Brief Report: Consecutive Alendronate Administration-Mediated Inhibition of Osteoclasts Improves Long-Term Engraftment Potential and Stress Resistance of HSCs.

Osteoclasts form a bone marrow (BM) cavity serving as a hematopoietic niche for the maintenance of hematopoietic stem cells (HSCs). However, the role of osteoclasts in the BM has been controversially reported and remains to be further understood. In the present study, we investigated how osteoclasts affect the modulation of hematopoietic stem/progenitor cells in the BM by administering bisphosphate alendronate (ALN) to B6 mice for 21 consecutive days to inhibit osteoclast activity. ALN treatment caused a reduction in the number of tartrate-resistant acid phosphate (TRAP)-positive osteoclast cells and an increase in bone mineral density, particularly in the trabecular zone, but not in the cortical zone of the BM. Osteoclast inhibition caused by ALN treatment decreased mitochondrial reactive oxygen species (ROS) generation and SA-ß-gal activity of CD150+ CD48- Lineage-Sca-1+ c-Kit+ (LSK) cells, eventually leading to an improvement in the engraftment potential and self-renewal activity of HSCs. Moreover, ALN-treated mice exhibited an enhanced resistance of HSCs in response to the genotoxic stress of 5-fluorouracil, as determined by mitochondrial ROS generation, SA-ß-gal activity, and p16INK4a expression in subsets of LSK and CD150+ CD48- LSK cells as well as competitive assay. Collectively, our findings indicate that inhibition of osteoclast activity improves the long-term engraftment potential and stress resistance of HSCs. Stem Cells 2016;34:2601-2607.

Testicular acid phosphatase induces odontoblast differentiation and mineralization.

Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in ß-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active ß-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active ß-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through ß-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration.

A (S)-(+)-decursin derivative, (S)-(+)-3-(3,4-dihydroxy-phenyl)-acrylic acid 2,2-dimethyl-8-oxo-3,4-dihydro-2H,8H-pyrano[3,2-g]-chromen-3-yl-ester, attenuates the development of atopic dermatitis-like lesions in NC/Nga mice.

(S)-(+)-decursin is a biological coumarin compound isolated from Angelica gigas Nakai. (S)-(+)-decursin and its analogue have a variety of pharmacological activities. In the present study, the anti-inflammatory effect of a (S)-(+)-decursin derivative, (S)-(+)-3-(3,4-dihydroxy-phenyl)-acrylic acid 2,2-dimethyl-8-oxo-3,4-dihydro-2H,8H-pyrano [3,2-g]-chromen-3-yl-ester (Compound 6, C6), on in vitro and in vivo atopic dermatitis was investigated. C6 suppressed the secretion of IL-6, IL-8, and monocyte chemotactic protein-1 increase by the house dust mite extract in the eosinophilic leukemia cell line and THP-1 cells. C6 inhibited the production of TARC, IL-6, and IL-8 increase by IFN-γ and TNF-α in the human keratinocyte cell line. In the in vivo experiment, NC/Nga mice were sensitized to 2,4-dinitrochlorobenzene, and then C6 or dexamethasone (Dex) were orally and dorsally administered for three weeks. C6 treatment reduced the skin severity score compared with that of the control group. C6 inhibited the thickening of the epidermis and inflammatory cell infiltration into the dermis by evaluating the histological examination. The serum immunoglobulin E (IgE) level decreased in the C6-treated group compared with that of the control group. The inhibitory effect of C6 on IgE concentration was similar to that of Dex. The levels of IL-4, IL-5, IL-13, and eotaxin increased after treatment with concanavalin A in mouse splenocytes. The cytokine levels of the C6-treated group were lower than those of the control group. Taken together, C6 may attenuate atopic dermatitis-like lesions through its anti-inflammatory effect, such as inhibition of IgE and inflammatory cytokines, and it may be valuable as a therapeutic drug for the treatment of atopic dermatitis.

The inhibitory effect of Duchesnea chrysantha extract on the development of atopic dermatitis-like lesions by regulating IgE and cytokine production in Nc/Nga mice.

