Open Access

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

  • Authors:
    • Karina Galoian
    • Silva Abrahamyan
    • Gor Chailyan
    • Amir Qureshi
    • Parthik Patel
    • Gil Metser
    • Alexandra Moran
    • Inesa Sahakyan
    • Narine Tumasyan
    • Albert Lee
    • Tigran Davtyan
    • Samvel Chailyan
    • Armen Galoyan
  • View Affiliations

  • Published online on: November 9, 2017     https://doi.org/10.3892/ijo.2017.4199
  • Pages: 139-154
  • Copyright: © Galoian et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

Introduction

Chondrosarcoma is cancer of the cartilage that eventually metastasize. The disease can affect multiple organs, such as long bones, spine, pelvis, larynx and head. Conventional therapies are not effective in this disease treatment and there is urgency in seeking new approaches (1,2). The signaling events resulting in mesenchymal cell transformation to sarcoma have yet to be fully elucidated. Proline rich polypeptide 1, (PRP-1), also known as (galarmin) is produced by the brain neurosecretory cells and comprised of 15 amino acids (3), and is a mTOR kinase (mTORC1) inhibitor in chondrosarcoma, which causes 80–90% inhibition of chondrosarcoma cell growth, halting G1/S phase cell cycle progression in chondrosarcoma (4,5) and other mesenchymal tumors (6). The ability of PRP-1 to upregulate tumor suppressor miRNAs and downregulate onco-miRNAs in human chondrosarcoma JJ012 cell line was demonstrated (7). The upregulation of most tumor suppressors in chondrosarcoma (8) including inflammation related TET1/2 and SOCS3 is one of the unique PRP-1 properties, however, it depends on which molecular pathway these tumor suppressors are part of (9). PRP-1 epigenetically downregulates embryonic stem cell marker miR-302c in human chondrosarcoma and its targets Nanog, c-Myc and Bmi1 (10). To understand better the mechanism of PRP-1 action and its potential as therapeutic agent in the future, it is very important to identify the receptor it binds to. In the present study, we present evidence that PRP-1 exerts its effect via interacting with toll like receptor family TLR1/2, TLR6 and mucin MUC5B. Innate immunity toll-like receptors (TLRs), or pattern recognition receptors are sensitive both to endogenous and exogenous ligands (11,12) and can be found both inside the cells and at the cell surface. Intracellular TLRs start their journey from the endoplasmic reticulum (ER) through the Golgi and eventually to endolysosomes (13). TLRs play active roles in carcinogenesis and tumor progression or its inhibition (14,15) where the activation of TLR signalling could regulate antitumor immunity of the host (16). The term alarmins is often used when referring to endogenous TLR ligands. The innate immune system can be activated by recognizing pathogen associated molecular patterns (PAMPs). The injured cells in their turn have ability to release danger-associated molecular patterns (DAMPs) and contribute to the activation of innate immune system. Thus, immune system is involved not only in fighting the infection by mobilizing the immunologic arsenal, but also in the process of tissue repair. Hence, the term non-infectious inflammation response, whenever TLR signaling is mediated by endogenous ligands, which secure autoimmune disease and tumorigenesis in addition to tissue repair and injury (17,18). TLRs1 (cluster of differentiation 281), 2, 4, 5 and 6 are expressed on the cell surface, whereas TLRs3, 7, 8 and 9 are intracellular nucleic acid receptors. The ligand for TLR10 remains to be found (19). The antitumorigenic role of TLR2 is recognized, its deficiency led to early intestinal tumor formation (20). Most of endogenous TLR ligands are agonists of TLR4 and TLR2 (21). There is a reported link berween TLR signaling andmucins (MUCs) leading to effective pathogen elimination (22-24). Mucins are glycosylated large extracellular proteins that are found not only in mucous cells but also in connective tissue and goblet cells. Mucin expression glycosylation alterations can lead to the development of cancer and cellular transformation (2531). Apomucin with the attached O-linked oligosaccharides is the protein backbone for mucin. There are 'secreted (gel-forming and non-gel-forming)' and 'membrane-bound' mucins, with transmemebrane domain (32). The goblet cells from the epithelium and mucous cells from submucosal glands generate secreted mucins. Secreted mucins on the chromosome 11p15 include MUC2, MUC5AC, MUC5B, MUC6 and MUC19. Some of the mucins can manifest themselves as tumor suppressors, for example MUC4 (33,34). MUC5B expression has protumorigenic (28,35) or antitumorigenic consequence for the cell growth (36,37) and was linked both to decreased survival or better prognosis in cancer patients correspondingly, depending on the disease and organ specificity. MUC5B was shown to have very beneficial effects in human airway defense (38). The epigenetic mechanism, hypermethylation of MUC5B promoter was attributed to the silencing of its tumor suppressor activity (39). Both overexpression and downregulation of mucins in different organs can contribute to cancer pathology and inflammation (26,40).

Materials and methods

PRP-1 initial isolation and chemical synthesis

Initially, PRP-1 was isolated from the neurosecretory granules of bovine neurohypophysis by the method described (3,41) followed by its chemical synthesis (42).

PRP-1 antiserum affinity chromatography purification

Antiserum for PRP1 was generated (43), then affinity chromatography purified, AminoLink Plus Immobilization kit instructions (44894; Thermo Fisher Scientific, Waltham, MA, USA) were followed for protein sample desalting with Zeba Spin columns (89891; Thermo Fisher Scientific).

Tissue culture

The human JJ012 chondrosarcoma cell line was received from Dr Joel Block's Laboratory (Rush University, Chicago IL, USA). JJ012 chondrosarcoma cells were cultured as previously described (8). The medium composition: Dulbecco's modified Eagle's medium (DMEM), supplemented with F12, 10% fetal bovine serum (FBS), 25 μg/ml ascorbic acid, 100 ng/ml insulin, 100 nM hydrocortisone and 1% penicillin/streptomycin.

Brief immunocytochemistry protocol

Adherent cells were grown directly on coverslips with 5×105 cells/coverslip in 6-well clusters, where they were cultured overnight at 37°C in an incubator. Twenty-four hours later the medium was removed and samples were fixed in 1 ml of 4% formaldehyde solution, (F8775; Sigma-Aldrich St. Louis, MO, USA) in phosphate-buffered saline (PBS), pH 7.4 1X Gibco, (10010-023) PBS for 15 min in the incubator. Samples were washed with PBS twice, then were permeabilized with PBS/Triton X-100 (T9284; Sigma-Aldrich), 1% for 5 min at room temperature. Detergent was removed and non-specific sites were blocked in PBS containing 2% bovine serum albumin (BSA, A2153; Sigma-Aldrich) at room temperature for 30 min. Samples were further incubated overnight in cold room along with all primary antibodies for the experiment, followed by two consecutive washes the next morning and incubation in BSA solution with secondary antibodies at room temperature for 2 h along with Zenon complex and two washes with PBS, for 10 min each. Second fixation step with formaldehyde for 15 min at room temperature in the dark was performed, followed by two washing steps.

Zenon complex formation

PRP-1 serum antibody and Zenon rabbit IgG, Alexa Fluor 488 (Z-25302; Molecular Probes, Eugene, OR, USA) were mixed according to the manual and the procedures. The mixture was incubated for 10 min at room temperature with labeling reagent A, then another 10 min incubation with the blocking reagent B and 1 ml of the resulting mixture was applied to each well. Cells were stained with 3 μM of 4′,6-diamino-2-phenylindole dihydrochloride (DAPI, D1306; Thermo Fisher Scientific) for nuclear staining or 10 min at room temperature, the washed with PBS twice. The samples on coverslips were mounted in Antifade mounting medium, followed by microscopy. ProLong Gold Antifade reagent (P10144; Life Technologies) was applied as a liquid mountant directly to fluorescently labeled cells on microscope slides. The reagent contains chemicals to protect fluorescent dyes from fading during fluorescence microscopy.

