MBTPS1 regulates proliferation of colorectal cancer primarily through its action on sterol regulatory element-binding proteins.

Among the main metabolic pathways implicated in cancer cell proliferation are those of cholesterol and fatty acid synthesis, both of which are tightly regulated by sterol regulatory element-binding proteins (SREBPs). SREBPs are activated through specific cleavage by membrane-bound transcription factor protease 1 (MBTPS1), a serine protease that cleaves additional substrates (ATF6, BDNF, CREBs and somatostatin), some of which are also implicated in cell proliferation. The goal of this study was to determine whether MBTPS1 may serve as a master regulator in proliferation of colorectal cancer (CRC). Tumors from CRC patients showed variable levels of MBTPS1 mRNA, which were in positive correlation with the levels of SREBPs and ATF6, and in reverse correlation with BDNF levels. Chemical inhibition of MBTPS1 activity in two CRC-derived cell lines resulted in a marked decrease in the levels of SREBPs, but not of its other substrates and a marked decrease in cell proliferation, which suggested that MBTPS1 activity is critical for proliferation of these cells. In accordance, CRISPR/Cas9 targeted knockout (KO) of the MBTPS1 gene resulted in the survival of only a single clone that presented a phenotype of severely attenuated proliferation and marked downregulation of several energy metabolism pathways. We further showed that survival of the MBTPS1 KO clone was dependent upon significant upregulation of the type-1 interferon pathway, the inhibition of which halted proliferation entirely. Finally, rescue of the MBTPS1 KO cells, resulted in partial restoration of MBTPS1 levels, which was in accordance with partial recovery in proliferation and in SREBP levels. These finding suggest that MBTPS1 plays a critical role in regulating colon cancer proliferation primarily through SREBP-associated lipid metabolism, and as such may serve as a possible therapeutic target in CRC.

Haematological patients' perception of home transfusions: Effect of the COVID-19 pandemic.

BACKGROUND AND OBJECTIVES: The COVID-19 pandemic has led to a growing interest in hospital-at-home programmes, including home transfusion services. We studied whether the pandemic had influenced patients' perception of home transfusions. MATERIALS AND METHODS: We conducted a survey among haematology patients who receive transfusions in the hospital day care facility. Patients were asked about the burden of day care transfusions and whether they would prefer receiving home transfusions. The survey was conducted during the COVID-19 pandemic, and the results were compared with a survey performed before the pandemic (baseline). RESULTS: Sixty patients were included in the COVID-19 cohort and 31 patients in the baseline cohort. There was a non-significant decrease in the proportion of patients willing to receive home transfusions during the pandemic compared with baseline (35% vs. 47%, respectively, p = 0.28). More patients in the COVID-19 cohort were afraid to receive home transfusions (60% compared with 48% at baseline, p = 0.29), and fewer patients believed that hospital transfusion impaired their quality of life (19% compared with 36% at baseline, p = 0.09). These unexpected results may be partly attributed to the shorter time needed to arrive at the hospital during the pandemic and a greater fear of having transfusion-related adverse effects at home. CONCLUSIONS: Our results show that the pandemic did not increase the willingness of patients to receive home transfusions, with a non-significant drift towards refusal of home transfusions. Patients' opinions should be taken into consideration when planning for future home transfusion services, by creating a comprehensive approach to patients' needs.

Acute social isolation and regrouping cause short- and long-term molecular changes in the rat medial amygdala.

Social isolation poses a severe mental and physiological burden on humans. Most animal models that investigate this effect are based on prolonged isolation, which does not mimic the milder conditions experienced by people in the real world. We show that in adult male rats, acute social isolation causes social memory loss. This memory loss is accompanied by significant changes in the expression of specific mRNAs and proteins in the medial amygdala, a brain structure that is crucial for social memory. These changes particularly involve the neurotrophic signaling and axon guidance pathways that are associated with neuronal network remodeling. Upon regrouping, memory returns, and most molecular changes are reversed within hours. However, the expression of some genes, especially those associated with neurodegenerative diseases remain modified for at least a day longer. These results suggest that acute social isolation and rapid resocialization, as experienced by millions during the COVID-19 pandemic, are sufficient to induce significant changes to neuronal networks, some of which may be pathological.

Willingness and concerns of transfusion-dependent hematological patients toward the option of home transfusion therapy.