Duchesnea chrysantha belongs to the Rosaceae family and has been used traditionally for the treatment of various diseases in Korea and other parts of East Asia. This study examined the antiinflammatory effect of Duchesnea chrysantha extract (DcE) on atopic dermatitis in vitro and in vivo. DcE inhibited the production of IL-6, IL-8 and MCP-1 in THP-1 cells and the release of IL-6 and MCP-1 in EoL-1 cells after treatment with house dust mite extract. In the in vivo experiment, Nc/Nga mice were sensitized to DNCB and then orally and dorsally administered DcE (50 mg/kg in PBS) for 3 weeks. The DcE administration significantly reduced the skin severity score when compared with the control group and inhibited the thickening of the epidermis and infiltration of inflammatory cells into the dermis. In addition, the serum IgE levels decreased markedly in the DcE-treated mice when compared with the control group. The synthesis of IL-5, IL-13, MCP-1 and eotaxin was also decreased in splenocytes of the DcE-treated group, while IFN-γ was increased in the Dc-administered group. These results may indicate that DcE attenuates the development of atopic dermatitis-like lesions by lowering the IgE and inflammatory cytokine levels, and that it is useful in drug development for the treatment of atopic dermatitis.

Suppression of ovalbumin-induced airway inflammatory responses in a mouse model of asthma by Mimosa pudica extract.

Asthma is an inflammatory airway disease. The pathogenic mechanisms of asthma include the infiltration of leukocytes and release of cytokines. Mimosa pudica (Mp) has been used traditionally for the treatment of insomnia, diarrhea and inflammatory diseases. Although Mp extract has various therapeutic properties, the effect of this extract on asthma has not yet been reported. This study investigated the suppressive effects of Mp extract on asthmatic responses both in vitro and in vivo. Mp extract was acquired from dried and powdered whole plants of M. pudica using 80% ethanol. BALB/c mice were used for the mouse model of asthma induced by ovalbumin. Mp extract significantly inhibited the HMC-1 cell migration induced by stem cell factor and blocked the release of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) in EoL-1 cells. Leukocytosis, eosinophilia and mucus hypersecretion in asthmatic lung were significantly suppressed by Mp extract. The release of ovalbumin-specific IgE in bronchoalveolar lavage fluid and serum was also decreased. Mp extract treatment resulted in no liver cytotoxicity. The Mp extract has inhibitory properties on asthma and may be used as a potent therapeutic agent for allergic lung inflammation.

Urinary Exosomal microRNA-21 as a Marker for Scrub Typhus-Associated Acute Kidney Injury.

Background: Urinary microRNA (miRNA)-21 is a biomarker for acute kidney injury (AKI). We conducted this study to determine if a urinary exosomal analysis for this biomarker could serve as a novel diagnostic approach for detecting kidney disease. Materials and Methods: We investigated the clinical significance of urinary exosomal miRNA-21 levels for AKI in scrub typhus patients. We collected 138 urine samples from scrub typhus patients at the time of admission. Urinary exosomal miRNA-21 was assessed in 25 age- and sex-matched scrub typhus patients with and without AKI. Results: The total leukocyte count was higher in AKI patients than in non-AKI patients (10.40 × 103/mL vs. 6.40 × 103/mL, p < 0.01). Urinary exosomal miRNA-21 levels were higher in the AKI group than in the non-AKI group (20.1 ± 1.2 vs. 17.8 ± 1.8 ΔCt value of miRNA-21, p < 0.01). Additionally, the miRNA-21 levels correlated directly with the total leukocyte counts and inversely with the estimated glomerular filtration rate. A receiver operating characteristic curve analysis demonstrated good discriminative power for the diagnosis of scrub typhus-associated AKI, with an area under the curve value of 0.907. Conclusion: Urinary exosomal miRNA-21 could be a surrogate marker for scrub typhus-associated AKI diagnosis.

Development of an in vitro test system measuring transcriptional downregulatory activities on IL-13.

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a wellknown antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells.

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C delta (PKC delta), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC delta, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5min and peaked at 30min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC delta (p<0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .

Suppressive effects of Houttuynia cordata Thunb (Saururaceae) extract on Th2 immune response.

