Keizo Kasono

Stimulation of prostaglandin E2 and bone resorption by recombinant human interleukin 1 alpha in fetal mouse bones

Recombinant human interleukin 1 alpha (rhIL-1 alpha) stimulates prostaglandin E2 and bone resorption in cultured forearm bones of fetal mouse in a dose-dependent manner: the minimal rhIL-1 alpha to elicit a significant bone resorption was 1.6 ng/ml (89 pM). The half maximal concentrations to elicit bone resorption and thymocyte proliferation were 3.3 ng/ml (183 pM) and 0.31 ng/ml (17 pM), respectively. The bone resorbing activity induced by IL-1 was partially inhibited by indomethacin and hydrocortisone, and completely inhibited by anti-IL 1-antibody. There was a good correlation between PGE2 production and bone resorption induced by IL-1 alpha. These results suggest that rhIL-1 alpha stimulates bone resorption at approximately 10 times the concentrations necessary for thymocyte proliferation and that PGE2 produced in the bone is at least in part involved in osteoclastic bone resorption.

Recombinant human interleukin 1 alpha and beta stimulate mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

The effects of interleukin 1 (IL-1) on MC3T3-E1 cells (clonal osteoblast-like cells established from mouse calvaria) were studied to elucidate the mechanism of IL-1-induced bone resorption. Recombinant human interleukin 1 alpha (rhIL-1 alpha) and beta (rhIL-1 beta) stimulated PGE2 production in MC3T3-E1 cells in a dose dependent manner. rhIL-1 alpha and 1 beta also stimulated MC3T3-E1 cells to produce macrophage-colony stimulating activity (M-CSA) in a dose-dependent manner. Indomethacin completely abolished PGE2 production but did not affect CSA. These results suggest that bone resorption induced by IL-1s is at least in part mediated by PGE2 produced by osteoblasts, and that M-CSA produced by osteoblasts may synergistically potentiate bone resorption by recruiting osteoclast precursors.

Tumor necrosis factor type α (cachectin) stimulates mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

Abstract To elucidate the mechanism of tumor necrosis factor alpha (TNF-α)-induced bone resorption, the effects of recombinant human TNF-α on mouse osteoblast-like cells (MC3T3-E1) were studied. TNF-α stimulated MC3T3-E1 cells to produce prostaglandin E2 (PGE2) and macrophage colony stimulating activity (M-CSA) in a dose-dependent manner. TNF decreased alkaline phosphatase (AL-P) activity of MC3T3-E1 cells. These TNF effects were observed at 1 ng/ml (∼6×10−11M). The inhibitory effect on AL-P activity was reversible and the cell growth of MC3T3-E1 cells was only slightly affected by TNF. These findings suggest that both PGE2 and M-CSA stimulated by TNF-α are possibly involved in osteoblast-mediated osteoclastic bone resorption, whereas inhibition of AL-P activity may lead to a decrease in bone formation.

Spermine, a Natural Polyamine, Suppresses LFA-1 Expression on Human Lymphocyte

Natural polyamines, spermine, spermidine, and putrescine, play a pivotal role in the regulation of gene expression; therefore, the age-dependent decreases and the disease-dependent increases in polyamine synthesis suggest a possible contribution of polyamines to the age-related and disease-associated changes in cellular function. In this study, we examined the effects of polyamines on the cellular function and the expression of adhesion molecules on human PBMCs from healthy volunteers. Flow cytometry revealed that PBMCs cultured with spermine decreased mean fluorescent intensities (MFIs) of CD11a and CD18 in the lymphocyte light-scattered region, but not in the monocyte region. This suppression was observed in a dose- and time-dependent manner and found nonspecifically on all cell subsets we tested (CD3+, CD4+, CD8+, CD19+, CD45RA+, CD45RO+, CD4+CD45RA+, CD4+CD45RO+, CD8+CD45RA+, CD8+CD45RO+). The decreases of CD11a and CD18 MFIs were accompanied by the decrease in adherent capacity of PBMCs to HUVECs. Spermine did not hinder cell activities or cell viability. Among 42 healthy volunteers (mean, 49.5 years old; from 26 to 69), blood spermine levels inversely correlated with the CD11a MFIs of cells in the lymphocyte region (r = −0.48; p = 0.001), but not with those in the monocyte region. The effects of spermidine seemed weaker than those of spermine, and blood spermidine levels had no correlation with CD11a MFIs of the lymphocyte region. Putrescine had no effect on the expressions of membrane molecules. Polyamines, especially spermine, decrease LFA-1 (CD11a/CD18) expression on human lymphocyte and adhesion capacity of PBMCs to HUVECs.

Inhibitory effect of interleukin-4 on osteoclast-like cell formation in mouse bone marrow culture.

