TGFβ 释放共培养试验

Emanuel Rognoni

doi:10.21769/BioProtoc.1314

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Peer-reviewed

TGFβ Release Co-culture Assay

TGFβ 释放共培养试验

Emanuel Rognoni email

发布: 2014年12月05日第4卷第23期 DOI: 10.21769/BioProtoc.1314 浏览次数: 11329

评审: Anonymous reviewer(s)

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Cover of Nature Medicine, featuring study using the protocol.

Apr 2014

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Abstract

TGFβ is a potent cytokine modulating various processes including proliferation, differentiation, ECM synthesis and apoptosis (Siegel and Massague, 2003). Thus in many tissues availability of TGFβ is tightly regulated. TGFβ is secreted as an inactive complex where it is encapsulated by the latency associated protein (LAP), a ligand trap protein, which inhibits TGFβ binding to its receptor and retains TGFβ in the extracellular matrix (ten Dijke and Arthur, 2007). TGFβ can be released from the matrix and converted into its biological active form by huge number of processes including heat, high and low pH, release of reactive oxygen species (ROS) or various proteases (e.g. plasmin, elastase, matrix metalloproteinase-2 and -9) (Barcellos-Hoff and Dix, 1996; Lyons et al., 1988; Taipale et al., 1994; Yu and Stamenkovic, 2000). However, under physiological conditions the interaction of αv-class integrins with the RGD tripeptide motif in the LAP protein represents the key factor for TGFβ release in vivo. The relevance of integrin mediated TGFβ release for in vivo development and homeostasis is further underlined by the observation that mice with the integrin-binding deficient LAP proteins (RGD motif mutated to RGE) recapitulate all major phenotypes of TGFβ1 null mice, including multi-organ inflammation and defects in vasculogenesis (Shull et al., 1992; Yang et al., 2007). This striking phenotype overlap with TGFβ deficient mice and phenotypes of mice lacking αv-class integrins (Aluwihare et al., 2009; Bader et al., 1998) demonstrates an essential interconnection of integrins with TGFβ signaling in vivo, while the role of non-integrin mediated release mechanisms (ROS, pH, proteolytic cleavage etc.) during development remains less clear.

The TGFβ release assay measures the ability of cells to release TGFβ from a matrix. The assay was developed by (Annes et al., 2004) and we further optimized the protocol for keratinocytes. For other cell types the cell culture medium and culturing conditions would need to be adapted accordingly.

In keratinocytes TGFβ release is mainly mediated by αvβ6 integrin but also integrin αvβ3, αvβ5 and αvβ8 have been shown to liberate TGFβ, while other RGD binding integrins, such as α5β1 or α8β1 cannot release TGFβ (Asano et al., 2005a, 2005b; Mu et al., 2002; Munger et al., 1999). Mechanistically, the interaction with αvβ3, αvβ5 or αvβ6 integrin induces a conformational change in the LAP-TGFβ by generating an actin cytoskeleton dependent pulling force, allowing TGFβ to access its receptors. For αvβ8 integrin mediated TGFβ release it was shown that proteolytic cleavage is involved [see (Mu et al., 2002) for blocking conditions of TGFβ release by proteolytic cleavage and αvβ8 integrin].

The following protocol is optimized for the study of αvβ6-integrin mediated TGFβ release in keratinocytes.

Materials and Reagents

Cell lines
1. CHO-LTBP1 TGFβ rich matrix producing cell line, generated by the Daniel Rifkin lab expresses high levels of LTBP1-TGFβ (Annes et al., 2004).
2. Transformed mink lung epithelial cells (tMLEC) TGFβ reporter cell line, stably expresses a luciferase reporter plasmid under control of a truncated plasminogen activator inhibitor type 1 promoter (PAI-1) (Abe et al., 1998).
  Note: CHO-LTBP1 and tMLEC are cultured in DMEM growth medium. tMLEC DMEM growth medium is supplemented with 250 mg/ml Geneticin (Life Technologies, InvitrogenTM, catalog number: 10131035 ).
Antibodies
1. αvβ6 integrin blocking antibody (Millipore, clone 10D5, catalog number: MAB2077Z )
2. TGFβ neutralizing antibody (R&D Systems, clone 1D11, catalog number: MAB1835 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
BrightGlo luciferase assay kit (Promega Corporation, catalog number: E2610 )
Dulbecco's minimum essential medium (DMEM) (Life Technologies, Gibco^®, catalog number: 11966-025 )
Fetal bovine serum (FBS) (Life Technologies, Gibco^®, catalog number: 10270-106 )
Calcium chloride (CaCl₂) (Carl Roth, catalog number: A119.1 )
Chelex 100 resin (Bio-Rad Laboratories, catalog number: 143-2832 )
Penicillin-streptomycin (pen-strep) (Life Technologies, Gibco^®, catalog number: 15070-063 )
Minimum essential medium (MEM) (Sigma-Aldrich, catalog number: M8167 )
Insulin (Sigma-Aldrich, catalog number: I5500 )
Epidermal Growth Factor (EGF) (Sigma-Aldrich, catalog number: E9644 )
Transferin (Sigma-Aldrich, catalog number: T8158 )
Phosphoethanolamine (Sigma-Aldrich, catalog number: P0503 )
Ethanolamine (Sigma-Aldrich, catalog number: E0135 )
Hydrocortisone (Calbiochem^®, catalog number: 386698 )
L-Glutamine (Life Technologies, Invitrogen^TM, catalog number: 25030-081 )
Trypsin powder (Life Technologies, Gibco^®, catalog number: 27250-018 )
0.5%Trypsin/EDTA (Life Technologies, Gibco^®, catalog number: 15400-054 )
Sodium chloride (NaCl) (Carl Roth, catalog number: P029 )
di-Sodium hydrogen phosphate (Na₂HPO₄) (Carl Roth, catalog number: T876 )
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
Potassium chloride (KCl) (Carl Roth, catalog number: HN02.3 )
Potassium dihydrogen phosphate (KH₂PO₄) (Carl Roth, catalog number: 3904 )
DMEM growth medium (see Recipes)
Keratinocyte growth medium for murine keratinocyte culture (KGM) (see Recipes)
Starving KGM (see Recipes)
0.4% keratinocyte trypsin (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)
PBS/EDTA (see Recipes)
0.1% trypsin/EDTA (see Recipes)
Chelated FBS (see Recipes)

Equipment

Flat bottom 96-well plates for cell culture (Corning, Costar^®, catalog number: 3595 )
Round bottom white 96-well plates (Corning, Costar^®, catalog number: 3789 )
1.5 ml Eppendorf tubes (Sigma-Aldrich, catalog number: T9661-1000EA )
Cell culture incubator (37 °C, 5% CO₂)
Standard centrifuge to spin down the cells
Luminometer, e.g. GloMax (Promega corporation)
Standard bright field microscope
Multichannel pipette (recommended)