Hypotonic stress induces RANKL via transient receptor potential melastatin 3 (TRPM3) and vaniloid 4 (TRPV4) in human PDL cells

Abstract

Bone remodeling occurs in response to various types of mechanical stress. The periodontal ligament (PDL) plays an important role in mechanical stress-mediated alveolar bone remodeling. However, the underlying mechanism at the cellular level has not been extensively studied. In this study, we investigated the effect of shear stress on the expression of bone remodeling factors, including receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG), as well as its upstream signaling pathway in primary human PDL cells. We applied hypotonic stress to reproduce shear stress to PDL cells. Hypotonic stress induced the messenger RNA (mRNA) and protein expression of RANKL but not OPG. It also increased intracellular Ca(2+) concentration ([Ca(2+)]i). Extracellular Ca(2+) depletion and nonspecific plasma membrane Ca(2+) channel blockers completely inhibited the increase in both [Ca(2+)]i and RANKL mRNA expression. We identified the expression and activation of transient receptor potential melastatin 3 (TRPM3) and vaniloid 4 (TRPV4) channels in PDL cells. Pregnenolone sulfate (PS) and 4α-phorbol 12, 13-didecanoate (4α-PDD), which are agonists of TRPM3 and TRPV4, augmented Ca(2+) influx and RANKL mRNA expression. Both pharmacological (2-aminoethoxydiphenyl borate [2-APB], ruthenium red [RR], ononetin [Ono], and HC 067047 [HC]) and genetic (small interfering RNA [siRNA]) inhibitors of TRPM3 and TRPV4 reduced the hypotonic stress-mediated increase in [Ca(2+)]i and RANKL mRNA expression. Our study shows that hypotonic stress induced RANKL mRNA expression via TRPM3- and TRPV4-mediated extracellular Ca(2+) influx and RANKL expression. This signaling pathway in PDL cells may play a critical role in mechanical stress-mediated alveolar bone remodeling.

Keywords: bone remodeling/regeneration; cell signaling; ion channels; mechanotransduction; osmotic stress; periodontal ligament.

Figure 1.

Effect of hypotonic stress on receptor activator of nuclear factor–kappa B (NF-κB) ligand (RANKL) messenger RNA (mRNA) expression and Ca²⁺ signaling in primary human PDL cells. (A–C) Increase in the expression of RANKL but not osteoprotegerin (OPG) by hypotonic stress. (A) Cells were treated with the hypotonic solution (Hypo; 215 mOsm) or adenosine triphosphate (100 µM) for 12 h. The mRNA levels of RANKL and OPG were analyzed by reverse transcription–polymerase chain reaction (RT-PCR). (B) The levels of RANKL and OPG mRNA were quantified after the value was normalized to GAPDH (n = 10). (C) Cells were treated with the hypotonic solution for 24 h. The protein levels of RANKL and OPG were analyzed by Western blot. (D) Increase in RANKL, but not OPG, mRNA expression by thapsigargin (Tg). Cells were treated with Tg (1 µM) for the indicated time. RANKL and OPG mRNA levels were analyzed by RT-PCR. (E–G) Increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) by hypotonic stress. After hypotonic stress was applied to fura-2/AM-stained cells once (E) or repetitively (F), the fluorescence intensity was measured at excitation wavelengths of 340 and 380 nm. (G) Summary of the effect of repetitive hypotonic solution application on [Ca²⁺]_i (n = 8). C, control; Hypo, hypotonic solution. The asterisks denote statistically significant differences between the compared values: ***P < 0.001.

Figure 2.

Effect of the hypotonic stress–induced increase in [Ca²⁺]_i on receptor activator of nuclear factor–kappa B (NF-κB) ligand (RANKL) messenger RNA (mRNA) expression. (A, B) Extracellular Ca²⁺ depletion and nonspecific plasma membrane Ca²⁺ channel blockers abolished the hypotonic stress–induced increase in [Ca²⁺]_i. The change in [Ca²⁺]_i in response to the hypotonic solution was measured after the application of a Ca²⁺-free solution (A) or pretreatment with gadolinium (Gd³⁺: 10 µM) or lanthanum (La³⁺: 100 µM) for 4 min (B). (C, D) Nonspecific plasma membrane Ca²⁺ channel blockers inhibited the effects of hypotonic stress on RANKL, but not osteoprotegerin (OPG), mRNA expression. Cells were pretreated with Gd³⁺ (10 µM), La³⁺ (100 µM), or the intracellular Ca²⁺-selective chelator, BAPTA-AM (5 µM), for 30 min and incubated with or without the hypotonic solution for 12 h. (C) The mRNA levels of RANKL and OPG were determined by reverse transcription–polymerase chain reaction (RT-PCR). (D) The mRNA expression levels of RANKL and OPG were quantified after normalized to GAPDH (n = 4). C, control; Hypo, hypotonic solution; –Ca²⁺, Ca²⁺-free solution. The asterisks denote statistically significant differences between the compared values: *P < 0.05, **P < 0.01.

Figure 3.

