Dihydroceramide desaturase functions as an inducer and rectifier of apoptosis : effect of retinol derivatives, antioxidants and phenolic compounds

Dihydroceramide desaturase (Degs1) catalyses the introduction of 4,5-trans double bond into dihydroceramide to form ceramide. We show here that Degs1 is polyubiquitinated in response to retinol derivatives, phenolic compounds or anti-oxidants in HEK293T cells. The functional predominance of native versus polyubiquitinated forms of Degs1 appears to govern cytotoxicity. Therefore, 4-HPR or celecoxib appear to stimulate the de novo ceramide pathway (with the exception of C24:0 ceramide), using native Degs1, and thereby promote PARP cleavage and LC3B-I/II processing (autophagy/apoptosis). The ubiquitin-proteasomal degradation of Degs1 is positively linked to cell survival via XBP-1s and results in a concomitant increase in dihydroceramides and a decrease in C24:0 ceramide levels. However, in the case of 4-HPR or celecoxib, the native form of Degs1 functionally predominates, such that the apoptotic programme is sustained. In contrast, 4-HPA or AM404 do not produce apoptotic ceramide, using native Degs1, but do promote a rectifier function to induce ubiquitin-proteasomal degradation of Degs1 and are not cytotoxic. Therefore, Degs1 appears to function both as an ‘inducer’ and ‘rectifier’ of apoptosis in response to chemical cellular stress, the dynamic balance for which is dependent on the nature of chemical stress, thereby determining cytotoxicity. The de novo synthesis of ceramide or the ubiquitin-proteasomal degradation of Degs1 in response to anti-oxidants, retinol derivatives and phenolic compounds appear to involve sensors, and for rectifier function, this might be Degs1 itself

Native and polyubiquitinated forms of dihydroceramide desaturase are differentially linked to human embryonic kidney cell survival

There is controversy concerning the role of dihydroceramide desaturase (Degs1) in regulating cell survival with studies showing that it can both promote and protect against apoptosis. We have therefore, investigated the molecular basis for these opposing roles of Degs1. Treatment of HEK293T cells with the sphingosine kinase inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) or fenretinide, but not the Degs1 inhibitor, GT11 (((N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octan-amide)) induced the polyubiquitination of Degs1 (Mr=40-140 kDa) via a mechanism involving oxidative stress, p38 MAPK and Mdm2 (E3 ligase). The polyubiquitinated forms of Degs1 exhibit ‘gain of function’ and activate pro-survival pathways, p38 MAPK, JNK and X-box protein-1s (XBP-1s). In contrast, another sphingosine kinase inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) at concentrations of 25-50 uM failed to induce formation of the polyubiquitinated forms of Degs1. In contrast with SKi, ABC294640 (25 uM) promotes apoptosis of HEK293T cells via a Degs1-dependent mechanism that is associated with increased de novo synthesis of ceramide. These findings are the first to demonstrate that the polyubiquitination of Degs1 appears to change its function from pro-apoptotic to pro-survival. Thus, polyubiquitination of Degs1 might provide an explanation for the reported opposing functions of this enzyme on cell survival/apoptosis

The role of sphingosine kinases and dihydroceramide desaturase in cell survival/apoptosis and inflammation pathways

