Plant miR171 modulates mTOR pathway in HEK293 cells by targeting GNA12.

Plant microRNAs have shown the capacity to regulate mammalian systems. The potential bioactivity of miR171vr, an isoform of the plant miR171, on human embryonic kidney 293 (HEK293) cells was investigated. Bioinformatics simulations revealed that human G protein subunit alpha 12 (GNA12) transcript could represent an excellent target for miR171vr. To confirm this prediction, in vitro experiments were performed using a synthetic microRNA designed on miR171vr sequence. MiR-treated cells showed a significant decrease of GNA12 mRNA and protein levels, confirming the putative cross-kingdom interaction. In addition, miR171vr determined the modulation of GNA12 downstream signaling factors, including mTOR, as expected. Finally, the effect of the plant miRNA on HEK293 cell growth and its stability in presence of several stressors, such as those miming digestive processes and procedures for preparing food, were evaluated. All this preliminary evidence would suggest that miR171vr, introduced by diet or as supplement in gene therapies, could potentially influence human gene expression, especially for treating disorders where GNA12 is over-expressed (i.e. oral cancer, breast and prostate adenocarcinoma) or mTOR kinase is down-regulated (e.g. obesity, type 2 diabetes, neurodegeneration).

The oncoprotein Myc controls the phosphorylation of S6 kinase and AKT through protein phosphatase 2A.

This study focuses on the effects of Myc oncoprotein on the translational apparatus of the cell. Translation is an energy consuming process that involves a large number of accessory factors. The production of components of the protein synthesis machinery can be regulated at the transcriptional level by specific factors. It has been shown that the product of the oncogene Myc, a transcription factor frequently activated in cancer, can control translational activity through an increase in the transcription of the eIF4F complex components (eIF4E, eIF4AI, and eIF4GI). However, additional effects at the posttranslational level have also been described. For instance, it has been shown that Myc upregulation can induce mammalian target of rapamycin (mTOR)-dependent 4E-binding protein 1 (4E-BP1) hyperphosphorylation. We induced overexpression or inhibition of Myc through transfection of complementary DNA constructs or specific small interfering RNA in PC3 (prostate carcinoma) and HeLa (cervical carcinoma) cells. We have observed that overexpression of Myc causes an increase in 4E-BP1 phosphorylation and activation of protein synthesis. Unexpectedly, we detected a parallel decrease in the phosphorylation level of S6 kinase (in PC3 and HeLa) and AKT (in HeLa). We report evidence that these changes are mediated by an increase in protein phosphatase 2A activity.

MDM4 actively restrains cytoplasmic mTORC1 by sensing nutrient availability.

BACKGROUND: Many tumor-related factors have shown the ability to affect metabolic pathways by paving the way for cancer-specific metabolic features. Here, we investigate the regulation of mTORC1 by MDM4, a p53-inhibitor with oncogenic or anti-survival activities depending on cell growth conditions. METHOD: MDM4-mTOR relationship was analysed through experiments of overexpression or silencing of endogenous proteins in cell culture and using purified proteins in vitro. Data were further confirmed in vivo using a transgenic mouse model overexpressing MDM4. Additionally, the Cancer Genome Atlas (TCGA) database (N = 356) was adopted to analyze the correlation between MDM4 and mTOR levels and 3D cultures were used to analyse the p53-independent activity of MDM4. RESULTS: Following nutrient deprivation, MDM4 impairs mTORC1 activity by binding and inhibiting the kinase mTOR, and contributing to maintain the cytosolic inactive pool of mTORC1. This function is independent of p53. Inhibition of mTORC1 by MDM4 results in reduced phosphorylation of the mTOR downstream target p70S6K1 both in vitro and in vivo in a MDM4-transgenic mouse. Consistently, MDM4 reduces cell size and proliferation, two features controlled by p70S6K1, and, importantly, inhibits mTORC1-mediated mammosphere formation. Noteworthy, MDM4 transcript levels are significantly reduced in breast tumors characterized by high mTOR levels. CONCLUSION: Overall, these data identify MDM4 as a nutrient-sensor able to inhibit mTORC1 and highlight its metabolism-related tumor-suppressing function.

Neuroprotection by rat Müller glia against high glucose-induced neurodegeneration through a mechanism involving ERK1/2 activation.

