Atsuto Inoue

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.

Nitric oxide accelerates the ascorbic acid-induced osteoblastic differentiation of mouse stromal ST2 cells by stimulating the production of prostaglandin E2

Nitric oxide (NO) promoted the differentiation of clonal stromal cells (ST2 cells) derived from mouse bone marrow to osteoblast-like cells. The level of expression of mRNA for osteocalcin, a marker of osteoblastic differentiation, and the formation of mineralized nodules, increased in ST2 cells treated with a donor of NO. We used the reverse transcriptase-polymerase chain reaction (RT-PCR) to identify the subtypes of NO synthase that were expressed in the ST2 cells and we detected the expression of an inducible NO synthase gene in response to tumor necrosis factor-alpha (TNF-alpha). In various types of cell, NO induces the synthesis of prostaglandin E(2) and cGMP, which are known as regulators of osteoblastic differentiation, by activating cyclooxygenases and soluble guanylate cyclase, respectively. Prostaglandin E(2) was generated in response to NO in ST2 cells, however, no synthesis of cGMP in response to NO was detected. Two inhibitors of cyclooxygenase-2, N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide (nimesulide) and 1-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid (indomethacin), inhibited the formation of mineralized nodules by ST2 cells. Our observations suggest that NO might promote osteoblastic differentiation of ST2 cells by stimulating the production of prostaglandin E(2).

Endothelins inhibit the mineralization of osteoblastic MC3T3-E1 cells through the A-type endothelin receptor

We examined the effects of various endothelins on the mineralization of mouse clonal preosteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed mRNAs for endothelin (ET)-1 and the A-type receptor for ET (ETA). A pharmacological study also demonstrated the predominant expression of the ETA receptor. Northern blotting analysis revealed that ETs decreased the expression of mRNA for osteocalcin, which is a marker protein for the maturation of osteoblastic cells. ET-1 also decreased in the deposition of calcium by MC3T3-E1 cells in a dose-dependent manner and it had an inhibitory effect even at 10-11 M. The rank order of potency of ETs was ET-1 = ET-2 > ET-3. Brief treatment with 10-7 M ET-1 on days 6- 8 alone suppressed mineralization. ET-1 enhanced the rate of production of inositol 1,4,5-trisphosphate (IP3) in MC3T3-E1 cells, but it had no effect on the rate of production of cAMP. Taken together, our data indicate that ET-1 might inhibit the mineralization of osteoblastic cells via an interaction with the ETA receptor, with generation of IP3 as the intracellular signal.

Molecular cloning and characteristics of a novel zinc finger protein and its splice variant whose transcripts are expressed during spermatogenesis

Testicular zinc finger protein (TZF) has a zinc finger motif of the Cys2-His2 type and its transcript is expressed predominantly in mouse spermatogenic cells. Using the fragment of TZF as a probe, we isolated the alternative splice variant form (TZF-L) from mouse testis cDNA library. Analysis of the open reading frame of each cDNA indicated that TZF and TZF-L were polypeptides of 942 and 2025 amino acid residues, respectively, and the N-terminal 902 amino acids of TZF-L were identical to those of TZF. The C-terminal region of TZF-L had more a zinc finger motif of the Cys2-His2 type and poly-Glu and poly-Pro regions. The mouse TZF/TZF-L gene spanned >20 kb and consisted of 11 exons. RT-PCR analysis of the expression level of mRNAs for mouse TZF and TZF-L showed that both transcripts are highly expressed in testis and moderately in kidney and ovary. Elevated expression of both transcripts during testicular development in mice was restricted to spermatocytes at the pachytene stage of meiotic prophase. Fusion proteins with GFP also demonstrated the nuclear localization of TZF and TZF-L. These experiments suggest that TZF and TZF-L may act to control the gene activity during spermatogenesis.

Testicular zinc finger protein recruits histone deacetylase 2 and suppresses the transactivation function and intranuclear foci formation of agonist-bound androgen receptor competitively with TIF2

We previously reported that testicular zinc finger protein (TZF) is a corepressor for androgen receptor (AR). The present study demonstrated that a central portion (amino acids 512-663) of TZF, TZF(512-663), is responsible for both binding to AR and repressing the transactivation. TZF recruited endogenous histone deacetylase 2 (HDAC2) and formed a complex with agonist-bound AR. Imaging analyses showed that TZF and TZF(512-663) were recruited by AR and simultaneously impaired distinct AR foci formation. Quantification of the foci number using a three-dimensional imaging method revealed that the number of intranuclear AR foci was related to its transactivation activity. Moreover, increased levels of TZF dissociated a coactivator, TIF2, from the AR foci and vice versa. These results indicate that the ligand-dependent transactivation function of AR is quantitatively related to its intranuclear foci formation, and suggest that corepressors, such as TZF, act on these intranuclear events competitively with coactivators.

Vasoactive peptide-regulated gene expression during osteoblastic differentiation.

