Expression of Slug in S100ß-protein-positive cells of postnatal developing rat anterior pituitary gland.

Among heterogeneous S100ß-protein-positive (S100ß-positive) cells, star-like cells with extended cytoplasmic processes, the so-called folliculo-stellate cells, envelop hormone-producing cells or interconnect homophilically in the anterior pituitary. S100ß-positive cells are known, from immunohistochemistry, to emerge from postnatal day (P) 10 and to proliferate and migrate in the parenchyma of the anterior pituitary with growth. Recent establishment of S100ß-GFP transgenic rats expressing specifically green fluorescent protein (GFP) under the control of the S100ß-promoter has allowed us to observe living S100ß-positive cells. In the present study, we first confirmed that living S100ß-positive cells in tissue cultures of S100ß-GFP rat pituitary at P5 were present prior to P10 by means of confocal laser microscopy and that they proliferated and extended their cytoplasmic processes. Second, we examined the expression of the Snail-family zinc-finger transcription factors, Snail and Slug, to investigate the mechanism behind the morphological changes and the proliferation of S100ß-positive cells. Interestingly, we detected Slug expression in S100ß-positive cells and its increase together with development in the anterior pituitary. To analyze downstream of SLUG in S100ß-positive cells, we utilized specific small interfering RNA for Slug mRNAs and observed that the expression of matrix metalloprotease (Mmp) 9, Mmp14 and chemokine Cxcl12 was down-regulated and that morphological changes and proliferation were decreased. Thus, our findings suggest that S100ß-positive cells express Slug and that its expression is important for subsequent migration and proliferation.

CXCL10/CXCR3 signaling mediates inhibitory action by interferon-gamma on CRF-stimulated adrenocorticotropic hormone (ACTH) release.

Secretion of hormones by the anterior pituitary gland can be stimulated or inhibited by paracrine factors that are produced during inflammatory reactions. The inflammation cytokine interferon-gamma (IFN-γ) is known to inhibit corticotropin-releasing factor (CRF)-stimulated adrenocorticotropin (ACTH) release but its signaling mechanism is not yet known. Using rat anterior pituitary, we previously demonstrated that the CXC chemokine ligand 10 (CXCL10), known as interferon-γ (IFN-γ) inducible protein 10 kDa, is expressed in dendritic cell-like S100ß protein-positive (DC-like S100ß-positive) cells and that its receptor CXCR3 is expressed in ACTH-producing cells. DC-like S100ß-positive cells are a subpopulation of folliculo-stellate cells in the anterior pituitary. In the present study, we examine whether CXCL10/CXCR3 signaling between DC-like S100ß-positive cells and ACTH-producing cells mediates inhibition of CRF-activated ACTH-release by IFN-γ, using a CXCR3 antagonist in the primary pituitary cell culture. We found that IFN-γ up-regulated Cxcl10 expression via JAK/STAT signaling and proopiomelanocortin (Pomc) expression, while we reconfirmed that IFN-γ inhibits CRF-stimulated ACTH-release. Next, we used a CXCR3 agonist in primary culture to analyze whether CXCL10 induces Pomc-expression and ACTH-release using a CXCR3 agonist in the primary culture. The CXCR3 agonist significantly stimulated Pomc-expression and inhibited CRF-induced ACTH-release, while ACTH-release in the absence of CRF did not change. Thus, the present study leads us to an assumption that CXCL10/CXCR3 signaling mediates inhibition of the CRF-stimulated ACTH-release by IFN-γ. Our findings bring us to an assumption that CXCL10 from DC-like S100ß-positive cells acts as a local modulator of ACTH-release during inflammation.

Proton receptor GPR68 expression in dendritic-cell-like S100ß-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification.

S100ß-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100ß-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100ß-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin ß-6) in the round type. Here, we further investigate the function of the subpopulation of S100ß-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100ß-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100ß-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

Isolation of dendritic-cell-like S100ß-positive cells in rat anterior pituitary gland.

S100ß-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100ß-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100ß-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100ß protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100ß-positive cells by means of their property of adhering to laminin.

Transcriptional termination control of a novel ABC transporter gene involved in antibiotic resistance in Bacillus subtilis.

