Human osteoblast-like cells (HOB) produce vascular endothelial growth factor (VEGF), the steady state level of which is stimulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. As osteoblasts and endothelial cells are proximally located in skeletal tissue, we investigated the anabolic effects of 1,25-(OH)2D3 and VEGF on HOB cocultured with endothelial cells. When HOB with high alkaline phosphatase (Al-P) activity and human umbilical vein endothelial cells (HUVEC) with little activity were cultured together, Al-P activity increased, accompanied by an increase in cell number. When HOB and HUVEC were cultured separately, 1,25-(OH)2D3 did not directly stimulate [3H]thymidine incorporation into HUVEC, but stimulated it in the presence of HOB. VEGF did not directly stimulate the Al-P activity of HOB but stimulated it in the presence of HUVEC. The conditioned medium of HOB stimulated the proliferation of HUVEC, and this was partially blocked by anti-VEGF antibody. Conversely, the conditioned medium of HUVEC increased Al-P activity and [3H]thymidine incorporation into HOB, and this was partially blocked by antiinsulin-like growth factor I antibody and BQ-123, a specific antagonist of the endothelin-1 (ET-1) receptor. 1,25-(OH)2D3 stimulated the release of VEGF and ET-1 from HOB and HUVEC, respectively. Furthermore, the 1,25-(OH)2D3-induced release of VEGF was enhanced in HOB cocultured with HUVEC. A quantitative reverse transcription-PCR study revealed that genes for VEGF receptors (Flt-1 and KDR) were expressed in HUVEC, but not in HOB, and that 1,25-(OH)2D3 increased the levels of expression of VEGF receptor genes in endothelial cells only when cocultured with HOB. In summary, we demonstrated that 1,25-(OH)2D3 exerts an anabolic effect on osteoblasts by enhancing their production of VEGF, which stimulates its receptors on endothelial cells, followed by increased production of osteotropic growth factors, such as insulin-like growth factor I and ET-1. These in vitro findings suggest that the VEGF/VEGF receptor system may be involved in both bone formation and bone remodeling in vivo.

To elucidate the pathogenesis of thyroid gland hypervascu-

In order to investigate the risk factors, pathogenesis and natural course of the osteoporosis frequently seen in anorexia nervosa, we measured the bone mineral density (BMD) of the lumbar spine using dual X-ray absorptiometry in 51 Japanese female patients with anorexia nervosa, and followed the change in BMD of 29 patients for 11 to 46 months. We also evaluated the serum osteocalcin and the urinary CrossLaps, degradation products of collagen I, in 103 samples obtained from 51 patients. There was a significant correlation between the spinal BMD and the duration of emaciation below a body mass index (BMI) of 15 kg/m 2 (r ¼ ¹0.652, P < 0.0001) and 16 kg/m 2 (r ¼ ¹0.647, P < 0.0001). The increase in BMD per year in the 29 patients significantly correlated with the BMI at the time of entry of each follow-up period (r ¼ 0.712, P < 0.0001). The critical BMI for a positive increase in BMD was 16.4 Ϯ 0.3 kg/m 2 (mean Ϯ S.E.M.). The serum osteocalcin declined, while the urinary CrossLaps increased in proportion to a decrease in BMI. Both markers were normalized in patients whose BMI was between 16.4 and 18.5 kg/m 2 . The ratio of urinary CrossLaps to serum osteocalcin correlated with BMI (r ¼ ¹0.664, P < 0.0001).We conclude that the body weight history is the most important predictor of the presence of osteoporosis as well as of recovery. The BMD of patients does not increase to the normal range even several years after the recovery from this disorder, and they remain a high-risk group for osteoporosis in the future.

To elucidate the mechanism of decreased 131I uptake by the thyroid gland in patients with subacute thyroiditis and painless thyroiditis, human thyroid follicles were cultured with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and/or interferon-gamma (IFN gamma), and the effects of these cytokines on thyroid function were studied in vitro. When human thyrocytes were cultured in RPMI-1640 medium containing 0.5% fetal calf serum and TSH for 5-8 days, the cells incorporated 125I, synthesized de novo [125I]iodotyrosines and [125I]iodothyronines, and secreted [125I]T4 and [125I]T3 into the medium. IL-1 alpha and IL-1 beta inhibited 125I incorporation and [125I]iodothyronine release in a concentration-dependent manner. The minimal inhibitory effect was detected at 10 pg/ml. Electron microscopic examination revealed a marked decrease in lysosome formation in IL-1-treated thyrocytes. TNF alpha and IFN gamma also inhibited thyroid function in a concentration-dependent manner. Furthermore, when thyrocytes were cultured with IL-1, TNF alpha and IFN gamma, these cytokines more than additively inhibited thyroid function. Although the main mechanism of 131I uptake suppression in the thyroid gland in subacute thyroiditis is due to cellular damage and suppression of TSH release, our present findings suggest that IL-1, TNF alpha, and IFN gamma produced in the inflammatory process within the thyroid gland further inhibit iodine incorporation and at least partly account for the decreased 131I uptake by the thyroid gland in destruction-induced hyperthyroidism.

