A Focus on Regulatory Networks Linking MicroRNAs, Transcription Factors and Target Genes in Neuroblastoma

Neuroblastoma (NB) is a tumor of the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. NB originates from neural crest cells (NCCs) undergoing a defective sympathetic neuronal differentiation and although the starting events leading to the development of NB remain to be fully elucidated, the master role of genetic alterations in key oncogenes has been ascertained: (1) amplification and/or over-expression of MYCN, which is strongly associated with tumor progression and invasion; (2) activating mutations, amplification and/or over-expression of ALK, which is involved in tumor initiation, angiogenesis and invasion; (3) amplification and/or over-expression of LIN28B, promoting proliferation and suppression of neuroblast differentiation; (4) mutations and/or over-expression of PHOX2B, which is involved in the regulation of NB differentiation, stemness maintenance, migration and metastasis. Moreover, altered microRNA (miRNA) expression takes part in generating pathogenetic networks, in which the regulatory loops among transcription factors, miRNAs and target genes lead to complex and aberrant oncogene expression that underlies the development of a tumor. In this review, we have focused on the circuitry linking the oncogenic transcription factors MYCN and PHOX2B with their transcriptional targets ALK and LIN28B and the tumor suppressor microRNAs let-7, miR-34 and miR-204, which should act as down-regulators of their expression. We have also looked at the physiologic role of these genetic and epigenetic determinants in NC development, as well as in terminal differentiation, with their pathogenic dysregulation leading to NB oncogenesis.

Role of SIRT1 and Progesterone Resistance in Normal and Abnormal Endometrium

Context Progesterone resistance, a known pathologic condition associated with a reduced cellular response to progesterone and heightened estrogen responses, appears to have a normal physiologic role in mammalian reproduction. The molecular mechanism responsible for progesterone resistance in normal and abnormal endometrium remains unclear. Objective To examine the roles of Sirtuin-1 (SIRT1) in normal endometrium as well as endometrium associated with infertility and endometriosis, as an epigenetic modulator associated with progesterone resistance. Methods SIRT1 expression was examined by Western blot, RT-qPCR and immunohistochemistry in mouse uterus and human endometrium. Mice with uterine specific Sirt1 overexpression were developed to examine SIRT1’s role in endometrial function and endometriosis development. EX-527, a SIRT1 inhibitor, and SRT1720, a SIRT1 agonist, were also used to evaluate SIRT1 effect on endometriosis. Results In normal healthy women, endometrial SIRT1 is expressed only during menses. SIRT1 was dramatically overexpressed in the endometrium from women with endometriosis in both the epithelium and strom. In mice, SIRT1 is expressed at the time of implantation between day 4.5 and 5.5 of pregnancy. Overexpression of SIRT1 (Sirt1 over) in the mouse uterus leads to subfertility due to implantation failure and decidualization defects and progesterone resistance. SIRT1 overexpression in endometriotic lesion promotes worsening endometriosis development. EX-527 (SIRT1 inhibitor) significantly reduced the number of endometriotic lesions in the mouse endometriosis model. Conclusions SIRT1 expression and progesterone resistance appears to play -roles in normal endometrial functions. Aberrant SIRT1 expression contributes to progesterone resistance and may participate in the pathophysiology of endometriosis. SIRT1 is a novel and targetable protein for the diagnosis as well as treatment of endometriosis and the associated infertility seen in this disease.

Reduced MC4R signaling alters nociceptive thresholds associated with red hair

Humans and mice with natural red hair have elevated basal pain thresholds and an increased sensitivity to opioid analgesics. We investigated the mechanisms responsible for higher nociceptive thresholds in red-haired mice resulting from a loss of melanocortin 1 receptor (MC1R) function and found that the increased thresholds are melanocyte dependent but melanin independent. MC1R loss of function decreases melanocytic proopiomelanocortin transcription and systemic melanocyte-stimulating hormone (MSH) levels in the plasma of red-haired (Mc1re/e) mice. Decreased peripheral α-MSH derepresses the central opioid tone mediated by the opioid receptor OPRM1, resulting in increased nociceptive thresholds. We identified MC4R as the MSH-responsive receptor that opposes OPRM1 signaling and the periaqueductal gray area in the brainstem as a central area of opioid/melanocortin antagonism. This work highlights the physiologic role of melanocytic MC1R and circulating melanocortins in the regulation of nociception and provides a mechanistic framework for altered opioid signaling and pain sensitivity in red-haired individuals.

