ◽

1990 ◽

Vol 75 (3) ◽

pp. 596-602 ◽

Cited By ~ 112

Author(s):

JR Keller ◽

IK Mcniece ◽

KT Sill ◽

LR Ellingsworth ◽

PJ Quesenberry ◽

...

Keyword(s):

Bone Marrow ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Bone Marrow Cells ◽

Dependent Manner ◽

Tgf Beta ◽

Hematopoietic Progenitor ◽

Growth Factor Beta ◽

Dose Dependent

Abstract We previously reported that transforming growth factor beta (TGF-beta) selectively inhibits colony-stimulating factor-driven hematopoietic progenitor cell growth. We report here that TGF-beta 1 can act directly on hematopoietic progenitors to inhibit the growth of the most primitive progenitors measurable in vitro. Highly enriched populations of hematopoietic progenitor cells were obtained by isolating lineage negative (Lin-), Thy-1-positive (Thy-1+) fresh bone marrow cells, or by isolating cells from interleukin-3 (IL-3) supplemented bone marrow cultures expressing Thy-1 antigen with the fluorescent activated cell sorter. TGF-beta 1 inhibited IL-3-induced Thy-1 expression on Thy-1- negative (Thy-1-) bone marrow cells in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. In addition, TGF-beta 1 inhibited the formation of multipotent and mixed colonies by isolated Thy-1+ cells, while single lineage granulocyte and macrophage colonies were not affected. The growth of Thy-1+ Lin- cells incubated as single cells in Terasaki plates in medium supplemented with IL-3 were inhibited by TGF-beta, demonstrating a direct inhibitory effect. Hematopoietic stem cells, which have a high proliferative potential (HPP) when responding to combinations of growth factors in vitro, have been detected in the bone marrow of normal mice and mice surviving a single injection of 5- fluorouracil. TGF-beta 1 inhibited the growth of all subpopulations of HPP colony forming cells (CFC) in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. Thus, TGF-beta directly inhibits the growth of the most immature hematopoietic cells measurable in vitro.

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Suppression of the TGF-β pathway by a macrolide antibiotic decreases fibrotic responses by ocular fibroblasts in vitro

Royal Society Open Science ◽

10.1098/rsos.200441 ◽

2020 ◽

Vol 7 (9) ◽

pp. 200441

Author(s):

Thomas Stahnke ◽

Beata Gajda-Deryło ◽

Anselm G. Jünemann ◽

Oliver Stachs ◽

Katharina A. Sterenczak ◽

...

Keyword(s):

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Macrolide Antibiotic ◽

Drug Repositioning ◽

Dependent Manner ◽

Glaucoma Filtration Surgery ◽

Concentration Dependent Manner ◽

Device Implantation

To elucidate and to inhibit post-surgical fibrotic processes after trabeculectomy in glaucoma therapy, we measured gene expression in a fibrotic cell culture model, based on transforming growth factor TGF-β induction in primary human tenon fibroblasts (hTFs), and used Connectivity Map (CMap) data for drug repositioning. We found that specific molecular mechanisms behind fibrosis are the upregulation of actins, the downregulation of CD34, and the upregulation of inflammatory cytokines such as IL6, IL11 and BMP6 . The macrolide antibiotic Josamycin (JM) reverses these molecular mechanisms according to data from the CMap, and we thus tested JM as an inhibitor of fibrosis. JM was first tested for its toxic effects on hTFs, where it showed no influence on cell viability, but inhibited hTF proliferation in a concentration-dependent manner. We then demonstrated that JM suppresses the synthesis of extracellular matrix (ECM) components. In hTFs stimulated with TGF-β1, JM specifically inhibited α-smooth muslce actin expression, suggesting that it inhibits the transformation of fibroblasts into fibrotic myofibroblasts. In addition, a decrease of components of the ECM such as fibronectin, which is involved in in vivo scarring, was observed. We conclude that JM may be a promising candidate for the treatment of fibrosis after glaucoma filtration surgery or drainage device implantation in vivo .

