Thioredoxin Interacting Protein Expressed in Osteoblasts Mediates the Anti-Proliferative Effects of High Glucose and Modulates the Expression of Osteocalcin

Article information

J Bone Metab. 2024;31(3):209-218

Publication date (electronic) : 2024 August 31

doi : https://doi.org/10.11005/jbm.2024.31.3.209

Sarah McGarry ¹^,²

, Karen Kover ¹^,²

, Francesco De Luca ¹^,²

¹Division of Endocrinology, Children’s Mercy Hospitals, Kansas City, MO, USA

²Department of Pediatrics, University of Missouri-Kansas City-School of Medicine, Kansas City, MO, USA

Corresponding author: Francesco De Luca, Division of Endocrinology, Children’s Mercy Hospitals, 2401 Gillham Rd, Kansas City, MO 64108, USA, E-mail: fdeluca59@gmail.com

Received 2024 March 13; Revised 2024 June 1; Accepted 2024 June 3.

Abstract

Background

Hyperglycemia is associated with impaired bone health in patients with diabetes mellitus. Although a direct detrimental effect of hyperglycemia on the bone has been previously reported, the specific molecular mediator(s) responsible for the inhibitory effect of high glucose levels on the bone remains unclear. We hypothesized that thioredoxin-interacting protein (Txnip), an essential mediator of oxidative stress, is such a mediator.

Methods

We cultured MG-63 cells (immortalized human osteoblasts) with normal or high glucose concentrations and transfected them with scrambled or Txnip-specific small interfering RNA (siRNA).

Results

High glucose levels increased Txnip expression and reduced MG-63 cell proliferation. The high-glucose level mediated reduction in cell proliferation was prevented in Txnip siRNA-transfected cells. In addition, we demonstrated that silencing Txnip mRNA expression in osteoblasts reduced the expression of the osteocalcin gene. Our results suggest that high glucose levels or silencing of Txnip mRNA expression may induce apoptosis in osteoblasts.

Conclusions

Our findings indicate that Txnip is an intracellular mediator of the anti-proliferative effects of extracellular high glucose levels on osteoblasts.

Keywords: Hyperglycemia; Thioredoxin interacting protein; Osteoblasts

GRAPHICAL ABSTRACT

INTRODUCTION

Diabetes mellitus (DM) is a very prevalent disease characterized by hyperglycemia, insulin deficiency and/or insulin resistance. Its main long-term complications consist of cardiovascular disease, retinopathy, and chronic kidney insufficiency.[1] In addition, individuals with DM experience an increased risk for bone fractures.[2,3]

In DM, it appears that osteoblast dysfunction is one of the factors contributing to impaired bone quality/mass.[2] An in vitro model has been previously utilized to study the effects of hyperglycemia on osteoblasts, culturing osteoblast-like cells in the presence of high glucose in the medium. Published evidence has revealed that high glucose inhibits mineralization and proliferation in MC3T3-E1 cells, an immortalized cell line derived from rat calvaria.[4–8] However, little is known about any possible molecular mediator directly responsible for the deleterious effect of high glucose on osteoblasts.

Oxidative stress is involved in bone metabolic changes and leads to the formation of high reactive oxygen species (ROS) levels. Excessive ROS activity and defects in the cellular antioxidant system lead to abnormal cellular bone metabolism.[9]

Thioredoxin (TRX)-interacting protein (Txnip), also known as TRX-binding protein-2, is a cytosolic protein that is an essential mediator of oxidative stress and inflammatory response.[10] Txnip is referred to as Txnip because it interacts with TRX, an important intracellular antioxidant protein, and negatively regulates TRX function in many mammalian tissues.[10] TRX, as a thiol oxidoreductase, exerts antioxidant properties through the scavenging of ROS.

In 2003, Kobayashi et al. [11] first reported that Txnip expression level was rapidly elevated following high glucose stimulation in cultured mesangial cells and kidneys of diabetic mice.

In vivo findings showed that the overexpression of TRX in TRX-transgenic mice partially restored the reduced bone mineral density (BMD) and prevented streptozotocin-induced bone formation and osteopenia in diabetes.[12]

Goto-Kakizaki rats, a model of type 2 DM, have significantly reduced bone mass, and the expression of Txnip in their bone tissue is significantly increased.[13]

Yet, there is no evidence that hyperglycemia directly affects the expression of Txnip in bone cells, and that Txnip in turn is responsible of bone cell dysfunction.

