Dental pulp cell-derived powerful inducer of TNF-α comprises PKR containing stress granule rich microvesicles

It is well known that dental pulp tissue can evoke some of the most severe acute inflammation observed in the human body. We found that dental pulp cells secrete a factor that induces tumor necrosis factor-α production from macrophages, and designated this factor, dental pulp cell-derived powerful inducer of TNF-α (DPIT). DPIT was induced in dental pulp cells and transported to recipient cells via microvesicles. Treatment of dental pulp cells with a PKR inhibitor markedly suppressed DPIT activity, and weak interferon signals were constitutively activated inside the cells. In recipient macrophages, stimulation with DPIT-containing supernatants from pulp cells resulted in activation of both nuclear factor-κB and MAP kinases like JNK and p38. Proteomics analyses revealed that many stress granule-related proteins were present in supernatants from dental pulp cells as well as microvesicle marker proteins like GAPDH, β-actin, HSPA8, HSPB1, HSPE1, and HSPD1. Furthermore, giant molecule AHNAK and PKR were detected in microvesicles derived from dental pulp cells, and gene silencing of AHNAK in dental pulp cells led to reduced DPIT activity. Thus, it appeared that the core protein of DPIT was PKR, and that PKR was maintained in an active state in stress granule aggregates with AHNAK and transported via microvesicles. The activity of DPIT for TNF-α induction was far superior to that of gram-negative bacterial endotoxin. Therefore, we, report for the first time, that active PKR is transported via microvesicles as stress granule aggregates and induces powerful inflammatory signals in macrophages.

. DPIT is a powerful TNF-α stimulator. (a) TNF-α gene expression and (b) TNF-α production was examined in differentiated THP-1 (dTHP-1) cells stimulated with the supernatants from DP-1 and primary dental pulp cells (priDPC)-1. dTHP-1 cells are stimulated with the supernatants from DP-1 (DP-1 sup) and priDPC (priDPC sup), LPS or Pam3CSK4 at indicated concentration for indicated time periods, and total RNAs and supernatants are collected. (a: right) dTHP-1 cells are stimulated for 2 hrs with various stimulants and gene expression was analyzed by qPCR. (b: right) Culture supernatants of dTHP-1 cells were collected 2 hrs after stimulation and TNF-α protein level was measured by ELISA. Amounts of TNF-α in culture supernatants of original dental pulp cells were also measured (b: right). changes from s spindle shape to a fried-egg shape (Fig. 5c) 7 . Taken together, we confirmed that culture supernatants from dental pulp cells contained a factor that could strongly induce pro-inflammatory reactions.
Thus, we, aimed to clarify the nature of this pro-inflammatory factor, designated DPIT. To determine the differences in gene expression between cells with and without DPIT activity, we compared gene expression profiles between DP-1 with DPIT activity and gingival tissue-derived fibroblasts without DPIT activity, and between PriDPC-1 with DPIT activity and gingival tissue-derived fibroblasts without DPIT activity by DNA microarray analyses (the entire DNA microarray data base is deposited at www.ncbi.nlm.nih.gov/geo, GSE124492). We selected genes that were commonly up-regulated in both DP-1 and PriDPC-1 associated with high DPIT activity (Table 1). Interestingly, many of the commonly up-regulated genes were family members of interferon-inducible genes, suggesting that dental pulp cells were constantly exposed to weak stress stimuli. Interferon signals are known to activate interferon-induced double-stranded RNA-activated protein kinase (EIF2AK2), also known as protein kinase R (PKR) 8 . In fact, we found in our microarray analyses that PKR expression was elevated by in DP-1 and PriDPC-1, respectively, compared with control gingival fibroblasts and the significant up-regulation of PKR expression in both DP-1 and PriDPC-1 was further confirmed by qPCR analysis utilizing the primer pairs targeting for PKR ( Supplementary Fig. 3).