Antibodies used for immunocytochemistry

For plasma membrane staining wheat germ agglutinin Alexa Fluor 594 conjugate was used (W11262; Thermo Fisher Scientific); TLR1 rabbit antibody (ab180798; Abcam); goat anti-rabbit H&L (DyLight 550) (ab96884; Abcam); mouse anti-MUC5B, Abcam (ab77995); goat anti-mouse IgG secondary antibody Alexa Fluor 647 (A2124; Life Technologies).

Imaging

Image acquisition was performed by the Analytical Imaging Core Facility at DRI/SCCC, University of Miami (FL, USA).

Zeiss 200M, ApoTome fluorescent microscope, DAPI 49, GFP 38HE, Cy3 43, Cy5 50 filter cubes, heated stage, Orca II ERG Hamamatsu b/w 14 bit camera and AxioVision acquisition software were used. The coverslips were placed in regular 35-mm Petri dishes and the cells grown on them, covered with medium. Once the cells were grown, the coverslips were taken out, the cells were fixed, stained and mounted on the glass slides. For imaging controls secondary antibodies were used without the primaries.

Human MUC5B ELISA and electrophoresis and western blotting

MUC5B protein was measured with human mucin -5 subtype (MUC5B) ELISA kit (MyBioSource, San Diego, CA, USA) (MBS 704534-48T).

The cells were trypsinized once they reached confluency and then seeded in 6-well clusters at a concentration of 1×106 cells/ml. PRP-1 was added only to the experimental samples but not to controls. The overnight incubation in 5% CO2 incubator at 37°C was followed by cell wash with ice-cold PBS with added protease inhibitor. The cell lysis buffer (C2978; Sigma-Aldrich) was supplemented with the protease inhibitor in a 1:100 ratio. The cells were collected with a scraper and centrifuged at 15,000 × g at 4°C. The supernatant was collected and the protein concentration was measured. The pellets were frozen at −80°C until loading on the gel (20 μg/lane). Polyacrylamide gel electrophoresis and western blotting reagents were supplied by Lonza, Inc. (Allendale, NJ, USA), and all the related procedures followed the company's protocol. The catalog numbers for the reagents and the suppliers are listed below for convenience, although they were reported in our previous communication (8). PAGEr™ Gold Precast Gels (59502; 10% Tris-Glycine; Lonza); ECL reagent (RPN2109; GE Healthcare, Little Chalfont, UK); Western Blocker solution (W0138; Sigma-Aldrich); ProSieve QuadColor Protein marker (4.6–300 kDa, 00193837; Lonza); 20X Reducing Agent for ProSieve ProTrack Dual Color Loading buffer (00193861; Lonza); ProTrack Loading buffer (00193861; Lonza); ProSieve ProTrack Dual Color Loading buffer EX running buffer (00200307; Lonza); ProSieve EX Western Blot Transfer buffer (00200309; Lonza); Immobilon®-P PVDF Membranes (P4188; Sigma-Aldrich).

Immunoblot antibodies

Rabbit polyclonal anti-TLR6 (ab37072), MW 92 kDa (Abcam); rabbit anti-TLR1 cell (2209), MW 86 kDa (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TLR1 (ab68158), MW 90 kDa (Abcam); mouse anti-TLR2 [TL2.1], (ab9100), MW 90 kDa (Abcam); Mouse anti-TLR3 (TLR3.7) (sc-32232), MW 104 kDa (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-TLR4 (25) (sc-293072), MW 95-120 kDa (Santa Cruz Biotechnology); mouse anti-TLR5 (19D759.2), (sc-57461), MW 110-120 kDa (Santa Cruz Biotechnology); rabbit anti-TLR7, (5632), MW 140 kDa (Cell Signaling Technology); mouse TLR 8 (9A6), (sc-135584), MW 119.8 kDa (Santa Cruz Biotechnology); rabbit anti-TLR9, (5845), MW 130 kDa (Cell Signaling Technology); mouse TLR10 (2A11), sc-293300, MW 90 kDa (Santa Cruz Biotechnology); mouse anti-tubulin, (T5168; Sigma-Aldrich); rabbit anti-TRIF/TICAM1, NBP2-31189, MW 75 kDa (Novus Biologicals, Littleton, CO, USA); mouse anti-TICAM2, MW 21 kDa (Santa Cruz Biotechnology); rabbit anti-TRAF6 (3566R-100), MW 54 kDa (BioVision, Inc., Milpitas, CA, USA); goat anti-rabbit IgG, HRP-linked (7074; Cell Signaling Technology); anti-mouse IgG, HRP-linked (7076; Cell Signaling Technology).

Lead Hunter discovery services (DiscoveRx)

Nuclear Hormone Receptor Assays: PathHunter® NHR Protein Interaction (Pro) and Nuclear Translocation (NT) assays monitor the activation of a nuclear hormone receptor in a homogeneous, non-imaging assay format using a technology developed by DiscoveRx called enzyme fragment complementation (EFC). The company described NHR Pro assay detects of protein-protein interactions between an actvated NHR protein and a nuclear fusion protein containing steroid receptor co-activator peptide (SRCP). When bound by ligand, the NHR will migrate to the nucleus and recruit the SRCP domain, whereby complementation occurs, generating a unit of active β-galactosidase (β-gal) and production of chemiluminescent signal.

Arrestin pathway

The PathHunter® β-arrestin assay based on activation of a GPCR using a method developed by DiscoveRx called enzyme fragment complementation (EFC) with β-galactosidase (β-gal) as the functional reporter (44). In brief, according to the manufacturer's protocol: the enzyme is split into two inactive complementary portions (EA for enzyme acceptor and ED for enzyme donor) expressed as fusion proteins in the cell. EA is fused to β-arrestin and ED is fused to the GPCR of interest. When the GPCR is activated and β-arrestin is recruited to the receptor, ED and EA complementation occurs, restoring β-gal activity which is measured using chemiluminescent PathHunter® detection reagents.

Data analysis

The GPCR max panel % agonist was calculated as 100% (mean of test samples - mean of vehicle control)/mean Max control ligand - mean of vehicle control). For antagonist mode assays, percentage inhibition was calculated using the following formula: % Inhibition =100% × (1 – (mean RLU of test sample − mean RLU of vehicle control)/(mean RLU of EC80 control − mean RLU of vehicle control). For the orphan max panel, % agonist activity was calculated as 100% × (mean of test sample − mean of vehicle control)/mean of vehicle control.

gpcrMAX and NHR - Agonist mode calculation

To determine if a compound is potentially acting as an agonist to activate the receptor and induce arrestin recruitment the following factors should be considered: Is the % activity >30%? If so, is the compound mean RLU >Baseline RLU + 3 × Baseline SD.

gpcrMAX and NHR - Antagonist mode

Inhibition of GPCR activation by a compound acting as an antagonist of ligand binding results in a decrease in β-arrestin recruitment to the target GPCR. The NHR panel measures agonist interactions during a 6-h period and antagonist are preincubated for 1 h prior to agonist challenge. To determine if a compound is potentially acting as an antagonist to inhibit receptor activation the following factors should be considered: Is the % inhibition >35%, if so, is the compound mean RLU <EC80 RLU − 3 × EC80 SD.

orphanMAX - Agonist mode

Activation of Orphan GPCR by a compound acting as an agonist will result in an increase in β-arrestin recruitment to the target orphan GPCR. To determine if a compound is potentially acting as an agonist to activate an orphan receptor and induce arrestin recruitment the following factor should be considered: Is the % activity >50%. If so, is the compound mean RLU >Baseline RLU + 3 × Baseline SD.

