Kaoru Fujiyama

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.

Molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor transcript.

In the present study, we report the molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor (hTSHR) mRNA transcript, which has previously been detected on Northern blot analysis of human thyroid cells. The smaller hTSHR cDNA, designated hTSHR cDNA-I, is approximately 1 kb in size and encodes a protein of 253 amino acids. Comparison of the nucleotide sequence of hTSHR cDNA-I with available hTSHR genomic sequence data reveals that the cDNA-I contains exons 1-8 and unidentified DNA tract, presumably an intron. Thus, the hTSHR cDNA-I encodes for the N-terminal half of the extracellular domain of the hTSHR (approximately 60%). The truncated TSHR-I may be secreted and function as a TSH binding protein.

Impairment of the TSH signal transduction system in human thyroid carcinoma cells

In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.

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.

Two elderly cases of familial mediterranean fever with rheumatoid arthritis

Familial Mediterranean fever (FMF) is a genetic autoinflammatory disorder that usually develops before 20 years of age and is characterized by periodic fever with serositis and arthritis. Both FMF and rheumatoid arthritis (RA) involve arthritis; however, their coexistence is rare. We describe two RA patients with an MEFV mutation in exon 2, who were diagnosed with FMF at an age of over 50 years. We also discuss the possibility that MEFV mutations could modulate RA disease activity.

Increased Fas-mediated cytotoxicity of CD4-positive T cells in patients with human T-lymphotropic virus type I-associated myelopathy

Using a 51Cr release assay, we investigated Fas-mediated cytotoxicity of peripheral blood CD4+ T cells of patients with human T-lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) against T98G, a glioblastoma cell line which expresses Fas. Cytotoxic activity of CD4+ T cells against T98G was significantly higher in HAM patients than in controls. Moreover, when CD4+ T cells of HAM patients were preincubated with a monoclonal antibody to human Fas ligand (FasL), cytotoxic activity against T98G was significantly suppressed. These results suggest that damage to nervous tissues by the Fas/FasL system is involved in the pathogenesis of HAM.

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.