HERC2 deficiency activates C-RAF/MKK3/p38 signalling pathway altering the cellular response to oxidative stress.

HERC2 gene encodes an E3 ubiquitin ligase involved in several cellular processes by regulating the ubiquitylation of different protein substrates. Biallelic pathogenic sequence variants in the HERC2 gene are associated with HERC2 Angelman-like syndrome. In pathogenic HERC2 variants, complete absence or marked reduction in HERC2 protein levels are observed. The most common pathological variant, c.1781C > T (p.Pro594Leu), encodes an unstable HERC2 protein. A better understanding of how pathologic HERC2 variants affect intracellular signalling may aid definition of potential new therapies for these disorders. For this purpose, we studied patient-derived cells with the HERC2 Pro594Leu variant. We observed alteration of mitogen-activated protein kinase signalling pathways, reflected by increased levels of C-RAF protein and p38 phosphorylation. HERC2 knockdown experiments reproduced the same effects in other human and mouse cells. Moreover, we demonstrated that HERC2 and RAF proteins form molecular complexes, pull-down and proteomic experiments showed that HERC2 regulates C-RAF ubiquitylation and we found out that the p38 activation due to HERC2 depletion occurs in a RAF/MKK3-dependent manner. The displayed cellular response was that patient-derived and other human cells with HERC2 deficiency showed higher resistance to oxidative stress with an increase in the master regulator of the antioxidant response NRF2 and its target genes. This resistance was independent of p53 and abolished by RAF or p38 inhibitors. Altogether, these findings identify the activation of C-RAF/MKK3/p38 signalling pathway in HERC2 Angelman-like syndrome and highlight the inhibition of RAF activity as a potential therapeutic option for individuals affected with these rare diseases.

Regulation of MAPK Signaling Pathways by the Large HERC Ubiquitin Ligases.

Protein ubiquitylation acts as a complex cell signaling mechanism since the formation of different mono- and polyubiquitin chains determines the substrate's fate in the cell. E3 ligases define the specificity of this reaction by catalyzing the attachment of ubiquitin to the substrate protein. Thus, they represent an important regulatory component of this process. Large HERC ubiquitin ligases belong to the HECT E3 protein family and comprise HERC1 and HERC2 proteins. The physiological relevance of the Large HERCs is illustrated by their involvement in different pathologies, with a notable implication in cancer and neurological diseases. Understanding how cell signaling is altered in these different pathologies is important for uncovering novel therapeutic targets. To this end, this review summarizes the recent advances in how the Large HERCs regulate the MAPK signaling pathways. In addition, we emphasize the potential therapeutic strategies that could be followed to ameliorate the alterations in MAPK signaling caused by Large HERC deficiencies, focusing on the use of specific inhibitors and proteolysis-targeting chimeras.

Octyl gallate decrease lymphocyte activation and regulates neutrophil extracellular traps release.

BACKGROUND: Inflammation is a complex mechanism with an objective to destroy and eliminate the invading microorganisms. During acute inflammation, the neutrophils are the major cells involved in this process and, although they defend the organism, must die to not generate damage. The two major mechanisms that drive neutrophils to death are: apoptosis and a novel mechanism recently discovered denominated NETosis. This process is a "suicidal mechanism", in which the cells release "neutrophil extracellular traps" (NETs) during the inflammatory response. Octyl gallate (OG) is one of the gallic acid derivates, with several protective effects, such as antioxidant and anti-inflammatory in cancer models. Thus, this study aimed to investigate the action of OG on the proliferation of lymphocytes, neutrophils activation, and its effectiveness in an experimental sepsis model. METHODS: Lymphocytes and neutrophils were obtained from healthy donors. Cell viability, apoptosis, NETs release and antioxidant capacity of OG were observed. In addition, survival was evaluated in an experimental model of sepsis in C57BL/6 mice. RESULTS: Our study demonstrated, for the first time, that the OG can act as an inhibitor of reactive oxygen species (ROS) release, NETs formation in primary human neutrophils and, modulates the lipopolysaccharide (LPS) effect in neutrophil apoptosis. The OG also inhibited peripheral blood mononuclear cells (PBMCs) proliferation in vitro. Despite the positive results, we did not observe an increase in the survival of septic animals. CONCLUSIONS: The pharmacological potential of OG, modulating activation of neutrophils and lymphocytes, suggests the use as an adjuvant therapeutic strategy in inflammatory diseases.

