Hiromi Hagiwara

Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid

The stromal cell line ST2, derived from mouse bone marrow, differentiated into osteoblast-like cells in response to ascorbic acid. Ascorbic acid induced alkaline phosphatase (ALPase) activity, the expression of mRNAs for proteins that are markers of osteoblastic differentiation, the deposition of calcium, and the formation of mineralized nodules by ST2 cells. We investigated the mechanism whereby ascorbic acid induced the differentiation of ST2 cells. Inhibitors of the formation of collagen triple helices completely blocked the effects of ascorbic acid on ST2 cells, an indication that matrix formation by type I collagen is essential for the induction of osteoblastic differentiation of ST2 cells by ascorbic acid. We furthermore examined the effects of bone morphogenetic proteins (BMPs) on the differentiation of ST2 cells induced by ascorbic acid. Ascorbic acid had no effect on the expression of mRNAs for BMP-4 and the BMP receptors. However, a soluble form of BMP receptor IA inhibited the induction of ALPase activity by ascorbic acid. These results suggest that ascorbic acid might promote the differentiation of ST2 cells into osteoblast-like cells by inducing the formation of a matrix of type I collagen, with subsequent activation of the signaling pathways that involve BMPs.

Cloning and sequence analysis of a cDNA encoding human non-selective type of endothelin receptor.

A cDNA encoding non-selective type (ETB) of endothelin receptor was isolated from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 442 amino acid residues with a relative Mr of 49,643. The deduced amino acid sequence of human ETB receptor was 88% and 64% identical to those of rat lung ETB receptor and bovine lung ET-1-specific (ETA) receptor, respectively, and contained a relatively long and proline-rich extracellular N-terminal region in addition to a significant similarity with the G protein-coupled receptor super-family with seven transmembrane segments.

Endothelin B receptors are expressed by astrocytes and regulate astrocyte hypertrophy in the normal and injured CNS

The ability of mammalian central nervous system (CNS) neurons to survive and/or regenerate following injury is influenced by surrounding glial cells. To identify the factors that control glial cell function following CNS injury, we have focused on the endothelin B receptor (ETBR), which we show is expressed by the majority of astrocytes that are immunoreactive for glial acid fibrillary protein (GFAP) in both the normal and crushed rabbit optic nerve. Optic nerve crush induces a marked increase in ETBR and GFAP immunoreactivity (IR) without inducing a significant increase in the number of GFAP‐IR astrocytes, suggesting that the crush‐induced astrogliosis is due primarily to astrocyte hypertrophy. To define the role that endothelins play in driving this astrogliosis, artificial cerebrospinal fluid (CSF), ET‐1 (an ETAR and ETBR agonist), or Bosentan (a mixed ETAR and ETBR antagonist) were infused via osmotic minipumps into noninjured and crushed optic nerves for 14 days. Infusion of ET‐1 induced a hypertrophy of ETBR/GFAP‐IR astrocytes in the normal optic nerve, with no additional hypertrophy in the crushed nerve, whereas infusion of Bosentan induced a significant decrease in the hypertrophy of ETBR/GFAP‐IR astrocytes in the crushed but not in the normal optic nerve. These data suggest that pharmacological blockade of astrocyte ETBR receptors following CNS injury modulates glial scar formation and may provide a more permissive substrate for neuronal survival and regeneration. GLIA 41:180–190, 2003.

Excess iron inhibits osteoblast metabolism

Hemochromatosis is an iron overload disorder associated with osteopenia and osteoporosis. To learn more about the effects of iron on bone cells, we examined the effects of ferric ion on the proliferation, differentiation, and mineralization of two types of cultured osteoblasts, the cell line MC3T3-E1 and rat calvarial osteoblast-like (ROB) cells. We used ferric ammonium citrate (FAC) as a donor of ferric ion, and FAC inhibited the proliferation of MC3T3-E1 cells in a dose-dependent manner. FAC (0.1-1 microg/ml) inhibited indices of osteoblast differentiation, such as the expression of type I collagen (mRNA and protein), the activity of alkaline phosphatase, and the deposition of calcium by osteoblasts. These results suggest that iron overload might give rise to osteoporosis by inhibiting osteoblast proliferation and differentiation.

