Histological and immunohistochemical studies of the fungiform and the circumvallate papillae through the life stages from 6- to 72-week-old Sprague-Dawley male rats.

Taste sensitivity decreases with age. Therefore, we investigated the histological and immunohistochemical changes in the receptive fields circumvallate papilla (CvP) and fungiform papilla (FfP) to explore the mechanism underlying age-related changes in taste sensitivity in 6- to 72-week-old rats. We analyzed papilla size, the thickness of the keratin layer of the papilla and stratified squamous epithelium, taste bud size, the keratin layer around the taste pores in the CvP and FfP, and the number and distribution of taste buds in the CvP coronal section. We further assessed the expression of marker proteins for Type II and III cells, phospholipase C subtype beta 2 (PLCß2), and synaptosomal-associated protein 25 (SNAP-25). The cellular activity of these taste cells was examined through co-localization with the senescence cell marker protein-30 (SMP30). There were no differences in the number of taste bud sections in the CvP among the age groups. However, the size of the CvP increased and the density of the taste bud area in the CvP area decreased with increasing age. In contrast, the number of cells with co-expression of SMP30, PLCß2, and SNAP-25 decreased with age. Furthermore, the morphological structures of the CvP, FfP, and taste buds in these regions changed with age, but not the overall taste bud number in the CvP coronal section. The decrease in cell count with co-expression of SMP30 and PLCß2, or SNAP-25 may indicate reduced cellular functions of taste cells with aging.

Lectin histochemistry of posterior lingual glands of developing rats.

The posterior lingual glands are classified as Weber and von Ebner glands. Glycans play an important role in salivary glands. Although the distribution of glycans can explain functional diversity and variation, there are many unknowns in the developing rat posterior lingual glands. The purpose of this study was to elucidate the relationship between the development and function of the posterior lingual gland in rats by histochemical analysis using lectins that bind to sugar residues. In adult rats, Arachis hypogaea (PNA), Glycine maximus (SBA), and Triticum vulgaris (WGA) were associated with serous cells and Dolichos biflorus (DBA) with mucous cells. In both Weber's and von Ebner's glands, all 4 lectins were bound to serous cells in early development, but as development progressed, DBA disappeared in serous cells and only the DBA remained in mucous cells. These results suggest that Galß (1,3) > Galß(1,4) > Gal, αGalNAc > αGal > ßGalNAc, NeuAc > (GalNAc)2-3>>>GlcNAc, and GalNAcα(1,3) are present in the early stage of development, but that GalNAcα(1,3) disappear in serous cells and only GalNAcα(1,3) are localized in mucous cells after maturation. These results indicate that Weber glands function as serous glands in the early postnatal stage when von Ebner glands have not matured.

Quantitative morphometric analysis of molar teeth and alveolar bone using micro-computed tomography in aged mice.

OBJECTIVES: Irreversible morphological regressions of the teeth or related structures in older people can diminish their overall health. However, research on human aging in dentistry is complicated by several confounding factors. In this study, we conducted a morphometric analysis of the mandibular second molars and surrounding alveolar bone in C57BL/6 mice to evaluate age-related changes in the oral cavity. METHODS: The animals were divided into five groups based on their age: 4 weeks (juvenile mice; n = 5); 20 weeks (n = 7), 50 weeks (n = 5), 77 weeks (n = 7), and 100 weeks (n = 5); changes were evaluated using micro-computed tomography. RESULTS: The molars of juvenile mice had sharp and pointed cusps and presented maximum heights. With age and occlusal wear, the cusp heights demonstrated a significant decrease (up to 75%) until the last stage of life. Conversely, apparent lesions were not observed on the basal portion of the crown, even in the most heavily worn teeth. The roots of the molars continued to grow in length at 4 weeks of age. Alveolar bone resorption begins to occur in middle age and continues throughout life. The proportion of vertical bone loss reached approximately 40% of the entire root length, demonstrating a remarkable increase between weeks 77 and 100. CONCLUSIONS: Overall, these morphological changes were similar to those observed in humans. Therefore, it might be appropriate to use aged mice as an experimental model for basic and clinical research in geriatric dentistry.

Senescence-accelerated mouse prone 8 (SAMP8) mice exhibit reduced entoconid in the lower second molar.

