Loss of Tuberous Sclerosis Complex 2 confers inflammation via dysregulation of Nuclear factor kappa-light-chain-enhancer of activated B cells.

Background: Aberrant activation of mTORC1 is clearly defined in TSC, causing uncontrolled cell growth. While mTORC1 inhibitors show efficacy to stabilise tumour growth in TSC, they are not fully curative. Disease facets of TSC that are not restored with mTOR inhibitors might involve NF-κB. The study aimed to characterise NF-κB in the context of TSC. Results: Enrichment of NF-κB-regulated genes was observed in TSC patient tumours, SEN/SEGAs, cortical tubers and a TSC tumour-derived cell line (621 - 101). Highlighting an inflammatory component of TSC, TSC cell models showed an elevated level of NF-κB and STAT3 activation. Herein, we report a dysregulated inflammatory phenotype of TSC2-deficient cells where NF-κB promotes autocrine signalling involving IL-6. Of importance, mTORC1 inhibition does not block this inflammatory signal to promote STAT3, while NF-κB inhibition was much more effective. Combined mTORC1 and NF-κB inhibition was potent at preventing anchorage-independent growth of TSC2-deficient cells, and unlike mTORC1 inhibition alone was sufficient to prevent colony regrowth after cessation of treatment. Conclusion: This study reveals autocrine signalling crosstalk between NF-κB and STAT3 in TSC cell models. Furthermore, the data presented indicate that NF-κB pathway inhibitors could be a viable adjunct therapy with the current mTOR inhibitors to treat TSC.

Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells.

Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.

Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours.

BACKGROUND: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. METHODS: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. RESULTS: MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. CONCLUSIONS: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.

Male infertility-linked point mutation reveals a vital binding role for the C2 domain of sperm PLCζ.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.

Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm.

STUDY QUESTION: Is it possible to improve clinical visualization of phospholipase C zeta (PLCζ) as a diagnostic marker of sperm oocyte activation capacity and male fertility? SUMMARY ANSWER: Poor PLCζ visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLCζ, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols. WHAT IS KNOWN ALREADY: Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLCζ. PLCζ represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLCζ antibody specificity and detection protocols. STUDY DESIGN, SIZE DURATION: Two PLCζ polyclonal antibodies, with confirmed PLCζ specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLCζ visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1-0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats. MAIN RESULTS AND THE ROLE OF CHANCE: Despite high specificity for native PLCζ following immunoblotting using epitope-specific polyclonal PLCζ antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLCζ compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLCζ visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLCζ visualization efficacy may be due to steric or conformational occlusion of native PLCζ, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLCζ fluorescence, and the proportion of sperm exhibiting detectable PLCζ fluorescence in sperm from different males. LIMITATIONS, REASONS FOR CAUTION: Direct correlation of fertility outcomes with the level of PLCζ in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLCζ visualization we propose would indeed reflect fertility status. WIDER IMPLICATIONS OF THE FINDINGS: We propose that AUM alters conformational interactions to enhance PLCζ epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLCζ-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLCζ and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLCζ appears to be kept in an inactive form in the sperm. LARGE SCALE DATA: Not applicable. STUDY FUNDING/COMPETING INTERESTS: J.K. is supported by a Health Fellowship award from the National Institute for Social Care and Health Research (NISCHR). M.N. is supported by a Marie Curie Intra-European Research Fellowship award. This work was also partly funded by a research grant from Cook Medical Technologies LLC. There are no competing financial interests to declare.

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2).

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca(2+) oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP2) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca(2+) oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP2, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca(2+) oscillation inducing activity and in vitro interaction with PIP2 without affecting its Ca(2+) sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca(2+) frequency and the binding ability of different PLCζ mutants to PIP2. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP2, leading to complete abolishment of its Ca(2+) oscillation inducing activity.

Human PLCζ exhibits superior fertilization potency over mouse PLCζ in triggering the Ca(2+) oscillations required for mammalian oocyte activation.