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Human interleukin-4 inhibits proliferation of megakaryocyte progenitor cells in culture

Blood ◽

10.1182/blood.v81.3.624.624 ◽

1993 ◽

Vol 81 (3) ◽

pp. 624-630 ◽

Cited By ~ 23

Author(s):

Y Sonoda ◽

Y Kuzuyama ◽

S Tanaka ◽

S Yokota ◽

T Maekawa ◽

...

Keyword(s):

Transforming Growth Factor Beta ◽

Colony Formation ◽

Neutralizing Antibodies ◽

Transforming Growth Factor ◽

Early Stage ◽

Interleukin 4 ◽

Dependent Manner ◽

Inhibitory Effects ◽

Necrosis Factor Alpha

Abstract We studied the effects of recombinant human interleukin-4 (rhIL-4) on megakaryocyte colony formation from enriched hematopoietic progenitors. IL-4 strongly inhibited pure and mixed megakaryocyte colony formation in a dose-dependent manner. Formation of erythroid bursts, eosinophil colonies, and erythrocyte-containing mixed colonies was not affected by the addition of IL-4 as reported previously (Sonoda Y, et al; Blood 75:1615, 1990). Delayed addition experiments suggested that IL-4 acts on an early stage of proliferation of megakaryocyte progenitors. Neutralizing antibodies (antisera) prepared against transforming growth factor beta, tumor necrosis factor alpha, interferon alpha (IFN alpha), and IFN gamma did not affect the inhibitory effects of IL-4 on pure and mixed megakaryocyte colony formation. In addition, the inhibitory effects of IL-4 was also seen in serum-free cultures and in cultures containing highly enriched CD34+, HLA-DR+ cells as a target population. These results indicate that IL-4 may function as one of the negative regulators in human megakaryocytopoiesis in vitro.

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BMP-4 inhibits follicle-stimulating hormone secretion in ewe pituitary

Journal of Endocrinology ◽

10.1677/joe.1.05988 ◽

2005 ◽

Vol 186 (1) ◽

pp. 109-121 ◽

Cited By ~ 69

Author(s):

M-O Faure ◽

L Nicol ◽

S Fabre ◽

J Fontaine ◽

N Mohoric ◽

...

Keyword(s):

Growth Factor ◽

Mrna Expression ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Hormone Secretion ◽

Inhibitory Effect ◽

Type I ◽

Mrna Levels ◽

Dependent Manner

Activins and inhibins, members of the transforming growth factor-beta family are able to stimulate and inhibit, respectively, FSH synthesis and release. Other members of this superfamily, the bone morphogenetic proteins (BMPs), may also affect FSH synthesis in the mouse. The aim of this work was to determine whether BMPs are expressed in the ovine pituitary and whether they play a role in the regulation of FSH release. The mRNAs encoding BMP-2, BMP-4, BMP-7 and the oocyte-derived growth factor, growth differentiation factor (GDF)-9 were detected in the pituitaries of cyclic ewes by reverse-transcriptase PCR, as well as the mRNAs encoding the BMP type I receptors, BMPR-IA (activin-receptor-like kinase (ALK)-3) and BMPR-IB (ALK-6), and type II receptors (BMPR-II). Immunolabeling of pituitary sections revealed the presence of BMPR-IA (ALK-3) and BMPR-II in gonadotrope cells. To investigate the potential effects of BMPs on FSH secretion, ewe pituitary cell cultures were treated with BMP-4 (10−11 M to 10−9 M) for 48 h. Interestingly, FSH release was decreased in a dose-dependent manner. At 10−9 M BMP-4 both FSH concentration and FSHβ mRNA expression were reduced by 40% of control values. In contrast, there was no inhibitory effect on either LH or LHβ mRNA expression. A similar result was found with BMP-6. BMP-4 triggered the phosphorylation of Smad1, suggesting that the effect of BMP-4 on FSH secretion is due to the activation of the BMPs signaling pathway. Furthermore, BMP-4 blocked the stimulatory effect of activin on both FSH release and FSHβ mRNA and amplified the suppression of FSH release and FSHβ mRNA levels induced by 17β-estradiol. These results indicate that a functional BMP system operates within the sheep pituitary, at least in vitro, to decrease FSH release and to modulate the effect of activin.

