Naohiko Hayakawa

IL-6 trans-signalling directly induces RANKL on fibroblast-like synovial cells and is involved in RANKL induction by TNF-α and IL-17

OBJECTIVES We investigated the influence of cytokines on the expression of RANK ligand (RANKL) in fibroblast-like synoviocytes from RA patients (RA-FLS). METHODS RA-FLS were stimulated by IL-6, TNF-alpha, IL-17 and IL-1beta with or without soluble IL-6 receptor (sIL-6R) for 24 h. The expression of RANKL was measured by real-time PCR, western blotting and immunostaining. In proliferation assay, RA-FLS were cultured with cytokines for 3 days. RA-FLS were co-cultured with RAW cell in the presence of IL-6/sIL-6R for 3 days and then NFATc1 mRNA expression in RAW cells was examined. RA-FLS was cultured with parthenolide [PAR, signal transducer and activator of transcription (STAT) inhibitor] or PD98059 (PD, mitogen-activated protein kinase inhibitor) in the presence of IL-6/sIL-6R and then the influence of these drugs on phosphorylation of STAT3 and ERK1/2, and RANKL expression was examined. RESULTS RANKL expression was induced by IL-6/sIL-6R (but not IL-6 alone) and by IL-1beta. On the other hand, TNF-alpha and IL-17 did not induce RANKL expression, although TNF-alpha, IL-17 or IL-1beta stimulated cell growth and IL-6 production. However, in the presence of sIL-6R, TNF-alpha or IL-17 induced RANKL expression. By the co-culture of RA-FLS, NFATc1 mRNA expression was induced in RAW cells. Finally, IL-6/sIL-6R induced phosphorylation of STAT3 and ERK1/2 in RA-FLS, and was completely inhibited by PAR and PD, respectively. PAR completely inhibited IL-6/sIL-6R-induced RANKL expression, but PD did not. CONCLUSIONS IL-6/sIL-6R directly induced RANKL expression in RA-FLS and it is essential for RANKL induction by TNF-alpha and IL-17. Moreover, RANKL induction by IL-6/sIL-6R is mediated by the janus kinase/STAT signalling pathway.

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. Youngs 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.

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.

In Vivo Dose-related Receptor Binding of the Vitamin D Analogue [3H]-1,25-dihydroxy-22-oxavitamin D3 (OCT) in Rat Parathyroid, Kidney Distal and Proximal Tubules, Duodenum, and Skin, Studied by Quantitative Receptor Autoradiography

1,25-Dihydroxy-22-oxavitamin D3 (OCT) is a new synthetic analogue of 1,25(OH)2D3 with a low calcemic effect. This study utilized quantitative receptor autoradiography to determine the dose-related receptor binding and saturation among the vitamin D target cells: parathyroid chief cells, kidney distal and proximal tubule epithelium, duodenal absorptive epithelium, and epidermal keratinocytes. Rats were injected with 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, or 16.0 μg/kg bw of [26-3H]-OCT and sacrificed 1 hr afterwards. Then autoradiographs were prepared under identical conditions. In these target cells, nuclear uptake of radioactivity increased with dose and then achieved a plateau. However, their saturation doses showed differences: parathyroid chief cells 1-2 μg; duodenal absorptive epithelium, distal tubule epithelium, and epidermal keratinocytes 4-6 μg; proximal tubule epithelium 8 μg (per kg bw). In contrast, in nontarget cells, such as liver and duodenal smooth muscle, radioactivity did not concentrate in the nuclei but increased in the cytoplasm with dose, without plateauing. These results provide the first information on the relative saturabilities of various target cell populations with a vitamin D ligand. Parathyroid chief cells required the relatively lowest receptor saturation dose. This suggests a high sensitivity and response to OCT treatment with related therapeutic potential for the regulation of parathyroid function.

Distribution of 1,25-dihydroxyvitamin D3[22-oxa] in vivo receptor binding in adult and developing skin.

Because of the therapeutic potential of oxacalcitriol (OCT, 22-oxa-dihydroxyvitamin D3), in vivo studies were conducted in adult and neonatal rats to identify the nuclear receptor sites of action in different tissues of the skin. Results were compared with those for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and oestradiol from previous studies. Autoradiograms were prepared from the dorsal skin of adult rats and the skin of the leg and head regions of neonatal rats 1 or 2 h after the injection of3H-OCT. Specific nuclear concentrations of radioactivity, eliminated by competition with unlabelled OCT or 1,25(OH)2D3, were found in cells of the epidermis, outer hair sheath, hair bulb and sebaceous glands, but were absent or low in most fibroblasts of the dermis and hypodermis. The strongest nuclear binding of OCT was conspicuous in outer hair sheaths, where it was 1.5 to 3.2 times higher than in keratinocytes of the epidermis. The distribution of nuclear receptors for OCT was similar to that for 1,25(OH)2D3 but in part dissimilar to that for oestradiol. Oestradiol binding was found in the epidermis and hair sheaths, and also predominantly in fibroblasts of the dermis and hair dermal papillae. The results suggest genomic regulatory effects of OCT, similar to the effects of vitamin D, on proliferation, differentiation and activity of keratinocytes, growth and maintenance of hair, and proliferation and secretion of sebaceous glands. This may be utilized therapeutically, since OCT has a lower calcaemic effect than 1,25(OH)2D3.

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

SummaryReceptor binding with3H-1,25(OH)2 vitamin D3 (vitamin D) and its oxygen analog3H-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 vita-min D and its analogue OCT, which is absent with3H-(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.

Skin research and drug localization with receptor microscopic autoradiography

Abstract:  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.

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.