2.1 Animals and tissue collection

To obtain timed utero-placental tissues, sexually mature Sprague Dawley female rats were caged overnight with fertile males. Day 0.5 of pregnancy was designated by the presence of sperm in the vaginal smear. Utero-placental tissues were collected from pregnant females on different days of pregnancy. Tissues were snap-frozen in liquid nitrogen for RNA and protein isolation and in dry ice-cooled hexane for cryo-sectioning. The IICB Animal Care and Use Committee approved all procedures for handling and experimentation with rodents in accordance with the regulations set forward by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India (http://cpcsea.nic.in).

2.2 Culture of A7r5 and HEK293T cell lines

Rat smooth muscle cell line, A7r5, was obtained from ATCC (USA). A7r5 cells were cultured in DMEM high glucose basal media (D7777, Sigma) supplemented with 10% FBS and 1% penicillin–streptomycin (Invitrogen). HEK293T were obtained from ATCC (USA). HEK293T cells were cultured in DMEM (D7777, Sigma) supplemented with 10% FBS, 2 mM l-glutamine and 1% penicillin–streptomycin (Invitrogen). All the cells were cultured in the presence of 5% CO₂ at 37°C in a humidified incubator.

2.3 Isolation of rat primary trophoblast cells and coculture with VSMC

Junctional zones, isolated from rat placentas on E16.5, were thoroughly washed with sterile DPBS and minced into fine pieces. They were further minced in the presence of 0.05% Trypsin-EDTA (0.5 mL) for 2 min. Then 4.5 mL 0.05% Trypsin-EDTA was added and the suspension was incubated in the presence of 5% CO₂ at 37°C for 15 min. Following incubation, the cell suspension was pipetted vigorously until a slurry-like appearance was obtained. Trypsin neutralization was then performed using 5 mL trophoblast media (RPMI-1640 (Sigma-Aldrich) supplemented with 20% FBS (Invitrogen), 1% penicillin–streptomycin (Invitrogen), 1% Glutamax (Invitrogen), 1 mM sodium pyruvate (Sigma-Aldrich), and 100 µM β-mercaptoethanol (Sigma-Aldrich)). Cells were then washed three times with DPBS and the pellet was resuspended in 5 mL RBC lysis buffer (1.5 M NH₄Cl, 140 mM NaHCO₃, 10 mM EDTA, pH 7.3), incubated for 5 min at room temperature. Ten milliliters of DPBS were added to the cell suspension and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in fresh TS media and strained using 70 µm cell strainer (cc, USA) to obtain single-cell suspension. 2 × 10⁵ cells were plated on each culture insert (BD Falcon), which were placed on companion plates (BD Falcon) containing 24 h culture of A7r5 cells. After 48 h of co-culture RNA and protein was isolated from the A7r5 cells. Protein from the primary trophoblast cells was extracted on the day of isolation.

2.4 RNA isolation, reverse transcription, and real-time PCR analysis

Total RNA from tissues or cells was isolated using TRIzol reagent (Invitrogen), as per the manufacturer's protocol. Tissue samples were homogenized and cultured cells were scraped in ice-cold Trizol. Phase separation was done by adding chloroform. RNA was precipitated from the aqueous phase using isopropanol, followed by 70% ethanol wash. The RNA pellet was dissolved in nuclease-free DEPC-treated water. First-strand cDNA was synthesized with 2.5 µg of total RNA using M-MLV Reverse Transcriptase enzyme using the standard protocol (Invitrogen).

Ten-fold dilution of cDNAs and Power SYBR GREEN PCR Master Mix (Applied Biosystems) was used in the real-time PCR reaction. Reactions were run using a 7500 Real-Time PCR System (Applied Biosystems). Conditions included an initial holding stage (95°C for 10 min), then 40 cycles (95°C for 15 s and 60°C for 1 min) followed by a dissociation stage (95°C for 15 s, 60°C for 1 min, and then 95°C for 30 s). Primers used for real-time PCR are listed in Table 1. The amount of a specific mRNA was normalized relative to the amount of rpL7 (∆Ct = Ct_gene − Ct_rpL7). Fold change of gene expression was measured by using $<math altimg="urn:x-wiley:00219541:media:jcp31130:jcp31130-math-0001" wiley:location="equation/jcp31130-math-0001.png" xmlns="http://www.w3.org/1998/Math/MathML"><mrow><mrow><msup><mn>2</mn><mrow><mo>\unicode{x02010}</mo><mo>\unicode{x02206}</mo><mo>\unicode{x02206}</mo><mi>Ct</mi></mrow></msup></mrow></mrow></math>$f, where ∆∆Ct denoted the change in ∆Ct values between samples and reference sample. At least three different biological replicates were used. Error bars represent the standard error of the mean from three different biological replicates.