TriCEPS technology

This technology from Dualsystems Biotech AG (Zurich, Switzerland) was implemented to detect PRP-1 receptor or interacting partners. Specific cell surface protein receptors are involved in the drug, peptide ligand mediated physiological responses. The TriCEPS method is based on the Ligand-based receptor capture (LRC) technology where special reagent can be coupled to a ligand of interest, which allows to capture the ligand when bound to corresponding receptors. One can picture TriCEPS with three arms: one that binds to amino group containing ligands, a second for the ligand-based capture of glycosylated and a third one with biotin tag for purifying receptor peptides to be analyzed by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., insulin) receptor.

TriCEPS protocol: Ligand coupling

This procedure implemented processing of ligand and the identification of receptor candidates (3 ligand and 3 control samples, 300 μg control ligand or 300 μg ligand of interest to 120 μl of 25 mM HEPES pH 8.2, 1.5 μl (150 μg) TriCEPS v.3 was added to both reactions and mixed immediately by pipetting up and down using a 200 μl pipette, then incubated at 20°C under gentle agitation (350 rpm) in a ThermoMixer for 90 min. Cell preparation and oxidation 1.2×108 cells were utilized for the experiment in triplicates. Cells were centrifuged at 300 × g for 5 min at 4°C, then were resuspended in 49 ml LRC buffer. The oxidation agent 1 ml (75 mM sodium metaperiodate) were added to a final concentration of 1.5 mM metaperiodate, followed by incubation for 15 min at 4°C. The mild oxidant sodium metaperiodate generates aldehydes from carbohydrates that link to the proteins of cell surface. When the ligand binds to the receptor, the hydrazine group formed a bond with the aldehyde for protein labeling.

Mass spectrometry

The LRC-TriCEPS samples were analyzed on a Thermo LTQ Orbitrap XL spectrometer. The samples were processed in data dependent acquisition mode in a 90-min gradient with 10 cm C18 packed column. The statistical ANOVA model was applied to the six remaining samples in the CaptiRec dataset. With models of Gaussian distribution the system tests each protein for differential abundance in all pairwise comparisons of ligand and control samples and calculates P-values. P-values are undergoing multiple comparisons to control the experiment-wide false discovery rate (FDR). Then, this adjusted P-value from each individual protein is plotted against the magnitude of the fold enrichment between the two experimental conditions. The receptor candidate space is defined based on the criteria where the area in the volcano plot that is limited by an enrichment factor of ≥4-fold and an FDR-adjusted P≤0.01.

RT2 qPCR primer assays

These custom designed assays by Qiagen (Valencia, CA, USA) served as sensitive gene expression profiling tool for real-time PCR analyses. Assay utilized RT2 SYBR-Green qPCR Master Mixes. Mature RNA isolated using RNA extraction according to the manufacturer's instructions. RNA quality was determined using a spectrophotometer and was reverse transcribed using a cDNA conversion. The cDNA in combination with RT2 SYBR-Green qPCR Master Mix (cat. no. 330529) was used with RT2 qPCR assays. Ct values were uploaded on web portal at http://www.qiagen.com/geneglobe. Samples were assigned to control and test groups. Ct values were normalized based on a manual selection of reference genes. The data analysis web portal calculates fold change/regulation using ∆∆Ct method, in which ∆Ct is calculated between gene of interest (GOI) and an average of housekeeping genes (HKG) followed by ∆∆Ct calculations [∆Ct(experiment) − ∆Ct(control)]. Fold change is then calculated using the 2−∆∆Ct formula.

Results

PRP-1 receptors are not G protein coupled, neither nuclear nor orphan receptors

The search for PRP-1 binding partners started with DiscoverX platform of G protein coupled receptors, (GPCR) in agonist, (Table I) and antagonist modes (Table II). There was no indication that PRP-1 was G protein coupled, neither that this peptide was agonist for orphan receptors (Table III) or agonist/antagonist for nuclear receptor in nhrMax panels (Table IV). MUC5B was identified as PRP-1 receptor binding partner in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. We proceeded further in the attempt to identify binding partners for PRP-1 using TriCEPS Ligand-receptor capture (LRC) technology from Dualsystems Biotech AG (45). LRC was used to identify novel ligand-receptor interactions. After the TriCEPS coupled ligand (PRP-1) bound to its targets in or at the cell membrane the second arm of TriCEPS coupled to the glycans of that target receptor. The third arm of TriCEPS was used to isolate the proteins that are bound to TriCEPS (Fig. 1A). In the next step the isolated proteins were subjected to a trypsin digest. The resulting peptides of the digest were identified and quantified using liquid chromatography, tandem mass spectrometry (LC-MS/MS) (45,46). Then, the quantified peptides from the control reaction (transferrin as ligand) were compared to the ligand of interest (PRP-1) reaction (labelled in the volcano plot as peptide). The proteins that were 4-fold enriched in one of the treatments compared to the other treatments were considered as the binding partners of the ligand used. When the cells were treated with TriCEPS coupled transferrin, the transferrin receptor protein (TFR1) was enriched (left side of the volcano plot), whereas in the ligand of interest treated samples the MUC5B was enriched. Thus, MUC5B was identified as receptor for PRP-1 (Fig. 1B). The data of the experiment was presented in biological triplicates. The P-value obtained for every protein was plotted against the log2 of the magnitude of the fold enrichment. The space for positive control receptors and high-confidence receptor candidates was designated and visualized based on (fold-change >4) significant enrichment (adjusted P<0.01). True positive receptor candidates that contain only few tryptic peptides can be enriched substantially but will rarely get adjusted P<0.01. For the final selection of receptor candidates for follow-up investigations, all proteins in the receptor space should be viewed based on the following parameters: proteins that were increased >4 times (log2=2 on the x-axis) with adjusted P<0.01 [−log (0.01)=2] were considered to be in the receptor space (white). The protter figure (Fig. 1C) usually displays peptides belonging to the ligand's receptor as being enriched (shown in highlighted stripes) compared with the control sample (no ligand). Protter is an interactive tool for protein data analysis (47). The experimental results with MUC5B ELISA confirmed the binding, PRP-1 at 1 μg/ml detected MUC5B presence at 440 ng/ml in the cell lysates of human JJ012 chondrosarcoma cells (Fig. 1D). Toll like receptors TLR1/2 and TLR6 were identified by western blot as binding interaction partners with PRP-1 in human chondrosarcoma JJ012 cell line lysates. Polyacrylamide gel electrophoresis, immunoblot results indicated that PRP-1 caused strong upregulation of TLR1 and TLR2 in comparison to untreated control. TLR3 expression was present but weak and data is not shown. TLR4 and TLR5 were expressed, but PRP-1 did not have any effect (Fig. 2). TLR6 protein expression was also increased in dose-dependent manner in PRP-1 treated samples. TLR7 was not expressed at all in the cell line, whereas TLR8, 9 and 10 were expressed but there was no indication of PRP-1 effect (Fig. 2). Thus, TLR1/2 and TLR6 were identified as interacting binding partners for PRP-1. Fig. 2 depicts the PRP-1 action on the protein expression of the adaptors TICAM1 (TRIF) and TICAM2 (TRAM). PRP-1 upregulated in dose-dependent manner the adaptor TICAM2 but not TICAM1. Toll like receptors TLR1/2 and TLR6 and MUC5B were detected with PRP-1 in the nucleus of human chondrosarcoma cells by immunocytochemistry. Since the experiments with immunoblot and TriCEPS indicated that cell surface receptors TLR1/2, TLR6 and gel forming mucin MUC5B were the binding partners for PRP-1, the immunocytochemistry experiments followed next; not only to prove PRP-1 endogenous presence in chondrosarcoma, but also its cellular colocalization with the binding partners. The images are displayed in Fig. 3. PRP-1 antibody, isolated from rabbit serum and affinity chromatography purified was labeled with Zenon Alexa Fluor 488 IgG complex (green) and manifested its presence in the nucleus (labeled with DAPI in blue) of the chondrosarcoma cells (Fig. 3A). The green speckles and dots can be seen both inside and outside the nucleus. In the separate experiment without PRP-1 we have demonstrated that MUC5B is present in the nucleus of these cells as well (Fig. 3B). The plasma membrane is seen in red and MUC5B, which was labeled with DyLight 488 is green. The composite image (Fig. 3C) demonstrates nuclear localization of both MUC5B (left panel) and PRP-1 (right panel) in aqua green color on the background of blue nucleus. Fig. 3D illustrates the presence of TLR1 receptor, (labeled in yellow with H&L DyLight 550) in the nucleus and around it. Fig. 3E depicts colocalization experiment of TLR6 and PRP-1 and whereas it was problematic to show TLR1 and PRP-1 colocalization in the previous figure due to spectral overlaps, here TLR6 receptor nuclear and cytoplasmic localization is demonstrated with PRP-1, which was stained with Zenon Alexa Fluor 488 IgG (green). H&L DyLight 550 was used as a secondary antibody (yellow) for TLR6. The immunoblot experiment with the nucleolin antibody, marker for nucleoli indicated that PRP-1 was not located in the nucleoli, as no changes in nucleolin protein expression was observed on PRP-1 treatment (Fig. 3F).

