1α,25(OH)2D3 downregulates gene expression levels of muscle ubiquitin ligases MAFbx and MuRF1 in human myotubes.

Clinical trials involving in patients with osteoporosis have reported that activated vitamin D3 (1α,25(OH)2D3, calcitriol) can prevent falling by acting on the skeletal muscles. However, pharmacological mechanisms of 1α,25(OH)2D3 with respect to skeletal muscle hypertrophy or atrophy are still poorly understood. Therefore, we examined changes in the expression of several related genes in human myotubes to test whether 1α,25(OH)2D3 influences hypertrophy and atrophy of skeletal muscle. Myotubes treated with 1α,25(OH)2D3 increased interleukin-6 (IL-6) expression and inhibited expression of tumor necrosis factor alpha (TNF-α), whereas the expression of insulin-like growth factor-1 (IGF-1) that is involved in muscle hypertrophy was not affected. However, 1α,25(OH)2D3 treatment significantly inhibited the expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), ubiquitin ligases involved in muscle atrophy. The analysis of pathways using microarray data suggested that 1α,25(OH)2D3 upregulates AKT-1 by inhibiting the expression of protein phosphatase 2 (PP2A), a phosphatase acting on AKT-1, in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, thereby inhibiting the expression of ubiquitin ligases. Thus, this study showed that 1α,25(OH)2D3 might have an inhibitory effect on the expression of MAFbx and MuRF1 in skeletal muscle and a suppressive effect on muscle degradation in patients with osteoporosis.

Effects of alfacalcidol on mechanical properties and collagen cross-links of the femoral diaphysis in glucocorticoid-treated rats.

Bone fragility is increased in glucocorticoid (GC)-induced osteopenia even though GC-treated patients have higher bone mineral density (BMD), suggesting that the impaired bone quality may affect bone strength. This study was conducted to clarify the effects of GC on bone strength and collagen cross-links of adult rats and the effect of coadministration of alfacalcidol (ALF), a prodrug of active vitamin D(3). Six-month-old male Wistar-Imamichi rats (n = 32) were divided into the following four groups with equal average body weight: (1) 4-week age-matched controls, (2) 4-week GC (prednisolone, 10 mg/kg daily, i.m.) with concomitant administration of vehicle, (3) 4-week GC with concomitant administration of ALF (0.05 µg/kg daily, p.o.), and (4) 4-week GC with concomitant administration of ALF (0.1 µg/kg daily, p.o.). At the end of treatment, BMD, collagen cross-links, mechanical properties of the femoral midshaft, bone metabolic markers, and biochemical parameters were analyzed. In the GC group, femoral bone strength decreased without any change of BMD. This was accompanied by a decrease in the content of enzymatic cross-links. ALF (0.1 µg/kg) inhibited the GC-induced reduction in bone strength. The content of mature cross-links in the 0.1-µg/kg ALF group was significantly higher than that in the GC group. GC treatment caused a decrease in bone metabolic markers and serum calcium levels, which was counteracted by ALF coadministration. Preventive treatment with ALF inhibited the deterioration of bone mechanical properties primarily in association with the restoration of enzymatic cross-link formation and amelioration of the adverse effects of GC treatment on calcium metabolism.

Comparison of effects of alfacalcidol and alendronate on mechanical properties and bone collagen cross-links of callus in the fracture repair rat model.

