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 evidences that BMPs and TGFβs/Activins also activate R-SMADs of the respective other sub-family, in a process termed as 'lateral signaling'^3,4,5,6,7,8 and that there are mixed SMAD complexes consisting of both, SMAD1/5 and SMAD2/3, members^3,9. Two activated R-SMADs subsequently form trimeric complexes with the common mediator SMAD4. These transcription factor complexes are then able to translocate into the nucleus and regulate the transcription of target genes. SMADs can interact with a variety of different transcriptional co-activators and co-repressors, leading to the diversification of the possibilities to regulate target genes¹⁰. Deregulation of SMAD signaling has severe implications in a variety of diseases. In line with this, unbalanced TGFβ/BMP signaling may lead to severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, or atherosclerosis^{3,11,12,13,14}.

Endothelial cells (ECs) form the innermost layer of blood vessels and are, therefore, exposed to shear stress (SS), a frictional force exerted by the viscous flow of the blood. Interestingly, ECs residing at the parts of the vasculature, which are exposed to high levels of uniform, laminar SS, are kept in a homeostatic and quiescent state. In contrast, ECs that experience low, non-uniform SS, e.g., at bifurcations or the lesser curvature of the aortic arch, are proliferative and activate inflammatory pathways¹⁵. In turn, sites of dysfunctional ECs are prone to develop atherosclerosis. Interestingly, ECs in these atheroprone areas display aberrantly high levels of activated SMAD2/3 and SMAD1/5^16,17,18. In this context, enhanced TGFβ/BMP signaling was found to be an early event in the development of atherosclerotic lesions¹⁹ and interference with BMP signaling was found to markedly reduce vascular inflammation, atheroma formation, and associated calcification²⁰.

Proximity Ligation Assay (PLA) is a biochemical technique to study protein-protein interactions in situ^21,22. It relies on the specificity of antibodies of different species that can bind target proteins of interest, allowing highly specific detection of endogenous protein interactions at a single-cell level. Here, primary antibodies have to bind to their target epitope at a distance of less than 40 nm to allow for the detection²³. Therefore, PLA is greatly beneficial over traditional co-immunoprecipitation approaches, where several million cells are needed to detect endogenous protein interactions. In PLA, species-specific secondary antibodies, covalently linked to DNA fragments (termed Plus and Minus probes), bind the primary antibodies and if the proteins of interest interact, Plus and Minus probes come in close proximity. The DNA gets ligated in the following step and the rolling circle amplification of the circular DNA is made possible. During amplification, fluorescently labeled complementary oligonucleotides bind to the synthesized DNA, allowing these protein interactions to be visualized by conventional fluorescence microscopy.

The protocol described here enables scientists to quantitatively compare the number of active SMAD transcription complexes at atheroprotective and atheroprone SS conditions in vitro using PLA. SS is generated via a programmable pneumatic pump system that is able to generate laminar unidirectional flow of defined levels and allows stepwise increases of flow rates. This method allows for the detection of interactions between SMAD1/5 or SMAD2/3 with SMAD4, as well as mixed-R-SMAD complexes. It can easily be expanded to analyze interactions of SMADs with transcriptional co-regulators or to transcription factor complexes other than SMADs. Figure 1 shows the major steps of the protocol presented below.

Figure 1: Schematic representation of the protocol described. (A) Cells seeded in 6-channel slides are exposed to shear stress with a pneumatic pump system. (B) Fixed cells are used for PLA experiment or for control conditions. (C) Images of PLA experiments are acquired with a fluorescence microscope and are analyzed using ImageJ analysis software. Please click here to view a larger version of this figure.