Figure 3

Immunocytochemistry results indicating nuclear localization of PRP-1 and nuclear colocalization with MUC5B, TLR1, TLR6 in human chondrosarcoma JJ012 cell line. (A) PRP-1 antibody localization in nucleus of human chondrosarcoma JJ012 cells. Dark blue color-DAPI was applied for nuclear staining. Zenon Alexa fluor rabbit 488IgG (green) was used for PRP-1 rabbit serum IgG antibody staining. (B) MUC5B receptor localization in the nucleus of human JJ012 chondrosarcoma cells. Alexa Fluor 594 wheat germ agglutinin (WGA) was used to label plasma membrane (red) at 1:200 dilution for 1 h. DAPI (dark blue) stained nucleus at 3 μM. Rabbit anti-MUC5B was adopted as primary, and green goat anti-rabbit IgG H&L (DyLight 488) was used as a secondary antibody. (C) Composite image of nuclear localization of MUC5B (left panel) and PRP-1 antibody (right panel) in human JJ012 chondrosarcoma cells. Dark blue color-DAPI was applied for nuclear staining. Zenon Alexa Fluor rabbit 488IgG (green) was used for PRP-1 rabbit serum IgG antibody detection. Rabbit anti-MUC5B was adopted as primary, and green goat anti-rabbit IgG H&L (DyLight 488) was used as a secondary antibody. (D) TLR1 receptor localization in the cytoplasm and the nucleus in human chondrosarcoma JJ012 cells. TLR1 rabbit antibody was used as a primary, whereas goat anti-rabbit H&L (DyLight 550) was applied as the secondary antibody (yellow color). (E) TLR6 receptor nuclear and cytoplasmic colocalization with PRP-1 is demonstrated, H&L DyLight 550 was used as a secondary antibody (yellow). PRP-1 was stained with Zenon Alexa Fluor 488 IgG (green). (F) Western blot of nucleolin protein expression in human chondrosarcoma cell line. PRP-1 did not have any effect on nucleolin expression, indicating the absence of PRP-1 location in nucleoli. The band corresponded to MW of 77 kDa.

Table I

PRP-1 effect on GPCR receptors (agonist mode).

Table I

PRP-1 effect on GPCR receptors (agonist mode).

GPCR IDAssay modeConc (μM)Mean RLU% Activity
ADCYAP1R1Agonist62712001
ADORA3Agonist62137001
ADRA1BAgonist63729001
ADRA2AAgonist63125000
ADRA2BAgonist63154004
ADRA2CAgonist62963000
ADRB1Agonist61843001
ADRB2Agonist6188003
AGTR1Agonist64249002
AGTRL1Agonist64293001
AVPR1AAgonist6236000
AVPR1BAgonist6352000
AVPR2Agonist68228000
BDKRB1Agonist6301001
BDKRB2Agonist66636000
BRS3Agonist62093000
C3AR1Agonist6559000
C5AR1Agonist61194000
C5L2Agonist61648000
CALCRAgonist6425001
CALCRL-RAMP1Agonist6928000
CALCRL-RAMP2Agonist62192001
CALCRL-RAMP3Agonist64252000
CALCR-RAMP2Agonist61395002
CALCR-RAMP3Agonist6286007
CCKARAgonist6446000
CCKBRAgonist68948000
CCR10Agonist6934000
CCR1Agonist65407007
CCR2Agonist6670000
CCR3Agonist62721002
CCR4Agonist61803000
CCR5Agonist6898000
CCR6Agonist61410000
CCR7Agonist67662001
CCR8Agonist6359000
CCR9Agonist61193001
CHRM1Agonist611811001
CHRM2Agonist6546001
CHRM3Agonist61663002
CHRM4Agonist678790016
CHRM5Agonist629951005
CMKLR1Agonist6819000
CNR1Agonist6800000
CNR2Agonist6315400−2
CRHR1Agonist63614001
CRHR2Agonist61611000
CRTH2Agonist61726000
CX3CR1Agonist63427001
CXCR1Agonist62199000
CXCR2Agonist61652001
CXCR3Agonist63879001
CXCR4Agonist6725002
CXCR5Agonist62309001
CXCR6Agonist6277002
CXCR7Agonist61945000
DRD1Agonist6730000
DRD2LAgonist6835000
DRD2SAgonist62472000
DRD3Agonist64147002
DRD4Agonist6228003
DRD5Agonist6210001
EBI2Agonist61501000
EDG1Agonist61655000
EDG3Agonist68779000
EDG4Agonist62343004
EDG5Agonist61743001
EDG6Agonist6574100−2
EDG7Agonist61544000
EDNRAAgonist6385000
EDNRBAgonist6689000
F2RAgonist6470700−2
F2RL1Agonist65662000
F2RL3Agonist6909900−1
FFAR1Agonist65608003
FPR1Agonist611339004
FPRL1Agonist6639000
FSHRAgonist6197900−2
GALR1Agonist62634001
GALR2Agonist63077001
GCGRAgonist62956000
GHSRAgonist65246002
GIPRAgonist617500−1
GLP1RAgonist61235000
GLP2RAgonist61014001
GPR1Agonist6584000
GPR103Agonist6451002
GPR109AAgonist64582004
GPR109BAgonist64104001
GPR119Agonist62893003
GPR120Agonist6278001
GPR35Agonist62871001
GPR92Agonist62576001
GRPRAgonist6390000
HCRTR1Agonist6456000
HCRTR2Agonist6699000
HRH1Agonist63456001
HRH2Agonist6913001
HRH3Agonist6471002
HRH4Agonist69103004
HTR1AAgonist68790000
HTR1BAgonist61214900−4
HTR1EAgonist629633003
HTR1FAgonist63459002
HTR2AAgonist64551001
HTR2CAgonist69216001
HTR5AAgonist69900000
KISS1RAgonist6458001
LHCGRAgonist6247000
LTB4RAgonist61873001
MC1RAgonist617000−2
MC3RAgonist6251003
MC4RAgonist625900−1
MC5RAgonist6549002
MCHR1Agonist61403001
MCHR2Agonist6628001
MLNRAgonist62278001
MRGPRX1Agonist68696001
MRGPRX2Agonist63558000
MTNR1AAgonist6791002
NMBRAgonist6628000
NMU1RAgonist6981001
NPBWR1Agonist6696003
NPBWR2Agonist61694001
NPFFR1Agonist61311003
NPSR1BAgonist6899002
NPY1RAgonist61061000
NPY2RAgonist63620000
NTSR1Agonist63134000
OPRD1Agonist6912000
OPRK1Agonist6364000
OPRL1Agonist62296000
OPRM1Agonist61370001
OXER1Agonist686900−2
OXTRAgonist6258000
P2RY1Agonist6123800−1
P2RY11Agonist6689001
P2RY12Agonist62505001
P2RY2Agonist63845002
P2RY4Agonist6397000−1
P2RY6Agonist63242000
PPYR1Agonist6349000
PRLHRAgonist6359004
PROKR1Agonist6496002
PROKR2Agonist6157000
PTAFRAgonist64809001
PTGER2Agonist6308004
PTGER3Agonist62467001
PTGER4Agonist6957001
PTGFRAgonist6164000
PTGIRAgonist61351001
PTHR1Agonist61291001
PTHR2Agonist61231000
RXFP3Agonist61490006
SCTRAgonist65026001
SSTR1Agonist615000−3
SSTR2Agonist6118000
SSTR3Agonist6820001
SSTR5Agonist61769001
TACR1Agonist67021001
TACR2Agonist64151001
TACR3Agonist61458000
TBXA2RAgonist62033001
TRHRAgonist6276001
TSHR(L)Agonist696004
UTR2Agonist6312003
VIPR1Agonist63998000
VIPR2Agonist63423001