2.5 Protein isolation, estimation, and western blot analysis

Tissue samples were homogenized and cells were scraped in ice-cold RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 0.2 mM PMSF, and 1 mM sodium orthovanadate) supplemented with protease inhibitor cocktail (Cell Signalling Technology). The homogenate was then centrifuged at 14,000 rcf for 10 min at 4°C and the supernatants were collected, aliquoted, and stored at −80°C until further use. Protein samples were quantified using the Bradford protein assay kit (Bio-Rad) as per the manufacturer's instructions.

The protein samples were denatured by boiling in a water bath for 5 min with SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2.5% SDS, 0.002% Bromophenol blue, 5% β-Mercaptoethanol, 10% glycerol) and then centrifuged briefly. Forty micrograms of protein samples were fractionated on SDS-polyacrylamide gel and transferred onto PVDF membrane. Membranes were blocked with either 2% or 5% skimmed milk for 1 h, then incubated overnight at 4°C with primary antibodies (diluted to the required concentration) on an orbital shaker. Membranes were rinsed and incubated with HRP-conjugated secondary antibodies for 1.5 h at room temperature on an orbital shaker. An ECL kit, Clarity Max^TM Western ECL Substrate (Bio-Rad) was used to develop chemiluminescence signals associated with protein bands. Images were acquired with the Chemidoc Imaging System (UVP).

2.6 Immuno-precipitation

Immuno-precipitation of proteins was done using Pure Proteome protein A/G magnetic beads (Millipore). Two hundred and fifty micrograms of protein were incubated with primary antibodies at required dilution overnight at 4°C. Twenty-five microliters of washed magnetic beads were then incubated with the protein-antibody solution for 2 h at room temperature. The beads were then washed three times for 30 s each with 500 µL of binding buffer. Following the last wash 40 µL of 1× SDS sample buffer was used to resuspend the beads and was heated at 70°C for 8 min. The beads were then captured with the magnetic stand and the supernatant was used for western blot analysis.

2.7 Immuno-histochemistry

Immuno-histochemical analyses were performed on 10-μm tissue sections prepared with the aid of a cryostat (Leica). Tissue sections were fixed in ice-cold acetone for 5 min, and incubated with 0.3% ice-chilled H₂O₂ in PBS for 5 min. Nonspecific binding was blocked by incubation with 1.5% normal goat serum in phosphate-buffered saline (PBS) for 1 h, and then exposed for 1 h to anti-PRNP antibodies in 1:600 dilution in PBS. Unbound antibodies were washed off with three changes of PBS, followed by incubation with biotinylated anti-rabbit secondary antibodies for 30 min (Vector laboratories) diluted in PBS. Samples were then washed three times with PBS for 5 min each and were incubated with ABC reagents (Vector Laboratories) for 30 min. Samples were washed again three times with PBS for 5 min each and were developed with AEC reagents (Vector Laboratories) in the dark and were monitored continuously under a microscope until specific brown color developed. Samples were washed with tap water and then counterstained with hematoxylin (Sigma). Stained tissue sections were examined and images were recorded with a Leica microscope equipped with a CCD camera and Leica Application Suite X (LAS X).

2.8 Immunofluorescence staining

Tissue sections were fixed in ice-cold acetone for 5–10 min, washed with PBS, then blocked with blocking buffer containing 5% goat serum and 0.3% Triton-X-100 in PBS for 1 h. Sections were then incubated with either H-caldesmon (1:200) or PRNP (1:200) for 2 h at room temperature and overnight at 4°, respectively. Samples were then washed three times with PBS, 5 min each, followed by incubation with FITC-conjugated anti-rabbit IgG (1:200 dilution) (Sigma) and TRITC-conjugated anti-rabbit IgG (1:1000 dilution) (Sigma) for 1.5 h at room temperature. Sections were again washed three times, followed by nuclear staining with Hoechst at a concentration of 2 µg/mL. Sections were again washed five times with PBS before mounting with Fluoroshield solution (Sigma). Images were captured with a Leica microscope equipped with a CCD camera and Leica Application Suite X (LAS X).

2.9 Designing, cloning of shRNA oligos in pLKO.1 vector, generation of lentiviral particles harboring shRNA, and transduction in VSMC

Broad institute portal https://portals.broadinstitute.org/gpp/public/seq/search was used to design shRNA oligos targeting coding exons. The oligos having higher intrinsic scores were selected and purchased from IDT. The sequence of the oligos is given in Table 2. Individual shRNAs were cloned in separate pLKO.1 vector (Addgene). Forward and reverse oligos were annealed by boiling for 4 min followed by slow cooling at room temperature using 20 µM of each of the oligos in 50 µL of NEB buffer2. The pLKO.1 vector was digested with EcoR1 and Age1 enzymes and ligated with shRNA oligos having compatible sticky ends in a 1:10 molar ratio using T4 DNA ligase (NEB) following standard ligation protocol. One Shot® Mach1™T1™ chemically competent Escherichia coli (Invitrogen) cells were transformed with the ligated mix. The transformants were then plated on LB agar plates containing ampicillin as a selection marker. Correctness of clones was confirmed by restriction digestion and sequencing.

For producing lentiviral particles, HEK293T cells (7 × 10⁵ cells) were plated on 6 cm tissue culture plates with antibiotic-free media and incubated at 37^°C, 5% CO₂ overnight. Next day, before transfection, transfection cocktail was prepared using 1 µg pLKO.1 shRNA plasmid (Sigma), 750 ng psPAX2 packaging plasmid, 250 ng pMD2.G envelope plasmid (Addgene) to a final volume of 100 µL serum free OPTI-MEM. In a separate tube 100 µL Master Mix was prepared with 6 µL Fugene and 94 µL OPTI-MEM and incubated for 5 min. The master mix was added to transfection cocktail to make a final volume of 200 µL and allowed to stand for 20–30 min. Following the incubation period DNA-Fugene mix was added drop wise on cells and spread by gentle swirling. Next day, transfection reagents containing media were removed and replaced with fresh complete growth media. Media containing viral particles were harvested after every 48 h in two rounds. Two batches of media containing viral particles were pooled, centrifuged, and stored in small aliquots at −80° C until used.

A7r5 cells were plated on 35 mm dishes and allowed to reach ~70% confluency overnight. Next day morning, the media was removed and fresh media supplemented with either lentiviral particles containing empty vector or a cocktail of shRNA 1,2,3 were added in separate dishes. After 24 h the lentiviral containing media was discarded and replenished with fresh medium and cells were harvested for further experiments.

2.10 Cloning and characterization of full-length rat Osr1 cDNA

Rat Osr1 full-length cDNA was cloned in pCAG-DsRed vector (Addgene) using the directional cloning strategy. RNA isolated from the heart was reverse-transcribed using MMLV-RT (Invitrogen) and full-length rat Osr1 (NM_001106716.3) cDNA was amplified from using LA-Taq DNA polymerase (TaKaRa, Japan). Primers used were Forward: 5′-TATAGGTACCAATGGGCA GCAAAACCT-3′ and Reverse: 5′-TATAGCGGCCGCTTAGCATTTGATCTTG-3′ having KpnI and Not1 restriction sites, respectively. The amplified product was cloned in-frame into pCAG-dsRed vector (Addgene) by deleting the dsRed using the standard protocol. Correctness of the clone was verified using restriction digestion and sequencing. Expression of OSR1 was assessed using the A7r5 cell line.

2.11 Scratch wound assay

2 × 10⁵ A7r5 cells were seeded onto a 35 mm dish and after 24 h cells were treated with either control vector or a cocktail of shRNAs to downregulate Prnp. Next day, the virus-containing media was replaced by fresh media and a scratch was made using sterile cell combs (cell comb scratch assay kit, Merck Millipore) to form a cell-free area of approximately 700 µm of width. Images of the scratch wound were recorded immediately after scratch (t = 0 h) and 24 h after scratch (t = 24 h) using a Leica DMi8 microscope.