In the present study, we have analyzed the effects of high glucose on Txnip expression and function in cultured osteoblast cell line MG-63 (immortalized human osteoblast cell line), which is a prototype of osteoblast phenotype widely utilized in bone research.

METHODS

1. Cell culture and reagents

MG-63 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) media supplemented with 10% fetal bovine serum (ATCC) and 1% penicillin/streptomycin. Cells were cultured at 37°C in a CO₂ incubator. DMEM high-glucose media was purchased from the ATCC.

2. Cell proliferation assays

Cell proliferation or viability analysis was carried out by applying the MTT assay. Cells were seeded into 12-well plates (1.5×10⁴ cells/well). After 48 or 72 hr of treatment, 10% MTT 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reagent (5 mg·mL–1) was added and incubated for 4 hr. The supernatant was removed, followed by the addition of 600 μL dimethyl sulfoxide (DMSO) to dissolve formazan crystals. Absorbance was measured at 570 nm. The amount of formazan produced was proportional to the number of viable cells.

3. Flow cytometry analysis

Cells were washed twice with phosphate-buffered saline (PBS), dissociated with 0.05% of trypsin, neutralized in DMEM media, washed twice in PBS and then resuspended in 300 μL Annexin Binding Buffer 5X (Thermo Fisher Scientific, Waltham, MA, USA) diluted 5:1 in PBS. A 3 μL of mouse IgG solution (1 mg/mL) was added and incubated for 5 min on ice. The cells were washed and then resuspended in 300 μL of diluted Annexin Binding Buffer, filtered through a 40 μm strainer, 3 μL (1 mg/mL) of Annexin-V (eBioscience, San Diego, CA, USA) was added and incubated for 15 min at room temperature followed by 3 μL (1 mg/mL) 7-aminoactinomycin D (eBioscience) to assess dead and apoptotic cells. Flow cytometry was performed on a BD LSRFortessa and data analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA).

4. Small interfering RNA (siRNA) knockdown

siRNAs for Txnip (#AM16708) and the Silencer Select Negative Control #1 siRNA (Scramble, #AM4611) were purchased from Thermo Fisher Scientific as SMARTpools. For siRNA transfections, cells were transfected with oligos using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After transfection, efficiency was determined through reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

5. RT-qPCR

Total RNAs were extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA) and RT-qPCR analysis was performed using Bio-Rad MyiQ (Bio-Rad, Hercules, CA, USA). The conditions for RT-qPCR reactions were: one cycle at 95°C for 20 sec followed by 45 cycles at 95°C for 3 sec and annealing at 60°C for 30 sec. Results were normalized to the housekeeping gene 18s ribosomal RNA. Relative expression levels of genes from different groups were calculated with the 2ΔΔCT method and compared with the expression level of appropriate control cells.

6. Statistical analysis

Statistical significance was determined via Student’s t-test. Results were considered significant when ^a)P<0.05, ^b)P<0.01, or ^c)P<0.001.

RESULTS

1. Effects of high glucose on Txnip expression in MG-63 cells

In MG-63 cells transfected with scramble siRNAs and cultured in the presence of high glucose (25 mM), the expression of Txnip analyzed after 48 hr in culture was significantly greater than in cells transfected with scramble siRNA cultured in the presence of normal glucose concentration (5 mM) (Fig. 1A).

Fig. 1

Effects of high glucose on thioredoxin-interacting protein (Txnip) expression in MG-63 cells. MG-63 cells were maintained in Dulbecco’s modified Eagle’s medium media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured in 37°C in a CO₂ incubator. Cells were transfected with a Txnip-specific small interfering RNA (siRNA) or a control, scramble siRNA (Silencer Select Negative Control siRNA), both purchased from Thermo Fisher Scientific (Waltham, MA, USA) as SMARTpools. After transfection, cells were cultured up to 72 hr with normal glucose concentration (5 mM) or high glucose concentration (25 mM) added to the culture medium. After 48 and 72 hr in culture, total RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA), reverse transcription-quantitative polymerase chain reaction analysis was performed and Txnip mRNA expressed measured after 48 hr (A) and 72 hr (B). Results were normalized to the housekeeping gene 18s ribosomal RNA. SN, cells transfected with scramble siRNA and cultured in normal glucose; SH, cells transfected with scramble siRNA and cultured in high glucose; TN, cells transfected with Txnip-specific siRNA and cultured in normal glucose; TH, cells transfected with Txnip-specific siRNA and cultured in high glucose. ^a)P<0.05, ^c)P<0.001.