Role of TRPM3 and TRPV4 in the Ca²⁺ influx and receptor activator of nuclear factor–kappa B (NF-κB) ligand (RANKL) messenger RNA (mRNA) expression. (A, B) TRPM3 and TRPV4 were expressed in periodontal ligament (PDL) cells. The mRNA and protein expression of TRPM3 and TRPV4 were examined using reverse transcription–polymerase chain reaction (RT-PCR) and Western blot, respectively. (C, D) Effect of TRPM3 and TRPV4 activation on [Ca²⁺]_i. [Ca²⁺]_i was measured after the treatment with the indicated concentration of pregnenolone sulfate (PS) (C) or 4α-phorbol 12, 13-didecanoate (4α-PDD) (D). (E, F) Increase of RANKL, but not osteoprotegerin (OPG), mRNA expression by TRPM3 or TRPV4 activators. Cells were treated with PS (50 µM) (E) or 4α-PDD (10 µM) (F) for 12 h. The mRNA levels of RANKL and OPG were determined by RT-PCR. (G, H) Induction of TRPM3 and TRPV4 expression by hypotonic stress. (G) Expression of TRPM3 and TRPV4 mRNA was analyzed by RT-PCR with RNA isolated from cells treated with hypotonic solution for 12 h. (H) Expression of TRPM3 and TRPV4 protein was analyzed by Western blot with protein isolated from cells treated with hypotonic solution for 24 h. (I–L) TRPM3 and TRPV4 were activated by hypotonic stress. After the application of hypotonic stress, current-voltage relations of TRPM3 (I, J) and TRPV4 (K, L) were determined by whole-cell patch clamp (–100 to +100 mV in 400-ms intervals; V_h = 0 mV) in immortalized human PDL cells (n = 3). C, control; Hypo, hypotonic solution.

Figure 4.

Role of TRPM3 and TRPV4 in the hypotonic stress–induced increase in [Ca²⁺]_i and receptor activator of nuclear factor–kappa B (NF-κB) ligand (RANKL) messenger RNA (mRNA) expression. (A, B) Effect of 2-aminoethoxydiphenyl borate (2-APB) and ruthenium red (RR) on hypotonic stress–mediated [Ca²⁺]_i. [Ca²⁺]_i was measured after pretreatment with 2-APB (75 µM) or RR (10 µM) for 4 min and quantified as peak value (n = 3). (C, D) Prevention of hypotonic stress–induced RANKL mRNA expression after the treatment of nonspecific transient receptor potential (TRP) channel blockers. (C) Cells were pretreated with 2-APB (75 µM) or RR (10 µM) for 30 min and incubated with or without the hypotonic solution for 12 h. The mRNA levels of RANKL and osteoprotegerin (OPG) were analyzed by reverse transcription–polymerase chain reaction (RT-PCR). (D) The levels of RANKL and OPG mRNA were quantified after being normalized to GAPDH (n = 4). (E, F) Effect of ononetin (Ono) and HC 067047 (HC) on hypotonic stress–mediated [Ca²⁺]_i. [Ca²⁺]_i was measured after pretreatment with Ono (10 µM) or HC (10 µM) for 4 min and quantified as peak value (n = 10). (G, H) Prevention of hypotonic stress–induced RANKL mRNA expression after the treatment of specific TRPM3 or TRPV4 blockers. (G) Cells were pretreated with Ono (10 µM) or HC (10 µM) for 30 min and incubated with or without the hypotonic solution for 12 h. The mRNA levels of RANKL and OPG were analyzed by RT-PCR. (H) The levels of RANKL and OPG mRNA were quantified after being normalized to GAPDH (n = 3). C, control; Hypo, hypotonic solution; Iono, ionomycin. The asterisks denote statistically significant differences between the compared values: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Effect of TRPM3 and TRPV4 knockdown with small interfering RNA (siRNA) on the hypotonic stress–induced increase in [Ca²⁺]_i and receptor activator of nuclear factor–kappa B (NF-κB) ligand (RANKL) messenger RNA (mRNA) expression. (A) Inhibition of TRPM3 and TRPV4 with siRNA. The reduced mRNA expression of TRPM3 and TRPV4 was measured by reverse transcription–polymerase chain reaction (RT-PCR) in periodontal ligament (PDL) cells. (B–D) Knockdown of TRPM3 or TRPV4 prevented TRPM3- or TRPV4-mediated Ca²⁺ influx. [Ca²⁺]_i was measured after treatment with pregnenolone sulfate (PS) (B) or 4α-phorbol 12, 13-didecanoate (4α-PDD) (C) and quantified as peak value (D; n = 5) in the siRNA-transfected PDL cells. (E–G) Effect of TRPM3 and TRPV4 knockdown on hypotonic stress–mediated increase in [Ca²⁺]_i. [Ca²⁺]_i was measured after TRPM3 and TRPV4 siRNA transfection and quantified as peak value (n = 4). (H, I) Suppression of hypotonic stress–induced RANKL mRNA expression by TRPM3 and TRPV4 knockdown. (H) Cells were transfected with siRNA of TRPM3 and TRPV4 and incubated with or without the hypotonic solution for 12 h. The mRNA levels of RANKL and osteoprotegerin (OPG) were analyzed by RT-PCR. (I) The levels of RANKL and OPG mRNA were quantified after being normalized to GAPDH (n = 4). C, control; NC (negative control), control siRNA; Hypo, hypotonic solution; siTRPM3, TRPM3 siRNA; siTRPV4, TRPV4 siRNA. The asterisks denote statistically significant differences between the compared values: *P < 0.05, **P < 0.01, ***P < 0.001.