Negative regulation of NaF-induced apoptosis by Bad-CAII complex.

Fluoride is used to prevent caries in dentistry. However, its mechanism of cytotoxicity induction is unclear. This study was undertaken to determine whether sodium fluoride (NaF) induces apoptosis in human oral cells and if so, whether Bad protein is involved in the process. NaF showed higher cytotoxicity and apoptosis-inducing activity against human oral squamous cell carcinoma cells (HSC-2) than against human gingival fibroblasts (HGF). Western blot analysis showed that NaF enhanced the expression and dephosphorylation of Bad protein. This study demonstrates for the first time that Bad protein forms a complex with carbonic anhydrase II (CAII), and NaF stimulates the detachment of CAII from the Bad-CAII complex and the replacement by the formation of Bad-Bcl-2 complex. Knockdown of Bad and CAII mRNA by siRNA inhibited and enhanced the NaF-induced caspase activation, respectively. The present study suggests that CAII negatively regulates the NaF-induced apoptosis by forming a complex with Bad.

Possible link between glycolysis and apoptosis induced by sodium fluoride.

Fluoride has been used to prevent caries in the dentition, but the possible underlying mechanisms of cytotoxicity induction by this compound are still unclear. Since fluoride is known as an inhibitor of glycolytic enzymes, we investigated the possible connection between NaF-induced apoptosis and glycolysis in human promyelocytic leukemia HL-60 cells. NaF-induced apoptotic cell death is characterized by caspase activation, internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, and production of apoptotic bodies. Higher activation of caspases-3 and -9, as compared with that of caspase-8, suggested the involvement of an extrinsic pathway. Utilization of glucose was nearly halted by NaF, whereas that of glutamine was rather enhanced. NaF enhanced the expression of Bad protein, but not that of Bcl-2 and Bax proteins, and reduced HIF-1alpha mRNA expression. Analysis of these data suggests a possible link between glycolysis and apoptosis.

Transient occurrence of 27 kDa heat-shock protein in the terminal tubule cells during postnatal development of the rat submandibular gland.

It has been suggested that the 27 kDa heat-shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27-immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3-week-old rats for 7 days, the number of Hsp27-positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3-4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants.

[Expression and localization of cell growth factors in the salivary gland: a review].

The salivary gland secretes not only digestive enzymes but also various cell growth factors. Especially in rodents, granular ducts which develop between striated and intercalated ducts, are known to secrete epidermal growth factor (EGF) and nerve growth factor (NGF). Out of newly discovered growth factors, we have examined hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2) and have demonstrated their expression and localization in the rat submandibular gland. HGF and TGF-beta show a very similar distribution pattern to EGF, i.e., exclusive localization in the secretory granules of granular duct cells. These factors are suggested to function as i) exocrine salivary factors, or ii) endocrine factors after reabsorption. In contrast, FGF-2 shows a different pattern of cellular and subcellular localization from EGF and others. FGF-2 is localized in cytoplasm of striated and excretory duct cells and pillar cells in granular ducts, but not in secretory granules of granular duct cells. Since the receptor for FGF also present in the same cells as its ligand, FGF is suggested to function in the salivary gland as an autocrine/paracrine factor. The possible physiological roles of salivary growth factors in the digestive organs are discussed based on our own data and an extensive literature.

The role of cyclic AMP response element-binding protein in testosterone-induced differentiation of granular convoluted tubule cells in the rat submandibular gland.

The postnatal development of granular convoluted tubules (GCT) in the duct system of the rodent submandibular gland is known to be androgen-dependent, but the underlying molecular mechanism is unclear. To test the possible role of the transcription factor, cyclic AMP response element-binding protein (CREB), in the androgen-induced differentiation of GCT, the effect of testosterone on the expression and localization of epidermal growth factor (EGF), a marker of GCT cells, and of CREB was examined in the submandibular glands of immature 3-week-old rats. Northern blotting demonstrated increases in both EGF and CREB mRNA 1-4 days after testosterone administration. Immunoprecipitation also indicated that CREB protein was increased in amount with testosterone administration, and that induced CREB was phosphorylated at the serine residue as in the active form of CREB. In situ hybridization demonstrated that cells with CREB mRNA signal first appeared in the distal portions of striated ducts at 1 day and had increased in number by 4 days after giving testosterone, when cells with EGF mRNA signal became evident in the same duct portions. Immunohistochemistry also showed the occurrence of CREB protein in the nuclei of duct epithelial cells before their differentiation into EGF-positive GCT cells. Finally, pieces of submandibular gland from immature rats were cultured in vitro and their expression of EGF mRNA analysed by the reverse transcriptase-polymerase chain reaction. Testosterone in the medium caused a marked enhancement of EGF expression in the gland in 1-4 days, which was attenuated by simultaneous administration of the antisense oligonucleotide for CREB as well as that for the androgen receptor. These results suggest the CREB is upregulated by androgen and has a crucial role in androgen-induced differentiation of GCT in the duct system of the rat submandibular gland.

