Involvement of P2X receptors in the regulation of insulin secretion, proliferation and survival in mouse pancreatic ß-cells.

In order to clarify the functional role of ionotropic purinergic (P2X) receptors in pancreatic ß-cells, we examined the effect of several P2 receptor agonists and antagonists on insulin secretion by mouse pancreatic islets, mouse Beta-TC6 cell proliferation and survival of dispersed islet cells in culture. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the expression of mRNAs of P2X(4) receptor in mouse islets and P2X(1), P2X(2), P2X(3), P2X(4), P2X(5) and P2X(7) receptors in Beta-TC6 cells. The presence of P2X(4) receptor proteins in islets and Beta-TC6 cells was confirmed by immunofluorescent staining and Western blot analysis. We have previously found that the functional P2Y(1) receptor but not P2Y(2) and P2Y(4) receptors was present in islets. In this study we found that a nonspecific P2 receptor agonist, ATP (1 µM) stimulated insulin secretion by islets in the presence of high glucose (20 mM) in culture. The effect of ATP was partially inhibited by a P2 receptor antagonist PPADS as well as a P2Y(1) receptor antagonist MRS2179. In addition, a P2X(4) receptor potentiator ivermectin per se augmented glucose-induced insulin secretion and slightly potentiated the effect of ATP. These results suggested the involvement of P2Y(1)and P2X receptors. We also found that ATP inhibited proliferation of Beta-TC6 cells in a concentration-dependent manner during 72 h culture. The inhibitory effect of ATP was completely reversed by PPADS and partially by treating cells with small interfering RNA targeted for P2X(4) receptor mRNA. Furthermore, we found that the phosphorylation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) was suppressed by treatment with ATP in Beta-TC6 cells. In addition, we found that ATP reduced the cell viability and DNA synthesis of islet cells in culture. The effect of ATP on the cell viability was blocked by PPADS or MRS2179. These results suggested that P2X receptors as well as the P2Y(1) receptor played a role in the modulation of insulin secretion, proliferation and cell viability in mouse pancreatic ß-cells.

The change of antizyme inhibitor expression and its possible role during mammalian cell cycle.

Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/midbody during telophase. Thereafter, AIn and antizyme were separated and present at different regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in the number of binucleated cells in HTC cells in culture. These findings suggested that AIn contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to complete mitosis during the cell cycle.

[Identification of novel molecules regulating differentiation and hormone secretion and clarification of their functional mechanisms in pancreatic endocrine cells].

In order to find novel bioactive molecules regulating differentiation and hormone secretion of pancreatic endocrine cells, the effects of various substances including purinergic receptor agonists and inhibitors of polyamine biosynthesis were examined in pancreatic islets and several pancreatic cell lines. The nicotinic alpha3beta4 receptor was found to be present and capable of increasing cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and insulin secretion in mouse pancreatic Beta-TC6 cells. Activation of both nicotinic and muscarinic M(3)/M(4) receptors resulted in reduction of insulin release when compared with stimulation of muscarinic receptor alone in Beta-TC6 cells. In mouse islets, purinergic P2Y(1) and P2Y(6) receptors, which are coupled to Gq proteins, were expressed and appeared to regulate insulin secretion through Ca(2+) mobilization from intracellular stores. Similar results were observed in Beta-TC6 cells. Spermidine, one of polyamines, was found to modulate insulin synthesis and [Ca(2+)](i) in Beta-TC6 cells by use of a specific spermidine synthesis inhibitor, trans-4-methylcyclohexylamine (MCHA). Antizyme, which binds to ornithine decarboxylase (ODC) and thereby reduces the cellular polyamine level, was found to be necessary for conversion of ASPC-1 cells, a pancreatic ductal tumor cell line, into alpha-cells forming the islet-like structure and expressing glucagon gene. These findings help advance our understanding of the complex mechanisms involved in the regulation of pancreatic endocrine cell function and develop new therapeutic agents in diabetes mellitus.

