Tatsuya Yoshizawa

Fgf10 is essential for limb and lung formation

The interactions between fibroblast growth factors (FGF) and their receptors have important roles in mediating mesenchymal-epithelial cell interactions during embryogenesis. In particular, Fgf10 is predicted to function as a regulator of brain, lung and limb development on the basis of its spatiotemporal expression pattern in the developing embryo. To define the role of Fgf10, we generated Fgf10-deficient mice. Fgf10-/- mice died at birth due to the lack of lung development. Trachea was formed, but subsequent pulmonary branching morphogenesis was disrupted. In addition, mutant mice had complete truncation of the fore- and hindlimbs. In Fgf10–/– embryos, limb bud formation was initiated but outgrowth of the limb buds did not occur; however, formation of the clavicles was not affected. Analysis of the expression of marker genes in the mutant limb buds indicated that the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) did not form. Thus, we show here that Fgf10 serves as an essential regulator of lung and limb formation.

Suppressive function of androgen receptor in bone resorption.

As locally converted estrogen from testicular testosterone contributes to apparent androgen activity, the physiological significance of androgen receptor (AR) function in the beneficial effects of androgens on skeletal tissues has remained unclear. We show here that inactivation of AR in mice using a Cre-loxP system-mediated gene-targeting technique caused bone loss in males but not in females. Histomorphometric analyses of 8-week-old male AR knockout (ARKO) mice showed high bone turnover with increased bone resorption that resulted in reduced trabecular and cortical bone mass without affecting bone shape. Bone loss in orchidectomized male ARKO mice was only partially prevented by treatment with aromatizable testosterone. Analysis of primary osteoblasts and osteoclasts from ARKO mice revealed that AR function was required for the suppressive effects of androgens on osteoclastogenesis supporting activity of osteoblasts but not on osteoclasts. Furthermore, expression of the receptor activator of NF-κB ligand (RANKL) gene, which encodes a major osteoclastogenesis inducer, was found to be up-regulated in osteoblasts from AR-deficient mice. Our results indicate that AR function is indispensable for male-type bone formation and remodeling.

Stimulation of Osteoclast Formation by 1,25-Dihydroxyvitamin D Requires Its Binding to Vitamin D Receptor (VDR) in Osteoblastic Cells: Studies Using VDR Knockout Mice

Previous studies have shown that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays important roles in the formation of osteoclasts through its actions on osteoblastic cells. We have generated mice lacking vitamin D receptor (VDR) by gene targeting (VDR-/-). These mice had tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and exhibited similar levels of parameters for bone resorption to those in wild type mice. The present studies were undertaken to clarify whether effects of 1,25(OH)2D on osteoclast formation require VDR in osteoblasts, and to examine mechanisms of the formation of osteoclasts without VDR-mediated actions using VDR-/- mice. When wild-type calvarial osteoblasts and spleen cells were co-cultured with 1,25(OH)2D, TRAP-positive osteoclasts were formed regardless of the genotypes of spleen cells. In contrast, when osteoblasts from VDR-/- mice were co-cultured, no osteoclasts could be formed even with wild-type spleen cells. Parathyroid hormone and interleukin-1alpha stimulated osteoclast formation by co-cultures from VDR-/- mice, and the generated osteoclasts showed resorbing activity. These results demonstrate that VDR-mediated actions of 1,25(OH)2D in osteoblasts are essential for osteoclast formation by 1,25(OH)2D, and that functionally intact osteoclasts can be formed without 1,25(OH)2D actions under stimulations by other agents. It is suggested that osteoclastic bone resorption can be maintained without 1,25(OH)2D actions by other stimulatory agents.

In vivo function of VDR in gene expression-VDR knock-out mice

Vitamin D exerts many biological actions through nuclear vitamin D receptor (VDR)-mediated gene expression. The transactivation function of VDR is activated by binding 1alpha,25-dihydroxyvitamin D3[1alpha,25(OH)2D3], an active form of vitamin D. Conversion from 25(OH)D3 is finely regulated in kidney by 25(OH)D3 1alpha-hydroxylase[25(OH)D 1alpha-hydroxylase], keeping serum levels of 1alpha,25(OH)2D3 constant. Deficiency of vitamin D and mutations in the genes like VDR (type II genetic rickets) are known to cause rickets like lowered serum calcium, alopecia and impaired bone formation. However, the molecular basis of vitamin D VDR system in the vitamin D action in intact animals remained to be established. In addition, the 1alpha-hydroxylase gene from any species had not yet been cloned, irrespective of its biological significance and putative link to the type I genetic rickets. We generated VDR-deficient mice (VDR KO mice). VDR KO mice grew up normally until weaning, but after weaning they developed abnormality like the type II rickets patients. These results demonstrated indispensability of vitamin D-VDR system in mineral and bone metabolism only in post-weaning life. Using a newly developed cloning system, we cloned the cDNA encoding a novel P450 enzyme, mouse and human 1alpha-hydroxylase. The study in VDR KO mice demonstrated the function of liganded VDR in the negative feed-back regulation of 1alpha,25(OH)2D3 production. Finally, from the analysis of type I rickets patients, we found missense genetic mutations in 1alpha-hydroxylase, leading to the conclusion that this gene is responsible for the type I rickets.

Aberrant Growth Plate Development in VDR/RXRγ Double Null Mutant Mice

VDR forms heterodimers with one of three RXRs, RXRα, RXRβ, and RXRγ, and it is thought that RXR ligands can also modulate the trans-activation function of VDR/RXR heterodimers. In the present study we generated VDR/RXRγ double null mutant mice to examine the convergent actions of vitamin D and vitamin A signaling and to explore the possibility of a functionally redundant VDR. Although RXRγ−/− mice exhibited no overt abnormalities, VDR−/−/RXRγ−/− mice appeared similar to VDR−/− mice, showing features typical of vitamin D-dependent rickets type II, including growth retardation, impaired bone formation, hypocalcemia, and alopecia. However, compared to VDR−/− mice, growth plate development in VDR−/−/RXRγ−/− mutant mice was more severely impaired. Normalizing mineral ion homeostasis through dietary supplementation with high calcium and phosphorous effectively prevented rachitic abnormalities, except for disarranged growth plates in VDR−/−/RXRγ−/− mutant mice, and alopecia in both VDR−/− and VDR−/−/RXRγ−/− muta...

Molecular genetics of vitamin D receptor acting in bone

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] is involved in biological actions such as calcium homeostasis, cell proliferation, and cell differentiation, to many target tissues [1,2]. In bone formation and metabolism, in particular, 1α,25(OH)2D3 has long been believed to be a prime regulatory factor [3]. That vitamin D deficiency causes rickets, with impaired bone formation and metabolism is well documented. The effects of vitamin D on bone tissues have been intensively studied. For example, by in vitro co-culturing with osteoblasts, cytodifferentiation of osteoclasts from their precursor cells in spleen has been shown to be induced by 1α,25(OH)2D3 [4,5], indicating a significant role of 1α,25(OH)2D3 in osteoclast differentiation. Thus, despite the accumulating findings of 1α,25(OH)2D3 action on bone, its direct effect on bone tissue in intact animals is still elusive because exogenous 1α,25(OH)2D3 has an indirect influence on bone tissue through fluctuating concentrations of serum calcium. To investigate the action of 1α,25(OH)2D3 on bone tissue of intact animals, we generated mice lacking the vitamm D receptor (VDR) by targeted gene disruption and analyzed the bone phenotype in these mice.