Constitutive expression of the 27-kDa heat-shock protein in neurons and satellite cells in the peripheral nervous system of the rat.

By use of reverse transcriptase-polymerase chain reaction, abundant expression of the mRNA of 27 kDa heat shock protein (Hsp27) was revealed in the sympathetic and parasympathetic ganglia as well as in the sensory ganglia of unstressed adult rats. In situ hybridization and immunohistochemistry further localized Hsp27 mRNA and protein to both neurons and satellite cells in all types of ganglia examined. Schwann cells in the ganglia and peripheral nerve fibers were devoid of Hsp27 signal. These results suggested that Hsp27 is constitutively expressed in neurons and satellite cells in the entire peripheral nervous system of the rat.

Nerve growth factor (NGF) supports tooth morphogenesis in mouse first branchial arch explants.

Posterior midbrain and anterior hindbrain neuroectoderm trans-differentiate into cranial neural crest cells (CNCC), emigrate from the neural folds, and become crest-derived ectomesenchyme within the mandibular and maxillary processes. To investigate the growth factor requirement specific for the initiation of tooth morphogenesis, we designed studies to test whether nerve growth factor (NGF) can support odontogenesis in a first branchial arch (FBA) explant culture system. FBA explants containing neural-fold tissues before CNCC emigration and the anlagen of the FBA were microdissected from embryonic day 8 (E8) mouse embryos, and cultured for 8 days in medium supplemented with 10% fetal calf serum only, or serum-containing medium further supplemented with either NGF or epidermal growth factor (EGF) at three different concentrations: 50, 100, or 200 ng/ml. Morphological, morphometric, and total protein analyses indicated that growth and development in all groups were comparable. Meckel's cartilage and tongue formation were also observed in all groups. However, odontogenesis was only detected in explants cultured in the presence of exogenous NGF. NGF-supplemented cultures were permissive for bud stage (50 ng/ml) as well as cap stage of tooth morphogenesis (100 and 200 ng/ml). Morphometric analyses of the volume of tooth organs showed a significant dose-dependent increase in tooth volume as the concentration of NGF increased. Whole-mount in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction for Pax9, a molecular marker of dental mesenchyme, further supported and confirmed the morphological data of the specificity and dose dependency of NGF on odontogenesis. We conclude that (1) E8 FBA explants contain premigratory CNCC that are capable of emigration, proliferation, and differentiation in vitro; (2) serum-supplemented medium is permissive for CNCC differentiation into tongue myoblasts and chondrocytes in FBA explants; and (3) NGF controls CNCC cell fate specification and differentiation into tooth organs.

Enhancement by hepatocyte growth factor of bone and cartilage formation during embryonic mouse mandibular development in vitro.

To elucidate the possible roles of hepatocyte growth factor (HGF) in the early development of mouse mandible, HGF was applied to an organ-culture system with chemically defined media. Mandibular arches microdissected from mouse embryos at the 10th day of gestation were cultured for 10 days with or without HGF, HGF plus HGF-receptor (c-met) antisense oligodeoxyribonucleotide, or HGF plus c-met sense oligodeoxyribonucleotide in the media. The cultured mandibles were then analysed, histologically in serial paraffin sections. In the absence of HGF, the tooth organs of bud stage, Meckel's cartilage and the tongue were formed, whereas only a slight amount of bone tissue was formed in the cultured mandible. The expression of intrinsic HGF and c-met in the cultured mandibles was confirmed by reverse transcriptase-polymerase chain reaction. Furthermore, immunohistochemistry demonstrated that both HGF and c-met were localized in areas of the mesenchymal tissue forming bone and cartilage. With HGF in the medium, the volume of both bone and cartilage increased significantly and dose-dependently. HGF also increased the rate of proliferation of osteogenic cells and chondrocytes. Addition of c-met antisense oligodeoxyribonucleotide partially inhibited the HGF-induced enhancement of bone and cartilage formation, whereas addition of c-met sense oligodeoxyribonucleotide had no effect. These results revealed that exogenous HGF enhances bone and cartilage morphogenesis in the cultured mandibles, suggesting physiological roles for intrinsic HGF in the early development of mouse mandible.

Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1.

The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues. Immunohistochemical analysis by use of the anti-MCT1 antibody showed that MCT1 is distributed throughout the upper and lower intestines, especially in the basolateral membrane and, to a lesser extent, in the brush-border membrane. When the transporter gene rat MCT1 was transfected into MDA-MB231 cells, transport of benzoic acid, a model weak organic acid that has been generally believed to be transported across the cell membranes by passive diffusion, and lactic acid in rat MCT1-transfected cells was significantly increased compared with transport in cells transfected with the expression vector pRc-CMV alone (mock cells). The observed transport was pH-dependent and activity increased between pH 7.5 and pH 5.5, whereas pH-dependence in mock cells was moderate. Rat MCT1-mediated benzoic acid uptake was saturable, with an apparent Km value of 3.05 mM. In addition, MCT1 increased the efflux of [14C]benzoic acid from the cells. Several weak organic acids were also transported by rat MCT1. These results show that pH-dependent intestinal absorption of weak organic acids, previously explained in terms of passive diffusion according to the pH-partition hypothesis, is at least partially accounted for by MCT1-mediated transport energized at acidic pH by utilization of the proton gradient as a driving force.

Occurrence and nuclear localization of cAMP response element-binding protein in the post-natal development of the rat submandibular gland.

Cyclic AMP response element-binding protein (CREB) is a 43-kDa polypeptide that binds a cAMP response element located at the 5' promoter region of cAMP regulatory genes. The spatial and temporal distribution of CREB in the postnatal development of the rat submandibular gland was investigated using immunohistochemistry with a specific antibody. At birth, cells of the terminal tubules and ducts in the submandibular gland showed a nuclear CREB immunoreactivity of moderate intensity. At 1-2 weeks after birth, an intense CREB immunoreactivity was localized primarily to acinar cells. When the beta-adrenergic agonist isoproterenol was administered to 2-week-old rats, a twofold transient increase in the number of immunoreactive acinar cells was induced. Beginning 3 weeks after birth, CREB immunoreactivity shifted from acini to the duct system and showed a clear localization in the cells of the intercalated ducts and distal portions of striated ducts, where the granular convoluted tubule develops after 4 weeks. Immunopositive materials were localized exclusively in the nuclei of both acinar and ductal immunoreactive cells. After the development of the granular convoluted tubules, CREB immunoreactivity was absent in the tubule cells and was gradually reduced in intensity over the entire gland. In order to examine a hypothesis that CREB is involved in the initial differentiation of the granular convoluted tubular cells, testosterone was administered to hypophysectomized adult rats. Whereas the tubular cells of hypophysectomized rats showed a complete regression, and no CREB immunoreactivity was found in any acinar or duct cells, administration of testosterone for a few days induced an intense CREB immunoreactivity in the nuclei of duct cells, followed by their differentiation into the granular convoluted tubular cells. These results suggested that CREB is involved not only in the growth and differentiation of acinar cells that are regulated by beta-adrenergic nerves but also in those of the duct system, and especially in the androgen-regulated differentiation of the granular convoluted tubular cells, during the post-natal development of the rat submandibular gland.

Transforming growth factor alpha up-regulates desmin expression during embryonic mouse tongue myogenesis.

Myogenesis is determined by a set of myogenic differentiation factors that are, in turn, regulated by a number of peptide growth factors. During embryonic mouse tongue formation, transforming growth factor alpha (TGF alpha), epidermal growth factor (EGF), and their cognate receptor (EGFR) are co-expressed spatially and temporally with desmin, a muscle-specific structural protein. This investigation tested the hypothesis that TGF alpha directly regulates the myogenic program in developing tongue myoblasts. Mandibular processes from the first branchial arch of embryonic day 10.5 (E10.5) mouse embryos were microdissected and explanted into an organ culture system using serumless chemically defined medium. Exogenous TGF alpha at 10 and 20 ng/ml specifically increased the amount of desmin expression and the number of desmin-positive cells without affecting the general growth and development of the mandibles. This inductive response was detected as early as 2 days after treatment and sustained up to 9 days in culture. EGFR antisense oligonucleotides (30 microM) as well as tyrphostin (80 microM) were able to negate TGF alpha-induced up-regulation of desmin expression. These data indicate that autocrine and/or paracrine action of TGF alpha promotes tongue myogenesis, and that this action is mediated through functional kinase activity of the EGFR. We speculate that the myogenic program in the developing mouse tongue is dependent upon growth factor mediated cell-cell communication of mesenchymal cells originating from the occipital somites and ectomesenchymal cells originating from the cranial neural crest.