ETHNOPHARMACOLOGICAL RELEVANCE: Houttuynia cordata Thunb (Saururaceae), known as 'E-Sung-Cho' in Korea, has been traditionally used for the treatment of herpes simplex, chronic sinusitis, and allergy. AIM OF THE STUDY: To investigate the inhibitory activity of Houttuynia cordata Thunb fractions (HcFs) on the T helper 2 (Th2) immune response, we evaluated the alternation of the release of Th2-type cytokines and chemokines such as interleukin (IL)-4 and IL-5, and thymus and activation-regulated chemokine (TARC/CCL17). MATERIALS AND METHODS: Ethanol fraction was obtained from dried and powdered whole plants of Houttuynia cordata Thunb using ethanol. The residue was diluted with water and was then successively partitioned with n-hexane, EtOAc and BuOH. HcFs include ethanol, n-hexane, EtOAc, BuOH and water fractions. RT-PCR and ELISA were performed to measure mRNA and protein expression of cytokines. RESULTS: HcFs inhibited the expression of IL-4 and IL-5 in response to phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (CaI) in Jurkat T cells and the human mast cell line, HMC-1. IL-4- and tumor necrosis factor-alpha (TNF-alpha)-induced TARC production was blocked by HcFs in skin fibroblast CCD-986sk cells, particularly by the ethanol extract of Hc. Stimulants included in PMA, phytohemagglutinin (PHA) and CaI, increased the mRNA level of CC chemokine receptor 4 (CCR4), a receptor of TARC, in Jurkat T cells, and the ethanol extract of HcF weakly blocked the increased mRNA level. However, the stimulants and ethanol extract had no effect on the CCR4 protein level. The ethanol extract inhibited TARC-induced migration, as well as basal migration of Jurkat T cells. CONCLUSIONS: This study may show the usefulness of HcFs in the ethnopharmacological treatment of Th2-mediated or allergic inflammation, through the down-regulation of the production of Th2 cytokines and TARC, as well as cell migration.

Inhibitory effects of Duchesnea chrysantha extract on ovalbumin-induced lung inflammation in a mouse model of asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Duchesnea chrysantha (D. chrysantha) is a herb with anti-oxidative, anti-inflammatory and immune-enhancing properties. AIM OF THE STUDY: Asthma is an inflammatory disease of the lungs, and the hallmarks of the disease are increased inflammatory cell infiltration into the airways and poor respiratory function. Although there is the possibility that D. chrysantha may have an inhibitory effect on lung inflammation, the effects of D. chrysantha on asthma have not been fully investigated. In the present study, we investigated the anti-inflammatory activity of D. chrysantha extract (Dc extract) on lung inflammation in a murine model of ovalbumin-induced asthma. MATERIALS AND METHODS: Dc extract was obtained from dried and powdered whole plants of D. chrysantha using 80% ethanol. BALB/c mice induced by ovalbumin sensitization and nebulization were used as a mouse model of asthma. RT-PCR and ELISA were performed to measure mRNA and protein expression of cytokines. We examined the effects of Dc extract on leukocyte infiltration and mucus secretion using periodic acid-Schiff staining as well as hematoxylin and eosin staining. RESULTS: Dc extract significantly inhibited leukocytosis and eosinophilia in the bronchoalveolar lavage (BAL) fluid (p<0.01). Dc extract significantly reduced the elevated infiltration of inflammatory cells (p<0.05) and inhibited the increased mucus secretion, despite the absence of significant value. Although Dc extract weakly inhibited the mRNA expression of IL-4, IL-5, IL-13, and eotaxin, it strongly inhibited the protein expression of IL-5 (p<0.05) and eotaxin (p<0.01) in BAL fluid. Ovalbumin-specific IgE levels in the serum and BAL fluid were blocked by Dc extract (p<0.05). CONCLUSIONS: These results suggest the possibility that Dc extract can exert suppressive effects on asthma and may provide evidence that Dc extract is a useful agent for the treatment of allergic airway disease.

Osterix regulates corticalization for longitudinal bone growth via integrin ß3 expression.

