GPER1 contributes to T3-induced osteogenesis by mediating glycolysis in osteoblast precursors

Cell culture and treatments

Clonal murine calvarial MC3T3-E1 Subclone 14 cells were purchased from ATCC (Rockville, MD) and cultured in α-MEM containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL), 100 U/mL penicillin and 100 μg/mL streptomycin (Hyclone, Logan, UT) at 37°C in a 5% CO₂ atmosphere. When the cells grew to 70–80% confluence, they were treated with 0, 10, and 100 nM 3,3,5-Triiodo-L-thyronine (T3) (Sigma-Aldrich, St. Louis, MO).

Cells underwent osteogenic differentiation for 7 or 21 d (d) in the induction of osteogenic medium (60 μg/mL ascorbic acid, 2 mM β-glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich)). Then, the expression of osteogenic markers, ALP staining and ALP activity were detected to evaluate osteogenic differentiation, and Alizarin red staining was performed to evaluate the mineralization level. After treatment with T3 for 48 h (h), the glycolytic potential and the production of LD were measured. After treatment with T3 for 6, 12, 24, and 48 h, the rate-limiting enzymes in glycolysis were measured. In addition, the selective antagonist of GPER1, G15 (10 μM), and the PFKFB3 inhibitor, 3PO (10 μM), were jointly intervened with T3.

Transcriptome analysis

MC3T3-E1 cells were cultured overnight and treated with T3 at 100 nM for 48 h. Total RNA was extracted from MC3T3-E1 cells using Trizol reagent (Invitrogen, CA, USA). Sequencing of total RNA was performed on an Illumina Hiseq 4000 instrument (LC Bio, Hangzhou, China). Six samples, including three pairs of controls and T3-treated groups, were sequenced.

The sequencing steps are summarized as follows: after total RNA passed quality inspection, eukaryotic mRNA is enriched with magnetic beads connected with oligo (DT). The extracted mRNA was randomly broken into short fragments by a fragmentation buffer. The fragmented mRNA was used as a template to synthesize cDNA strands with six base random primers, and then reaction buffer, dNTPs, RNaseH, and DNA polymerase I were added for two-strand cDNA synthesis. The double-stranded product was purified by AMP Pure XP beads. The sticky end of the DNA was repaired to a flat end by using T4 DNA polymerase and Klenow DNA polymerase activities. Base A was added to the 3′ end, and a connector was added. AMP Pure XP beads were used for fragment selection. Finally, polymerase chain reaction (PCR) amplification was performed to obtain the final sequencing library. Illumina Novaseq was employed after the library passed the quality inspection. After sequencing, the selected read lengths were 2*150 bp (pe150).

Metabolite extraction and liquid chromatography tandem-mass spectrometry (LC-MS/MS)

Under the same culture conditions as for transcriptome sequencing, the MC3T3-E1 cells were divided into control and T3 treatment groups. The samples in each group were repeated seven times and cultured for 48 h. When the cultured cells reach the experimental end time, 500 μL precooled extractant (80% methanol aqueous solution, including internal standard) was added, and samples were vortexed for 2 min. The samples were quick-frozen in liquid nitrogen for 5 min, thawed for 5 min, vortexed for 2 min, and mixed well; this cycle was repeated three times. Afterwards, the samples were centrifuged for 10 min at 4°C and 12,000g. Then, 400 µL supernatant was concentrated in 1.5 ml microcentrifuge tubes at 4°C until the solvent volatilized completely. Next, 160 μL 50% acetonitrile water (containing 0.03% formic acid) was added, vortexed for 3 min, and centrifuged for 5 min at 12,000g and 4°C. The supernatant was then put into the injection bottle for liquid chromatography tandem-mass spectrometry (LC-MS/MS).

The data acquisition system included Ultra Performance Liquid Chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A,htps:ǁMw.shimadzu, com/) and Tandem mass spectrometry (MS/MS)(QTRAP^®, https://sciex.com/). Based on the self-built targeted standard database MWDB (metware database), the information and secondary spectrum data were qualitatively analyzed according to the retention time (RT) of the detected substance and the parent and daughter ions. The Software Analyst 1.6.3 was used to process mass spectrometry data. Using the local energy metabolism database, the metabolites of the samples were analyzed qualitatively and quantitatively by mass spectrometry. Multivariate statistical analysis was performed, including principal component analysis (PCA), cluster analysis (CA) and orthogonal partial least squares discriminant analysis (opls-da). Based on the results of opls-da, the metabolites with fold change ⩾ 2 and fold change ⩽ 0.5 were selected. If the difference between the test group and the control group was more than two fold, or less than 0.5 fold, the difference was considered to be significant. The China Hangzhou Lianchuan Biotechnology Co., Ltd. Illumina Hiseq 4000 (LC bio, China) laboratory was used for sequencing and metabolomic analysis.