Immunolocalization and pharmacological relevance of oligopeptide transporter PepT1 in intestinal absorption of beta-lactam antibiotics.

A polyclonal antibody (anti-PepT1/C) was raised against the rabbit intestinal H(+)-coupled oligopeptide transporter, PepT1. Anti-PepT1/C detected 70-80-kDa protein in crude membranes obtained from rabbit duodenum, jejunum and ileum. PepT1 was localized in the brush-border of the absorptive epithelial cells by subcellular fractionation of membranes on a sucrose density gradient and by immunohistochemistry using light and electron microscopy. Transport activity for cephalosporins and dipeptide expressed in Xenopus laevis oocytes injected with total mRNA obtained from rabbit small intestine was eliminated completely by prehybridization of the mRNA with antisense oligonucleotide against the 5'-coding region of rabbit PepT1 cDNA.

Immunohistochemical localization of two types of fatty acid-binding proteins in rat ovaries during postnatal development and in immature rat ovaries treated with gonadotropins.

BACKGROUND: The ovary of adult rats expresses two types of cytoplasmic fatty acid binding proteins (FABP), i.e., heart FABP (H-FABP) and intestinal 15 kDa protein (I-15P). We studied immunohistochemically the cellular localizations of these FABPs in the ovaries of rats at various postnatal ages and in the ovaries of immature (3-week-old) rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG). METHODS: The cryosections of ovaries were incubated with polyclonal antibodies against H-FABP and I-15P, and the immunoreactions were visualized at both light and electron microscopic levels. RESULTS: The immunoreactivity for H-FABP occurred temporarily in the follicular epithelial (granulosa) cells from 3 days to 2 weeks post partum, and then was localized exclusively to the theca/interstitial gland cells from 2 weeks to adulthood. In contrast, the immunoreactivity for I-15P appeared temporarily in a small subset of theca/interstitial gland cells from 2 to 3 weeks, disappeared at 4 weeks, and was localized exclusively to the corpus luteum cells after the onset of ovulation in the animal around 5 weeks. In the immature rat ovaries induced to ovulate by treatment with gonadotropins, I-15P-immunoreactive cells were first recognized in the luteinized granulosa layer of large preovulatory follicles, and increased in number progressively in the developing corpora lutea after the ovulation. CONCLUSIONS: Two types of FABPs are expressed in distinct steroid-producing cell types of rat ovary, and their expressions seem to be regulated in coincidence with the expressions of respective steroid hormones. These results suggest that FABPs play specific roles in the ovarian hormone synthesis.

Expression and localization of hepatocyte growth factor in rat submandibular gland.

By combination of in situ hybridization and immunohistochemical techniques, the expression of hepatocyte growth factor (HGF) was demonstrated in the submandibular gland of rats. Both the mRNA signal and immunoreactivity for HGF were localized exclusively to the epithelial cells of granular convoluted tubules, whereas they were absent from the other components of the submandibular gland. In the granular convoluted tubule cells, HGF-immunoreactivity was localized to the apical secretory granules, which was further substantiated by immunoelectron microscopy. These results added HGF to the list of many growth factors that are produced in the rat submandibular gland and secreted into the saliva.

Basic fibroblast growth factor in rat salivary glands.

We studied the occurrence and localization of basic fibroblast growth factor (bFGF) in rat salivary glands using a specific monoclonal antibody. It was shown that the extract of rat salivary glands has a pronounced stimulatory activity on the growth of bovine capillary endothelial cells, which is blocked by the addition of an antibody against bFGF. The concentration of bFGF in the submandibular/sublingual gland, as determined by radioimmunoassay, was approximately 80% that in the brain. Immunocytochemistry revealed bFGF-immunoreactivity localized primarily in the epithelial cells lining the striated ducts and excretory ducts of the parotid, sublingual and submandibular glands. In addition, intense bFGF-immunoreactivity was observed in the granular convoluted tubule of the submandibular gland, localized predominantly in the agranular pillar cells, which lay in small numbers among the majority of weakly immunostained cells containing many apical secretory granules. At the electron-microscopic level, the immunoreactive material was distributed diffusely in the cytoplasmic matrix and nuclei of all immunoreactive cells, whereas it was absent from all cytoplasmic organelles including the secretory granules. These results indicate that bFGF is localized in different cellular and subcellular compartments from those of other growth factors in the duct system of rat salivary glands.