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 humidified incubator.
4. Add 60 µL of pre-warmed M199 full medium to each of the reservoirs.
5. Culture for 2 days, with a gentle medium exchange once a day, at 37 °C in a humidified incubator.
   1. Aspirate the reservoirs completely, add 120 µL of pre-warmed M199 full medium in one of the reservoirs, and aspirate from the other side.
   2. Add 60 µL of pre-warmed M199 full medium to both reservoirs.
6. Assemble and start the flow set-up as detailed in the reference²⁵.
   1. Mount tubing on fluidic units. Here, silicone tubing with a diameter of 0.8 mm and 1.6 mm are used to apply shear stress of 1 dyn/cm² and 30 dyn/cm², respectively.
      NOTE: The material and tubing length should remain constant, as changes could influence the resulting shear stress. In general, other combinations of pump systems and tubing can be used, as long as the resulting shear stress is known, and the pump creates a steady laminar flow.
   2. Fill the reservoirs with an appropriate amount of pre-warmed M199 full medium (minimum 10 mL).
   3. Connect fluidic units with the tubing to the pump system and perform a pre-run without cells to equilibrate the medium and to remove any remaining air²⁵.
   4. Serially connect the single channels on the 6-channel slide to one another by using serial connection tubing. The first and the last channel on the slide will be connected to the tubing assembled in 1.6.1 (see Figure 1A for a scheme). Be careful not to introduce any air into the system as this could severely harm the cells. Further information on the serial connection can be found in reference²⁶.
   5. For the exposure of cells to high levels of shear stress (>20 dyn/cm²), use a ramp phase, i.e., increase the shear stress stepwise with adaptation phases. Steps can be set in increments of 5 dyn/cm² per 30 min.

2. Fixation

1. Detach slides from the pumps after the fluid SS exposure. Use clamps on the tubing when detaching, to avoid the medium spill in the incubator.
2. Immediately transfer flow slides on ice, while the remaining tubing is detached sequentially. When removing the tubing from reservoirs, the reservoir on the other side should be kept closed with a finger to avoid trapping air bubbles in the channel. This might interfere with fixation steps.
3. Keeping the cells on ice, aspirate the medium carefully from the reservoirs but not from the channel where the cells reside. Subsequently, wash samples with cold sterile PBS (4 °C) with three times the channel volume (90 µL). Add PBS in one reservoir and aspirate carefully from the other reservoir. Repeat this step in each of the 6 channels per slide.
   NOTE: For all washing and incubation steps the respective solution is added in one of the reservoirs which leads to an exchange of solutions in the channel. To allow for complete substitution of solutions in the channel, the excess solution is then aspirated from the other reservoir. Solution on the top of the cells in the channel is not removed. Cells should not dry at any time. Therefore, it is important to wash carefully without any air bubble insertion into the slides.
4. Fix the cells by adding 90 µL of buffered 4% PFA solution in the same reservoir where the PBS was added beforehand and similarly aspirate the liquid from the other reservoir. Repeat this step in each channel in each slide. After the addition of PFA solution, transfer the samples from ice to room temperature (RT) and incubate for 20 min.
   CAUTION: PFA is toxic and should be handled carefully. Use gloves and work under a fume hood.
5. Wash cells 3x with PBS (RT) by adding it in one reservoir and aspirating carefully from the other reservoir. Empty just the reservoirs, ensuring not to dry out the channel. Repeat this step for each of the 6 channels per slide.
6. Quench the PFA-fixation by adding 90 µL of ambient 50 mM ammonium chloride in PBS in one of the reservoirs. Aspirate excess solution from the other reservoir. Repeat for each channel in the slide. Incubate the samples for 10 min at RT.
7. Wash as described in step 2.5.
   ​NOTE: At this point, the samples may be stored at 4 °C overnight, or the protocol can be immediately continued with blocking and primary antibody incubation (see step 3).