BACKGROUND: One of the main obstacles of providing home-based palliative care to transfusion-dependent hematology patients is the lack of home transfusions services. While healthcare professionals are concerned with safety and cost of home transfusions, the attitude of the patients toward home transfusions are mostly unknown. AIM: To obtain quantitative data regarding the willingness and concerns of transfusion-dependent patients with hematological diseases toward the option of home transfusions. DESIGN: A cross sectional survey including a self-administered questionnaire in one of the three main spoken languages in Israel was administered to patients in 17 hospital hematology outpatient clinics between May 2019 and March 2020. RESULTS: About 52% of 385 patients that participated in the survey preferred home transfusions to hospital transfusions. Gender, age, education, or type of disease were not associated with preference for home transfusions, nor were hospital location or its size. The likelihood to prefer home transfusions was significantly higher among the Hebrew-speakers and those who had not experienced adverse effects previously. The most significant factor associated with preference of home transfusions was a perceived negative effect of hospital-based transfusion on quality of life. The main reason to reject home transfusions was fear of possible adverse effects and concerns over losing contact with the medical staff at the treating hospital. CONCLUSION: These data suggest that a significant portion of transfusion-dependent patients in Israel view home transfusions as a preferred treatment option and that its successful implementation requires maintaining ongoing contact with the treating hospital.

Limited Proteolysis of Cyclooxygenase-2 Enhances Cell Proliferation.

Accumulating evidence suggests that the cyclooxygenase-2 (COX-2) enzyme has additional catalytic-independent functions. Here we show that COX-2 appears to be cleaved in mouse and human tumors, which led us to hypothesize that COX-2 proteolysis may play a role in cell proliferation. The data presented herein show that a K598R point mutation at the carboxyl-terminus of COX-2 causes the appearance of several COX-2 immunoreactive fragments in nuclear compartments, and significantly enhances cell proliferation. In contrast, insertion of additional mutations at the border of the membrane-binding and catalytic domains of K598R COX-2 blocks fragment formation and prevents the increase in proliferation. Transcriptomic analyses show that K598R COX-2 significantly affects the expression of genes involved in RNA metabolism, and subsequent proteomics suggest that it is associated with proteins that regulate mRNA processing. We observe a similar increase in proliferation by expressing just that catalytic domain of COX-2 (ΔNT- COX-2), which is completely devoid of catalytic activity in the absence of its other domains. Moreover, we show that the ΔNT- COX-2 protein also interacts in the nucleus with ß-catenin, a central regulator of gene transcription. Together these data suggest that the cleavage products of COX-2 can affect cell proliferation by mechanisms that are independent of prostaglandin synthesis.

Conserved statin-mediated activation of the p38-MAPK pathway protects Caenorhabditis elegans from the cholesterol-independent effects of statins.

OBJECTIVE: Statins are a group of medications that reduce cholesterol synthesis by inhibiting the activity of HMG-CoA reductase, a key enzyme in the mevalonate pathway. The clinical use of statins to lower excess cholesterol levels has revolutionized the cardiovascular field and increased the survival of millions, but some patients have adverse side effects. A growing body of data suggests that some of the beneficial and adverse effects of statins, including their anti-inflammatory, anti-tumorigenic, and myopathic activities, are cholesterol-independent. However, the underlying mechanisms for these effects of statins are not well defined. METHODS: Because Caenorhabditis elegans (C. elegans) lacks the cholesterol synthesis branch of the mevalonate pathway, this organism is a powerful system to unveil the cholesterol-independent effects of statins. We used genetic and biochemical approaches in C. elegans and cultured macrophage-derived murine cells to study the cellular response to statins. RESULTS: We found that statins activate a conserved p38-MAPK (p38) cascade and that the protein geranylgeranylation branch of the mevalonate pathway links the effect of statins to the activation of this p38 pathway. We propose that the blockade of geranylgeranylation impairs the function of specific small GTPases we identified as upstream regulators of the p38 pathway. Statin-mediated p38 activation in C. elegans results in the regulation of programs of innate immunity, stress, and metabolism. In agreement with this regulation, knockout of the p38 pathway results in the hypersensitivity of C. elegans to statins. Treating cultured mammalian cells with clinical doses of statins results in the activation of the same p38 pathway, which upregulates the COX-2 protein, a major regulator of innate immunity in mammals. CONCLUSIONS: Statins activate an evolutionarily conserved p38 pathway to regulate metabolism and innate immunity. Our results highlight the cytoprotective role of p38 activation under statin treatment in vivo and propose that this activation underlies many of the critical cholesterol-independent effects of statins.

Substrate-inactivated cyclooxygenase-2 is disposed of by exosomes through the ER-Golgi pathway.