Müller cell activation is an early finding in diabetic retinopathy (DR), but its physiopathologic role in the disease is still unclear, especially in the early phases. We investigated on Müller glial activation in primary rat retinal cultures, exposed to High Glucose (HG), and in retinas from streptozotocin (stz)-induced diabetic rats. First of all, we checked if the presence of Müller glia influenced HG neurotoxicity. In mixed glial/neuronal retinal cultures, a single HG administration (sHG) for 48 h induced activation of Müller glia, in absence of neuronal damage. In contrast, in pure neuronal cultures, a marked neurotoxic effect was detected, suggesting that in this cell model Müller glia protect neurons from HG neurotoxicity. To better mimic the diabetic milieu, where retinal cells are constantly bathed in hyperglycemic fluid, and to further characterize astrocytic neuroprotective ability, mixed retinal cultures were exposed to repeated daily replacement of HG (rHG). In this paradigm, starting from 48 h, increased apoptosis and synaptic loss were observed, even in the presence of Müller cells. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whose activation triggers a prosurvival pathway, was increased by sHG, while it was down-regulated by rHG, suggesting that ERK1/2 activation is involved in neuroprotection. Consistently, in presence of ERK1/2 inhibitor PD98059, sHG exerted a proapoptotic effect also in glial/neuronal retinal cultures. In line with the in vitro data, early changes in diabetic retinas from stz-injected rats included Müller cell activation and increased pERK1/2 levels, but no signs of neuronal damage. These results suggest that, in the early phases of DR, Müller glial activation does not contribute to neurodegeneration, but may indeed have a neuroprotective activity against HG-induced neurotoxicity through a mechanism involving pERK1/2.

Articular Cartilage Regeneration by Hyaline Chondrocytes: A Case Study in Equine Model and Outcomes.

Cartilage injury defects in animals and humans result in the development of osteoarthritis and the progression of joint deterioration. Cell isolation from equine hyaline cartilage and evaluation of their ability to repair equine joint cartilage injuries establish a new experimental protocol for an alternative approach to osteochondral lesions treatment. Chondrocytes (CCs), isolated from the autologous cartilage of the trachea, grown in the laboratory, and subsequently arthroscopically implanted into the lesion site, were used to regenerate a chondral lesion of the carpal joint of a horse. Biopsies of the treated cartilage taken after 8 and 13 months of implantation for histological and immunohistochemical evaluation of the tissue demonstrate that the tissue was still immature 8 months after implantation, while at 13 months it was organized almost similarly to the original hyaline cartilage. Finally, a tissue perfectly comparable to native articular cartilage was detected 24 months after implantation. Histological investigations demonstrate the progressive maturation of the hyaline cartilage at the site of the lesion. The hyaline type of tracheal cartilage, used as a source of CCs, allows for the repair of joint cartilage injuries through the neosynthesis of hyaline cartilage that presents characteristics identical to the articular cartilage of the original tissue.

Arthroscopic Treatment of a Subchondral Bone Cyst via Stem Cells Application: A Case Study in Equine Model and Outcomes.

Subchondral bone cysts in horses represent one of the main causes of lameness that can occur in different anatomical locations. The study describes the treatment in regenerative therapy of the intracystic implantation of adipose tissue mesenchymal stromal cells (AMSCs) included in platelet-rich plasma (PRP). The ability of AMSCs to differentiate in osteogenic cells was tested in vitro and in vivo. Given the aim to investigate the application of AMSCs in bone defects and orthopedic pathologies in horses, a four-year-old male thoroughbred racing horse that had never raced before was treated for lameness of the left hind leg caused by a cyst of the medial femoral condyle. The horse underwent a new surgery performed with an arthroscopic approach in which the cystic cavity was filled with AMSCs contained in the PRP. Radiographs were taken 3, 5, and 10 months after the surgery to assess the development of newly regenerated bone tissue in the gap left by the cyst. Twelve months after the operation and after six months of regular daily training, the horse did not show any symptoms of lameness and started a racing career. According to the study, the use of AMSCs and PRP suggests promising benefits for treating subchondral bone cysts.

Estrogens Counteract Platinum-Chemosensitivity by Modifying the Subcellular Localization of MDM4.

