Littérature scientifique sur le sujet « DIFFERENTIATION, NGF, TUBULOGENESIS, PERIPHERIN »

Peripherin is the major neuronal intermediate filament (IF) protein in PC12 cells and both its synthesis and amount increase during nerve growth factor (NGF) promoted neuronal differentiation. To address the question of the biological function of peripherin in neurite initiation we have used an antisense oligonucleotide complementary to the 5' region of peripherin mRNA to specifically inhibit its transcription. The oligonucleotide blocks both the synthesis of peripherin and its increase in response to NGF. Peripherin was found to be a stable protein with a cellular half-life of approximately 7 d. 6 wk of incubation with the oligonucleotide decreases peripherin to 11% of the level in naive control cells and to 3% of that in NGF-treated control cells. Despite the depletion, NGF elicits apparently normal neurite outgrowth from the oligonucleotide-treated cells. As evaluated by EM, there are few IFs in these cells, either in the cell bodies or neurites. There is no compensatory increase in NF-M, NF-L, or vimentin levels as a result of the inhibition of peripherin synthesis. These findings suggest that peripherin is not required for neurite formation, but is necessary for the formation of a cellular IF network which could be involved in process stability. They also demonstrate the utility of antisense oligonucleotides for the study of proteins with long half-lives.

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.

Receptor tyrosine kinases play essential roles in morphogenesis and differentiation of epithelia. Here we examined various tyrosine kinase receptors, which are preferentially expressed in epithelia (c-met, c-ros, c-neu, and the keratin growth factor [KGF] receptor), for their capacity to induce cell motility and branching morphogenesis of epithelial cells. We exchanged the ligand-binding domain of these receptors by the ectodomain of trkA and could thus control signaling by the new ligand, NGF. We demonstrate here that the tyrosine kinases of c-met, c-ros, c-neu, the KGF receptor, and trkA, but not the insulin receptor, induced scattering and increased motility of kidney epithelial cells in tissue culture. Mutational analysis suggests that SHC binding is essential for scattering and increased cell motility induced by trkA. The induction of motility in epithelial cells is thus an important feature of various receptor tyrosine kinases, which in vivo play a role in embryogenesis and metastasis. In contrast, only the c-met receptor promoted branching morphogenesis of kidney epithelial cells in three-dimensional matrices, which resemble the formation of tubular epithelia in development. Interestingly, the ability of c-met to induce morphogenesis could be transferred to trkA, when in a novel receptor hybrid COOH-terminal sequences of c-met (including Y14 to Y16) were fused to the trkA kinase domain. These data demonstrate that tubulogenesis of epithelia is a restricted activity of tyrosine kinases, as yet only demonstrated for the c-met receptor. We predict the existence of specific substrates that mediate this morphogenesis signal.

Introduction: Type 2 diabetes mellitus (T2D) in association with critical limb ischemia (CLI) is detrimental to skeletal muscle function and metabolism and compromises muscle intrinsic regenerative ability. Hypothesis: Assess whether T2D+CLI negatively impact on muscular regenerative cells such as muscle pericytes (MPs) functional properties. Methods: Skeletal muscle biopsies were obtained under ethical approval at occasion of osteosarcoma diagnosis (H-MPs, n=8 ) or major amputation (T2D+CLI-MPs, n=11). MPs were characterized by flow cytometry (CD90, CD44, and Alkaline Phosphatase-AP), immunofluorescence (PDGFR-β, α-SMA, CD146, and NG2) and immunicytochemistry (AP). MP proliferation (BrdU assay), migration (scratch assay), apoptosis (caspase assay), myogenic differentiation (myosine heavy chain-MyHC staining), HUVECs tubulogenesis (Matrigel) and ROS production (MitoSox-Red) were assessed in vitro in at least n=3 donors per group. Molecular analysis were performed by RT-PCR. Results: Both H-MPs and T2D+CLI-MPs expressed typical pericyte markers, but T2D+CLI-MPs proliferated slowly (0.16±0.06 vs. 1.98±0.46 vs. H-MPs) and differentiated less into MyHC positive fibres (4.78±0.34 vs. 16.15±5.032 vs. H-MPs) while migration and apoptosis were not modified. The addition of T2D+CLI-MPs or their conditioned media to HUVECs impaired endothelial tubulization on Matrigel (respectively 10.40±1.39 vs. 13.73±0.64, and 11.88±1.04 vs. 16±1.4, vs. HUVECs alone). Furthermore ROS content increased in T2D+CLI-MPs (3.70±0.45 vs. 0.57±0.24, vs. H-MPs) and was associated with a down-regulation of SOD-1 (0.53±0.11 vs. 1.03±0.11) and catalase (0.41±0.1 vs. 0.97±0.2 vs. H-MPs) and an up-regulation of p66Shc (2.84±0.6 vs. 0.81±0.02 vs. H-MPs). Antioxidant treatment with N-acetylcysteine reverted altered proliferation (1.55±0.06 vs. 1.21±0.02) and antiangiogenic effect (13.63±1.00 vs. 7.367±0.75) compared to untreated counterparts. For all experiments significance was p<0.05. Conclusions: T2D+CLI hampers MPs biological functions relevant to muscular repair. Reversion of T2D+CLI-MPs alterations by antioxidant treatment suggests possible therapeutic targets for attenuation of peripheral complications.

