整合素αvβ8介导的细胞固有TGF-β活化测定

Robert Ian Seed; Stephen Lloyd Nishimura

doi:10.21769/BioProtoc.4385

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Measurement of Cell Intrinsic TGF-β Activation Mediated by the Integrin αvβ8

整合素αvβ8介导的细胞固有TGF-β活化测定

RS Robert Ian Seed email

SN Stephen Lloyd Nishimura

发布: 2022年04月20日第12卷第8期 DOI: 10.21769/BioProtoc.4385 浏览次数: 2660

评审: Julie WeidnerJohn P PhelanAnonymous reviewer(s)

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Abstract

Transforming growth factor beta (TGF-β) is a multi-functional cytokine that plays a significant role in multiple diseases, including fibrosis and tumor progression. Whilst the biologic effects of TGF-β are well characterized, it is unclear how TGF-β signaling is regulated to impart specific responses within certain cell types. One mechanism of regulation may be through TGF-β activation, since TGF-β is always expressed in a latent form (L-TGF-β). Campbell et al. recently presented a new structural model to demonstrate how the integrin α_vβ₈ might specifically control TGF-β activation and signaling. In this model, α_vβ₈ binds to cell surface L-TGF-β1 to induce a conformational change, which exposes mature TGF-β peptide to TGF-β receptors (TGF-βRs), allowing initiation of TGF-β signaling from within the latent complex. This model also predicts that TGF-β signaling would be directed specifically towards the TGF-β expressing cell surface. We sought to test the validity of the new structural model by creating a cell-based assay which utilizes luciferase TGF-β reporter cells (TMLC). TMLC cells express high levels of TGF-βRs, but do not express cell surface L-TGF-β. We modified TMLC reporter cells to express cell surface L-TGF-β1 in a mutant form, which prevents the release of mature TGF-β from the latent complex. The newly generated cell lines were then used in a novel functional assay to investigate whether integrin α_vβ₈ could potentiate cell intrinsic TGF-β signaling from within the latent complex in vitro.

Keywords: TGF-β (TGF-β)

Integrin (整联蛋白)

Reporter assay (报告基因分析)

Background

TGF-β is a potent cytokine with a plethora of biologic functions (Kubiczkova et al., 2012; Chen and ten Dijke, 2016; Fenton et al., 2017; French et al., 2020). For instance, TGF-β suppresses immune responses by facilitating the differentiation and immunosuppressive function of regulatory T-cells (Treg) (Stockis et al., 2017; Seed et al., 2021). TGF-β also plays a pivotal role in vascular physiology, amongst many other reported roles (Parichatikanond et al., 2020). Most cell types are known to express TGF-β and its receptors, so the mechanisms whereby TGF-β selectively and specifically functions within distinct cell types are not well understood. However, TGF-β is always expressed in a latent form (L-TGF-β), demonstrating that activation of TGF-β from L-TGF-β is an important event for the regulation of TGF-β function (Khalil, 1999). Therefore, understanding the mechanisms whereby TGF-β is activated may provide insights into how TGF-β signaling is controlled, even though it appears ubiquitous.

Many molecules have been associated with TGF-β activation, the most specific of which are reported to be the integrins α_vβ₆ and α_vβ₈ (Munger et al., 1998; Mu et al., 2002). A new structural model of α_vβ₈ mediated TGF-β activation proposes that α_vβ₈ expressed on the surface of one cell activates TGF-β presented on the surface of an opposing cell, via the adaptor molecule glycoprotein A repetitions dominant (GARP) (Campbell et al., 2020). In this case, TGF-β signaling is directed towards the TGF-β presenting cell. This model further predicts that conformational changes imparted on L-TGF-β1 by α_vβ₈ may expose mature TGF-β, so that TGF-βRs expressed on the same cell surface can bind to mature TGF-β when it is still present within the latent complex, thus allowing TGF-β signaling. Testing the validity of this model using cell-based assays is of biologic significance as TGF-β activation is commonly thought to require physical release of mature TGF-β from the latent complex, where it would be free to diffuse to other cell surfaces. Therefore, the new structural model could in part explain how TGF-β signaling is tightly controlled to function intrinsically within specific cell types (Campbell et al., 2020).

To construct a cell-based model to test the structural hypothesis, we employed the widely used TGF-β reporter cell line (TMLC) (Figure 1A). TMLC cells are a subclone of mink lung epithelial cells transfected to induce stable expression of a luciferase reporter gene fused to a truncated plasminogen activator inhibitor-1 (PAI-1) promoter sequence (Abe et al., 1994; Campbell et al., 2020). In addition to the reported abundance of TGF-βRs and high sensitivity and specificity for assessing TGF-β signaling, these cells do not present L-TGF-β at the cell surface (Abe et al., 1994; Campbell et al., 2020). This allows for the generation of a cell intrinsic reporter system using ectopic expression GARP and L-TGF-β1. GARP allows for cell surface presentation of L-TGF-β1 in TMLC cells (Figure 1A). Furthermore, we mutated the furin cleavage motif in L-TGF-β1 [to L-TGF-β(R249A)], which ensures that mature TGF-β remains covalently linked to latent peptides therefore preventing its release. We then subsequently assessed the ability of α_vβ₈ to activate cell intrinsic TGF-β from both wild-type (WT) and non-releasable forms of L-TGF-β1 (Figure 1A–D). These new models, accompanied by assessment of expression patterns in human tumors, provide functional support for the model proposed by Campbell et al. (Campbell et al., 2020; Seed et al., 2021), and a suitable in vitro cell based assay to measure cell intrinsic TGF-β signaling. It may be possible to adapt this protocol to test the ability of other known activators of TGF-β to initiate cell intrinsic TGF-β signaling.

