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TUFT1 , a novel candidate gene for metatarsophalangeal osteoarthritis, plays a role in chondrogenesis on a calcium-related pathway

View Article: PubMed Central - PubMed

ABSTRACT

Osteoarthritis (OA) is the most common degenerative joint disorder and genetic factors have been shown to have a significant role in its etiology. The first metatarsophalangeal joint (MTP I) is highly susceptible to development of OA due to repetitive mechanical stress during walking. We used whole exome sequencing to study genetic defect(s) predisposing to familial early-onset bilateral MTP I OA inherited in an autosomal dominant manner. A nonsynonymous single nucleotide variant rs41310883 (c.524C>T, p.Thr175Met) in TUFT1 gene was found to co-segregate perfectly with MTP I OA. The role of TUFT1 and the relevance of the identified variant in pathogenesis of MTP I OA were further assessed using functional in vitro analyses. The variant reduced TUFT1 mRNA and tuftelin protein expression in HEK293 cells. ATDC5 cells overexpressing wild type (wt) or mutant TUFT1 were cultured in calcifying conditions and chondrogenic differentiation was found to be inhibited in both cell populations, as indicated by decreased marker gene expression when compared with the empty vector control cells. Also, the formation of cartilage nodules was diminished in both TUFT1 overexpressing ATDC5 cell populations. At the end of the culturing period the calcium content of the extracellular matrix was significantly increased in cells overexpressing mutant TUFT1 compared to cells overexpressing wt TUFT1 and control cells, while the proteoglycan content was reduced. These data imply that overexpression of TUFT1 in ATDC5 inhibits chondrogenic differentiation, and the identified variant may contribute to the pathogenesis of OA by increasing calcification and reducing amount of proteoglycans in the articular cartilage extracellular matrix thus making cartilage susceptible for degeneration and osteophyte formation.

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Expression of chondrocyte differentiation and hypertrophy marker genes, and TUFT1 in ATDC5 cells.(A) Sox9, Col2a1, and Agc1 are markers for chondrocyte differentiation and (B) Runx2, Col10a1, Mmp13, and Alpl markers for chondrocyte hypertrophy. In addition, expression of TUFT1 was determined (C). Expressions were studied by real-time qPCR in mixed population clones overexpressing wt or mutant TUFT1 (ATDC5-wtTUFT1, ATDC5-mutTUFT1) and in empty vector controls (ATDC5-ctrl) at three time points during 15 days of differentiation. Hprt and Ppia were used as reference genes. Results are represented as means of three groups of quadruplet samples and relative gene expression denotes log(x+1) transformed fold changes. P-values relate to the confidence of whether the change in the relative gene expression over time is different between the ATDC5-wtTUFT1, ATDC5-mutTUFT1 and ATDC5-ctrl cell populations (S2 Table).
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pone.0175474.g003: Expression of chondrocyte differentiation and hypertrophy marker genes, and TUFT1 in ATDC5 cells.(A) Sox9, Col2a1, and Agc1 are markers for chondrocyte differentiation and (B) Runx2, Col10a1, Mmp13, and Alpl markers for chondrocyte hypertrophy. In addition, expression of TUFT1 was determined (C). Expressions were studied by real-time qPCR in mixed population clones overexpressing wt or mutant TUFT1 (ATDC5-wtTUFT1, ATDC5-mutTUFT1) and in empty vector controls (ATDC5-ctrl) at three time points during 15 days of differentiation. Hprt and Ppia were used as reference genes. Results are represented as means of three groups of quadruplet samples and relative gene expression denotes log(x+1) transformed fold changes. P-values relate to the confidence of whether the change in the relative gene expression over time is different between the ATDC5-wtTUFT1, ATDC5-mutTUFT1 and ATDC5-ctrl cell populations (S2 Table).

Mentions: Overexpression of both wt and mutant TUFT1 significantly influenced the expression of Col2a1 and Agc1 (P = 2.95x10-15 and P = 1.86x10-12, respectively, Fig 3A, S2 Table), but did not affect the expression of Sox9 (P = 0.461, Fig 3A, S2 Table): in ATDC5-mutTUFT1 cells Col2a1 expression differed from control cells on all the measurement days whereas in ATDC5-wtTUFT1 cells the difference reached statistical significance on days 8 and 15 (S3 Table). Additionally, Col2a1 expression was significantly lower in ATDC5-mutTUFT1 cells in comparison to ATDC5-wtTUFT1 cells on days 12 and 15 (P = 0.008 and P = 0.045, respectively, Fig 3A, S3 Table). Agc1 expression in both ATDC5-mutTUFT1 and ATDC5-wtTUFT1 cells deviated from the expression seen in control cells, while there was no statistically significant difference between the mutant and wt cells (S3 Table).


