Visualization and Quantification of TGFβ/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Summary

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Abstract

Transforming Growth Factor β (TGFβ)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGFβ/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGFβ/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).

Active TGFβ/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGFβ and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.

The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls.

The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGFβ/BMP signaling in ECs, both quantitatively and spatially.

Introduction

Proteins of the transforming growth factors beta (TGFβ) superfamily are pleiotropic cytokines with a variety of members, including TGFβs, bone morphogenetic proteins (BMPs), and Activins^1,2. Ligand binding induces the formation of receptor oligomers leading to the phosphorylation and, thereby, activation of cytosolic regulatory SMAD (R-SMAD). Depending on the sub-family of ligands, different R-SMADs are activated^1,2. While TGFβs and Activins mainly induce phosphorylation of SMAD2/3, BMPs induce SMAD1/5/8 phosphorylation. However, there are accumulating....

Protocol

1. Cell culture and fluid shear stress exposure

NOTE: Human umbilical vein ECs (HUVECs) were used as an example to study SS induced interaction of SMADs. The protocol described below can be applied to every SS responsive cell type.

1. Coat 6-channel slide with 0.1% porcine skin gelatin in PBS for 30 min at 37 °C.
2. Seed HUVECs in pre-coated 6-channel slides at a density of 2.5 x 10⁶ cells per mL in 30 µL of M199 full medium.
   NOTE: For further information on how to seed cells in the flow slide, see reference²⁴.
3. Let cells adhere for 1 h at 37 °C in a humidifie....

Results

We have previously used PLA to detect interactions of different SMAD proteins³ and analyzed shear stress induced changes in SMAD phosphorylation²⁸.

Here, both methods were combined with the protocol described above. HUVECs were subjected to shear stress of 1 dyn/cm² and 30 dyn/cm² and analyzed for interactions of SMAD transcription factors. We show that, when compared to the high shear stress (30 dyn/cm²), the low.......

Discussion

The PLA based protocol described here offers an efficient way to determine close proximity of two proteins (e.g., their direct interaction) in ECs exposed to shear stress with quantitative and spatial resolution. By using flow slides with multiple parallel channels, several different protein interactions can be examined at the same time in cells under identical mechanical conditions. In contrast, custom-build flow chamber systems often make use of a single channel that is built around a glass coverslip, which would allow.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
µ-Slide VI 0.4	ibidi	80606	6-channel slide
Ammonium Chloride	Carl Roth	K298.1	Quenching
Bovine Serum Albumin	Carl Roth	8076.4	Blocking
DAPI	Sigma Aldrich/ Merck	D9542	Stain DNA/Nuclei
DPBS	PAN Biotech	P04-53500	PBS
Duolink In Situ Detection Reagents Green	Sigma Aldrich/ Merck	DUO92014	PLA kit containing Ligase, ligation buffer, polymerase and amplification buffer (with green labeled oligonucleotides)
Duolink In Situ PLA Probe Anti-Mouse MINUS	Sigma Aldrich/ Merck	DUO92004	MINUS probe
Duolink In Situ PLA Probe Anti-Rabbit PLUS	Sigma Aldrich/ Merck	DUO92002	PLUS probe
Duolink In Situ Wash Buffers, Fluorescence	Sigma Aldrich/ Merck	DUO82049	PLA wash buffers A and B
Endothelial Cell Growth Supplement	Corning		supplement for medium (ECGS)
Fetal calf Serum			supplement for medium
FIJI			Image Analysis software
Formaldehyde solution 4% buffered	KLINIPATH/VWR	VWRK4186.BO1	PFA
Full medium			M199 basal medium +20 % FCS +1 % P/S + 2 nM L-Glu +  25 µg/mL Hep +   50 µg/mL ECGS
Gelatin from porcine skin, Type A	Sigma Aldrich	G2500	Use 0.1% in PBS for coating of flow channels
GraphPad Prism v.7	GarphPad		Statistical Program used for the Plots and statistical calculations
Heparin sodium salt from porcine intestinal mucosa	Sigma Aldrich	H4784-250MG	supplement for medium (Hep)
HUVECs
ibidi Mounting Medium	ibidi	50001	Liquid mounting medium
ibidi Pump System	ibidi	10902	pneumatic pump
Leica TCS SP8	Leica		confocal microscope
L-Glutamin 200mM	PAN Biotech	P04-80100	supplement for medium (L-Glu)
Medium 199	Sigma Aldrich	M2154	Base medium
mouse anti- SMAD1 Antibody	Abcam	ab53745	Suited for PLA
mouse anti- SMAD2/3 Antibody	BD Bioscience	610843	Not suited for PLA in combination with CST 9515
mousee anti- SMAD4 Antibody	Sanata Cruz Biotechnology	sc-7966	Suited for PLA
Penicillin 10.000U/ml /Streptomycin 10mg/ml	PAN Biotech	P06-07100	supplement for medium (P/S)
Perfusion Set WHITE	ibidi	10963	Tubings used for 1 dyn/cm2
Perfusion Set YELLOW and GREEN	ibidi	10964	Tubings used for 30 dyn/cm2
rabbit anti- phospho SMAD1/5 Antibody	Cell Signaling Technologies	9516	Suited for PLA
rabbit anti- SMAD2/3 XP Antibody	Cell Signaling Technologies	8685	Suited for PLA
rabbit anti- SMAD4 Antibody	Cell Signaling Technologies	9515	Not suited for PLA in combination with BD 610843
Serial Connector for µ-Slides	ibidi	10830	serial connection tubes
Triton X-100	Carl Roth	6683.1	Permeabilization