Evolution of Fc Receptor-Like Scavenger in Mammals

Fc receptor-like (FCRL) molecules comprise a large family of receptors, homologous to the receptors for the Fc portion of immunoglobulins (FCR). Within this family, an unusual gene known to exist in mice, rats and dogs, termed FCRLS, encodes a chimeric protein with both Ig-like FCRL and type B scavenger-receptor cysteine-rich (SRCR)-like domains. In mice, FCRLS is located next to the CD5L and KIRREL1 genes. Here, we show that the curious FCRLS gene is actually present across major mammalian groups, but its annotation is generally incorrect or absent. Anchored on mouse FCRLS and FCRL2 genomic sequence alignments, phylogenetic analyses demonstrated that many mammalian sequences currently annotated as FCRL2 cluster with FCRLS, supported by a conserved genetic synteny among organisms. This analysis shows that FCRLS is present in Rodentia, some Carnivora (Canidae and Ursidae), Chiroptera, Arctiodactyla, Proboscidae, and some Primata. Thus, the FCRLS most likely originated in a eutherian mammal ancestor since it is not present in Monotremata or Marsupialia. FCRLS has a peculiar distribution pattern across mammalian lineages, being present in some species, but absent in others from the same family, as in carnivores for example. The most parsimonious hypothesis to explain this FCRLS evolution is that it was convergently lost in several independent mammalian lineages. Analyses of branch-specific nucleotide evolutionary rates, show that FCRL2 and FCRLS have similar ranges of rates across mammals, suggesting that both genes have crucial, but separate functions in the immune system. Bayesian estimates of evolutionary rates for FCRLS in mammalian lineages revealed that carnivores display the highest mutation rate after rodents. Additionally, positive diversifying selection was detected for both FCRL2 and FCRLS. Our results show that the presence of the FCRLS gene is older and more widespread across mammals than previously thought and appears to be functional, being under positive selection. Its precise physiologic role should thus be investigated.

High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells

Mortality of patients with end-stage renal disease tremendously exceeds that of the general population due to excess cardiovascular morbidity. Large middle-sized molecules (LMM) including pro-inflammatory cytokines are major drivers of uremic cardiovascular toxicity and cannot be removed sufficiently by conventional high-flux (HFL) hemodialysis. We tested the ability of plasma from 19 hemodialysis patients participating in a trial comparing HFL with high cut-off (HCO) membranes facilitating removal of LMM to induce calcification in mesenchymal stromal cells (MSC) functioning as vascular progenitors. HCO dialysis favorably changed plasma composition resulting in reduced pro-calcific activity. LMM were removed more effectively by HCO dialysis including FGF23, a typical LMM we found to promote osteoblastic differentiation of MSC. Protein-bound uremic retention solutes with known cardiovascular toxicity but not LMM inhibited proliferation of MSC without direct toxicity in screening experiments. We could not attribute the effect of HCO dialysis on MSC calcification to distinct mediators. However, we found evidence of sustained reduced inflammation that might parallel other anti-calcifying mechanisms such as altered generation of extracellular vesicles. Our findings imply protection of MSC from dysfunctional differentiation by novel dialysis techniques targeted at removal of LMM. HCO dialysis might preserve their physiologic role in vascular regeneration and improve outcomes in dialysis patients.

Vitis vinifera bZIP14 functions as a transcriptional activator and enhances drought stress resistance via suppression of reactive oxygen species

Background: Drought stress affects grapevine growth and development and reduces berry yield and quality. Identifying genes that are involved in the plant response to drought stress will enable the development of new grape strains that are tolerant to drought. Objective: We cloned the VvibZIP14 gene from Vitis vinifera and analyzed its role in drought resistance. Methods: Gene expression was analyzed by quantitative real-time PCR. Subcellular localization was assessed with a transient expression assay. The transactivation activity of the protein was evaluated in yeast. The physiologic role of VvibZIP14 was analyzed by overexpressing VvibZIP14 in Arabidopsis following drought stress. Hydrogen peroxide accumulation in Arabidopsis was visualized by diaminobenzidine staining. Results: Drought stress caused the accumulation of VvibZIP14, which was localized in the nucleus and had transcriptional activity. Transgenic plants showed improved resistance to drought stress and reduced electrolyte leakage compared to plants overexpressing empty vector, whereas chlorophyll content, photosystem II maximal photochemical efficiency, and net photosynthetic rate were higher. Catalase, peroxidase, and superoxide dismutase activities were also increased in VvibZIP14-overexpressing plants subjected to drought stress. Conclusions: VvibZIP14 functions as a transcription factor that confers resistance to drought stress in grape by enhancing the antioxidant response.