It is very unlikely that PKR alone is transported extracellularly from dental pulp cells to recipient macrophages. We, therefore, hypothesized that PKR may be transported from dental pulp cells to macrophages via extracellular vesicles. To test this possibility, we collected extracellular vesicles from DP-1 treated with a PKR inhibitor, and examined their DPIT activity. We found that DPIT activity was inhibited after treatment of DP-1 with the PKR inhibitor in a dose-dependent manner (Fig. 6a). Pre-incubation of dTHP-1 cells with PKR inhibitors also suppressed TNF-α production in dTHP-1 cells following dental pulp cell-derived supernatant stimulation (Fig. 6b). Interestingly, however, down-regulation of endogenous PKR in dTHP-1 cells by small interfering RNA (siRNA) transfection did not decrease TNF-α production after stimulation with DP-1 supernatant (Fig. 6c), suggesting that TNF-α expression was not a result of endogenous PKR activation in recipient macrophages. Culture supernatants from dental pulp cells, both DP-1 and PriDPC-1, markedly induced phosphorylation of p38 MAP kinase (p38), c-Jun N-terminal kinase (JNK), and nuclear factor (NF)-κB in dTHP-1 cells (Fig. 6d). LPS-dose-dependent phosphorylation of these kinases has been confirmed to verify the validity and quality of THP-1 cells used in this study ( Supplementary Fig. 4). Because PKR was reported to activate p38, JNK and NF-κB 8-10 , we hypothesized The production of IL-1β, IL-6, and MCP-1, and (b) the gene expression of IL-6 and MCP-1 (b) in differentiated THP-1 (dTHP-1) cells stimulated with the supernatants from DP-1 and primary dental pulp cells (priDPC) were evaluated. dTHP-1 cells are stimulated with the supernatants from DP-1 (DP-1 sup) and priDPC (priDPC sup), LPS or Pam3CSK4 at indicated concentration, and total RNAs and supernatants are collected. Culture supernatants of dTHP-1 cells were collected 2 hrs after the stimulation and IL-1β, IL-6, and MCP-1 protein levels were measured by ELISA. For gene expression analysis, dTHP-1 cells were stimulated with various stimulants for 2 hrs. Endogenous amounts of these cytokines in culture supernatants of dental pulp cells were also measured (a).
www.nature.com/scientificreports www.nature.com/scientificreports/ that DPIT activity was mediated by PKR derived from dental pulp cells, and that the possible transport machinery was extracellular particles.
As mentioned above, it is very unlikely that a signaling molecule alone is secreted from a certain cell type in its intact form and transferred to another target cell. We, therefore, hypothesized that important signaling molecules may be packed and transported inside the extracellular microparticles. To test this possibility, we first separated the contents of cell culture supernatants from dental pulp cells using different pore-size filters and found that DPIT activity was completely lost in a fraction with a particle size below 220 nm (Fig. 7a). The most abundant microvesicles from dental pulp cells isolated by ultra-centrifugation had a particle size of 230-250 nm, and round-shaped morphology of microvesicles was identified by transmission electron microscopy (TEM) analysis (Fig. 7b,c). To confirm the activity of these vesicles, we compared the TNF-α-inducing activity of these particles with those of microvesicles derived from Hek293 cells (Hek293-MVs) and MCF7 cells (MCF7-MVs) isolated in a similar manner, and confirmed that only dental pulp cell-derived microvesicles (DP-1-MVs and priDPC-1-MVs) exhibited TNF-α inducing activity at both the gene and protein levels (Fig. 7d,e). Next, we examined MAP kinase and NF-κB activation in dTHP-1 cells stimulated with isolated microvesicles, and confirmed increased phosphorylation of these signaling molecules from 30 min after the stimulation with DP-1-MVs and priDPC-1-MVs but not Hek293-MVs or MCF7-MVs (Fig. 8a). Furthermore, pre-treatment of dTHP-1 cells with NF-κB, ERK1/2, p38, and JNK1/2 inhibitors led to reduced TNF-α gene and protein expression after DP-1-MVs stimulation. These reductions were marked, especially when the cells were pre-treated with the JNK inhibitor, while an inhibitor for receptor-interacting protein kinase 1 (RIPK1) had no inhibitory effect, suggesting that JNK is an important signaling molecule that mediates TNF-α induction (Fig. 8b,c). Further, we stimulated DP-1 cells with several concentrations of DP-1-MVs in fresh serum-free medium to clarify the autocrine effects of dental pulp cell-derived www.nature.com/scientificreports www.nature.com/scientificreports/ microvesicles and found that the gene expression