OBJECTIVE: The position and size of the major cusps in mammalian molars are arranged in a characteristic pattern that depends on taxonomy. In humans, the cusp which locates distally within each molar is smaller than the mesially located cusp, which is referred to as "distal reduction". Although this concept has been well-recognized, it is still unclear how this reduction occurs. Current study examined whether senescence-accelerating mouse prone 8 (SAMP8) mice could be a possible animal model for studying how the mammalian molar cusp size is determined. DESIGN: SAMP8 mice were compared with parental control (SAMR1) mice. Microcomputed tomography images of young and aged mice were captured to observe molar cusp morphologies. Cusp height from cement-enamel junction and mesio-distal length of molars were measured. The statistical comparison of the measurements was performed by Mann-Whitney U test. RESULTS: SAMP8 mice showed reduced development of the disto-lingual cusp (entoconid) of lower second molar when compared with SAMR1 mice. The enamel thickness and structure was disturbed at entoconid, and aged SAMP8 mice displayed severe wear of the entoconid in lower second molar. These phenotypes were observed on both sides of the lower second molar. CONCLUSIONS: In addition to the general senescence phenotype observed in SAMP8 mice, this strain may genetically possess molar cusp phenotypes which is determined prenatally. Further, SAMP8 mice would be a potential model strain to study the genetic causes of the distal reduction of molar cusp size.

Deficiency of C1q/TNF-related protein 3 (CTRP3) decreases adipose tissue weight in diet-induced obesity mice.

Adipokines are important regulators of lipid and glucose metabolism. A family of adiponectin paralogs is known as C1q and tumor necrosis factor (TNF)-related proteins (CTRPs). One line of Ctrp3-deficient mice shows reduced liver size in response to obesity. We generated and characterized another line of Ctrp3 knockout (KO) mice to reveal novel physiological functions of CTRP3. Interestingly, high fat diet (HFD)-fed Ctrp3 KO mice displayed a decrease in the epididymal white adipose tissue (WAT) weight to total body weight ratio. Histologically, adipocyte size was significantly smaller in the epididymal WAT of HFD-fed Ctrp3 KO mice than wild-type (WT) controls. The expression of several genes involved in lipogenesis, lipolysis and adipogenesis in the epididymal WAT of Ctrp3 KO mice fed a HFD was decreased. The present findings provide new insight into the role of CTRP3 as adipokine in the regulation of adipose tissue in obesity.

Kruppel-like factor 4 upregulates matrix metalloproteinase 13 expression in chondrocytes via mRNA stabilization.

Matrix metalloproteinase 13 (MMP13) is indispensable for normal skeletal development and is also a principal proteinase responsible for articular joint pathologies. MMP13 mRNA level needs to be tightly regulated in both positive and negative manners to achieve normal development and also to prevent joint destruction. We showed previously that Kruppel-like factor 4 (KLF4) strongly induces the expression of members of the MMP family of genes including that for MMP13 in cultured chondrocytes. Through expression-based screening of approximately 400 compounds, we identified several that efficiently downregulated MMP13 gene expression induced by KLF4. Compounds grouped as topoisomerase inhibitors (transcriptional inhibitors) downregulated MMP13 expression levels, which proved the validity of our screening method. In this screening, trichostatin A (TSA) was identified as one of the most potent repressors. Mechanistically, increased MMP13 mRNA levels induced by KLF4 were not mainly caused by increased rates of RNA polymerase II-mediated MMP13 transcription, but arose from escaping mRNA decay. TSA treatment almost completely blunted the effect of KLF4. Importantly, KLF4 was detected in chondrocytes at the joint destruction sites in a rodent model of osteoarthritis. Our results partially explain how KLF4 regulates numerous proteinase gene expressions simultaneously in chondrocytes. Also, these observations suggest that modulation of KLF4 activity or expression could be a novel therapeutic target for osteoarthritis.

Osteoblastic lysosome plays a central role in mineralization.

Mineralization is the most fundamental process in vertebrates. It is predominantly mediated by osteoblasts, which secrete mineral precursors, most likely through matrix vesicles (MVs). These vesicular structures are calcium and phosphate rich and contain organic material such as acidic proteins. However, it remains largely unknown how intracellular MVs are transported and secreted. Here, we use scanning electron-assisted dielectric microscopy and super-resolution microscopy for assessing live osteoblasts in mineralizing conditions at a nanolevel resolution. We found that the calcium-containing vesicles were multivesicular bodies containing MVs. They were transported via lysosome and secreted by exocytosis. Thus, we present proof that the lysosome transports amorphous calcium phosphate within mineralizing osteoblasts.

Kruppel-Like Factor 4 represses osteoblast differentiation via ciliary Hedgehog signaling.