Expression, localization and developmental regulation of insulin-like growth factor I mRNA in rat submandibular gland.

The mRNA for insulin-like growth factor I (IGF-I) in the submandibular gland of mature and developing rats was examined by Northern blotting and in situ hybridization with an oligonucleotide probe. In the mature adult rat, IGF-I mRNA was expressed at a higher level in the submandibular gland than in the liver, and was localized primarily in the granular convoluted tubule (GCT) cells. A 4.7-kb mRNA on Northern blots, which was expressed only slightly in the liver, proved to be the predominant size species of IGF-I transcripts in the GCT cells, and its level increased progressively with the postnatal development of GCTs in the gland. In addition, a 1.8-kb mRNA for IGF-I was also expressed at a much lower level throughout the acinar and duct systems, irrespective of age. These results have shed a light on the status of IGF-I as one of the many biologically active polypeptides that are produced in the rodent submandibular gland.

Immunocytochemical localization of basic fibroblast growth factor in the rat pituitary gland.

Immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in the rat pituitary gland at light and electron microscopic levels using a specific monoclonal antibody. In the anterior lobe, bFGF-immunoreactivity was found mostly in a cell population without secretory granules, which was identified as the folliculo-stellate (FS) cell by its ultra-structure and expression of S-100 protein. The subcellular localization of the immunoreactivity in FS cell was strictly nuclear. Besides the FS cell, a minor cell population containing small granules of 60-100 nm diameter, which seemed to represent immature endocrine cells, was immunostained in the nuclei with the anti-bFGF antibody. In the intermediate lobe, a subpopulation of the S-100 protein-positive cells lining the pituitary cleft was immunoreactive for bFGF. These results imply that the agranular cell types are the main producer and/or target of rat pituitary bFGF, and that bFGF may play a role in the differentiation of pituitary endocrine cells.

Expression and localization of intestinal 15 kDa protein in the rat.

Rat intestinal 15 kDa protein (I-15P) is highly homologous to porcine gastrotropin. We studied the occurrence, distribution and subcellular localization of I-15P in the entire rat body, using the immunocytochemistry to localize protein and in situ hybridization to localize mRNA. Both techniques demonstrated the expression of I-15P in the enterocytes of ileum, luteal cells of ovary and a subpopulation of steroid-endocrine cells of adrenal gland. Immuno-electron microscopy further demonstrated that I-15P is localized in both the cytoplasmic and nuclear matrix regions of these cells. The present results suggest roles of I-15P not only in the transport of bile salts but also in the metabolisms of certain steroid hormones.

Cloning of a cDNA encoding rat intestinal 15 kDa protein and its tissue distribution.

A cDNA encoding rat intestinal 15 kDa protein was isolated and sequenced from a rat ileum-specific cDNA library. This cDNA was found to contain an open reading frame of 384 nucleotides as well as 5' (27 nucleotides) and 3' (46 nucleotides) non-coding regions. The deduced sequence of 127 amino acids was identical to that of rat I-15P which was purified from rat intestinal epithelium. The nucleotide sequence of the open reading frame exhibited 79% identity to that of the porcine gastrotropin. Northern blot analysis indicated that the same size of transcript as that of the ileum was detected in the ovary, suggesting that I-15P or a homologous protein might be involved in the metabolism of steroids in steroid hormone-producing tissues.

Immunocytochemical localization of rat intestinal 15 kDa protein, a member of cytoplasmic fatty acid-binding proteins.

Rat intestinal 15 kDa protein (I-15P) is a member of the family of cytoplasmic fatty acid-binding proteins. Using a specific antiserum against I-15P, we studied the tissue distribution and subcellular localization of this protein in the entire rat body. By immunoblot analysis of cytosolic proteins, I-15P was detected not only in the distal portion of small intestine but also in the ovary and adrenal gland. Immunohistochemically, I-15P was localized to the absorptive epithelial cells as well as a subpopulation of enterochromaffin cells in the intestine, the lutein cells in the ovary, and subpopulations of cortical cells in the adrenal gland. Furthermore, I-15P-like immunoreactivity was also demonstrated in the surface mucous cells of stomach and the granular convoluted tubule cells of submandibular gland. Immuno-electron microscopy showed that the immunoreactivity was confined to the cytoplasmic matrix region, except in the enterochromaffin cells and granular convoluted tubule cells, where it was localized in the secretory granules. The present findings suggest that I-15P plays a role in the cellular metabolism of steroids.