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Attenuated RANKL-induced cytotoxicity by Portulaca oleracea ethanol extract enhances RANKL-mediated osteoclastogenesis
- Munkhsoyol Erkhembaatar†1,
- Eun-Joo Choi†2,
- Hak-Yong Lee3,
- Choong Hun Lee4,
- Young-Rae Lee5, 6 and
- Min Seuk Kim1, 4Email author
© Erkhembaatar et al. 2015
Received: 25 February 2015
Accepted: 7 July 2015
Published: 14 July 2015
Portulaca oleracea (PO) has been widely used as traditional medicine because of its pharmacological activities. However, the effects of PO on osteoclasts that modulate bone homeostasis are still elusive.
In this study, we examined the effects of PO ethanol extract (POEE) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated Ca2+ mobilization, nuclear factor of activated T-cell c1 (NFATc1) amplification, tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cell (MNC) formation, and cytotoxicity.
Our results demonstrated that POEE suppressed RANKL-induced Ca2+ oscillations by inhibition of Ca2+ release from internal Ca2+ stores, resulting in reduction of NFATc1 amplification. Notably, POEE attenuated RANKL-mediated cytotoxicity and cleavage of polyadenosine 5′-diphosphate-ribose polymerase (PARP), resulted in enhanced formation of TRAP+ MNCs.
These results present in vitro effects of POEE on RANKL-mediated osteoclastogenesis and suggest the possible use of PO in treating bone disorders, such as osteopetrosis.
Portulaca oleracea (PO), also known as verdolaga and pigweed, has been widely used not only as food, but also as traditional medicine treating insect bites, bacillary dysentery, diarrhea, and hemorrhoids. The chemical constituents of PO have been repeatedly reported to have diverse pharmacological activities. For instance, polysaccharides and betacyanins isolated from PO have antifatigue effects and improve cognition deficits in mice, respectively [1, 2]. Importantly, PO ethanol extract (POEE) is known to have protective effects against ultraviolet-induced apoptosis in keratinocytes and fibroblasts , whereas POEE elicits cytotoxicity in cancer cells . These accumulated evidences suggest the multiple effects of PO, which plays different roles dependent on cell type.
Bone is constantly being remodeled by the delicate balance between the activities of osteoblasts, which are in charge of bone mineralization, and osteoclasts, which resorb bone matrix. In the process of bone remodeling, receptor activator of nuclear factor-κB ligand (RANKL), a key molecule expressed in osteoblasts, mediates osteoclastogenesis resulting in breakdown of bone. Contact between RANKL and receptor activator of nuclear factor-κB (RANK), expressed on osteoclast precursor cells, mediates differentiation-related signals through nuclear factor of activated T-cell c1 (NFATc1) activation . Notably, repeated reports have clearly showed that RANKL-induced free cytosolic Ca2+ ([Ca2+]i) oscillations modulate NFATc1 activity [5–7]. Generation of long-lasting [Ca2+]i oscillations sequentially activates Ca2+/calmodulin-dependent protein kinase, calcineurin, and NFATc1. Activated NFATc1 accumulates inside the cell nucleus, eliciting the induction of gene expression necessary for osteoclastogenesis. On the other hand, RANKL is also known to inhibit cell proliferation and induce apoptosis through a tumor necrosis factor receptor-associated factor 6 (TRAF6)-dependent but NF-κB-independent mechanism . However, the signal pathways underlying these opposing effects of RANKL-RANK contact are less well understood.
In this study, we report in vitro effects of POEE, which attenuates RANKL-induced cytotoxicity and enhances RANKL-mediated osteoclastogenesis. Our findings may suggests the possible use of PO to modulate the activity of osteoclast.
Cell culture and reagents
BMMs isolation was conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Wonkwang University (committee member: Sung Yeon Kim, Jungkee Kwon, Hong Geun Oh, Hong-Seob So, Okjin Kim, Chun-Soo Ko). Primary BMMs were cultured in alpha-modified minimum essential medium (α-MEM; Sigma-Aldrich, MO, USA) supplemented with 10 % fetal bovine serum (FBS) and 30 ng/mL macrophage colony-stimulating factor (M-CSF) and incubated in 5 % CO2. PO collected at a local farm (Duhak-dong, Jecheon-si, Chungcheongbuk-do, Republic of Korea) was purchased from the University Oriental Herbal Drugstore (Iksan, Republic of Korea), and it was authenticated by Oriental pharmacologist Jang-Ho Ko (Huvet Inc., Iksan, Republic of Korea). A voucher specimen has not been deposited in a public herbarium. Dried PO was ground and extracted with ethanol for 3 h at 70 °C. After filtering, the precipitate was collected and vaccum-dried at 78 °C and then used for each experiment. Soluble recombinant mouse RANKL and M-CSF were purchased from KOMA Biotech (Seoul, Korea). Cyclopiazonic acid (CPA) and adenosine triphosphate (ATP) were obtained from TOCRIS Bioscience (Bristol, US) and Sigma Aldrich (MO, USA), respectively. Antibodies against polyadenosine 5′-diphosphate-ribose polymerase (PARP), NFATc1, and β-actin were purchased from Cell Signaling Technology (MA, USA), Santa Cruz Biotechnology (TX, USA), and Sigma Aldrich (MO, USA), respectively. Fura-2-acetoxymethyl ester (Fura-2 AM) was obtained from TEFLabs (TX, USA).