Real-time PCR

Total RNA was extracted from a single cell suspension using Easyspin Plus according to the manufacturer (Aidlab, Beijing, China). cDNA was synthesized from the total RNA samples by using a PCR Reverse Transcription Kit (Takara, Dalian, China). Real-time quantitative PCR experiments were performed using SYBR Premix Ex Taq II according to the manufacturer’s protocol (Takara). The primer sequences used are list in Table 1. The primers were synthesized by Chongqing Qingke Biological Co., Ltd. (Chongqing, China). All analyses were performed in triplicate.

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Table 1.

Sequences of primers.

Gene	Sequence
COL1	-F	5′-AGAACAGCGTGGCCT-3′
	-R	5′-TCCGGTGTGACTCGT-3′
Runx2	-F	5′-CCCAGCCACCTTTACCTACA-3′
	-R	5′-TATGGAGTGCTGCTGGTCTG-3′
Osx	-F	5′-ATGGCGTCCTCTCTGCTTG-3′
	-R	5′-TGAAAGGTCAGCGTATGGCTT-3′
Hk2	-F	5′-AGAGAACAAGGGCGAGGAG-3′
	-R	5′-GGAAGCGGACATCACAATC-3′
Pkm	-F	5′-CTGTGGAGATGCTGAAGGAG-3′
	-R	5′-CAACAGGACGGTAGAGAATG-3′
Pkfkb3	-F	5′-CTACCTCAACTGGATAGGTGTTC-3′
	-R	5′-AGGGCGGAAGAAGTTGTAAG-3′
Ldh	-F	5′-TGGAAGACAAACTCAAGGGCGAGA-3′
	-R	5′-TGACCAGCTTGGAGTTCGCAGTTA-3′
Gper1	-F	5′-CCCTGGACTTAAGAAGATATACTTGCA-3′
	-R	5′-CCCGAAGTCCCGGCATA-3′
Gadph	-F	5′-AGGTCGGTGTGAACGGATTTG-3′
	-R	5′-TGTAGACCATGTAGTTGAGGTCA-3′

Western blot analysis

Aliquots of cell lysates containing 30 μg of proteins were separated by 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked with TBST buffer containing 8% skimmed milk and incubated with primary antibodies against RUNX2 (1:1000), COL1 (1:1000), HK2 (1:1000), PFKFB3(1:1000), PKM (1:1000) LDH (1:1000), β-actin (1:5000)(all from Abcam, Cambridge, UK), and OSX (1:1000, Santa Cruz Biotechnology, Dallas, TX). Blots were incubated overnight at 4°C, followed by the addition of horseradish peroxidase-linked secondary antibodies (ZSGB-GIO Beijing, China) and visualization of the Electrochemiluminescence bands. Quantification of the bands was carried out using densitometry analysis software, Quantity One (Bio-Rad). β-actin expression was used as an internal control to normalize the expressions of other proteins; and all analyses were performed in triplicate.

Immunofluorescent staining

The treated cells were fixed using 4% paraformaldehyde (PFA), permeated with 0.5% Triton X-100, and blocked using 1% bovine serum albumin (BSA) for 15 min at room temperature. Then, cells were incubated with primary antibodies (anti-HK2, PFKFB3, PKM, and LDH, Abcam, Cambridge, MA, 1:1000) and then with fluorescent-dye-conjugated secondary antibodies to detect the localization of target proteins in MC3T3-E1 cells. Finally, fluorescence was observed using a Leica confocal microscope (Heidelberg, Germany).

Glycolysis assay

Around 5,000 cells/well were plated in a 96-well plate and incubated overnight in 100 μl of cultured media. Cells were treated as described above. The glycolysis assay was performed using Cayman’s Glycolysis Cell-Based Assay Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. The absorbance of each well was measured using a plate reader with a test wavelength of 490 nm. All analyses were performed in triplicate.

Lactate assay

Around 5,000 cells/well were plated in a 96-well plate and treated with T3, as described above. The LD content of the cell culture supernatant was determined using a Lactate Assay Kit (Jiangsu KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instruction. The absorbance of each well was measured using a microculture plate reader with a test wavelength of 530 nm. All analyses were performed in triplicate. The concentration of lactate was expressed according to the following equation: Concentration = (OD cell well – OD blank well) × standard concentration (3 mM)/(OD standard well – OD blank well) × 100%.