Table II

PRP-1 effect on GPCR receptors (antagonist mode).

Table II

PRP-1 effect on GPCR receptors (antagonist mode).

GPCR IDAssay modeConc (μM)Mean RLU% Inhibition
ADCYAP1R1Antagonist61676100−6
ADORA3Antagonist6891300−3
ADRA1BAntagonist620762000
ADRA2AAntagonist610757000
ADRA2BAntagonist68561002
ADRA2CAntagonist61511200−4
ADRB1Antagonist6674600−5
ADRB2Antagonist6179900−3
AGTR1Antagonist625685000
AGTRL1Antagonist622636000
AVPR1AAntagonist6789300−1
AVPR1BAntagonist6258900−2
AVPR2Antagonist634870000
BDKRB1Antagonist6225900−8
BDKRB2Antagonist642939005
BRS3Antagonist612488004
C3AR1Antagonist61933800−2
C5AR1Antagonist620293003
C5L2Antagonist6547000−5
CALCRAntagonist63582001
CALCRL-RAMP1Antagonist613950004
CALCRL-RAMP2Antagonist6892100−1
CALCRL-RAMP3Antagonist622936001
CALCR-RAMP2Antagonist66982000
CALCR-RAMP3Antagonist659000−8
CCKARAntagonist61490300−3
CCKBRAntagonist63697600−3
CCR10Antagonist61295800−3
CCR1Antagonist61214200−3
CCR2Antagonist61380800−3
CCR3Antagonist61136200−3
CCR4Antagonist620751002
CCR5Antagonist62359300−2
CCR6Antagonist614877002
CCR7Antagonist63413600−2
CCR8Antagonist61376000−4
CCR9Antagonist61538300−6
CHRM1Antagonist62892100−12
CHRM2Antagonist6540500−6
CHRM3Antagonist61187400−6
CHRM4Antagonist61390200−18
CHRM5Antagonist64585200−4
CMKLR1Antagonist63258400−2
CNR1Antagonist63773002
CNR2Antagonist65805005
CRHR1Antagonist641038001
CRHR2Antagonist62968600−2
CRTH2Antagonist61149200−8
CX3CR1Antagonist632070002
CXCR1Antagonist63818300−1
CXCR2Antagonist6565700−3
CXCR3Antagonist61392600−1
CXCR4Antagonist61397002
CXCR5Antagonist61098600−8
CXCR6Antagonist6101400−6
CXCR7Antagonist62856200−2
DRD1Antagonist6696300−5
DRD2LAntagonist63938002
DRD2SAntagonist612420006
DRD3Antagonist61263000−15
DRD4Antagonist665500−2
DRD5Antagonist6149600−8
EBI2Antagonist62425000−1
EDG1Antagonist69367000
EDG3Antagonist645485001
EDG4Antagonist66575005
EDG5Antagonist62180100−8
EDG6Antagonist611692004
EDG7Antagonist61432500−3
EDNRAAntagonist69533000
EDNRBAntagonist61248000−1
F2RAntagonist61612200−16
F2RL1Antagonist636381003
F2RL3Antagonist63027600−1
FFAR1Antagonist610705000
FPR1Antagonist63077200−4
FPRL1Antagonist62991600−2
FSHRAntagonist6621700−2
GALR1Antagonist61759100−4
GALR2Antagonist61686800−11
GCGRAntagonist63115100−4
GHSRAntagonist62068100−6
GIPRAntagonist679600−24
GLP1RAntagonist61983100−9
GLP2RAntagonist6727800−11
GPR1Antagonist61076400−5
GPR103Antagonist61038006
GPR109AAntagonist61141200−4
GPR109BAntagonist62871700−6
GPR119Antagonist6521500−1
GPR120Antagonist6130400−6
GPR35Antagonist6910400−6
GPR92Antagonist685450012
GRPRAntagonist61570000−7
HCRTR1Antagonist632423000
HCRTR2Antagonist62611400−1
HRH1Antagonist619122000
HRH2Antagonist63478001
HRH3Antagonist6180200−6
HRH4Antagonist62264500−9
HTR1AAntagonist62576600−5
HTR1BAntagonist623344004
HTR1EAntagonist654870005
HTR1FAntagonist69825002
HTR2AAntagonist63103200−5
HTR2CAntagonist64188400−3
HTR5AAntagonist645365000
KISS1RAntagonist62709003
LHCGRAntagonist6144000−15
LTB4RAntagonist618448000
MC1RAntagonist669300−2
MC3RAntagonist6153200−6
MC4RAntagonist6128200−6
MC5RAntagonist6179900−5
MCHR1Antagonist61023000−4
MCHR2Antagonist6530900−3
MLNRAntagonist62032800−7
MRGPRX1Antagonist63890200−2
MRGPRX2Antagonist61945500−8
MTNR1AAntagonist6241200−9
NMBRAntagonist6720100−5
NMU1RAntagonist6985900−3
NPBWR1Antagonist61882000
NPBWR2Antagonist61070300−3
NPFFR1Antagonist6290800−3
NPSR1BAntagonist66999000
NPY1RAntagonist69736004
NPY2RAntagonist63477300−2
NTSR1Antagonist62192400−1
OPRD1Antagonist6795800−2
OPRK1Antagonist6215800−12
OPRL1Antagonist61062000−7
OPRM1Antagonist62828200−2
OXER1Antagonist6246400−16
OXTRAntagonist65400001
P2RY1Antagonist65306001
P2RY11Antagonist6484300−12
P2RY12Antagonist66996008
P2RY2Antagonist61093000−4
P2RY4Antagonist613232000
P2RY6Antagonist618256003
PPYR1Antagonist6276600−4
PRLHRAntagonist6122100−3
PROKR1Antagonist6499800−2
PROKR2Antagonist61115008
PTAFRAntagonist63532600−8
PTGER2Antagonist672100−11
PTGER3Antagonist6967100−2
PTGER4Antagonist68524004
PTGFRAntagonist64530001
PTGIRAntagonist6380600−2
PTHR1Antagonist62941200−2
PTHR2Antagonist62882300−1
RXFP3Antagonist6330200−3
SCTRAntagonist63424900−2
SSTR1Antagonist638300−14
SSTR2Antagonist6797000−11
SSTR3Antagonist6771000−5
SSTR5Antagonist61327400−11
TACR1Antagonist64862800−2
TACR2Antagonist623311000
TACR3Antagonist62745900−1
TBXA2RAntagonist61032900−10
TRHRAntagonist6301600−5
TSHR(L)Antagonist683000−4
UTR2Antagonist6156000−7
VIPR1Antagonist63344300−9
VIPR2Antagonist63604300−3

