ST5 Positively Regulates Osteoclastogenesis Via Src/Syk/Calcium Signaling Pathways
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Molecules and Cells ST5 Positively Regulates Osteoclastogenesis via Src/Syk/Calcium Signaling Pathways Min Kyung Kim, Bongjun Kim, Jun-Oh Kwon, Min-Kyoung Song, Suhan Jung, Zang Hee Lee, and Hong- Hee Kim* Department of Cell and Developmental Biology, BK21 Program and Dental Research Institute (DRI), School of Dentistry, Seoul National University, Seoul 03080, Korea *Correspondence: [email protected] https://doi.org/10.14348/molcells.2019.0189 www.molcells.org For physiological or pathological understanding of bone INTRODUCTION disease caused by abnormal behavior of osteoclasts (OCs), functional studies of molecules that regulate the generation Bone is a dynamic organ that is constantly being disrupted and action of OCs are required. In a microarray approach, and restructured for maintenance of homeostasis (Zaidi, we found the suppression of tumorigenicity 5 (ST5) gene 2007). The process of bone resorption is controlled by ac- is upregulated by receptor activator of nuclear factor-κB tion of osteoclasts (OCs), which are multinucleated cells ligand (RANKL), the OC differentiation factor. Although the (MNCs) generated from a monocyte/macrophage lineage of roles of ST5 in cancer and β-cells have been reported, the hemopoietic cells (Boyle et al., 2003; Rho et al., 2004). The function of ST5 in bone cells has not yet been investigated. action of excessive OCs induces an imbalance in homeostasis, Knockdown of ST5 by siRNA reduced OC differentiation triggering pathological bone diseases, such as osteoporosis from primary precursors. Moreover, ST5 downregulation (Zaidi, 2007). decreased expression of NFATc1, a key transcription factor Osteoclastogenesis is a multi-step process including com- for osteoclastogenesis. In contrast, overexpression of ST5 mitment of OC precursor cells to mononucleated preosteo- resulted in the opposite phenotype of ST5 knockdown. In clasts (pOCs), fusion for formation of multinucleated OCs, immunocytochemistry experiments, the ST5 protein is co- and maturation of OCs for bone resorption activity. The localized with Src in RANKL-committed cells. In addition, process requires the action of two essential factors, mac- ST5 enhanced activation of Src and Syk, a Src substrate, in rophage-colony-stimulating factor (M-CSF) and receptor response to RANKL. ST5 reduction caused a decrease in activator of nuclear factor-κB ligand (RANKL) (Boyle et al., RANKL-evoked calcium oscillation and inhibited translocation 2003; Rho et al., 2004). The binding of RANKL to its receptor of NFATc1 into the nucleus. Taken together, these findings (RANK) initiates recruitment of tumor necrosis factor (TNF) re- provide the first evidence of ST5 involvement in positive ceptor-associated factor 6 (TRAF6) (Darnay et al., 1999) and regulation of osteoclastogenesis via Src/Syk/calcium signaling. activates downstream signaling molecules including MAPKs (ERK, JNK, and p38), NFκB, and AP-1 complex (a protein Keywords: calcium, NFATc1, osteoclasts, RANKL, Src, complex of c-Jun and c-Fos) (Park et al., 2017; Takayanagi, suppression of tumorigenicity 5, Syk 2005). Finally, these signaling pathways induce expression of the nuclear factor of activated T cells c1 (NFATc1), which is a key transcription factor for osteoclastogenesis. NFATc1 Received 23 August, 2019; revised 24 September, 2019; accepted 25 September, 2019; published online 11 November, 2019 eISSN: 0219-1032 ©The Korean Society for Molecular and Cellular Biology. All rights reserved. cc This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. 810 Mol. Cells 2019; 42(11): 810-819 The Role of ST5 in Osteoclastogenesis Min Kyung Kim et al. induces OC-specific genes, including tartrate-resistant acid Bone marrow-derived macrophage (BMM) isolation and phosphatase (TRAP) and cathepsin K (Kim and Kim, 2014). OC differentiation Calcium signaling is known to play a role in NFATc1 activation Bone marrow from the tibiae and femurs of 5-week-old by promoting its nuclear translocation. When RANKL triggers ICR mice was flushed with α-MEM (Welgene, Korea). After a signal for induction of osteoclastogenesis, calcium signaling removing erythrocytes with red blood cell lysis buffer (Invit- is stimulated via activation of PLCγ, which leads to generation rogen), cells were cultured in α-MEM containing 10% fetal of inositol 1,4,5-trisphosphate (IP3) and its binding to IP3 bovine serum overnight in culture dishes. Non-adherent bone receptors on the endoplasmic reticulum (ER) (Kim and Kim, marrow cells were collected and cultured in the presence 2014; Koga et al., 2004). In addition, Src- and Src substrate of M-CSF (30 ng/ml) for three days in 100 mm-Petri dishes. Syk-dependent signaling is involved in activation of PLCγ by Adherent cells (BMMs) were considered to be OC precursors RANKL (Tomomura et