3. Blocking and primary antibody incubation

1. To permeabilize the cells, add 90 µL of 0.3% Triton-X-100 in PBS in an emptied reservoir, and aspirate from the other reservoir for each channel. Incubate for 10 min at RT.
2. Wash as described in step 2.5.
3. Add 90 µL of sterile PLA blocking solution in one reservoir of a channel and aspirate from the other side. Repeat this step for each channel. Block for 1 h at 37 °C in a humidified chamber.
   1. To make a humidified chamber, use a 10 cm dish with wet tissue sealed with wax film and place the dish in the incubator. Alternatively, other humidity chamber formats can be used that supply a humid atmosphere.
      NOTE: Alternatively, self-made blocking solution can be used (e.g., 3% (w/v) BSA in PBS, sterile filtered).
4. Prepare primary antibodies (1:100) in PLA antibody diluent. Prepare 30 µL of the solution per channel. Add both primary antibodies simultaneously and vortex.
   NOTE: Alternatively, self-made antibody diluent can be used (e.g., 1% (w/v) BSA in PBS). Antibodies used here are combinations of SMAD1-SMAD2/3, SMAD2/3-SMAD4 and phospho-SMAD1/5-SMAD4. Detailed information can be found in the Table of Materials.
5. Before the application of primary antibodies, aspirate the blocking solution from the reservoirs and, also, carefully from the channel. Pipette 30 µL of the primary antibody solution immediately into the empty channel by tilting the channel while adding the solution.
   NOTE: Perform the removal of the blocking solution and addition of the antibody solution channel-by-channel to ensure cells do not dry out in between.
6. Incubate samples with the primary antibodies overnight in humidified chambers at 4 °C.
   ​NOTE: The incubation can also be performed for 1 h at room temperature, if interested in continuing with the following steps on the same day.

4. PLA probe incubation

NOTE: For all steps in section 4.1-7.3, the washing buffers A and B are stored at 4 °C and need to be warmed to RT prior to the use.

1. Dilute PLA-probes (+)-mouse and (-)-rabbit to 1:5 in PLA antibody diluent (or 1% BSA) solution. Prepare 30 µL per channel.
2. Wash samples 2x for 5 min using 90 µL of the wash buffer A at RT by adding it in one of the reservoirs and aspirating carefully from the other reservoir. Repeat this step for each of the 6 channels per slide.
3. Aspirate the wash buffer A carefully and add 30 µL of PLA probe solution (prepared in step 4.1), similar to the addition of primary antibodies in step 3.5.
4. Incubate samples for 1 h at 37 °C in a humidified chamber.

5. Ligation

1. Wash samples 2x for 5 min using 90 µL of the wash buffer A at RT, as described in 4.2.
2. Prepare a 1:5 dilution of the ligation buffer in deionized water. Use this buffer to dilute the ligase enzyme to 1:40 (on ice). Use 30 µL per channel.
3. Aspirate the wash buffer A completely and add the ligation solution as described in 3.5.
4. Incubate samples for 30 min at 37 °C in a humidified chamber.

6. Amplification

1. Wash samples 2x for 2 min using 90 µL of wash buffer A at RT, as described in 4.2.
2. Prepare the amplification buffer by diluting it 1:5 in deionized water and use it to dilute the polymerase enzyme to 1:80 (on ice). Protect from light. Prepare 30 µL per channel.
3. Aspirate the wash buffer A completely and immediately add the prepared amplification solution into the empty channel, as described in 3.5. Incubate samples for 100 min at 37 °C in a humidified chamber.

7. Mounting

1. Wash samples 2x for 10 min using 90 µL of Wash Buffer B at RT as described in 4.2. Add DAPI (1:500) from 1 mg/mL stock solution (in deionized water) in the first wash to stain nuclei. Do not dry the channel.
2. Dilute the wash buffer B in deionized water (1:10) and wash 1x with 90 µL of 0.1x buffer B solution as described in 4.2.
3. Aspirate the wash buffer B completely and immediately add 2-3 drops of the liquid mounting medium into one reservoir. Distribute it in the channel by tilting the slide. Store samples at 4 °C in a humidified environment until imaging.

8. Image acquisition

1. Acquire images using a fluorescence microscope. Ensure that the respective filters fitting the fluorescent PLA probes are available.
   NOTE: It is beneficial to make use of a confocal microscope, if possible, as obtained PLA spots are more defined. This also supports further image processing and data analysis.

9. Image analysis and quantification using ImageJ/FIJI

1. Process exported images (.tiff) with an image processing program, such as ImageJ²⁷.
   NOTE: All scripts used within this study and that are necessary for the automatic counting of cellular, nuclear, and all PLA events (per cell) can be found in a GitHub repository: https://github.com/Habacef/Proximity-Ligation-Assay-analysis. Perform statistical analysis using any suitable program or tool.

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

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