DLK1 Expressed in Mouse Orexin Neurons Modulates Anxio-Depressive Behavior but Not Energy Balance

Lateral hypothalamic area (LHA) neurons expressing the neuropeptide orexin (OX) are implicated in obesity and anxio-depression. However, these neurons release OX as well as a host of other proteins that might contribute to normal physiology and disease states. We hypothesized that delta-like homolog 1 (DLK1), a protein reported to be co-expressed by all OX neurons, contributes to the regulation of energy balance and/or anxio-depression. Consistent with previous reports, we found that all rat OX neurons co-express DLK1. Yet, in mice and humans only a subset of OX neurons co-expressed DLK1. Since human OX-DLK1 distribution is more similar to mice than rats, mice are a comparable model to assess the human physiologic role of DLK1. We therefore used a viral lesion strategy to selectively delete DLK1 within the LHA of adult mice (DLK1Null) to reveal its role in body weight and behavior. Adult-onset DLK1 deletion had no impact on body weight or ingestive behavior. However, DLK1Null mice engaged in more locomotor activity than control mice and had decreased anxiety and depression measured via the elevated plus maze and forced swim tests. These data suggest that DLK1 expression via DLK1-expressing OX neurons primarily contributes to anxio-depression behaviors without impacting body weight.

The Collision of Meta-Inflammation and SARS-CoV-2 Pandemic Infection

The coronavirus disease 2019 (COVID-19) pandemic has forced us to consider the physiologic role of obesity in the response to infectious disease. There are significant disparities in morbidity and mortality by sex, weight, and diabetes status. Numerous endocrine changes might drive these varied responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, including hormone and immune mediators, hyperglycemia, leukocyte responses, cytokine secretion, and tissue dysfunction. Studies of patients with severe COVID-19 disease have revealed the importance of innate immune responses in driving immunopathology and tissue injury. In this review we will describe the impact of the metabolically induced inflammation (meta-inflammation) that characterizes obesity on innate immunity. We consider that obesity-driven dysregulation of innate immune responses may drive organ injury in the development of severe COVID-19 and impair viral clearance.

ALKBH7 mediates necrosis via rewiring of glyoxal metabolism

ABSTRACTAlkb homolog 7 (ALKBH7) is a mitochondrial α-ketoglutarate dioxygenase required for necrotic cell death in response to DNA alkylating agents, but its physiologic role within tissues remains unclear. Herein, we show that ALKBH7 plays a key role in the regulation of dialdehyde metabolism, which impacts cardiac survival in response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we do not find evidence that ALKBH7 functions as a prolyl-hydroxylase. However, we do find that mice lacking ALKBH7 exhibit a significant increase in glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Consistent with increased dialdehyde production, metabolomics analysis reveals rewiring of metabolic pathways related to the toxic glycolytic by-product methylglyoxal (MGO), as well as accelerated glycolysis and elevated levels of MGO protein adducts, in mice lacking ALKBH7. Consistent with roles for both necrosis and glycative stress in cardiac IR injury, hearts from male but not female Alkbh7-/- mice are protected against IR, although somewhat unexpectedly this protection does not appear to involve modulation of the mitochondrial permeability transition pore. Highlighting the importance of MGO metabolism for the observed protection, removal of glucose as a metabolic substrate or pharmacologic inhibition of GLO-1 both abrogate cardioprotection in ALKBH7 deficient mice. Integrating these observations, we propose that ALKBH7 plays a role in the regulation of glyoxal metabolism, and that protection against necrosis and IR injury bought on by ALKBH7 deficiency originates from hormetic signaling in response to elevated MGO stress.

The Peroxisomal PTS1-Import Defect of PEX1- Deficient Cells Is Independent of Pexophagy in Saccharomyces cerevisiae

The important physiologic role of peroxisomes is shown by the occurrence of peroxisomal biogenesis disorders (PBDs) in humans. This spectrum of autosomal recessive metabolic disorders is characterized by defective peroxisome assembly and impaired peroxisomal functions. PBDs are caused by mutations in the peroxisomal biogenesis factors, which are required for the correct compartmentalization of peroxisomal matrix enzymes. Recent work from patient cells that contain the Pex1(G843D) point mutant suggested that the inhibition of the lysosome, and therefore the block of pexophagy, was beneficial for peroxisomal function. The resulting working model proposed that Pex1 may not be essential for matrix protein import at all, but rather for the prevention of pexophagy. Thus, the observed matrix protein import defect would not be caused by a lack of Pex1 activity, but rather by enhanced removal of peroxisomal membranes via pexophagy. In the present study, we can show that the specific block of PEX1 deletion-induced pexophagy does not restore peroxisomal matrix protein import or the peroxisomal function in beta-oxidation in yeast. Therefore, we conclude that Pex1 is directly and essentially involved in peroxisomal matrix protein import, and that the PEX1 deletion-induced pexophagy is not responsible for the defect in peroxisomal function. In order to point out the conserved mechanism, we discuss our findings in the context of the working models of peroxisomal biogenesis and pexophagy in yeasts and mammals.