In MG-63 cells transfected with Txnip-specific siRNA, whether cultured in the presence of high glucose or normal glucose, the Txnip expression was significantly reduced when compared to cells transfected with scramble siRNAs and cultured in the presence of high glucose or normal glucose (Fig. 1A).

After 72 hr in culture, the mean Txnip expression in scramble siRNA-transfected MG-63 cells cultured with high glucose was greater than in cells cultured with normal glucose (Fig. 1B). However, this difference did not reach statistical significance.

2. Effects of glucose and Txnip on MG-63 cell proliferation

We then assessed the effects of high glucose on MG-63 cell proliferation by MTT assay. After 48 and 72 hr in culture, MG-63 cells transfected with scramble siRNA and cultured in the presence of high glucose exhibited a significantly decreased cell proliferation compared to cell cultured with normal glucose (Fig. 2). In contrast, at 48 and 72 hr cell proliferation was increased in cells previously transfected with Txnip siRNA and cultured in normal glucose (compared to cells previously transfected with scramble siRNA), with the difference being significant only at 48 hr (Fig. 2).

Fig. 2

Effects of high glucose and thioredoxin-interacting protein (Txnip) on MG-63 cell proliferation. MG-63 cells were maintained in Dulbecco’s modified Eagle’s medium media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured in 37°C in a CO₂ incubator. Cells were transfected with a Txnip-specific small interfering RNA (siRNA) or a control, scramble siRNA. After transfection, cells were cultured up to 72 hr with normal glucose concentration (5 mM) or high glucose concentration (25 mM) added to the culture medium. Cell proliferation or viability was measured after 48 hr (A) and 72 hr (B) by utilizing the MTT assay. After 48 or 72 hr in culture, 10% MTT was added and incubated for 4 hr. The supernatant was removed, followed by the addition of 600 μL dimethyl sulfoxide to dissolve formazan crystals. Absorbance was measured at 570 nm. The amount of formazan generated was proportional to the number of viable cells. Results are expressed in folds relative to the amount of formazan generated in the culture medium of the SN cells. SN, cells transfected with scramble siRNA and cultured in normal glucose; SH, cells transfected with scramble siRNA and cultured in high glucose; TN, cells transfected with Txnip-specific siRNA and cultured in normal glucose; TH, cells transfected with Txnip-specific siRNA and cultured in high glucose. ^a)P<0.05, ^b)P<0.01, ^c)P<0.001.

At 72 hr, cell proliferation was increased in Txnip siRNA-transfected MG-63 cells cultured in high glucose compared to cells transfected with scramble siRNAs and cultured in high glucose (Fig. 2B).

3. Effects of glucose and Txnip on BGLAP and Runx2 mRNA expression in MG-63 cells

To study the effects of Txnip on osteoblast differentiation, we analyzed the mRNA expression of BGLAP and Runx2 in MG-63 cells transfected with scramble or Txnip siRNA and cultured in the presence of normal or high glucose.

MG-63 transfected with Txnip siRNA (when compared to cells transfected with scramble siRNAs) and cultured in normal glucose exhibited a decreased BGLAP expression at 48 and 72 hr (Fig. 3A, B), with the difference being statistically significant at 48 hr (Fig. 3A). High glucose in the culture medium was associated with decreased BGLAP expression in MG-63 cells transfected with Txnip siRNA (compared to cells transfected with scramble siRNAs and cultured in high glucose), but this difference did not reach statistical significance. Glucose concentration in the culture medium or type of siRNA did not affect the expression of Runx2 (Fig. 3C, D).