In vitro osteoclast formation
Murine BMMs were prepared from the femur and tibia of 4- to 6-week-old mice, as previously described . Briefly, bone marrow was flushed out with culture medium (α-MEM) and collected. After removal of red blood cells, whole marrow cells were plated on uncoated Petri dishes in the presence of M-CSF (10 ng/mL). The following day, the BMMs were collected and then seeded on designated culture dishes for each experiment. Henceforth, M-CSF was supplemented in the culture medium at a concentration of 30 ng/mL. To generate osteoclasts, the BMMs were treated with RANKL (50 ng/mL) for the indicated time. To evaluate the formation of multi-nucleated cells (MNCs) and tartrate-resistant acid phosphatase (TRAP) activity, BMMs were plated on 24-well culture dishes at a density of 1.2 × 105 cells per well. Cytochemical staining for TRAP expression was performed using a leukocyte acid phosphate assay kit (Sigma-Aldrich, MO, USA), following the manufacturer’s procedures. For measurement of total TRAP activity, p-nitrophenyl phosphate (Sigma-Aldrich, MO, USA) substrate was added to the culture medium containing whole lysates of BMMs. Optical density was measured at an absorbance of 405 nm.
Isolated BMMs, as described in “In vitro osteoclast formation,” were plated in 96-well plates at a density of 1 × 104 cells per well. RANKL and POEE were then added to each well at the indicated concentrations. Following incubation with POEE for the indicated length of time, cytotoxicity was determined using the Vybrant cytotoxicity assay kit (Sigma Aldrich, MO, USA), following the manufacturer’s procedure. Optical density was measured using a microplate reader at 530/590 nm (Ex/Em).
Western blot analysis
Isolated BMMs were plated on 60-mm dishes at a density of 1 × 106 cells. Following incubation under the stated conditions, each sample was lysed in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 % NP-40, 1 % sodium deoxycholate, 0.1 % SDS) containing protease inhibitors, and the collected whole cell lysates were cleared by centrifugation at 14,000 × g for 10 min at 4 °C. Proteins in the total lysates were separated by size using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with the indicated antibodies overnight, and immunoreactive proteins were detected using an electrochemiluminescent (ECL) detection system on the following day.
[Ca2+]i was determined using the Ca2+-sensitive fluorescent dye Fura-2, as described previously . Briefly, isolated BMMs were plated on cover glass at approximately 80 % confluence (6 × 105 cells per 35-mm dish) and cultured in α-MEM medium. After stimulation with RANKL for 2 d, cells plated on cover glass were used as samples for [Ca2+]i measurement. The cells on a cover glass were transferred to HEPES buffer containing 10 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 10 mmol/L glucose, adjusted to pH 7.4 and 310 mOsm, and then loaded with Fura-2 fluorescent Ca2+ indicator for 50 min at RT and placed in a chamber connected to a perfusion system. Cells were briefly washed out with regular HEPES buffer and continuously perfused with HEPES buffer without RANKL and M-CSF. Each of the indicated chemicals was diluted in HEPES buffer or Ca2+-free HEPES buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L EGTA, and 10 mmol/L glucose, adjusted to pH 7.4 and 310 mOsm) and perfused for the designated length of time. Under continuous perfusion with regular HEPES buffer (37 °C), intracellular fluorescence was excited at dual wavelengths (340 and 380 nm) and emitted fluorescence (510 nm) was captured using a charge-coupled device (CCD) camera. Captured images were digitized and analyzed using MetaFluor software, with data expressed as the ratio of fluorescence intensities (F340/F380).
Results were analyzed using Student’s 2-tailed t-test. Data are presented as mean ± SEM of the stated number of observations obtained from the indicated number of independent experiments. P-values less than 0.05 were considered statistically significant (*P < 0.05, **P < 0.01).
Results and discussion
POEE acutely abolished RANKL-induced [Ca2+]i oscillations
POEE reduced receptor-mediated Ca2+ release from internal Ca2+ stores, but not extracellular Ca2+ influx via store-operated Ca2+ channels
POEE enhanced RANKL-mediated TRAP+ MNC formation
POEE attenuated RANKL-mediated cytotoxicity
According to our results and other reports, RANKL is known to not only elicit Ca2+ - Ca2+/calmodulin-dependent kinase – calcineurin – NFATc1 signal pathway, which induces differentiation-related gene expression , but also suppress cell proliferation and induce apoptosis through TRAF6-dependent mechanism . These reports obviously show that these conflicting signal pathways are both involved in osteoclastogenesis. Notably, NFATc1 activation occurs in relatively early stage of osteoclastogenesis, whereas apoptosis is induced in relatively late stage of osteoclastogenesis . Considering these, our data clearly demonstrate that POEE has multiple effects on RANKL-mediated osteoclastogensis. In early stage, POEE suppresses RANKL-induced Ca2+ oscillation and NFATc1 expression, whereas POEE attenuates RANKL-mediated cytotoxicity in late stage, resulting in enhanced multi-nucleated cells formation. Taken together, anti-apoptotic function of POEE is more essential and dominant effect on RANKL-mediated osteoclastogenesis.
Our results demonstrate that POEE has dual and opposite effects on RANKL-mediated osteoclastogenesis. POEE not only inhibits the generation of RANKL-induced [Ca2+]i oscillations and NFATc1 activation but also markedly enhances RANKL-mediated osteoclastogenesis by attenuating RANKL-induced cytotoxicity. Our findings suggest that the RANKL-induced cytotoxicity caused by Ca2+ release from internal Ca2+ store is attenuated by POEE, which resulted in enhanced RANKL-mediated osteoclastogenesis.
This research was supported by Wonkwang University in 2014.
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