The Role of Galectin-3 in 1α,25(OH)2D3-Regulated Osteoclast Formation from White Leghorn Chickens In Vitro

Bones play an important role in maintaining the level of calcium in blood. They provide support for soft tissues and hematopoiesis and undergo continuous renewal throughout life. In addition, vitamin D is involved in regulating bone and calcium homeostasis. Galectin-3 (Gal-3) is a β-galactoside-binding protein that can regulate bone cell differentiation and function. Here, we aimed to study the regulatory effects of Gal-3 on vitamin-D-regulated osteoclastogenesis and bone resorption in chicken. Gal-3 expression in bone marrow stromal cells (BMSCs) from 18-day-old chicken embryos was inhibited or overexpressed. BMSCs were then co-cultured with bone marrow monocytes/macrophages (BMMs) with or without addition of 1α,25(OH)2D3. The results showed that 1α,25(OH)2D3 upregulated the expression of Gal-3 mRNA and receptor activator of nuclear-factor κB ligand (RANKL) expression in BMSCs and promoted osteoclastogenesis, as shown by the upregulated expression of osteoclast (OC) markers (CtsK, CAII, MMP-9, and TRAP) and increased bone resorption, a method for measuring the bone resorption area in vitro. Knockdown of Gal-3 by small-interfering RNA (siRNA) in BMSCs downregulated the expression of RANKL mRNA and attenuated the effects of 1α,25(OH)2D3 on osteoclastogenesis and bone resorption. Conversely, overexpression of Gal-3 in BMSCs enhanced the effects of osteoclastogenesis and bone resorption by increasing the expression of RANKL mRNA. These results demonstrated that Gal-3 mediates the differentiation and bone resorption of osteoclasts regulated by 1α,25(OH)2D3.


Introduction
Bone homeostasis is achieved by osteogenesis and bone resorption, which are regulated by osteoblasts (OBs) and osteoclasts (OCs), respectively [1,2]. OCs are specifically responsible for physiological and pathological bone resorption. Excessive increase or decrease in their quantity and activity is detrimental to bone and calcium (Ca) homeostasis [3,4]. Vitamin D is a sterol derivative and has long been considered to promote osteogenesis by promoting the intestinal transport of Ca and osteolysis, ensuring the stability of serum calcium homeostasis. Previous studies have shown that 1α,25-dihydroxyvitamin D 3 (1α, 25[OH] 2 D 3 ) can upregulate receptor activator of nuclear-factor κB ligand (RANKL) expression in OBs and bone marrow stromal cells (BMSCs) in mammalians, indirectly inducing bone marrow monocytes/macrophages (BMMs) to differentiate into OCs for bone resorption [5][6][7].
Galectin-3 (Gal-3) is a member of the β-galectin family and presents in the nucleus. Gal-3 can regulate cell migration, adhesion, apoptosis, and gene expression [8,9]. Studies have shown that Gal-3 is expressed in chondrocytes, OBs, BMSCs, and OCs and regulates osteocyte differentiation and function [10,11]. In Gal-3 (Gal-3 −/− ) knockout mice, OB and OC differentiation and bone resorption were impaired [12]. It has also been confirmed that Gal-3 could overlap with the transcription factor Runx2, which regulates OB differentiation [13]. Furthermore, Gal-3 can regulate bone marrow mesenchymal stem cell migration through RhoA-GTP signaling and may be a potential target for treating bone reconstruction-related diseases [14]. Aubin et al. found that 1α,25(OH) 2 D 3 promoted Gal-3 expression in a rat OB-derived sarcoma cell line, ROS 17/2.8 [15]. Simon et al. reported that knockout Gal-3 in OBs displayed higher osteoclastogenesis, independently of the RANKL signaling pathway [16]. However, the role of Gal-3 in 1α,25(OH) 2 D 3 -regulated osteoclastogenesis and bone resorption and whether it affects vitamin D regulation of osteoclastogenesis in poultry (chicken) is unclear.
Chicken bones have been used in bone development and bone injury studies for a long time as they are similar to those of humans and other vertebrates, and vitamin D has been found to regulate bone development in chickens [17][18][19]. Our previous study showed that 1α,25(OH) 2 D 3 promoted osteoclastogenesis in a chicken BMSC-BMM co-culture system in a dose-dependent manner, with 10 −8 mol/L having the most significant effect [20]. In addition, 10 −8 mol/L 1α,25(OH) 2 D 3 was used in the current study to further examine the effects of vitamin D on Gal-3 expression in BMSCs, BMMs, and BMSC-BMM co-culture. Small-interfering RNA (siRNA) and gene overexpression were used to knockdown or overexpress Gal-3 to observe the effects of 1α,25(OH) 2 D 3 on osteoclastogenesis, bone resorption, and RANKL signaling and to examine whether Gal-3 affects 1α,25(OH) 2 D 3 regulation of osteoclastogenesis through RANKL signaling. These results will provide a foundation for studies on vitamin D regulation of bone and calcium homeostasis in poultry.