ABSTRACT Background: Leucine-Rich Repeat (LRR) proteins play a key role in several systems, from immune response to neuritogenesis. Some years ago, our laboratory identified a gene located in the q27 region of the human X Chromosome that encodes for a LRR-enriched protein. Interestingly, this cytogenetic band contains the gene responsible for the Fragile X syndrome, the most common form of inherited intellectual disability, and other several tissue-specific genes, in particular several brain-specific genes. The function of the gene identified in our laboratory, located to on the q27.3 region and named Slitrk2, is to date largely unknown. The goal of this work was to characterize structurally and functionally Slitrk2. Principal Findings: Slitrk2 is a member of the Slitrk family. Slitrk family has 6 members whose function is still unknown. In order to achieve a better understanding about Slitrk2 function, we carried out integrated analysis at the transcriptomic, proteomic and bioinformatic level. At a transcriptomic level, we found that Slitrk2 expression is mainly restricted to the Central Nervous System. Slitrk2 is also expressed, although at low level compared to brain, in mammary gland, pancreas, prostate, testis. This expression pattern suggest a role for Slitrk2 not only in brain, but in general in organogenesis and morphogenesis. Slitrk2 expression during mouse embryogenesis starts at Embryonic Day 11, indicating that Slitrk2 acts in post-gastrulation mechanisms. By in situ hybridization in adult mouse brain, we found that Slitrk2 is expressed mainly in the Dentate Gyrus of the hippocampal formation. The in silico analysis of the protein reveals that Slitrk2 is likely to be located at the plasma-membrane. Some Trans-Golgi retrivial signals were found in the Slitrk2 sequence. With a multilevel computer-assisted approach we identified two potential phosphorylation sites: Threonine 806 and Tyrosine 832. Tyrosine 832 is in an aminoacidic environment able to be recognised by the COP-I coatomer, indicating that a Golgi-Retrivial of Slitrk2 is possible. The three-dimensional structure of Slitrk2 obtained with Phyre and I-TASSER calculations showed that the predicted extracellular region of Slitrk2 folded similarly to the ectodomain of other key neuronal protein such as Lingo-1 and NogoReceptor. With in vitro analyses, we found that Slitrk2 protein is mainly located intracellularly, being polarized to one side of the perinuclear boundary in PC12 cells. Slitrk2 expression is regulated by Nerve Growth Factor during PC12 cells differentiation, being downregulated after NGF exposure. The forced expression of Slitrk2 during NGF differentiation of PC12 cells lead to a reduction of the neuritic network caused by an impairment in Intermediate Filaments assembly (Peripherin). During NGF deprivation of neurotrophin-differentiated PC12 cells Slitrk2 transcript is upregulated. The knocking-down of Slitrk2 with short-interfering RNA recovered the drop in cell viability caused by neurotrophin depletion in NGF-differentiated PC12 cells. Outside the nervous system, Slitrk2 is expressed at low levels also in mammary gland. Also in mammary cell lines, both normal and tumoral (MCF10A and LA7/3F12, respectively), Slitrk2 forced expression determined impairment in differentiation reducing tubulogenesis. In both the above-mentioned mammary cell lines, immunofluorescence analysis showed that Slitrk2 localization is mainly intracellularly as seen in PC12 cells. Significance: In this work we performed: i) the transcriptional analysis of Slitrk2 in neuronal and non-neuronal systems; ii) the bioinformatic analysis of Slitrk2 topology, phosphorylations and three-dimensional structure and iii) the functional analysis of Slitrk2 in both neuronal and mammary gland cells. The main findings of this work are related to: i) the in vivo expression of Slitrk2 in adult mouse brain; ii) the homology modelling of the three-dimensional structure of Slitrk2 with the mapping of the predicted potential phosphorylation sites, potential N-glycosylation site and predicted ligand-binding sites and iii) the involment of Slitrk2 in the regulation of Intermediate Filament assembly in neuronal cells and the involment of Slitrk2 as a regulator of tubulogenesis in mammary cells. Future directions: In order to complete this work, we are currently analysing the role of Slitrk2 in embryosgenesis by using Danio rerio (Zebrafish) as a model of development. We are also studying more in deep the pathways by which Slitrk2 regulates neuronal cells differentiation. As last, we are now studying at the molecular level the role of Slitrk2 in tubulogenesis not only in mammary gland but also in kidney.