Quality evaluation of health foods containing licorice in the Japanese Market.

Focusing on licorice, a highly used raw material in health foods, quantitative analysis of functional/medicinal components and a safety and functional evaluation was carried out for herbal medicines, health food ingredients, and so-called health foods. A functional component, glabridin, was detected in herbal medicines from Glycyrrhiza glabra and G. inflata, health food ingredients, and in commercially available health foods that contain licorice. Likewise, glycyrrhizin, a medicinal component, was detected in these sources, except in licorice oil extract. Estrogen activity in vitro was detected in some of the herbal medicines, health food ingredients, and in health foods containing licorice. In the in vivo study, liver weight in ovariectomized (OVX) mice treated with licorice oil extract was significantly higher than that in OVX and sham mice in a dose dependent manner. These results suggest that excessive intake of licorice oil extract from health foods should be avoided, even though these ingredients might be beneficial for medical use in order to maintain bone health in postmenopausal women. Measurement of hepatic cytochrome P-450 (CYP) activity, reproductive organ weight, and fat and bone mass in OVX mice was considered useful for evaluating the safety and efficacy of estrogenic health food ingredients derived from herbal medicines.

Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

DNA unwinding of autonomously replicating sequence 1 (ARS1) from the yeast Saccharomyces cerevisiae was investigated. When a negatively supercoiled plasmid DNA containing ARS1 was digested with single-strand-specific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The regions containing the core consensus A domain and the 3'-flanking B domain of ARS1 were weakly digested. When the DNA was incubated with the multisubunit single-stranded DNA-binding protein (SSB, also called RPA [replication protein A]) from human and yeast cells prior to mung bean nuclease digestion, the cleavage in the A and B domains was greatly increased. Furthermore, a region corresponding to the 5'-flanking C domain of ARS1 was digested. These results indicate that three domains of ARS1, each of which is important for replication in yeast cells, closely correspond to the regions where the DNA duplex is easily unwound by torsional stress. SSB may stimulate the unwinding of the ARS1 region by its preferential binding to the destabilized three domains. Mung bean nuclease digestion of the substitution mutants with mutations of ARS1 (Y. Marahrens and B. Stillman, Science 255:817-823, 1992) revealed that the sequences in the B2 and A elements are responsible for the unwinding of the B domain and the region containing the A domain, respectively.

DNA replication from initiation zones of mammalian cells in a model system.

We reported that DNA replication initiates from the region containing an autonomously replicating sequence from Saccharomyces cerevisiae when negatively supercoiled plasmid DNA is incubated with the proteins required for simian virus 40 DNA replication (Y. Ishimi and K. Matsumoto, Proc. Natl. Acad. Sci. USA 90:5399-5403, 1993). In this study, the DNAs containing initiation zones from mammalian cells were replicated in this model system. When negatively supercoiled DNA containing an initiation zone (2 kb) upstream of the human c-myc gene was incubated with simian virus 40 T antigen as a DNA helicase, HSSB (also called replication protein A), and DNA polymerase alpha-primase complex isolated from HeLa cells, DNA replication was specifically initiated from the center of the initiation zone, which was elongated bidirectionally in the presence of a DNA swivelase. Without HSSB, the level of DNA synthesis was significantly reduced and the localized initiation could not be detected, indicating that HSSB plays an essential role in the initiation of DNA replication. The digestion of negatively supercoiled template DNA with a single-strand-specific nuclease revealed that HSSB stimulated DNA unwinding in the center of the initiation zone where the DNA duplex is relatively unstable. In contrast, DNA replication started from a broad region of an initiation zone downstream of the dihydrofolate reductase gene from chinese hamster ovary cells, but the center of the region was mapped near the origin of bidirectional DNA replication. These results suggested that this system mimics a fundamental process of initiation of eukaryotic DNA replication. The mechanism of initiation is discussed.

Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro.

The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.

Induction of DNA replication by transcription in the region upstream of the human c-myc gene in a model replication system.