Animals
The white leghorn chicken embryos used here were fertilized SPF-grade eggs from Single Comb White Leghorn (Yigida Biotechnology, Jining, China). The SPF-grade eggs were incubated at 37 • C and 60% humidity until they were 18 days old. The animal use was approved by the Animal Care and Use Committee of Yangzhou University (SYXK [Su] 2016-0020).

Isolation of BMSCs and BMMs
Tibias and femurs were separated, and bone marrow cells were filtered with a 200mesh sieve and then centrifuged at 1200 r/min for 5 min. The cells were subsequently resuspended in α-MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (EallBio, Beijing, China) and then incubated in an incubator (5% CO 2 , 37 • C). After 2 days, the BMSCs were adherent and the BMMs were non-adherent.

Overexpression of Gal-3 Plasmids
Gal-3 homologous recombination was performed with PEXP-RB-MAM-EGFP transient vector (RiboBio, Guangzhou, China). The gene EGFP-Gal-3 was used for cell transfection, and the empty carrier control was named EGFP. Transfection was performed using Lipofectamine TM 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions, as previously reported [21].

Co-Culture of BMSCs and BMMs
BMSCs were seeded into cell cultural plates or bone resorption cultural plates at a ratio of 1:100 (BMSCs:BMMs). Cells were then treated with 10 −8 mol/L 1α,25(OH) 2 D 3 (Sigma-Aldrich, USA) for 5 d (control group without 1α,25(OH) 2 D 3 ). The medium was changed every 2 days. At the end of the incubation period, the medium was decanted from the cell cultures. BMSCs were transfected with NC siRNA and Gal-3 siRNA for 10 h. BMMs were seeded into these transfected BMSC cultures at a ratio of 1:100 (BMSCs:BMMs).
At the end of the incubation period, the medium was decanted from the cell cultures. BMSCs were transfected with EGFP plasmid and EGFP-Gal-3 for 10 h. BMMs were transferred into BMSCs at a ratio of 100:1 (BMMs:BMSCs). Then, the medium was changed to α-MEM (containing 10% FBS) for 5 d. The medium was changed every 2 days.

Identification of Osteoclastogenesis
At the end of the incubation period, the medium was decanted from the cell cultures. Cells were fixed with 4% paraformaldehyde for 10 min (New Cell & Molecular Biotech Co., Ltd., Hangzhou, China). Tartrate-resistant Acid Phosphatase (TRAP) staining solution was added according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA), as previously described [22].

Identification of Bone Resorption
At the end of the incubation period, the medium was decanted from the cell cultures. PBS was used to repeatedly wash the plates, and photographs were subsequently taken under an inverted microscope. Image-Pro Plus software was used for calculation of the area of the bone resorption pits.

ELISA
The supernatants of cells were collected, and the levels of CtsK, MMP-9, RANKL, and OPG were measured using chicken ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The OD value was measured at 450 nm within 15 min. Protein content was calculated based on the OD.