A new homozygous HERC1 gain-of-function variant in MDFPMR syndrome leads to mTORC1 hyperactivation and reduced autophagy during cell catabolism.

The giant 532 kDa HERC1 protein is a ubiquitin ligase that interacts with tuberous sclerosis complex subunit 2 (TSC2), a negative upstream regulator of the mammalian target of rapamycin complex 1 (mTORC1). TSC2 regulates anabolic cell growth through its influence on protein synthesis, cell growth, proliferation, autophagy, and differentiation. TSC subunit 1 (TSC1) stabilizes TSC2 by inhibiting the interaction between TSC2 and HERC1, forming a TSC1-TSC2 complex that negatively regulates mTORC1. HERC1-TSC2 interaction destabilizes and degrades TSC2. Recessive mutations in HERC1 have been reported in patients with intellectual disability. Some patients exhibit epilepsy, macrocephaly, somatic overgrowth, and dysmorphic facial features as well. Here we describe two sisters from a consanguineous marriage with a novel homozygous missense variant in the C-terminal HECT domain of HERC1 [chr15:g63,907,989C>G GRCh37.p11 | c.14,072G>C NM_003922 | p.(Arg4,691Pro)]. Symptoms compris global developmental delay, macrocephaly, somatic overgrowth, intellectual disability, seizures, schizoaffective disorder, and pyramidal tract signs. We functionally assessed the HERC1 mutation by investigation of patient and control fibroblasts under normal and nutrient starving conditions. During catabolic state, mTORC1 activity remained high in patient fibroblasts, which stands in stark contrast to its downregulation in controls. This was corroborated by an abnormally high phosphorylation of S6K1-kinase, a direct downstream target of mTORC1, in patients. Moreover, autophagy, usually enhanced in catabolic states, was down-regulated in patient fibroblasts. These data confirm that the missense variant found in both patients results in a gain-of-function for the mutant HERC1 protein.

CPBMF65, a synthetic human uridine phosphorylase-1 inhibitor, reduces HepG2 cell proliferation through cell cycle arrest and senescence.

Hepatocellular carcinoma (HCC) is the most prevalent type of tumor among primary liver tumors and is the second highest cause of cancer-related deaths worldwide. Current therapies are controversial, and more research is needed to identify effective treatments. A new synthetic compound, potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65), is a potent inhibitor of the human uridine phosphorylase-1 (hUP1) enzyme, which controls the cell concentration of uridine (Urd). Urd is a natural pyrimidine nucleoside involved in cellular processes, such as RNA synthesis. In addition, it is considered a promising biochemical modulator, as it may reduce the toxicity caused by chemotherapeutics without impairing its anti-tumor activity. Thus, the objective of this study is to evaluate the effects of CPBMF65 on the proliferation of the human hepatocellular carcinoma cell line (HepG2). Cell proliferation, cytotoxicity, apoptosis, senescence, autophagy, intracellular Urd levels, cell cycle arrest, and drug resistance were analyzed. Results demonstrate that, after incubation with CPBMF65, HepG2 cell proliferation decreased, mainly through cell cycle arrest and senescence, increasing the levels of intracellular Urd and maintaining cell proliferation reduced during chronic treatment. In conclusion, results show, for the first time, the ability of a hUP1 inhibitor (CPBMF65) to reduce HepG2 cell proliferation through cell cycle arrest and senescence.

