Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Developmental expression of glial fibrillary acidic protein and glutamine synthetase in serum-free aggregating cell cultures of fetal rat telencephalon.

Serum-free aggregating cell cultures of fetal rat telencephalon were examined by a combined biochemical and double-labeling immunocytochemical study for the developmental expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). It was found that these two astroglial markers are co-expressed at different developmental stages in vitro. During the phase of cellular maturation (i.e. between days 14 and 34), GFAP levels and GS activity increase rapidly and in parallel. At the same time, the number of immunoreactive cells increase while the long and thick processes staining in early cultures gradually disappear. The present results demonstrate that in this particular cell culture system only one type of astrocytes develops which expresses both GFAP and GS and which attains a relatively high degree of maturation.

NEUROPATHOPHYSIOLOGICAL MECHANISMS IN GLUTARIC ACIDURIA TYPE I: 3-HYDROXYGLUTARIC ACID LEADS TO AMMONIA INCREASE AND NON-APOPTOTIC CELL DEATH

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in glutaric aciduria type I (GA-I). 1 mM glutarate (GA) or 3-hydroxyglutarate (3OHGA) were repeatedly added to the culture media at two different time points. In cultures treated with 3OHGA, we observed an increase in lactate in the medium, pointing to a possible inhibition of Krebs cycle and respiratory chain. We further observed that 3OHGA and to a lesser extend GA induced an increase in ammonia production with concomitant decrease of glutamine concentrations, which may suggest an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease which has deleterious effects on early stages of brain development. By immunohistochemistry we showed that 3OHGA increased non-apoptotic cell death. On the cellular level, 3OHGA and to a lesser extend GA led to cell swelling and loss of astrocytic fibers whereas a loss of oligodendrocytes was only observed for 3OHGA. We conclude that 3OHGAwas the most toxic metabolite in our model for GA-I. 3OHGA induced deleterious effects on glial cells, an increase of ammonia production, and resulted in accentuated cell death of non-apoptotic origin.

T Cell Priming by Activated Nlrc5-Deficient Dendritic Cells Is Unaffected despite Partially Reduced MHC Class I Levels.

NLRC5, a member of the NOD-like receptor (NLR) protein family, has recently been characterized as the master transcriptional regulator of MHCI molecules in lymphocytes, in which it is highly expressed. However, its role in activated dendritic cells (DCs), which are instrumental to initiate T cell responses, remained elusive. We show in this study that, following stimulation of DCs with inflammatory stimuli, not only did NLRC5 level increase, but also its importance in directing MHCI transcription. Despite markedly reduced mRNA and intracellular H2-K levels, we unexpectedly observed nearly normal H2-K surface display in Nlrc5(-/-) DCs. Importantly, this discrepancy between a strong intracellular and a mild surface defect in H2-K levels was observed also in DCs with H2-K transcription defects independent of Nlrc5. Hence, alongside with demonstrating the importance of NLRC5 in MHCI transcription in activated DCs, we uncover a general mechanism counteracting low MHCI surface expression. In agreement with the decreased amount of neosynthesized MHCI, Nlrc5(-/-) DCs exhibited a defective capacity to display endogenous Ags. However, neither T cell priming by endogenous Ags nor cross-priming ability was substantially affected in activated Nlrc5(-/-) DCs. Altogether, these data show that Nlrc5 deficiency, despite significantly affecting MHCI transcription and Ag display, is not sufficient to hinder T cell activation, underlining the robustness of the T cell priming process by activated DCs.