Effects of nicotine on cultured cells suggest that it can influence the formation and resorption of bone

The acute effects of nicotine [1-methyl-2-(3-pyridyl)pyrrolidine] on the formation and resorption of bone were examined in cultures of clonal rat calvarial osteogenic cells (ROB-C26) and clonal mouse calvarial preosteoblastic cells (MC3T3-E1), as well as in osteoclast-like cells formed during coculture of mouse bone marrow cells and clonal stromal cells from mouse bone marrow, ST2 cells, at concentrations that occur in the saliva of smokeless tobacco users. Nicotine stimulated the rate of deposition of Ca(2+) by ROB-C26 cells, as well as the alkaline phosphatase activity of these cells, in a dose-dependent manner. However, both activities decreased in MC3T3-E1 cells that had been exposed to nicotine. These results indicate that nicotine affected osteoblastic differentiation in osteoblast-like cells. By contrast, nicotine reduced, in a dose-dependent manner, the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and the formation of pits on slices of dentine, both of which are typical characteristics of osteoclasts. Our results suggest that nicotine might have critical effects on bone metabolism.

Endothelin induces two types of contractions of rat uterus: Phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using 125I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.

Olive polyphenol hydroxytyrosol prevents bone loss

Polyphenols reportedly exert physiological effects against diseases such as cancer, arteriosclerosis, hyperlipidemia and osteoporosis. The present study was designed to evaluate the effects of oleuropein, hydroxytyrosol and tyrosol, the major polyphenols in olives, on bone formation using cultured osteoblasts and osteoclasts, and on bone loss in ovariectomized mice. No polyphenols markedly affected the proliferation of osteoblastic MC3T3-E1 cells at concentrations up to 10μM. Oleuropein and hydroxytyrosol at 10 to 100μM had no effect on the production of type I collagen and the activity of alkaline phosphatase in MC3T3-E1 cells, but stimulated the deposition of calcium in a dose-dependent manner. In contrast, oleuropein at 10 to 100μM and hydroxytyrosol at 50 to 100μM inhibited the formation of multinucleated osteoclasts in a dose-dependent manner. Furthermore, both compounds suppressed the bone loss of trabecular bone in femurs of ovariectomized mice (6-week-old BALB/c female mice), while hydroxytyrosol attenuated H(2)O(2) levels in MC3T3-E1 cells. Our findings indicate that the olive polyphenols oleuropein and hydroxytyrosol may have critical effects on the formation and maintenance of bone, and can be used as effective remedies in the treatment of osteoporosis symptoms.

Peroxisome proliferator-activated receptor δ as a molecular target to regulate lung cancer cell growth

It has been assumed that prostaglandin (PG)I2 signaling contributes to the negative growth control of lung cancer cells; however, the mechanism remains unresolved. PGI2 functions through a cell surface G protein‐coupled receptor (prostaglandin I2‐binding receptor, IP) and also exerts an effect by interacting with a nuclear hormone receptor, peroxisome proliferator‐activated receptor δ (PPARδ). We found that PPARδ was a key molecule of PGI2 signaling to give negative growth control of lung cancer cells (A549), using carbarprostacyclin, a PGI2 agonist for IP and PPARδ, and L‐165041, a PPARδ agonist. Furthermore, PPARδ‐induced cell growth control was reinforced by the inhibition of cyclooxygenase. These results suggest that PPARδ activation under the suppression of PG synthesis is important to regulate lung cancer cell growth.

Sitosterol-stimulative production of plasminogen activator in cultured endothelial cells from bovine carotid artery

The endothelial cell is a rich source of plasminogen activator that is associated with fibrinolytic activity in blood vessel. Addition of sitosterol to the culture medium of endothelial cells from bovine carotid artery gave rise to a marked increment in the activity of plasminogen activator. Removal of sitosterol from the culture medium resulted in a decrease of plasminogen activator activity back to normal levels. Enhancement of plasminogen activator activity in cultured endothelial cells was not observed by cholesterol, 5-androsten-3 beta-ol and others.

Treatment of Myoblastic C2C12 Cells with BMP-2 Stimulates Vitamin D-induced Formation of Osteoclasts

The interaction of osteoclast precursors with osteoblasts and/or stromal cells is essential for the formation of mature osteoclasts and the resorption of bone. We found that myoblastic C2C12 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells in the presence of 10−7 M 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and that C2C12 cells that had been treated with bone morphogenetic protein-2 (BMP-2) dose-dependently stimulated the formation of osteoclasts. The newly developed TRAP-positive multinucleated cells were capable of resorbing mineralized tissues. Treatment of C2C12 cells with BMP-2 for 24 h enhanced the subsequent expression in C2C12 cells of mRNA for the receptor activator of nuclear factor-κB ligand (RANKL) in the presence of 1α,25(OH)2D3. Since the formation of osteoclasts was inhibited dose-dependently by exogenous OPG, the expression of RANKL in response to BMP-2 appeared to be critical for the formation of osteoclasts. Our findings suggest that BMP-2 might play an important role in the differentiation of cells that support osteoclastogenesis.