Corticalization, coalescence of trabecular bone into the metaphyseal cortex, is important for the longitudinal growth of long bones. However, little is known about the molecular mechanisms controlling corticalization. To understand the molecular mechanisms underlying corticalization, we analyzed osteoblast-specific Osterix-knockout mice (Col-OMT). In control mice, corticalization was initiated after 7 postnatal days, and the number of osteoblasts in the peripheral spongiosa was increased compared to the number in the central spongiosa. In contrast, in Col-OMT mice, corticalization was delayed, and the number of osteoblasts in peripheral zones was unchanged compared to the central zone. Furthermore, femoral length was decreased in Col-OMT mice at 1 month. Because Col-OMT mice exhibited impaired matrix coalescence and osteoblast migration, we evaluated integrin signaling in Col-OMT mice. Osterix bound to the Itgb3 promoter and increased transcription of the Itgb3 gene in osteoblast cells. Interestingly, the inner and outer cortical bones were separated in Itgb3-null mice at postnatal day 7. In Itgb3-null mice, the number of osteoblasts in peripheral zones was not changed, and the femoral length was decreased. Taken together, these results indicate that Osterix regulates corticalization for longitudinal bone growth via the control of integrin ß3 expression in osteoblasts. Our findings imply that the ability to control osteoblast function during corticalization may help in the treatment of short stature.

p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells.

9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.

Effects of lactose-beta-sitosterol and beta-sitosterol on ovalbumin-induced lung inflammation in actively sensitized mice.

Asthma is a disease marked by chronic lung inflammation and the number of patients suffering from asthma increases annually. Both beta-sitosterol (BS) and beta-sitosterol glucoside exist in a variety of plants and have anti-tumor, anti-microbial, and immunomodulatory activities. However, the precise role of BS and beta-sitosterol glucoside in asthma has not been well understood. The aim of this study was to investigate the inhibitory effects of BS and lactose-BS (L-BS) on the pathophysiological process in ovalbumin-induced asthmatic mice. The total cells and eosinophils in the bronchoalveolar lavage (BAL) fluid markedly decreased (p<0.05) after L-BS or BS administration (1 mg/kg; i.p.), and the ROS production also decreased in comparison to the asthma control. Histopathological features were detected by performing histochemistry, including H&E and alcian blue & P.A.S staining. Both L-BS and BS mitigated the inflammation by eosinophil infiltration and mucus hypersecretion by goblet hyperplasia. These effects of L-BS were superior to those of BS. L-BS and BS inhibited the increased mRNA and protein expression of IL-4 and IL-5 in the lung tissue and BAL fluid, respectively. The IgE concentration in the BAL fluid and serum was measured by performing ELISA and the ovalbumin-specific IgE in the BAL fluid was uniquely inhibited by L-BS (p<0.05). The splenocytes were isolated from the normal and asthmatic mice and incubated in the absence and presence of 100 microg/ml ovalbumin, respectively. L-BS blocked the survival rate of the splenocytes of the mice (p<0.01). This finding indicates the possibility of L-BS and BS as potential therapeutic molecules in asthma and may contribute to the need to improve current therapeutic drugs.

The inhibitory effect of Houttuynia cordata extract on stem cell factor-induced HMC-1 cell migration.

Hottuynia cordata Thunb (Saururaceae; HC) is known as a therapeutic drug that has been used in traditional oriental medicine for the treatment of allergy. Mast cells play an important role in a variety of inflammatory diseases, and specifically asthma and atopy. In the present study, we investigated the effect of HC extracts on the migration of the human mast cell line, HMC-1, in response to stem cell factor (SCF). Treatment with HC extracts at a concentration of 10mug/ml for 24h showed no significant decrease in the survival rate of the HMC-1 cells. SCF showed the typical bell-shape curve for the HMC-1 cell chemoattraction with the peak of the curve at the SCF concentration of 100ng/ml. HC-1, which was the whole plant (Houttuynia cordata) extracted with 80% EtOH, and HC-3, which was the residue successively partitioned with EtOAc, both had inhibitory effects on HMC-1 cell movement. After the treatment with 10mug/ml HC-1 extract for 6 and 24h, the chemotactic index (CI) of HMC-1 cells decreased up to 74 and 63%, respectively. HC-3 extract treatment for 6 and 24h lowered the CI to 72 and 44%, respectively. The HC-1 and HC-3 extracts had no inhibitory effect on the mRNA and surface protein expressions of c-kit, SCF receptor. SCF mediated the chemotaxis signaling via NF-kappaB activation, and both extracts inhibited the activation. Therefore, our results indicate that HC-1 and HC-3 extracts decrease the chemotactic ability of HMC-1 cells in response to SCF by inhibiting the NF-kappaB activation, and these substances may be useful for treating mast cell-induced inflammatory diseases.

cDNA sequences of two biliproteins, BP1 and BP2, from the cabbage white butterfly, Pieris rapae and their tissue- and stage-specific accumulation.

