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Expression and immunolocalization of Gpnmb, a glioma-associated glycoprotein, in normal and inflamed central nervous systems of adult rats.

Huang JJ, Ma WJ, Yokoyama S - Brain Behav (2012)

Bottom Line: Double immunofluorescence staining showed that these cells were co-stained most frequently with the microglia/macrophage marker OX42, and occasionally with the radial glia marker RC2 or the neuronal marker NeuN.Furthermore, an intraperitoneal injection of bacterial endotoxin lipopolysaccharide increased the number of Gpnmb and OX42 double-positive cells in the area postrema, which is one of the circumventricular organs, indicating infiltration of hematogenous macrophages.These results suggest that Gpnmb, which is expressed in microglia and macrophages in non-tumorous neural tissues, plays an important role in the regulation of immune/inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine Kanazawa 920-8640, Japan.

ABSTRACT
Glycoprotein nonmetastatic melanoma B (Gpnmb) is a type I transmembrane protein implicated in cell differentiation, inflammation, tissue regeneration, and tumor progression. Gpnmb, which is highly expressed in glioblastoma cells, is a potential therapeutic target. However, little is known about its expression, cellular localization, and roles in non-tumorous neural tissues. In this study, we examined Gpnmb expression in the central nervous system of adult rats under both normal and inflammatory conditions. Reverse transcription-polymerase chain reaction analysis revealed that Gpnmb mRNA was expressed in the cerebrum, cerebellum, brain stem, and spinal cord of normal adult rats. Immunoperoxidase staining revealed that Gpnmb-immunoreactive cells were widely distributed in the parenchyma of all brain regions examined, with the cells being most prevalent in the hippocampal dentate gyrus, cerebellar cortex, spinal dorsal horn, choroid plexus, ependyma, periventricular regions, and in layers II and III of the cerebral cortex. Double immunofluorescence staining showed that these cells were co-stained most frequently with the microglia/macrophage marker OX42, and occasionally with the radial glia marker RC2 or the neuronal marker NeuN. Furthermore, an intraperitoneal injection of bacterial endotoxin lipopolysaccharide increased the number of Gpnmb and OX42 double-positive cells in the area postrema, which is one of the circumventricular organs, indicating infiltration of hematogenous macrophages. These results suggest that Gpnmb, which is expressed in microglia and macrophages in non-tumorous neural tissues, plays an important role in the regulation of immune/inflammatory responses.

No MeSH data available.


Related in: MedlinePlus

Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Gpnmb mRNA in CNS of adult rats. (A) Schematic representation of the recognition sites of Gpnmb-specific PCR primers (arrows), the predicted sizes of the amplification products, and the probe used for Southern blot analysis. The exon–intron organization is depicted based on GenBank accession number NC_005103. Exons are indicated by rectangles and introns and 5′- and 3′-flanking regions are indicated by lines. Open and filled rectangles represent untranslated and protein-coding regions, respectively. (B) Photomicrographs of Southern blot analysis. Total cellular RNA from the indicated regions was subjected to RT-PCR. The amplified cDNAs were separated on 1.2% agarose gels, transferred to nylon membranes, and hybridized with HRP-conjugated Gpnmb- or GAPDH-specific probes. The absence of reverse transcriptase is indicated as [RT (-)]. GAPDH cDNA was amplified as a positive control. Size markers are given on the right.
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fig01: Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Gpnmb mRNA in CNS of adult rats. (A) Schematic representation of the recognition sites of Gpnmb-specific PCR primers (arrows), the predicted sizes of the amplification products, and the probe used for Southern blot analysis. The exon–intron organization is depicted based on GenBank accession number NC_005103. Exons are indicated by rectangles and introns and 5′- and 3′-flanking regions are indicated by lines. Open and filled rectangles represent untranslated and protein-coding regions, respectively. (B) Photomicrographs of Southern blot analysis. Total cellular RNA from the indicated regions was subjected to RT-PCR. The amplified cDNAs were separated on 1.2% agarose gels, transferred to nylon membranes, and hybridized with HRP-conjugated Gpnmb- or GAPDH-specific probes. The absence of reverse transcriptase is indicated as [RT (-)]. GAPDH cDNA was amplified as a positive control. Size markers are given on the right.

Mentions: To examine whether Gpnmb mRNA was expressed in rat CNS, we first performed RT-PCR analysis. Primers were designed to distinguish between the amplified product from cDNA and an amplified product derived from contaminating genomic DNA. As shown in Fig. 1A, sense and antisense primers were made to recognize exons 6 and 11, respectively. PCR products from cDNA and genomic DNA were predicted to be 993 bp and 4.4 kb, respectively. Furthermore, specificity of PCR products was confirmed by Southern blot analysis using an internal probe (Fig. 1A). Gpnmb mRNA expression was detected in all brain regions examined; GAPDH cDNA was used to confirm the integrity of RNA preparations (Fig. 1B).