Mesenchymal stem cells improves survival in LPS-induced acute lung injury acting through inhibition of NETs formation.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are syndromes of acute hypoxemic respiratory failure resulting from a variety of direct and indirect injuries to the gas exchange parenchyma of the lungs. During the ALI, we have an increase release of proinflammatory cytokines and high reactive oxygen species (ROS) formation. These factors are responsible for the release and activation of neutrophil-derived proteases and the formation of neutrophil extracellular traps (NETs). The excessive increase in the release of NETs cause damage to lung tissue. Recent studies have studies involving the administration of mesenchymal stem cells (MSCs) for the treatment of experimental ALI has shown promising results. In this way, the objective of our study is to evaluate the ability of MSCs, in a lipopolysaccharide (LPS)-induced ALI model, to reduce inflammation, oxidative damage, and consequently decrease the release of NETs. Mice were submitted lung injury induced by intratracheal instillation of LPS and subsequently treated or not with MSCs. Treatment with MSCs was able to modulate pulmonary inflammation, decrease oxidative damage, and reduce the release of NETs. These benefits from treatment are evident when we observe a significant increase in the survival curve in the treated animals. Our results demonstrate that MSCs treatment is effective for the treatment of ALI. For the first time, it is described that MSCs can reduce the formation of NETs and an experimental model of ALI. This finding is directly related to these cells modulate the inflammatory response and oxidative damage in the course of the pathology.

Mesenchymal stem cells cannot affect mRNA expression of toll-like receptors in different tissues during sepsis.

OBJECTIVE AND DESIGN: Experimental animal models and human clinical studies support a crucial role for TLRs in infectious diseases. The aim of this study was to test the ability of MSCs, which have immunomodulatory effects, of altering the mRNA expression of toll-like receptors during a experimental model of sepsis in different tissues. MATERIALS AND METHODS: Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 106 cells/animal). Lungs, cortex, kidney, liver and colon tissue were dissected after 12 h of sepsis induction and TLR2/3/4/9 mRNA were evaluated by RT-qPCR. RESULTS: We observed a decrease of TLR2 and 9 mRNA expression in the liver of the sepsis group, while TLR3 was decreased in the lung and liver. No change was found between the sepsis group and the sepsis + MSC group. CONCLUSIONS: In this model of experimental sepsis the MSCs were unable to modify the mRNA expression of the different toll-like receptors evaluated.

Fructose-1,6-bisphosphate reverts iron-induced phenotype of hepatic stellate cells by chelating ferrous ions.

Hepatic fibrosis is an extracellular matrix deposition by hepatic stellate cells (HSC). Fibrosis can be caused by iron, which will lead to hydroxyl radical production and cell damage. Fructose-1,6-bisphosphate (FBP) has been shown to deliver therapeutic effects in many pathological situations. In this work, we aimed to test the effects of FBP in HSC cell line, GRX, exposed to an excess of iron (Fe). The Fe-treatment increased cell proliferation and FBP reversed this effect, which was not due to increased necrosis, apoptosis or changes in cell cycle. Oil Red-O staining showed that FBP successfully increased lipid content and lead GRX cells to present characteristics of quiescent HSC. Fe-treatment decreased PPAR-γ expression and increased Col-1 expression. Both effects were reversed by FBP which also decreased TGF-ß1 levels in comparison to both control and Fe groups. FBP, also, did not present scavenger activity in the DPPH assay. The treatment with FBP resulted in decreased proliferation rate, Col-1 expression and TGF-ß1 release by HSC cells. Furthermore, activated PPAR-γ and increased lipid droplets induce cells to become quiescent, which is a key event to reversion of hepatic fibrosis. FBP also chelates iron showing potential to improve Cell redox state.

Therapeutic ultrasound stimulates MC3T3-E1 cell proliferation through the activation of NF-κB1, p38α, and mTOR.

BACKGROUND AND OBJECTIVES: As the population ages, osteometabolic diseases and osteoporotic fractures emerge, resulting in substantial healthcare resource utilization and impaired quality of life. Many types of mechanical stimulation have the potential of being recognized by bone cells after a mechanical sign is transformed into a biological one (a process called mechanotransduction). The therapeutic ultrasound (TU) is one of several resources capable of promoting bone cell mechanical stimulation. Therefore, the main purpose of present study was to evaluate the effect of TU on the proliferation of pre-osteoblasts using in vitro bioassays. STUDY DESIGN/MATERIALS AND METHODS: We used MC3T3-E1 pre-osteoblast lineage cells kept in Alpha medium. Cells were treated using pulsed mode therapeutic ultrasound, with frequency of 1 MHz, intensity of 0.2 W/cm(2) (SATA), duty cycle of 20%, for 30 minutes. Nifedipine and rapamycin were used to further investigate the role of L-type Ca(2+) channels and mTOR pathway. Intracellular calcium, TGF-ß1, magnesium, and the mRNA levels of osteopontin, osteonectin, NF-κB1, p38α were evaluated. RESULTS: The results show that TU stimulates the growth of MC3T3-E1 cells and decreases the supernatant calcium and magnesium content. Also, it increases intracellular calcium, activates NF-κB1 and mTOR complex via p38α. Moreover, TU promoted a decrease in the TGF-ß1 synthesis, which is a cell growth inhibitor. CONCLUSIONS: Therapeutic ultrasound, with frequency of 1 MHz, intensity of 0.2 W/cm(2) (SATA) and pulsed mode, for 30 minutes, was able to increase the proliferation of preosteoblast-like bone cells. This effect was mediated by a calcium influx, with a consequent activation of the mTOR pathway, through increased NF-κB1 and p38α.