Statistical Analysis
Results are expressed as the mean ± SD from at least three independent experiments. Significance was calculated by one-way analysis of variance (ANOVA) using SPSS 19.0 software. p-values lower than 0.05 were considered statistically significant.
In addition, the area of bone resorption pits was significantly (p < 0.01) increased in the 10 −8 mol/L 1α,25(OH) 2 D 3 group ( Figure 1E,F). These results show that 10 −8 mol/L 1α,25(OH) 2 D 3 promotes osteoclastogenesis and bone resorption in the chicken BMSC-BMM co-culture system.  Figure 1E,F). These r 1α,25(OH)2D3 promotes osteoclastogenesis and bone resor BMM co-culture system. BMSCs and BMMs were co-cultured on 6-well plates with or without (Con) addition of 10 −8 mol/L 1α,25(OH) 2 D 3 . Cells were treated for 5 d, and 10 fields were randomly selected. The number of TRAP-positive multinucleated OCs (black arrows) was counted. Bar = 100 µm. (C,D) BMSCs and BMMs were co-cultured on 6-well plates with or without (Con) addition of 10 −8 mol/L 1α,25(OH) 2 D 3 . Cells were treated for 5 d, and qRT-PCR was used to quantitate the mRNA expression of OC marker genes (CtsK, TRAP, MMP-9, and CAII). Chicken ELISA kits were used to measure CtsK and MMP-9 protein levels in the cell culture supernatant (n = 3 per group). (E,F) BMSCs alone, no cells, and BMSC-BMM were added to a 96-well bone resorption culture plate. After that, 10 −8 mol/L 1α,25(OH) 2 D 3 was added or not added (Con) to BMSC-BMM co-culture wells for 5 d. An inverted microscope was used to observe 10 randomly selected fields. Image-Pro Plus 6.0 software was used to analyze the area of bone resorption pits (red arrows). Bar = 50 µm. (Graph bars show mean ± SD. * p < 0.05 and ** p < 0.01 show significant difference compared with the control group).

Knockdown of Gal-3 Inhibited Osteoclastogenesis
BMSCs were transfected with targeting of Gal-3 siRNA for 36, 72, and 120 h, and the expression of Gal-3 mRNA was significantly (p < 0.01) decreased in the si-Gal-3 group at each time point ( Figure 4A).
As shown in Figure 4B  BMSCs were seeded in 6-well plates with or without (Con) 1α,25(OH) 2 D 3 . After 5 d, qRT-PCR was used to quantitate the expression of RANKL and OPG mRNA, and the RANKL/OPG ratio was calculated. (B) BMSCs were seeded in 6-well plates with or without (Con) 1α,25(OH) 2 D 3 . After 5 d, the cell supernatant was collected, and ELISA kits were used to measure RANKL and OPG protein levels. (Graph bars show mean ± SD. * p < 0.05 and ** p < 0.01 show significant difference compared with the control group; n = 3 per group.).

Knockdown of Gal-3 Inhibited Osteoclastogenesis
BMSCs were transfected with targeting of Gal-3 siRNA for 36, 72, and 120 h, and the expression of Gal-3 mRNA was significantly (p < 0.01) decreased in the si-Gal-3 group at each time point ( Figure 4A). (B,C) After Gal-3 knockdown in BMSCs, BMSCs and BMMs were co-cultured on 6-well plates with or without addition of 1α,25(OH)2D3. Cells were cultured for 5 days. NC was the blank control. Ten fields were randomly selected. The number of TRAP-positive multinucleated OCs (black arrows) was counted. Bar = 200 μm. (D) After Gal-3 knockdown, BMSCs were co-cultured with BMMs with or without addition of 1α,25(OH)2D3. Cells were cultured for 5 d. NC was the blank control. Quantitative RT-PCR was used to quantitate the mRNA expression of OC marker genes (CtsK, CAII, MMP-9, and TRAP) (n = 3 per group). (E,F) After BMSCs were seeded in 96-well bone resorption culture plates and Gal-3 was knocked down, BMSCs and BMMs were co-cultured with or without addition of 1α,25(OH)2D3. Cells were cultured for 5 days. NC was the blank control. An inverted microscope was used for observation of 10 randomly selected fields. Image-Pro Plus 6.0 software was used to analyze the area of bone resorption pits (red arrows). Bar = 100 μm. (Graph bars show mean ± SD. * p < 0.05 and ** p < 0.01 show significant difference compared with the NC control group. # p < 0.05 and ## p < 0.01 show significant difference compared with the 1α,25(OH)2D3-treated si-Gal-3 group).