Two similar full-length cDNAs of biliprotein were isolated and shown to encode the two isoelectric forms of biliverdin-binding proteins (BPs): BP1 and BP2 in Pieris rapae. Sequence analysis of two cDNA clones shows that both BPs contain a 567-bp open reading frame which predicts a 189-amino acid protein and a 15-amino acid signal peptide. The calculated isoelectric points are pI=7.25 (BP1) and 6.74 (BP2), respectively. Comparison of two sequences of BP1 and BP2 reveals 12 base differences in the open reading frame, of which three nucleotide changes lead to two amino acid substitutions. The 5'-UTR from the two clones shows no difference, but an additional 45-bp fragment is inserted in the 3'-UTR of BP2 making its message a little longer than that of BP1. Northern blot analysis confirmed that the BP mRNAs are expressed from the late 4th instar to the adult stage with exception of prepupae and newly ecdysed pupae. While the BP1 transcript was prevalent in the larval stage, the BP2 transcript was abundant in the whole body only after the pupal stage in P. rapae. Both BPs were detected in a stage-specific pattern in the epidermis, testis, hindgut, wing, brain, and egg, with a lesser amount in the fat body. Two-dimensional gel electrophoresis and Western blot analyses revealed that BP1 was dominant in tissues from larvae, BP2 was dominant in tissues from pupal stages, and both BPs appeared in tissues from the adult stage, though BP2 was predominant.

Differential regulation of CC chemokine receptors by 9-cis retinoic acid in the human mast cell line, HMC-1.

Mast cells are well known as effector cells in a variety of inflammatory diseases, including asthma as well as other allergic disorders. The precise role of 9-cis retinoic acid (9CRA) in mast cells is not understood despite the accepted fact that 9CRA regulates inflammatory responses and neutrophil differentiation. In this study, we investigated the effects of 9CRA on the expression of CC chemokine receptors in the human mast cell line, HMC-1. 9CRA selectively inhibits the CCR2 mRNA level and increases the CCR3 mRNA level in both a time and dose dependent manner. Other CC chemokine receptors, including CCR1, CCR4 and CCR5 are not altered by treatment with 9CRA. Both TNF-alpha and LPS, known pro-inflammatory molecules, have no effect on mRNA levels of CC chemokine receptors. For surface expression, 9CRA decreased the CCR2 level but had no effect on the CCR3 level. 9CRA inhibited the chemotactic activity in response to the CCR2-dependent chemokine, MCP-1/CCL2 but not in response to CCR3-specific chemokine, eotaxin/CCL11. 9CRA decreased spontaneous homotype clustering. Therefore, our results demonstrate that 9CRA differentially decreases both CCR2 expression and chemotactic ability of HMC-1 cells, and may regulate the inflammatory effects of mast cells.

Requirement of Smad4-mediated signaling in odontoblast differentiation and dentin matrix formation.

Dentin is the major part of tooth and formed by odontoblasts. Under the influence of the inner enamel epithelium, odontoblasts differentiate from ectomesenchymal cells of the dental papilla and secrete pre-dentin which then undergo mineralization into dentin. Transforming growth factor-beta (TGF-ß)/bone morphogenetic protein (BMP) signaling is essential for dentinogenesis; however, the precise molecular mechanisms remain unclear. To understand the role of TGF-ß/BMP signaling in odontoblast differentiation and dentin formation, we generated mice with conditional ablation of Smad4, a key intracellular mediator of TGF-ß/BMP signaling, using Osr2 or OC-Cre mice. Here we found the molars of Osr2CreSmad4 mutant mice exhibited impaired odontoblast differentiation, and normal dentin was replaced by ectopic bone-like structure. In Osr2CreSmad4 mutant mice, cell polarity of odontoblast was lost, and the thickness of crown dentin was decreased in later stage compared to wild type. Moreover, the root dentin was also impaired and showed ectopic bone-like structure similar to Osr2CreSmad4 mutant mice. Taken together, our results suggest that Smad4-dependent TGF-ß/BMP signaling plays a critical role in odontoblast differentiation and dentin formation during tooth development.

Maturation of cortical bone suppresses periosteal osteoprogenitor proliferation in a paracrine manner.

