Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis

Summary

The study demonstrates a quantitative PCR-based assay for primary cilia-mediated Sonic Hedgehog (SHH) signaling pathway activation using SHH agonist in a cellular model of ciliogenesis.

Abstract

Ciliopathy refers to a collection of multisystemic and polygenic human diseases and disorders that arise due to aberration of structure or function of motile or non-motile (primary) cilia that are microtubule-based cell protrusions. Primary cilia (PC) are present in most human epithelial and endothelial tissues and serve as a hub for physiological signal transduction, including Sonic Hedgehog (SHH) signaling. Various situations demand examining the function of PC, which may be achieved by measuring the canonical SHH signaling pathway that is almost exclusively mediated by PC. Here, a quantitative PCR (qPCR) based technique is developed to measure the transcriptional level of SHH pathway genes in quiescent hTERT-RPE1 (or RPE1) cells that mostly contain PC. Quiescence in RPE1 cells is achieved by serum starvation for 48 h, while activation of the SHH pathway is promoted by a specific agonist. This cell culture-based assay is easy to follow and sensitive. Successful demonstration of this assay in ciliated RPE1 cells indicates its immense potential as an in vitro assay to examine the proper function of PC upon genetic or epigenetic alteration of one or multiple genes, which may be associated with various ciliopathies.

Introduction

A cilium is a microtubule-based and membrane-ensheathed protrusion from the cell surface, which is assembled on a basal body¹. In vertebrates, two types of cilia, namely motile and non-motile, are observed. Motile cilia are located in specialized tissues and confer motility. Non-motile primary cilia (PC) are present in most vertebrate cells, and perform sensory functions and transduce critical signals from the extracellular milieu to the interior of the cells². Once assembled, ciliary elongation is helped by intraflagellar transport (IFT) machinery, which is a bidirectional transport system carrying cargo driven by kinesins (anterograde movement) and dynein-II motor proteins (retrograde movement)^3,4. Structural or functional defects in motile or primary cilia may lead to a plethora of multisystemic human diseases that are collectively known as ciliopathies^5,6.

PC serves as the hub for various developmental signaling processes, such as Wingless (Wnt), G-protein-coupled receptors (GPCR), receptor-tyrosine kinases (RTKs), Transforming growth factor β (TGFβ), and Platelet-derived growth factor (PDGF) signaling^7,8. In mammals, Sonic Hedgehog (SHH) signaling is a critical one, which is almost exclusively transduced by PC in cells containing PC^9,10,11. Aberrant structure or function of PC leads to attenuated signaling pathways that are associated with diseases like congenital heart defects, polycystic kidney disease, craniofacial abnormalities, retinal dystrophy, and a few rare diseases such as Bardet-Biedl Syndrome (BBS), Joubert Syndrome (JBTS), Alström Syndrome, Jeune Asphyxiating Thoracic Dysplasia (JATD)^12,13,14,15. Most of these are polygenic diseases of epithelial or endothelial tissues, which render the challenges in early diagnosis and treatment of these diseases. Determining how such mutations contribute to attenuated PC-mediated signaling in a cell culture model may not only provide insight into the molecular intricacy of that signaling pathway but also facilitate the diagnosis of various ciliopathies and may suggest therapeutic interventions. With this notion, a quantitative PCR (qPCR) based assay is developed to determine alteration in PC-mediated SHH signaling pathway in human telomerase reverse transcriptase (hTERT) immortalized retinal pigment epithelial cells (hTERT-RPE1, referred to here as RPE1). RPE1 cells are near-diploid, non-transformed human cells that are widely used as the cellular model for ciliogenesis since approximately 75%-80% of these cells assemble PC upon serum starvation (incubating cells in serum-free medium) for 24-48 h, compared to only 15%-18% ciliation in an asynchronously growing culture of RPE1 cells¹⁶.

Hedgehog signaling (HH) is conserved among vertebrates for embryonic development¹⁷. In mammals, three different types of Hedgehog signaling are witnessed: Sonic (SHH), Indian (IHH), and Desert (DHH), of which SHH and IHH show overlapping functions at the tissue level¹¹. SHH signaling plays key roles during embryonic development by regulating the patterning of limbs and cell type specification in the nervous system and maintaining adult tissue homeostasis^10,11,18. Although SHH signaling is complex, there is increasing evidence to suggest that canonical SHH signaling is reliant on PC^7,11,19, which is mediated by collective functions of Gli transcription factors (Gli1, Gli2, and Gli3). It has been suggested that HH signaling and PC have coevolved, with critical signaling components concentrated in the compact volume at the cilium's tip to enable effective responses to low ligand ratios¹¹.

