DPP4 Regulates DHCR24-Mediated Cholesterol Biosynthesis to Promote Methotrexate Resistance in Gestational Trophoblastic Neoplastic Cells

Metabolic reprogramming could promote cellular adaptation in response to chemotherapeutic drugs in cancer cells. Herein, we aimed to characterize the metabolomic profiles regulated by Dipeptidyl Peptidase 4 (DPP4) in methotrexate (MTX)-resistant gestational trophoblastic neoplastic (GTN) cells. A total of eighty metabolites were found to be commonly altered in DPP4-depleted JAR/MTX and JEG3/MTX cells. Cholesterol biosynthesis-related metabolites were markedly impacted by DPP4 knockdown in MTX-resistant sublines. Manipulation of DPP4 expression remarkably affected the level of cellular cholesterol in GTN cells. Our analysis also identified 24-Dehydrocholesterol Reductase (DHCR24) as a potential downstream effector of DPP4. Manipulation of DHCR24 expression affected cellular cholesterol level, reactive oxygen species (ROS) accumulation, and chemosensitivity to MTX in GTN cell models. In addition, over-expression of DHCR24 could markedly restore cellular cholesterol level and rescue cell survival in DPP4-depleted MTX-resistant GTN cells. Highly correlated expression of DPP4 and DHCR24 was observed in clinical GTN specimens. Further, DPP4 inhibitor sitagliptin effectively inhibited cholesterol biosynthesis, reduced DHCR24 expression and enhanced MTX-induced cytotoxicity in vitro and in vivo. In conclusion, our findings suggested that DPP4 might regulate DHCR24-mediated cholesterol biosynthesis to promote methotrexate resistance in GTN cells. Targeting DPP4/DHCR24 signaling might help to sensitize MTX-resistant GTN to MTX treatment.

Structural basis for sterol sensing by Scap and Insig

The sterol regulatory element-binding protein (SREBP) pathway senses the cellular cholesterol level through sterol regulated association between Scap and Insig. Despite the recent structural determination of the transmembrane domains of human Scap and Insig-2 bound to 25-hydroxycholesterol (25HC), the structure and regulatory mechanism of the luminal domains of Scap by cholesterol remains elusive. Here, combining cryo-EM analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex in the presence of digitonin instead of 25HC. Despite the lack of sequence similarity, the structure of the luminal domain Loop 1 and a co-folded segment in Loop 7 of Scap resembles that of the luminal/extracellular domain in NPC1 and related proteins. Comparison of the sterol-loaded structures of these proteins provides clues of the regulation of Loop 1/7 interaction by cholesterol. We also show that the structure of Scap(D428A), which suppresses SREBP activation under sterol depletion, is identical to WT when complexed with Insig-2, although the gain of function may also involve a later step in protein trafficking.

Taurine Decreases Cellular Cholesterol Level in HepG2 Cells Partly through Upregulating Calcineurin

Objective: Taurine exerts cholesterol-lowering effect through inducing CYP7A1 and promoting the biotransformation of cholesterol into bile acids in livers, but its molecular mechanism remains unclear. Taurine also suppresses the expression of MCIP1, a calcineurin inhibitory protein. Here we aimed to explore whether calcineurin involves in the cholesterol-lowering effect and upregulation of CYP7A1 by taurine. Methods: High cellular cholesterol conditions were obtained by incubating with 0.2mM cholesterol contained DMEM in HepG2 cells. FK506, a calcineurin inhibitor, was used to depress cellular calcineurin. CnAb-/- cells are the HepG2 cells of which calcineurin was knocked down. Taurine was cultured in wild type, high-cholesterol conditions, calcineurin inhibition or deficiency HepG2 cells respectively for 24h or 48h. The levels of intracellular total cholesterol were determined by an enzymatic method and the expressions of CYP7A1, calcineurin, MEK1/2, c-Jun/p-c-Jun and SHP-1 were detected by western blotting. Results: High cellular cholesterol conditions in HepG2 cells were established and resulted in increased CYP7A1 and calcineurin expression. Taurine exhibited the decreasing-cholesterol effects on HepG2 cells regardless of whether cells with high cholesterol conditions or inhibited / deleted intracellular calcineurin. However, the extent of decreasing cholesterol after calcineurin repression or deficiency was much less than that of controls. Taurine could induce the expression of CYP7A1 but this induction was abolished when the cellular calcineurin was inhibited or deleted. Taurine was able to suppress MEK1/2, p-c-Jun and SHP-1, which are several key molecules in one inhibitory pathway of CYP7A1 transcription, whereas this suppression on MEK1/2 but not p-c-Jun or SHP-1 was reversed after completely knocking down calcineurin. Conclusions: Calcineurin was found to be required partly in taurine-decreasing cholesterol effect through inhibiting MEK1/2 which resulted in CYP7A1 upregulation.

Modulation of Tissue Factor-Factor VIIa Coagulant and Signaling Functions by Membrane Cholesterol and Caveolae.

Cholesterol in the membrane not only regulates flexibility and mechanical stability of the membrane but also plays a critical role in differentiating and maintaining cell surface microdomains of differing lipid composition, particularly sphingolipid rafts. Cholesterol- and sphingolipid-rich rafts in association with a structural protein, caveolin, form caveolae, flask-shaped invaginations in the plasma membrane. Lipid rafts and caveolae are shown to regulate various cellular functions, including receptor function, endocytosis, intracellular trafficking of receptors and signaling pathways. In the present study, we investigated the role of membrane cholesterol and caveolae in modulating coagulant and signaling functions of tissue factor (TF)-factor VIIa (VIIa) complexes on tumor cells. Breast carcinoma cells, MDA-MB-231, were treated with ß-methyl cyclodextrin (CD), which depletes membrane cholesterol and thereby disrupts caveolae, or with filipin, which disrupts caveolae without depleting the membrane cholesterol. TF-VIIa coagulant function was measured in factor X activation assay and the signaling function was evaluated in IP3 hydrolysis and IL-8 gene induction. As expected, CD (10 mM) treatment of tumor cells depleted cellular cholesterol level by more than 70% whereas filipin (5 μg/ml) treatment did not reduce the cellular cholesterol level. Both the treatments had no effect on the cell viability as measured in trypan blue exclusion method and MTT assay. CD treatment, in a dose-pendent manner (1 to 10 mM CD), impaired both TF-VIIa coagulant function and the signaling function (both IP3 hydrolysis and IL-8 gene expression). In contrast, varying effects were observed with filipin treatment. Filipin treatment (5 μg/ml) increased TF-VIIa coagulant function, reduced the TF-VIIa-induced IP3 hydrolysis and no effect on TF-VIIa-induced IL-8 gene expression. Detergent (Triton X-100) extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF was distributed both in lipid rafts and soluble fractions. CD and filipin treatments slightly reduced TF association with lipid rafts. Overall these data suggest that the membrane cholesterol modulates TF-VIIa proteolytic function and hence the TF-VIIa protease-induced signaling. In contrast, disruption of caveolae uncouples selective signaling pathways activated by TF-VIIa without impairing TF-VIIa protease activity.