Takeshi Kiriyama

Tumor necrosis factor-α and interleukin-1β increase the Fas-mediated apoptosis of human osteoblasts☆☆☆★

Abstract Our recent work demonstrated functional Fas expression on human osteoblasts, and the histologic examination of the periarticular osteoporosis region in patients with rheumatoid arthritis (RA) showed apoptosis in osteoblasts. High concentrations of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6—which are thought to increase bone resorption—have been determined in RA synovium. We investigated the effect of these cytokines on the Fas-mediated apoptosis of human osteoblasts. The human osteoblastic cell line MG63 and human primary osteoblast-like cells from bone biopsy specimens were used as human osteoblasts. Fas expression on these cells was examined by flow cytometry, and Fas-mediated apoptosis induced by anti-Fas immunoglobulin M (IgM) was determined by a chromium 51 release assay, the presence of cells with hypodiploid DNA, staining with Hoechst 33258 dye, and the detection of DNA fragmentation on agarose gel electrophoresis. The proliferation of osteoblasts was analyzed by a tritiated thymidine incorporation assay. Spontaneous apoptosis was not found on cultured osteoblasts. The apoptosis of human osteoblasts was not induced by TNF-α, IL-1β, or IL-6 alone in the absence of anti-Fas IgM. In addition, proliferation of the cells was not affected by these cytokines. Fas was constitutively expressed on unstimulated osteoblasts, and treatment of these cells with IL-1β or TNF-α significantly augmented Fas expression. Human osteoblasts were committed to apoptosis with anti-Fas IgM, and the treatment of both IL-1β and TNF-α markedly increased Fas-mediated apoptosis. TNF-α augmented both Fas expression and Fas-mediated apoptosis more efficiently than did IL-1β. In addition, an additive effect on both Fas expression and Fas-mediated apoptosis was demonstrated when TNF-α and IL-1β were added to osteoblasts. IL-6 influenced neither Fas expression nor the Fas-mediated apoptosis of osteoblasts. Furthermore, no synergistic effect of IL-6 with IL-1β or TNF-α was observed. IL-1β, TNF-α, or IL-6 did not change Bcl-2 expression. Our results suggest that IL-1β and TNF-α regulate osteoblast cell number by up-regulating the Fas-mediated apoptosis of osteoblasts, one of the putative mechanisms inducing periarticular osteoporosis in patients with RA. (J Lab Clin Med 1999;134:222-31)

Fas and Fas Ligand Interaction Is Necessary for Human Osteoblast Apoptosis

We investigated the cellular and humoral interactions between peripheral blood mononuclear cells (PBMCs) and human osteoblasts, leading to apoptosis of osteoblasts. Human osteoblastic cell line MG63 and human primary osteoblast‐like cells obtained from biopsy specimens were used in this study. PBMCs were isolated from healthy donors and cultured with or without stimulation by recombinant interleukin‐2 followed by 12‐o‐tetradecanoyl‐phorbol 13‐acetate with ionomycin. Fas was functionally expressed on MG63 and primary osteoblast‐like cells. Activated PBMCs expressed Fas ligand (FasL) strongly on their surface and killed MG63 and primary osteoblast‐like cells. Cultured supernatants of activated PBMCs also induced apoptotic cell death of MG63 and primary osteoblast‐like cells. In contrast, both unstimulated PBMCs and cultured supernatants of unstimulated PBMCs did not induce apoptosis of these cells. Furthermore, the cytotoxic effect and induction of apoptosis against MG63 and primary osteoblast‐like cells by activated PBMCs and cultured supernatants were inhibited significantly by human Fas chimeric protein. Our data showed that human osteoblasts expressed Fas fuctionally and both membrane‐type and soluble form FasL from activated PBMCs induced apoptosis of these cells, providing the one possible mechanism of bone loss in inflammatory diseases such as rheumatoid arthritis.

Alfacalcidol reduces accelerated bone turnover in elderly women with osteoporosis