Materials and Reagents

96 well culture plate flat, white (Costar, catalog number: 3917)
96 well immulon 4 HBX plates, flat (Fisher Scientific, catalog number: 3855)
Note: We have obtained comparable datasets if immulon 4HBX plates are substituted for standard 96 well cell culture plates (Corning, catalog number: 3599).
8-well, straight form multi-channel aspiration manifold (Drummond, catalog number: 3-000-093)
Multi-channel Pipette 30–300 µL (Gilson)
15 mL Falcon tube (Corning, catalog number: 352097)
1.5 mL sterile microcentrifuge tube (Eppendorf, EP022363344)
Sterile filter pipette tips (Axygen, catalog numbers: 350-rs, 250-rs, 30-rs)
Serological pipettes (Fisherbrand, catalog numbers: 13-676-10J and 13-676-10k)
Reagent reservoirs (Corning, catalog number: 4872)
Sterile Aeraseal cell culture plate sealer (EXCEL Scientific, catalog number: B-100)
Easyseal transparent plate sealers (Greiner Bio-One, catalog number: 676001)
Firefly luciferase assay kit 2.0 (Biotium, catalog number: 30085-2)
G418 sulfate (Geneticin) (Thermo Scientific, catalog number: 11811031)
Blasticidin-HCL (Corning, catalog number: 30-100-RB)
Puromycin dihydrochloride (Sigma-Aldrich, catalog number: P8833)
0.25% trypsin-EDTA solution (CCF Media Production, catalog number: CCFGP003)
Dulbecco's Modification of Eagles Medium (DMEM) with 4.5 g/L glucose and L-glutamine, without sodium pyruvate (Fisher Scientific, catalog number: 10-017-CV)
Fetal bovine serum (FBS) qualified (Gibco, catalog number: 26140-079)
Penicillin/streptomycin 100× (CCF Media Production, catalog number: CCFGK004)
Amphotericin B (Fungizone) 100× (Fisher Scientific, catalog number: 12002-032)
Recombinant human TGF-β1 (R&D Systems, catalog number: 240-B-002/CF)
Phosphate buffered saline W/O calcium and magnesium (CCF Media Production, catalog number: CCFAL003-216T02)
Bovine serum albumin (BSA) essentially globulin free, low endotoxin (Sigma-Aldrich, catalog number: A2934-100G)
TMLC wild-type (WT), TMLC L-TGF-β1, TMLC L-TGF-β1(R249A) TMLC GARP, TMLC L-TGF-β1/GARP and TMLC L-TGF-β1(R249A)/GARP cell lines. Development of TMLC lines is described in Campbell et al. (2020). Available with MTA approval from Stephen Nishimura, Dept. Of Pathology, University of California San Francisco, San Francisco, California.
Recombinant Human α_vβ₈, α_vβ₆, and α_vβ₃ ectodomain. Available with an MTA approval from Stephen Nishimura, Dept. Of Pathology, University of California San Francisco, San Francisco, California. Development and purification of these reagents is described in Campbell et al. (2020).
MACS separation buffer (Miltenyi, catalog number: 130-091-221)
EZ-Link^TM Sulfo-NHS-LC-Biotin (ThermoFisher Scientific, catalog number: 21335)
Anti-HA antibody (Genscript, catalog number: A01244-100)
Anti-latency associated peptide (LAP) antibody, clone AF246 (R&D Systems, catalog number: AF-246-NA)
TMLC basal media (see Recipes)
5 mg/mL Blasticidin-HCl stock solution (see Recipes)
50 mg/mL G418 sulfate stock solution (see Recipes)
10 mg/mL puromycin dihydrochloride stock solution (see Recipes)
20 μg/mL recombinant human TGF-β1 stock solution (see Recipes)
1 μg/mL α_vβ₃ or α_vβ₈ ectodomain coating solution (see Recipes)
1 μg/mL anti-LAP coating solution (see Recipes)
1% BSA blocking solution (see Recipes)
1× luciferase cell lysis buffer (see Recipes)
1× luciferase assay buffer (see Recipes)

Equipment

37°C humidified incubator, 5% CO₂
Jouan C4i benchtop centrifuge or equivalent, for spinning 15–50 mL tubes (Thermo Scientific, model: Jouan C4i)
Flow cytometer (BD Biosciences, model: LSR II)
Cell sorter (BD Biosciences, model: FACS ARIA II)
Luciferase assay plate reader (Promega, model: Glomax Explorer)
Luna automated cell counter or equivalent (Luna, model: L10001-LG)
Hoefer RED ROCKER rocking platform or equivalent (Hoefer Scientific Instruments)

Software

GraphPad Prism 9 (https://www.graphpad.com/scientific-software/prism/)

Procedure

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Seed, R. I. and Nishimura, S. L. (2022). Measurement of Cell Intrinsic TGF-β Activation Mediated by the Integrin αvβ8. Bio-protocol 12(8): e4385. DOI: 10.21769/BioProtoc.4385.