Spermidine regulates insulin synthesis and cytoplasmic Ca(2+) in mouse beta-TC6 insulinoma cells.

In order to assess the functional role of the polyamines spermidine and spermine in pancreatic beta-cells, we examined the effect of spermidine and spermine synthase inhibitors, trans-4-methylcyclohexylamine (MCHA) and N-(3-aminopropyl)cyclohexylamine (APCHA), on cellular polyamine and insulin contents, insulin secretion, and cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in mouse insulin-secreting Beta-TC6 cells. The cellular spermidine and spermine contents were reduced 90% and 64% by cultivation of cells in the presence of MCHA and APCHA for 3 days, respectively. Addition of spermidine or spermine reversed the polyamine level reduced by MCHA or APCHA, respectively. Insulin secretion was decreased 40~60% in the cells treated with MCHA or APCHA. The reduction by MCHA was reversed to the untreated level by adding spermidine exogenously, while the effect of APCHA was not reversed by treatment with spermine. The cellular insulin content was also reduced by treatment with MCHA but not the expression of insulin 1 and 2 genes, suggesting that spermidine was involved in the translation of insulin mRNAs. The elevation of [Ca(2+)](i), a key event triggering insulin secretion induced by glucose, was reduced in Beta-TC6 cells by MCHA treatment. The spermidine synthase inhibitor also augmented the sustained [Ca(2+)](i) rise induced by carbamylcholine but not by a high concentration of KCl or nicotine. These results suggested that spermidine rather than spermine plays an important role in the regulation of insulin synthesis and the glucose-induced [Ca(2+)](i) rise in Beta-TC6 cells.

Antizyme is necessary for conversion of pancreatic tumor cells into glucagon-producing differentiated cells.

Human pancreatic tumor cell lines - AsPC-1, PANC-1, MIA paca2, KP-1 and KP-59 cells - can be induced to differentiate into pancreatic hormone-producing cells by brief trypsin treatment and subsequent culture in a serum-free, chemically defined medium. During culture, AsPC-1 cells formed cell clusters resembling the pancreatic islets, expressed genes associated with the pancreatic development and produced glucagon but not insulin. When PANC-1, MIA paca2, KP-1 and KP-59 cells were treated and cultured the same way, they underwent similar morphological changes and produced insulin and glucagon. We used these systems to identify intracellular regulatory molecules involved in the conversion of pancreatic tumor cells into glucagon-producing cells. We found that the expression of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase, was increased and its localization was altered from the nucleus to the cytoplasm during AsPC-1 cell differentiation. Transient transfection of AsPC-1 cells with AZ1 siRNA resulted in inhibition of the morphological and functional cell differentiation as well as the specific suppression of AZ1 expression. By contrast, constitutive overexpression of AZ1 in AsPC-1 cells led to the enhancement of glucagon production. We also found that PANC-1 cells reduced the expression of glucagon mRNA when treated with AZ1 siRNA. These results suggested that AZ1 was necessary for the conversion of pancreatic tumor cells into glucagon-producing cells. Glucagon production in AsPC-1 cells was not affected by addition of putrescine, suggesting that the polyamines were not directly involved in the AZ1-mediated conversion of pancreatic tumor cells to differentiated state.

Activation of beta-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation.

