Ilginatinib

Interleukin 15 participates in Jagged1-induced mineralization in human dental pulp cells

Chatvadee Kornsuthisopona, Jeeranan Manokawinchokea, Opor Sonpoungb, Thanaphum Osathanona,c, Damrong Damrongsric,*

A B S T R A C T

Objectives: Crosstalk between Notch and other cell signaling molecules has been implicated to regulate the osteogenic differentiation. Understanding the interaction between Notch and IL15 is essential to reveal molecular mechanism. Thus, the objective of the present study was to investigate whether IL15 participates in the Notch signaling-induced mineral deposition in human dental pulp cells (hDPs).
Methods: hDPs were explanted from dental pulp tissues. To activate Notch signaling, the cells were seeded on Jagged1-immobilized surfaces. The mRNA expression was evaluated using real-time polymerase chain reaction. hDPs were treated with 5–50 ng/mL IL15. Cell viability and proliferation were determined using an MTT assay. Mineral deposition was examined using alizarin red s and Von Kossa staining. In some experiments, the cells were pretreated with a JAK inhibitor prior to stimulation.
Results: Jagged1 induced IL15 and IL15RA expression in hDPs. IL15 treatment significantly increased mineral deposition at 14 d and upregulated ALP, OCN, OSX, ANKH, and ENPP1 mRNA expression. IL15-induced mineralization was attenuated by JAK inhibitor pretreatment. Further, JAK inhibitor pretreatment inhibited the effect of Jagged1 on hDP mineral deposition.
Conclusion: IL15 promoted the osteogenic differentiation in hDPs. Moreover, IL15 participated in the Jagged1- induced mineralization in hDPs.

Keywords:
Human dental pulp cells
Notch signaling Interleukin 15
Mineralization
Differentiation