To evaluate the effects of alfacalcidol on bone turnover in elderly women with osteoporosis, an open-label, prospective, calcium-controlled study was conducted. A total of 80 patients with osteoporosis were divided into two groups: the control group, group C (mean age, 78.0 years), in which patients were given calcium, and group D (mean age, 77.4 years), in which the patients were given alfacalcidol 1 µg/day together with calcium for 6 months. Calcium regulation, lumbar bone mineral density (LBMD), and markers for bone turnover were assessed. A significant increase in urinary calcium/creatinine ratio (90% increase from baseline at 3 months; P = 0.0083, and 60% at 6 months; P = 0.0091) and a significant decrease in serum parathyroid hormone (30% decrease from baseline at 6 months; P < 0.0001) was observed in group D compared with the corresponding changes in group C. Significant decreases of bone resorption markers (deoxypyridinoline and N-telopeptide) at 6 months (about 15% decrease from the baseline values) were observed in group D compared with the corresponding changes in group C. The changes in bone formation markers (bone-derived alkaline phosphatase and osteocalcin) in group D were significantly different at 6 months (−21.5%; P = 0.0047 and −13.4%; P = 0.0032, respectively) from the values in group C. The magnitudes of the decrease in bone turnover markers were highly correlated with the corresponding baseline values, suggesting that alfacalcidol treatment effectively reduces bone turnover in patients with high bone turnover rates. The LBMD in group D increased by 1.7% and that in group C decreased by 1.6% (P = 0.0384). The changes in calcium metabolism and LBMD were in good agreement with those in previous reports. Although the changes in bone turnover markers in group D were slight, significant reduction in bone turnover with alfacalcidol treatment, together with the change in calcium metabolism, may account for the effects of alfacalcidol on BMD and on fracture prevention reported previously. In conclusion, alfacalcidol reduces bone turnover in elderly women with high-bone-turnover osteoporosis, and it may have beneficial effects on bone.

Acquired nephrogenic diabetes insipidus secondary to distal renal tubular acidosis and nephrocalcinosis associated with Sjögren’s syndrome

A 52-year-old woman was referred to our hospital because of 16-year history of polyuria and polydipsia. Hyposthenuria, hyperchloremic metabolic acidosis and the inabilities to acidify the urine after acid-loading test and to concentrate the urine in responses to water-deprivation and antidiuretic hormone administration allowed us to diagnose renal tubular acidosis and nephrogenic diabetes insipidus. Radiographic examinations revealed bilateral nephrocalcinosis. The patient was also found to have clinical and laboratory findings characteristic for Sjögren’s syndrome. Thus the longstanding, poorly monitored distal renal tubular acidosis associated with Sjögren’s syndrome was considered to result in very rare renal complications-nephrocalcinosis and nephrogenic diabetes insipidus. In patients with renal tubular acidosis and/or nephrogenic diabetes insipidus of unknown etiology, therefore, Sjögren’s syndrome should be considered as one of primary disorders.

Central pressor effect of parathyroid hormone-related protein in conscious rats

Although the parathyroid hormone-related protein gene is widely expressed in the central nervous system, the role of this protein in blood pressure is unknown. This article examines whether parathyroid hormone-related protein is involved in the central regulation of blood pressure. An intraventricularly injected solution of parathyroid hormone-related protein elicited a dose-dependent increase of mean arterial pressure accompanied by a decrease of heart rate in conscious Sprague-Dawley rats. An anti-parathyroid hormone-related protein monoclonal antibody, given in an intraventricularly injected solution, blocked the pressor effect of parathyroid hormone-related protein. Furthermore, this pressor effect of parathyroid hormone-related protein was also abolished after pretreatment by intravenous administration of either hexamethonium bromide or doxazosin mesylate. These results suggest that central parathyroid hormone-related protein is implicated in the regulation of blood pressure, and that this effect may be mediated through sympathetic activation.

Parathyroid hormone-related protein (PTHrP) and blood pressure

Parathyroid hormone-related protein (PTHrP) is one of the main factors from tumors causing malignancy associated hypercalcemia. It is also expressed in various kinds of normal tissues including vascular smooth muscle systems. Colocalization of the PTH/PTHrP receptor and locally produced PTHrP and its vasorelaxant activities suggest that PTHrP has a regulatory role in the modulation of blood pressure (BP). There have been many evidences that indicate the importance of circulating PTH and PTH/PTHrP receptors in the regulation of BP in various conditions. These data suggest that, under normal physiological conditions, locally produced PTHrP is a real factor that regulates these smooth musele systems in a paracrine or autocrine fashion. The central nervous system also has a very important role to regulate the BP. The evidence that PTHrP is expressed in the central nervous system and the presence of the PTHrP immunoreactivity in human cerebrospinal fluid lead to the possibility that PTHrP has some role in the central regulation of BP. We have found that intraventricularly administered PTHrP had a pressor effect on systemic BP which support the previous possibility.

Subnormal secretion of parathyroid hormone prevents age-related bone loss

Parathyroid hormone (PTH) is a key factor involved in the systemic regulation of bone resorption. It is well known that a high turnover of bone occurred together with the reduced bone mass in patients with hyperparathyroidism. However, the effect of subnormal secretion of PTH on age-related bone loss has not been extensively investigated. Recently, some investigators and us have focused on the effect of suppressed PTH secretion and have demonstrated that patients with subnormal secretion of PTH preserved a relatively higher bone mineral densities than age- and sex-matched controls. We believe that these results will give a new insight into the mechanism of age-related bone loss or osteoporosis. Further studies are needed to evaluate the mechanisms of this protective effect of suppressed PTH secretion on bone mass.