levels of IL-6, MCP-1, and TNF-α were unchanged in DP-1 cells in contrast to the dTHP-1 cells, in which these gene expression levels were drastically increased by the stimulation with the DP-1-MVs ( Supplementary Fig. 5). These results indicated that DP-1-MVs did not have autocrine effect. We then labeled dental pulp DP-1-MVs with a fluorescent dye and observed their cellular uptake by dTHP-1 cells. After 100 min of incubation with microvesicles, cellular uptake was observed to be initiated (Fig. 9a). Cellular intake appeared to be inhibited by nystatin treatment, but not by methyl-β-cyclodextrin (MBCD) treatment. In fact, TNF-α production from dTHP-1 cells stimulated with DP-1-MVs for 2 hrs was completely inhibited by nystatin treatment, an inhibitor of membraneous cholesterol-mediated endocytosis, but not by MBCD treatment, a reagent for cholesterol depletion, suggesting that microvesicles were incorporated by dTHP-1 cells and promoted TNF-α synthesis (Fig. 9b). Further, to exclude the possibility that cholesterol depletion might disrupt the integrity of microvesicles, we incubated DP-1-MVs with the equivalent concentration of nystatin and MBCD utilized for treatment of dTHP-1 cells and found that the leakage of microvesicle-proteins such as HSP90α and GAPDH into the solution after 100 min incubation was not obvious when treated with either nystatin or MBCD unlike the DP-1-MVs treated with TritonX-100 (1%), which induced lysis of microvesicles ( Supplementary Fig. 6). . THP-1 cell activation by DPIT is independent of PKC activation. (a) Floating unTHP-1 cells were stimulated for 3 hrs with DP-1 sup or PMA with a PKC inhibitor (GF109203X: 10 μM) and cell attachment was visualized with phase-contrast microscopy. (b) Simultaneously, total RNAs are collected to see TNF-α gene expression (b). Although PKC inhibitor markedly suppressed unTNP-1 cell attachment to the culture plate and TNF-α gene expression in unTHP-1 cells in the presence of PMA, no inhibitory effect was observed when the cells were stimulated DP-1 sup. Data represents means ± SD of at least two independent experiments with identical results. Statistical analyses were performed by Student's t-test. **p < 0.01, ***p < 0.001, significantly difference versus cells untreated with PKC inhibitor.
www.nature.com/scientificreports www.nature.com/scientificreports/ Certain Toll like receptors (TLRs) have been suggested to act as receptors for exosomal or microvesicle-derived miRNA 11,12 . We therefore examined the involvement of these receptors in cellular uptake of the microvesicles. However, neither gene silencing nor selective inhibitors of TLR signaling and their down-stream molecules such as MyD88, TRAF6, TAK1, RIPK1, and TRADD resulted in decreased TNF-α secretion from dTHP-1 cells after stimulation with DP-1-MVs ( Supplementary Fig. 7). Generally, the observed particle size was larger than that of regular exosomes and corresponded to slightly larger microvesicle 13,14 . Therefore, we performed proteomics analyses using the LC-MS/MS method to determine the kinds of molecules included in culture supernatants from DP-1 with DPIT activity, and identified several marker proteins typical of microvesicles such as GAPDH, β-actin, HSPA8, HSPB1, HSPE1, and HSPD1 14 (Table 2). Furthermore, the most abundantly detected protein in the culture supernatants was neuroblast differentiation-associated protein AHNAK, with a molecular size of 700 kDa. AHNAK is a giant protein associated with pseudopod formation and is often seen in extracellular particles [15][16][17] . In fact, AHNAK protein was detected in the cytosol of dental pulp cells, but not Hek293 cells (Fig. 10a), and also in dental pulp tissue (Fig. 10b). In addition, AHNAK was detected in culture supernatants from dental pulp cells (Fig. 10c). Thus, we examined the possible roles of this molecule in active molecule transportation. Gene silencing of AHNAK in DP-1 with siRNAs was successful, with significant reduction in microvesicle production ( Fig. 10d-g). Treatment of DP-1 with the siRNA resulted in abolition of AHNAK in microvesicles, and led to decreased TNF-α gene expression and protein production from recipient dTHP-1 cells challenged with the equal amounts of these . These cells were stimulated with DP-1 sup for indicated time periods and total RNAs were collected. (c) Induction of morphological changes of normal human macrophages to a "fried egg-like" appearance. Differentiated normal human macrophages were stimulated with DP-1 sup for indicated time periods and adhered cells were visualized with phase-contrast microscopy.