1. Introduction

The regulation of lineage-specific differentiation has been highlighted in tissue regeneration research (Xie et al., 2020). To induce the osteogenic lineage commitment, the differentiation via several signaling pathways has been studied, e.g., bone morphogenetic protein (Bilem et al., 2016; Mishina et al., 2004), Wnt (Boyan, Olivares-Navarrete,
Berger, Hyzy, & Schwartz, 2018; Cook, Fellgett, Pownall, O’Shea, & Genever, 2014), and Notch (Kamalakar et al., 2020; Ugarte et al., 2009). Notch is a crucial conserved signaling pathway and is involved in a diverse range of physiological processes during embryonic development and adult tissue homeostasis (Lai, 2004). In mammalian cells, Notch receptors (Notch1, Notch2, Notch3, and Notch4) and Notch ligands (Delta-like1, Delta-like3, Delta-like4, Jagged1, and Jagged2) activate the canonical Notch pathway (Kopan & Ilagan, 2009). The role of Notch signaling in osteogenic differentiation has been reported in several types of cells, however, the effects occur in a context-specific and cell type-specific manner (Cao et al., 2017; Manokawinchoke, Nattasit et al., 2017; Ndong, Stephenson, Davis, Garcia, & Goudy, 2018; Osathanon et al., 2019; Semenova et al., 2020).
Jagged1 is a membrane bound receptor in canonical Notch pathway. Jagged1 but not Dll1 promotes mineralization in the cells isolated from human exfoliated deciduous teeth (Sukarawan, Peetiakarawach, Pavasant, & Osathanon, 2016). Notch signaling activation using immobilized Jagged1 upregulates osteogenic-related gene expression and induces mineral deposition in human dental pulp cells (hDPs) (Manokawinchoke, Nattasit et al., 2017), implying that Notch signaling promotes osteogenic differentiation in hDPs. These effects of Jagged1 in hDPs are attenuated in NOTCH2 knockdown cells, suggesting the role of NOTCH2. Although the effect of Jagged1 in hDPs differentiation has been elucidated, the intracellular mechanism is yet unclear. Global gene expression profile of Jagged1-treated hDPs was examined (Manokawinchoke, Nattasit et al., 2017). The further experiment is required to elucidate the participated molecules/pathways in Jagged1 induced mineralization in hDPs.
Crosstalk between Notch and other cell signaling molecules has been reported during the osteogenic differentiation of various tissue types (Cao et al., 2017; Liao et al., 2017; Wang et al., 2019). Interaction between Notch and IL15 has been reported. During lymphoid development, IL15 downregulation was mediated by Notch signaling, driving the maturation of hematopoietic stem cells and commitment to a T cell fate (Colpitts et al., 2013). IL15 acts in concert with Notch signals to induce the innate lymphoid cell development (Ettersperger et al., 2016). These studies evaluated the IL15-Notch relationship in regard to the immunity. However, the crosstalk that occurs between Notch and IL15 during the osteogenic differentiation remains unresolved.
Interleukin 15 (IL15) is a pro-inflammatory cytokine belonging to the IL2 family (Waldmann, 2006). IL15 participates in various physiological and pathological processes, including the immune response and cell differentiation (Allard-Chamard et al., 2020; Loro et al., 2017; Shi, Li, Miao, Guo, & Yi, 2019). IL15 binds to several types of receptors.
IL15RA is one of the receptors that exhibit an affinity to IL15 (Allard-Chamard et al., 2020). This receptor uses various intracellular mechanisms to differentially regulate cell responses, depending on the cell type. These signal transduction pathways include the Janus tyrosine kinase-signal transducer and activator of transcription (JAK/STAT) pathway (Krolopp, Thornton, & Abbott, 2016). IL15 and IL15RA upregulation are detected in association with inflammation. For example, in inflammatory arthritis (Warner et al., 2020). Several cell types express IL15 and IL15RA, including cardiomyocytes, fibroblasts, adipocytes, bone marrow stromal cells, and immune cells (Guo et al., 2020).
IL15 regulates cell differentiation towards the osteogenic lineage. IL15 treatment decreased the adipogenic differentiation potential of bovine bone marrow mesenchymal stem cells. The results demonstrated that the expression of peroxisome proliferator activated receptor γ (PPARG), a key regulator of adipose tissue differentiation, was decreased along with the reduced intracellular lipid accumulation (Shi et al., 2019). The decrease in preadipocyte differentiation of skeletal muscle tissue was observed in transgenic mice overexpressing IL15 mRNA, leading to the reduced body fat (Quinn, Anderson, Strait-Bodey, Stroud, & Argiles, 2009´ ). In contrast, IL15RA knockout mice exhibited a significant decrease in femur cortical tissue mineral density compared with the wild-type mice (Loro et al., 2017). Correspondingly, reduced mineral deposition was observed in bone marrow stromal cells isolated from IL15RA knockout mice (Loro et al., 2017).
Taken together, we hypothesized that IL15 might interact with Notch during osteogenesis. Therefore, the aim of the present study was to investigate the participation of IL15 in the Jagged1-induced mineralization in hDPs.

