The calcium channel TRPV6 is a novel regulator of RANKL-induced osteoclastic differentiation and bone absorption activity through the IGF-PI3K-AKT pathway

Abstract

Objectives: Calcium ion signals are important for osteoclast differentiation. Transient receptor potential vanilloid 6 (TRPV6) is a regulator of bone homeostasis. However, it was unclear whether TRPV6 was involved in osteoclast formation. Therefore, the aim of this study was to evaluate the role of TPRV6 in bone metabolism and to clarify its regulatory role in osteoclasts at the cellular level.

Materials and methods: Bone structure and histological changes in Trpv6 knockout mice were examined using micro-computed tomography and histological analyses. To investigate the effects of Trpv6 on osteoclast function, we silenced or overexpressed Trpv6 in osteoclasts via lentivirus transfection, respectively. Osteoclast differentiation and bone resorption viability were measured by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assays. The expression of osteoclast marker genes, including cathepsin k, DC-STAMP, Atp6v0d2 and TRAP, was measured by qRT-PCR. Cell immunofluorescence and Western blotting were applied to explore the mechanisms by which the IGF-PI3K-AKT pathway was involved in the regulation of osteoclast formation and bone resorption by Trpv6.

Results: We found that knockout of Trpv6 induced osteoporosis and enhanced bone resorption in mice, but did not affect bone formation. Further studies showed that Trpv6, which was distributed on the cell membrane of osteoclasts, acted as a negative regulator for osteoclast differentiation and function. Mechanistically, Trpv6 suppressed osteoclastogenesis by decreasing the ratios of phosphoprotein/total protein in the IGF-PI3K-AKT signalling pathway. Blocking of the IGF-PI3K-AKT pathway significantly alleviated the inhibitory effect of Trpv6 on osteoclasts formation.

Conclusions: Our study confirmed the important role of Trpv6 in bone metabolism and clarified its regulatory role in osteoclasts at the cellular level. Taken together, this study may inspire a new strategy for the treatment of osteoporosis.

Keywords: IGF-PI3K-AKT; TRPV6; osteoclast; osteoporosis.

FIGURE 1

Knockout of Trpv6 induced osteoporosis in mice. (A) qRT‐PCR analysis of Trpv2, Trpv4, Trpv5 and Trpv6 expression in BMMs and OCP from Trpv6−/− mice and WT mice. Summarized data showed that Trpv6 expression was significantly decreased in BMMs from Trpv6‐/‐ mice. n = 3, **P < .01. Trpv2, Trpv4 and Trpv5 expression levels were not significantly changed in BMMs and OCP derived from Trpv6‐/‐ mice and WT mice. n = 3. (B) BMD at the distal end of the intact femurs of each experimental group. n = 5, **P < .01. (C) Tb.N in the distal end of the intact femurs of each experimental group. n = 5, **P < .01. (D) BV/TV at the distal end of the intact femurs of each experimental group. n = 5, **P < .01. (E) Representative figures of micro‐CT analysis of the distal end of intact femurs from Trpv6−/− and WT mice. (F) Haematoxylin‐eosin staining was performed to identify histological structures at the distal end of the intact femurs of Trpv6−/− and WT mice. Scale bars are 50 μm

FIGURE 2

In Trpv6 knockout mice, bone absorption was enhanced and bone formation remained unchanged (A) Histological sections of femurs were subjected to Masson's trichrome staining. Scale bars are 50 μm. (B) Quantitative analysis of osteoclast number/bone surface (N.Ob/BS). n = 5, **P < .01. (C) Tetracycline labelling was observed by fluorescence light microscopy in the slices of the tibia of each experimental group. Scale bars are 50 μm. (D) Quantitative analysis of mineral apposition rate. n = 5, **P < .01. (E) Serum levels of ALP of Trpv6‐/‐ mice and WT mice. n = 5, **P < .01. (F) Histological sections of femurs stained for TRAP activity. (G) Quantitative analysis of TRAP‐stained area in femur sections of mice. n = 5, **P < .01. (H) Serum levels of CTX‐I of Trpv6‐/‐ mice and WT mice. n = 5, **P < .01