www.nature.com/scientificreports www.nature.com/scientificreports/ microvesicles (Fig. 10h,j). Similarly, significantly decreased signaling as evaluated by JNK phosphorylation was observed (Fig. 10j). These data suggest that AHNAK had a role in the transportation of DPIT. AHNAK has been detected in exosomes from certain cancer cells such as breast cancer cells 17 . However, we did not detect any TNF-α-inducing activity in supernatants from these cancer cells (Fig. 11a,b). We also investigated the presence of PKR in extracellular vesicles from both dental pulp cells and the breast cancer cells MCF-7, and confirmed its presence in cancer cell-derived microvesicles (Fig. 11c).
Because the breast cancer cells used in our experiments also contained PKR in extracellular compartments, we examined the differences between cells with and without DPIT activity. The possibility was that dental pulp cell-derived microvesicles contained activated or at least pre-activated PKR in their compartments without activation of target substrates inside the vesicles and that the active kinase was trans-introduced to the recipient monocyte/macrophage cell line. We carefully re-examined the proteomics data and confirmed the detection of many recently reported stress granule-associated proteins in the culture supernatants from pulp cells (Supplementary Table 1) [18][19][20][21][22][23][24][25][26][27][28] . In particular, eukaryotic translation initiation factor 4 (EIF4), caprin 1, and Ras GTPase-activating protein-binding protein (G3BP), all of which were reported to directly bind to PKR in stress granules 29 , were detected with other stress granule proteins (Table 3). Large stress granules were reported to retain www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ PKR in an active state, but in a non-auto-phosphorylated form 29 . However, we also detected a PKR substrate protein, eukaryotic translation initiation factor 2 (EIF2), in DP-1 supernatant (Table 3). Thus, we treated the culture supernatants with a PKR inhibitor and examined their DPIT activity. Surprisingly, treatment of the culture supernatants with a low concentration of the PKR inhibitor up-regulated DPIT activity (Supplementary Fig. 8). These results suggested that the kinase inhibitor suppressed PKR ejection from stress granule aggregates and subsequent auto-phosphorylation within the vesicles, thereby maintaining PKR in a pre-active state and inhibiting activation of EIF2. Furthermore, as soon as the inhibitor-treated vesicles encountered recipient macrophages, they released considerable amounts of active PKR into the recipient cells. Endogenous PKR activator, a protein activator of interferon-induced PKR, also known as PACT, was reported to be a stress granule protein 30 . Therefore, we examined whether DP-1 supernatants contained PACT in their vesicles. However, we were unable to detect PACT protein in DP-1-MVs, although it was detected in DP-1 lysate (Supplementary Fig. 9). Moreover, down-regulation of endogenous PACT with a siRNA in DP-1 did not result in decreased DPIT activity ( Supplementary Fig. 10), suggesting that a "factor X" other than PACT was acting as an endogenous activator of PKR inside the large stress granules 29 . Future study is needed to identify "factor X" and, in fact, MCF-7 cells as well as MCF-7-MVs appear to be good tools to search for the candidate molecule of "factor X" by utilizing exogenous overexpression and inhibition of endogenous expression strategies since MCF7 cell-derived vesicles appear to contain other necessary components than "factor X". Based on these experiments, the following mechanism is proposed. In dental pulp cells, a sustained weak stress signal is evoked, resulting in activation of a series of interferon-inducible genes and PKR. Activated PKR continuously promotes the formation and aggregation of stress granules containing active PKR, and the dental pulp cells release stress granule aggregates together with AHNAK into microvesicles. These microvesicles maintain PKR in a pre-active state inside the stress granule aggregates and eventually inject PKR into target cells such as monocytes and macrophages. PKR becomes auto-phosphorylated just after ejection from the stress granule aggregates and exhibits kinase activity. A scheme explaining this cascade is shown in Fig. 11d.
In conclusion, an extremely powerful endogenous TNF-α inducer from macrophages was discovered. The TNF-α-inducing activity of this factor was superior to that of other known TNF-α inducers like bacterial endotoxin. Furthermore, we showed that this strong innate inflammatory activator was transported via extracellular particles. If similar phenomena are observed in serious autoimmune-or neuro-degenerative diseases, the current findings will greatly contribute to our understanding for the patho-physiology of these diseases.