TUFT1 , a novel candidate gene for metatarsophalangeal osteoarthritis, plays a role in chondrogenesis on a calcium-related pathway
Expression of chondrocyte differentiation and hypertrophy marker genes, and TUFT1 in ATDC5 cells.(A) Sox9, Col2a1, and Agc1 are markers for chondrocyte differentiation and (B) Runx2, Col10a1, Mmp13, and Alpl markers for chondrocyte hypertrophy. In addition, expression of TUFT1 was determined (C). Expressions were studied by real-time qPCR in mixed population clones overexpressing wt or mutant TUFT1 (ATDC5-wtTUFT1, ATDC5-mutTUFT1) and in empty vector controls (ATDC5-ctrl) at three time points during 15 days of differentiation. Hprt and Ppia were used as reference genes. Results are represented as means of three groups of quadruplet samples and relative gene expression denotes log(x+1) transformed fold changes. P-values relate to the confidence of whether the change in the relative gene expression over time is different between the ATDC5-wtTUFT1, ATDC5-mutTUFT1 and ATDC5-ctrl cell populations (S2 Table).
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pone.0175474.g003: Expression of chondrocyte differentiation and hypertrophy marker genes, and TUFT1 in ATDC5 cells.(A) Sox9, Col2a1, and Agc1 are markers for chondrocyte differentiation and (B) Runx2, Col10a1, Mmp13, and Alpl markers for chondrocyte hypertrophy. In addition, expression of TUFT1 was determined (C). Expressions were studied by real-time qPCR in mixed population clones overexpressing wt or mutant TUFT1 (ATDC5-wtTUFT1, ATDC5-mutTUFT1) and in empty vector controls (ATDC5-ctrl) at three time points during 15 days of differentiation. Hprt and Ppia were used as reference genes. Results are represented as means of three groups of quadruplet samples and relative gene expression denotes log(x+1) transformed fold changes. P-values relate to the confidence of whether the change in the relative gene expression over time is different between the ATDC5-wtTUFT1, ATDC5-mutTUFT1 and ATDC5-ctrl cell populations (S2 Table).
Mentions: Overexpression of both wt and mutant TUFT1 significantly influenced the expression of Col2a1 and Agc1 (P = 2.95x10-15 and P = 1.86x10-12, respectively, Fig 3A, S2 Table), but did not affect the expression of Sox9 (P = 0.461, Fig 3A, S2 Table): in ATDC5-mutTUFT1 cells Col2a1 expression differed from control cells on all the measurement days whereas in ATDC5-wtTUFT1 cells the difference reached statistical significance on days 8 and 15 (S3 Table). Additionally, Col2a1 expression was significantly lower in ATDC5-mutTUFT1 cells in comparison to ATDC5-wtTUFT1 cells on days 12 and 15 (P = 0.008 and P = 0.045, respectively, Fig 3A, S3 Table). Agc1 expression in both ATDC5-mutTUFT1 and ATDC5-wtTUFT1 cells deviated from the expression seen in control cells, while there was no statistically significant difference between the mutant and wt cells (S3 Table).

View Article: PubMed Central - PubMed

ABSTRACT

Osteoarthritis (OA) is the most common degenerative joint disorder and genetic factors have been shown to have a significant role in its etiology. The first metatarsophalangeal joint (MTP I) is highly susceptible to development of OA due to repetitive mechanical stress during walking. We used whole exome sequencing to study genetic defect(s) predisposing to familial early-onset bilateral MTP I OA inherited in an autosomal dominant manner. A nonsynonymous single nucleotide variant rs41310883 (c.524C>T, p.Thr175Met) in TUFT1 gene was found to co-segregate perfectly with MTP I OA. The role of TUFT1 and the relevance of the identified variant in pathogenesis of MTP I OA were further assessed using functional in vitro analyses. The variant reduced TUFT1 mRNA and tuftelin protein expression in HEK293 cells. ATDC5 cells overexpressing wild type (wt) or mutant TUFT1 were cultured in calcifying conditions and chondrogenic differentiation was found to be inhibited in both cell populations, as indicated by decreased marker gene expression when compared with the empty vector control cells. Also, the formation of cartilage nodules was diminished in both TUFT1 overexpressing ATDC5 cell populations. At the end of the culturing period the calcium content of the extracellular matrix was significantly increased in cells overexpressing mutant TUFT1 compared to cells overexpressing wt TUFT1 and control cells, while the proteoglycan content was reduced. These data imply that overexpression of TUFT1 in ATDC5 inhibits chondrogenic differentiation, and the identified variant may contribute to the pathogenesis of OA by increasing calcification and reducing amount of proteoglycans in the articular cartilage extracellular matrix thus making cartilage susceptible for degeneration and osteophyte formation.

No MeSH data available.


Related in: MedlinePlus