The literature details significant controversy regarding the role of dihydroceramide desaturase (Degs1) in regulating cell survival/apoptosis and therefore this study primarily examined the molecular basis of the reported opposing roles of Degs1. The sphingosine kinase inhibitor SKi [2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole] or the Degs1 inhibitor fenretinide promoted the polyubiquitination of Degs1 (Mr = 40-140 kDa) through a mechanism involving p38 mitogen-activated protein kinase (MAPK), oxidative stress, and Mdm2 (E3 ligase) in HEK293T cells.;The polyubiquitinated forms of Degs1 acquire a 'gain of function' and activate pro-survival pathways, p38 MAPK/c-Jun N-terminal kinase (JNK), and X-box protein 1s (XBP-1s) in HEK293T cells. In contrast, the sphingosine kinase inhibitor, ABC294640 [3-(4- chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide] (25 to 50 μM), did not promote formation of polyubiquitinated Degs1 forms and induced apoptosis of HEK293T cells via a mechanism involving native Degs1. Native Degs1 appears to function in this context via substrate induction to increase de novo synthesis of apoptotic ceramides.;These results were achieved using siRNA transfections, western protein analysis of protein expression, immunoprecipitation, [3H]-Thymidine incorporation assay, and mass spectrometry. These novel findings are the first to reveal that the polyubiquitinated forms of Degs1 exhibit opposing function compared with the native form, which could explain the controversy concerning the role of this enzyme in apoptosis versus cell survival described within literature.;The study next investigated the role of sphingosine kinases (SK1, SK2) in regulating p53, stress activated protein kinases, and XBP-1s in HEK293T cells since SK inhibitors are known to promote p53-dependent cell death. SKi stimulated polyubquitination of p53 to form two higher molecular mass proteins (63 and 90 kDa), which might represent inactive forms of p53. The formation of p63/p90 in response to SKi was enhanced by completely eliminating SK1 from HEK293T cells using a combination of SK1 siRNA and SKi.;In addition, sphingolipids measurements showed a decrease in the levels of S1P and increase in the levels of sphingosine under these conditions. However, SK2 or Degs1 had no role in regulating the formation of p63/p90. In addition, the complete elimination of SK1 enhanced the activation of p38 MAPK/JNK pro-survival pathways in response to SKi.;Taken together with the effect on p53, these findings suggest that SK1 opposes pro-survival signalling pathways in HEK293T cells. The proteasome inhibitor, MG132 also induced expression of the pro-survival protein XBP-1s and this was enhanced when HEK293T cells were treated with SKi. SK2 siRNA reduced XBP-1s levels in response to MG132/SKi while p53 siRNA promoted this effect. These findings were achieved using siRNA and transient plasmid transfections, western protein analysis of protein expression, immunoprecipitation, [3H]-Thymidine incorporation assay, mass spectrometry, and immunofluorescence microscopy which suggest that SK2 opposes the death promoting function of p53.;Lastly, the role of SK1 and SK2 in regulating inflammation-based transcriptional factors in keratinocytes was investigated using western protein analysis of protein expression and luciferase reporter assays. SK2 inhibitors, such as ABC294640 or SKi or K145 or (R)-methylether FTY720 (ROMe) were shown to reverse the degradation of inhibitor kappa B (IκB) and transcriptional regulation of nuclear factor kappa B (NF-κB) in response to TNFα. In contrast, the potent SK1 inhibitor, PF-543 did not reverse IκB degradation and only weakly inhibited transcriptional regulation of NF-κB at a concentration that is 50-fold higher than the Ki for inhibition of SK1 activity.;Thus SK2 and not SK1 is proposed to regulate NF-κB signalling and transcription. The effect of the sphingosine kinases on transcriptional regulation of Activator Protein-1 (AP-1) was also investigated. SK2 inhibitors, ABC294640 or K145 reduced phorbol myristate acetate (PMA) stimulated phosphorylation of JNK and ERK-1/2 and transcriptional activity of AP-1. In contrast, other SK inhibitors including PF-543, SKi and ROMe did not inhibit JNK and ERK-1/2 signalling and only produced a minimal reduction in AP-1 transcriptional activity, thereby suggesting that the effects of SK2 inhibitors on JNK/ERK signalling and AP-1 transcriptional activity are likely to be 'off-target'.;These novel significant findings provide improved understanding of the role of Degs1, SK1 and SK2 in regulating cell survival and inflammation and this might ultimately aid in the identification of novel signalling networks and therapeutic targets for treatment of various diseases, including cancer.The literature details significant controversy regarding the role of dihydroceramide desaturase (Degs1) in regulating cell survival/apoptosis and therefore this study primarily examined the molecular basis of the reported opposing roles of Degs1. The sphingosine kinase inhibitor SKi [2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole] or the Degs1 inhibitor fenretinide promoted the polyubiquitination of Degs1 (Mr = 40-140 kDa) through a mechanism involving p38 mitogen-activated protein kinase (MAPK), oxidative stress, and Mdm2 (E3 ligase) in HEK293T cells.;The polyubiquitinated forms of Degs1 acquire a 'gain of function' and activate pro-survival pathways, p38 MAPK/c-Jun N-terminal kinase (JNK), and X-box protein 1s (XBP-1s) in HEK293T cells. In contrast, the sphingosine kinase inhibitor, ABC294640 [3-(4- chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide] (25 to 50 μM), did not promote formation of polyubiquitinated Degs1 forms and induced apoptosis of HEK293T cells via a mechanism involving native Degs1. Native Degs1 appears to function in this context via substrate induction to increase de novo synthesis of apoptotic ceramides.;These results were achieved using siRNA transfections, western protein analysis of protein expression, immunoprecipitation, [3H]-Thymidine incorporation assay, and mass spectrometry. These novel findings are the first to reveal that the polyubiquitinated forms of Degs1 exhibit opposing function compared with the native form, which could explain the controversy concerning the role of this enzyme in apoptosis versus cell survival described within literature.;The study next investigated the role of sphingosine kinases (SK1, SK2) in regulating p53, stress activated protein kinases, and XBP-1s in HEK293T cells since SK inhibitors are known to promote p53-dependent cell death. SKi stimulated polyubquitination of p53 to form two higher molecular mass proteins (63 and 90 kDa), which might represent inactive forms of p53. The formation of p63/p90 in response to SKi was enhanced by completely eliminating SK1 from HEK293T cells using a combination of SK1 siRNA and SKi.;In addition, sphingolipids measurements showed a decrease in the levels of S1P and increase in the levels of sphingosine under these conditions. However, SK2 or Degs1 had no role in regulating the formation of p63/p90. In addition, the complete elimination of SK1 enhanced the activation of p38 MAPK/JNK pro-survival pathways in response to SKi.;Taken together with the effect on p53, these findings suggest that SK1 opposes pro-survival signalling pathways in HEK293T cells. The proteasome inhibitor, MG132 also induced expression of the pro-survival protein XBP-1s and this was enhanced when HEK293T cells were treated with SKi. SK2 siRNA reduced XBP-1s levels in response to MG132/SKi while p53 siRNA promoted this effect. These findings were achieved using siRNA and transient plasmid transfections, western protein analysis of protein expression, immunoprecipitation, [3H]-Thymidine incorporation assay, mass spectrometry, and immunofluorescence microscopy which suggest that SK2 opposes the death promoting function of p53.;Lastly, the role of SK1 and SK2 in regulating inflammation-based transcriptional factors in keratinocytes was investigated using western protein analysis of protein expression and luciferase reporter assays. SK2 inhibitors, such as ABC294640 or SKi or K145 or (R)-methylether FTY720 (ROMe) were shown to reverse the degradation of inhibitor kappa B (IκB) and transcriptional regulation of nuclear factor kappa B (NF-κB) in response to TNFα. In contrast, the potent SK1 inhibitor, PF-543 did not reverse IκB degradation and only weakly inhibited transcriptional regulation of NF-κB at a concentration that is 50-fold higher than the Ki for inhibition of SK1 activity.;Thus SK2 and not SK1 is proposed to regulate NF-κB signalling and transcription. The effect of the sphingosine kinases on transcriptional regulation of Activator Protein-1 (AP-1) was also investigated. SK2 inhibitors, ABC294640 or K145 reduced phorbol myristate acetate (PMA) stimulated phosphorylation of JNK and ERK-1/2 and transcriptional activity of AP-1. In contrast, other SK inhibitors including PF-543, SKi and ROMe did not inhibit JNK and ERK-1/2 signalling and only produced a minimal reduction in AP-1 transcriptional activity, thereby suggesting that the effects of SK2 inhibitors on JNK/ERK signalling and AP-1 transcriptional activity are likely to be 'off-target'.;These novel significant findings provide improved understanding of the role of Degs1, SK1 and SK2 in regulating cell survival and inflammation and this might ultimately aid in the identification of novel signalling networks and therapeutic targets for treatment of various diseases, including cancer