Catalysis of arachidonic acid (AA) by cyclooxygenase-2 (COX-2) gives rise to a single product that serves as a precursor for all prostaglandins, which are central mediators of inflammation. Rapid up-regulation of COX-2 expression in response to pro-inflammatory stimuli is a well-characterized means of generating the large pool of prostaglandins necessary for inflammation. However, an efficient inflammatory process must also terminate rapidly and thus requires cessation of COX-2 enzymatic activity and removal of excess protein from the cell. Previous studies showed that COX-2 that has not been exposed to AA ('naive') degrades in the cellular proteasome. However, continuous exposure to AA induces suicide inactivation of COX-2 and its elimination no longer occurs in neither the proteasomal nor lysosomal machineries. In the present study, we show that either overexpressed or endogenously induced COX-2 is secreted via exosomes through the endoplasmic reticulum-Golgi pathway. We further find that excretion of COX-2 is significantly enhanced by prolonged exposure to AA. Genetic or chemical inhibition of COX-2 enzymatic activity has no effect on its secretion in the absence of substrate, but prevents the additional activity-dependent secretion. Finally, transfer of COX-2 to target cells only occurs in the absence of AA stimulation. Together, these results suggest that exosomal secretion of AA-activated COX-2 constitutes a means to remove damaged inactive COX-2 from the cell.

The Heparanase Inhibitor PG545 Attenuates Colon Cancer Initiation and Growth, Associating with Increased p21 Expression.

Heparanase activity is highly implicated in cellular invasion and tumor metastasis, a consequence of cleavage of heparan sulfate and remodeling of the extracellular matrix underlying epithelial and endothelial cells. Heparanase expression is rare in normal epithelia, but is often induced in tumors, associated with increased tumor metastasis and poor prognosis. In addition, heparanase induction promotes tumor growth, but the molecular mechanism that underlines tumor expansion by heparanase is still incompletely understood. Here, we provide evidence that heparanase down regulates the expression of p21 (WAF1/CIP1), a cyclin-dependent kinase inhibitor that attenuates the cell cycle. Notably, a reciprocal effect was noted for PG545, a potent heparanase inhibitor. This compound efficiently reduced cell proliferation, colony formation, and tumor xenograft growth, associating with a marked increase in p21 expression. Utilizing the APC Min+/- mouse model, we show that heparanase expression and activity are increased in small bowel polyps, whereas polyp initiation and growth were significantly inhibited by PG545, again accompanied by a prominent induction of p21 levels. Down-regulation of p21 expression adds a novel feature for the emerging pro-tumorigenic properties of heparanase, while the potent p21 induction and anti-tumor effect of PG545 lends optimism that it would prove an efficacious therapeutic in colon carcinoma patients.

Down-regulation of cyclooxygenase-2 by the carboxyl tail of the angiotensin II type 1 receptor.

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT1) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT1 also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or ß-arrestin-dependent signaling. Instead, AT1 enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequence alone is sufficient to down-regulate COX-2. Collectively these results propose a new role for AT1 in regulating COX-2 expression in a mechanism that deviates from its canonical signaling pathways. Down-regulation of COX-2 by a short peptide that originates from AT1 may present as a basis for novel therapeutic means of eliminating excess COX-2 protein.

ß1-Adrenergic receptor downregulates the expression of cyclooxygenase-2.

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the generation of prostanoids, and is thus one of the key players in the inflammatory process. Contrary to the constitutively expressed isoform COX-1, the expression of COX-2 is rapidly and transiently upregulated following pathological stimuli but little is known about pathways that mediate its degradation. Here we show that co-expression of COX-2 together with the ß1 adrenergic receptor (ß1AR) specifically lowers the expression of COX-2 in a dose-dependent manner. We further find that stimulation of the receptor for prolonged periods of time does not reverse the ß1AR-induced decrease in COX-2, suggesting that this effect does not occur via classical ß1-mediated signaling pathways. Rather we find that the half-life of COX-2 is significantly decreased in the presence of ß1AR and that inhibition of the proteasome reverses the effect of the receptor on COX-2. Together these findings ascribe a new role for ß1AR in the downregulation of COX-2.

Underwater trauma causes a long-term specific increase in the expression of cyclooxygenase-2 in the ventral CA1 of the hippocampus.

The pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is regularly expressed in the hippocampal neurons, but its role in emotional trauma is not known. Here we show that a single acute stress caused by a near-drowning experience results in heightened anxiety-like behavior one month after the trauma. Biochemical analyses of dorsal and ventral hippocampal CA1, CA3 and dentate gyrus revealed decreased ubiquitination and elevated levels of COX-2 in the traumatized animals only in the ventral CA1. To reveal the identity of the ubiquitin E3 ligase that targets COX-2, we tested the effect of several representative E3 ligases on COX-2 expression in vitro. We found that while AIP4 and Nedd4 had no effect, Mdm2 lowered COX-2 expression by nearly 50%, an effect that was not observed by its dominant negative form. To test whether this also occurs in the hippocampus, we immunoprecipitated Mdm2 from dorsal and ventral CA1 of traumatized and control animals and probed for the presence of COX-2. Our results showed that the levels of Mdm2 were not affected by the trauma but there was significantly less COX-2 associated with Mdm2 in the ventral but not dorsal CA1 of the traumatized animals. Together these data propose that an increase in COX-2 expression in ventral CA1 following trauma is likely due to its attenuated degradation. Unraveling the pathways and mechanisms that control hippocampal COX-2 degradation is important to boost the development of novel therapeutic approaches designed to treat stress-related pathologies.

Upregulation of prostaglandin receptor EP1 expression involves its association with cyclooxygenase-2.

While many signals cause upregulation of the pro-inflammatory enzyme cyclooxygenase -2 (COX-2), much less is known about mechanisms that actively downregulate its expression. We have recently shown that the prostaglandin EP1 receptor reduces the expression of COX-2 in a pathway that facilitates its ubiquitination and degradation via the 26S proteasome. Here we show that an elevation of COX-2 intracellular levels causes an increase in the endogenous expression of prostaglandin EP1. The increase in EP1 levels does not occur at the transcriptional level, but is rather associated with complex formation between the receptor and COX-2, which occurs both in vitro and in mammalian tissues. The EP1-COX-2 complex is disrupted following binding of arachidonic acid to COX-2 and accompanied by a parallel reduction in EP1 levels. We propose that a transient interaction between COX-2 and EP1 constitutes a feedback loop whereby an increase in COX-2 expression elevates EP1, which ultimately acts to downregulate COX-2 by expediting its proteasomal degradation. Such a post translational mechanism may serve to control both the ligand-generating system of COX-2 and its reception system.

Brain region-specific methylation in the promoter of the murine oxytocin receptor gene is involved in its expression regulation.

Oxytocin is a nine amino acid neuropeptide that is known to play a critical role in fetal expulsion and breast-feeding, and has been recently implicated in mammalian social behavior. The actions of both central and peripheral oxytocin are mediated through the oxytocin receptor (Oxtr), which is encoded by a single gene. In contrast to the highly conserved expression of oxytocin in specific hypothalamic nuclei, the expression of its receptor in the brain is highly diverse among different mammalian species or even within individuals of the same species. The diversity in the pattern of brain Oxtr expression among mammals is thought to contribute to the broad range of social systems and organizations. Yet, the mechanisms underlying this diversity are poorly understood. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression levels of the Oxtr in individuals with autism. Here we hypothesize that DNA methylation is involved in the expression regulation of Oxtr in the mouse brain. By combining bisulfite DNA conversion and Next-Generation Sequencing we found that specific CpG sites are differentially methylated between distinct brain regions expressing different levels of Oxtr mRNA. Some of these CpG sites are located within putative binding sites of transcription factors known to regulate Oxtr expression, including estrogen receptor α (ERα) and SP1. Specifically, methylation of the SP1 site was found to positively correlate with Oxtr expression. Furthermore, we revealed that the methylation levels of these sites in the various brain regions predict the relationship between ERα and Oxtr mRNA levels. Collectively, our results suggest that brain region-specific expression of the mouse Oxtr gene is epigenetically regulated by DNA methylation of its promoter.

Prostaglandin receptor EP1-mediated differential degradation of cyclooxygenases involves a specific lysine residue.

The cyclooxygenase (COX) enzyme isoforms COX-1 and COX-2 catalyze the main step in the generation of prostanoids that mediate major physiological functions. Whereas COX-1 is a ubiquitously expressed stable protein, COX-2 is transiently upregulated in many pathologies and is often associated with a poor prognostic outcome. We have recently shown that an interaction of COX-2 with the prostaglandin EP1 receptor accelerates its degradation via a mechanism that augments its level of ubiquitination. Here we show that the sensitivity of both COX-1 and COX-2 to EP1 is altered upon modification of one lysine residue. A point mutation of lysine to-arginine in position 432 of COX-2 (K432R) yields an enzyme with decreased sensitivity to EP1 -mediated degradation. In contrast, insertion of a putative ubiquitination site into the corresponding position of COX-1 (H446K') yields an enzyme with higher levels of ubiquitination and reduced expression. Furthermore, compared to wild type COX-1, H446K' is significantly more sensitive to downregulation by EP1 . Together these data suggest that distinctive ubiquitination of COX-1 and COX-2 may be responsible for their different sensitivity to EP1 -mediated degradation.

DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

Selective increase in the association of the ß2 adrenergic receptor, ß Arrestin-1 and p53 with Mdm2 in the ventral hippocampus one month after underwater trauma.

Chronic infusion of mice with a ß2 adrenergic receptor (ß2AR) analog was shown to cause long-term DNA damage in a pathway which involves ß Arresin-1-mediated activation of Mdm2 and subsequent degradation of the tumor suppressor protein p53. The objective of the present study was to test whether a single acute stress, which manifests long lasting changes in behavior, affects the interaction of Mdm2 with p53, ß2AR, and ß Arrestin-1 in the dorsal and ventral hippocampal CA1. Adult rats were subject to underwater trauma, a brief forceful submersion under water and tested a month later for behavioral and biochemical changes. Elevated plus maze tests confirmed that animals that experienced the threat of drowning present heightened levels of anxiety one month after trauma. An examination of the CA1 hippocampal areas of the same rats showed that underwater trauma caused a significant increase in the association of Mdm2 with ß2AR, ß Arrestin-1, and p53 in the ventral but not dorsal CA1. Our results provide support for the idea that stress-related events may result in biochemical changes restricted to the ventral 'emotion-related' parts of the hippocampus.

Prostaglandin EP1 receptor down-regulates expression of cyclooxygenase-2 by facilitating its proteasomal degradation.

The enzyme cyclooxygenase-2 (COX-2) is rapidly and transiently up-regulated by a large variety of signals and implicated in pathologies such as inflammation and tumorigenesis. Although many signals cause COX-2 up-regulation, much less is known about mechanisms that actively down-regulate its expression. Here we show that the G protein-coupled receptor prostaglandin E(1) (EP(1)) reduces the expression of COX-2 in a concentration-dependent manner through a mechanism that does not require receptor activation. The reduction in COX-2 protein is not due to decreased protein synthesis and occurs because of enhancement of substrate-independent COX-2 proteolysis. Although EP(1) does not interfere with the entry of COX-2 into the endoplasmic reticulum-associated degradation cascade, it facilitates COX-2 ubiquitination through complex formation. Blockade of proteasomal activity results in degradation of the receptor and concomitant recovery in the expression of COX-2, suggesting that EP(1) may scaffold an unknown E3 ligase that ubiquitinates COX-2. These findings propose a new role for the EP(1) receptor in resolving inflammation through down-regulation of COX-2.

Inhibition of exocytosis or endocytosis blocks activity-dependent redistribution of synapsin.

The synaptic vesicle cycle encompasses the pre-synaptic events that drive neurotransmission. Influx of calcium leads to the fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter, closely followed by endocytosis. Vacated release sites are repopulated with vesicles which are then primed for release. When activity is intense, reserve vesicles may be mobilized to counteract an eventual decline in transmission. Recently, interplay between endocytosis and repopulation of the readily releasable pool of vesicles has been identified. In this study, we show that exo-endocytosis is necessary to enable detachment of synapsin from reserve pool vesicles during synaptic activity. We report that blockage of exocytosis in cultured mouse hippocampal neurons, either by tetanus toxin or by the deletion of munc13, inhibits the activity-dependent redistribution of synapsin from the pre-synaptic terminal into the axon. Likewise, perturbation of endocytosis with dynasore or by a dynamin dominant-negative mutant fully prevents synapsin redistribution. Such inhibition of synapsin redistribution occurred despite the efficient phosphorylation of synapsin at its protein kinase A/CaMKI site, indicating that disengagement of synapsin from the vesicles requires exocytosis and endocytosis in addition to phosphorylation. Our results therefore reveal hitherto unidentified feedback within the synaptic vesicle cycle involving the synapsin-managed reserve pool.

Tyrosine phosphorylation of the 2B subunit of the NMDA receptor is necessary for taste memory formation.

We aimed to test whether tyrosine phosphorylation of the NMDA receptor (NMDAR) in the insular cortex is necessary for novel taste learning. We found that in rats, novel taste learning leads to elevated phosphorylation of tyrosine 1472 of the NR2B subunit of the NMDAR and increases the interaction of phosphorylated NR2B with the major postsynaptic scaffold protein PSD-95. Injection of the tyrosine kinase inhibitor genistein directly into the insular cortex of rats before novel taste exposure prevented the increase in NR2B tyrosine phosphorylation and behaviorally attenuated taste-memory formation. Functionally, tyrosine phosphorylation of NR2B after learning was found to determine the synaptic distribution of the NMDAR, since microinjection of genistein to the insular cortex altered the distribution pattern of NMDAR caused by novel taste learning.