Table III

PRP-1 effect on orphan receptors (agonist mode).

Table III

PRP-1 effect on orphan receptors (agonist mode).

GPCR IDAssay modeConc (μM)Mean RLU% Inhibition
ADCYAP1R1Antagonist61676100−6
ADORA3Antagonist6891300−3
ADRA1BAntagonist620762000
ADRA2AAntagonist610757000
ADRA2BAntagonist68561002
ADRA2CAntagonist61511200−4
ADRB1Antagonist6674600−5
ADRB2Antagonist6179900−3
AGTR1Antagonist625685000
AGTRL1Antagonist622636000
AVPR1AAntagonist6789300−1
AVPR1BAntagonist6258900−2
AVPR2Antagonist634870000
BDKRB1Antagonist6225900−8
BDKRB2Antagonist642939005
BRS3Antagonist612488004
C3AR1Antagonist61933800−2
C5AR1Antagonist620293003
C5L2Antagonist6547000−5
CALCRAntagonist63582001
CALCRL-RAMP1Antagonist613950004
CALCRL-RAMP2Antagonist6892100−1
CALCRL-RAMP3Antagonist622936001
CALCR-RAMP2Antagonist66982000
CALCR-RAMP3Antagonist659000−8
CCKARAntagonist61490300−3
CCKBRAntagonist63697600−3
CCR10Antagonist61295800−3
CCR1Antagonist61214200−3
CCR2Antagonist61380800−3
CCR3Antagonist61136200−3
CCR4Antagonist620751002
CCR5Antagonist62359300−2
CCR6Antagonist614877002
CCR7Antagonist63413600−2
CCR8Antagonist61376000−4
CCR9Antagonist61538300−6
CHRM1Antagonist62892100−12
CHRM2Antagonist6540500−6
CHRM3Antagonist61187400−6
CHRM4Antagonist61390200−18
CHRM5Antagonist64585200−4
CMKLR1Antagonist63258400−2
CNR1Antagonist63773002
CNR2Antagonist65805005
CRHR1Antagonist641038001
CRHR2Antagonist62968600−2
CRTH2Antagonist61149200−8
CX3CR1Antagonist632070002
CXCR1Antagonist63818300−1
CXCR2Antagonist6565700−3
CXCR3Antagonist61392600−1
CXCR4Antagonist61397002
CXCR5Antagonist61098600−8
CXCR6Antagonist6101400−6
CXCR7Antagonist62856200−2
DRD1Antagonist6696300−5
DRD2LAntagonist63938002
DRD2SAntagonist612420006
DRD3Antagonist61263000−15
DRD4Antagonist665500−2
DRD5Antagonist6149600−8
EBI2Antagonist62425000−1
EDG1Antagonist69367000
EDG3Antagonist645485001
EDG4Antagonist66575005
EDG5Antagonist62180100−8
EDG6Antagonist611692004
EDG7Antagonist61432500−3
EDNRAAntagonist69533000
EDNRBAntagonist61248000−1
F2RAntagonist61612200−16
F2RL1Antagonist636381003
F2RL3Antagonist63027600−1
FFAR1Antagonist610705000
FPR1Antagonist63077200−4
FPRL1Antagonist62991600−2
FSHRAntagonist6621700−2
GALR1Antagonist61759100−4
GALR2Antagonist61686800−11
GCGRAntagonist63115100−4
GHSRAntagonist62068100−6
GIPRAntagonist679600−24
GLP1RAntagonist61983100−9
GLP2RAntagonist6727800−11
GPR1Antagonist61076400−5
GPR103Antagonist61038006
GPR109AAntagonist61141200−4
GPR109BAntagonist62871700−6
GPR119Antagonist6521500−1
GPR120Antagonist6130400−6
GPR35Antagonist6910400−6
GPR92Antagonist685450012
GRPRAntagonist61570000−7
HCRTR1Antagonist632423000
HCRTR2Antagonist62611400−1
HRH1Antagonist619122000
HRH2Antagonist63478001
HRH3Antagonist6180200−6
HRH4Antagonist62264500−9
HTR1AAntagonist62576600−5
HTR1BAntagonist623344004
HTR1EAntagonist654870005
HTR1FAntagonist69825002
HTR2AAntagonist63103200−5
HTR2CAntagonist64188400−3
HTR5AAntagonist645365000
KISS1RAntagonist62709003
LHCGRAntagonist6144000−15
LTB4RAntagonist618448000
MC1RAntagonist669300−2
MC3RAntagonist6153200−6
MC4RAntagonist6128200−6
MC5RAntagonist6179900−5
MCHR1Antagonist61023000−4
MCHR2Antagonist6530900−3
MLNRAntagonist62032800−7
MRGPRX1Antagonist63890200−2
MRGPRX2Antagonist61945500−8
MTNR1AAntagonist6241200−9
NMBRAntagonist6720100−5
NMU1RAntagonist6985900−3
NPBWR1Antagonist61882000
NPBWR2Antagonist61070300−3
NPFFR1Antagonist6290800−3
NPSR1BAntagonist66999000
NPY1RAntagonist69736004
NPY2RAntagonist63477300−2
NTSR1Antagonist62192400−1
OPRD1Antagonist6795800−2
OPRK1Antagonist6215800−12
OPRL1Antagonist61062000−7
OPRM1Antagonist62828200−2
OXER1Antagonist6246400−16
OXTRAntagonist65400001
P2RY1Antagonist65306001
P2RY11Antagonist6484300−12
P2RY12Antagonist66996008
P2RY2Antagonist61093000−4
P2RY4Antagonist613232000
P2RY6Antagonist618256003
PPYR1Antagonist6276600−4
PRLHRAntagonist6122100−3
PROKR1Antagonist6499800−2
PROKR2Antagonist61115008
PTAFRAntagonist63532600−8
PTGER2Antagonist672100−11
PTGER3Antagonist6967100−2
PTGER4Antagonist68524004
PTGFRAntagonist64530001
PTGIRAntagonist6380600−2
PTHR1Antagonist62941200−2
PTHR2Antagonist62882300−1
RXFP3Antagonist6330200−3
SCTRAntagonist63424900−2
SSTR1Antagonist638300−14
SSTR2Antagonist6797000−11
SSTR3Antagonist6771000−5
SSTR5Antagonist61327400−11
TACR1Antagonist64862800−2
TACR2Antagonist623311000
TACR3Antagonist62745900−1
TBXA2RAntagonist61032900−10
TRHRAntagonist6301600−5
TSHR(L)Antagonist683000−4
UTR2Antagonist6156000−7
VIPR1Antagonist63344300−9
VIPR2Antagonist63604300−3
ADCYAP1R1Agonist62712001
ADORA3Agonist62137001
ADRA1BAgonist63729001
ADRA2AAgonist63125000
ADRA2BAgonist63154004
ADRA2CAgonist62963000
ADRB1Agonist61843001
ADRB2Agonist6188003
AGTR1Agonist64249002
AGTRL1Agonist64293001
AVPR1AAgonist6236000
AVPR1BAgonist6352000
AVPR2Agonist68228000
BDKRB1Agonist6301001
BDKRB2Agonist66636000
BRS3Agonist62093000
C3AR1Agonist6559000
C5AR1Agonist61194000
C5L2Agonist61648000
CALCRAgonist6425001
CALCRL-RAMP1Agonist6928000
CALCRL-RAMP2Agonist62192001
CALCRL-RAMP3Agonist64252000
CALCR-RAMP2Agonist61395002
CALCR-RAMP3Agonist6286007
CCKARAgonist6446000
CCKBRAgonist68948000
CCR10Agonist6934000
CCR1Agonist65407007
CCR2Agonist6670000
CCR3Agonist62721002
CCR4Agonist61803000
CCR5Agonist6898000
CCR6Agonist61410000
CCR7Agonist67662001
CCR8Agonist6359000
CCR9Agonist61193001
CHRM1Agonist611811001
CHRM2Agonist6546001
CHRM3Agonist61663002
CHRM4Agonist678790016
CHRM5Agonist629951005
CMKLR1Agonist6819000
CNR1Agonist6800000
CNR2Agonist6315400−2
CRHR1Agonist63614001
CRHR2Agonist61611000
CRTH2Agonist61726000
CX3CR1Agonist63427001
CXCR1Agonist62199000
CXCR2Agonist61652001
CXCR3Agonist63879001
CXCR4Agonist6725002
CXCR5Agonist62309001
CXCR6Agonist6277002
CXCR7Agonist61945000
DRD1Agonist6730000
DRD2LAgonist6835000
DRD2SAgonist62472000
DRD3Agonist64147002
DRD4Agonist6228003
DRD5Agonist6210001
EBI2Agonist61501000
EDG1Agonist61655000
EDG3Agonist68779000
EDG4Agonist62343004
EDG5Agonist61743001
EDG6Agonist6574100−2
EDG7Agonist61544000
EDNRAAgonist6385000
EDNRBAgonist6689000
F2RAgonist6470700−2
F2RL1Agonist65662000
F2RL3Agonist6909900−1
FFAR1Agonist65608003
FPR1Agonist611339004
FPRL1Agonist6639000
FSHRAgonist6197900−2
GALR1Agonist62634001
GALR2Agonist63077001
GCGRAgonist62956000
GHSRAgonist65246002
GIPRAgonist617500−1
GLP1RAgonist61235000

