TMEM175 plays a crucial role in osteoblast differentiation by regulating lysosomal function and autophagy

Abstract

Bone provides structural support, enables movement, protects internal organs, regulates calcium and phosphorus levels, and contains bone marrow essential for hematopoiesis. Osteoblasts are specialized cells responsible for bone formation through the secretion of extracellular matrix components. Transmembrane protein 175 (TMEM175), which functions as an endosomal/lysosomal K⁺ channel and a lysosomal H⁺ channel, regulates lysosomal function and autophagy. Despite the recognized importance of lysosomes and autophagy in osteoblast differentiation, the specific role of TMEM175 in osteoblast differentiation has not been revealed. In this study, we investigated whether TMEM175 is associated with human bone mineral density and fracture and examined the role of TMEM175 in osteoblast differentiation. In analyses of single nucleotide polymorphisms of pore ion channel genes using the mouse2human database, a significant correlation between TMEM175 single nucleotide polymorphisms and human bone mineral density and fracture was identified. TMEM175 expression levels were found to increase during osteoblast differentiation from bone chip-derived mesenchymal stem cells (BMSCs). Knockdown of TMEM175 in BMSCs suppressed osteoblast differentiation, as evidenced by decreased matrix mineralization and lower expression levels of osteoblast marker genes. Further analysis indicated that TMEM175 deficiency leads to lysosomal dysfunction and partially impairs autophagic clearance during osteoblast differentiation. Moreover, the TMEM175 inhibitor 4-aminopyridine decreased osteoblast differentiation of BMSCs. Taken together, this study reveals that TMEM175 plays an important role in osteoblast differentiation by regulating lysosomal function and autophagic clearance.

Keywords: Autophagy; Lysosome; Osteoblast differentiation; Transmembrane protein 175.

Fig. 1

TMEM175 SNPs are associated with human BMD and fracture. The association of SNPs of 140 pore ion channel genes with human BMD and fracture was analyzed using z-scores and P-values from the mouse2human database. (A and B) The bubble charts show the z-scores and P-values of SNPs for human BMD (A) and human fracture (B). In these charts, the Y-axis indicates the z-scores, and the color represents P-values (-log₁₀p). BMD, bone mineral density.

Fig. 2

TMEM175 is upregulated during osteoblast differentiation. Osteoblast differentiation of BMSCs was induced with the osteogenic medium containing dexamethasone, ascorbic acid, and β-glycerophosphate for up to 8 days. (A) The mRNA expression levels of Tmem175 and osteoblast marker genes Alpl, Sp7, and Bglap were measured by qRT-PCR on days 0, 2, 4, 6, and 8. (B and C) The expression levels of TMEM175 proteins on days 0, 4, and 8 were analyzed by western blotting (B) and quantified with ImageJ software (C). qRT-PCR, quantitative reverse transcription- polymerase chain reaction; mRNA, messenger RNA.

Fig. 3

Knockdown of TMEM175 in BMSCs reduces osteoblast differentiation. BMSCs were transfected with either negative control siRNA or TMEM175 siRNA with lipofectamine RNAiMAX, followed by treatment with the osteogenic medium to induce osteoblast differentiation. (A-C) The efficiency of TMEM175 knockdown was assessed by qRT-PCR (A) and western blot analysis (B and C). (D) Proliferation of the siCtrl and siTMEM175 groups during osteoblast differentiation was measured using the WST-8 cell viability assay on the indicated days. (E and F) Matrix mineralization was evaluated using alizarin red staining on days 5 and 8 for the nontreated, siCtrl, and siTMEM175 groups (E). Day 8 samples were quantified (F). (G) The mRNA expression levels of osteoblast marker genes in the siCtrl and siTMEM175 groups were analyzed using qRT-PCR up to day 8. (H and I) The protein levels of osteocalcin, an osteoblast marker, were detected by western blot analysis on day 6 in the siCtrl and siTMEM175 groups. BMSC, bone chip-derived mesenchymal stem cell. qRT-PCR, quantitative reverse transcription- polymerase chain reaction; mRNA, messenger RNA; siRNA, small interference RNA; WST-8, water-soluble tetrazolium salt 8.

Fig. 4

TMEM175 deficiency induces lysosomal dysfunction. (A) The mRNA expression levels of lysosome-related genes Tfeb, Lamp1, Lamp2, Ctsb, and Ctsd were analyzed using qRT-PCR. (B and C) The expression levels of cathepsin B and cathepsin D were assessed by western blot analysis (B) and quantified with ImageJ software (C). (D) The activity of cathepsin B and cathepsin D was measured on day 3 using respective activity assay kits. qRT-PCR, quantitative reverse transcription- polymerase chain reaction; mRNA, messenger RNA.

Fig. 5

TMEM175 deficiency partially impairs autophagic clearance. (A) The mRNA expression levels of autophagy-related genes Atg5, Atg7, Atg10, Becn1, and Map1lc3b were analyzed using qRT-PCR. (B and C) Beclin1 and LC3 protein levels were detected by western blot analysis on day 2. The autophagic clearance inhibitor bafilomycin A1 was used to accumulate formed autophagosomes. (C) The protein expression levels of Beclin1 and LC3 were quantified with ImageJ software. (D and E) LC3 was assessed by immunofluorescence (D). The number and size of LC3 puncta were quantified with ImageJ software, and LC3 intensity was measured with the Zen software (E). qRT-PCR, quantitative reverse transcription- polymerase chain reaction; mRNA, messenger RNA.

Fig. 6

4-AP suppresses osteoblast differentiation through lysosomal dysfunction and autophagic impairment. (A and B) Matrix mineralization was evaluated by alizarin red staining on day 7 and quantified. (C) The mRNA expression levels of osteoblast differentiation marker genes were evaluated using qRT-PCR on the indicated day. (D) The activity of cathepsin B and cathepsin D was assessed on day 3 using the respective activity assay kit. (E and F) Protein levels of Beclin1 and LC3 were analyzed by western blot analysis. (F) The expression levels of Beclin1 and LC3 were quantified using ImageJ software. (G and H) LC3 was evaluated by immunofluorescence microscopy (G). The number and size of LC3 puncta were quantified using ImageJ software, and LC3 intensity was measured using the Zen software (H). 4-AP, 4-aminopyridine. qRT-PCR, quantitative reverse transcription- polymerase chain reaction; mRNA, messenger RNA.