Both bone density and quality are important determinants of bone strength. Bone quality is prescribed by matrix characteristic including collagen cross-linking and bone structural characteristics and is important in reinforcement of bone strength. We investigated the effects of alfacalcidol (ALF), a prodrug of calcitriol, and alendronate (ALN), a bisphosphanate, on the mechanical properties and content of enzymatic cross-links in femoral bone using a fracture repair rat model. Forty 3-month-old female Wistar-Imamichi rats were randomized into 4 groups: SHAM (sham-operated+vehicle), OVX (ovariectomy+vehicle), ALF (ovariectomy+ALF, 0.1 microg/kg/d, p.o.) and ALN (ovariectomy+ALN, 10 microg/kg/d, s.c.). Treatment began immediately after SHAM or OVX surgery. Three weeks later, all animals underwent transverse osteotomies at the midshaft of the left femur. Treatment was continued and rats were sacrificed at 12 weeks post-fracture for evaluation by X-ray radiography, micro-CT, pQCT, biomechanical testing and bone histomorphometry. In the ALN group, no new cortical shell appeared and the callus diameter was significantly larger than in the OVX group (p<0.05). Stiffness of fractured callus in the ALF group, but not in the ALN group, was significantly higher than in the OVX group. Young's modulus in the ALN group was significantly decreased compared to the OVX group. Moreover, micro-CT analysis showed that ALN treatment increased the lowly mineralized bone in the callus by, resulting in the highest content of woven bone area and lowest content of lamellar bone. The total amount of enzymatic cross-links in both the ALF and ALN groups was significantly higher than in the OVX control group. Of particular interest, the Pyr-to-Dpyr ratio was significantly decreased by ALF administration, suggesting that ALF but not ALN normalized the enzymatic cross-link patterns in fractured bone to the control level. In conclusion, ALN and ALF treatment increased bone strength via the distinctive effect on bone mass and quality. ALN formed larger calluses and increased enzymatic cross-links despite delayed woven bone remodeling into lamellar bone, whereas ALF treatment induced lamellar bone formation coincided with increasing in the enzymatic cross-linking and normalizing the cross-link pattern in callus to native bone pattern.

IL-6/sIL-6R trans-signalling, but not TNF-alpha induced angiogenesis in a HUVEC and synovial cell co-culture system.

Angiogenesis in synovia is a characteristic of RA patients. We examined whether IL-6 or TNF-alpha induce tubule formation in a co-culture system of fibroblast-like synovial cells from RA patients (RA-FLS) and human umbilical vein endothelial cells (HUVEC). The effects of IL-6 and TNF-alpha on the expression of angiogenic factors in RA-FLS and HUVEC, and the proliferation of HUVEC were also studied. IL-6 + sIL-6R induced tubule formation, whereas IL-6 alone did not. IL-6/sIL-6R-induced tubule formation was completely suppressed by the addition of either anti-IL-6R or anti-VEGF antibody. TNF-alpha did not induce tubule formation. On the contrary, it decreased CD31-positive area compared with the control. IL-6 + sIL-6R augmented VEGF production in RA-FLS, whereas IL-6 alone did not. Anti-IL-6R antibody suppressed IL-6/sIL-6R-induced VEGF production, but not spontaneous VEGF production. In contrast, TNF-alpha did not induce VEGF production from RA-FLS and HUVEC. IL-6 + sIL-6R stimulation of RA-FLS strongly induced mRNA expression of VEGF, but not of other angiogenic factors, such as EGF, bFGF, TGF-beta, IL-1, TNF-alpha and IL-8. Neither IL-6 nor IL-6/sIL-6R promoted HUVEC proliferation, whereas TNF-alpha significantly inhibited VEGF-induced HUVEC proliferation. In conclusion, IL-6/sIL-6R complex showed angiogenic activity via the production of VEGF from RA-FLS, but TNF-alpha was anti-angiogenic in our experimental system.

Anti-IL-6 receptor antibody increases blood IL-6 level via the blockade of IL-6 clearance, but not via the induction of IL-6 production.

We explored the mechanism for the increase of blood IL-6 level after anti-IL-6 receptor (IL-6R) antibody injection. First, we examined whether anti-IL-6R antibody stimulates IL-6 production. Single injection of tocilizumab (anti-IL-6R antibody) in monkeys with collagen-induced arthritis (CIA) caused a marked increase in blood IL-6 and IL-6R levels, but did not increase IL-6 mRNA and IL-6R mRNA expression in liver, spleen, lymph nodes, synovium or whole blood 1, 3 and 7 days later. This suggests that tocilizumab did not induce IL-6 and IL-6R production. Second, we investigated whether anti-IL-6R antibody releases IL-6 from IL-6 complexes in the blood. When plasma from CIA monkeys was incubated with tocilizumab, the IL-6 concentration was not affected. Finally, we studied whether anti-IL-6R antibody affects the clearance of IL-6 from the blood. When MR16-1 (anti-mouse IL-6R antibody) was injected into IL-6-deficient mice continuously infused with human IL-6, blood human IL-6 levels significantly increased. These results suggest that the elevation of blood IL-6 after the administration of anti-IL-6R antibody is the result of inhibition of the clearance of IL-6 due to IL-6R blockade, and that it is not the result of induction of IL-6 production or release of IL-6 from complexes.