al., 2012; Yamasaki et al., 2014). and harvested. BMMs were seeded at 3.5 × 104 per well Suppression of tumorigenicity 5 (ST5) can exist in three in 48-well plates or 3.5 × 105 per well in 6-well plates with isoforms including p70 (isoform3), p82 (isoform2), and p126 M-CSF (30 ng/ml) and cultured with M-CSF (30 ng/ml) and (isoform1) encoded by alternatively spliced transcripts (2.8 RANKL (150 ng/ml) for 4 or 5 days to induce OC formation. kb, 3.1 kb, 4.6 kb mRNA, respectively) (Lichy et al., 1992). Cells were fixed in 3.7% formaldehyde and permeabilized The shortest form ST5 p70 is related with low tumorigenicity using 0.1% Triton X-100. TRAP staining was performed in and is the reason for the naming of the ST5 gene (Lichy et al., the dark for 10 minutes according to the manufacturer’s pro- 1992), whereas the longest ST5 p126 activates ERK signaling tocol. Multinucleated TRAP-positive cells were captured and in response to EGF on COS-7 cells (Majidi et al., 1998). Phys- counted under an Olympus BX51 microscope (Japan). iologically, the N-terminal domain of p126 binds the Src-ho- mology 3 (SH3) domain of c-Abl, leading to activation of the Microarray MAPK/ERK2 kinase. Moreover, the N-terminal domain of ST5 BMMs were cultured in α-MEM with M-CSF (30 ng/ml) and p126 also binds the SH3 domain of Src (Majidi et al., 1998). RANKL (150 ng/ml). Total RNA was prepared from untreated The C-terminal region of ST5 p126 binds and catalyzes the cells (day 0), and cells treated with RANKL (day 3) and was exchange of GDP to GTP of the Rab 13 protein, which pro- then hybridized with Mouse WG-6 v2.0 BeadChips (Illumina, motes metastatic behavior (Ioannou et al., 2015). In addition USA). Microarray data were analyzed with the Genome Stu- to its function in cancer, the downregulation of ST5 has been dio Gene Expression Module (Gx) v3.2 (Illumina). correlated with mutation of hepatocyte nuclear factor-4α (HNF-4α), a key regulator of β-cell proliferation (Ou et al., ST5 gene knockdown by siRNA 2018). However, to our knowledge, there is no report of ST5 BMMs were transfected with 25 nM ST5 siRNA (sc-153863; expression or function in the skeletal system. Santa Cruz Biotechnology). In brief, siRNA and transfection In this report, we found that ST5 is induced by RANKL reagent HiPerFect (Qiagen, The Netherlands) were mixed during OC differentiation. In loss- and gain-of-function stud- with serum-free α-MEM medium. After the mixtures were ies, we revealed that ST5 promotes OC differentiation by up- incubated for 20 min at room temperature, they were added regulating NFATc1 via activation of Src/Syk/calcium signaling. to the cells. MATERIALS AND METHODS Bone resorption assay To determine osteoclast lytic activity, BMMs (1 × 104) were Reagents seeded on dentine discs (Osteosite Dentine Discs; Immu- Recombinant human M-CSF and RANKL were purchased nodiagnostic Systems, UK) in 96-well plate and transfected from PeproTech (USA). Anti-NFATc1 was acquired from BD with ST5 siRNA or control siRNA. Cells were cultured with Biosciences (USA). Anti-c-Fos anti-α-tubulin were purchased M-CSF (30 ng/ml) and RANKL (150 ng/ml) for 7 to 9 days. from Santa Cruz Biotechnology (USA). Polyclonal antibodies The plates were rinsed with distilled water to remove cells against ERK, JNK, p38, IκB, phospho-ERK, phospho-JNK, for 15 min, and the dentine discs were carefully wiped with phospho-p38, phospho-IκB, phospho-Src family, and phos- a cotton swab. The discs were scanned with a Carl Zeiss LSM pho-Syk were purchased from Cell Signaling Technology 5-PASCAL laser-scanning microscope (Carl Zeiss Microimag- (USA). A monoclonal antibody against β-actin was obtained ing, Germany). The areas and depths of 50 resorption pits in from Sigma Aldrich (USA). The NE-PER Nuclear and Cytoplas- each sample were determined with image analysis software mic Extraction Reagents Kit was obtained from Invitrogen (LSM 5 Image Browser; Carl Zeiss Microimaging). The assay (Carlsbad, CA, USA). The TRAP staining kit was purchased was performed in triplicate wells per group. from Sigma Aldrich. Reverse transcription-polymerase chain reaction (RT-PCR) Animals and real-time PCR The ICR mice used for preparation of OC precursor cells were After extraction of total RNA using Trizol (Invitrogen), 3 μg of purchased from OrientBio (Korea). All animal experiments RNA was reverse transcribed into cDNA using Superscript II were performed with approval of the Institutional Animal (Invitrogen) according to the manufacturer's instructions. The Care and Use Committee at Seoul National University (SNU- PCR reaction was performed as follows: denaturation at 95oC 160105-3-5) and conducted according to the Guidelines for for 15 s and annealing at 60oC or 57oC for 30 s. PCR prod- Animal Experimentation. ucts were loaded onto 1.5% agarose gels. The Hprt gene Mol. Cells 2019; 42(11): 810-819 811 The Role of ST5 in Osteoclastogenesis Min Kyung Kim et al.