2.12 Cell invasion assay

2 × 10⁵ A7r5 cells were seeded onto a 35 mm dish and after 24 h cells were treated with either control vector or a cocktail of shRNAs and were cultured for 24 h after transduction. Cells were trypsinized and seeded on ECMatrix^TM inserts (Cell Invasion Assay kit, ECM550, Merk Millipore) at a density of 1 × 10⁵ cells per insert in 300 µL of serum-free media. Media with 10% FBS was added to the lower chamber. After 24 h of migration, the residual media as well as non-invaded cells were removed from the inner surface of the inserts using cotton swabs. The inserts were dipped in staining solution for 25 min and the background was destained with several changes of distilled water. Images of invaded cells were taken using a Leica DMi8 microscope. Invaded cells were counted from five to six different microscopic fields from each biological replicates. Invaded cells were also quantified by dissolving the stain at the lower surface in 10% acetic acid and measuring the absorbance at 560 nm using a multi-mode plate reader (PerkinElmer).

2.13 Flow cytometric analysis to detect early apoptosis using Annexin-V-PI staining

A7r5 cells transduced with either control or shRNA lentivirus cocktail were trypsinized, washed, and resuspended at a concentration of 3 × 10⁵/mL. The cell suspension was again centrifuged, and the pellet was dissolved in 96 µL of Annexin-V binding buffer followed by Annexin-V and PI staining as per the manufacturer's protocol (Cell Signaling Technology). The percentage of cells undergoing early apoptosis was analyzed using a Flow cytometer (LSR, Fortessa, BD).

To detect TRAIL-induced apoptosis, A7r5 cells were transduced for 24 h with either control or sh-Prnp lenti-particles. Both control and Prnp knockdown cells were then treated with increasing doses (10, 50, and 100 ng/mL) of recombinant murine TRAIL (Preprotech) and incubated for 24 h at 37°C. Apoptosis was then analyzed by Annexin-V and PI staining.

2.14 Antibodies

Anti-PRNP (14025), anti-GAPDH (5174), anti-FAK (3285), anti-PDGFR-β (3169), anti-Caspase 3 (9665), anti-cleaved caspase-3 (9664), anti-Caspase-8 (4790), anti-Bcl-xL (2764), anti-caldesmon (12503 S) rabbit monoclonal antibody, anti-Bax (2772) rabbit polyclonal antibody were purchased from Cell Signaling Technology, anti-Caspase-8 (cleaved, AHP967) antibody was purchased from Bio-Rad laboratories, anti-OSR1 mouse monoclonal antibody (sc-376545) was purchased from Santa Cruz Biotechnology. Anti-DR4 rabbit polyclonal antibody (BML-SA225-0100) was purchased from Enzo Life Sciences. HRP-conjugated anti-rabbit (A120-101P) and anti-mouse (A90-134P) secondary antibodies were purchased from Bethyl Laboratories and used at a dilution of 1:10,000 for western blot. FITC-conjugated anti-rabbit IgG (F0382) and TRITC-conjugated anti-rabbit IgG (T6778) were purchased from Sigma.

2.15 Statistical analysis

All experiments were performed at least three times with three different biological replicates. The standard error of the mean was calculated using Graph Pad Prism software. Statistical significance among experiment groups was calculated by unpaired Student's t-test and Mann–Whitney U test for non-parametric variables.

3.1 Identification and spatiotemporal distribution of cellular prion in the metrial gland

The metrial gland (MG) marks the entry point of maternal blood vessels and the site for SAR in the rat. We carefully isolated rat metrial glands on embryonic days 11.5, 13.5, 16.5, and 19.5 without contaminating the surrounding smooth muscle layer. Quantitative real-time PCR was performed to evaluate the temporal expression pattern of Prnp transcripts with the progression of gestation. Prnp mRNA levels gradually increased from E11.5 to E 16.5, followed by a sharp decline in E19.5 (Figure 1a). These data were further affirmed by analyzing the PRNP protein levels in these tissue lysates using western blot analysis (Figure 1b). NIH ImageJ software was used to quantify the protein bands using GAPDH as endogenous control (Figure 1c).