Primary cilia are appendages observed in most types of cells, and serve as cellular antennae for sensing environmental signals. Evidence is accumulating that correct ciliogenesis and ciliary functions are indispensable for normal skeletal development by regulating signaling pathways important for bone development. However, whether ciliogenesis is regulated by bone-related factors in osteoblasts is largely unknown. Here we show that Kruppel-Like Factor 4 (KLF4), which is known to repress osteoblast differentiation, supports the formation and maintenance of cilia in cultured osteoblasts; however, the length of the cilia observed in KLF4-induced cells were significantly shorter compared to the control cells. Basal Hedgehog signaling was repressed by KLF4. Significantly, activating Hedgehog signaling using a Smoothened agonist significantly rescued osteoblast mineralization and osteoblastic gene expressions. Global gene expression analysis showed that KLF4 induced number of genes including the nuclear receptor, Pregnane X receptor (PXR), and PXR repressed calvarial osteoblast mineralization and repressed Gli1 expression similar as the effect observed by inducing KLF4. Our results implicate that KLF4 plays important roles for maintaining osteoblasts in an immature state by repressing basal activation of the Hedgehog signaling.

Decreased sensory nerve excitation and bone pain associated with mouse Lewis lung cancer in TRPV1-deficient mice.

Bone pain is one of the most common and life-limiting complications of cancer metastasis to bone. Although the mechanism of bone pain still remains poorly understood, bone pain is evoked as a consequence of sensitization and excitation of sensory nerves (SNs) innervating bone by noxious stimuli produced in the microenvironment of bone metastases. We showed that bone is innervated by calcitonin gene-related protein (CGRP)+ SNs extending from dorsal root ganglia (DRG), the cell body of SNs, in mice. Mice intratibially injected with Lewis lung cancer (LLC) cells showed progressive bone pain evaluated by mechanical allodynia and flinching with increased CGRP+ SNs in bone and augmented SN excitation in DRG as indicated by elevated numbers of pERK- and pCREB-immunoreactive neurons. Immunohistochemical examination of LLC-injected bone revealed that the tumor microenvironment is acidic. Bafilomycin A1, a selective inhibitor of H+ secretion from vacuolar proton pump, significantly alleviated bone pain, indicating that the acidic microenvironment contributes to bone pain. We then determined whether the transient receptor potential vanilloid 1 (TRPV1), a major acid-sensing nociceptor predominantly expressed on SNs, plays a role in bone pain by intratibially injecting LLC cells in TRPV1-deficient mice. Bone pain and SN excitation in the DRG and spinal dorsal horn were significantly decreased in TRPV1 -/- mice compared with wild-type mice. Our results suggest that TRPV1 activation on SNs innervating bone by the acidic cancer microenvironment in bone contributes to SN activation and bone pain. Targeting acid-activated TRPV1 is a potential therapeutic approach to cancer-induced bone pain.

Kruppel-like factor 4 regulates matrix metalloproteinase and aggrecanase gene expression in chondrocytes.

Kruppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays crucial roles during the development and maintenance of multiple organs. We and others have previously shown that KLF4 is involved in bone modeling and remodeling but roles played by KLF4 during skeletogenesis are still not fully understood. Here, we show that KLF4 is expressed in the epiphyseal growth plate and articular chondrocytes. Most articular chondrocytes expressed KLF4 in embryos but it localized only in a subset of superficial zone cells in postnatal mice. When KLF4 was overexpressed in chondrocytes in vitro, it severely repressed chondrocytic gene expressions. Global gene expression profiling of KLF4-transduced chondrocytes revealed matrix degrading proteinases of the matrix metalloproteinase and disintegrin and metalloproteinase with thrombospondin-1 domain families within the group of upregulated genes. Proteinase induction by KLF4 was alleviated by Trichostatin A treatment suggesting the possible involvement of epigenetic mechanisms on proteinase induction by KLF4. These results indicate the possible involvement of KLF4 in physiological and pathological aspects during cartilage development and maintenance.

c-Fos expression in the parabrachial nucleus following intraoral bitter stimulation in the rat with dietary-induced zinc deficiency.