2. Materials and methods

2.1. Cell isolation and culture

The cell isolation protocol was approved by the Human Research Ethics Committee, Faculty of Dentistry, Chulalongkorn University (No. 020/2018). Third molar impacted teeth that required surgical removal based on the patient’s treatment plan were collected for cell isolation. Tissue explantation was employed to isolate the cells. Briefly, the dental pulp tissues were gently removed from the teeth and minced into small pieces (size 2 × 2 mm). The tissues were placed on the 35-mm tissue culture dishes with 1000 μL growth medium. After confluence, the cells were trypsinized and seeded on the 60-mm tissue culture dishes and this population was considered passage 1. Subculturing was performed at a 1:3 ratio when the cells reached confluence. The cells from passage 3–6 were used in the experiments. The cells were maintained in the growth medium consisting of Dulbecco’s Modified Eagle Medium (DMEM, cat. no. 11960, Gibco, USA) supplemented with 10 % fetal bovine serum (FBS, cat. no. 10270, Gibco, USA), 1 % L-glutamine (GlutaMAX-1, cat. no. 35050, Gibco, USA), 100 unit/mL penicillin, 100 μg/mL streptomycin, and 250 ηg/mL amphotericin B (Antibiotic–Antimycotic, cat. no. 15240, Gibco, USA). The cell culture experiments were maintained in an incubator in a 5 % CO2 humidified atmosphere at 37 ◦C.
For Jagged1 treatment, the cells were seeded at a density of 25,000 cells per well in the 24-well plates. The immobilization was performed as previously described (Manokawinchoke, Nattasit et al., 2017). For osteogenic differentiation, the cells were cultured in the osteogenic medium, which was growth in the medium supplemented with 50 μg/mL L-ascorbic acid (cat. no. A-4034, Sigma-Aldrich, USA), 5 mM β-glycerophosphate (cat. no. G9422, Sigma-Aldrich, USA), and 250 nM dexamethasone (cat. no. D8893, Sigma-Aldrich, USA). In some experiments, the cells were treated with 5–50 ng/mL recombinant human IL15 (cat. no. 247-ILB-005, R&D Systems, USA). In the inhibition experiments, the cells were pretreated with 15 nM JAK Inhibitor I (CAS 457081-03-7, Calbiochem, USA) 30 min prior to seeding on the tissue culture plates.

2.2. Bioinformatic analysis

To evaluate effect of Jagged1 on IL15 and IL15RA gene expression, the NCBI Gene Expression Omnibus series GSE94989 was downloaded (Manokawinchoke, Nattasit et al., 2017). The dataset was the RNA sequencing of hDPs treated with Jagged1 in growth medium for 24 h. Human IgG, Fc fragment (hFc) was used as the immobilization control condition. In another condition, the hDPs were pretreated with 20 μM γ-secretase inhibitor (DAPT, cat. no. D5942, Sigma-Aldrich, USA) to inhibit Notch signaling prior to Jagged1 exposure. The raw expression was analyzed using NetworkAnalyst (Xia, Gill, & Hancock, 2015; Zhou et al., 2019). The RNA read counts of IL15 and IL15RA were identified and an expression heatmap was generated using Heatmapper (Babicki et al., 2016).

2.3. Cell proliferation assay

Cell proliferation was indirectly examined using a cell metabolic activity assay (MTT assay). The cells were seeded at a density of 12,500 cells per well in the 24-well plates. At day 1, 3, and 7 after treatment, the cells were incubated with a 0.5 mg/mL MTT solution for 15 min at 37 ◦C to allow the formation of a formazan crystal. The formazan crystal was solubilized in a dimethyl sulfoxide and glycine buffer. The absorbance at 570 nm was measured using a microplate reader (Biotek ELX800, USA) and the percentage of cell number was calculated.

2.4. Mineralization assay

In vitro mineralization was examined using alizarin red s staining. Briefly, the cells were fixed with cold methanol and gently rinsed with deionized water. The cells were incubated with alizarin red s solution (1 % w/v, Sigma Aldrich) with gentle agitation at room temperature for 3 min. The excess stain was washed twice with deionized water. The staining was imaged using Microscope ECLIPSE Ts2, Nikon-DS-Fi3 (Japan). Subsequently, the staining was solubilized with 10 % cetylpyridium chloride monohydrate (Sigma Aldrich, USA) and the absorbance at 570 nm was measured using a microplate reader (Biotek ELX800, USA). For Von Kossa staining, the cells were fixed with 4% paraformaldehyde solution and gently rinsed with deionized water. The cells were stained with 1% silver nitrate solution for 20 min under UV light and subsequently incubated with 5% sodium thiosulfate for 5 min.