ALP staining

Around 5 × 10⁵ MC3T3-E1 cells were seeded in a six-well plate and allowed to grow to 70–80% confluence. The cells were treated, as above, for 7 days in osteogenic medium. The medium was replaced every 2–3 days. The cells were washed with phosphate buffered saline (PBS) three times. According to the instructions of the ALP staining kit (Beyotime, Shanghai, China), the samples were covered by BCIP/NBT working solution and incubated for 5–30 min at room temperature, until the color developed to the desired shade. The colorimetric reaction was terminated by washing with distilled water 1–2 times.

ALP activity assay

Cells were treated the same as for the ALP staining assay (above) followed by adding 100 μL 0.1%Triton-X100 for cell lysis with shaking for 30 min. ALP activity was determined using an ALP activity kit (Beyotime) according to the manufacturer’s instruction. The absorbance of each well was measured using a plate reader with a test wavelength of 405 nm. The blank wells were used as control. All analyses were performed in triplicate.

Alizarin red S (ARS) staining

Around 5 × 10⁵ MC3T3-E1 cells were seeded in six-well plates and cultured for 21 days in α-MEM with osteogenic medium. For alizarin red staining, the medium was discarded and the cells were washed twice with PBS, followed by fixing with 95% ethanol for 30 min. Cells were then rinsed three times with ddH2O to remove the ethanol completely. Next, 300 µl of 1% alizarin red stain (AR-S) solution was added to each well and the calcium deposits were labeled at 37°C water bath for 30–60 min and observed and photographed under an inverted phase contrast microscope (Olympus, Tokyo, Japan). Staining intensity was determined using ImageJ software (version 1.47, National Institutes of Health, Bethesda, MD).

Statistical analyses

The SPSS 22.0 statistical package (IBM Corp., Armonk, NY) was used for all analysis. All values are expressed as mean ± SD. Levene’s test was used to assess the equality of variances (test standard 0.10). One-Way analysis of variance (ANOVA) and least significant difference test were used to analyze the mean of each group with normal distribution and homogeneity of variance. Bonferroni test was used for Post Hoc multiple comparisons. Kruskal–Wallis H test was used when homogeneity of variance was not met. A p value of P < 0.05 was used to indicate a statistically significant difference.

T3 promoted osteoblastic differentiation and mineralization

We first observed the effect of T3 on the osteogenic capacities of MC3T3-E1 osteoblast precursors. As shown in Figure 1(A) to (D), treatment with 10 nM or 100 nM T3 promoted the protein expression of RUNX2, OSX, and COL1 in MC3T3-E1 cells after 7 days of osteogenic induction (Figure 1(A) to (D)). However, there was no significant difference between the efficiency of T3 at either of the two treatment concentrations (Figure 1(A) to (D)). In addition, treatment with 10 nM or 100 nM T3 enhanced both ALP staining intensity and ALP activity after 7 days of osteogenic induction (Figure 1(E) and (F)), supporting the idea that T3 promotes osteoblastic differentiation. Alizarin red staining also revealed that treatment with 10 nM or 100 nM T3 increased the number of mineralized nodules and mineralized areas after 21 days of osteogenic induction (Figure 1(G) and (H)), supporting the idea that T3 promotes matrix mineralization. These results confirm the effectiveness of our experimental model in evaluating the roles of T3.

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Figure 1.

T3 promoted osteoblastic differentiation and mineralization. (A to D) MC3T3-E1 cells were treated with 0, 10, and 100 nM T3 for 7 days under osteogenic induction, and the protein expression of COL1, RUNX2, and OSX proteins were analyzed. Bar chart showing the protein levels of COL1, RUNX2, and OSX normalized to β-actin level. (E and F) ALP staining and activity in MC3T3-E1 cells treated with the indicated treatments for 7 days under osteogenic induction (Unit of ALP activity: King U/GPROT). (G and H) Alizarin red staining in MC3T3-E1 cells treated with the indicated treatments for 21 days under osteogenic induction.

Transcriptomic and metabolomic analysis of T3-treated MC3T3-E1 cells

Our transcriptomic analysis revealed that treatment with T3 resulted in the upregulation of 522 genes and downregulation of 526 genes in MC3T3-E1 cells (Figure 2A). Through a transcriptomic analysis, we learned that T3 administration significantly increased the mRNA expression of GPER1 and PFKFB3 (Figure 2(B) and (C)). As expected, we confirmed that treatment with 10 nM or 100 nM T3 significantly upregulated the protein expression of GPER1 in MC3T3-E1 cells via Western blot assays (Figure 2(D) and (E)), which verified the transcriptomic analysis results.