Zinc deficiency causes various symptoms including taste disorders. In the present study, changes in expression of c-Fos immunoreactivity in neurons of the parabrachial nucleus (PBN), one of the relay nuclei for transmission of gustatory information, after bitter stimulation to the dorsal surface of the tongue were examined in zinc-deficient rats. Experimental zinc-deficient animals were created by feeding a low-zinc diet for 4weeks, and showed the following symptoms of zinc deficiency: low body weight, low serum zinc content and behavioral changes to avoid bitter stimulation. In normal control animals, intraoral application of 1mM quinine caused increased numbers of c-Fos-immunoreactive (c-Fos-IR) neurons in the external lateral subnucleus and external medial subnucleus of the PBN (elPBN and emPBN, respectively) compared with application of distilled water. However, in the zinc-deficient animals, the numbers of c-Fos-IR neurons in the elPBN and emPBN did not differ significantly between application of quinine and distilled water. After feeding the zinc-deficient animals a normal diet for 4weeks, the symptoms of zinc deficiency recovered, and the expression of c-Fos-IR neurons following intraoral bitter stimulation became identical to that in the normal control animals. The present results indicate that dietary zinc deficiency causes alterations to neuronal activities in the gustatory neural circuit, and that these neuronal alterations can be reversed by changing to a normal diet.

Syntaxin 4a Regulates Matrix Vesicle-Mediated Bone Matrix Production by Osteoblasts.

Osteoblasts secrete matrix vesicles and proteins to bone surfaces, but the molecular mechanisms of this secretion system remain unclear. The present findings reveal the roles of important genes in osteoblasts involved in regulation of extracellular matrix secretion. We especially focused on "soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor" (SNARE) genes and identified notable Syntaxin 4a (Stx4a) expression on the basolateral side of the plasma membrane of osteoblasts. Furthermore, Stx4a overexpression was found to increase mineralization by osteoblastic cells, whereas Stx4a knockdown reduced levels of mineralization. Also, BMP-4 and IGF-1 induced the localization of Stx4a to the basolateral side of the cells. To examine the function of Stx4a in osteoblasts, we generated osteoblast-specific Stx4a conditional knockout mice, which demonstrated an osteopenic phenotype due to reduced matrix secretion. Bone mineral density, shown by peripheral quantitative computed tomography (pQCT), was reduced in the femur metaphyseal and diaphyseal regions of Stx4a osteoblast-specific deficient mice, whereas bone parameters, shown by micro-computed tomography (µCT) and bone histomorphometric analysis, were also decreased in trabecular bone. In addition, primary calvarial cells from those mice showed decreased mineralization and lower secretion of matrix vesicles. Our findings indicate that Stx4a plays a critical role in bone matrix production by osteoblasts. © 2016 American Society for Bone and Mineral Research.

C1q/TNF-related protein 3 expression and effects on adipocyte differentiation of 3T3-L1 cells.

Adipose tissue-derived adipokines influence a number of organs critical for energy homeostasis and metabolism. One of the most extensively studied adipokines is adiponectin, which exerts anti-diabetic, anti-inflammatory, and anti-atherogenic functions on various cell types. CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor-related protein (CTRP) superfamily. CTRP3 reduces hepatic triglyceride levels in diet-induced obese (DIO) mice. However, the physiological role of CTRP3 in adipocytes is largely unknown. In the course of our investigation of expression profiles of CTRPs during adipocyte differentiation, we found that CTRP3 expression pattern is different from that previously reported. Therefore, we examined the effect of CTRP3 on adipogenesis using 3T3-L1 cells. The expression level of CTRP3 was markedly decreased during the differentiation of 3T3-L1 cells. Recombinant CTRP3 (rCTRP3) treatment significantly reduced intracellular lipid content and decreased expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα), adiponectin, and fatty acid binding protein 4 (FABP4) in 3T3-L1 cells. Furthermore, rCTRP3 induced the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Akt in differentiated 3T3-L1 adipocytes. These results suggest that CTRP3 may negatively regulate lipid metabolism during adipocyte differentiation.

Immunohistochemical localization of SNARE core proteins in intrapulpal and intradentinal nerve fibers of rat molar teeth.

OBJECTIVE: The present study was designed to elucidate whether three soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) core proteins, syntaxin-1, synaptosomal-associated protein of 25kDa (SNAP-25), and vesicle-associated membrane protein-2 (VAMP-2), are present in the dental pulp of the rat molar at both the light and electron microscopic levels. DESIGN: Immunohistochemistry for protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, syntaxin-1, SNAP-25, and VAMP-2 was performed on decalcified rat molars for light and electron microscopic analyses. Double-immunolabeling of PGP 9.5 and the SNARE core proteins, as well as combinations of the SNARE core proteins, was also carried out. RESULTS: PGP 9.5-immunoreactive nerve fibers ran toward the coronal region, ramified at the subodontoblast layer, and formed the subodontoblastic nerve plexus. Most nerve fibers penetrated the predentin and dentin along the dentinal tubules. Most, if not all, nerve fibers displayed immunoreactivity for syntaxin-1, SNAP-25, and VAMP-2. Immunoelectron microscopic analyses confirmed the presence of immunoreactivity for the SNARE core proteins within the intradental axonal elements. CONCLUSIONS: The present findings suggest that, since SNARE core proteins participate in the docking and exocytosis of synaptic vesicles in the central nervous system, they may contribute to vesicle exocytosis from the dental nerve fibers even though there are no apparent synapses.