In members of one of the subfamilies of the bacterial ATP binding cassette (ABC) transporters, the two nucleotide binding domains are fused as a single peptide and the proteins have no membrane-spanning domain partners. Most of the ABC efflux transporters of this subfamily have been characterized in actinomycetes, producing macrolide, lincosamide, and streptogramin antibiotics. Among 40 ABC efflux transporters of Bacillus subtilis, five proteins belong to this subfamily. None of these proteins has been functionally characterized. We examined macrolide, lincosamide, and streptogramin antibiotic resistance in insertional disruptants of the genes that encode these proteins. It was found that only a disruptant of vmlR (formerly named expZ) showed hypersensitivity to virginiamycin M and lincomycin. Expression of the vmlR gene was induced by the addition of these antibiotics in growth medium. Primer extension analysis revealed that transcription of the vmlR gene initiates at an adenosine residue located 225 bp upstream of the initiation codon. From the analysis of the vmlR and lacZ fusion genes, a 52-bp deletion from +159 to +211 resulted in constitutive expression of the vmlR gene. In this region, a typical rho-independent transcriptional terminator was found. It was suggested that the majority of transcription ends at this termination signal in the absence of antibiotics, whereas under induced conditions, RNA polymerase reads through the terminator, and transcription continues to the downstream vmlR coding region, resulting in an increase in vmlR expression. No stabilization of vmlR mRNA occurred under the induced conditions.

Increased stability of bmr3 mRNA results in a multidrug-resistant phenotype in Bacillus subtilis.

A spontaneous mutant isolated in the presence of a high concentration of puromycin acquired a multidrug-resistant phenotype. Expression of the bmr3 gene was dramatically increased. A base substitution, T to A at the +4 position, detected in the mutant resulted in the stabilization of bmr3 mRNA.

The BceRS two-component regulatory system induces expression of the bacitracin transporter, BceAB, in Bacillus subtilis.

BceA and bceB encode a nucleotide-binding domain (NBD) and membrane-spanning domain (MSD) subunit, respectively, of an ATP-binding cassette (ABC) transporter in Bacillus subtilis. Disruption of these genes resulted in hypersensitivity to bacitracin, a peptide antibiotic that is non-ribosomally synthesized in some strains of Bacillus. Northern hybridization analyses showed that expression of the bceAB operon is induced by bacitracin present in the growth medium. The bceRS genes encoding a two-component regulatory system are located immediately upstream of bceAB. Deletion analyses of the bceAB promoter together with DNase I footprinting experiments revealed that a sensor kinase, BceS, responds to extracellular bacitracin either directly or indirectly and transmits a signal to a cognate response regulator, BceR. The regulator binds directly to the upstream region of the bceAB promoter and upregulates the expression of bceAB genes. The bcrC gene product is additionally involved in bacitracin resistance. The expression of bcrC is dependent on the ECF sigma factors, sigmaM and sigmaX, but not on the BceRS two-component system. In view of these results, possible roles of BceA, BceB and BcrC in bacitracin resistance of B. subtilis 168 are discussed.

A bacitracin-resistant Bacillus subtilis gene encodes a homologue of the membrane-spanning subunit of the Bacillus licheniformis ABC transporter.

Bacitracin is a peptide antibiotic nonribosomally produced by Bacillus licheniformis. The bcrABC genes which confer bacitracin resistance to the bacitracin producer encode ATP binding cassette (ABC) transporter proteins, which are hypothesized to pump out bacitracin from the cells. Bacillus subtilis 168, which has no bacitracin synthesizing operon, has several genes homologous to bcrABC. It was found that the disruption of ywoA, a gene homologous to bcrC, resulted in hypersensitivity to bacitracin. Resistance to other drugs such as surfactin, iturin A, vancomycin, tunicamycin, gramicidin D, valinomycin and several cationic dyes were not changed in the ywoA disruptant. Spontaneous bacitracin-resistant mutants (Bcr-1 and -2) isolated in the presence of bacitracin have a single base substitution from A to G in the ribosome binding region. Northern hybridization analysis and determination of the expression of ywoA-LacZ transcriptional fusion gene revealed that the transcription of the ywoA gene was dependent on extracytoplasmic function (ECF) sigma factors sigma(M) and sigma(X). Preincubation of wild-type cells in the presence of a low concentration of bacitracin induced increased resistance to bacitracin about two- to threefold, although the mechanism of this induction has not yet been elucidated. It has been reported that a commercially available bacitracin is a mixture of several components and also contains impurity. Bacitracin A was purified by reverse phase high-performance liquid chromatography (HPLC). Similar results were obtained with bacitracin A as those with crude bacitracin, indicating that contaminating substances were not responsible for the results obtained in this study.