A mutation in SLC20A2 (c.C1849T) promotes proliferation while inhibiting hypertrophic differentiation in ATDC5 chondrocytes

Abstract

Aims: This study aimed to investigate the effect of solute carrier family 20 member 2 (SLC20A2) gene mutation (identified from a hereditary multiple exostoses family) on chondrocyte proliferation and differentiation.

Methods: ATDC5 chondrocytes were cultured in insulin-transferrin-selenium medium to induce differentiation. Cells were transfected with pcDNA3.0 plasmids with either a wild-type (WT) or mutated (MUT) SLC20A2 gene. The inorganic phosphate (Pi) concentration in the medium of cells was determined. The expression of markers of chondrocyte proliferation and differentiation, the Indian hedgehog (Ihh), and parathyroid hormone-related protein (PTHrP) pathway were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting.

Results: The expression of SLC20A2 in MUT group was similar to WT group. The Pi concentration in the medium of cells in MUT group was significantly higher than WT group, which meant the SLC20A2 mutation inhibited Pi uptake in ATDC5 chondrocytes. The proliferation rate of ATDC5 chondrocytes in MUT group was greater than WT group. The expression of aggrecan (Acan), α-1 chain of type II collagen (COL2A1), and SRY-box transcription factor 9 (SOX9) were higher in MUT group than WT group. However, the expression of Runt-related transcription factor 2 (Runx2), α-1 chain of type X collagen (COL10A1), and matrix metallopeptidase 13 (MMP13) was significantly decreased in the MUT group. Similar results were obtained by Alcian blue and Alizarin red staining. The expression of Ihh and PTHrP in MUT group was higher than WT group. An inhibitor (cyclopamine) of Ihh/PTHrP signalling pathway inhibited the proliferation and restored the differentiation of chondrocytes in MUT group.

Conclusion: A mutation in SLC20A2 (c.C1948T) decreases Pi uptake in ATDC5 chondrocytes. SLC20A2 mutation promotes chondrocyte proliferation while inhibiting chondrocyte differentiation. The Ihh/PTHrP signalling pathway may play an important role in this process. Cite this article: Bone Joint Res 2020;9(11):751-760.

Keywords: Chondrocyte; Differentiation; Phosphate; Proliferation; SLC20A2.

Fig. 1

Sanger sequencing confirmed the a) cloned wild-type and b) mutant mouse solute carrier family 20 member 2 (SLC20A2) complementary DNA (cDNA) (NM_011394). c) and d) This mutation causes a change of an amino acid in SLC20A2 protein (R > C). The arrows indicate the site of variance.

Fig. 2

a) An immunofluorescence examination (magnification: 40×) was performed to determine the transfection efficiency of the cell lines. Three different quantities of pcDNA3.0 plasmids were used to achieve the multiplicity of infection (MOI) of 100, 200, and 300. The results indicate that all the cell lines with a MOI of 300 had the best transfection efficiency (the green fluorescence shows the transfected plasmids). Scale bar = 1,000 μm. b) and c) Expression of solute carrier family 20 member 2 (SLC20A2), as determined by b) quantitative real-time polymerase chain reaction (qRT-PCR) and c) western blot. There was no significant difference in SLC20A2 expression level between the wild-type (WT) and mutant (MUT) groups. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA.

Fig. 3

The inorganic phosphate (Pi) concentration in the culture medium, as measured using a Malachite Green Phosphate Assay Kit and microplate spectrophotometer. A high Pi concentration in the medium reflected low Pi uptake in the cells. The Pi concentration in the mutant group (MUT) was significantly higher than that in the wild-type group (WT) on days 7, 14, and 21.

Fig. 4

The evaluation of cell proliferation of chondrocytes. The expression of markers of chondrocyte proliferation (aggrecan (Acan), α-1 chain of type II collagen (COL2A1), and SRY-box transcription factor 9 (SOX9)), as measured by: a) to c) quantitative real-time polymerase chain reaction (qRT-PCR); and d) western blot, were significantly higher in the mutant (MUT) group than in the wild-type (WT) group. e) The proliferation rate (optical density (OD)) of the chondrocytes in the MUT group was significantly higher than that in the WT group on days 7, 14, and 21 (*p < 0.05 vs WT group). f) and g) The results of Alcian blue staining (magnification: 100×) on day 21 also showed a significant increase of cartilage glycosaminoglycans (stained blue) in the MUT group when compared with the WT group. f) and g) Cyclopamine caused a decrease in cartilage glycosaminoglycans in the MUT group. Scale bar = 50 μm. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA.

Fig. 5

The evaluation of chondrocyte differentiation. The expression of markers of chondrocyte differentiation (α-1 chain of type X collagen (COL10A1), matrix metallopeptidase 13 (MMP13), and runt-related transcription factor 2 (Runx2)), as measured by: a) to c) quantitative real-time polymerase chain reaction (qRT-PCR); and d) western blot, were significantly lower in the mutant (MUT) group compared with the wild-type (WT) group. e) and f) The results of Alizarin red staining (magnification: 100×) on day 21 also showed that calcium deposition (stained red) was significantly decreased in the MUT group compared with the WT group, and cyclopamine caused a significant increase in calcium deposition in the MUT group. Scale bar = 50 μm. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA.

Fig. 6

The expression of Indian hedgehog (Ihh)/parathyroid hormone-related protein (PTHrP) signalling pathway. The expression of Ihh and PTHrP, as measured by: a) and b) quantitative real-time polymerase chain reaction (qRT-PCR); and c) western blot, were significantly higher in the mutant (MUT) group compared with the wild-type (WT) group. Cyclopamine significantly inhibited the expression of Ihh and PTHrP in the MUT group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA.