Spontaneous Fractures in the Mouse Mutant sfx Are Caused by Deletion of the Gulonolactone Oxidase Gene, Causing Vitamin C Deficiency

Abstract

Using a mouse mutant that fractures spontaneously and dies at a very young age, we identified that a deletion of the GULO gene, which is involved in the synthesis of vitamin C, is the cause of impaired osteoblast differentiation, reduced bone formation, and development of spontaneous fractures.Introduction: A major public health problem worldwide, osteoporosis is a disease characterized by inadequate bone mass necessary for mechanical support, resulting in bone fracture. To identify the genetic basis for osteoporotic fractures, we used a mouse model that develops spontaneous fractures (sfx) at a very early age. Materials and Methods: Skeletal phenotype of the sfx phenotype was evaluated by DXA using PIXImus instrumentation and by dynamic histomorphometry. The sfx gene was identified using various molecular genetic approaches, including fine mapping and sequencing of candidate genes, whole genome microarray, and PCR amplification of candidate genes using cDNA and genomic DNA as templates. Gene expression of selected candidate genes was performed using real-time PCR analysis. Osteoblast differentiation was measured by bone marrow stromal cell nodule assay. Results: Femur and tibial BMD were reduced by 27% and 36%, respectively, in sfx mice at 5 weeks of age. Histomorphometric analyses of bones from sfx mice revealed that bone formation rate is reduced by >90% and is caused by impairment of differentiated functions of osteoblasts. The sfx gene was fine mapped to a 2 MB region containing ∼30 genes in chromosome 14. By using various molecular genetic approaches, we identified that deletion of the gulonolactone oxidase (GULO) gene, which is involved in the synthesis of ascorbic acid, is responsible for the sfx phenotype. We established that ascorbic acid deficiency caused by deletion of the GULO gene (38,146-bp region) contributes to fractures and premature death because the sfx phenotype can be corrected in vivo by treating sfx mice with ascorbic acid and because osteoblasts derived from sfx mice are only able to form mineralized nodules when treated with ascorbic acid. Treatment of bone marrow stromal cells derived from sfx/sfx mice in vitro with ascorbic acid increased expression levels of type I collagen, alkaline phosphatase, and osteocalcin several-fold. Conclusion:The sfx is a mutation of the GULO gene, which leads to ascorbic acid deficiency, impaired osteoblast cell function, and fractures in affected mice. Based on these and other findings, we propose that ascorbic acid is essential for the maintenance of differentiated functions of osteoblasts and other cell types.

Follicle-stimulating hormone stimulates TNF production from immune cells to enhance osteoblast and osteoclast formation

“…Our data provide a plausible explanation of these findings: decreased levels of ascorbic acid would elevate bone turnover causing increases in both osteoclastic and osteoblastic function, but with a net bone loss. Indeed, sfx mice deficient in an enzyme essential for the production of ascorbic acid undergo spontaneous fractures at an early age (32). Thus, we speculate that the treatment of postmenopausal osteoporosis might benefit from supplementation with ascorbic acid.…”

Nuclear Factor-E2-related Factor-1 Mediates Ascorbic Acid Induction of Osterix Expression via Interaction with Antioxidant-Responsive Element in Bone Cells

“…This article must therefore be hereby marked "advertisement" in accordance with 18vincing evidence that AA also influences differentiation of other cell type lineages, such as neuronal cells, that produce little or no matrix (16). Furthermore, the gulonolactone oxidase knock-out mice have normal levels of proline hydroxylation and collagen production in the tail, mammary gland, and tumors, whereas these mice show widespread abnormalities in multiple organs besides the bone (2,17). In addition, it has been demonstrated that AA can stimulate the expression of a number of osteogenic marker genes in the presence of collagen synthesis inhibitors and can induce chondrocyte hypertrophy independent of production of a collagen-rich matrix (18).…”

“…In addition, several epidemiological studies have shown that a lower level of blood AA, also known as vitamin C, may substantially increase the risk of hip fracture in smokers and elderly patients, whereas supplementation of antioxidant vitamins greatly reduces the frequency of osteoporotic fractures (6 -8). These genetic studies together with clinical observations have reinforced the critical role of antioxidant vitamins in maintaining bone health (2). Although it is well known that AA is required for osteoblast differentiation, the molecular mechanisms by which AA regulates bone formation and mineralization remain to be elucidated.…”

“…In order to identify candidate genes that may be important in the development of osteoporotic fractures, we previously studied a mutant mouse that develops spontaneous fracture (sfx) 2 at the very early age of 5-7 weeks (2, 3). We and others found that deletion of the gulonolactone oxidase gene, which is involved in the synthesis of ascorbic acid (AA), was responsible for AA deficiency and impairment of differentiated functions of osteoblast, bone fracture, and premature death in sfx mice (2,4). Treatment of sfx mice with AA in drinking water completely rescued the bone phenotypes in vivo and prevented them from premature death (2).…”

“…We and others found that deletion of the gulonolactone oxidase gene, which is involved in the synthesis of ascorbic acid (AA), was responsible for AA deficiency and impairment of differentiated functions of osteoblast, bone fracture, and premature death in sfx mice (2,4). Treatment of sfx mice with AA in drinking water completely rescued the bone phenotypes in vivo and prevented them from premature death (2). Consistent with our findings, a point mutation from G to A at nucleotide 182 in the L-gulono-␥-lactone oxidase gene in rats, which alters an amino acid residue from cysteine to tyrosine, has also been reported to reduce AA synthesis and impair bone formation (5).…”