2.5. Polymerase chain reaction (PCR)

Total RNA was isolated using Trizol reagent (RiboEx solution, cat. no. 301-001, GeneAll, Korea). The RNA purity and amount were evaluated using NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific, USA). Aliquots of 1 μg RNA were used for complimentary DNA conversion using a reverse transcriptase kit (ImProm-II Reverse Transcription System, cat. no. A3800, Promega, USA). Real- time PCR was conducted in a CFX connect Real-Time PCR machine (Bio-Rad, Singapore) with FastStart Essential DNA Green Master kit (cat. no. 06402712001, Roche Life Science, USA). The mRNA expression levels were calculated using the 2− ΔΔCT method. Melt curve analysis was performed to confirm a product specificity. The expression of 18S mRNA levels was used as the normalization reference. The oligonucleotide sequences are presented in Supplementary Table 1.

2.6. Western blot analysis

Radioimmunoprecipitation buffer containing a cocktail of protease inhibitor (Sigma) was used for total protein collection. Samples were subjected to electrophoresis in a 12 % sodium dodecyl sulfate- polyacrylamide gels and subsequently transferred to the nitrocellulose membranes. The membranes were incubated with primary antibody (Jak1 antibody, #3332, Cell signaling Technology, USA, dilution 1:500 or actin antibody, #A2066, Sigma, USA, dilution 1:2000) for overnight at 4 ◦C. Anti-rabbit IgG, HRP-linked antibody (#7074, Cell Signaling Technology, USA) was incubated with the membranes at dilution 1:5000 for 1.5 h. SuperSignal® West Pico Chemiluminescent Substrate   (#34579, ThermoFisher Scientific, USA) was utilized to enhance substrate for detecting horseradish peroxidase (HRP) on immunoblots. The presence of target protein was evaluated by chemiluminescence (Pierce Biotechnology, IL, USA).

2.7. Statistical analyses

The experiments were conducted with the cells isolated from at least four different donors. In the graphs, each dot represents an individual data value. The horizontal lines represent median values. The statistical difference between groups was evaluated using the Mann Whitney U test All illustrations and statistical analyses were performed using GraphPad Prism version 8.4.3 (GraphPad Software, CA, USA). A statistically significant difference was considered when p < 0.05.

3. Results

3.1. Jagged1 induced IL15 and IL15RA expression

Bioinformatic analysis of the RNA sequencing data from the Jagged1-treated hDPs demonstrated that Jagged1 upregulated IL15 and IL15RA expression compared with the hFc control condition (Fig. 1A). This inductive effect was attenuated by pretreatment with DAPT, confirming the influence of Notch signaling. The effect of Jagged1 on IL15 and IL15RA mRNA expression was confirmed using real-time PCR. hDPs were treated with Jagged1 and maintained in the growth medium for 24 h. The results demonstrated that Jagged1 activated the Notch target gene, HES1, expression in a dose-dependent manner (Fig. 1B). Correspondingly, Jagged1 treatment led to the upregulation of IL15 and IL15RA in a dose-dependent manner (Fig. 1C and D). A significant increase in IL15 and IL15RA mRNA expression was observed when the hDPs were treated with 10 nM Jagged1.
In the time course experiment, the cells were seeded on Jagged1- immobilized surfaces and maintained in the growth medium. The mRNA expression levels were examined at day 3 and 7. Jagged1 continuously activated Notch signaling as determined by the significant upregulation of HES1 at day 3 and 7 (Fig. 1E). However, the significant upregulation of IL15 and IL15RA mRNA levels was observed only at day 3 while there was no significant difference at day 7 compared with the hFc control (Fig. 1F and G).