Skin research and drug localization with receptor microscopic autoradiography.

For the localization of drugs and related functional characterization, cellular-subcellular resolution can be achieved with radiolabelled compounds of high specific activity and receptor microscopic autoradiography, which is the method of choice for high-resolution qualitative and quantitative imaging. Detailed information together with integrative surveys can be obtained which is impossible with other methods. The history of discoveries of drug targets testifies to the utility and potential of receptor microscopic autoradiography that was designed to preserve in vivo conditions by excluding any liquid treatment during tissue preparation and to avoid translocation and loss of diffusible compounds. Examples of autoradiograms include in vivo applications of 3H-vitamin D, 3H-OCT (oxacalcitriol), 3H-oestradiol and 3H-retinoic acid. For topical applications, routes of delivery and sites of deposition and retention are demonstrated.

Salivary glands epithelial and myoepithelial cells are major vitamin D targets.

Receptor binding with 3H-1,25(OH)2 vitamin D3 (vitamin D) and its oxygen analog 3H-OCT is demonstrated in rat, hamster, and mice submandibular, sublingual and parotid glands, using receptor microautoradiography high-resolution imaging. Nuclear uptake and retention of radiolabeled compound exist strongest in epithelial cells of striated ducts, granular convoluted tubules and in myoepithelial cells throughout, scattered in epithelial cells of intercalated ducts and relatively low in cells of serous and mucous acini. Deposition and retention of radiolabeled compound is also observed in interstitial spaces. The specific nuclear localization with vitamin D and its analogue OCT, which is absent with 3H-(OH) vitamin D3 and in competition with excess non-radioactive vitamin D, indicates involvement of vitamin D in the multi-hormonal regulation of salivary gland secretion, excretion, and cell proliferation. These data--together with previously recognized similar receptor binding in esophagus, gastric glands, entero-endocrine cells, pyloric muscle, and generative and absorptive epithelium of the small intestine and colon, point to the importance of vitamin D for the digestive system regulation of functions and maintenance with related therapeutic potentials.

Binding of highly concentrated maxacalcitol to the nuclear vitamin D receptors of parathyroid cells.

BACKGROUND: Injection of maxacalcitol (OCT) directly into the parathyroid gland (PTG) is a clinically safe and effective treatment for advanced secondary hyperparathyroidism (A-SHPT) resistant to conventional medical treatment. In the present study, the degree of nuclear localization of directly injected OCT in parathyroid cells (PTC) was investigated by microautoradiography (mARG) in a model of A-SHPT. METHODS: The 5/6 nephrectomized Sprague-Dawley rats were fed a high-phosphate and low-calcium diet for 8 weeks and consequently the level of vitamin D receptor (VDR) in their PTC severely decreased. The bilateral PTG were surgically exposed and only the left gland were directly injected with 3H-OCT (DI-3H-OCT). The time course of the changes in both radioactivity and localization of 3H-OCT in the bilateral glands was analysed using a bioimaging analyser system and mARG, respectively. A very high dose of unlabelled calcitriol was administered intravenously (IV-1,25D3) prior to DI-3H-OCT, as a competitive study. RESULTS: Peak radioactivity levels in the directly injected and intact PTG occured immediately and 1 h, respectively, after DI-3H-OCT, and the difference was about 50-fold higher in the treated gland. The of mARG showed a marked concentration of silver grains in the nuclei of PTC in the gland treated with DI-3H-OCT and that concentration was significantly suppressed by IV-1,25D3. CONCLUSIONS: Direct injection of OCT into the PTG enables the administration of the highly concentrated drug for specific binding to nuclear vitamin D binding sites, including VDR of PTC, which markedly suppresses the parathyroid hormone, improves the response to calcium and vitamin D and induces apoptosis in PTC.

