Cytokine regulation of syndecan-1 and-2 gene expression in human periodontal fibroblasts and osteoblasts

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta(1)), and interleukin (IL-1beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta(1), separately and in combination, in the prolonged presence of IL-1beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta(1) and decreased levels in response to IL-1beta. The effect of IL-1beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta(1), separately or in combination, in the presence of IL-1beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta(1) or IL-1beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta(1) on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Dissertation to obtain a Master Degree in Biotechnology

TOLL-LIKE RECEPTORS (TLR) 2 AND 4 EXPRESSION OF KERATINOCYTES FROM PATIENTS WITH LOCALIZED AND DISSEMINATED DERMATOPHYTOSIS

There are few studies on the role of innate immune response in dermatophytosis. An investigation was conducted to define the involvement of Toll-Like Receptors (TLRs) 2 and 4 in localized (LD) and disseminated (DD) dermatophytosis due to T. rubrum. Fifteen newly diagnosed patients, eight patients with LD and seven with DD, defined by involvement of at least three body segments were used in this study. Controls comprised twenty skin samples from healthy individuals undergoing plastic surgery. TLR2 and TLR4 were quantified in skin lesions by immunohistochemistry. A reduced expression of TLR4 in the lower and upper epidermis of both LD and DD patients was found compared to controls; TLR2 expression was preserved in the upper and lower epidermis of all three groups. As TLR4 signaling induces the production of inflammatory cytokines and neutrophils recruitment, its reduced expression likely contributed to the lack of resolution of the infection and the consequent chronic nature of the dermatophytosis. As TLR2 expression acts to limit the inflammatory process and preserves the epidermal structure, its preserved expression may also contribute to the persistent infection and limited inflammation that are characteristic of dermatophytic infections.

Einfluss von ZNS-Verletzung auf die m-RNA-Expression der Protease-Aktivierenden Rezeptoren (PARs) und Indentifizierung eines Interaktionspartners für PAR-2 in Retina

Protease-activated receptor, optic nerve crush, focal ischemia, protein-protein interaction, alpha crystallin A

Functional expression of P2Y 2 receptor and trafficking of the receptor

P2Y, Endocytosis, UTP, Purinergic receptors

Bedeutung der Expression der G-Protein-gekoppelten-Rezeptoren BRS 3, CCKA, CXCR 4, GHRH, GRPR, NK 1, NMBR, NT 2, PAC 1, VPAC 1 und VPAC 2 bei kolorektalen Tumoren in Abhängigkeit von Tumorstadium und Differenzierungsgrad

Magdeburg, Univ., Med. Fak., Diss., 2012

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

PGC1alpha expression is controlled in skeletal muscles by PPARbeta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes.

Mice in which peroxisome proliferator-activated receptor beta (PPARbeta) is selectively ablated in skeletal muscle myocytes were generated to elucidate the role played by PPARbeta signaling in these myocytes. These somatic mutant mice exhibited a muscle fiber-type switching toward lower oxidative capacity that preceded the development of obesity and diabetes, thus demonstrating that PPARbeta is instrumental in myocytes to the maintenance of oxidative fibers and that fiber-type switching is likely to be the cause and not the consequence of these metabolic disorders. We also show that PPARbeta stimulates in myocytes the expression of PGC1alpha, a coactivator of various transcription factors, known to play an important role in slow muscle fiber formation. Moreover, as the PGC1alpha promoter contains a PPAR response element, the effect of PPARbeta on the formation and/or maintenance of slow muscle fibers can be ascribed, at least in part, to a stimulation of PGC1alpha expression at the transcriptional level.

Variation d'expression des récepteurs hormonaux et d'HER-2 dans l'évolution du cancer du sein : quelles implications en pratique clinique [Hormone receptors and HER-2 changes during breast cancer progression: clinical implications].

Breast cancer remains a major public health problem. Even if there is an increase in this cancer curability, metastatic breast cancer remains a lethal disease in the vast majority of cases. Therapeutic advances in the chemotherapeutic and targeted therapies fields induced an increase in survival, however the proportion of long survivors remains low. Phenotypic instability, an early process initiated during tumour progression, and continued on the metastatic stage of the disease, can be one of the putative hypotheses explaining these results. An increasing amount of scientific data are pledging for a reanalysis of the phenotypic profile regarding hormone receptors and HER-2 status of metastatic lesions in order to identify drugable targets and allow individualisation of the treatment of these metastatic breast cancer patients. Phenotypic changes between the primary tumour and the paired metastatic lymph nodes are a challenging pitfall, raising the question of which site has to be assessed in the adjuvant treatment decision process. This article presents a comprehensive analysis of the frequency of theses phenotypic changes altogether with new modalities to evaluate this phenotypic status.

Constitutive expression of a chimeric receptor that delivers IL-2/IL-15 signals allows antigen-independent proliferation of CD8+CD44high but not other T cells.

We have prepared transgenic mice whose T cells constitutively express a chimeric receptor combining extracellular human IL-4R and intracellular IL-2Rbeta segments. This receptor can transmit IL-2/IL-15-like signals in response to human, but not mouse, IL-4. We used these animals to explore to what extent functional IL-2R/IL-15R expression controls the capacity of T cells to proliferate in response to IL-2/IL-15-like signals. After activation with Con A, naive transgenic CD8+ and CD4+ T cells respond to human IL-4 as well as to IL-2. Without prior activation, they failed to proliferate in response to human IL-4, although human IL-4 did prolong their survival. Thus, IL-2-induced proliferation of activated T cells requires at least one other Ag-induced change apart from the induction of a functional IL-2R. However, a fraction of CD8+CD44high T cells proliferate in human IL-4 without antigenic stimulation or syngeneic feeder cells. In contrast, CD4+CD44high T cells are not constitutively responsive to human IL-4. We conclude that although all transgenic T cells express a functional chimeric receptor, only some CD8+CD44high T cells contain all molecules required for entry into the cell cycle in response to human IL-4 or IL-15.

Expression of the gene encoding the high-Km glucose transporter 2 by the early postimplantation mouse embryo is essential for neural tube defects associated with diabetic embryopathy.

AIMS/HYPOTHESIS: Excess glucose transport to embryos during diabetic pregnancy causes congenital malformations. The early postimplantation embryo expresses the gene encoding the high-Km GLUT2 (also known as SLC2A2) glucose transporter. The hypothesis tested here is that high-Km glucose transport by GLUT2 causes malformations resulting from maternal hyperglycaemia during diabetic pregnancy. MATERIALS AND METHODS: Glut2 mRNA was assayed by RT-PCR. The Km of embryo glucose transport was determined by measuring 0.5-20 mmol/l 2-deoxy[3H]glucose transport. To test whether the GLUT2 transporter is required for neural tube defects resulting from maternal hyperglycaemia, Glut2+/- mice were crossed and transient hyperglycaemia was induced by glucose injection on day 7.5 of pregnancy. Embryos were recovered on day 10.5, and the incidence of neural tube defects in wild-type, Glut2+/- and Glut2-/- embryos was scored. RESULTS: Early postimplantation embryos expressed Glut2, and expression was unaffected by maternal diabetes. Moreover, glucose transport by these embryos showed Michaelis-Menten kinetics of 16.19 mmol/l, consistent with transport mediated by GLUT2. In pregnancies made hyperglycaemic on day 7.5, neural tube defects were significantly increased in wild-type embryos, but Glut2+/- embryos were partially protected from neural tube defects, and Glut2-/- embryos were completely protected from these defects. The frequency of occurrence of wild-type, Glut2+/- and Glut2-/- embryos suggests that the presence of Glut2 alleles confers a survival advantage in embryos before day 10.5. CONCLUSIONS/INTERPRETATIONS: High-Km glucose transport by the GLUT2 glucose transporter during organogenesis is responsible for the embryopathic effects of maternal diabetes.

Brain-derived neurotrophic factor enhances the expression of the monocarboxylate transporter 2 through translational activation in mouse cultured cortical neurons.

MCT2 is the predominant neuronal monocarboxylate transporter allowing lactate use as an alternative energy substrate. It is suggested that MCT2 is upregulated to meet enhanced energy demands after modifications in synaptic transmission. Brain-derived neurotrophic factor (BDNF), a promoter of synaptic plasticity, significantly increased MCT2 protein expression in cultured cortical neurons (as shown by immunocytochemistry and western blot) through a translational regulation at the synaptic level. Brain-derived neurotrophic factor can cause translational activation through different signaling pathways. Western blot analyses showed that p44/p42 mitogen-activated protein kinase (MAPK), Akt, and S6 were strongly phosphorylated on BDNF treatment. To determine by which signal transduction pathway(s) BDNF mediates its upregulation of MCT2 protein expression, the effect of specific inhibitors for p38 MAPK, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), p44/p42 MAPK (ERK), and Janus kinase 2 (JAK2) was evaluated. It could be observed that the BDNF-induced increase in MCT2 protein expression was almost completely blocked by all inhibitors, except for JAK2. These data indicate that BDNF induces an increase in neuronal MCT2 protein expression by a mechanism involving a concomitant stimulation of PI3K/Akt/mTOR/S6, p38 MAPK, and p44/p42 MAPK. Moreover, our observations suggest that changes in MCT2 expression could participate in the process of synaptic plasticity induced by BDNF.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Messenger RNA expression of transporter and ion channel genes in undifferentiated and differentiated Caco-2 cells compared to human intestines.

PURPOSE: The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. METHOD: Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. RESULTS: Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. CONCLUSIONS: Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.