Expression and immunolocalization of Gpnmb, a glioma-associated glycoprotein, in normal and inflamed central nervous systems of adult rats.

Huang JJ, Ma WJ, Yokoyama S - Brain Behav (2012)

Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Gpnmb mRNA in CNS of adult rats. (A) Schematic representation of the recognition sites of Gpnmb-specific PCR primers (arrows), the predicted sizes of the amplification products, and the probe used for Southern blot analysis. The exon–intron organization is depicted based on GenBank accession number NC_005103. Exons are indicated by rectangles and introns and 5′- and 3′-flanking regions are indicated by lines. Open and filled rectangles represent untranslated and protein-coding regions, respectively. (B) Photomicrographs of Southern blot analysis. Total cellular RNA from the indicated regions was subjected to RT-PCR. The amplified cDNAs were separated on 1.2% agarose gels, transferred to nylon membranes, and hybridized with HRP-conjugated Gpnmb- or GAPDH-specific probes. The absence of reverse transcriptase is indicated as [RT (-)]. GAPDH cDNA was amplified as a positive control. Size markers are given on the right.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3345354&req=5

fig01: Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Gpnmb mRNA in CNS of adult rats. (A) Schematic representation of the recognition sites of Gpnmb-specific PCR primers (arrows), the predicted sizes of the amplification products, and the probe used for Southern blot analysis. The exon–intron organization is depicted based on GenBank accession number NC_005103. Exons are indicated by rectangles and introns and 5′- and 3′-flanking regions are indicated by lines. Open and filled rectangles represent untranslated and protein-coding regions, respectively. (B) Photomicrographs of Southern blot analysis. Total cellular RNA from the indicated regions was subjected to RT-PCR. The amplified cDNAs were separated on 1.2% agarose gels, transferred to nylon membranes, and hybridized with HRP-conjugated Gpnmb- or GAPDH-specific probes. The absence of reverse transcriptase is indicated as [RT (-)]. GAPDH cDNA was amplified as a positive control. Size markers are given on the right.
Mentions: To examine whether Gpnmb mRNA was expressed in rat CNS, we first performed RT-PCR analysis. Primers were designed to distinguish between the amplified product from cDNA and an amplified product derived from contaminating genomic DNA. As shown in Fig. 1A, sense and antisense primers were made to recognize exons 6 and 11, respectively. PCR products from cDNA and genomic DNA were predicted to be 993 bp and 4.4 kb, respectively. Furthermore, specificity of PCR products was confirmed by Southern blot analysis using an internal probe (Fig. 1A). Gpnmb mRNA expression was detected in all brain regions examined; GAPDH cDNA was used to confirm the integrity of RNA preparations (Fig. 1B).

Bottom Line: Double immunofluorescence staining showed that these cells were co-stained most frequently with the microglia/macrophage marker OX42, and occasionally with the radial glia marker RC2 or the neuronal marker NeuN.Furthermore, an intraperitoneal injection of bacterial endotoxin lipopolysaccharide increased the number of Gpnmb and OX42 double-positive cells in the area postrema, which is one of the circumventricular organs, indicating infiltration of hematogenous macrophages.These results suggest that Gpnmb, which is expressed in microglia and macrophages in non-tumorous neural tissues, plays an important role in the regulation of immune/inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine Kanazawa 920-8640, Japan.

ABSTRACT
Glycoprotein nonmetastatic melanoma B (Gpnmb) is a type I transmembrane protein implicated in cell differentiation, inflammation, tissue regeneration, and tumor progression. Gpnmb, which is highly expressed in glioblastoma cells, is a potential therapeutic target. However, little is known about its expression, cellular localization, and roles in non-tumorous neural tissues. In this study, we examined Gpnmb expression in the central nervous system of adult rats under both normal and inflammatory conditions. Reverse transcription-polymerase chain reaction analysis revealed that Gpnmb mRNA was expressed in the cerebrum, cerebellum, brain stem, and spinal cord of normal adult rats. Immunoperoxidase staining revealed that Gpnmb-immunoreactive cells were widely distributed in the parenchyma of all brain regions examined, with the cells being most prevalent in the hippocampal dentate gyrus, cerebellar cortex, spinal dorsal horn, choroid plexus, ependyma, periventricular regions, and in layers II and III of the cerebral cortex. Double immunofluorescence staining showed that these cells were co-stained most frequently with the microglia/macrophage marker OX42, and occasionally with the radial glia marker RC2 or the neuronal marker NeuN. Furthermore, an intraperitoneal injection of bacterial endotoxin lipopolysaccharide increased the number of Gpnmb and OX42 double-positive cells in the area postrema, which is one of the circumventricular organs, indicating infiltration of hematogenous macrophages. These results suggest that Gpnmb, which is expressed in microglia and macrophages in non-tumorous neural tissues, plays an important role in the regulation of immune/inflammatory responses.

No MeSH data available.


Related in: MedlinePlus