Recently, interleukin 4 (IL-4) was reported to inhibit bone resorption in mouse long bone culture. To test the effect of IL-4 on the formation of osteoclast-like cells, we used a mouse bone marrow culture system that formed mononuclear and multinucleated cells with osteoclast characteristics. Recombinant mouse IL-4 dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells [TRAP(+)MNC] induced by 1,25(OH)2D3, PTH(1-34) or Interleukin-1 alpha (IL-1 alpha). The minimum effective inhibitory concentration was 0.01 ng/ml, and 1 ng/ml IL-4 completely inhibited TRAP(+)MNC formation. IL-4 also dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive mononuclear cells. Treatment of cultures with IL-4 for the first or last 48 h of an 8-day culture period inhibited TRAP(+)MNC formation to the same extent, whereas IFN-gamma and calcitonin suppressed TRAP(+)MNC formation mainly at the early and the late phase, respectively. IL-4, as a macrophage fusion factor, at higher concentrations (0.1-10 ng/ml), increased formation of tartrate-resistant acid phosphatase-negative multinucleated cells [TRAP(-)MNC] with giant macrophage characteristics. The half-maximal concentrations inhibiting TRAP(+)MNC formation and stimulating TRAP(-)MNC formation were 0.05 ng/ml and 2 ng/ml, respectively. These results demonstrate that IL-4 inhibits bone resorption by inhibiting the recruitment of osteoclast precursor and formation of multinucleated osteoclast-like cells, and by stimulating the formation of macrophage polykaryons.

Effects of Glucocorticoids and Calcitonin on Parathyroid Hormone‐related Protein (PTHrP) Gene Expression and PTHrP Release in Human Cancer Cells Causing Humoral Hypercalcemia

Glucocorticoids are widely used for the treatment of malignancy‐associated hypercalcemia to delay the occurrence of an escape phenomenon inherent in calcitonin therapy. Using parathyroid hormone‐related protein (PTHrP)‐producing squamous carcinoma cells (T3M‐1 and EC‐GI) established in our laboratory, we investigated the in vitro effects of glucocorticoids and calcitonin on PTHrP mRNA expression in the cells and release of PTHrP into the culture medium. The PTHrP gene was constitutively expressed in the logarithmic growth phase in both squamous carcinoma cell lines. When these cells became superconfluent, PTHrP mRNA expression was greatly diminished in T3M‐1 cells but was not distinctly diminished in EC‐GI cells. Hydrocortisone inhibited the PTHrP mRNA expression in T3M‐1 cells and EC‐GI cells in a dose‐dependent manner. In accordance with the decreased expression of PTHrP mRNA, the release of immunoreactive as well as bioactive PTHrP also decreased in the conditioned medium of glucocorticoid‐treated cells. The minimal effective concentration of prednisolone was about 10 ‐7 M, which is readily attainable in the serum of patients treated with the agent. Calcitonin and indotnethacin did not affect the PTHrP mRNA expression or PTHrP release into the medium. Calcitonin did not modulate the hydrocortisone‐induced inhibition of PTHrP production. These in vitro findings suggest that the combined use of glucocorticoids and calcitonin plays a beneficial role in the treatment of malignancy‐associated hypercalcemia, since the steroid hormone can suppress PTHrP mRNA expression and release of bioactive PTHrP in certain PTHrP‐producing tumors.

Hypotonicity reduces the activity of murine aquaporin-2 promoter induced by dibutyryl cAMP

The present study was undertaken to determine whether hypotonicity regulates the aquaporin‐2 (AQP‐2) gene in vitro. The 5′‐flanking region of the AQP‐2 gene contains the tonicity‐response enhancer (TonE) promoter located between −570 and −560 bp, and another distinct hypertonicity‐responsive region between −6.1 and −4.3 kb of the AQP‐2 gene. The 5′‐flanking region of murine AQP‐2 gene up to −9.5 kb was cloned into a luciferase (Luc) reporter plasmid. The constructs, which have TonE and/or the hypertonicity‐responsive region, together with the murine AQP‐2 gene, were co‐transfected into murine IMCD3 cells. When the cells were co‐transfected with the construct containing more than 1.1 kb of the 5′‐flanking region of murine AQP‐2 gene (–9.5AQP2, −6.1AQP2 and −1.1AQP2) and the AQP‐2 gene, 24 h exposure to 5 μmol l−1 dibutyryl cAMP (DBcAMP) significantly increased the Luc activity by 2.3‐fold in the isotonic medium (300 mosmol kg−1). In the hypotonic medium (225 mosmol kg−1), basal activity was not altered, and the response of Luc activity to 24 h exposure to 5 μmol l−1DBcAMP was abolished. Similar findings were obtained in isosmotic, urea‐supplemented medium (estimated tonicity, 225 mosmol kg−1). The response of Luc activity to 5 μmol l−1 DBcAMP in the hypotonic medium was not affected in cells either transfected with 0.36 kb of the 5′‐flanking region of AQP‐2 or co‐transfected with −1.1AQP2 and a dominant‐negative TonE binding protein (pDNTonEBP). Pre‐incubation of cells with 1 μmol l−1 SP600125, an inhibitor of c‐Jun N‐terminal kinase (JNK), restored the response of Luc activity to 5 μmol l−1 DBcAMP under hypotonic conditions. These findings may indicate that hypotonicity reduces the cAMP‐induced AQP‐2 promoter activity mediated via TonE by activating JNK kinase.