The formation of bone occurs via a series of events that are regulated by various hormones and cytokines. We previously reported that endothelin (ET) inhibits the mineralization by osteoblastic cells and natriuretic peptide (NP) promotes osteoblastic differentiation. Therefore, we attempted to identify the genes induced by ET or NP in mouse preosteoblastic MC3T3-E1 cells using the method known as differential display-polymerase chain reaction (DD-PCR) to understand the bone metabolism further. Consequently, we found that expression levels of mRNAs for fibronectin, type XII collagen, p160 Rho-associated kinase (ROCK), caldesmon, calpain, nucleolin and a novel gene with a zinc finger motif are downregulated in osteoblasts by ET stimuli. We also found that expression levels of mRNAs for eIF-4A and a novel gene are increased by C-type natriuretic peptide (CNP) stimuli.

Identification of a novel osteoblastic gene, inducible by C-type natriuretic peptide, whose transcript might function in mineralization as a noncoding RNA.

We reported previously that C-type natriuretic peptide (CNP) promotes the differentiation and mineralization of osteoblast-like cells. However, little information is available about the mechanism of action of CNP in differentiating osteoblastic cells. In this study, using the technique known as differential display-polymerase chain reaction, we identified a novel cDNA fragment that corresponded to a transcript whose level was increased by CNP in mouse clonal preosteoblastic MC3T3-E1 cells. Northern blotting analysis revealed transcripts of 1.3 kb and 2.3 kb in MC3T3-E1 cells. Both these transcripts were also expressed at high levels in the heart and stomach. We isolated a full-length cDNA (2,135 bp) from a cDNA library derived from MC3T3-E1 cells using the original cDNA fragment. Analysis of the sequence and of products of transcription and translation in vitro indicated that the transcript of the gene did not include any extensive open reading frames. Enhanced expression, after transfection, of transcript in MC3T3-E1 cells stimulated the deposition of calcium of these cells and the formation of mineralized nodules, but did not affect the activity of alkaline phosphatase. Our results suggest that CNP promotes the expression of a novel transcript, which might stimulate the mineralization of MC3T3-E1 cells.

Effects of ONO-6950, a novel dual cysteinyl leukotriene 1 and 2 receptors antagonist, in a guinea pig model of asthma.

We assessed in this study the anti-asthmatic effects of ONO-6950, a novel cysteinyl leukotriene 1 (CysLT1) and 2 (CysLT2) receptors dual antagonist, in normal and S-hexyl glutathione (S-hexyl GSH)-treated guinea pigs, and compared these effects to those of montelukast, a CysLT1 selective receptor antagonist. Treatment with S-hexyl GSH reduced animals LTC4 metabolism, allowing practical evaluation of CysLT2 receptor-mediated airway response. ONO-6950 antagonized intracellular calcium signaling via human and guinea pig CysLT1 and CysLT2 receptors with IC50 values of 1.7 and 25 nM, respectively (human receptors) and 6.3 and 8.2 nM, respectively (guinea pig receptors). In normal guinea pigs, both ONO-6950 (1 or 0.3 mg/kg, p.o.) and the CysLT1 receptor antagonist montelukast (0.3 or 0.1 mg/kg, p.o.) fully attenuated CysLT1-mediated bronchoconstriction and airway vascular hyperpermeability induced by LTD4. On the other hand, in S-hexyl GSH-treated guinea pigs ONO-6950 at 3 mg/kg, p.o. or more almost completely inhibited bronchoconstriction and airway vascular hyperpermeability elicited by LTC4, while montelukast showed only partial or negligible inhibition of these airway responses. In ovalbumin sensitized guinea pigs, treatment with S-hexyl GSH on top of pyrilamine and indomethacin rendered antigen-induced bronchoconstriction sensitive to both CysLT1 and CysLT2 receptor antagonists. ONO-6950 strongly inhibited this asthmatic response to the level attained by combination therapy with montelukast and BayCysLT2RA, a selective CysLT2 receptor antagonist. These results clearly demonstrate that ONO-6950 is an orally active dual CysLT1/LT2 receptor antagonist that may provide a novel therapeutic option for patients with asthma.

Endothelins inhibit mineralization of rat calvarial osteoblast-like cells

We examined the effects of members of the endothelin (ET) family on mineralization of rat calvarial osteoblast-like cells. The accumulation of calcium in cells and cell layers was attenuated by ETs with the rank order of potency ET-1 = ET-2 > ET-3. We stained the mineralized nodules by von Kossa staining and measured the number and area of mineralized nodules. The inhibitory effects of ET-1 and ET-2 on the formation of mineralized nodules were stronger than those of ET-3. Our data suggest that ET-1 may inhibit the mineralization process of osteoblastic cells through the ETA receptor.

Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice.

Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor‐mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318‐null male mice exhibited infertility, whereas Zfp318‐null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318‐null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.