3.2. IL15 did not affect cell proliferation but promoted mineralization in hDPs

A previous report found that Jagged1 inhibited hDP cell proliferation while inducing the odonto/osteogenic differentiation (Manokawinchoke, Nattasit et al., 2017). To further evaluate whether Jagged1-induced IL15 expression was involved in these responses, hDPs were treated with 5–50 ng/mL recombinant human IL15. IL15 was not toxic to the cells after 24 h (Fig. 2A). Further, hDPs proliferated as determined by the increase in cell number at day 7 compared with day 1. IL15 treatment had no effect on cell proliferation in all experimental conditions (5–50 ng/mL) (Fig. 2B). hDPs were maintained in the osteogenic medium with or without IL15 supplementation (20 ng/mL). A significant increase in in vitro mineral deposition was observed in the IL15-treated group compared with the control group at day 14 after induction (Fig. 2C–G). The mRNA expression of osteogenic marker genes and mineralization-related genes was evaluated at day 7. IL15 supplementation in the osteogenic medium significantly upregulated ALP, OCN, OSX, ANHK, and ENPP1 mRNA levels compared with the control condition, while a slight increase was observed for COL1A1 and RUNX2 mRNA expression after IL15 treatment (Fig. 2H – N). In addition, pretreating the cells with a JAK inhibitor prior to IL15 exposure attenuated the effect of IL15-induced mineralization in hDPs at day 14 as shown by alizarin red s and Von Kossa staining (Fig. 3A). The JAK inhibitor also significantly inhibited the influence of IL15 on mineral deposition (Fig. 3B).

3.3. Jagged1 induced mineralization in hDPs via the JAK-STAT pathway

hDPs were seeded on Jagged1 immobilized surface for 6 h. The JAK1 protein expression was examined using western blot assay. Jagged1 treatment result in the increased trend of JAK1 expression (Fig. 4A and B). To determine the participation of IL15 on Jagged1-induced mineralization in hDPs, the cells were pretreated with a JAK inhibitor (15 nM) for 30 min prior to seeding on Jagged1-immobilized surfaces. The cells were maintained in the osteogenic medium for 14 d. The results demonstrated that JAK inhibitor treatment abolished the Jagged1- induced mineralization in hDPs (Fig. 4C–D), demonstrating the involvement of the JAK-STAT pathway.

3.4. IL15 did not affect the mRNA expression of Notch related genes

To further examine whether Jagged1-induced IL15 expression exhibited a feedback regulation on Notch signaling, hDPs were treated with 20 ng/mL IL15 in the normal growth medium for 7 d. IL15 did not affect the mRNA expression levels of Notch target genes, HES1 or HEY1, or the Notch ligand (JAG1) (Fig. 5A–C).