An important relationship between transcription and initiation of DNA replication in both eukaryotes and prokaryotes has been suggested. In an attempt to understand the molecular mechanism of this interaction, we examined whether transcription can induce DNA replication in vitro by constructing a system in which both replication and transcription were combined. Relaxed circular DNA possessing a replication initiation zone located upstream of the human c-myc gene and a T7 promoter near the P1 promoter of the gene was replicated in the presence of T7 RNA polymerase. In our model system, replication was carried out with the proteins required for simian virus 40 DNA replication. DNA synthesis, which was dependent on both T7 RNA polymerase and the replication proteins, was detected mainly in the promoter and upstream regions of the c-myc gene. Blocking RNA synthesis at the initial stage of the reaction severely reduced DNA synthesis, suggesting that RNA chain elongation is required to induce DNA synthesis. The results indicated that transcription can induce DNA replication in the upstream region of the transcribed gene, most likely by introducing negative supercoiling into the region, which results in unwinding of the DNA duplex.

Biochemical analysis of the intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity.

Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2, 4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex. Biochemical analyses of these mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated.

Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells.

DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the alpha subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.

Biological activities of 24-fluoro-1 alpha,25-dihydroxyvitamin D-2 and its 24-epimer.

Biological activities of two epimeric 24-fluorinated vitamin D-2 analogs, 24-fluoro-1 alpha,25-dihydroxyvitamin D-2 [24-F-1,25-(OH)2D2] and its 24-epimer [24-epi-24-F-1,25-(OH)2D2], were studied and compared with 1 alpha,25-dihydroxyvitamin D-3 [1,25-(OH)2D3] and 1 alpha,25-dihydroxyvitamin D-2 [1,25-(OH)2D2]. 24-F-1,25-(OH)2D2 was nearly as active as 1,25-(OH)2D3 and 1,25-(OH)2D2 both in regulating calcium metabolism in vivo including bone mineral mobilization and intestinal calcium transport and in inducing differentiation of HL-60 cells. While 24-epi-24-F-1,25-(OH)2D2 showed distinct properties in these two types of the actions. Though the 24-epimer was nearly as potent as 1,25-(OH)2D3 in inducing differentiation of HL-60 cells, it showed little activity in regulating calcium metabolism in vivo. The fluorine atom introduced at the 24-position of either 1,25-(OH)2D2 or its 24-epimer had no potentiating effect. This is in sharp contrast with the cases of 24- and 26,27-multifluorinated analogs of active vitamin D-3.

Synthesis of simian virus 40 C-family catenated dimers in vivo in the presence of ICRF-193.

The effect of ICRF-193, a non-cleavable, complex-stabilizing type of topoisomerase II inhibitor, on SV40 DNA replication in vivo was examined. As analyzed by one and two-dimensional gel electrophoresis, C-family catenated dimers, each composed of two intertwined, covalently closed SV40 DNAs, were mainly synthesized in the presence of the drug. On removal of the drug these C-family dimers were segregated into monomers. These results indicate that topoisomerase II is required for the segregation of replicated daughter molecules, but it is not absolutely required for the replication of DNA molecules up to the C-family dimers.

Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.

Effects of Soy Phytoestrogens and New Zealand Functional Foods on Bone Health.

New Zealand is a rich source of food components that may have bioactivity on bone. Docosahexaenoic acid (DHA) from fish oil has been shown to maintain bone in ovariectomised (OVX) rats. Kiwifruit, a source of fibre and carotenoids, may also affect bone via a prebiotic as well as direct cell-based mechanisms. We aimed to 1) ascertain the effects of DHA on two cell models, including interactions with soy isoflavones; 2) and investigate the specific effects of carotenoids from kiwifruit as well as whole kiwifruit in cell-based and rodent models as well as in a human study. RAW 264.7 mouse monocytes or mouse bone marrow was used to generate osteoclasts (OC). Cells were exposed to the agents between 5 and 21 d and formation and activity of OC measured, including molecular markers. DHA inhibited OC formation in both cell models, including expression of cathepsin K, NFATc1 as well as actin ring formation. Combination with isoflavones enhanced these effects. In OVX rats and mice fed with kiwifruit for 8 wk, green kiwifruit reduced the rate of bone loss after OVX, and in mice it reduced C-telopeptide of Type 1 collagen (CTX) levels and RANKL expression while in menopausal women, green kiwifruit affected blood lipids and bone markers positively.

Regulation by calcium and 1,25-(OH)2D3 of cell proliferation and function of bovine parathyroid cells in culture.