Willingness to get HPV vaccination among female university students in Kuwait and its relation to vaccine conspiracy beliefs

A substantial burden of high-risk human papillomavirus (HPV) infections and HPV-related cancers can be mitigated by vaccination. The current study aimed to investigate the willingness of female students at the University of Kuwait to get HPV vaccination and its possible association with general vaccine conspiracy beliefs (VCBs). This cross-sectional survey study was conducted during September–November 2022 using a validated VCB scale as the survey instrument. The final sample comprised 611 respondents with a median age of 22 y and a majority of Arab ethnicity (n = 600, 98.2%). Only 360 respondents (56.9%) heard of HPV before participation and these students showed an above-average level of HPV knowledge (mean knowledge score of 12.7 ± 2.6 out of 16 as the maximum score), of whom only 33 self-reported HPV vaccine uptake (9.2%). The willingness to accept free-of-charge HPV vaccination was seen among 69.8% of the participants, with 20.1% who were hesitant and 10.1% who were resistant. The acceptance of HPV vaccination if payment is required was 23.1%. Reasons for HPV vaccine hesitancy/resistance included complacency to the HPV disease risks, lack of confidence in HPV vaccination, and inconvenience. The embrace of VCBs was associated with significantly higher odds of HPV vaccine hesitancy/resistance. The current study showed the detrimental impact of endorsing vaccine conspiracy beliefs manifested in lower intention to get HPV vaccination among female university students in Kuwait. This should be considered in vaccine promotion efforts aiming to reduce the burden of HPV cancers

Monkeypox Knowledge and Confidence in Diagnosis and Management with Evaluation of Emerging Virus Infection Conspiracies among Health Professionals in Kuwait

