Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene C-MYC. We found that AMBRA1 favors the interaction between C-MYC and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of C-MYC correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.
In zebrafish, ovulated oocytes contain both maternal cortisol and the mRNA for the glucocorticoid receptor (gr), which is spread as granular structures throughout the ooplasm. At 0.2 hpf, this transcript is relocated in the blastodisc area and partitioned among blastomeres. At 6-8 hpf, it is replaced by zygotic transcript. We used morpholinos to block translation of both maternal and zygotic gr transcripts, and a missplicing morpholino to block post-transcriptionally the zygotic transcript alone. Only knockdown of translation produced an increase of apoptosis and subsequent craniofacial and caudal deformities with severe malformations of neural, vascular, and visceral organs in embryos and 5-dpf larvae. Such defects were rescued with trout gr2 mRNA. Microarray analysis revealed that 114 and 37 highly expressed transcripts were up-and down-regulated, respectively, by maternal Gr protein deficiency in 5-hpf embryos. These results indicate that the maternal gr transcript and protein participate in the maternal programming of zebrafish development. Developmental Dynamics 240:874-889,
Glucocorticoids (GCs) play important roles in developmental and physiological processes through the transcriptional activity of their cognate receptor (Gr). Using CRISPR/Cas9 technology, we established a zebrafish null Gr mutant line and compared its phenotypes with wild type and a zebrafish line with partially silenced gr (grs357/s357). Homozygous gr−/− larvae are morphologically inconspicuous and, in contrast to GR−/− knockout mice, viable through adulthood, although with reduced fitness and early life survival. Mutants gr−/− are fertile, but their reproductive capabilities fall at around 10 months of age, when, together with cardiac and intestinal abnormalities already visible at earlier stages, increased fat deposits are also observed. Mutants show higher levels of whole-body cortisol associated with overstimulated basal levels of crh and pomca transcripts along the HPI axis, which is unresponsive to a mechanical stressor. Transcriptional activity linked to immune response is also hampered in the gr−/− line: after intestinal damage by dextran sodium sulphate exposure, there are neither inflammatory nor anti-inflammatory cytokine gene responses, substantiating the hypothesis of a dual-action of the GC-GR complex on the immune system. Hence, the zebrafish gr mutant line appears as a useful tool to investigate Gr functions in an integrated in vivo model.
Amputated tails of lizards regenerate while limbs form scars which histological structure is very different from the original organs. Lizards provide useful information for regenerative medicine and some hypotheses on the loss of regeneration in terrestrial vertebrates. Analysis of tail and limb transcriptomes shows strong downregulation in the tail blastema for immunoglobulins and surface B and T receptors, cell function, and metabolism. In contrast, in the limb blastema genes for myogenesis, muscle and cell function, and extracellular matrix deposition but not immunity are variably downregulated. The upregulated genes show that the regenerating tail is an embryonic organ driven by the Wnt pathway and non-coding RNAs. The strong inflammation following amputation, the non-activation of the Wnt pathway, and the upregulation of inflammatory genes with no downregulation of immune genes indicate that the amputated limb does not activate an embryonic program. Intense inflammation in limbs influences in particular the activity of genes coding for muscle proteins, cell functions, and stimulates the deposition of dense extracellular matrix proteins resulting in scarring limb outgrowths devoid of muscles. The present study complements that on upregulated genes, and indicates that the regenerating tail requires immune suppression to maintain this embryonic organ connected to the rest of the tail without be rejected or turned into a scar. It is hypothesized that the evolution of the adaptive immune system determined scarring instead of organ regeneration in terrestrial vertebrates and that lizards evolved the process of tail regeneration through a mechanism of immuno-evasion.
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