Table IV

PRP-1 activity with nhrMAX panel.

Table IV

PRP-1 activity with nhrMAX panel.

Assay formatAssay targetConc (μM)Value 1Value 2Average valueSD% Efficacy
AgonistAR6360032003400282.84−0.3
AntagonistAR6172001740017300141.424.3
AgonistERalpha6504005000050200282.840.6
AntagonistERalpha62936002882002909003818.4−2.8
AntagonistERRalpha66580061200635003252.7−4.7
Inverse agonistERRalpha613460012000012730010324−7.2
AgonistFXR64000620051001555.60.9
AntagonistFXR695400103400994005656.9−4.5
AgonistGR61520016800160001131.40.3
AntagonistGR69998001086200104300061094−9.7
AgonistLXRalpha623280021680022480011314−0.6
AntagonistLXRalpha6166000018076001733800104370−10.7
AgonistLXRbeta6311400330200320800132941.6
AntagonistLXRbeta6120820013792001293700120920−4
AgonistMR61620018000171001272.80.3
AntagonistMR62954003038002996005939.7−0.7
AgonistPPARalpha6146001500014800282.840.9
AntagonistPPARalpha61084001050001067002404.2−3.3
AgonistPPARdelta61077800124700011624001196400.9
AntagonistPPARdelta63209000288480030469002292402.2
AgonistPPARgamma6440058005100989.950.3
AntagonistPPARgamma6296002840029000848.536.5
AgonistPRalpha62580022800243002121.3−1.6
AntagonistPRalpha61854001904001879003535.5−3.1
AgonistPRbeta6320024002800565.691.4
AntagonistPRbeta62860033600311003535.5−0.6
AgonistRARalpha629400448003710010889−3.3
AntagonistRARalpha61040001068001054001979.914
AgonistRARbeta626360024360025360014142−3.9
AntagonistRARbeta647500057220052360068731−0.5
AgonistRXRalpha62260002300002280002828.4−3.8
AntagonistRXRalpha6851800821400836600214960
AgonistRXRgamma63678003542003610009616.7−2.7
AntagonistRXRgamma612666001310200128840030830−1.2
AgonistTHRalpha64340034600390006222.5−0.2
AntagonistTHRalpha638300041580039940023193−4.8
AgonistTHRbeta6732400819600776000616609.3
AntagonistTHRbeta612740001246600126030019375−5.2

[i] SD, standard deviation.

RT2 qPCR primer assays show the effect of PRP-1 on gene expression of TLR receptors and MUC5B

RT2 qPCR custom designed primer assays were performed by Qiagen to understand the effect of PRP-1 on gene expression of TLR receptors and MUC5B. Mature RNA was isolated using RNA extraction according to the manufacturer's instructions. RNA was subjected to spectrophotometrical quality control and then reverse transcribed to cDNA. RT2 SYBR-Green qPCR Master Mix was used with RT2 qPCR assays. In this study, 7 genes (TLR1, TLR2, TLR6, MUC5B, c-Myc and 2 housekeeping genes GAPDH and ACTB) were profiled on three samples with technical triplicates. c-Myc was included, as we wanted to confirm its drastic downregulation after PRP-1 treatment in luciferase assay (4) and western blot experiments, reported earlier (10). The heat map, clustergram of average Ct values across the gene of each sample, with the magnitude of gene expression scale below, is presented in Fig. 4. As evident from the figure, there is dose-dependent effect of PRP-1 on the expression of the above mentioned genes, except the control housekeeping genes. The TLR1 receptor was well expressed in the cells treated with 10 μg/ml, whereas no expression was detected at 1 μg/ml peptide treatment. TLR2, TLR6, MUC5B demonstrated high expression with 1 μg/ml treatment when compared to nontreated control. c-Myc expression went down drastically when treated with 10 μg/ml PRP-1. The data analysis web portal calculated fold change/regulation using ∆∆Ct method, in which ∆Ct is calculated between gene of interest (GOI) and an average of housekeeping genes (HKG) followed by ∆∆Ct calculations (∆Ct (experiment) − ∆Ct (control). Fold change is then calculated using the 2−∆∆Ct formula. The system detected only statistically significant upregulation (P<0.0001) of TLR2 for the samples treated with 1 μg/ml PRP-1, with 5.26-fold upregulation when calculated with the ∆∆Ct.