As the 2022 human monkeypox (HMPX) multi-country outbreak is spreading, the response of healthcare workers (HCWs) is central to mitigation efforts. The current study aimed to evaluate HMPX knowledge and confidence in diagnosis and management among HCWs in Kuwait. We used a self-administered questionnaire distributed in July–August 2022 through a snowball sampling approach. The survey items evaluated HMPX knowledge, confidence in diagnosis and management of the disease, and the belief in conspiracies regarding emerging virus infections (EVIs). The sample size was 896 HCWs: nurses (n = 485, 54.1%), pharmacists (n = 154, 17.2%), physicians (n = 108, 12.1%), medical technicians/allied health professionals (MT/AHP, n = 96, 10.7%), and dentists (n = 53, 5.9%). An overall low level of HMPX knowledge was noticed for items assessing virus transmission and non-cutaneous symptoms of the disease, with higher knowledge among physicians. Approximately one-fifth of the study sample agreed with the false notion that HMPX is exclusive to male homosexuals (n = 183, 20.4%), which was associated with lower knowledge with higher frequency among MT/AHP compared to nurses, physicians, and pharmacists. Confidence levels were low: confidence in diagnosis based on diagnostic tests (n = 449, 50.1%), confidence in the ability to manage the HMPX (n = 426, 47.5%), and confidence in the ability to diagnose HMPX clinically (n = 289, 32.3%). Higher confidence levels were found among nurses and participants with postgraduate degrees. Higher embrace of conspiracy beliefs regarding EVIs was noticed among participants with lower knowledge, and among those who agreed or were neutral/had no opinion regarding the false idea of HMPX exclusive occurrence among male homosexuals, while lower levels of belief in conspiracies were noticed among physicians, dentists, and pharmacists compared to MT/AHP. Variable levels of HMPX knowledge were observed in this study per item, with low level of knowledge regarding virus transmission. Differences in knowledge and confidence levels in diagnosis and management of HMPX should be considered in education and training aiming to prepare for outbreak response. The relatively high prevalence of embracing conspiratorial beliefs regarding EVIs is worrisome and needs proper interventions. The attitude towards male homosexuals’ role in monkeypox spread should be evaluated in future studies considering the possibility of stigma and discrimination in this most-at-risk group

The regulation of p53, p38 MAPK, JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knock down of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knock down of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knock down of SK2 and increased by knock down of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells

The Role of Psychological Factors and Vaccine Conspiracy Beliefs in Influenza Vaccine Hesitancy and Uptake among Jordanian Healthcare Workers during the COVID-19 Pandemic

Vaccination to prevent influenza virus infection and to lessen its severity is recommended among healthcare workers (HCWs). Health professionals have a higher risk of exposure to viruses and could transmit the influenza virus to vulnerable patients who are prone to severe disease and mortality. The aim of the current study was to evaluate the levels of influenza vaccine acceptance and uptake as well as its determinants, among Jordanian HCWs over the last influenza season of 2021/2022. This study was based on a self-administered electronic survey that was distributed in March 2022. Psychological determinants of influenza vaccine acceptance and vaccine conspiracy beliefs were assessed using the previously validated 5C scale questionnaire (confidence, complacency, constraints, calculation and collective responsibility) and the vaccine conspiracy beliefs scale. The study sample comprised a total of 1218 HCWs: nurses (n = 412, 33.8%), physicians (n = 367, 30.1%), medical technicians (n = 182, 14.9%), pharmacists (n = 161, 13.2%) and dentists (n = 87, 7.1%), among others. About two-thirds of the study sample expressed willingness to receive influenza vaccination if provided free of charge (n = 807, 66.3%), whereas less than one-third were willing to pay for the vaccine (n = 388, 31.9%). The self-reported uptake of the influenza vaccine in the last influenza season was 62.8%. The following factors were significantly associated with higher acceptance of influenza vaccination if provided freely, as opposed to vaccine hesitancy/rejection: male sex; physicians and dentists among HCW categories; higher confidence and collective responsibility; and lower complacency, constraints and calculation. Higher influenza vaccine uptake was significantly correlated with nurses and physicians among HCW categories, older age, a higher monthly income, higher confidence and collective responsibility, lower complacency and constraints and lower embrace of general vaccine conspiracy beliefs. The results of the current study can provide helpful clues to improve influenza vaccine coverage among HCWs in Jordan. Consequently, this can help to protect vulnerable patient groups and reserve valuable resources in healthcare settings. Psychological determinants appeared to be the most significant factors for vaccine acceptance and uptake, whereas the embrace of general vaccine conspiracy beliefs was associated with lower rates of influenza vaccine uptake, which should be considered in educational and interventional measures aiming to promote influenza vaccination