Pathological Role of Aquaporin-2 in Impaired Water Excretion and Hyponatremia

In the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), inappropriately elevated secretion of vasopressin can result in a reduction of antidiuretic efficacy: a phenomenon known as ‘vasopressin escape’. We compared experimental SIADH with 1‐deamino‐8‐d‐arginine vasopressin (dDAVP)‐excess rats, where both groups received continuous subcutaneous administration of dDAVP by osmotic minipump but the SIADH rats also received a liquid diet that induced hyponatraemia. The SIADH rats, but not the dDAVP excess rats, showed a marked attenuation of urinary concentrating ability. Vasopressin V2 receptor binding capacity and mRNA expression were similar between the two groups, but the SIADH rats showed a diminished up‐regulation of aquaporin‐2 (AQP‐2) mRNA and protein expression. These findings indicate the presence of tonicity‐response regions in the AQP‐2 promoter gene, and that either hypervolemia or hypotonicity may attenuate the postreceptor signalling of vasopressin in renal collecting duct cells in SIADH rats.

Prolonged and ubiquitous inhibition by interferon γ of bone resorption induced by parathyroid hormone-related protein, 1,25-dihydroxyvitamin D3, and interleukin 1 in fetal mouse forearm bones.

SummaryTo investigate the mechanism of the inhibitory effects of interferon-γ (IFN-γ) on bone resorption, the effects of murine IFN-γ on45Ca release from prelabeled fetal mouse forearm bones were investigated. Murine IFN-γ usually did not affect basal45Ca release but almost completely and equipotently inhibited bone resorption induced by PTH(1-34), PTH-rP(1-34), 1,25(OH)2D3, and interleukin 1 (IL-1). The half-maximal concentration for inhibition of bone resorption induced by IL-1α was 25.8±14.6 U/ml (mean±SD for 13 experiments), which is not different from those for PTH, PTH-rP, and 1,25(OH)2D3. There was no correlation between prostaglandin E2 concentration in the conditioned medium and45Ca release from the IFN-γ-treated forearm bones. The inhibitory effect of IFN-γ on bone resorption induced by PTH-rP (1-34) or IL-1α continued during 6 days of culture, whereas that of calcitonin disappeared after 2 days of culture. These findings suggest that IFN-γ nonpreferentially inhibits bone resorption induced by various bone-resorbing factors in fetal mouse forearm bones via a PGE2-independent mechanism. As no escape phenomenon developed in IFN-γ-treated bones, the cytokine may be potentially useful for treatment of certain patients with malignancy-associated hypercalcemia.

Thiazolidinediones increase the number of platelets in immune thrombocytopenic purpura mice via inhibition of phagocytic activity of the reticulo-endothelial system

Thiazolidinediones (TZDs) have broad spectrum of actions, including immunomodulating effects that are dependent or independent of the target nuclear receptor, peroxisome proliferator activated receptor-gamma (PPAR-gamma). In this study, we investigated the effect of TZDs on the platelet numbers in male immune thrombocytopenic purpura (ITP) model mice, (NZW x BXSB)F(1) (W/BF(1)) in vivo, and attempted to clarify the mechanism of action. Seven-day treatment with troglitazone increased platelet counts by 66% compared with those of controls. Within two weeks after the termination of the treatment period, the numbers of platelets were decreased to the level in controls. Pioglitazone showed only weak increasing effect on platelet counts in short-term experiment. However, long-term treatment with the drug resulted in a more pronounced up-regulation of platelets. We next assayed the platelet-associated antibodies (PAA) and the survival rate of antibody-sensitized mouse erythrocytes (Ab-mRBC) in W/BF1 mice. Pioglitazone slightly decreased the production of PAA and significantly elongated the survival period of Ab-mRBC in vivo. These drugs showed dose-dependent inhibitory effects on the cell proliferation and Fcgamma receptor (FcgammaR)-mediated phagocytic activity of macrophage-like cells in vitro. These results suggest that TZDs improve platelet counts in this mouse model mainly by suppressing systemic reticulo-endothelial phagocytic function.