Mesenchymal stem cells decrease splenocytes apoptosis in a sepsis experimental model.

OBJECTIVE AND DESIGN: Mesenchymal stem cells (MSCs) are potent modulators of immune responses. Sepsis is the association of a systemic inflammatory response with an infection. The aim of this study was to test the ability of MSCs derived from adipose tissue, which have immunomodulatory effects, and to inhibit the septic process in an experimental model of mice. METHODS: Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 10(6) cells/animal). RESULTS: In the control group, there were no deaths; in the untreated septic group, the mortality rate was 100 % within 26 h; in the septic group treated with MSCs, the mortality rate reached 40 % within 26 h. The group treated with MSCs was able to reduce the markers of tissue damage in the liver and pancreas. The treated group had a reduction of inflammatory markers. Furthermore, the MSCs-treated group was able to inhibit the increase of apoptosis in splenocytes observed in the untreated septic group. CONCLUSIONS: Our data showed that MSCs ameliorated the immune response with decrease of inflammatory cytokines and increase anti-inflammatory IL-10; moreover, inhibited splenocytes apoptosis and, consequently, inhibited tissue damage during sepsis.

HERC1 deficiency causes osteopenia through transcriptional program dysregulation during bone remodeling.

Bone remodeling is a continuous process between bone-forming osteoblasts and bone-resorbing osteoclasts, with any imbalance resulting in metabolic bone disease, including osteopenia. The HERC1 gene encodes an E3 ubiquitin ligase that affects cellular processes by regulating the ubiquitination of target proteins, such as C-RAF. Of interest, an association exists between biallelic pathogenic sequence variants in the HERC1 gene and the neurodevelopmental disorder MDFPMR syndrome (macrocephaly, dysmorphic facies, and psychomotor retardation). Most pathogenic variants cause loss of HERC1 function, and the affected individuals present with features related to altered bone homeostasis. Herc1-knockout mice offer an excellent model in which to study the role of HERC1 in bone remodeling and to understand its role in disease. In this study, we show that HERC1 regulates osteoblastogenesis and osteoclastogenesis, proving that its depletion increases gene expression of osteoblastic makers during the osteogenic differentiation of mesenchymal stem cells. During this process, HERC1 deficiency increases the levels of C-RAF and of phosphorylated ERK and p38. The Herc1-knockout adult mice developed imbalanced bone homeostasis that presented as osteopenia in both sexes of the adult mice. By contrast, only young female knockout mice had osteopenia and increased number of osteoclasts, with the changes associated with reductions in testosterone and dihydrotestosterone levels. Finally, osteocytes isolated from knockout mice showed a higher expression of osteocytic genes and an increase in the Rankl/Opg ratio, indicating a relevant cell-autonomous role of HERC1 when regulating the transcriptional program of bone formation. Overall, these findings present HERC1 as a modulator of bone homeostasis and highlight potential therapeutic targets for individuals affected by pathological HERC1 variants.

Simvastatin attenuates inflammatory process on LPS-induced acute lung injury in mice.