The effects of high calcium and 1,25-(OH)2D3 on parathyroid cell growth, PTH secretion, and steady-state levels of pre-proPTH mRNA in proliferating bovine parathyroid cells were examined. Cells were established in primary tissue culture and then tested in passages 2 and 5. Cell proliferation was suppressed by 10(-9)-10(-7) M 1,25-(OH)2D3 but not by high calcium (2.5 mM). Cells at passages 2 and 5 were grown to subconfluence and then exposed for 72 h to 2.5 mM calcium or 10(-7) M 1,25-(OH)2D3. Pre-proPTH mRNA was decreased to approximately 50% of control by 2.5 mM calcium compared with 0.3 and 1.0 mM calcium. PTH secretion, as tested by low calcium stimulation for 1 h at the end of 72 h incubation, was inhibited by 50% in cells that had been exposed to high calcium compared with control. Incubation with 10(-7) M 1,25-(OH)2D3 caused a decrease in the levels of pre-proPTH mRNA and PTH release to 50% of control at 72 h. These results suggest that cultured bovine parathyroid cells, at least in early passages, have responses to high calcium and 1,25-(OH)2D3 similar to those in primary nonproliferating cultures studied earlier and that 1,25-(OH)2D3 inhibits the proliferation of parathyroid cells in a dose-responsive fashion.

A differentiation-inducing factor produced by the osteoblastic cell line MC3T3-E1 stimulates bone resorption by promoting osteoclast formation.

We have reported that the differentiation-inducing factor (DIF) is present in conditioned medium of mouse osteoblast-like cell (MC3T3-E1) cultures. In the present study, the DIF from conditioned medium of MC3T3-E1 cells was partially purified and its biologic activity was examined. The DIF was purified by monitoring the induction of phagocytic activity of mouse myeloblastic leukemia cells (M1). The DIF induced differentiation of not only M1 cells but also mouse myelomonocytic cells (WEHI-3). Furthermore, the DIF increased the in vitro bone-resorbing activity and the osteoclast number in mouse calvaria. The increases were inhibited by the addition of either salmon calcitonin or indomethacin. When mouse bone marrow cells were cultured with the DIF for 8 days, formation of osteoclast-like multinucleated cells was stimulated dose dependently. The DIF from MC3T3-E1 cells appeared to be different from interleukin-1 (IL-1), tumor necrosis factor (TNF), and transforming growth factor beta (TGF-beta). These results suggest that the DIF partially purified from osteoblast-like cell cultures stimulates osteoclastic bone resorption by promoting differentiation and fusion of osteoclast progenitors to form multinucleated osteoclasts.

Cooperative effects of exercise training and genistein administration on bone mass in ovariectomized mice.

We reported that genistein, a soybean isoflavone, prevents bone loss caused by estrogen deficiency, without undesirable effects on the uterus. In this study, we examined cooperative effects of genistein administration and running exercise on bone mass in ovariectomized (OVX) mice. Female mice aged 7 weeks were either sham-operated or OVX and divided into six groups: (1) sham; (2) OVX; (3) OVX, treated with genistein at a submaximal dose (0.4 mg/day) subcutaneously (G); (4) OVX, exercised on a treadmill daily for 30 minutes/day at 12 m/minute on a 10 degree uphill slope (Ex); (5) OVX, given genistein and exercised (ExG); and (6) OVX, treated with 17beta-estradiol (0.03 microg/day) in the same manner as genistein (E2). Four weeks after intervention, bone mass was estimated by dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Bone mineral density (BMD) of the whole femur measured by DXA was higher in both the G and the Ex groups than in the OVX group. Furthermore, BMD in the ExG group was significantly higher than that in the groups receiving either intervention alone. Bone area in distal region of the femur was significantly higher in Ex and ExG groups as compared with those in the OVX and G groups. pQCT analysis showed that the cross-sectional areas (CSAs) and periosteum perimeter at midshaft of the femur did not differ in the sham and OVX groups but were significantly higher in Ex and ExG groups. Histomorphometric analysis showed that bone formation rate/bone surface (BFR/BS) was significantly higher in both Ex and ExG groups as compared with that in non-exercised groups. The bone volume (BV/TV) in the distal femoral cancellous bone was lower in the OVX than that in the sham group, and it was restored completely in the ExG group, as in the E2 group. Thickness of the trabecular bone (Tb.Th) was higher in Ex and ExG groups than that in the OVX and G groups. These results indicate that the combined intervention of moderate exercise and the submaximal dose of genistein administration show a cooperative effect in preventing bone loss in OVX mice.