Fig. 3

Effects of thioredoxin-interacting protein (Txnip) on MG-63 cell BGLAP and Runx2 mRNA expression. MG-63 cells were maintained in Dulbecco’s modified Eagle’s medium media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured in 37°C in a CO₂ incubator. Cells were transfected with a Txnip-specific small interfering RNA (siRNA) or a control, scramble siRNA. After transfection, cells were cultured up to 72 hr with normal glucose concentration (5 mM) or high glucose concentration (25 mM) added to the culture medium. After 48 hr and 72 hr in culture, total RNA was extracted, reverse transcription-quantitative polymerase chain reaction analysis was performed and the mRNA expression of BGLAP (A, B) and Runx2 (C, D) (two markers of osteoblast differentiation) measured. Results were normalized to the housekeeping gene 18s ribosomal RNA. SN, cells transfected with scramble siRNA and cultured in normal glucose; SH, cells transfected with scramble siRNA and cultured in high glucose; TN, cells transfected with Txnip-specific siRNA and cultured in normal glucose; TH, cells transfected with Txnip-specific siRNA and cultured in high glucose. ^c)P<0.001.

4. Effects of glucose and Txnip on MG-63 cell apoptosis

We then studied the effects of high glucose and Txnip on apoptosis by flow cytometry. When compared to MG-63 cells transfected with scramble siRNA and cultured in the presence of normal glucose, at 48 hr high glucose revealed a two-fold increase in the percentage of cell apoptosis of MG-63 cells transfected with scramble siRNA (Fig. 4A). In addition, cells transfected with Txnip siRNA and cultured with normal glucose exhibited a three-fold mean increase in apoptosis, compared to cells transfected with scramble siRNA cultured in normal glucose (Fig. 4A).

Fig. 4

Effects of high glucose and thioredoxin-interacting protein (Txnip) on MG-63 cell apoptosis. Cell apoptosis was analyzed after 48 hr (A) and 72 hr (B) by using flow cytometry. After 48 hr and 72 hr in culture, cells were washed twice with phosphate-buffered saline (PBS), dissociated with 0.05% of trypsin, neutralized in Dulbecco’s modified Eagle’s medium media, washed twice in PBS and then resuspended in 300 μL Annexin Binding Buffer 5X diluted 5:1 in PBS. 3 μL of mouse immunoglobulin G solution (1 mg/mL) was added and incubated for 5 min on ice. The cells were washed and then resuspended in 300 μL of diluted Annexin Binding Buffer, filtered through a 40 μm strainer, 3 μL (1 mg/mL) of Annexin V was added and incubated for 15 min at room temperature followed by 3 μL (1 mg/mL) 7-aminoactinomycin D to assess apoptotic cells. Flow cytometry was performed on a BD LSRFortessa and data analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA). siRNA, small interfering RNA; SN, cells transfected with scramble siRNA and cultured in normal glucose; SH, cells transfected with scramble siRNA and cultured in high glucose; TN, cells transfected with Txnip-specific siRNA and cultured in normal glucose; TH, cells transfected with Txnip-specific siRNA and cultured in high glucose.

At 72 hr in culture, high glucose or silenced Txnip expression did not induce any obvious change in percentage of cell apoptosis compared to cells transfected with scramble siRNA and cultured in normal glucose (Fig. 4B).

The significant effects of high glucose and Txnip silenced expression in osteoblasts are summarized in Figure 5.

Fig. 5

Schematic summary of our findings. MG-63 cells cultured in the presence of high glucose (25 mM) in the medium exhibited increased thioredoxin-interacting protein (Txnip) mRNA expression and decreased cell proliferation. After transfection with Txnip small interfering RNA (siRNA), decreased Txnip mRNA expression resulted in increased cell proliferation and decreased BGLAP mRNA expression.

DISCUSSION

Several lines of evidence indicate that DM is associated with impaired bone health. The prevalence of osteoporotic fractures is increased in patients with type 1 DM or type 2 DM.[14] While some studies have suggested a reduced BMD in subjects with DM,[15,16] others have found no change or even increased BMD, implying bone fragility/decreased bone quality as a primary causative mechanism of increased predisposition to bone fractures.[17–19] Hyperglycemia and hypoglycemia, which are both common metabolic derangements in DM, have been associated with increased fracture risk.[20–24]

Prior studies have implied a direct detrimental role of hyperglycemia on osteoblast proliferation. High concentration of glucose in the culture media inhibits proliferation of osteoblast-like cells [25,26] as well as of mesenchymal stem cells.[27]