Estrogen activity towards cancer-related pathways can impact therapeutic intervention. Recent omics data suggest possible crosstalk between estrogens/gender and MDM4, a key regulator of p53. Since MDM4 can either promote cell transformation or enhance DNA damage-sensitivity, we analysed in vivo impact of estrogens on both MDM4 activities. In Mdm4 transgenic mouse, Mdm4 accelerates the formation of fibrosarcoma and increases tumor sensitivity to cisplatin as well, thus confirming in vivo Mdm4 dual mode of action. Noteworthy, Mdm4 enhances chemo- and radio-sensitivity in male but not in female animals, whereas its tumor-promoting activity is not affected by mouse gender. Combination therapy of transgenic females with cisplatin and fulvestrant, a selective estrogen receptor degrader, was able to recover tumor cisplatin-sensitivity, demonstrating the relevance of estrogens in the observed sexual dimorphism. Molecularly, estrogen receptor-α alters intracellular localization of MDM4 by increasing its nuclear fraction correlated to decreased cell death, in a p53-independent manner. Importantly, MDM4 nuclear localization and intra-tumor estrogen availability correlate with decreased platinum-sensitivity and apoptosis and predicts poor disease-free survival in high-grade serous ovarian carcinoma. These data demonstrate estrogen ability to modulate chemo-sensitivity of MDM4-expressing tumors and to impinge on intracellular trafficking. They support potential usefulness of combination therapy involving anti-estrogenic drugs.

Author Correction: Lymphoblastoid cell lines from Diamond Blackfan anaemia patients exhibit a full ribosomal stress phenotype that is rescued by gene therapy.

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Lymphoblastoid cell lines from Diamond Blackfan anaemia patients exhibit a full ribosomal stress phenotype that is rescued by gene therapy.

Diamond Blackfan anaemia (DBA) is a congenital bone marrow failure syndrome characterised by selective red cell hypoplasia. DBA is most often due to heterozygous mutations in ribosomal protein (RP) genes that lead to defects in ribosome biogenesis and function and result in ribosomal stress and p53 activation. The molecular mechanisms underlying this pathology are still poorly understood and studies on patient erythroid cells are hampered by their paucity. Here we report that RP-mutated lymphoblastoid cell lines (LCLs) established from DBA patients show defective rRNA processing and ribosomal stress features such as reduced proliferation, decreased protein synthesis, and activation of p53 and its target p21. These phenotypic alterations were corrected by gene complementation. Our data indicate that DBA LCLs could be a useful model for molecular and pharmacological investigations.

PIM1 destabilization activates a p53-dependent response to ribosomal stress in cancer cells.

Defects in ribosome biogenesis triggers a stress response (ribosomal stress) that can lead to growth arrest and apoptosis. Signaling pathways activated by ribosomal stress are specifically involved in the pathological mechanism of a group of disorders defined as ribosomopathies. However, more generally, the quality control of ribosome synthesis is part of the regulatory circuits that control cell metabolism. A number of studies identified tumor suppressor p53 as a central player in ribosomal stress. We have previously reported that the kinase PIM1 plays a role as a sensor for ribosome deficiency. In this report we address the relationship between PIM1 and p53 in cancer cell lines after depletion of a ribosomal protein. We identified a novel signaling pathway that includes the kinase AKT and the ubiquitin ligase MDM2. In fact, our results indicate that the lower level of PIM1, induced by ribosomal stress, causes inactivation of AKT, inhibition of MDM2 and a consequent p53 stabilization. Therefore, we propose that activation of p53 in response to ribosomal stress, is dependent on the pathway PIM1-AKT-MDM2. In addition, we report evidence that PIM1 level may be relevant to assess the sensitivity of cancer cells to chemotherapeutic drugs that induce ribosomal stress.

Depletion of ribosomal protein S19 causes a reduction of rRNA synthesis.

Ribosome biogenesis plays key roles in cell growth by providing increased capacity for protein synthesis. It requires coordinated production of ribosomal proteins (RP) and ribosomal RNA (rRNA), including the processing of the latter. Here, we show that, the depletion of RPS19 causes a reduction of rRNA synthesis in cell lines of both erythroid and non-erythroid origin. A similar effect is observed upon depletion of RPS6 or RPL11. The deficiency of RPS19 does not alter the stability of rRNA, but instead leads to an inhibition of RNA Polymerase I (Pol I) activity. In fact, results of nuclear run-on assays and ChIP experiments show that association of Pol I with the rRNA gene is reduced in RPS19-depleted cells. The phosphorylation of three known regulators of Pol I, CDK2, AKT and AMPK, is altered during ribosomal stress and could be involved in the observed downregulation. Finally, RNA from patients with Diamond Blackfan Anemia (DBA), shows, on average, a lower level of 47S precursor. This indicates that inhibition of rRNA synthesis could be one of the molecular alterations at the basis of DBA.