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Published on: July 29, 2016
A mutation in SLC20A2 (c.C1849T) promotes proliferation while inhibiting hypertrophic differentiation in ATDC5
YiQiang Li1, XueMei Lin1, MingWei Zhu1
1Department of Pediatric Orthopaedics, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China.
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.
Insights
A mutation in the solute carrier family 20 member 2 (SLC20A2) gene impairs phosphate uptake in chondrocytes. This SLC20A2 mutation increases cell proliferation but reduces differentiation, potentially involving the Ihh/PTHrP pathway.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Hereditary multiple exostoses is linked to genetic mutations.
- Solute carrier family 20 member 2 (SLC20A2) plays a role in phosphate transport.
- Understanding SLC20A2's function is crucial for bone development disorders.
Purpose of the Study:
- To investigate the impact of an SLC20A2 gene mutation on chondrocyte proliferation and differentiation.
- To analyze the role of the Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP) signaling pathway.
Main Methods:
- ATDC5 chondrocytes were cultured and induced to differentiate.
- Cells were transfected with wild-type (WT) or mutated (MUT) SLC20A2.
- Phosphate uptake, gene/protein expression (qRT-PCR, Western blotting), and pathway activity were assessed.
Main Results:
- The SLC20A2 mutation significantly reduced phosphate uptake in chondrocytes.
- Chondrocyte proliferation increased, while differentiation markers (e.g., Acan, COL2A1, SOX9) were upregulated, and terminal differentiation markers (e.g., Runx2, COL10A1, MMP13) were downregulated.
- The Ihh/PTHrP pathway was upregulated in the MUT group and its inhibition restored normal chondrocyte function.
Conclusions:
- A specific SLC20A2 mutation (c.C1948T) impairs phosphate transport in chondrocytes.
- This mutation promotes chondrocyte proliferation and inhibits differentiation.
- The Ihh/PTHrP signaling pathway is implicated in the observed effects of the SLC20A2 mutation.