Cell attachment and morphology studies. Frequencies of adherent cells and morphological changes
were observed using an inverted phase-contrast light microscope (DMIRB; Leica, Bensheim, Germany). Floating undifferentiated THP-1 cells were seeded in 6-well plates at a density of 1 × 10 6 cells/well, and stimulated with or without 100 nM PMA in RPMI growth medium or supernatants from DP-1 cells. The plates were incubated in a and protein production (right) in dTHP-1 cells, while necrostatin treatment to suppress RIP-1 had no effect. Data represent means ± SD of at least two independent experiments with identical results. Statistical analyses were performed by one-way ANOVA, followed by Tukey's test. **p < 0.01, ***p < 0.001. significant difference versus dTHP-1 cells treated with DMSO before stimulation with MVs (b,c). Figure 9. Uptake of dental pulp cell-derived MVs by recipient dTHP-1 cells. (a,b) DP-1-MVs uptake triggers TNF-α production in dTHP-1 cells. PKH67-labeled DP-1-MVs are incorporated by dTHP-1 cells (a), while cellular uptake (a) and TNF-α production (b) are suppressed by pre-treatment of dTHP-1 cells with nystatin, but not MBCD (a,b). DP-1 cell-derived MVs are labeled with PKH67 and cellular uptake by dTHP-1 cells were observed by fluorescent microscopy as described in "Methods". The cells were pretreated with or without nystatin or MBCD to suppress lipid raft mediated endocytosis or to achieve cholesterol depletion from culture medium. Data represent means ± SD of at least two independent experiments with identical results. Statistical analyses were performed by one-way ANOVA, followed by Dunnett's test (b). *p < 0.05, **p < 0.01, ***p < 0.001. significant difference versus dTHP-1 cells stimulated with MVs in the absence of nystatin or MBCD (b).
www.nature.com/scientificreports www.nature.com/scientificreports/ humidified atmosphere (37°C, 5% CO 2 ) for specified periods, and the cells were washed twice with PBS before micrographs were taken. proteomics analyses. Conditioned medium (total: 10 L) was collected from immortalized dental pulp cells (DP-1). The conditioned medium was concentrated using a C18 preparative cartridge and fractions were eluted with acetonitrile. Following evaporation, the fractions were subjected to LC-MS/MS analyses (Filgen Ltd., Nagoya, Japan).
Microarray analyses. mRNAs were isolated from sub-confluent immortalized dental pulp cells, primary dental pulp cells and control gingival fibroblasts. Equal amounts (10 μg) of the mRNAs were subjected to microarray analyses using Agilent DNA chips. Data analyses were performed as described previously 33 .

Matrix vesicle purification.
Microvesicle-deprived serum was generated by filtration through an 220 nm filter (Millipore, Billerica, MA). DP-1, priDPC-1, HEK293, and MCF-7 cells were maintained until they reached 80% confluency and cultured in fresh medium containing microvesicle-deprived serum for 24 h. On the following day, the supernatants were collected, centrifuged at 2000 rpm for 20 min to remove debris and large apoptotic bodies, and filtered through an 800-nm filter (Millipore). Matrix vesicles were sedimented by centrifugation at 16,000 × g for 45 min. washed twice with DPBS, and resuspended in DPBS containing 0.3 mM EDTA. The amounts of matrix vesicles were quantified by a QuantiPro BCA Assay Kit (Sigma-Aldrich). Dynamic light scattering measurements were performed by UBE Scientific Analysis Laboratory (Tokyo, Japan) with a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Matrix vesicles were suspended in PBS. The machine settings were reflective index of 1.334 and viscosity (cP) of 0.8900 at room temperature. The intensities were recorded three times. Transmission electron microscopy (TEM) analyses were performed by Hanaichi Ultrastructure Research Institute (Okazaki, Japan). The microvesicles in PBS were placed on carbon-film grids for 2 min, from which excess PBS was removed by touching one end of the grid with the filter paper. Then, the grid was touched onto a drop of 2% uranyl acetate and excess liquid was drained off. The grid was allowed to dry for several minutes and then examined using a JEM-1200 EX microscope (JEOL, Akishima, Japan) at 80 kiloelectron volts.