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Cachexia often causes deterioration in the quality of life in cancer patients; however, its mechanism remains poorly understood. Cachexia has often been observed in experimental animals with bone metastases, and parathyroid hormonerelated protein (PTHrP) plays an important role in the formation of such metastases. We therefore investigated the possible involvement of PTHrP in an experimental cachexia model using human lung-cancer cells (HARA-B Cachexia often accompanies various cancers in advanced stages and tends to deteriorate the quality of life in such patients while also affecting the survival period and/or response to chemotherapy. 1 As a result, understanding the mechanism of cancer cachexia could lead to the development of appropriate types of therapeutic intervention for these patients; however, its mechanism remains poorly understood. Cachexia is thought to be induced by soluble factors produced and released from hematopoietic cells and/or cancer cells. Several cytokines, i.e., TNF, 2,3 IL-1, 4 IFN-␥, 5 IL-6, 6,7 leukemia inhibitory factor (LIF) 8 and TGF-␤, 9 induce cachexia in different models. However, exactly how these cytokines are involved in the mechanism of cachexia induction remains to be elucidated.In the present report, we describe an experimental cachexia model using a human lung cancer-derived cell line (HARA-B), in which parathyroid hormone-related protein (PTHrP) mRNA is strongly expressed. Using this model, the involvement of PTHrP in the mechanism of cachexia induction was investigated. MATERIAL AND METHODS CellsHARA-B cells were established from a bone lesion formed after the intracardiac inoculation of HARA cells. 10 Cells were maintained in RPMI-1640 supplemented with 10% FBS at 37°C in humidified atmosphere of 5% CO 2 . Cells were grown as a monolayer culture in a flask, and a single-cell suspension of cells was obtained by trypsin treatment. MiceMale 5-week-old BALB/c nu/nu mice (Nippon CLEA, Tokyo, Japan), kept in a specific pathogen-free environment, were used. Measurement of cachexic parametersFor the in vivo experiment on cachexia, a single-cell suspension of HARA-B cells (5 ϫ 10 6 ) was inoculated s.c. into the right flank of nude mice. Body weight and tumor size [length (a) and width (b)] were thereafter measured periodically. Then, mice were killed 8 weeks after cell inoculation. At this time, blood was collected from the heart and the epididymal adipose tissue and gastrocnemius muscle were weighed. Serum samples, obtained after centrifugation, were stored at -30°C until serum glucose, calcium and PTHrP levels were determined. Serum glucose and calcium levels were measured using the enzyme-reaction method (glucose C⌸ test; Wako, Osaka, Japan) and a color-chelate reaction method (Calcium C-test, Wako), respectively. The serum PTHrP level was measured using a radioimmunoassay kit specific for the C-terminal portion of PTHrP (Daiichi, Tokyo, Japan). The serum mouse IL-6 level was measured using an ELISA kit (Endogen, Woburn, MA). Tumor weight was estimated by the formula (ab 2...

No abstract

The mechanism of bone resorption induced by interleukin 1 (IL-1) was examined in mice using three different in vitro assay systems: a fetal long bone organ culture system, a bone marrow culture system, and a coculture system of primary osteoblastic cell populations and spleen cells. In the organ culture system, recombinant human IL-1 alpha (rhIL-1 alpha) increased both bone resorption and osteoclast number. Both were partially suppressed in the presence of indomethacin. In the marrow culture, both rhIL-1 alpha and rhIL-1 beta stimulated osteoclastlike cell formation, which was completely inhibited by adding indomethacin concurrently. Furthermore, there was a good correlation between the number of osteoclastlike cells formed and the amount of prostaglandin E2 (PGE2) released into the culture media. This indicates that PGE2 is involved in the mechanism of IL-1-mediated osteoclastlike cell formation. In the coculture of primary osteoblastic cell populations and spleen cells, rhIL-1 again stimulated osteoclastlike cell formation, which was inhibited by adding indomethacin. In the cocultures in which direct interaction between osteoblastic cells and spleen cells was inhibited, PGE2 synthesis was similarly increased but no osteoclastlike cells were formed. These results indicate that IL-1 induces osteoclast formation by a mechanism involving PG (most likely PGE2). Furthermore, direct interaction between osteoclast progenitors and osteoblastic cells is required in the osteoclast recruitment induced by IL-1.