4. Discussion

The present study described the participation of IL15 in Jagged1- induced mineralization in hDPs. Jagged1 treatment resulted in the upregulation of IL15 and IL15RA mRNA expression. IL15 treatment resulted in a significant increase in mineral deposition and this effect was abolished by pretreatment with a JAK inhibitor. Further, JAK inhibitor treatment attenuated the effect of Jagged1 on mineralization in hDPs. These results imply the involvement of IL15 in Jagged1- induced mineralization in hDPs.
Among canonical Notch receptors, Jagged1 have been shown to effectively promote the osteogenic differentiation. Jagged1 treatment significantly enhanced mineralization higher than Dll1 treatment (Sukarawan et al., 2016). Role of Jagged1 in the osteogenic differentiation and mineralization is previously reported (Hansamuit, Osathanon, & Suwanwela, 2020; Manokawinchoke, Pavasant, & Osathanon, 2017, 2020; Osathanon et al., 2019). Jagged1 interacted with NOTCH2 receptor and regulated the osteogenic differentiation of human periodontal ligament cells (hPDLs) and hDPs (Manokawinchoke, Nattasit et al., 2017, 2020). In this respect, NOTCH2 knockdown cells exhibited decreased mineralization after Jagged1 treatment. Mechanistically, Jagged1 treatment resulted in the increased binding between notch intracellular domain and protein kinase C delta (PKCδ) (Zhu, Sweetwyne, & Hankenson, 2013). Additionally, Jagged1 downregulated the expression of transcription factor TWIST, a negative regulator for the osteogenic differentiation. (Osathanon, Ritprajak et al., 2013; Yen, Ting, & Maxson, 2010). However, the effect of Jagged1 is cell-type specific. For example, Jagged1 directly increased the osteoclast differentiation from progenitor cells. Nevertheless, the partial regulation of Jagged1 on the osteogenic differentiation was demonstrated. Jagged1 partially promoted the osteogenic differentiation in murine gingival fibroblast derived induced pluripotent stem cells (Osathanon, Manokawinchoke, Egusa, & Pavasant, 2017). In addition, Jagged1 treatment inhibited osteoprotegerin expression of hPDLs, which indirectly promoted the osteoclast differentiation (Manokawinchoke, Sumrejkanchanakij, Subbalekha, Pavasant, & Osathanon, 2016). These results indicate the crucial role of Jagged1 in bone homeostasis.
A previous report demonstrated that the mineral density of femur cortical tissue was significantly decreased in IL15RA knockout mice compared with the wild-type control, while the trabecular structure was not different between these groups (Loro et al., 2017). Further, it was noted that osteoclast and osteocyte cell numbers were reduced in the IL15RA knockout mice (Loro et al., 2017). Moreover, bone marrow stromal cells isolated from the IL15RA knockout mice exhibited a decreased mineralization without a significant change in Alp, Ocn, or Col1a1 gene expression (Loro et al., 2017). Mechanistically, Il15ra silencing decreased ENPP1 activity in MC3T3 cells (Loro et al., 2017), which may participate in the reduced mineral deposition in the cells isolated from the IL15RA knockout mice. Correspondingly, the present study found that IL15 supplementation resulted in a significant increase in ALP, ANKH, and ENPP1 mRNA expression. These genes regulate phosphate metabolism and control mineralization. ENPP1 breaks down adenosine triphosphate into pyrophosphate and ANKH transports intracellular pyrophosphate to the extracellular space (Foster et al., 2008). The function of these 2 genes leads to the extracellular accumulation of pyrophosphate. However, ALP cleaves pyrophosphate into inorganic phosphate (Foster et al., 2008). Hence, the increased expression of ALP could result in the accumulation of inorganic phosphate extracellularly. Previous reports demonstrated that the accumulation of extracellular inorganic phosphate promoted mineralization in various cell types (Nowwarote, Sukarawan, Pavasant, Foster, & Osathanon, 2018; Osathanon, Nowwarote, Manokawinchoke, & Pavasant, 2013). Our study observed that IL15 treatment upregulated OSX, RUNX2, and OCN expression. OSX and RUNX2 are important transcription factors that regulate the osteoblast differentiation, which is first step in bone formation (Huang, Yang, Shao, & Li, 2007). Osteoblasts express OCN gene, which regulates matrix mineralization (Zoch, Clemens, & Riddle, 2016). Based on these results, IL15 regulates phosphate metabolism regulatory genes and osteoblast differentiation-related genes, leading to increased mineralization in hDPs.
IL15 utilized the JAK-STAT signaling pathway to regulate cell responses. It has been shown in CD4+ cells that IL15-induced SAMHD1 phosphorylation is JAK pathway dependent (Manganaro et al., 2018). A JAK/STAT signaling inhibitor (573108) abolished the effect of IL15 on PPARG expression in mesenchymal stem cells after the adipogenic induction (Shi et al., 2019). IL15 stimulation activates specific JAK/STAT pathway components. IL15 stimulates glucose uptake in C2C12 cells via the Jak3/Stat3 pathway because IL15 did not affect the levels of phospho-Jak1 or phospho-Stat5 (Krolopp et al., 2016). The IL-15/STAT3/STAT5 axis is crucially involved in natural killer cell biology (Gotthardt, Trifinopoulos, Sexl, & Putz, 2019). In the present study, the IL15-induced mineralization in hDPs was abolished Ilginatinib by JAK inhibitor treatment, confirming the involvement of the JAK/STAT pathway. Further, we demonstrated that Jagged1-treated hDPs exhibited the increased trend of JAK1 expression. Although, the phosphorylated JAK1 was not examined in the present study. JAK inhibition in Jagged1-treated hDPs resulted in the decreased mineral deposition, implying the participation of the JAK/STAT pathway in Jagged1-induced mineralization in hDPs.

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