The relationship between fusion and proliferation in mouse alveolar macrophages.

We have reported that protein factors separated from conditioned media of Concanavalin A-stimulated spleen cell cultures induce growth and fusion of mouse alveolar macrophages. A macrophages growth factor (MGF) was purified and, at the final step of purification on HPLC, eluted at the same position as a colony-stimulating factor (CSF), suggesting that MGF is identical with CSF. In the present study, we examined the relationship between proliferation and fusion of macrophages using purified CSF (MGF) and 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. The latter was used in place of a macrophage fusion factor which is supposed to be contained in the same conditioned medium, since a macrophage fusion factor has not yet been isolated. Adding less than 5% unfractionated conditioned medium from Concanavalin A-stimulated spleen cells markedly induced proliferation of alveolar macrophages without inducing fusion. In contrast, adding the same unfractionated conditioned medium at concentrations of 10% or more suppressed proliferation dose dependently, whereas it induced fusion reciprocally. Proliferation of macrophages was similarly enhanced by adding purified CSF or retinoic acid. Fusion of macrophages was induced by 1 alpha,25-(OH)2D3, but not by purified CSF or retinoic acid. Adding 1 alpha,25-(OH)2D3 together with purified CSF or retinoic acid completely suppressed the increase of proliferation induced by either growth factor, whereas that treatment rather potentiated the fusion induced by 1 alpha,25-(OH)2D3 alone. These results indicate that the fusion and proliferation of macrophages occur in a reciprocal fashion.

Transcriptional regulation of the production of the third component of complement (C3) by 1 alpha,25-dihydroxyvitamin D3 in mouse marrow-derived stromal cells (ST2) and primary osteoblastic cells.

We have purified a 190-kDa protein produced by mouse marrow-derived stromal cells (ST2) in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] and unequivocally identified it as mouse complement C3 (C3). In this study we examined the regulation by 1 alpha,25-(OH)2D3 of C3 production in ST2 cells at both the transcriptional and translational levels. 1 alpha,25-(OH)2D3 greatly increased the protein production of C3 at 24 h, and it attained a maximum at 72 h. C3 mRNA stimulation by 1 alpha,25-(OH)2D3 was initiated at 12 h and reached a maximum at 48 h. 1 alpha,25-(OH)2D3 increased the expression of C3 mRNA dose-dependently, ranging from 10(-10)-10(-8) M. The increase in the C3 production in response to 1 alpha,25-(OH)2D3 appeared to occur at a transcriptional level, since actinomycin-D completely inhibited both mRNA expression and protein production of C3 induced by 1 alpha,25-(OH)2D3. Besides 1 alpha,25-(OH)2D3, local bone-resorbing agents, such as interleukin-1 alpha, tumor necrosis factor-alpha, and lipopolysaccharides, also stimulated the expression of C3 mRNA, not only in ST2 cells, but also in primary osteoblastic cells. C3 production by hepatocytes occurred regardless of the presence or absence of 1 alpha,25-(OH)2D3. These results clearly indicate that 1 alpha,25-(OH)2D3 tissue-specifically regulates the synthesis of C3 in bone. Bone C3 may play an important role in bone metabolism.

Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency.

Genistein, an isoflavone abundantly present in soybeans, has structural similarity to estrogen, suggesting that genistein may act as a phytoestrogen. To examine the possible role of genistein in hemopoiesis and bone metabolism, female mice were either sham-operated or ovariectomized (OVX), and selected OVX mice were administered genistein for 2-4 weeks (0.1-0.7 mg/day) or 17beta-estradiol (E2; 0.01-0.1 microg/day) s.c., using a miniosmotic pump (Alza Corp., Palo Alto, CA). In OVX mice, uterine weight declined but was completely restored by E2 administration. In contrast, genistein did not demonstrate a reversal of the OVX-induced uterine atrophy. The number of bone marrow cells markedly increased, 2-4 weeks after OVX, and most of these were B220-weakly positive pre-B cells. The increased B-lymphopoiesis was completely restored, not only by E2 but also by genistein administration. In OVX mice, the trabecular bone volume of the femoral distal metaphysis, measured by microcomputed tomography scanning and dual-energy x-ray absorptiometry, was markedly reduced; and genistein restored this, as did E2. These results indicate that genistein exhibits estrogenic action in bone and bone marrow, to regulate B-lymphopoiesis and prevent bone loss, without exhibiting estrogenic action in the uterus. Phytoestrogens may be useful for preventing bone loss caused by estrogen deficiency in females.