Postnatal morphological and lectin histochemical observation of the submucosal glands of rat nasopharynx.

The development of submucosal glands of rat nasopharynx was studied with respect to their morphological maturation and glycoprotein alterations during the postnatal period. This study examined the histological morphology with hematoxylin-eosin and the binding pattern of lectins, soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin-I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), and succinylated WGA (sucWGA) on frozen sections from newborn into adulthood. At birth, nasopharyngeal glands consisted of rudimentary secretory units which by postnatal day 3 (PN3) showed the characteristic features of salivary glands comprised of mixed mucous and serous cells. With maturation, serous cells increased in number and were arranged in clusters. Lectin reactivity at birth was detected at the acinar cell basal membranes for DBA, SBA, VVA, UEA-1 and PNA. At PN3, lectins labeled the apical cytoplasm and basolateral membranes of mucous cells and progressively with maturation, extended from the apical to basal portions of the cytoplasm with variable reactivity of VVA, PNA and sucWGA. Serous cells were labeled by UEA-1 starting from PN10 and also by PNA in adults. Ducts showed variable lectin reaction on the luminal membrane with strong reactivity of DBA and UEA-1 at PN21. Taken together, lectin histochemistry indicated the transitional occurrence of glycoproteins depending on the stage of maturation of the glands. Moreover, these results emphasize the difference in the morphology and lectin histochemistry between the nasopharyngeal and palatine glands.

Fate mapping of Trps1 daughter cells during cardiac development using novel Trps1-Cre mice.

Tricho-rhino-phalangeal syndrome (TRPS) is a rare congenital disorder that is characterized by abnormal hair growth and skeletal deformities. These result in sparse hair, short stature, and early onset of joint problems. Recent reports have shown that a relatively high proportion of patients with TRPS exhibit a broad range of congenital heart defects. To determine the regulation of Trps1 transcription in vivo, we generated novel transgenic mice, which expressed Cre recombinase under the murine Trps1 proximal promoter sequence (Trps1-Cre). We crossed these mice with Cre reporter mice to identify Trps1 daughter cells. Labeled cells were observed in the appendicular joint tissue, dermal papilla of the hair follicles, cardiac valves, aortic sinus, atrial walls, and the interventricular septum. In situ analysis showed restricted Trps1 expression, which was observed in endocardial cushions of the outflow tract, and in leaflets of all mature cardiac valves. These results suggest that the Trps1 proximal promoter sequence contains some of the tissue-specific Trps1 regulatory region. Further, our findings partially explain why patients with TRPS show a broad range of congenital cardiac defects, although Trps1 expression is observed in a more restricted fashion. genesis 54:379-388, 2016. © 2016 Wiley Periodicals, Inc.

A novel adipokine C1q/TNF-related protein 3 is expressed in developing skeletal muscle and controls myoblast proliferation and differentiation.

Several hormones and growth factors, including adipokines, play important roles during muscle development and regeneration. CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor-related protein (CTRP) superfamily. CTRP3 is a novel adipokine previously reported to reduce glucose output in hepatocytes and lower glucose levels in mice models. In the present study, we provide the first evidence for a physiological role of the CTRP3 in myogenesis using C2C12 myoblasts. CTRP3 was expressed in developing skeletal muscle tissues, and the expression level of CTRP3 was increased during myogenic differentiation of C2C12 cells. Recombinant CTRP3 (rCTRP3) promoted the proliferation of undifferentiated C2C12 myoblasts and this response required activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. In contrary, rCTRP3 inhibited myogenic differentiation and fusion of C2C12 cells by suppressing the expression of myogenic marker genes (myogenin and myosin heavy chain). CTRP3 mRNA expression was increased in C2C12 myoblasts treated with transforming growth factor-ß3 (TGF-ß3), suggesting that TGF-ß3 is one of the extracellular factors regulating CTRP3 expression during myogenesis. These results indicate a novel physiological role for CTRP3 during skeletal myogenesis.

