An Assay for Transforming Growth Factor-β Using Cells Transfected with a Plasminogen Activator Inhibitor-1 Promoter-Luciferase Construct

Abé, Mayumi; Harpel, John G.; Metz, Christine N.; Nunes, Irene; Loskutoff, David J.; Db, Rifkin

doi:10.1006/abio.1994.1042

Cited by 715 publications

(793 citation statements)

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“…Human recombinant TGF‐β1 and the neutralizing monoclonal mouse anti‐TGF‐β antibody (anti‐TGF‐β) recognizing all three isoforms, was obtained from R&D Systems. Activity of this antibody was determined using the mink lung epithelial cell system (MLEC) (Abe et al,1994; Krieglstein et al, 2000). 4‐aminopyridine (4AP), Bicuculline methiodide, SP600125, and SB431542 were purchased from Tocris Bioscience (Bristol, UK).…”

Section: Methodsmentioning

confidence: 99%

“…To detect the latent part of TGF‐ βs, the conditioned medium has to be acidified first to convert latent TGF‐ βs into activated ones. To evaluate the levels of activated TGF‐ βs in extracellular environment after 4AP treatment, the MLEC assay was performed as described by Abe et al (1994). MLECs were placed into 96‐well plates at the density of 2 × 10 4 cells per well and treated with collected conditioned medium either with or without acidification with 1 M HCL and pH adjustment with 1 M NaOH (to activate latent TGF‐ βs) for 18 hr.…”

Section: Methodsmentioning

confidence: 99%

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TGF‐β signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes

Khakipoor

Ophoven

Schrödl-Häußel

et al. 2017

Glia

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The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) expressed in astrocytes regulates intracellular and extracellular pH. Here, we introduce transforming growth factor beta (TGF‐β) as a novel regulator of NBCe1 transcription and functional expression. Using hippocampal slices and primary hippocampal and cortical astrocyte cultures, we investigated regulation of NBCe1 and elucidated the underlying signaling pathways by RT‐PCR, immunoblotting, immunofluorescence, intracellular H(+) recording using the H(+) ‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, mink lung epithelial cell (MLEC) assay, and chromatin immunoprecipitation. Activation of TGF‐β signaling significantly upregulated transcript, protein, and surface expression of NBCe1. These effects were TGF‐β receptor‐mediated and suppressed following inhibition of JNK and Smad signaling. Moreover, 4‐aminopyridine (4AP)‐dependent NBCe1 regulation requires TGF‐β. TGF‐β increased the rate and amplitude of intracellular H+ changes upon challenging NBCe1 in wild‐type astrocytes but not in cortical astrocytes from Slc4a4‐deficient mice. A Smad4 binding sequence was identified in the NBCe1 promoter and Smad4 binding increased after activation of TGF‐β signaling. The data show for the first time that NBCe1 is a direct target of TGF‐β/Smad4 signaling. Through activation of the canonical pathway TGF‐β acts directly on NBCe1 by binding of Smad4 to the NBCe1 promoter and regulating its transcription, followed by increased protein expression and transport activity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

TGF‐β signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes

Khakipoor

Ophoven

Schrödl-Häußel

et al. 2017

Glia

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“…Rifkin, New York Medical Center, New York, NY). 42 Mink lung epithelial cells were plated in 24-well plates at a density of 2 ϫ 10 5 cells per well with incubation medium (4.5 g/mL glucose, 43 Dulbecco's modified Eagle's medium ϩ 10% fetal bovine serum, 2 mmol/L L-glutamine, and 200 g/mL G418 sulfate). Cells were allowed to attach for 4 hours at 37°C and then were washed with serum-free medium.…”

Section: Plasminogen Activator Inhibitor-1 Luciferase Assay For Detecmentioning

confidence: 99%

Blockade of TSP1-Dependent TGF-β Activity Reduces Renal Injury and Proteinuria in a Murine Model of Diabetic Nephropathy

Miao

Schoeb

et al. 2011

The American Journal of Pathology

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Transforming growth factor-␤ (TGF-␤) is key in the pathogenesis of diabetic nephropathy. Thrombospondin 1 (TSP1) expression is increased in diabetes, and TSP1 regulates latent TGF-␤ activation in vitro and in diabetic animal models. Herein, we investigate the effect of blockade of TSP1-dependent TGF-␤ activation on progression of renal disease in a mouse model of type 1 diabetes (C57BL/6J-Ins2 Akita ) as a targeted treatment for diabetic nephropathy. Akita and control C57BL/6 mice who underwent uninephrectomy received 15 weeks of thrice-weekly i.p. treatment with 3 or 30 mg/kg LSKL peptide, control SLLK peptide, or saline. The effects of systemic LSKL peptide on dermal wound healing was assessed in type 2 diabetic mice (db/db). Proteinuria (urinary albumin level and albumin/creatinine ratio) was significantly improved in Akita mice treated with 30 mg/kg LSKL peptide. LSKL treatment reduced urinary TGF-␤ activity and renal phospho-Smad2/3 levels and improved markers of tubulointerstitial injury (fibronectin) and podocytes (nephrin). However, LSKL did not alter glomerulosclerosis or glomerular structure. LSKL did not increase tumor incidence or inflammation or impair diabetic wound healing. These data suggest that selective targeting of excessive TGF-␤ activity through blockade of TSP1-dependent TGF-␤ activation represents a therapeutic strategy for treating diabetic nephropathy that preserves the homeostatic functions of TGF-␤.

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“…Cells were cultured with experimental supernatants and were compared with a standard curve generated using recombinant human TGF-b1 (R&D). Cells were lysed and luciferase activity was measured using a Luciferase Reporter Assay (Biotium) [36].…”

Section: Tgf-b Bioassaymentioning

confidence: 99%

Importance of TLR2 in the direct response of T lymphocytes to Schistosoma mansoni antigens

et al. 2010

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The immunomodulatory effect of Schistosoma mansoni antigens has often been attributed to interaction with PRR expressed on APC. Our previous work has shown that S. mansonisoluble egg antigen (SEA) can induce, together with a Th2 response, TGF-b-dependent Foxp3 expression in naïve CD4 1 T cells from NOD mice. We found that SEA can directly upregulate the expression of surface-bound TGF-b in purified CD4 1 T cells in the absence of accessory cell interactions. In this study, we show that the C-type lectin receptors DEC-205 and galectin-3 were involved in the direct interaction between S. mansoni antigens and CD4 1 T cells. SEA was able to enhance CD4 1 T-cell secretion of bioactive TGF-b in response to TLR2 ligand stimulation, in the absence of APC. We also show that TLR2 expressed on CD4 1 T cells was important for the Foxp3 expression induced by SEA.

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An Assay for Transforming Growth Factor-β Using Cells Transfected with a Plasminogen Activator Inhibitor-1 Promoter-Luciferase Construct

Cited by 715 publications

References 0 publications

TGF‐β signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes

TGF‐β signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes

Blockade of TSP1-Dependent TGF-β Activity Reduces Renal Injury and Proteinuria in a Murine Model of Diabetic Nephropathy

Importance of TLR2 in the direct response of T lymphocytes to Schistosoma mansoni antigens

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