In the absence of SHH ligands, Patched1 (Ptch1), which is localized on the PC membrane, inhibits the activity of Smoothened (Smo) through a poorly understood mechanism that likely involves lipid transport, thus keeping the pathway repressed^7,20. The constitutively active ciliary GPR161, which is a G-coupled protein receptor and a negative regulator of the system, ensures the repression is maintained by preserving high levels of ciliary cyclic AMP (cAMP) that increases the activity of protein kinase A (PKA)²¹, and by maintaining ciliary localization of PKA⁷. PKA activity, along with casein kinase 1 (CK1) and glycogen synthase kinase 3 β (GSK3β), phosphorylates multiple residues of Gli proteins, Gli2 and Gli3, inside PC. Gli2 and Gli3 can act as both activators and repressors of transcription of target genes. PKA-mediated phosphorylation causes inhibition of the activator function of Gli2, while phosphorylated Gli3 is cleaved by proteases to produce the repressor form Gli3R²². Overall, the expression of SHH target genes is repressed in such conditions.

When SHH ligand is present, Ptch1 binds to it, and GPR161 leaves PC, and Smo, being phosphorylated by G protein-coupled receptor (GPCR) kinase 2 (GPRK2) and CK1, is trafficked into PC, together with β-arrestin and the microtubule motor KIF3A²³. In concert, the localization of Gli2/3, Suppressor of Fused (SUFU), and Kif7 at the ciliary tip is promoted and is linked to decreased Gli2/3 cleavage, thereby increasing activator function of Gli2/3^24,25. Once activated, Gli2/3 induces the expression of Gli1, which can only function as an activator, and thereby amplifies the response of the SHH pathway²². The binding of SHH to Ptch1 is facilitated by CAM-related/downregulated by oncogenes (CDO), brother of CDO (BOC), growth arrest-specific 1 (GAS1), and low-density lipoprotein receptor-related protein 2 (LRP2). Conversely, Hedgehog-interacting protein (HHIP) can sequester SHH by directly interacting with it and also competes with Ptch1 from interacting with SHH²⁶. Importantly, GLI1, HHIP, and PTCH1 are transcribed upon SHH pathway activation, whereas the last two participate in the negative regulation of the SHH pathway.

Previous studies demonstrated that the transcript levels of SHH target genes may be measured to assess the SHH pathway, though it may vary significantly in different cell types^27,28. Such variation in SHH pathway response may be attributed to different combinations of PC-mediated SHH signaling and SHH signaling that is mediated by the cell membrane, likely in the absence of PC. SAG is a Smo agonist that is used in this method to mimic SHH signaling activation in serum-starved RPE1 cells that are majorly ciliated. The transcript levels of SHH target genes such as HHIP, GLI1, and PTCH1 and a non-target gene SMO were measured using qPCR in control and SAG-treated cells and were compared to determine the activation of the SHH pathway. Successful demonstration of this cell culture-based sensitive assay to measure PC-mediated SHH signaling suggests its promising application in biomedical research associated with primary cilia function.

Protocol

1. Cell culture

1. Passage a near-confluent T-25 cell culture flask of RPE1 cells at 1:5 dilution of the original culture into a fresh T-25 cell culture flask containing 5 mL of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin-streptomycin mix (final concentration- 100 U/mL penicillin G and 100 µg/mL streptomycin). This medium is referred to as the complete medium. Grow cells at 37 °C in the presence of 5% CO₂.
   NOTE: RPE1 cells are non-transformed cells commonly used to study the molecular regulation of PC assembly and disassembly in cell culture. Approximately 80% of RPE1 cells assemble PC upon serum starvation, which is again disassembled upon re-addition of serum¹⁶.
2. Prepare the 12 mm round glass coverslips by incubating them in 50 mL of 1 N HCl in a covered glass beaker overnight without shaking at 60 °C in a water bath or incubator.
3. Next day, using a 25 mL pipette, pipette out the acid solution and discard it properly in a chemical hazard container. Next, wash the coverslips 3x in 100 mL of distilled water by incubating for 15 min with occasional shaking, followed by pipetting out.
4. Repeat the washing steps with 70% ethanol and 95% ethanol. Dry the coverslips by individually placing them on a laboratory blotting paper in a biosafety cabinet. Once dried, sterilize with UV radiation for 60 min.