We have hypothesized that Txnip is a mediator of the direct effects of hyperglycemia on osteoblasts. Txnip is a critical mediator of intracellular oxidative stress [28–30]; it functions by interacting and neutralizing TRX, an important intracellular antioxidant protein. It has been demonstrated that Txnip expression is consistently increased in skeletal muscle cells of diabetic and prediabetic patents.[31] High glucose enhances Txnip expression in cultured renal proximal tubule cell lines,[31] in glomerular mesangial cells,[32] and in breast-cancer-derived cells.[33] In addition, Txnip is expressed in bone.[28,34,35]

To test our hypothesis, we utilized MG-63 cells, an immortalized human osteoblast cell line.[25] In our study, we have demonstrated that high glucose in the culture media inhibits MG-63 cell proliferation and increases Txnip expression. The increased expression of Txnip has a causative role in the high glucose-mediated decreased cell proliferation. Indeed, Txnip silenced expression by siRNA in MG-63 cells prevents the inhibitory effects of high glucose on proliferation.

An anti-proliferative role of Txnip has also been shown in other cells. HTR-8/SVneo cells (a human trophoblast cell line) treated with high glucose exhibit reduced proliferation, with the knockdown of Txnip reversing the effects of high glucose on HTR-8/Svneo cells.[36] The reduced proliferation induced by interleukin-1β in cultured articular chondrocytes is prevented by silencing of Txnip via siRNA.[37]

In our study, we have also demonstrated a reduced expression of BGLAP (the gene encoding osteocalcin) in MG-63 cells transfected with Txnip siRNA, which suggests a regulatory role of Txnip on osteocalcin expression in osteoblasts. Osteocalcin, or bone γ-carboxyglutamic acid protein, is a factor expressed and secreted only by osteoblasts.[36] It is the most abundant noncollagenous protein present in bone. Osteocalcin is initially synthesized as a prohormone (95 amino acids) and then cleaved to form the mature peptide. In humans, the mature peptide has 49 amino acids and is carboxylated at positions 17, 21, and 24. Carboxylation increases the affinity of osteocalcin to the bone extracellular matrix. Osteoclast resorption, on the other hand, creates an acidic environment where osteocalcin is decarboxylated. Undercarboxylated osteocalcin has lower affinity to bone matrix and is released into the bloodstream and it can function as a hormone.[38] Evidence provided by studies on osteocalcin knock-out mice revealed a role, possibly marginal, for osteocalcin in regulating bone formation and mineralization.[39] In contrast, more recent evidence suggests a complex regulatory role for the systemic form of Osteocalcin released from bone into circulation on energy expenditure, glucose homeostasis, insulin secretion, and brain function.[40] Our finding of a modulatory effect of Txnip on BGLAP-osteocalcin expression is novel and suggests a possible, indirect, contribution of Txnip in regulating a variety of metabolic pathways in mammals. A previous study has described an increased expression of osteocalcin in osteoblasts transfected with Txnip siRNA,[34] which contradicts our finding. One possible explanation is the different cell lines used (MG-63 in our study, human fetal osteoblast cell line in the previous study).

Our experiments on the effect of high glucose and Txnip on cell apoptosis suggested (after 48 hr in culture) a trend of increased apoptosis induced by high glucose or by the silencing of Txnip expression in MG-63 cells. However, none of these effects was significant.

Previous reports suggest a stimulatory effect of high glucose on cell apoptosis in other cell types,[36,41] with this effect being prevented by the knockdown of Txnip expression. In another study on human osteoblasts, high glucose increased Caspase 3 activity (with Caspase 3 being a marker of apoptosis). However, this effect was observed after 7 days in culture.[42]

In conclusion, our study has demonstrated that high glucose induces reduced proliferation of cultured immortalized human osteoblast cells, and that this effect is mediated by the increased expression and activity of intracellular Txnip. Furthermore, we have shown that Txnip in osteoblasts modulates the expression of the osteocalcin gene, with the silencing of Txnip expression resulting in reduced osteocalcin gene expression. The trend of increased apoptosis in osteoblasts cultured in high glucose or after transfection with Txnip siRNA is not significant.

Notes

Funding

The authors received no external financial support for this article.

Ethics approval and consent to participate

This study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

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