Gene silencing using siRNAs. Stealth TM RNAi duplexes, comprising mixtures of three different siR-NAs against human AHNAK (HSS149070, HSS149071 and HSS149072) and PACT (HSS112561, HSS189437, and HSS189438) were obtained from Invitrogen Life Technologies. Stealth TM RNAi negative control duplex (Medium GC Duplex; Invitrogen Life Technologies) was used as a control. The RNA sequences for targeting PKR, TLR1, TLR3, TLR4, MyD88, TRAF6, TAK1, RIP1, TNFR, TRADD, and IL1BR by siRNAs were selected by Enhanced siDirect, a web-based target-specific siRNA design software. All siRNAs were generated by RNAi Inc. (Tokyo, Japan). The control siRNA used in the study was provided by RNAi Inc. All RNA sequences used for siRNA duplexes are shown in Supplementary Table 3. The siRNAs were reverse-transfected into DP-1 cells and forward-transfected into differentiated THP-1 cells at final concentrations of 10 and 50 nM, respectively, using Lipofectamine RNAiMAX reagent (Life Technologies) and incubated for 48 h.
www.nature.com/scientificreports www.nature.com/scientificreports/ Abcam). The immunoreactive proteins were identified using a Westernbright Quantum Kit (K-12042-D20; Advansta, Menlo Park, CA). Uptake analysis. Matrix vesicles were stained with PKH67 fluorescent dye (Sigma-Aldrich). washed with DPBS, and centrifuged at 16,000 × g for 45 min. Differentiated THP-1 cells were pretreated with nystatin or Figure 11. The role of PKR in DPIT activity. (a-c) Cancer cell-derived supernatants failed to induce TNF-α gene expression in either undifferentiated (a) or differentiated (b) THP-1 cells, although PKR protein was identified in both DP-1-MVs and MCF7-MVs (c). (d) Schematic representation of the role of DPTIF in activation of innate immune responses. In dental pulp cells, sustained, weak stress signals constitutively activate interferon-inducible signals, resulting in activation of PKR. Activated PKR and stress granule proteins form stress granule complexes that eventually meet giant protein AHNAK. Large stress granule/AHNAK complexes are released from dental pulp cells in microvesicles. The released microvesicles maintain PKR in an active state, and eventually deliver active PKR into recipient monocytes/macrophages. www.nature.com/scientificreports www.nature.com/scientificreports/ MBCD for 30 min. Next, 80 ng of PKH67-labeled matrix vesicles was incubated with 2 × 10 5 primed THP-1 cells for 100 min. Uptake of PKH67-labeled matrix vesicles was analyzed with an FV1000-D (Olympus, Tokyo, Japan).
Immunofluorescence analyses. DP-1 and priDPC cells were seeded into 8-well non-coated glass chamber slides (SCS-N08; Matsunami, Osaka, Japan), fixed with 3.7% formaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in DPBS for 5 min, blocked with 1% BSA in DPBS for 1 h at room temperature, and incubated with anti-AHNAK antibody (1:200 dilution) overnight at 4°C. On the following day, the cells were incubated with Cy3-conjugated goat anti-rabbit secondary antibody (1:1000 dilution) for 1 h at room temperature and embedded with SlowFade ™ Diamond Antifade Mountant containing DAPI (Life Technologies). Immunofluorescence signals were observed using an FV1000-D microscope (Olympus). Human dental pulp tissues dissected from healthy teeth extracted for orthodontic purposes were fixed with 3.7% formaldehyde for 24 h, sequentially immersed in 15% sucrose in PBS overnight and 30% sucrose overnight, embedded in OCT compound, and sectioned at 10-μm thickness. Rehydrated sections were treated as described above to detect AHNAK expression in human dental pulp tissues.
Mtt assay. MTT cell proliferation assays were performed to calculate the numbers of surviving cells using an MTT Kit (Roche, Basel, Switzerland) following the manufacturer's protocol. The solubilized formazan product was spectrophotometrically quantified by measuring the absorbances at 575 nm and 650 nm on a microtiter plate reader (Multiskan).

Proteins
Description Score  Table 3. Stress granule-associated proteins which possibly bind to PKR.