The Wnt/beta-catenin signaling pathway is critical for normal mammalian development, the specification of epidermal cells and neoplastic transformation of intestinal epithelium. However, precise molecular information regarding cell-specific responses to beta-catenin signaling has been limited. This question was addressed using a mouse model in which exon 3 of the beta-catenin gene was deleted in several cell types with loxP-mediated recombination utilizing a Cre transgene under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). The stabilization of beta-catenin in prostate epithelium resulted in hyperplasias and extensive transdifferentiation into epidermal-like structures, which expressed keratins 1 and 6, filaggrin, loricrin and involucrin. The cell-specific loss of NKCC1 protein and reduced nuclear Stat5a is further suggestive of a loss of prostate epithelial characteristics. In addition to the prostate, hyperplasias and squamous metaplasias were detected in epithelia of the epididymis, vas deferens, coagulating gland, preputial gland and salivary gland. However, and in contrast to a recent study, no lesions reminiscent of high-grade prostate intraepithelial neoplasia were detected. Since beta-catenin was activated in several cell types and impinged upon the viability of these mice, it was not possible to evaluate the cumulative effect over more than 3 months. To assess long-term consequences of beta-catenin activation, mutant and control prostate tissues were transplanted into the mammary fat pads of wild-type males. Notably, squamous metaplasias, intra-acinous hyperplasia and possible neoplastic transformation were observed after a total of 18 weeks of beta-catenin stimulation. This suggests that the transdifferentiation into squamous metaplasias is an early response of endoderm-derived cells to beta-catenin, and that the development of intra-acinous hyperplasias or neoplastic foci is a later event.

Involvement of Oct-1 in transcriptional regulation of beta-casein gene expression in mouse mammary gland.

Mouse beta-casein gene promoter contains a region termed block C which is crucial for its gene transcription induced by lactogenic hormones. Nuclear extracts from mouse mammary glands contain at least two binding complexes (DS1 and DS2) which specifically bind to double-stranded block C region DNA. The binding sequence of these complexes was identified to be 5'-AAATTAGCATGT-3' which contains a sequence element related to the consensus octamer motif's complement ATTTGCAT. In the present study, we demonstrate that this sequence element indeed is the binding site for octamer-binding transcription factors (Octs) and Octs represent the double-stranded DNA binding proteins specifically binding to the block C region. Formation of the specific double-stranded binding complexes can be completely blocked by Oct binding motif oligonucleotides and anti-rOct-1 antiserum. We also show that Oct-1B represents at least partial, if not all, double-stranded binding protein, DS1, in mammary nuclear extract. Oct-1B may function as a transcriptional activator on casein gene promoter. The Oct binding activity to beta-casein gene promoter in the mammary gland is affected under influence of hormones both in vitro and in vivo. The DS1 binding activity can be induced by the combination of lactogenic hormones insulin, hydrocortisone and prolactin in organ culture of virgin mouse mammary gland. The binding activity in vivo can be induced by injection of progesterone or its combination with estradiol in virgin mice.

Cloning, genomic organization, expression, and effect on beta-casein promoter activity of a novel isoform of the mouse Oct-1 transcription factor.

The ubiquitously expressed transcription factor Oct-1, a member of the POU domain factors, is involved in the regulation of expression of many tissue-specific and house-keeping genes. Multiple alternatively spliced isoforms of Oct-1 have been identified in human and mouse cells. The expression patterns of these isoforms and the analysis of their genomic organization and structure have suggested that the structural variation of Oct-1 isoforms may be important in conferring target and tissue specificity to its transcriptional activity. In this study, we have cloned and sequenced a new mouse Oct-1 isoform, named mOct-1Z. This novel isoform differs markedly at the C-terminus from the previously identified Oct-1 isoforms A, B, and C. It is generated by alternative splicing from the Oct-1 gene and its transcript exhibits a frameshift followed by an early stop codon, thus, its predicted protein has a distinct, much shorter C-terminal tail. However, this truncated isoform could still effectively bind to a consensus Oct-1 motif oligonucleotide and, like Oct-1B, activated the basal promoter activity of the mouse beta-casein gene. Oct-1Z is another ubiquitously expressed Oct-1 isoform, its transcript being detected in all mouse tissues examined, including the mammary gland, liver, lung, kidney, spleen, small intestine mucosa, uterus, and ovary.

Co-existence of muscarinic and nicotinic receptors and their functional interaction in mouse Beta-TC6 cells.

