Korean J Physiol Pharmacol.  2013 Oct;17(5):427-433. 10.4196/kjpp.2013.17.5.427.

Activation of G Proteins by Aluminum Fluoride Enhances RANKL-Mediated Osteoclastogenesis

Affiliations
  • 1Department of Oral Biology, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 120-752, Korea. dmshin@yuhs.ac
  • 2Department of Pediatric Dentistry, Yonsei University College of Dentistry, Seoul 120-752, Korea.
  • 3Department of Oral Physiology, College of Dentistry, Wonkwang University, Iksan 570-749, Korea. happy1487@wku.ac.kr

Abstract

Receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular Ca2+ mobilization in a form of oscillations, which plays essential roles by activating sequentially Ca2+/calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether Ca2+ mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated Ca2+ mobilization induced by aluminum fluoride (AlF4-), a G-protein activator, with or without RANKL and the effects of AlF4- on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that AlF4- induces intracellular Ca2+ concentration ([Ca2+]i) oscillations, which is dependent on extracellular Ca2+ influx. Notably, co-stimulation of AlF4- with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by AlF4-. Taken together, these results demonstrate that G-protein would be a novel modulator responsible for [Ca2+]i oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis.

Keyword

AlF4-; Ca2+ signaling; G protein; MAPK activation; Osteoclastogenesis

MeSH Terms

Acid Phosphatase
Aluminum Compounds
Animals
Calcineurin
Fluorides*
GTP-Binding Proteins*
Isoenzymes
Macrophages
Mice
Osteoclasts
Protein Kinases
Receptor Activator of Nuclear Factor-kappa B
Acid Phosphatase
Aluminum
Aluminum Compounds
Calcineurin
Fluorides
GTP-Binding Proteins
Isoenzymes
Protein Kinases
Receptor Activator of Nuclear Factor-kappa B

Figure

  • Fig. 1 Effects of the [Ca2+]i increases mediated by RANKL and AlF4- in BMMs. BMMs were treated with RANKL (50 ng/ml) and/or AlF4- (1.25 mM NaF+25 nM AlCl3). After 48 h of the stimulations, [Ca2+]i oscillations in BMMs were measured using Fura-2 fluorescence dye. Compared to RANKL alone, application of AlF4- similarly increased frequency of the [Ca2+]i oscillations in the presence or absence of RANKL (left panels). These induction of [Ca2+]i oscillations were diminished by the removal of extracellular Ca2+ (right panel).

  • Fig. 2 Increased expression of NFATc1 in BMMs induced by RANKL and AlF4-. Whole cells lysates were collected from cells stimulated with RANKL and/or AlF4- for indicated time. NFATc1 and actin were blotted with its antibody (upper panels) and then showed means of protein expression levels between RANKL and AlF4- (lower panels). Expression level was significantly increased for NFATc1 in 48 h and 72 h of RANKL and AlF4- treatments (n=3). Data were expressed as the mean±SEM. **p<0.01, ***p<0.001 compared with RANKL treated group.

  • Fig. 3 RANKL and AlF4--induced osteoclast differentiation in BMMs. (A) To confirm the formation of multinucleated cells (MNCs, number of nuclear>3), each sample was incubated for 6 days in (a) control and the presence of (b) RANKL, (c) RANKL and AlF4-, and (d) AlF4-. TRAP staining was performed as described in methods. And then MNCs in each well was counted (n=3, scale bar, 50 µm). Application of RANKL and AlF4- in BMMs was significantly increased rates of TRAP+ MNCs formation more than the application of RANKL alone (lower panel). (B) Actin staining of TRAP+ MNCs in application of RANKL and AlF4- was similarly showed normal actin ring formations during osteoclastogenesis compared to RANKL treated BMM cells. Bar graph shows relative MNCs of BMMs treated with RANKL only and RANKL+ AlF4-. Data were normalized to the number of MNCs in RANKL treated BMMs and expressed as the mean±SEM. *p<0.05 compared with RANKL treated group.

  • Fig. 4 Phosphorylation of MAPK in BMMs induced by RANKL and AlF4-. To confirm the phosphorylation of MAPK in response of RANKL and/or AlF4-, ERK and JNK were blotted with its antibody. Applications of RANKL and/or AlF4- in BMMs were induced phosphorylation of ERK and JNK in a time-dependent manner.


Cited by  1 articles

Humanin suppresses receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation via AMP-activated protein kinase activation
Namju Kang, Ki Woo Kim, Dong Min Shin
Korean J Physiol Pharmacol. 2019;23(5):411-417.    doi: 10.4196/kjpp.2019.23.5.411.


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