2. Cell growth, serum starvation, and SAG treatment

1. Resuspend the cells in the T-25 flask in a pre-warmed complete medium after dislodging the cells by the enzymatic activity of pre-warmed 0.25% Trypsin-EDTA solution. Determine the cell density of the suspension by counting cells using a hemocytometer.
2. Prepare two 35 mm cell culture dishes and place two sterile 12 mm coverslips in each of them. Passage 2 x 10⁵ asynchronously growing RPE1 cells in each 35 mm dish containing coverslips and grow the cells in 2 mL of complete medium for 24 h.
3. After 24 h, remove the medium of growing RPE1 cells and wash the cells 1x with pre-warmed 1 mL of DPBS buffer. Add 2 mL of pre-warmed DMEM media containing 1% penicillin-streptomycin but devoid of FBS (serum-free medium) to each dish and incubate the cells for 24 h at 37 °C in the presence of 5% CO₂.
4. After 24 h, replace the medium with the serum-free medium containing either SAG to a final concentration of 250 nM or DMSO as the control solvent and incubate the cells for another 24 h. A cartoon diagram of the cell culture protocol is shown in Figure 1A.

3. Cell collection

1. Transfer the coverslips to a 24-well plate with one coverslip in each well. Add 0.5 mL of chilled methanol to each well and incubate the plate at -20 °C for 10 min to fix the cells on the coverslips. Immediately after, wash the coverslips 3x with 0.5 mL of wash buffer (1x PBS containing 0.5 mM MgCl₂ and 0.05% Triton-X 100).
2. Discard the media and add 0.5 mL of trizol reagent directly to each dish. Let it evenly distribute and keep for 5 min. Pipette up and down a few times to make a homogeneous mixture and collect it in 1.5 mL nuclease-free microcentrifuge tubes.

4. Preparation of total RNA and cDNA

1. Add 0.1 mL of chloroform to each tube containing lysed cells and incubate for 2-3 min. Centrifuge each sample for 15 min at 12,000 x g at 4 °C. Transfer the aqueous phase containing the RNA to fresh microcentrifuge tubes.
2. Add 0.25 mL of isopropanol to the aqueous phase and incubate for 10 min. Centrifuge for 10 min at 12,000 x g at 4°C. The RNA precipitate forms a white gel-like pellet. Using a micropipette, discard the supernatant.
3. Resuspend the RNA pellet in 0.5 mL of freshly diluted 75% ethanol. Vortex the samples briefly, and then centrifuge for 5 min at 7500 x g at 4°C. Discard the supernatant with a micropipette.
4. Air dry the RNA pellet for 10 min and resuspend in 25 µL of nuclease-free water. Determine the concentration of total RNA by measuring the absorbance at 260 nm using a microvolume spectrophotometer.
5. Use 1-3 µg of total RNA from both samples to prepare cDNAs following the manufacturer's instructions for a cDNA synthesis kit. Store cDNAs at -20 °C.

5. Quantitative PCR

1. Set the qPCR reactions (in triplicate, Table 1) using 1:5 diluted cDNAs from each sample with primer sets of GLI1, PTCH1, HHIP, SMO, and β-ACTIN (as housekeeping gene; Table 2) genes with a qPCR master mix containing suitable DNA polymerase, dNTPs, and buffer, in a suitable multi-well, fluorescence-sensitive PCR plate.
2. Cover the plate with a suitable sealer, place it in a suitable qPCR instrument, and run a standardized qPCR program with 40 amplification cycles and a melting curve analysis step at the end of the program. The standardized 40-cycle qPCR program, including a melting curve analysis, is schematically shown in Figure 1B.
3. When the qPCR program is completed, check the melt curve of each amplicon for a single peak at the desired temperature.
   NOTE: At the end of amplification cycles, the associated melt curve analysis measures the melting temperature of the amplicon by increasing the temperature of each well from 65°C to 95°C, with 0.5°C increment per step of 5 s duration.
4. Export the amplification data to a spreadsheet and calculate the relative expression (2^−ΔΔCT method) of each transcript by normalizing against that of the β-ACTIN gene for each sample. Plot the graph and calculate the statistical significance of the changes in the relative expression of each transcript using an unpaired T-Test.