EGFP-Gal-3 Promoted OC Differentiation
BMSCs were transfected with EGFP-Gal-3 or EGFP for 36, 72, and 120 h. Compared with the EGFP group, the expression of Gal-3 mRNA significantly (p < 0.01) increased following treatment with EGFP-Gal-3 ( Figure 5A). In addition, the amount of TRAP; the expression of CtsK, TRAP, MMP-9, and CAII mRNA; and the area of bone resorption were significantly (p < 0.01 or p < 0.05) increased in the EGFP-Gal-3 group (Figure 5B-F). These results indicate that overexpression of Gal-3 in BMSCs could upregulate osteoclastogenesis and bone resorption. BMSCs and BMMs were co-cultured on 6-well plates with or without addition of 1α,25(OH) 2 D 3 . Cells were cultured for 5 days. NC was the blank control. Ten fields were randomly selected. The number of TRAP-positive multinucleated OCs (black arrows) was counted. Bar = 200 µm. (D) After Gal-3 knockdown, BMSCs were co-cultured with BMMs with or without addition of 1α,25(OH) 2 D 3 . Cells were cultured for 5 d. NC was the blank control. Quantitative RT-PCR was used to quantitate the mRNA expression of OC marker genes (CtsK, CAII, MMP-9, and TRAP) (n = 3 per group). (E,F) After BMSCs were seeded in 96-well bone resorption culture plates and Gal-3 was knocked down, BMSCs and BMMs were co-cultured with or without addition of 1α,25(OH) 2 D 3 . Cells were cultured for 5 days. NC was the blank control. An inverted microscope was used for observation of 10 randomly selected fields. Image-Pro Plus 6.0 software was used to analyze the area of bone resorption pits (red arrows). Bar = 100 µm. (Graph bars show mean ± SD. * p < 0.05 and ** p < 0.01 show significant difference compared with the NC control group. # p < 0.05 and ## p < 0.01 show significant difference compared with the 1α,25(OH) 2 D 3 -treated si-Gal-3 group).
As shown in Figure 4B

EGFP-Gal-3 Promoted OC Differentiation
BMSCs were transfected with EGFP-Gal-3 or EGFP for 36, 72, and 120 h. Compared with the EGFP group, the expression of Gal-3 mRNA significantly (p < 0.01) increased following treatment with EGFP-Gal-3 ( Figure 5A). In addition, the amount of TRAP; the expression of CtsK, TRAP, MMP-9, and CAII mRNA; and the area of bone resorption were significantly (p < 0.01 or p < 0.05) increased in the EGFP-Gal-3 group (Figure 5B-F). These results indicate that overexpression of Gal-3 in BMSCs could upregulate osteoclastogenesis and bone resorption.

Effects of Si-Gal-3 and EGFP-Gal-3 on RANKL Expression in BMSCs
As shown in Figure 6, si-Gal-3 significantly (p < 0.01) inhibited the expression of RANKL mRNA, but there was no significant (p > 0.05) difference in the expression of OPG mRNA and the ratio of RANKL/OPG. Conversely, overexpression of Gal-3 significantly (p (D) After Gal-3 was overexpressed in BMSCs, BMSCs were co-cultured with BMMs. Quantitative RT-PCR was used to quantitate the mRNA expression of OC marker genes (CtsK, CAII, MMP-9, and TRAP) (n = 3 per group). (E,F) After BMSCs were seeded in 96-well bone resorption culture plates and Gal-3 was overexpressed, BMSCs and BMMs were co-cultured for 5 days. An inverted microscope was used for observation of 10 randomly selected fields. Image-Pro Plus 6.0 software was used to analyze the area of bone resorption pits (red arrows). Bar = 100 µm. (Graph bars show mean ± SD. * p < 0.05 and ** p < 0.01 show significant difference compared with the control group).