Changes in the Distribution of Periodontal Nerve Fibers during Dentition Transition in the Cat.

The periodontal ligament has a rich sensory nerve supply which originates from the trigeminal ganglion and trigeminal mesencephalic nucleus. Although various types of mechanoreceptors have been reported in the periodontal ligament, the Ruffini ending is an essential one. It is unknown whether the distribution of periodontal nerve fibers in deciduous teeth is identical to that in permanent teeth or not. Moreover, morphological changes in the distribution of periodontal nerve fibers during resorption of deciduous teeth and eruption of successional permanent teeth in diphyodont animals have not been reported in detail. Therefore, in this study, we examined changes in the distribution of periodontal nerve fibers in the cat during changes in dentition (i.e., deciduous, mixed and permanent dentition) by immunohistochemistry of protein gene product 9.5. During deciduous dentition, periodontal nerve fibers were concentrated at the apical portion, and sparsely distributed in the periodontal ligament of deciduous molars. During mixed dentition, the periodontal nerve fibers of deciduous molars showed degenerative profiles during resorption. In permanent dentition, the periodontal nerve fibers of permanent premolars, the successors of deciduous molars, increased in number. Similar to permanent premolars, the periodontal nerve fibers of permanent molars, having no predecessors, increased in number, and were densely present in the apical portion. The present results indicate that the distribution of periodontal nerve fibers in deciduous dentition is almost identical to that in permanent dentition although the number of periodontal nerve fibers in deciduous dentition was low. The sparse distribution of periodontal nerve fibers in deciduous dentition agrees with clinical evidence that children are less sensitive to tooth stimulation than adults.

O/W nano-emulsion formation using an isothermal low-energy emulsification method in a mixture of polyglycerol polyricinoleate and hexaglycerol monolaurate with glycerol system.

We investigated how phase behavior changes by replacing water with glycerol in water/mixture of polyglycerol polyricinoleate (PGPR) and hexaglycerol monolaurate (HGML) /vegetable oil system, and studied the effect of glycerol on o/w nano-emulsion formation using an isothermal low-energy method. In the phase behavior study, the liquid crystalline phase (Lc) + the sponge phase (L3) expanded toward lower surfactant concentration when water was replaced with glycerol in a system containing surfactant HLP (a mixture of PGPR and HGML). O/W nano-emulsions were formed by emulsification of samples in a region of Lc + L3. In the glycerol/surfactant HLP/vegetable oil system, replacing water with glycerol was responsible for the expansion of a region containing Lc + L3 toward lower surfactant concentration, and as a result, in the glycerol/surfactant HLP/vegetable oil system, the region where o/w nano-emulsions or o/w emulsions could be prepared using an isothermal low-energy emulsification method was wide, and the droplet diameter of the prepared o/w emulsions was also smaller than that in the water/surfactant HLP/vegetable oil system. Therefore, glycerol was confirmed to facilitate the preparation of nano-emulsions from a system of surfactant HLP. Moreover, in this study, we could prepare o/w nano-emulsions with a simple one-step addition of water at room temperature without using a stirrer. Thus, the present technique is highly valuable for applications in several industries.

Kruppel-like factor 4 expression in osteoblasts represses osteoblast-dependent osteoclast maturation.

Kruppel-like factor 4 (KLF4) is a zinc-finger-type transcription factor with a restricted expression pattern during skeletal development. We have previously shown that KLF4 represses osteoblast mineralization concomitant with a down-regulation in the expression of a number of osteoblastic genes, both in vivo and in vitro. In addition to the cell-autonomous effects of KLF4 in osteoblasts, transgenic osteoblastic-KLF4 mice show severe defects in osteoclast maturation. Wild-type bone-marrow-derived macrophages co-cultured with KLF4-expressing osteoblasts exhibit reduced formation of multinuclear osteoclasts as compared with control cultures overexpressing green fluorescent protein. Significantly, the transduction of Runx2, a master regulator of osteoblastogenesis, together with KLF4 into osteoblasts restores the reduction in osteoclastogenesis induced by KLF4 alone. Various extracellular matrix molecules are down-regulated by KLF4 overexpression but this down-regulation can be partially restored by the co-transduction of Runx2. These results suggest that osteoblastic-KLF4 affects osteoclast maturation by regulating cell-matrix interactions and reinforce the importance of the regional down-regulation of KLF4 expression in the subset of osteoblasts for normal skeletal modeling and remodeling.