A sperm-specific phospholipase C-zeta (PLCζ) is believed to play an essential role in oocyte activation during mammalian fertilization. Sperm PLCζ has been shown to trigger a prolonged series of repetitive Ca(2+) transients or oscillations in oocytes that precede activation. This remarkable intracellular Ca(2+) signalling phenomenon is a distinctive characteristic observed during in vitro fertilization by sperm. Previous studies have notably observed an apparent differential ability of PLCζ from disparate mammalian species to trigger Ca(2+) oscillations in mouse oocytes. However, the molecular basis and confirmation of the apparent PLCζ species difference in activity remains to be provided. In the present study, we provide direct evidence for the superior effectiveness of human PLCζ relative to mouse PLCζ in generating Ca(2+) oscillations in mouse oocytes. In addition, we have designed and constructed a series of human/mouse PLCζ chimeras to enable study of the potential role of discrete PLCζ domains in conferring the enhanced Ca(2+) signalling potency of human PLCζ. Functional analysis of these human/mouse PLCζ domain chimeras suggests a novel role of the EF-hand domain in the species-specific differences in PLCζ activity. Our empirical observations are compatible with a basic mathematical model for the Ca(2+) dependence of generating cytoplasmic Ca(2+) oscillations in mammalian oocytes by sperm PLCζ.

Phospholipase Cζ binding to PtdIns(4,5)P2 requires the XY-linker region.

Phospholipase C-zeta (PLCζ) is a strong candidate for the mammalian sperm-derived factor that triggers the Ca(2+) oscillations required for egg activation at fertilization. PLCζ lacks a PH domain, which targets PLCδ1 to the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) substrate in the plasma membrane. Previous studies failed to detect PLCζ in the plasma membrane, hence the means of PLCζ binding to PtdIns(4,5)P(2) is unclear. We find that the PLCζ XY linker, but not the C2 domain, exhibits robust binding to PtdIns(4,5)P(2) or to liposomes containing near-physiological levels of PtdIns(4,5)P(2). The role of positively charged residues within the XY linker was addressed by sequentially substituting alanines for three lysine residues, K374, K375 and K377. Microinjection of these mutants into mouse eggs enabled their Ca(2+) oscillation-inducing activities to be compared with wild-type PLCζ. The XY-linker mutant proteins were purified and the in vitro PtdIns(4,5)P(2) hydrolysis and binding properties were monitored. Successive reduction of net positive charge within the PLCζ XY linker significantly affects both in vivo Ca(2+)-oscillation-inducing activity and in vitro PtdIns(4,5)P(2) interaction of mouse PLCζ. Our data suggest that positively charged residues within the XY linker play an important role in the PLCζ interaction with PtdIns(4,5)P(2), a crucial step in generating the Ca(2+) activation signal that is essential for fertilization in mammals.

Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP(2) hydrolysis activity of sperm PLCζ.

A male infertility-linked human PLCζ (phospholipase Cζ) mutation introduced into mouse PLCζ completely abolishes both in vitro PIP(2) (phosphatidylinositol 4,5-bisphosphate) hydrolysis activity and the ability to trigger in vivo Ca2+ oscillations in mouse eggs. Wild-type PLCζ initiated a normal pattern of Ca2+ oscillations in eggs in the presence of 10-fold higher mutant PLCζ, suggesting that infertility is not mediated by a dominant-negative mechanism.

Inflammation and frailty measures in older people.