Effects of Si-Gal-3 and EGFP-Gal-3 on RANKL Expression in BMSCs
As shown in Figure 6, si-Gal-3 significantly (p < 0.01) inhibited the expression of RANKL mRNA, but there was no significant (p > 0.05) difference in the expression of OPG mRNA and the ratio of RANKL/OPG. Conversely, overexpression of Gal-3 significantly (p < 0.05) upregulated the expression of RANKL mRNA and the ratio of RANKL/OPG but significantly inhibited OPG expression (p < 0.01) ( Figure 6B). These results show that Gal-3 could regulate RANKL expression in BMSCs. osteoclastogenesis, indicating that Gal-3 is involved in the differentiation of osteoclasts [16]. Our research found that knockdown of Gal-3 in BMSCs co-cultured with BMMs could suppress osteoclastogenesis and bone resorption. However, overexpression of Gal-3 could reverse it. In this study, we did not investigate the role of Gal-3 in regulating RANK in monocyte/macrophage since: (1) our study focused on the role of Gal-3 in regulating RANKL expression and osteoclastogenesis; (2) RANKL is expressed at very low levels in BMM; (3) 1α,25(OH) 2 D 3 increased Gal-3 expression only weakly in BMM (2-fold) but dramatically in BMSC (>10-fold). In addition, the transfection efficiency is extremely low in macrophages, and it is technically challenging to conduct experiments in macrophages. Next, the regulatory role of Gal-3 in the expression of RANKL was demonstrated in BMSCs. The results confirmed that the expression of RANKL mRNA was downregulated by knockdown of Gal-3, and the expression of RANKL mRNA and the ratio of RANKL/OPG were upregulated by overexpression of Gal-3. The current results are different from those of Simon et al., who reported that inhibition of osteoclastogenesis was regulated by Gal-3 in mice independently of the RANKL/OPG axis. This might be related to differences in species variation or the different degrees of knockdown or overexpression of Gal-3 used.
Our present study shows that 1α,25-(OH) 2 D 3 increased the levels of Gal-3 and RNAKL mRNA and promoted osteoclast differentiation and activation. Gal-3 knockdown led to decreased RANKL mRNA expression and blocked the effect of 1α,25-(OH) 2 D 3 on osteoclast differentiation. Gal-3 overexpression increased RANKL mRNA levels. It is well documented that Gal-3 present in the nucleus and can bind several transcription factors and enhance their DNA-binding activity [31]. We propose that 1α,25(OH) 2 D 3 -induced Gal-3 expression affects osteoclast differentitation through RANKL signaling in chicken BMSC (Figure 7). However, the mechanisms by which 1α,25-(OH) 2 D 3 regulates Gal-3 expression and Gal-3 regulates RANKL expression need to be further investigated in detail.
Vet. Sci. 2021, 8, 234 10 of Next, the regulatory role of Gal-3 in the expression of RANKL was demonstrated BMSCs. The results confirmed that the expression of RANKL mRNA was downregulate by knockdown of Gal-3, and the expression of RANKL mRNA and the ratio RANKL/OPG were upregulated by overexpression of Gal-3. The current results are di ferent from those of Simon et al., who reported that inhibition of osteoclastogenesis wa regulated by Gal-3 in mice independently of the RANKL/OPG axis. This might be relate to differences in species variation or the different degrees of knockdown or overexpre sion of Gal-3 used.
Our present study shows that 1α,25-(OH)2D3 increased the levels of Gal-3 and RNAK mRNA and promoted osteoclast differentiation and activation. Gal-3 knockdown led decreased RANKL mRNA expression and blocked the effect of 1α,25-(OH)2D3 o osteoclast differentiation. Gal-3 overexpression increased RANKL mRNA levels. It is we documented that Gal-3 present in the nucleus and can bind several transcription facto and enhance their DNA-binding activity [31]. We propose that 1α,25(OH)2D3-induce Gal-3 expression affects osteoclast differentitation through RANKL signaling in chicke BMSC (Figure 7). However, the mechanisms by which 1α,25-(OH)2D3 regulates Gal expression and Gal-3 regulates RANKL expression need to be further investigated detail.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors.