Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.

Whole tumor cell lysates (TCLs) obtained from cancer cells previously killed by treatments able to promote immunogenic cell death (ICD) can be efficiently used as a source of tumor-associated antigens for the development of highly efficient dendritic cell (DC)-based vaccines. Herein, the potential role of the interferon (IFN)-inducible protein phospholipid scramblase 1 (PLSCR1) in influencing immunogenic features of dying cancer cells and in enhancing DC-based vaccine efficiency was investigated.
PLSCR1 expression was evaluated in different mantle-cell lymphoma (MCL) cell lines following ICD induction by 9-cis-retinoic acid (RA)/IFN-α combination, and commercial kinase inhibitor was used to identify the signaling pathway involved in its upregulation. A Mino cell line ectopically expressing PLSCR1 was generated to investigate the potential involvement of this protein in modulating ICD features. Whole TCLs obtained from Mino overexpressing PLSCR1 were used for DC loading, and loaded DCs were employed for generation of tumor antigen-specific cytotoxic T lymphocytes.
The ICD inducer RA/IFN-α combination promoted PLSCR1 expression through STAT1 activation. PLSCR1 upregulation favored pro-apoptotic effects of RA/IFN-α treatment and enhanced the exposure of calreticulin on cell surface. Moreover, DCs loaded with TCLs obtained from Mino ectopically expressing PLSCR1 elicited in vitro greater T-cell-mediated antitumor responses compared with DCs loaded with TCLs derived from Mino infected with empty vector or the parental cell line. Conversely, PLSCR1 knock-down inhibited the stimulating activity of DCs loaded with RA/IFN-α-treated TCLs to elicit cyclin D1 peptide-specific cytotoxic T lymphocytes.
Our results indicate that PLSCR1 improved ICD-associated calreticulin exposure induced by RA/IFN-α and was clearly involved in DC-based vaccine efficiency as well, suggesting a potential contribution in the control of pathways associated to DC activation, possibly including those involved in antigen uptake and concomitant antitumor immune response activation.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and it has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the levels of human cytomegalovirus (HCMV) plaque formation in OUMS-36T-3 (36T-3) cells with high basal expression of PLSCR1 were significantly lower than those in human embryonic lung (HEL) cells with low basal expression of PLSCR1. In addition, the levels of HCMV plaque formation and replication in PLSCR1-knockout (KO) 36T-3 cells were significantly higher than those in parental 36T-3 cells and were comparable to those in HEL cells. Furthermore, compared to that in PLSCR1-KO cells, the expression of HCMV major immediate early (MIE) proteins was repressed and/or delayed in parental 36T-3 cells after HCMV infection. We also showed that PLSCR1 expression decreased the levels of the cAMP-responsive element (CRE)-binding protein (CREB)•HCMV immediate early protein 2 (IE2) and CREB-binding protein (CBP)•IE2 complexes, which have been suggested to play important roles in the IE2-mediated transactivation of the viral early promoter through interactions with CREB, CBP, and IE2. Interestingly, PLSCR1 expression repressed CRE- and HCMV MIE promoter-regulated reporter gene activities. These observations reveal, for the first time, that PLSCR1 negatively regulates HCMV replication by repressing the transcription from viral MIE and early promoters, and that PLSCR1 expression may contribute to the IFN-mediated suppression of HCMV infection. IMPORTANCE Because several IFN-stimulated genes (ISGs) have been reported to suppress HCMV replication, HCMV replication is thought to be regulated by an IFN-mediated host defense mechanism, but the mechanism remains unclear. PLSCR1 expression is induced in response to viral infection and IFN treatment, and PLSCR1 has been reported to play an important role in IFN-dependent antiviral responses. Here, we demonstrate that HCMV plaque formation and major immediate early (MIE) gene expression are significantly increased in PLSCR1-KO human fibroblast cells. PLSCR1 reduces levels of the CREB•IE2 and CBP•IE2 complexes, which have been suggested to play important roles in HCMV replication through its interactions with CREB, CBP, and IE2. In addition, PLSCR1 expression represses transcription from the HCMV MIE promoter. Our results indicate that PLSCR1 plays important roles in the suppression of HCMV replication in the IFN-mediated host defense system.

Phospholipid scramblase 1 (PLSCR1) serves a function in the pathogenesis and progression of various types of cancer. However, the role of PLSCR1 in human primary liver cancer remains unknown. The aim of the present study was to evaluate the expression of PLSCR1 in primary liver cancer and analyse the clinical significance. In addition, the present study detected and compared the biological behaviours of HepG2 cells with different levels of activated PLSCR1 or silenced PLSCR1. PLSCR1 expression in primary liver cancer tissue samples was examined using immunohistochemistry. Cultured HepG2 cells were infected with lentiviruses to suppress or activate PLSCR1 expression. Reverse transcription-quantitative PCR and western blotting were performed to analyse the effects of silencing or activating PLSCR1 in cell lines at the mRNA and protein levels, respectively. The effects of PLSCR1 expression on cell proliferation, adhesion, migration and invasion were subsequently determined using Cell Counting Kit 8, adhesion, and Transwell migration and invasion assays. PLSCR1 expression in primary liver cancer tissue samples was higher compared with that in adjacent non-cancerous liver tissue samples and normal tissue samples, and positively correlated with the clinical stage. PLSCR1 was effectively downregulated or overexpressed in HepG2 cells using small interfering RNA and lentivirus techniques, respectively. PLSCR1 upregulation promoted cell proliferation, invasion and migration, while PLSCR1 downregulation inhibited these effects. PLSCR1 is highly expressed in primary liver cancer and associated with the clinical stage. Downregulating the expression of PLSCR1 significantly inhibited the proliferation, adhesion, migration and invasion of cancer cells, suggesting that PLSCR1 may be a potential therapeutic target for preventing the progression of primary liver cancer.

The biological function and underlying mechanism of microRNA-628-5p (miR-628-5p) remains to be clarified in the growth and progression of pancreatic ductal adenocarcinoma (PDAC). Here, the expression levels of miR-628-5p in PDAC tissues and cells were detected by quantitative reverse transcriptase polymerase chain reaction and in situ hybridization. The relationship between miR-628-5p expression and clinicopathologic characteristics was examined in human PDAC tissue samples. Gain- and loss-of-function and the putative targets of miR-628-5p were evaluated in PDAC cell lines. The upstream and downstream signals of miR-628-5p in PDAC were also examined. MiR-628-5p was lowly expressed in PDAC tissues and cell lines, and low miR-628-5p expression in PDAC tissues was associated with poor clinicopathological characteristics and shorter overall survival. Functionally, restoration of miR-628-5p expression decreased PDAC cell proliferation, migration, invasion, and promoted cell apoptosis, whereas miR-628-5p silencing abolished these biological behaviors. MiR-628-5p was found to target and negatively regulate phospholipid scramblase 1 and insulin receptor substrate 1 expression, which resulted in the inhibition of the AKT/NF-κB signaling pathway. MYC knockdown led to miR-628-5p upregulation, whereas MYC overexpression repressed miR-628-5p expression. These findings indicate that miR-628-5p functions as a tumor-suppressive microRNA in PDAC and implicate miR-628-5p as a potential therapeutic target for PDAC patients.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and PLSCR1 has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the basal expression of PLSCR1 was significantly elevated in Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC). PLSCR1 was observed to directly interact with the EBV immediate-early transactivator BZLF1 in vitro and in vivo, and this interaction repressed the BZLF1-mediated transactivation of an EBV lytic BMRF1 promoter construct. In addition, PLSCR1 expression decreased the BZLF1-mediated up-regulation of lytic BMRF1 mRNA and protein expression in WT and PLSCR1-knockout EBV-infected NPC cells. Furthermore, we showed that PLSCR1 represses the interaction between BZLF1 and CREB-binding protein (CBP), which enhances the BZLF1-mediated transactivation of EBV lytic promoters. These results reveal for the first time that PLSCR1 specifically interacts with BZLF1 and negatively regulates its transcriptional regulatory activity by preventing the formation of the BZLF1-CBP complex. This interaction may contribute to the establishment of latent EBV infection in EBV-infected NPC cells.

