Yumiko Nagai

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.

Regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in osteoblastic cells.

In addition to their stimulating function on osteoclastic bone resorption, bone resorptive factors may regulate proteinases and related factors in osteoblastic cells to degrade bone matrix proteins. This study investigated the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in the cultures of mouse osteoblastic MC3T3‐E1 cells, mouse primary osteoblastic (POB) cells, and neonatal mouse calvariae. Expression of either MMP‐2, ‐3, ‐9, ‐11, ‐13, and ‐14 or TIMP‐1, ‐2, and ‐3 was detected in MC3T3‐E1 cells and POB cells. When the bone resorptive factors parathyroid hormone, 1,25‐dihydroxyvitamin D3, prostaglandin E2, interleukin‐1β (IL‐1β), and tumor necrosis factor‐α (TNF‐α) were added to the cell cultures, MMP‐13 mRNA levels were found predominantly to increase by all resorptive factors in the three cultures. mRNA levels of either MMP‐3 and ‐9 or TIMP‐1 and ‐3 were found to increase mainly by the cytokines IL‐1β and TNF‐α. BB94, a nonselective MMP inhibitor, neutralized the 45Ca release stimulated by these resorptive factors to an extent similar to that of calcitonin, strongly suggesting that bone resorptive factors function at least partly through MMP formation. We propose that MMP‐13 mRNA expression in osteoblastic cells may play an important role in stimulating matrix degradation by both systemic and local resorptive factors, whereas either MMP‐3 and ‐9 or TIMP‐1 and ‐3 might modulate matrix degradation by local cytokines only. J. Cell. Physiol. 185:207–214, 2000.

Transcriptional Induction of Matrix Metalloproteinase‐13 (Collagenase‐3) by 1α,25‐Dihydroxyvitamin D3 in Mouse Osteoblastic MC3T3‐E1 Cells

The removal of unmineralized matrix from the bone surface is essential for the initiation of osteoclastic bone resorption because osteoclasts cannot attach to the unmineralized osteoid. Matrix metalloproteinases (MMPs) are known to digest bone matrix. We recently reported that among the MMPs expressed in mouse osteoblastic cells, MMP‐13 (collagenase‐3) was the one most predominantly up‐regulated by bone resorbing factors including 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3]. In this study, we examined the mechanism of regulation of MMP‐13 expression by 1α,25(OH)2D3 in mouse osteoblastic MC3T3‐E1 cells. 1α,25(OH)2D3 increased steady‐state messenger RNA (mRNA) and protein levels of MMP‐13. De novo protein synthesis was essential for the induction because cycloheximide (CHX) decreased the effect of 1α,25(OH)2D3 on the MMP‐13 mRNA level. 1α,25(OH)2D3 did not alter the decay of MMP‐13 mRNA in transcriptionally arrested MC3T3‐E1 cells; however, it increased the MMP‐13 heterogeneous nuclear RNA (hnRNA) level and MMP‐13 transcriptional rate. The binding activity of nuclear extracts to the AP‐1 binding site, but not to the Cbfa1 binding site, in the MMP‐13 promoter region was up‐regulated by 1α,25(OH)2D3, suggesting the mediation of AP‐1 in this transcriptional induction. To determine the contribution of MMPs to bone resorption by 1α,25(OH)2D3, the inhibitory effect of BB94, an MMP inhibitor, on resorbed pit formation by mouse crude osteoclastic cells was examined on either an uncoated or collagen‐coated dentine slice. BB94 did not prevent resorbed pit formation on uncoated dentine whereas it did on collagen‐coated dentine. We therefore propose that the transcriptional induction of MMP‐13 in osteoblastic cells may contribute to the degradation of unmineralized matrix on the bone surface as an early step of bone resorption by 1α,25(OH)2D3.

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...

In vivo administration of 1,25‐dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose‐dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)2D3 was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 μg/kg of 1,25(OH)2D3 inhibited the PTH‐induced RANKL mRNA expression, but 0.5 μg/kg of the vitamin did not inhibit it. Regulator of G protein signaling‐2 (RGS‐2) gene expression was suppressed by 1,25(OH)2D3 dose‐dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)2D3 suppressed PTH‐induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)2D3 stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH‐induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor‐mediated signaling. J. Cell. Biochem. 90: 267–277, 2003.

Role of Interleukin-6 in Uncoupling of Bone In Vivo in a Human Squamous Carcinoma Coproducing Parathyroid Hormone-Related Peptide and Interleukin-6

OCC tumor has been established from a human squamous carcinoma associated with humoral hypercalcemia of malignancy (HHM) and shown to overproduce parathyroid hormone‐related peptide (PTHrP) and cause aggressive hypercalcemia when implanted into nude rats. In the present study, we have demonstrated by reverse transcription‐polymerase chain reaction and Northern blot analysis that OCC tumor also overexpressed interleukin 6 (IL‐6) mRNA and that tumor‐bearing animals exhibited a marked increase in plasma IL‐6 as well as PTHrP concentrations. When a monoclonal antibody against human IL‐6 was injected to block the activities of tumor‐derived IL‐6, bone loss in tumor‐bearing animals was significantly prevented. Quantitative bone histomorphometric analysis revealed that treatment with anti–IL‐6 antibody caused a substantial decrease in both osteoclast number and eroded surface (as parameters of bone resorption) and also a significant increase in the mineral apposition rate, but little effect on the osteoblastic surface. These results provide in vivo evidence suggesting that in tumors coproducing IL‐6 and PTHrP, IL‐6 is involved not only in the acceleration of osteoclastic bone resorption but also, at least in part, in the suppression of osteoblastic functions in HHM syndrome.

Parathyroid hormone increases the expression level of matrix metalloproteinase-13 in vivo.

Abstract Parathyroid hormone (PTH) increases serum calcium (Ca) by enhancing bone resorption and renal Ca reabsorption. However, detailed mechanisms of enhanced bone resorption by PTH remain to be elucidated. Although PTH has been shown to increase the expression level of osteoblastic matrix metalloproteinase (MMP)-13 in vitro, only limited results are available regarding the in vivo regulation of MMP expression. In the present study, we have examined expression levels of MMPs in PTH-infused rats. Infusion of 1.5 or 2.0 nmol/kg/day rat PTH(1–34) for 3 days resulted in a dose-dependent increase in serum Ca. PTH infusion also decreased serum phosphate levels and increased urinary excretion of Ca and phosphate. Infusion of PTH for 7 days resulted in less severe hypercalcemia and hypophosphatemia. Urinary Ca and phosphate excretion in rats infused for 7 days was less than that in rats infused for 3 days. Northern blot analysis showed that PTH infusion increased the expression level of MMP-13 in calvaria, although it did not affect MMP-2 expression. Furthermore, the time-course and severity of hypercalcemia and hypercalciuria correlated with the expression level of MMP-13. In situ hybridization also showed that PTH infusion increased the expression level of MMP-13 in femora. These results indicate that PTH enhances MMP-13 expression in vivo and suggest that PTH stimulates bone resorption at least partly by enhancing MMP-13 expression.