Keratinocytes as Depository of Ammonium-Inducible Glutamine Synthetase: Age- and Anatomy-Dependent Distribution in Human and Rat Skin

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 ß-catenin. Inhibition of, glycogen synthase kinase 3β in cultured keratinocytes and HaCaT cells, however, did not support a direct role of ß-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

The balance between the intronic miR-342 and its host gene Evl determines hematopoietic cell fate decision

Protein-coding and non-coding genes like miRNAs tightly control hematopoietic differentiation programs. Although miRNAs are frequently located within introns of protein-coding genes, the molecular interplay between intronic miRNAs and their host genes is unclear. By genomic integration site mapping of gamma-retroviral vectors in genetically corrected peripheral blood from gene therapy patients, we identified the EVL/MIR342 gene locus as a hotspot for therapeutic vector insertions indicating its accessibility and expression in human hematopoietic stem and progenitor cells. We therefore asked if and how EVL and its intronic miRNA-342 regulate hematopoiesis. Here we demonstrate that overexpression (OE) of Evl in murine primary Lin- Sca1+ cKit+ cells drives lymphopoiesis whereas miR-342 OE increases myeloid colony formation in vitro and in vivo, going along with a profound upregulation of canonical pathways essential for B-cell development or myelopoietic functions upon Evl or miR-342 OE, respectively. Strikingly, miR-342 counteracts its host gene by targeting lymphoid signaling pathways, resulting in reduced pre-B-cell output. Moreover, EVL overexpression is associated with lymphoid leukemia in patients. In summary, our data show that one common gene locus regulates distinct hematopoietic differentiation programs depending on the gene product expressed, and that the balance between both may determine hematopoietic cell fate decision

The balance between the intronic miR-342 and its host gene Evl determines hematopoietic cell fate decision

Protein-coding and non-coding genes like miRNAs tightly control hematopoietic differentiation programs. Although miRNAs are frequently located within introns of protein-coding genes, the molecular interplay between intronic miRNAs and their host genes is unclear. By genomic integration site mapping of gamma-retroviral vectors in genetically corrected peripheral blood from gene therapy patients, we identified the EVL/MIR342 gene locus as a hotspot for therapeutic vector insertions indicating its accessibility and expression in human hematopoietic stem and progenitor cells. We therefore asked if and how EVL and its intronic miRNA-342 regulate hematopoiesis. Here we demonstrate that overexpression (OE) of Evl in murine primary Li