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Myostatin regulates glucose uptake in BeWo cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00331.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. E1296-E1302 ◽

Cited By ~ 26

Author(s):

Nisha Antony ◽

John J. Bass ◽

Christopher D. McMahon ◽

Murray D. Mitchell

Keyword(s):

Glucose Uptake ◽

Cell Lines ◽

Transforming Growth Factor ◽

Muscle Growth ◽

Inhibitory Effect ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bewo Cells ◽

Placental Cells

Myostatin is a member of the transforming growth factor (TGF)-β superfamily, known for its ability to inhibit muscle growth. It can also regulate metabolism and glucose uptake in a number of tissues. To determine the mechanism of myostatin's effect on glucose uptake, we evaluated its actions using choriocarcinoma cell lines that are widely used as models for placental cells. Protein and mRNA were determined using immunoblotting and RT-PCR/PCR, respectively. Glucose uptake was assessed by uptake of radiolabeled deoxyglucose in vitro. All choriocarcinoma cell lines tested i.e., BeWo, JEG, and Jar, are used as models of placental cells, and all expressed myostatin protein and mRNA. Treatment of BeWo cells with myostatin resulted in inhibition of glucose uptake in a concentration-dependent manner ( P < 0.01). At all concentrations tested, follistatin, a functional inhibitor of myostatin, completely blocked the inhibitory effect of myostatin (40 nM) on glucose uptake by BeWo cells (0.4 nM, P < 0.05). Follistatin treatment alone also increased glucose uptake (0.4 and 4 nM, P < 0.001; 40 nM, P < 0.05). Because BeWo cells proliferated and greater cell densities were achieved, glucose uptake declined irrespective of treatment. Myostatin treatment of BeWo cells did not alter the levels of myostatin receptor, ActRII A/B proteins. The levels of glucose transport proteins also remained unaltered in BeWo cells with myostatin treatment. This study has shown that myostatin specifically inhibits glucose uptake into BeWo cells, suggesting that locally produced myostatin may control glucose metabolism within the placenta.

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miR-4732-3p in Extracellular Vesicles From Mesenchymal Stromal Cells Is Cardioprotective During Myocardial Ischemia

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.734143 ◽

2021 ◽

Vol 9 ◽

Author(s):

Rafael Sánchez-Sánchez ◽

Marta Gómez-Ferrer ◽

Ignacio Reinal ◽

Marc Buigues ◽

Estela Villanueva-Bádenas ◽

...

Keyword(s):

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Transforming Growth Factor Beta ◽

Stromal Cells ◽

Transforming Growth Factor ◽

Lentiviral Vector ◽

Glucose Deprivation ◽

Scar Tissue

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are an emerging alternative to cell-based therapies to treat many diseases. However, the complexity of producing homogeneous populations of EVs in sufficient amount hampers their clinical use. To address these limitations, we immortalized dental pulp-derived MSC using a human telomerase lentiviral vector and investigated the cardioprotective potential of a hypoxia-regulated EV-derived cargo microRNA, miR-4732-3p. We tested the compared the capacity of a synthetic miR-4732-3p mimic with EVs to confer protection to cardiomyocytes, fibroblasts and endothelial cells against oxygen-glucose deprivation (OGD). Results showed that OGD-induced cardiomyocytes treated with either EVs or miR-4732-3p showed prolonged spontaneous beating, lowered ROS levels, and less apoptosis. Transfection of the miR-4732-3p mimic was more effective than EVs in stimulating angiogenesis in vitro and in vivo and in reducing fibroblast differentiation upon transforming growth factor beta treatment. Finally, the miR-4732-3p mimic reduced scar tissue and preserved cardiac function when transplanted intramyocardially in infarcted nude rats. Overall, these results indicate that miR-4732-3p is regulated by hypoxia and exerts cardioprotective actions against ischemic insult, with potential application in cell-free-based therapeutic strategies.