Discussion

Metastatic chondrosarcoma is fatal because of metastatic spread and absence of the effective therapies. Therefore, search for new approaches is of the utmost importance. PRP-1, inhibits chondrosarcoma cell growth by >80% (4,6) it halts cell cycle progression in G1/S transition (6). This mTORC1 inhibitor, cytostatic peptide is potent upregulator of tumor suppressors and inhibitor of oncoproteins and embryonic stem cell markers (79). We have demonstrated also that intracellular expression of PRP-1 is associated with the early stages of lymphocyte activation by phytohemagglutinin, (PHA) (Fig. 5). However, the interacting partners or receptors for this important peptide has not been identified. Using triCEPS (ligand based receptor capture technology), we were able to identify MUC5B, one of the members of mucin family as the receptor for PRP-1. Notably, proline rich proteins in saliva, different from the neuropeptide PRP-1, were found in reported literature to interact with mucins (48). Immunoblot results of this study indicated that TLR1, TLR2 (which are usually dimerized) and TLR6 are binding interaction partners for PRP-1, as their expression increased in dose-response manner upon PRP-1 treatment. Indeed, the link between TLR1/2 and TLR6 was documented in the literature. TLR1 and TLR6 was shown to pair with TLR2 and that interaction was needed for pattern recognition of pathogens (49,50). TLR7 was not expressed in human JJ012 cell line at all, but all the other TLR groups were present. No changes in the expression of TLR10 were observed with PRP-1, although sometimes it was reported that in certain cases TLR10 is able to homodimerize or heterodimerize with TLR1 and TLR2, but its ligand remains unknown (19). We have demonstrated that PRP-1 upregulates the expression of adaptor protein TICAM2 (TRAM) but not of TICAM1 (TRIF). Most TLRs share a common signaling pathway in which myeloid differentiation factor 88 (MyD88) plays a central role (51). It is known also that TLR2 can be TRAM dependent in addition to the MyD88-dependent pathway with certain MyD88 independent exceptions (51). TLR2 is also internalized following ligand binding, but in this case, MyD88-dependent signaling continues from an intracellular location away from the plasma membrane and stimulates type I IFN production through an as yet unknown mechanism (52). Due to the importance of TLR signaling in tumorigenesis, TLR agonists have potential for antitumor therapy (5357). Both TLR and mucins have important role in host defense mechanism, however, the link between two of them in non-infectious conditions and cancer pathology deserves attention. Understanding connecting crosstalk between two of them will open new avenues for therapeutic intervention. Cancer cells might use the TLR signaling pathways much in the same way to upregulate the expression of MUCs which in turn may also regulate TLR signaling (22). Mucins are a class of major differentially expressed proteins between normal and cancer cells, which makes them a potential target for anticancer therapies. As a class of glycoproteins, MUCs are recognized as potential markers of disease progression or inhibition (58) and are currently investigated as therapeutic targets for cancer (59). Our experimental results indicated the nuclear localization of both MUC5B and TLR1/2. Indeed the evidence of their nuclear translocation was reported in the literature (24,60). Due to the importance of TLR signaling in tumorigenesis, TLR agonists have potential for antitumor therapy (22,5458). The fact that PRP-1 has receptors of innate immunity explains observed antibacterial properties of PRP (61). The biochemical evidence for the direct interaction of TLRs or MUC5B with endogenous stimulators is limited. There is no doubt that it is of great significance to identify those ligands and elucidate their biological functions, especially if upon their binding with the ligand, the antiproliferative effect in tumor is manifested. The western blot analysis and immunocytochemistry data indicated upregulated protein expression of TLR1, TLR6, MUC5B after the treatment with PRP-1 in JJ012 human chondrosarcoma cell line, the custom designed RT2 qPCR primer assays proved that PRP-1 indeed has effect on expression levels of its interacting partner genes as well. However, depending on the method it showed some dose response differences. For example, in case of TLR2 the protein expression upregulation with 1 and 10 μg/ml PRP-1 was observed in dose-response manner in western blot experiments. However, the heat map and qRT-PCR ∆Ct calculations proved that the highest upregulation of TLR2 expression is taking place at 1 μg/ml (>5-fold upregulation). On the gene expression level, the qRT-PCR did not report significant fold change in ∆Ct for TLR1 or TLR6, whereas in western blot experiments we saw obvious upregulation of protein expression for these respective receptors after the dose response treatment with PRP-1. MUC5B on the heat map demonstrated the most upregulation after the treatment with 1 μg/ml, which coincided with MUC5B ELISA results, though TriCEPS technology detected MUC5B as binding partner with 10 μg/ml PRP-1 treatment. c-Myc results demonstrated downregulation of its gene expression in dose-response manner with PRP-1, being downregulated very strongly at 10 μg/ml, which is concordant with our previous results (10). Despite these differences, it is important to mention that most of inhibitory responses caused by PRP-1 treatment on cell growth of tumor cell lines or upregulation of tumor suppressors and down-regulation of oncoproteins, were maximally observed when treated with 1–20 μg/ml PRP-1 range. PRP-1 is a compound naturally produced in the body and the fact it was detected in the nucleus of chondrosarcoma cells and that it upregulated TLR1/2 dimer and TLR6 possibly indicates PRP-1 as an endogenous ligand.

The ability of TLRs to recognize endogenous stimulators appears to be essential to their function in regulating noninfectious (sterile) inflammation. TLR-induced innate immune responses regulate non-infectious sterile inflammation and subsequently, adaptive immune response. The endogenous TLR ligands and their receptors can be localized in different cellular compartments and cannot interact physiologically. However, when the tissue is injured, the passive release of endogenous ligand or its active transport utilizing non-conventional lysosomal route. In the present study, we were able to identify the pattern recognition receptors of adaptive immunity TLR1, TLR2 and TLR6, and secreted mucin MUC5B as binding partners for cytostatic PRP-1 peptide. The mentioned results allow to understand the immunomodulatory, antibacterial effect of PRP-1, reported by our group before (3,42,61). From oncologic standpoint it is important information that immune receptors play antitumorigenic role when bound to PRP-1 ligand.

Acknowledgments

The present study was supported in part by a gift from the Ratcliffe Foundation to Miami Center of Orthopedic Research and Education. We would like to thank Qiagen Inc., Service Core for the qRT-PCR experiments, the Analytical Imaging Core Facility at DRI/SCCC, University of Miami, which provided immunocytochemistry imaging service. Our thanks to Ms. Maria Boulina for the help and guidance with the imaging. We would like to thanks Dr Paul Helbling and Dr Florian Marty from Dual Systems Biotech (Zurich), Switzerland, for their productive collaboration and triCEPS methodology experiments.

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January-2018
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Spandidos Publications style
Galoian K, Abrahamyan S, Chailyan G, Qureshi A, Patel P, Metser G, Moran A, Sahakyan I, Tumasyan N, Lee A, Lee A, et al: Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma. Int J Oncol 52: 139-154, 2018
APA
Galoian, K., Abrahamyan, S., Chailyan, G., Qureshi, A., Patel, P., Metser, G. ... Galoyan, A. (2018). Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma. International Journal of Oncology, 52, 139-154. https://doi.org/10.3892/ijo.2017.4199
MLA
Galoian, K., Abrahamyan, S., Chailyan, G., Qureshi, A., Patel, P., Metser, G., Moran, A., Sahakyan, I., Tumasyan, N., Lee, A., Davtyan, T., Chailyan, S., Galoyan, A."Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma". International Journal of Oncology 52.1 (2018): 139-154.
Chicago
Galoian, K., Abrahamyan, S., Chailyan, G., Qureshi, A., Patel, P., Metser, G., Moran, A., Sahakyan, I., Tumasyan, N., Lee, A., Davtyan, T., Chailyan, S., Galoyan, A."Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma". International Journal of Oncology 52, no. 1 (2018): 139-154. https://doi.org/10.3892/ijo.2017.4199