Periosteum contains enriched pools of osteogenic progenitors and is highly proliferative, thus giving this tissue a pivotal role in maintaining the diameter of the diaphyseal cortex and in recovery from fractures. Although periosteal proliferation has not been detected in normal bone, intense periosteal proliferation has been observed in pathologic states such as fracture, inflammation, and bone tumors. However, the mechanism by which periosteal osteoprogenitor proliferation is regulated remains poorly understood. To investigate this regulation mechanism, osteoblast/osteocyte-specific conditional knockout mice were developed lacking Smad4 and Osx, two factors that are essential for osteoblast differentiation and matrix mineralization. In Smad4 (Col) and Osx (Col) mice, osteocalcin, Dmp-1, and sclerostin expression were significantly decreased in the cortical bone. Interestingly, although Cre activity was not observed in the periosteum, the proliferation of periosteal osteoprogenitors was enhanced in Smad4 (Col) and Osx (Col) mice, as assessed by 5'-bromo-2'deoxyuridine incorporation and proliferating cell nuclear antigen localization. Since Wnt signaling is a major factor affecting periosteal proliferation, we evaluated Wnt signaling in the periosteum. The expression levels of ß-catenin and Lef-1 were increased in the periosteal osteoprogenitors. Moreover, the mRNA levels of ß-catenin, cyclin D1, Lef-1, and Axin2, all of which are Wnt target genes, were significantly increased in the periosteum of both Smad4 (Col) and Osx (Col) mice. These results indicated that extracellular proteins secreted by mature osteoblasts and osteocytes suppress the proliferation of periosteal osteoprogenitors by blocking Wnt signaling in a paracrine manner. Our data suggest a new concept of periosteal bone healing and periosteal bone formation.

Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability.

Regulation of osteoblast and osteocyte viability is essential for bone homeostasis. Smad4, a major transducer of bone morphogenetic protein and transforming growth factor-ß signaling pathways, regulates apoptosis in various cell types through a mitochondrial pathway. However, it remains poorly understood whether Smad4 is necessary for the regulation of osteoblast and osteocyte viability. In this study, we analyzed Smad4Δ(Os) mice, in which Smad4 was subjected to tissue-specific disruption under the control of the 2.3-kb Col1a1 promoter, to understand the functional significance of Smad4 in regulating osteoblast/osteocyte viability during bone formation and remodeling. Smad4Δ(Os) mice showed a significant increase in osteoblast number and osteocyte density in the trabecular and cortical regions of the femur, whereas osteoclast activity was significantly decreased. The proliferation of osteoblasts/osteocytes did not alter, as shown by measuring 5'-bromo-2'deoxyuridine incorporation. By contrast, the percentage of TUNEL-positive cells decreased, together with a decrease in the Bax/Bcl-2 ratio and in the proteolytic cleavage of caspase 3, in Smad4Δ(Os) mice. Apoptosis in isolated calvaria cells from Smad4Δ(Os) mice decreased after differentiation, which was consistent with the results of the TUNEL assay and western blotting in Smad4Δ(Os) mice. Conversely, osteoblast cells overexpressing Smad4 showed increased apoptosis. In an apoptosis induction model of Smad4Δ(Os) mice, osteoblasts/osteocytes were more resistant to apoptosis than were control cells, and, consequently, bone remodeling was attenuated. These findings indicate that Smad4 has a significant role in regulating osteoblast/osteocyte viability and therefore controls bone homeostasis.

Accumulation of 23 kDa lipocalin during brain development and injury in Hyphantria cunea.

The cDNA corresponding to a novel lipocalin was identified from the fall webworm, Hyphantria cunea. This lipocalin cDNA encodes a 194 residue protein with a calculated molecular mass of 23 kDa. Sequence analyses revealed that the 23 kDa lipocalin cDNA is most similar to Drosophila lazarillo, human apolipoprotein D, and Bombyrin. Northern blot analyses showed that 23 kDa lipocalin transcript is expressed in the whole body only in 4- and 6-day-old pupae. By Western blot analysis it was confirmed that 23 kDa lipocalin is mainly accumulated in brain and subesophageal ganglion, though it is detected in a small amount in fat body and epidermis of Hyphantria cunea. The accumulation of 23 kDa lipocalin in brain tissue was upregulated in response to injury. The putative function of 23 kDa lipocalin in brain is discussed.