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Liver enzymes elevation due to concurrent dexamethasone and lapatinib: Clinical and in vitro evidence.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e13023 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e13023-e13023

Author(s):

Yi Ling Teo ◽

Manit Saetaew ◽

Suthan Chanthawong ◽

Yoon Sim Yap ◽

Eric Chun Yong Chan ◽

...

Keyword(s):

Liver Enzyme ◽

Transforming Growth Factor ◽

Liver Enzymes ◽

Clinical Effects ◽

Dependent Manner ◽

Liver Enzyme Elevation ◽

Concentration Dependent Manner ◽

Transforming Growth Factor Α ◽

Enzyme Elevation

e13023 Background: Concomitant usage of lapatinib (L) and dexamethasone (D), which are cytochrome P450 (CYP) 3A4 substrate and inducer respectively, may increase the formation of potentially hepatotoxic reactive L metabolites. This study aims to evaluate the effect of D on the occurrence of liver enzyme elevation and to ascertain its role using a parallel in vitro experiment. Methods: Clinical effects of D on L-induced liver enzyme elevation were evaluated in a nested case-control study. Liver enzyme elevation is defined as the first clinically significant change (from baseline) of either total bilirubin (TB), alanine aminotransferase (ALT) or aspartate aminotransferase (AST) by at least 1 grade, based on the Common Terminology Criteria for Adverse Events (CTCAE) version 4.02. In the in vitro study, metabolically-competent transforming growth factor α mouse hepatocytes (TAMH) cells were treated with L and viabilities were compared in the presence or absence of D. Results: Among 97 patients, liver enzyme elevation episodes (87.5% vs. 60.3%, p=0.01) and ALT elevation (41.7% vs. 19.2%, p=0.03) occurred more frequently among patients receiving concomitant L+D comparing to L only. After adjusting for confounders (age, liver and brain metastasis, baseline liver function test (TB, AST, ALT, alkaline phosphatase), underlying liver disease, concurrent use of hepatotoxic medications), patients receiving concomitant L+D were 3.48 times (95% CI 1.24-9.80, p=0.02) and 4.57 times (95% CI 1.23-16.88, p=0.02) more likely to develop a change in ALT and among all liver enzymes respectively. Parallel to our clinical findings, treatment of TAMH cells with L reduced viability in a concentration-dependent manner. The introduction of D further reduced the viability. At 5 µM of L, the introduction of 10 µM and 20 µM of D resulted in a 19% and 59% decrease in viability respectively. Conclusions: The concomitant usage of L+D was associated with an increased occurrence of liver enzyme elevation. The in vitro findings have provided commensurating evidence on the role of D that increases the formation of L-derived hepatotoxic metabolites.

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Inhibition of the Combinatorial Signaling of Transforming Growth Factor-Beta and NOTCH Promotes Myotube Formation of Human Pluripotent Stem Cell-Derived Skeletal Muscle Progenitor Cells

Cells ◽

10.3390/cells10071649 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1649

Author(s):

In Young Choi ◽

Ho Tae Lim ◽

Young Hyun Che ◽

Gabsang Lee ◽

Yong Jun Kim

Keyword(s):

Notch Signaling ◽

Small Molecules ◽

Signaling Pathways ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Dependent Manner ◽

Healthy Human ◽

Myotube Formation ◽

Multinucleated Myotubes

Understanding the signaling pathways that regulate the final differentiation of human myoblasts is essential for successful cell transplantation and drug screening for the treatment of muscular dystrophy. In an effort to improve myotube formation from hiPSC-derived myoblasts, we validated a collection of 13 small molecules in a newly established in vitro screening platform for the assessment of myotube formation. The analysis of myotube formation as measured by the fusion index showed that the combinational inhibition of the TGFβ signaling with NOTCH signaling enhances the ability of multi-nucleated myotube production. Combinational treatment of inhibitors for TGFβ and NOTCH signaling pathways improved myotube formation in a dose-dependent manner. This effect was achieved by inhibiting the combinatorial mechanism of signaling. The combination treatment of small molecules effective in inducing multinucleated myotubes was validated in healthy human primary myoblasts. In addition, it was also applied to DMD patient iPSC-derived myoblasts to enhance the generation of multinucleated myotubes.

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