Receptor microscopic autoradiography for the study of percutaneous absorption, in vivo skin penetration, and cellular-intercellular deposition.

INTRODUCTION: Microscopic autoradiography with cellular resolution and preservation of in vivo conditions is potentially the method of choice to gain detailed information about sites of deposition and retention in the epidermis and of penetration to the dermis after topical application of drugs. We tested this using (3)H-Maxacalcitol. METHODS: Dorsal skin of adult rats was treated in vivo with ointment containing 1 or 40 microg/kg body weight of the vitamin D analogue (3)H-Maxacalcitol for periods of 0.5, 2, 8, 24, 48, or 168 h. Samples of skin exposed to the ointment and control samples remote from the treatment site were excised and freeze-mounted, and 4-microm frozen sections were exposed to nuclear emulsion. RESULTS: Two penetration routes to the dermis could be distinguished: one via epidermal cell layers and the other via hair follicles. Highest uptake and retention of radiolabeled steroid was observed in stratum corneum and in intercellular spaces of stratum granulosum. By contrast, cell boundaries and intercellular spaces in the stratum spinosum and basale contained low levels of radioactivity. Keratinocytes in these layers showed high concentration in the cytoplasm at early time intervals, when surrounding radioactivity levels were high, but high nuclear and low or no cytoplasmic concentration at late time intervals, when surrounding radioactivity levels were low. DISCUSSION: The autoradiographic method provides detailed information on time- and dose-related distribution of radiolabeled compound at the cellular level that is not obtainable with common radioassays and biochemical procedures. A sustained concentration and retention of radiolabeled steroid in the stratum corneum and intercellular space of the stratum granulosum indicate a selective deposition in components of secreted-membrane-coating granules and suggest a temporary barrier and depot for slow release. The differential cytoplasmic-nuclear distribution in the stratum Malpighi suggests functional correlation to a toxic-hormetic reversal of action on cell proliferation, from high-dose inhibitory effects associated with high extranuclear concentration as utilized in the treatment of psoriasis, to low-dose stimulatory effects associated with high nuclear and low cytoplasmic concentration as applicable in wound healing.

In vivo time-course of receptor binding in the parathyroid gland of the vitamin D analogue [(3)H]1,25-dihydroxy-22-oxavitamin D(3) compared with [(3)H]1,25-dihydroxyvitamin D(3), determined by micro-autoradiography.

1,25-Dihydroxy-22-oxavitamin D(3) (22-oxacalcitriol, OCT), is a new synthetic analogue of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3), calcitriol), to be used in the treatment of secondary hyperparathyroidism. This study used receptor micro-autoradiography in the parathyroid gland to determine and compare the time-course of receptor binding between OCT and 1,25(OH)(2)D(3). Mice were injected with 4 microg/kg of [26-(3)H]OCT or [26,27-methyl-(3)H]1,25(OH)(2)D(3), and killed at 5, 15, 30 min, 1, 2, 4, 8, 12, and 24 h afterwards. Thyroid-parathyroid tissue was excised and autoradiograms were prepared. Under identical conditions of dose and adjusted specific radioactivity between [(3)H]OCT and [(3)H]1,25(OH)(2)D(3), the plasma concentration of [(3)H]OCT was much lower than that of [(3)H]1,25(OH)(2)D(3). In the parathyroid at all time points, chief cell nuclei were labelled with varying degrees while connective tissue cells remained unlabelled. Nuclear receptor binding of [(3)H]OCT appeared equal to or higher than that of [(3)H]1,25(OH)(2)D(3). Nuclear uptake of [(3)H]OCT was maximal at 15 min and higher than that of [(3)H]1,25(OH)(2)D(3), which was maximal at 1 h after injection. Low levels of nuclear retention of the two compounds were still similarly detectable at 12 h. The results indicate the high affinity of OCT to parathyroid cells, and suggest that OCT has a higher therapeutic potential than 1,25(OH)(2)D(3), especially under clinical conditions, at which OCT with its lower calcaemic effect would allow treatment with a dose several times higher than 1,25(OH)(2)D(3).