Inflammation in patients defined as frail by Fried's phenotypic definition may be related to sarcopenia. This study aimed to investigate inflammation in older patients across different frailty criteria. Frailty status was determined in 110 patients aged over 75 years (mean 83.9 years) according to function (dependent, intermediate, independent); Fried (three or more items of exhaustion, weight loss, slow walking speed, low handgrip strength, low physical activity) and Frailty Index (a measure of accumulated deficits). With increasing patient frailty as defined by function and by Fried phenotype, tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and C-reactive protein (CRP) increased significantly. Albumin was lowest in the frailest subjects by each definition. The greatest differences were seen between intermediate and dependent groups and between the pre-frail and frail. Adjustment for multiple covariates (age, sex, BMI category, smoking status, number of co-morbidities and number of prescribed medications) did not account for any of the observed differences in levels of inflammatory markers. The Frailty Index correlated significantly with log-transformed CRP (r= 0.221, P < 0.05), log-transformed IL-6 (r= 0.369, P < 0.01), TNF-alpha (r= 0.379, P < 0.01) and inversely with albumin (r=- 0.545, P < 0.01). This study provides further evidence linking inflammation and frailty in older people, an association that seems consistent across different frailty measures.

Plasma esterases and inflammation in ageing and frailty.

OBJECTIVES: Esterases are enzymes of drug metabolism known to be reduced in frail older people and during acute illness. The mechanism for this is unknown. The aim of this study was to examine esterase activity and inflammation in ageing and frailty. METHODS: Thirty frail patients (mean age 84.9 years) dependent on continuing inpatient care, 40 patients of intermediate frailty attending Day Hospital (84.2 years), 40 fit older controls (82.7 years) and 30 young controls (23.3 years) were studied. Frailty indicators, plasma esterase activities and markers of inflammation were measured. RESULTS: With increasing patient frailty, C-reactive protein (CRP), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) increased significantly and esterase activity, with the exception of aspirin esterase, fell significantly (p < 0.005). There were significant negative correlations between log-transformed IL-6 and acetylcholinesterase (r = -0.354, p < 0.01), butyrylcholinesterase (r = -0.392, p < 0.01) and benzoylcholinesterase activity (r = -0.241, p < 0.05) and significant negative correlations between TNF-alpha and acetylcholinesterase (r = -0.223, p < 0.01), butyrylcholinesterase (r = -0.279, p < 0.01) and benzoylcholinesterase activity (r = -0.253, p < 0.01). Aspirin esterase activity did not correlate with IL-6 or TNF- alpha. CONCLUSION: Frailty was associated with higher inflammatory markers and lower esterase activity. There was a weak but significant negative correlation between both IL-6 and TNF-alpha and the activity of three of four esterases. The negative correlation between esterase activity and inflammatory markers may have a causal basis, comparable to the inflammatory suppression of cytochrome P-450 enzymes.

Nutrition, inflammation, and leptin levels in aging and frailty.

OBJECTIVES: To examine nutritional indices and levels of leptin and inflammatory markers across age and frailty. DESIGN: Observational study. SETTING: Continuing care wards and a day hospital in Cardiff, South Wales, United Kingdom. PARTICIPANTS: Thirty dependent patients (mean age 84.9) needing continuing inpatient care, 40 patients with falls attending a day hospital (mean age 84.2), 40 independent controls (mean age 82.7), and 30 young controls (mean age 23.3). MEASUREMENTS: Functional status, including the five frailty indicators proposed by Fried et al., anthropometry, and serum markers of nutrition and inflammation. RESULTS: The continuing care patients were frail, all having three to five frailty indicators. Day hospital patients were of intermediate frailty (mean Fried score 2.97), and the independent group was fittest (0.83). Body mass index, triceps skinfold thickness (TSF), and mid-arm muscle area were lowest in continuing care patients. With increasing patient frailty, albumin levels fell significantly (P<.005) and C-reactive protein (CRP) levels increased significantly (P<.005). Continuing care patients had significantly lower leptin levels (P<.005) and significantly higher interleukin (IL)-6 levels (P<.005). There was a significant correlation between log transformed leptin and TSF for each patient group. CONCLUSION: The frailest older people displayed features of cachexia. Their leptin levels were appropriately low given their low body fat, and IL-6 and CRP levels were high. The mechanism of their cachexia may therefore be similar to that proposed in heart failure and cancer: disturbed hypothalamic feedback of leptin or effects of proinflammatory cytokines.