6. Immunostaining and image acquisition using fluorescence microscopy

1. Incubate the fixed cells on coverslips in 200 µL of blocking buffer (2% BSA and 0.1% TritonX-100 in 1x PBS) for 30 min.
2. Incubate the cells with a mix of primary antibodies (typically an anti-mouse against acetylated α-tubulin of PC axoneme and anti-rabbit antibodies against ciliary membrane and centriole, respectively) diluted in blocking buffer (see Table of Materials for final dilution) overnight at 4 °C in a humidified chamber.
   1. Make a humidified chamber by putting a wet paper towel in the bottom half of an empty 1 mL pipette tip box. Lay a strip of transparent film on the rack surface and spot a droplet (20 -25 µL) of the antibody solution onto the film, one for each coverslip to be incubated. Place a coverslip on the droplets so that cells are immersed, and close the lid of the tip box.
3. Next day, invert the coverslips again and return them to the 24-well dish. Wash the coverslips with the wash buffer and then incubate them in 150 µL of secondary antibody mixture with a pre-standardized dilution of antibodies (here, green-fluorescent dye-conjugated anti-mouse and red-fluorescent dye-conjugated anti-rabbit diluted in blocking buffer) and nuclear DNA staining reagents for 1 h at room temperature. Wash the coverslips 3x.
4. Spot a droplet (roughly 3-5 µL) of the mounting solution containing antifade reagent on a glass microscope slide. Place a coverslip, with the cell side facing down, onto the mounting solution. Wipe excess liquid by gently pressing a clean wipe. Apply transparent nail polish along the edge of the coverslip to seal it onto the slide.
5. Put a slide on the microscope (see Table of Materials) attached with a motorized stage and a camera capable of digital imaging. Use a 60x Plan Apo oil immersion objective (with a 1.4 numerical aperture) to acquire the images of serum-starved RPE1 cells at ambient temperature, to visualize PC in cells, as marked by the mentioned antibodies.
6. Acquire images of the different samples on separate coverslips using identical exposure time for different fluorophores along the Z-axis using a digital microscopy imaging software package. Count the number of PC-containing cells from at least 500 cells to determine the percentage ciliation in 48 h serum-starved condition.

Results

The activation of the PC-mediated SHH signal transduction pathway is a way to understand the proper function of PC⁷. Here, the relative expression of SHH pathway target genes are measured using RT-qPCR assay upon SAG-mediated activation of the SHH pathway in RPE1 cells that are mostly ciliated. The SHH pathway's direct transcriptional outputs GLI1 and PTCH1 are frequently employed to gauge the activity of this pathway¹¹. This protocol also includes the HHIP transcr...

Discussion

Examining the functionality of PC is a crucial step towards understanding the dynamics of ciliation and diseases or disorders associated with the dysfunction of PC. Apart from examining ciliation by immunostaining for ciliary markers or fluorophore-tagged ciliary structural components, the function of PC is commonly determined by assaying the canonical SHH pathway activation by measuring the ciliary translocation of Smo in tissues and cells using fluorescence microscopy^27,

Materials

Name	Company	Catalog Number	Comments
DMSO, sterile filtered	Sigma	D2650
Donkey, anti-Mouse IgG (H+L) Alexa Fluor 488	ThermoFisher Scientific	A21202
Donkey, anti-Rabbit IgG (H+L) Alexa Fluor 568	ThermoFisher Scientific	A10042
Dulbecco's Modified Eagle Medium (DMEM)	Gibco, ThermoFisher Scientific	41965039
Dulbecco's phosphate-buffered saline (DPBS)	Gibco, ThermoFisher Scientific	14190144
Fetal bovine serum (FBS)	Himedia	RM1112
Fluorescence Microscope fitted with 60X objective	Carl Zeiss		Axio observer colibri 5
hTERT-RPE1 cells	ATCC	CRL-4000
iScript cDNA Synthesis Kit	Bio-Rad Laboratories	1708891
Mouse, monoclonal anti-acetylated tubulin antibody	Milipore Sigma-Aldrich	T7451	Final dilution 1:1000
Penicillin-Streptomycin, 100X	Gibco, ThermoFisher Scientific	15140122
Quantitaive PCR machine, CFX 96	Bio-Rad Laboratories
Rabbit, polyclonal Arl13B antibody	ProteinTech	17711-1-AP	Final dilution 1:250
Rabbit, polyclonal Cep135 antibody	ProteinTech	24428-1-AP	Final dilution 1:500
SlowFade gold Antifade moutant	ThermoFisher Scientific	S36940
Smoothened Agonist (SAG)	Abmole	M4865	Stock solution: in DMSO, 5 mM
SsoAdvanced Universal SYBR Green Supermix	Bio-Rad Laboratories	1725271
TRIzol Reagent	Invitrogen	15596026
TrypLE	Gibco, ThermoFisher Scientific	12605028