TCF7L2 regulates GPX4 to resist ferroptosis and enhance osteogenesis in mouse mesenchymal stem cells

Abstract

Critical-sized bone defects, characterized by poor spontaneous healing capacity, remain a common clinical challenge, and stem cell and gene therapies are key strategies for bone repair and regeneration. Transcription factor 7-like 2 (TCF7L2) is a key regulator of the Wnt signaling pathway, with potential applications in gene editing. However, the role of TCF7L2 in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) remains poorly understood. We knocked down or overexpressed TCF7L2 to evaluate its effect on erastin-induced ferroptosis in BMSCs. Simultaneously, we assessed the impact of TCF7L2 overexpression on the osteogenic capacity of BMSCs. To confirm the involvement of glutathione peroxidase 4 (GPX4), we conducted rescue experiments by knocking down GPX4 expression. A mouse cranial defect model was established to analyze the effect of TCF7L2 overexpression on cranial bone healing. The results showed that TCF7L2 knockdown promoted, while TCF7L2 overexpression inhibited, erastin-induced ferroptosis in BMSCs. Mechanistic studies revealed that TCF7L2 knockdown reduced, while TCF7L2 overexpression enhanced, GPX4 expression, thereby regulating ferroptosis. Conversely, GPX4 knockdown significantly attenuated the regulatory effects of TCF7L2 overexpression on cell proliferation and ferroptosis inhibition. Furthermore, TCF7L2 overexpression promoted cell proliferation, osteogenic differentiation, and mineralization in vitro, while enhancing cranial defect healing in vivo. This study is the first to reveal the dual role of TCF7L2: regulating ferroptosis in BMSCs via GPX4, while promoting BMSC proliferation and osteogenic differentiation. These findings provide novel molecular targets and theoretical foundations for the treatment of bone defects.

Keywords: BMSCs; Ferroptosis; GPX4; Osteogenesis; Osteogenic differentiation; TCF7L2.

Fig. 1

Sustained expression of TCF7L2 during osteogenic differentiation of BMSCs and the establishment of a ferroptosis cell model. A Western blot analysis showing sustained expression of TCF7L2 protein during the osteogenic differentiation of BMSCs, with grayscale semi-quantitative analysis. B Microscopic images showing dose-dependent induction of BMSCs death by erastin (scale bar: 400 μm). C CCK-8 assay showing cell viability of BMSCs treated with different concentrations of erastin. D CCK-8 assay showing cell viability of BMSCs treated with different concentrations of Fer-1. E Cell viability of BMSCs treated with erastin and erastin + Fer-1 for 24 h. F–H Relative levels of intracellular MDA, Fe²⁺, and GSH in BMSCs treated with erastin and erastin + Fer-1 for 24 h. I Western blot analysis of intracellular SLC7A11 and GPX4 protein expression with grayscale semi-quantitative analysis. Data are presented as mean ± SD, n = 3; ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

A Representative images of ROS staining detected by DCFH-DA and semi-quantitative analysis of fluorescence intensity (scale bar: 200 μm). B Representative images of mitochondrial membrane potential changes detected by JC-1 staining and semi-quantitative analysis of fluorescence intensity (scale bar: 200 μm). Data are presented as mean ± SD, n = 3; ns not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3

Knockdown of TCF7L2 Increases Sensitivity of BMSCs to Ferroptosis. A Relative mRNA expression levels of TCF7L2 in BMSCs after siRNA knockdown. B Proliferation of BMSCs after TCF7L2 knockdown, assessed by CCK-8 assay. C Relative intracellular GSH levels. D Relative intracellular MDA levels. E Relative intracellular Fe²⁺ levels. F Representative images of ROS staining detected by DCFH-DA and semi-quantitative analysis of fluorescence intensity (scale bar: 200 μm). G Representative images of mitochondrial membrane potential changes detected by JC-1 staining and semi-quantitative analysis of fluorescence intensity (scale bar: 200 μm). Data are presented as mean ± SD, n = 3; ns not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001

Fig. 4

TCF7L2 Overexpression Inhibits Ferroptosis in BMSCs. A Relative mRNA levels of TCF7L2 in BMSCs treated with shRNA. B Proliferation capacity of TCF7L2-overexpressing BMSCs assessed by CCK-8 assay. C Relative intracellular GSH levels. D Relative intracellular MDA levels. E Relative intracellular Fe²⁺ levels. F Representative images of ROS staining detected by DCFH-DA and semi-quantitative analysis of fluorescence intensity (scale bar: 200 μm). G Representative images of mitochondrial membrane potential changes detected by JC-1 staining and semi-quantitative analysis of fluorescence intensity (scale bar: 200 μm). Data are presented as mean ± SD, n = 3; ns not significant, ****P < 0.0001

Fig. 5

TCF7L2 Overexpression Inhibits BMSCs Ferroptosis by Activating GPX4. A Western blot analysis of TCF7L2 and GPX4 protein levels in BMSCs from each group after TCF7L2 overexpression, with densitometric quantification. B Western blot analysis of TCF7L2 and GPX4 protein levels in BMSCs from each group after TCF7L2 knockdown, with densitometric quantification. C Western blot analysis of TCF7L2 and GPX4 protein levels in BMSCs from each group after GPX4 knockdown, with densitometric quantification. D Western blot analysis of the effects of GPX4 knockdown on TCF7L2 and GPX4 protein levels in BMSCs after TCF7L2 overexpression, with densitometric quantification. E Representative images of ROS staining detected by DCFH-DA in TCF7L2 overexpression BMSCs with GPX4 knockdown, along with semi-quantitative analysis of fluorescence intensity. (scale bar: 200 μm). F Relative expression levels of MDA in cells. Data are presented as mean ± SD, n = 3; ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

TCF7L2 Overexpression Promotes Osteogenic Differentiation and Mineralization. A Western blot analysis of OPN and RUNX2 protein in BMSCs from each group, with densitometric quantification. B Relative mRNA levels of ALP, RUNX2, OPN, and OCN in BMSCs from each group. C Representative images and semi-quantitative analysis of ALP staining on day 7 (scale bar: 1000 μm). D Representative images and semi-quantitative analysis of Alizarin Red staining on day 21 (Scale bars: 1000 μm and 500 μm). Data are presented as mean ± SD, n = 3; ns not significant, ***P < 0.001, ****P < 0.0001

Fig. 7

In vivo overexpression of TCF7L2 promotes the repair of bone defects. A Representative images and micro-CT three-dimensional reconstruction images of each group six weeks post-surgery in mice with cranial defects. B–C Western blot analysis of TCF7L2 protein expression in cranial defect tissues of each group two weeks post-surgery, along with semi-quantitative analysis of grayscale values (n = 3). D Bone volume-to-total volume ratio (BV/TV). E Quantitative analysis of cranial defect area (%). F Quantitative analysis of cranial defect diameter (%). G H&E staining, OCN staining and GPX4 staining results of the bone defect area six weeks post-surgery (scale bars: 400 μm, 200 μm, and 100 μm). HB host bone. NB new bone. The black arrows: the positive staining with OCN or GPX4. Data are presented as mean ± SD, n = 5; ns: not significant, ****P < 0.0001

Fig. 8

TCF7L2 targeting regulates the mechanism by which GPX4 resists iron death and promotes osteogenesis in mouse MSCs