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Figure 2.

Transcriptomic and metabolomic analysis of T3-treated MC3T3-E1 cells. (A) Volcano plot representing differential expression analysis of genes regarding transcriptome sequencing between control and T3-treated groups (n = 3, each group). The X-axis showed log2-fold change in expression, and the negative log10 of the P value was plotted on the Y-axis. Each gene was represented by one point on the graph. (B) Heat map represented the differential expression analysis of GPER1 and PFKFB3 between two groups (n = 3, each group). (C) The mRNA levels of GPER1 and PFKFB3 in the two groups displayed by transcriptome sequencing. (D and E) MC3T3-E1 cells were treated with 0, 10, and 100 nM T3 for 48 h, and the protein expression of GPER1 were analyzed. Bar chart showing the protein level of GPER1 normalized to β-actin level. (F) Volcano plot representing differential expression metabolites in metabolomic analysis between control and T3-treated groups (n = 7, each group). The X-axis showed log2-fold change in expression, and the variable importance in projection (VIP) was plotted on the Y-axis. Each metabolite was represented by one point on the graph. (G) Heat map represented the differential expression analysis of DHAP and LD between two groups (n = 7, each group). (H and I) The metabolite levels DHAP and LD in the two groups displayed by metabolomic analysis.

Our metabolomic analysis showed that treatment with T3 resulted in increases in the levels of seven metabolites and the reduction in the level of one metabolite in MC3T3-E1 cells (Figure 2(F)). Through the metabolomic analysis, we learned that after T3 administration, the levels of DHAP and LD significantly increased (Figure 2(G) to (I)), which suggested that glycolysis may be involved with T3 in osteoblast formation.

The promotion of osteogenic activities by T3 was reversed by G15 treatment

T3 upregulates GPER1 expression in osteoblast precursors. Accordingly, we evaluated the roles of GPER1 in T3-regulated osteogenic activities. The specific GPER1 inhibitor G15 blocked the T3-increased mRNA levels of osteoblastic markers, including RUNX2, OSX, and COL1, but did not directly affect the mRNA expression of osteoblastic markers (Figure 3(A) to (C)). In addition, G15 treatment blocked the promoting effects of T3 on ALP staining intensity, ALP activity, mineralized nodule, and mineralized area formation while not directly affecting the aforementioned osteogenic activities (Figure 3(D) to (G)). These results suggested that GPER1 participates in T3-regulated osteoblastic differentiation and mineralization.

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Figure 3.

The promotion of osteogenic activities by T3 was reversed by G15 treatment. (A to C) MC3T3-E1 cells were treated with 100 nM T3 along with or without 10 μM G15 for 7 days under osteogenic induction, and the mRNA levels of COL1, RUNX2, and OSX were analyzed. (D and E) ALP staining and activity in MC3T3-E1 cells treated with the indicated treatments for 7 days under osteogenic induction (Unit of ALP activity: King U/GPROT). (F) Alizarin red staining in MC3T3-E1 cells treated with the indicated treatments for 21 days under osteogenic induction. (G) Bar chart showing the percentage of positive areas stained.

T3 upregulated glycolytic activities in MC3T3-E1 cells

The metabolomic analysis suggested that T3 affects the glycolysis of osteoblast precursors. As expected, treatment with 10 nM or 100 nM T3 significantly upregulated the glycolytic potential in MC3T3-E1 cells (Figure 4A). Treatment with 10 nM or 100 nM T3 promoted the production of LD in MC3T3-E1 cells (Figure 4B). However, no significant difference between the efficiency of T3 at the aforementioned concentrations two levels was observed (Figure 4(A) and (B)). We further observed the effects of T3 on several rate-limiting enzymes in osteoblast precursors. Following treatment with 100 nM T3 for the indicated times, HK2 mRNA levels were increased from 12 to 48 h, and the mRNA levels of PFKFB3, PKM, and LDH were increased at all time points (Figure 4(C) to (F)). Following treatment with 100 nM T3 for the indicated times, the protein levels of HK2 and PFKFB3 were increased at all time points (Figure 4(G) to (I)). With the same interventions, PKM protein levels were increased from 12 to 24 h, and LDH protein levels were increased at 24 and 48 h (Figure 4(G), (J) and (L)). Furthermore, immunofluorescence staining showed that the fluorescence intensity of the aforementioned enzymes in the cytoplasm increased after T3 administration (Figure 5(A) to (D)). These results confirmed that glycolysis is involved in T3-regulated osteogenesis.

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Figure 4.

