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Keratinocytes as depository of ammonium-inducible glutamine synthetase: age- and anatomy-dependent distribution in human and rat skin.

Danielyan L, Zellmer S, Sickinger S, Tolstonog GV, Salvetter J, Lourhmati A, Reissig DD, Gleiter CH, Gebhardt R, Buniatian GH - PLoS ONE (2009)

Bottom Line: While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age.Inhibition of, glycogen synthase kinase 3beta in cultured keratinocytes and HaCaT cells, however, did not support a direct role of beta-catenin in regulation of GS.Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacology, University Hospital of Tübingen, Tübingen, Germany.

ABSTRACT
In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component beta-catenin. Inhibition of, glycogen synthase kinase 3beta in cultured keratinocytes and HaCaT cells, however, did not support a direct role of beta-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8-10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia.

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Presence of GS in human foreskin of children and regional variations in its distribution revealed in co-localization studies with GFAP, or with SMAA.(A–D) Expression of GS (A) and GFAP (B) and merged micrograph of GS and GFAP (C) immune reaction in human foreskin sections. (D–F) Expression of GS (D) and SMAA (E) and merged micrograph of GS and SMAA immune reaction (F) in a human skin section. The line connecting the D–F clearly shows strong expression of SMAA (E) in stratum granulosum and its absence of stratum corneum. The latter is strongly stained for GS (D). GS and SMAA merge at the interface of stratum corneum boarder and stratum granulosum (F). Binding of specific antibodies was visualized with FITC-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG. Blue arrows point to stratum corneum, magenta arrows to stratum basale white arrows to fibroblasts and yellow arrows to small and middle-sized vessels. Scale bar: A–D, H 200 μm; E–G 100 μm.
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pone-0004416-g001: Presence of GS in human foreskin of children and regional variations in its distribution revealed in co-localization studies with GFAP, or with SMAA.(A–D) Expression of GS (A) and GFAP (B) and merged micrograph of GS and GFAP (C) immune reaction in human foreskin sections. (D–F) Expression of GS (D) and SMAA (E) and merged micrograph of GS and SMAA immune reaction (F) in a human skin section. The line connecting the D–F clearly shows strong expression of SMAA (E) in stratum granulosum and its absence of stratum corneum. The latter is strongly stained for GS (D). GS and SMAA merge at the interface of stratum corneum boarder and stratum granulosum (F). Binding of specific antibodies was visualized with FITC-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG. Blue arrows point to stratum corneum, magenta arrows to stratum basale white arrows to fibroblasts and yellow arrows to small and middle-sized vessels. Scale bar: A–D, H 200 μm; E–G 100 μm.

Mentions: Double labeling of sections of human foreskin with polyclonal antibodies (pAb) directed against GS (green, Figure 1A) and monoclonal antibodies (mAb) against bovine GFAP (red, Figure 1B) revealed almost complete co-localization of both proteins in all layers of the epidermis: stratum basale (magenta arrows throughout Figure 1), stratum spinosum, stratum granulosum and stratum corneum (blue arrows throughout Figure 1). Similar spatial distribution of GS and GFAP was illustrated by intense yellow staining of epidermis in merged micrographs (Figure 1C). In young foreskin, the stratum corneum is very thin. The reaction of GS was particularly strong in stratum granulosum and stratum corneum and slightly weaker in stratum basale and stratum spinosum. In the dermis of these skin samples (white arrows), GS was expressed weaker than in epidermis (cf. structures indicated by blue and white arrows in Figures 1A, C). Similar findings for GS were obtained also in older foreskin (not shown).


Keratinocytes as depository of ammonium-inducible glutamine synthetase: age- and anatomy-dependent distribution in human and rat skin.

Danielyan L, Zellmer S, Sickinger S, Tolstonog GV, Salvetter J, Lourhmati A, Reissig DD, Gleiter CH, Gebhardt R, Buniatian GH - PLoS ONE (2009)

Presence of GS in human foreskin of children and regional variations in its distribution revealed in co-localization studies with GFAP, or with SMAA.(A–D) Expression of GS (A) and GFAP (B) and merged micrograph of GS and GFAP (C) immune reaction in human foreskin sections. (D–F) Expression of GS (D) and SMAA (E) and merged micrograph of GS and SMAA immune reaction (F) in a human skin section. The line connecting the D–F clearly shows strong expression of SMAA (E) in stratum granulosum and its absence of stratum corneum. The latter is strongly stained for GS (D). GS and SMAA merge at the interface of stratum corneum boarder and stratum granulosum (F). Binding of specific antibodies was visualized with FITC-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG. Blue arrows point to stratum corneum, magenta arrows to stratum basale white arrows to fibroblasts and yellow arrows to small and middle-sized vessels. Scale bar: A–D, H 200 μm; E–G 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637544&req=5

pone-0004416-g001: Presence of GS in human foreskin of children and regional variations in its distribution revealed in co-localization studies with GFAP, or with SMAA.(A–D) Expression of GS (A) and GFAP (B) and merged micrograph of GS and GFAP (C) immune reaction in human foreskin sections. (D–F) Expression of GS (D) and SMAA (E) and merged micrograph of GS and SMAA immune reaction (F) in a human skin section. The line connecting the D–F clearly shows strong expression of SMAA (E) in stratum granulosum and its absence of stratum corneum. The latter is strongly stained for GS (D). GS and SMAA merge at the interface of stratum corneum boarder and stratum granulosum (F). Binding of specific antibodies was visualized with FITC-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG. Blue arrows point to stratum corneum, magenta arrows to stratum basale white arrows to fibroblasts and yellow arrows to small and middle-sized vessels. Scale bar: A–D, H 200 μm; E–G 100 μm.
Mentions: Double labeling of sections of human foreskin with polyclonal antibodies (pAb) directed against GS (green, Figure 1A) and monoclonal antibodies (mAb) against bovine GFAP (red, Figure 1B) revealed almost complete co-localization of both proteins in all layers of the epidermis: stratum basale (magenta arrows throughout Figure 1), stratum spinosum, stratum granulosum and stratum corneum (blue arrows throughout Figure 1). Similar spatial distribution of GS and GFAP was illustrated by intense yellow staining of epidermis in merged micrographs (Figure 1C). In young foreskin, the stratum corneum is very thin. The reaction of GS was particularly strong in stratum granulosum and stratum corneum and slightly weaker in stratum basale and stratum spinosum. In the dermis of these skin samples (white arrows), GS was expressed weaker than in epidermis (cf. structures indicated by blue and white arrows in Figures 1A, C). Similar findings for GS were obtained also in older foreskin (not shown).

Bottom Line: While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age.Inhibition of, glycogen synthase kinase 3beta in cultured keratinocytes and HaCaT cells, however, did not support a direct role of beta-catenin in regulation of GS.Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacology, University Hospital of Tübingen, Tübingen, Germany.

ABSTRACT
In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component beta-catenin. Inhibition of, glycogen synthase kinase 3beta in cultured keratinocytes and HaCaT cells, however, did not support a direct role of beta-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8-10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia.

Show MeSH
Related in: MedlinePlus