Acute lung injury (ALI) is a disease of high prevalence and is characterized by the excessive production of inflammatory mediators in the lungs of people sick. Inflammation is the major characteristic of ALI and studies report that inhibition of inflammatory cytokines could be an alternative treatment. Statins such as Simvastatin (SV) are known to their use for cholesterol reduction but also for inflammatory and immunoregulatory processes. In this study, we evaluated the effects of SV on LPS-induced alveolar macrophages and in ALI mice model. Our study has demonstrated the protective effects of SV on LPS-activated alveolar macrophages RAW 264.7 and LPS-induced ALI in mice. SV treatment significantly inhibited the alveolar macrophages activation by decreasing the iNOS, IL-1ß, and IL-6 gene expression in vitro and in vivo. The treatment also decreased the inflammatory cells migration and the cytokines gene expression. Our findings suggest that SV can act as an anti-inflammatory agent for acute lung injury.

Gestational stress alters maternal behavior and inflammatory markers in the olfactory bulb of lactating mice.

Inflammatory markers represent important candidates responsible for the altered behavior and physiology observed after stressful experiences. In the maternal brain, the olfactory bulb (OB) is a key constituent of the neural circuit that mediates the reciprocal interaction between mother and infant. This study aimed to investigate the effects of stress during pregnancy on maternal behavior and inflammatory changes in the olfactory bulb of lactating mice. Female Balb/c mice were divided into two groups: control (CT) and restraint stress (RS). Maternal behavior was performed during the first 8 days of life of the offspring. On the 10th day after parturition, corticosterone, gene, and protein expression were assessed. Stress during pregnancy decreased the maternal index at postnatal day 4 and the nuclear factor-κB 1 (NFκB1) gene expression in the OB. Moreover, females from the RS group showed increased interleukin (IL-1ß) protein expression. In contrast, stressed females exhibited a decreased tumor necrosis factor (TNF-α) protein expression in the OB. In conclusion, exposure to stress during pregnancy was able to induce specific postnatal effects on maternal behavior and balance of inflammatory mediators in the OB.

Sex-dependent metabolic effects of pregestational exercise on prenatally stressed mice.

Stressful events during the prenatal period have been related to hyperactive hypothalamic-pituitary-adrenal (HPA) axis responses as well as metabolic changes in adult life. Moreover, regular exercise may contribute to the improvement of the symptoms associated with stress and stress-related chronic diseases. Therefore, this study aims to investigate the effects of exercise, before the gestation period, on the metabolic changes induced by prenatal stress in adult mice. Female Balb/c mice were divided into three groups: control (CON), prenatal restraint stress (PNS) and exercise before the gestational period plus PNS (EX + PNS). When adults, the plasmatic biochemical analysis, oxidative stress, gene expression of metabolic-related receptors and sex differences were assessed in the offspring. Prenatal stress decreased neonatal and adult body weight when compared to the pregestational exercise group. Moreover, prenatal stress was associated with reduced body weight in adult males. PNS and EX + PNS females showed decreased hepatic catalase. Pregestational exercise prevented the stress-induced cholesterol increase in females but did not prevent the liver mRNA expression reduction on the peroxisome proliferator-activated receptors (PPARs) α and γ in PNS females. Conversely, PNS and EX + PNS males showed an increased PPARα mRNA expression. In conclusion, pregestational exercise prevented some effects of prenatal stress on metabolic markers in a sex-specific manner.

NRF2 function in osteocytes is required for bone homeostasis and drives osteocytic gene expression.

Osteocytes, the most abundant bone cell type, are derived from osteoblasts through a process in which they are embedded in an osteoid. We previously showed that nutrient restriction promotes the osteocyte transcriptional program and is associated with increased mitochondrial biogenesis. Here, we show that increased mitochondrial biogenesis increase reactive oxygen species (ROS) levels and consequently, NRF2 activity during osteocytogenesis. NRF2 activity promotes osteocyte-specific expression of Dmp1, Mepe, and Sost in IDG-SW3 cells, primary osteocytes, and osteoblasts, and in murine models with Nfe2l2 deficiency in osteocytes or osteoblasts. Moreover, ablation of Nfe2l2 in osteocytes or osteoblasts generates osteopenia and increases osteoclast numbers with marked sexual dimorphism. Finally, treatment with dimethyl fumarate prevented the deleterious effects of ovariectomy in trabecular bone masses of mice and restored osteocytic gene expression. Altogether, we uncovered the role of NRF2 activity in osteocytes during the regulation of osteocyte gene expression and maintenance of bone homeostasis.

The ubiquitin ligase HERC1 regulates cell migration via RAF-dependent regulation of MKK3/p38 signaling.