T3 upregulated glycolytic activities in MC3T3-E1 cells. (A and B) MC3T3-E1 cells were treated with 0, 10, and 100 nM T3 for 48 h, and the glycolytic potential and LD production in the supernatant were analyzed. (C to F) The mRNA expression of HK2, PFKFB3, PKM, and LDH in MCT3-E1 cells treated with indicated treatments. (G) The protein expression of HK2, PFKFB3, PKM, and LDH in MCT3-E1 cells treated with indicated treatments. (H to K) The bar chart shows the relative expression of HK2, PFKFB3, PKM, and LDH normalized to β-actin level.

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Figure 5.

The immunofluorescent results of altered glycolytic activities in MC3T3-E1 cells. After treatment with 0 or 100 nM T3 for 48 h, and the fluorescent intensity of HK2, PFKFB3, LDH, and PKM in the cytoplasm were evaluated by immunofluorescence staining (scale = 20 μm).

T3 upregulation of glycolytic activities in of MC3T3-E1 cells were reversed by G15 treatment

As a specific antagonist of GPER1, G15 was used as a pharmacological tool for our study. ¹³ Although the direct application of G15 did not reduce the glycolytic potential or LD production, the addition of G15 blocked the increase in glycolytic potential and LD production induced by T3 administration in MC3T3-E1 cells (Figure 6(A) and (B)). Moreover, we observed that although the direct application of G15 did not result in a decrease in the mRNA and protein expression of HK2, PFKFB3, PKM, and LDH, the addition of G15 blocked the enhancement of the mRNA and protein expression of the aforementioned rate-limiting enzymes induced by T3 administration in MC3T3-E1 cells (Figure 6(C) to (K)). These findings indicated that GPER1 participates in T3-regulated glycolysis of osteoblast precursors.

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Figure 6.

T3 upregulation of glycolytic activities in of MC3T3-E1 cells were reversed by G15 treatment. (A and B) MC3T3-E1 cells were treated with 100 nM T3 along with or without 10 μM G15 for 48 h, and the glycolytic potential and LD production in the supernatant were analyzed. (C to F) The mRNA expression of HK2, PFKFB3, PKM, and LDH in MCT3-E1 cells treated with indicated treatments as described in (A and B). (G) The protein expression of HK2, PFKFB3, PKM, and LDH in MCT3-E1 cells treated with indicated treatments as described in (A and B). (H–K) The bar chart shows the relative expression of HK2, PFKFB3, PKM, and LDH normalized to β-actin level.

T3 promoted osteogenic activities were blocked by 3PO addition

Finally, we evaluated the significance of glycolysis in T3-regulated osteogenic cell differentiation and mineralization. As a specific inhibitor of the PFKFB3 isozyme, 3PO was applied as a pharmacological tool to inhibit glycolysis. As shown in Figure 7(A), 3PO addition not only directly inhibited the glycolytic potential in MC3T3-E1 cells but also blocked the glycolytic potential that had been promoted by T3 administration. In addition, although the direct application of 3PO did not affect LD production in MC3T3-E1 cells, 3PO addition reversed the LD production promoted by T3 administration (Figure 7(B)). These results indicated the reliability of our experimental system. It was observed that 3PO treatment led to the recovered of the mRNA expression of RUNX2, OSX and COL1 in MC3T3-E1 cells that had been promoted by T3 administration while not directly affecting the expression levels of the aforementioned osteogenic genes (Figure 7(C) to (E)). Moreover, 3PO addition attenuated the ALP staining intensity and ALP activity that had been enhanced by T3 administration but did not directly affect the aforementioned osteogenic activities (Figure 7(F) and (G)). Moreover, Alizarin red staining showed results similar to those of ALP staining-related for osteogenic activities (Figure 7(H) and (I)). These results supported the idea that that glycolysis is involved in T3-regulated osteogenesis.

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Figure 7.

T3-promoted osteogenic activities were blocked by 3PO addition. (A and B) MC3T3-E1 cells were treated with 100 nM T3 along with or without 10 μM 3PO for 48 h, and the glycolytic potential and LD production in the supernatant were analyzed. (C to E) The mRNA levels of COL1, RUNX2, and OSX in MCT3-E1 cells treated with 100 nM T3 along with or without 10 μM 3PO for 7 days under osteogenic induction. (F and G) ALP staining and activity in MC3T3-E1 cells treated with the indicated treatments for 7 days under osteogenic induction (Unit of ALP activity: King U/GPROT). (H) Alizarin red staining in MC3T3-E1 cells treated with the indicated treatments for 21 days under osteogenic induction. (I) Bar chart showing the percentage of positive areas stained.