Dysregulated gene expression in the primary osteoblasts and osteocytes isolated from hypophosphatemic Hyp mice.

Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH), Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT) control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi) co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH)2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH)2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.

Vinculin functions as regulator of chondrogenesis.

To identify the genes involved in chondrocytic differentiation, we applied gene trap mutagenesis to a murine mesenchymal chondrogenic cell line ATDC5 and isolated a clone in which the gene encoding vinculin was trapped. The trapped allele was assumed to express a fusion protein containing a truncated vinculin lacking the tail domain and the geo product derived from the trap vector. The truncated vinculin was suggested to exert a dominant negative effect. Impaired functioning of vinculin caused by gene trapping in ATDC5 cells or knockdown in primary chondrocytes resulted in the reduced expression of chondrocyte-specific genes, including Col2a1, aggrecan, and Col10a1. The expression of Runx2 also was suppressed by the dysfunctional vinculin. On the other hand, the expression of Sox9, encoding a key transcription factor for chondrogenesis, was retained. Knockdown of vinculin in metatarsal organ cultures impaired the growth of the explants and reduced the expression of Col2a1 and aggrecan. Gene trapping or knockdown of vinculin decreased the phosphorylation of ERK1/2 but increased that of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) and Akt during chondrocytic differentiation, suggesting a disturbance of signaling by insulin-like growth factor I (IGF-I). Knockdown of vinculin in the metatarsal organ culture abrogated the IGF-I-induced growth and inhibited the up-regulation of Col2a1 and aggrecan expression by IGF-I. Loss of vinculin function in differentiating chondrocytes impaired the activation of the p38 MAPK pathway also, suggesting its involvement in the regulation of chondrogenesis by vinculin. Our results indicate a tissue-specific function of vinculin in cartilage whereby it controls chondrocytic differentiation.

Signaling of extracellular inorganic phosphate up-regulates cyclin D1 expression in proliferating chondrocytes via the Na+/Pi cotransporter Pit-1 and Raf/MEK/ERK pathway.

As chondrocytes mature, the concentration of inorganic phosphate (Pi) increases in the extracellular milieu. It was demonstrated that the progressive accumulation of Pi started from the proliferative zone and peaked in the hypertrophic zone of growth plate. Although extracellular Pi is reported to be involved in the apoptosis and mineralization of mature chondrocytes, its role in proliferating chondrocytes remains unclear. Here we investigated this role utilizing ATDC5, an established cell model of chondrocytic differentiation. In proliferating ATDC5 cells, we found that the expression of cyclin D1 was up-regulated, and that of alkaline phosphatase (ALP) was down-regulated in response to an increase in extracellular Pi within 24h. Moreover, an increase in extracellular Pi-induced activation of the Raf/MEK/ERK pathway, and treatment with a MEK inhibitor PD98059 abolished the effects on the expression of cyclin D1 and ALP, indicating that extracellular Pi regulates the expression of these genes through the Raf/MEK/ERK pathway. Consistent with its up-regulation of cyclin D1 expression, the extracellular Pi facilitated the proliferation of ATDC5 cells. Treatment with phosphonoformic acid (PFA), an inhibitor of sodium/phosphate (Na(+)/Pi) cotransporters, abrogated the activation of the Raf/MEK/ERK pathway and gene expression induced by the increase in extracellular Pi. Knocking down of the type III Na(+)/Pi cotransporter Pit-1 diminished the responsiveness of ATDC5 cells to the increase in extracellular Pi. Interestingly, the increased extracellular Pi induced the phosphorylation of fibroblast growth factor receptor substrate 2α (FRS2α), which was also cancelled by knocking down of the expression of Pit-1. In primary chondrocytes isolated from mouse rib cages as well, increased extracellular Pi induced the phosphorylation of ERK1/2 and alterations in the expression of cyclin D1 and ALP, both of which were abolished by treatment with PFA. These results suggest that signaling by extracellular Pi is mediated by Pit-1 and FRS2α, and leads to activation of the Raf/MEK/ERK pathway and increased expression of cyclin D1, which facilitates the proliferation of immature chondrocytes.

Involvement of nuclear factor I transcription/replication factor in the early stage of chondrocytic differentiation.

Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. To identify the genes involved in chondrocytic differentiation, we applied this method to a murine mesenchymal cell line, ATDC5, which differentiate into mature chondrocytes in the presence of insulin, and isolated a clone in which the gene encoding a transcription/replication factor, nuclear factor I-B (NFIB), was trapped. In this particular clone, named #7-57, the trap vector pPT1-geo was inserted into intron 6 of the NFIB gene in one of the alleles. As a result, both wild-type NFIB and a mutant protein lacking the carboxyl-terminal transactivation/repression domain were expressed in the clone. Immunoprecipitation/Western blotting confirmed the interaction between wild-type NFIB and the truncated protein derived from the trapped allele, suggesting that the mutant protein formed a heterodimer with wild-type NFI proteins. When cultured in the differentiation medium, #7-57 exhibited impaired nodule formation and less accumulation of cartilageous matrices compared with the parental ATDC5 cells. In addition, the expression of marker genes for proliferating chondrocytes, including type II collagen (Col2a1), matrillin-1, and PTHrP, was reduced in the clone. The expression of SOX9 was also slightly decreased in the clone #7-57 compared with the parental cells. The overexpression of wild-type NFIB in parental ATDC5 cells resulted in the increased expression of Col2a1, and a series of reporter assays using a Col2a1 promoter/enhancer-luciferase construct demonstrated the transcriptional regulation of the gene by NFIB and the dominant-negative effect of the truncated mutant derived from the trapped allele. Interestingly, mutation in the SOX9-binding site in the 48-bp cis-element located in intron 1 failed to abolish the transactivation of Col2a1 gene by NFIB, suggesting that NFI regulates the transactivation of Col2a1, at least in part, independently of SOX9. These results indicate the critical roles of NFI family transcription/replication factors in the early stage of chondrocytic differentiation.