Mouse Beta-TC6 insulinoma cells possessing nicotinic receptor [Ohtani, M., Oka, T., Badyuk, M., Xiao, Y., Kellar, KJ., Daly, JW., 2006. Mouse beta-TC6 insulinoma cells: high expression of functional alpha3beta4 nicotinic receptors mediating membrane potential, intracellular calcium, and insulin release. Mol. Pharmacol. 69, 899-907.] also expressed M(3) and M(4) muscarinic receptors. Carbamylcholine, a mixed muscarinic/nicotinic receptor agonist, or oxotremorine M, a selective muscarinic agonist, elicited an elevation of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and release of insulin. The maximal [Ca(2+)](i) response induced by carbamylcholine was larger than that of oxotremorine M or that of nicotine, suggesting that carbamylcholine enhanced the [Ca(2+)](i) response by stimulating two types of receptor. M(3) and M(4) muscarinic receptor antagonists inhibited the [Ca(2+)](i) responses to carbamylcholine and oxotremorine M, suggesting the involvement of these muscarinic receptors in the regulation of [Ca(2+)](i). In addition, pretreatment with carbamylcholine inhibited the [Ca(2+)](i) responses to oxotremorine M or nicotine, indicating that the effect of carbamylcholine on [Ca(2+)](i) was mediated by both muscarinic and nicotinic receptors. A phospholipase C (PLC) inhibitor U73122, a protein kinase C (PKC) inhibitor chelerythrine and a phospholipase A(2) (PLA(2)) inhibitor AACOCF(3) inhibited the [Ca(2+)](i) response to carbamylcholine or oxotremorine M, while these inhibitors did not block the effect of nicotine. Carbamylcholine induced a smaller extent of insulin secretion than oxotremorine M, suggesting that concomitant stimulation of muscarinic and nicotinic receptors by carbamylcholine resulted in the negative type of the receptor interaction.

Evidence for the possible involvement of the P2Y(6) receptor in Ca (2+) mobilization and insulin secretion in mouse pancreatic islets.

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca(2+)](i)) and insulin release in mouse pancreatic beta-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca(2+)](i) rise via cytoplasmic Ca(2+) mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 microM) transiently increased [Ca(2+)](i) in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y(1) receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y(6) receptor agonist UDP (200 microM) transiently increased [Ca(2+)](i) and reduced insulin secretion at high glucose, whereas the P2Y(2/4) receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca(2+)](i) transients induced by 2-MeSADP and UDP were antagonized by suramin (100 microM), U73122 (2 microM, PLC inhibitor), and 2-APB (10 or 30 microM, IP(3) receptor antagonist), but neither by staurosporine (1 microM, PKC inhibitor) nor depletion of extracellular Ca(2+). The effect of 2-MeSADP on [Ca(2+)](i) was also significantly inhibited by MRS2500, a P2Y(1) receptor antagonist. These results suggested that P2Y(1) and P2Y(6) receptor subtypes are involved in Ca(2+) mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca(2+)](i) and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y(1) and P2Y(6), but not P2Y(2) and P2Y(4) receptors.

Electrospray ionization and time-of-flight mass spectrometric method for simultaneous determination of spermidine and spermine.

A sensitive method for the determination of polyamines in mammalian cells was described using electrospray ionization and time-of-flight mass spectrometer. This method was 50-fold more sensitive than the previous method using ionspray ionization and quadrupole mass spectrometer. The method employed the partial purification and derivatization of polyamines, but allowed a measurement of multiple samples which contained picomol amounts of polyamines. Time required for data acquisition of one sample was approximately 2 min. The method was successfully applied for the determination of reduced spermidine and spermine contents in cultured cells under the inhibition of aminopropyltransferases. In addition, a new proper internal standard was proposed for the tracer experiment using (15)N-labeled polyamines.

Mouse beta-TC6 insulinoma cells: high expression of functional alpha3beta4 nicotinic receptors mediating membrane potential, intracellular calcium, and insulin release.