Owing to the heterogeneous nature of the metrial gland, we sought to decipher the specific cellular source of PRNP within this tissue by immuno-histological methods. Immuno-staining of PRNP protein on 10 µm cryosections revealed that the primary site of PRNP synthesis is the smooth muscle cells in the metrial gland on E11.5 (Figure 1e) and much less in the VSMCs (Figure 1f, see inset). On gestation day 13.5, specific signals were found in the smooth muscle layer (Figure 1h) as well as in the VSMCs that seem to migrate away from the arterial wall (Figure 1i,j, see inset). By E16.5, the maximum population of the VSMCs expresses prion and more migration was observed (Figure 1l–n). Staining the E13.5 metrial gland with either a smooth muscle marker, caldesmon (green, Figure 1q), or PRNP (red, Figure 1r) revealed that VSMCs surrounding almost all the arteries express caldesmon (Figure 1q). Upon merging two fluorescent images of the boxed region shown in Figure 1p, some cells were also found to express PRNP (Figure 1s), thus verifying these cells to be indeed VSMCs. On closer examination of individual arteries, it was observed that only the cells that are still strongly attached to the tunica of the arteries happen to express both caldesmon and PRNP, as the color of the cells is yellow denoting co-localization of both markers (Figure 1t–v, white asterisk). On the other hand, the VSMCs, which are in the process of migration and invasion, around their respective arteries, exclusively express prion and not caldesmon (Figure 1t–v, white arrowheads).

3.2 PRNP potentiates migration and invasion of VSMCs

The compelling presence of PRNP in migrating VSMC in the metrial gland, especially following trophoblast invasion into the metrial gland led to subsequent experimentation to understand the function of PRNP in VSMC. We therefore tested the A7r5 VSMC cell line for the presence of PRNP. As expected, PRNP was found to be abundantly expressed in the A7r5 cell line and the level of expression was comparable to that in the E16.5 metrial gland (Figure 2a). Furthermore, the immunocytochemical analysis showed that PRNP is expressed in the membrane of the A7r5 cells (Figure 2b).

The presence of PRNP in migratory VSMCs in the metrial gland led us to investigate the influence of PRNP in VSMC migration and invasion. Prnp was knocked down in A7r5 with a cocktail of lentivirus containing three short hairpin oligos targeting different exons (Supporting Information: Figure S1B–E). Scratch wound assay was used to evaluate VSMC migration. Photo-micrographic images depicting the width of the wound at the beginning of the scratching and after 24 h is shown in Figure 2c. Cells transduced with empty vector showed around 62.2% of wound healing capacity, whereas PRNP knocked-down cells showed only 23.3% of wound closure after 24 h (Figure 2d).

A similar migration-promoting role of PRNP was observed in VSMC invasion toward the chemoattractant. VSMCs were plated on trans-well inserts pre-coated with extracellular matrix proteins, collagen, and elastin. The ability of cells to invade the matrix and attach to the underside of the trans-well membrane was scored as well as quantified. Representative photo-micrographic images of stained cells on the underside of the trans-well are shown in Figure 2e. At least five different fields per well were captured from three biological replicates and the cells in each field were counted. A significant decrease in the number of invaded cells was observed in cells with PRNP knockdown. The relative percentage of cell count between the two groups indicated that nearly 30%–35% fewer cells have invaded when PRNP was knocked down (Figure 2f). Colorimetric quantification of the stained invaded cells also showed that there was a 40% reduction in the absorbance in PRNP-knocked down cells as compared with the control (Figure 2g).

3.3 PRNP forms a termolecular complex with FAK and PDGF receptor β

We sought to investigate the plausible mechanism by which PRNP might influence VSMC migration. Activation of FAK through the PDGF–PDGFRβ signaling pathway, is known to play a key role in VSMC migration. PRNP is known to be attached to the outer leaflet of the plasma membrane through a GPI-anchor. This information led us to hypothesize that PRNP might act as a co-receptor to the PDGFRβ–FAK axis. In line with our hypothesis, both FAK (Figure 3a) and PDGFRβ (Figure 3b) were found to be co-immunoprecipitated with PRNP from the E16.5 metrial gland lysate. To further confirm this interaction, a co-immunoprecipitation assay was performed using VSMC lysate and it was found that PRNP form an immune complex with both FAK and PDGFRβ (Figure 3c).

3.4 Odd-skipped-related 1 (Osr1) regulates Prnp in VSMCs

To understand regulation of Prnp expression promoter analysis of rat Prnp gene (ENSRNOG00000021259) was performed. Two putative binding sites for Odd-skipped related-1 transcription factor (GCT/CNCTG; O'Brien et al., 2018) were found at 732 bp and 3.2 kb upstream of the transcription start site (Figure 4a) of Prnp. Full-length Osr1 cDNA (NM_001106716.3) was cloned and expressed in A7r5 cells (Supporting Information: Figure S2A). Elevated levels of Osr1 mRNA and proteins were confirmed by real-time and western blot analysis, respectively (Figure 4b,c). Ectopic overexpression of OSR1 led to elevated levels of Prnp transcripts and protein in A7r5 cells (Figures 4b,c). Figure 4d depicts quantification of western blot analysis shown in Figure 4c. To further authenticate the overexpression data, Osr1 knocked down in A7r5 cells using short-hairpin RNA containing lentiviral particles (Figure 4e and Supporting Information: Figure S2b). As expected, downregulation of Osr1 (Figure 4f,g) resulted in abrogation of Prnp transcripts and protein expression (Figure 4f,g). Figure 4h depicts quantification of western blot analysis shown in Figure 4g.