FIGURE 3

Trpv6 negatively regulates osteoclast differentiation and fusion and inhibits osteoclast formation. (A) Western blotting showed a time‐dependent decrease in Trpv6 expression during osteoclast differentiation. n = 3, *P < .05, **P < .01. (B) Confocal laser scanning microscopy showed Trpv6 staining (arrowhead) predominantly at the cell membrane. (C‐D) TRAP staining of osteoclasts precursors at different time points. Quantification of TRAP + multinucleated cells. Scale bars = 20μm. n = 5, **P < .01. (E‐F) Patterns of resorption pits on bovine cortical bone slices. Quantification of resorption area per view area. n = 3, **P < .01. (G‐J) qRT‐PCR analysis of the osteoclast formation‐specific genes Cathepsin K, DC‐STAMP, Atp6v0d2 and TRAP in each group under 50 ng/mL RANKL for seven days. n = 5, **P < .01

FIGURE 4

Silencing of Trpv6 enhanced osteoclast formation and bone resorption. (A) All cells expressed GFP, indicating that the cells were successfully infected by lentivirus. (B) Verified Trpv6 knockdown or overexpression by lentivirus‐mediated transduction of primary culture osteoclasts precursors. n = 5, **P < .01. (C‐F) qRT‐PCR analysis of the osteoclast formation‐specific genes Cathepsin K, DC‐STAMP, Atp6v0d2 and TRAP with Trpv6 knockdown or overexpression. n = 5, **P < .01. (G‐H) TRAP staining of osteoclasts precursors with Trpv6 knockdown or overexpression. Quantification of TRAP + multinucleated cells. Scale bars = 20μm. n = 5, *P < .05, **P < .01

FIGURE 5

The IGF pathway was involved in the negative regulation of osteoclast formation and bone resorption by Trpv6. (A‐B) qRT‐PCR analysis showed that transcription levels of IGF1R and IGFBP1 in osteoclasts were significantly increased after Trpv6 silencing. n = 5, **P < .01. (C‐D) Western blot depicting that the protein abundance levels of IGF1R and IGFBP1 in osteoclasts were significantly increased after Trpv6 silencing. n = 5, **P < .01. (E‐F) TRAP staining revealed that adding the IGF1R antagonist NVP‐AEW541 to block the IGF signalling pathway significantly inhibited the osteoclast differentiation induced by Trpv6 silencing. n = 3, *P < .05, **P < .01. (G‐H) After the addition of the IGF1R blocker, the ability of bone resorption of osteoclasts induced by Trpv6 silencing was significantly inhibited, as evidenced by the bone resorption lacuna experiment. n = 3, *P < .05, **P < .01

FIGURE 6

Trpv6 negatively regulates osteoclast formation and bone resorption by inhibiting the IGF1R‐PI3K‐AKT signalling pathway. (A) Immunofluorescence images of Trpv6 and IGF1R upon osteoclast induction with RANKL. Scale bars = 50μm. (B) At 0, 5, 15 and 30 min of treatment with RANKL, cells were harvested and analysed by Western blotting using anti‐P85, anti‐pP85, anti‐PDK1, anti‐pPDK1, anti‐AKT, anti‐pAKT and anti‐actin antibodies. (C‐E) Quantitative analysis showed that the ratio of p‐P85/P85, p‐PDK1/PDK and p‐AKT/AKT was increased in cells isolated from Trpv6−/− mice compared to WT mice. n = 5, *P < .05, **P < .01. (F‐G) Immunofluorescence of p‐AKT in osteoclast isolated from Trpv6‐/‐ mice and WT mice. n = 3, **P < .01. (H‐I) After NVP‐AEW541 blocking of the IGF signalling pathway, Western blotting revealed that there was no significant difference in the abundance ratio of p‐AKT/AKT between the osteoclast derived from TRPV6‐/‐ mice and WT mice. n = 5, *P < .05, **P < .01

FIGURE 7

Illustrations of TRPV6 as a critical regulator in osteoclastic bone resorption. TRPV6 negatively regulates osteoclast formation and bone resorption by inhibiting the IGF/PI3K/AKT signalling pathway