Nicotine elicited membrane depolarization, elevation of intracellular calcium, rubidium efflux, and release of insulin from mouse beta-TC6 insulinoma cells. Such responses were blocked by the nicotinic antagonist mecamylamine but not by the muscarinic antagonist atropine. Neither the selective alpha4beta2 antagonist dihydro-beta-erythroidine nor the selective alpha7 antagonist methyllycaconitine significantly blocked the nicotine-elicited depolarization or the calcium response. The elevation of intracellular calcium did not occur in calcium-free media, indicating that the increase in intracellular calcium was due to the influx of calcium. The rank order of potency for nicotinic agonists was as follows: epibatidine > nicotine = 3-(azetidinylmethoxy)pyridine (A-85380), cytisine, dimethylphenylpiperazinium (DMPP). Cytisine and DMPP seemed to be partial agonists. The density of nicotinic receptors measured by [3H]epibatidine binding was 7-fold higher in membranes from beta-TC6 cells than in rat brain membranes. No binding of 125I-A-85380 was detected, indicating the absence of beta2-containing receptors. Reverse transcription-polymerase chain reaction analyses indicated the presence of mRNA for alpha3 and alpha4 subunits and beta2 and beta4 subunits in beta-TC6 cells. The binding and functional data suggest that the major nicotinic receptor is composed of alpha3 and beta4 subunits. The beta-TC6 cells thus provide a model system for pharmacological study of such nicotinic receptors.

Regulation of type II deiodinase expression by EGF and glucocorticoid in HC11 mouse mammary epithelium.

Thyroid hormones are important for mammary gland growth and development. The iodothyronine deiodinases play a key role in thyroid hormone metabolism. We have showed that type II 5'-deiodinase (5'D2) activity and mRNA are present in the mouse mammary gland and that their levels are reduced in the lactating gland. To investigate the regulatory mechanism of mouse 5'D2 gene (mdio2) expression in mammary epithelium, we employed the HC11 cell line, which is derived from mouse mammary epithelial cells and retains the ability to express differentiated function. HC11 cells were treated with combinations of insulin, glucocorticoid (GC, dexamethasone), prolactin, and epidermal growth factor (EGF), and 5'D2 activity and the D2-to-GAPDH mRNA ratio were measured by (125)I(-) release from (125)I-labeled thyroxine and semiquantitative RT-PCR, respectively. EGF increased both 5'D2 activity and mRNA levels about twofold. GC reduced both 5'D2 activity and mRNA in a dose-dependent manner, and their levels were decreased to approximately one-tenth and one-fifth, respectively, of control levels. These data demonstrated that mdio2 expression in HC11 cells is upregulated by EGF mainly at the pretranslational level and downregulated by GC at both pre- and posttranslational levels. Furthermore, we showed that GC reduced the promoter activity of the 627- bp 5'-upstream region of the mdio2/luciferase chimeric reporter gene, suggesting that GC exerts its effect, at least in part, at the transcriptional level.

Bcl-x is not required for maintenance of follicles and corpus luteum in the postnatal mouse ovary.

It has been proposed that Bcl-x is a key survival factor in many cell types, and that the bcl-x gene is activated by the transcription factor Stat5 through cytokine signals. In support of this, it has been demonstrated that the survival of mouse primordial germ cells during embryogenesis depends on the presence of Bcl-x. We have now investigated whether, in the mouse, Bcl-x is required for the postnatal maintenance of follicles and luteal cells, and whether Stat5 activates the bcl-x gene. The bcl-x gene was deleted in these cells within the mouse using Cre-loxP recombination. Loss of the bcl-x gene did not affect the numbers of primordial, primary, and antral follicles. Furthermore, expression of the bcl-x gene in the ovary was independent of Stat5 and its activating hormone, prolactin. To determine whether the prolactin receptor (PrlR), Stat5, and Bcl-x were required for establishment and maintenance of the corpus luteum, we induced pseudopregnancies in the respective gene-deletion mice. Whereas luteal cells underwent apoptosis in the absence of the PrlR, no changes were observed in the absence of Stat5 or Bcl-x.