3.5 Trophoblast cells influence Osr1 and PRNP expression in VSMCs

Invasive trophoblast cells are known to influence the phenotypic changes in metrial gland VSMCs located surrounding the uterine spiral arteries (Nandy et al., 2020). We, therefore, assessed the influence of trophoblast cells on OSR1 and PRNP expression by VSMCs by co-culturing VSMCs with trophoblast cells (Figure 5a). Co-culture of VSMCs with rat primary trophoblast cells for 48 h led to downregulating Osr1 and Prnp transcripts (Figure 5b) and protein (Figure 5c,d). As it is evident from the immunoblot assay (Figure 5e,f) that rat primary trophoblast cells do not express PRNP protein, therefore, the observed alteration of Prnp transcript and protein levels during co-culture can be attributed to the VSMC pool only. These data indicate that trophoblast cells might inhibit VSMC migration through OSR1–PRNP axis.

3.6 RNA interference of Prnp promotes apoptosis in VSMC

Apoptosis of VSMCs during pregnancy-related vascular reorganization is a well-characterized phenomenon (Bulmer et al., 2012; Harris et al., 2006; Helwig & Le Bouteiller, 2007; Liu et al., 2022; Whitley & Cartwright, 2009). Leading on from PRNP's ability to promote migratory and invasive properties of A7r5 cells, we postulated that PRNP facilitates the VSMCs to migrate away from the arterial wall, thus averting them from undergoing apoptosis. To test this hypothesis, Prnp was downregulated in A7r5 cells using RNA interference, and morphologically many dead cells were observed in the culture plate as compared to control (Figure 6a). Furthermore, early signs of apoptosis by Annexin-V-PI staining were tested in PRNP-knocked down A7r5 cells using flow cytometric analysis. Knockdown of PRNP substantially increased the percentage of Annexin-V positive and PI negative cells (9.2%) as compared to control (2.3%) cells (Figure 6b,c). Analysis of apoptotic markers was done to further validate this observation. Substantial elevation of the cleaved form of caspase-3 and caspase-8 was observed in PRNP-downregulated cells (Figure 6d–g). Even though total caspase3 was observed to increase in PRNP-downregulated cells, the change is nonsignificant and does not affect the overall rise of its cleaved isoform. However, this phenomenon also aptly points out that overall apoptotic signals are activated in these cells when PRNP is depleted. In line with this, proapoptotic marker Bax increased, although in less amount, and antiapoptotic marker Bcl-XL decreased in PRNP-knocked down VSMCs (Figure 6d–g).

3.7 PRNP inhibits VSMC apoptosis by blocking TRAIL-DR4 ligand–receptor complex formation

Previous studies from our laboratory demonstrated that trophendothelial cells secrete TRAIL, which causes selective demise of endothelial cells but not trophoblast cells (Paul et al., 2022). We, therefore, tested whether PRNP has any role in TRAIL-induced apoptosis. PRNP, being located in the outer leaflet of the plasma membrane, we first tested whether it interacts with TRAIL receptor DR-4. Interestingly, DR4 co-immunoprecipitated with PRNP in A7r5 cells (Figure 7a). This led us hypothesize that PRNP protects VSMCs from TRAIL-mediated apoptosis. Treatment of A7r5 cells with increasing doses of TRAIL (10, 50, and 100 ng/mL) induced more apoptotic death in PRNP-downregulated cells (~15%–20% more) as compared to control (Figure 7b–g). These data indicate that PRNP might prohibit TRAIL binding to its receptor on VSMCs leading to their protection from TRAIL-induced apoptosis.

To summarize, cellular Prion protein expression in the metrial gland of rat is initiated on E11.5. It peaks on E16.5, whereby its interaction with pro-migratory proteins PDGFRβ and FAK potentiate cell migration. Its antiapoptotic property appears to be mediated via its antagonistic effect toward TRAIL-DR4 signaling. An overview of the above concepts has been illustrated in Figure 8.