Granulomatosis with polyangiitis (GPA) is an autoimmune vasculitis associated with anti-neutrophil-cytoplasmic antibodies (ANCA) against proteinase 3 leading to kidney damage. Neutrophils from those patients have increased expression of membrane proteinase 3 during apoptosis. Here we examined whether neutrophils from patients with GPA have dysregulated protein expressions associated with apoptosis. A global proteomic analysis was performed comparing neutrophils from patients with GPA, with healthy individuals under basal conditions and during apoptosis. At disease onset, the cytosolic proteome of neutrophils of patients with GPA before treatment was significantly different from healthy controls, and this dysregulation was more pronounced following ex vivo apoptosis. Proteins involved in cell death/survival were altered in neutrophils of patients with GPA. Several proteins identified were PR3-binding partners involved in the clearance of apoptotic cells, namely calreticulin, annexin-A1 and phospholipid scramblase 1. These proteins form a platform at the membrane of apoptotic neutrophils in patients with GPA but not healthy individuals and this was associated with the clinical presentation of GPA. Thus, our study shows that neutrophils from patients with GPA have an intrinsic dysregulation in proteins involved in apoptotic cell clearance, which could contribute to the unabated inflammation and autoimmunity in GPA. Hence, harnessing these dysregulated pathways could lead to novel biomarkers and targeted therapeutic opportunities to treat kidney disease.

Microglia are the intrinsic immune sentinels of the central nervous system. Their activation restricts tissue injury and pathogen spread, but in some settings, including viral infection, this response can contribute to cell death and disease. Identifying mechanisms that control microglial responses is therefore an important objective. Using replication-incompetent adenovirus 5 (Ad5)-based vectors as a model, we investigated the mechanisms through which microglia recognize and respond to viral uptake. Transgenic, immunohistochemical, molecular-genetic, and fluorescence imaging approaches revealed that phosphatidylserine (PtdSer) exposure on the outer leaflet of transduced cells triggers their engulfment by microglia through TAM receptor-dependent mechanisms. We show that inhibition of phospholipid scramblase 1 (PLSCR1) activity reduces intracellular calcium dysregulation, prevents PtdSer externalization, and enables months-long protection of vector-transduced, transgene-expressing cells from microglial phagocytosis. Our study identifies PLSCR1 as a potent target through which the innate immune response to viral vectors, and potentially other stimuli, may be controlled.

Mantle cell lymphoma (MCL) is an aggressive haematological malignancy in which the response to therapy can be limited by aberrantly activated molecular and cellular pathways, among which autophagy was recently listed. Our study shows that the 9-cis-retinoic acid (RA)/Interferon(IFN)-α combination induces protective autophagy in MCL cell lines and primary cultures reducing the extent of drug-induced apoptosis. The treatment significantly up-regulates phospholipid scramblase 1 (PLSCR1), a protein which bi-directionally flips lipids across membranes. In particular, RA/IFN-α combination concomitantly increases PLSCR1 transcription and controls PLSCR1 protein levels via lysosomal degradation. Herein we describe a new function for PLSCR1 as negative regulator of autophagy. Indeed, PLSCR1 overexpression reduced MCL cell susceptibility to autophagy induced by RA/IFN-α, serum deprivation or mTOR pharmacological inhibition. Moreover, PLSCR1 can bind the ATG12/ATG5 complex preventing ATG16L1 recruitment and its full activation, as indicated by co-immunoprecipitation experiments. The combination of doxorubicin or bortezomib with RA/IFN-α strengthened PLSCR1 up-regulation and enhanced apoptosis, as a likely consequence of the blockade of RA/IFN-α-induced autophagy. Immunohistochemical analysis of 32 MCL biopsies revealed heterogeneous expression of PLSCR1 and suggests its possible implication in the response to anticancer therapies, especially to drugs promoting protective autophagy.

Phospholipid scramblase 1 involve in biological processes including phospholipid movement, proliferation, and apoptosis. Treatment with an antiphospholipid scramblase 1 antibody (NP1) has been demonstrated to inhibit cell proliferation in colorectal cancer. This study aimed to explore the role of NP1 treatment in the apoptosis of colorectal cancer cells. Results showed that NP1 treatment significantly increases the apoptosis of colorectal cancer cells via the activation of caspase 8, caspase 9, and caspase 3. Moreover, pretreatment with a caspase 8 inhibitor did not fully prevent the apoptotic effects of NP1. Taken together, these data indicate NP1 induces cell apoptosis primary through the intrinsic apoptotic pathway. NP1 may serve as a potential therapeutic agent.