The specific production of the third component of complement by osteoblastic cells treated with 1 alpha,25-dihydroxyvitamin D3.

A 190 kDa protein was purified from conditioned media of mouse marrow-derived stromal cell (ST2) cultures treated with 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) and identified as the third component of mouse complement (C3). Northern and Western blot analysis revealed that the production of C3 by ST2 and primary osteoblastic cells was strictly dependent on 1 alpha,25(OH)2D3, but the production by hepatocytes was not. Adding 1 alpha,25(OH)2D3 together with mouse C3 antibody to bone marrow cultures greatly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated cells. Adding C3 alone induced no TRAP-positive cell formation. These results suggest that, in bone tissues, C3 is specifically produced by osteoblasts in response to 1 alpha,25(OH)2D3 and somehow involved in inducing differentiation of bone marrow cells into osteoclasts in concert with other factors produced by osteoblasts in response to 1 alpha,25(OH)2D3.

Genistein, a soybean isoflavone, affects bone marrow lymphopoiesis and prevents bone loss in castrated male mice.

Soybean isoflavones exhibit selective effects on bone metabolism in postmenopausal women as well as in ovariectomized animals. Recently, the role of estrogen in bone metabolism in men has also received attention, because a man with a mutated estrogen receptor-alpha (ER(alpha)) gene will exhibit osteoporotic phenotypes. To examine the possible role of genistein, a soybean isoflavone, in bone marrow hemopoiesis and bone metabolism in men, male mice were orchidectomized (orx) and treated with genistein (0.4-0.8 mg/day) or 17beta-estradiol (E(2); 0.03 microg/day) subcutaneously for 3 weeks. In orx mice, seminal vesicle weight decreased markedly, and it was not affected by the administration of genistein or E(2). The number of bone marrow cells was markedly increased after orx, and the majority was B-220 weakly positive pre-B cells. Increased B-lymphopoiesis was restored completely by E(2) or genistein administration. In orx mice, bone mineral density of the femur decreased markedly, and this bone loss was prevented to a significant extent by treatment with genistein as well as E(2). Histomorphometry showed that the trabecular bone volume in the femoral distal metaphysis decreased markedly after orx, and genistein and E(2) prevented this bone loss. These results suggest that soybean isoflavones prevent bone loss due to androgen deficiency in males.

Interleukin 1 regulates the expression of osteopontin mRNA by osteoblasts.

Osteopontin is a matrix protein which belongs to the integrin superfamily and is involved in cell adhesion. In the present study, we examined the regulation of the mRNA expression of osteopontin by interleukin 1 alpha (IL-1 alpha) in osteoblasts. IL-1 alpha greatly increased the steady-state level of osteopontin mRNA in both a mouse osteoblastic cell line (MC3T3-E1) and mouse primary osteoblast-like cells. The increase in the osteopontin mRNA expression by IL-1 alpha was dose-dependent at a range of 0.004-0.2 nM. This was most likely due to an increase in the transcriptional rate, not to an increase in the stability of osteopontin mRNA. The in vitro nuclear transcription experiment showed that IL-1 alpha-treated MC3T3-E1 cells increased the synthesis of osteopontin mRNA. Besides IL-1 alpha, tumor necrosis factor alpha (TNF-alpha), lipopolysaccharides (LPS) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) increased the osteopontin mRNA expression in both the clonal osteoblasts (MC3T3-E1) and the primary osteoblast-like cells. In response to such bone-resorbing agents, primary osteoblast-like cells expressed osteopontin mRNA much more strongly than primary fibroblast-like cells isolated from mouse calvaria. Both IL-1 alpha and 1 alpha,25(OH)2D3 greatly increased the production of 68 and 62 kDa phosphoproteins in conditioned media of MC3T3-E1 cell cultures, which probably correspond to osteopontin. These results suggest that osteopontin plays an important role in bone remodeling, in particular bone resorption.