Protein modifications by phosphorylation or ubiquitylation have been selected throughout evolution as efficient regulatory mechanisms of cellular processes. Cell migration is a complex, highly coordinated process where these mechanisms must participate in an integrated manner to transmit signaling during migration. In this study, we show that the ubiquitin ligase HERC1 regulates the p38 signaling pathway, and that this regulation is mediated by the MAPK kinase MKK3. Moreover, we demonstrate a crosstalk between RAF and MKK3/p38 pathways where RAF acts upstream of MKK3. Mechanistically, HERC1 regulates the protein levels of C-RAF and MKK3. Thus, HERC1 ubiquitylates C-RAF, targeting it for proteasomal degradation, and RAF proteins regulate MKK3 mRNA levels. Accordingly, HERC1 knockdown induces C-RAF stabilization and activation of RAF proteins; in turn, this activation increases MKK3, which phosphorylates and activates p38. The importance of these observations is demonstrated by HERC1 regulation of cell migration through regulation of p38 signaling via a RAF-dependent mechanism. Thus, HERC1 plays an essential role as a regulator of crosstalk between RAF/MKK3/p38 signaling pathways during cell migration.

The HERC1 ubiquitin ligase regulates presynaptic membrane dynamics of central synapses.

HERC1 is a ubiquitin ligase protein, which, when mutated, induces several malformations and intellectual disability in humans. The animal model of HERC1 mutation is the mouse tambaleante characterized by: (1) overproduction of the protein; (2) cerebellar Purkinje cells death by autophagy; (3) dysregulation of autophagy in spinal cord motor neurons, and CA3 and neocortical pyramidal neurons; (4) impairment of associative learning, linked to altered spinogenesis and absence of LTP in the lateral amygdala; and, (5) motor impairment due to delayed action potential transmission, decrease synaptic transmission efficiency and altered myelination in the peripheral nervous system. To investigate the putative role of HERC1 in the presynaptic dynamics we have performed a series of experiments in cultured tambaleante hippocampal neurons by using transmission electron microscopy, FM1-43 destaining and immunocytochemistry. Our results show: (1) a decrease in the number of synaptic vesicles; (2) reduced active zones; (3) less clathrin immunoreactivity and less presynaptic endings over the hippocampal main dendritic trees; which contrast with (4) a greater number of endosomes and autophagosomes in the presynaptic endings of the tambaleante neurons relative to control ones. Altogether these results show an important role of HERC1 in the regulation of presynaptic membrane dynamics.

HERC Ubiquitin Ligases in Cancer.

HERC proteins are ubiquitin E3 ligases of the HECT family. The HERC subfamily is composed of six members classified by size into large (HERC1 and HERC2) and small (HERC3-HERC6). HERC family ubiquitin ligases regulate important cellular processes, such as neurodevelopment, DNA damage response, cell proliferation, cell migration, and immune responses. Accumulating evidence also shows that this family plays critical roles in cancer. In this review, we provide an integrated view of the role of these ligases in cancer, highlighting their bivalent functions as either oncogenes or tumor suppressors, depending on the tumor type. We include a discussion of both the molecular mechanisms involved and the potential therapeutic strategies.

HERCing: Structural and Functional Relevance of the Large HERC Ubiquitin Ligases.

Homologous to the E6AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing proteins (HERCs) belong to the superfamily of ubiquitin ligases. HERC proteins are divided into two subfamilies, Large and Small HERCs. Despite their similarities in terms of both structure and domains, these subfamilies are evolutionarily very distant and result from a convergence phenomenon rather than from a common origin. Large HERC genes, HERC1 and HERC2, are present in most metazoan taxa. They encode very large proteins (approximately 5,000 amino acid residues in a single polypeptide chain) that contain more than one RCC1-like domain as a structural characteristic. Accumulating evidences show that these unusually large proteins play key roles in a wide range of cellular functions which include neurodevelopment, DNA damage repair, and cell proliferation. To better understand the origin, evolution, and function of the Large HERC family, this minireview provides with an integrated overview of their structure and function and details their physiological implications. This study also highlights and discusses how dysregulation of these proteins is associated with severe human diseases such as neurological disorders and cancer.