Overexpression Of Sterol Regulatory Element-binding Protein-1 Research Articles

Sodium acetate promotes fat synthesis by suppressing TATA element modulatory factor 1 in bovine mammary epithelial cells

Short-chain fatty acids are important nutrients that regulate milk fat synthesis. They regulate milk synthesis via the sterol regulatory element binding protein 1 (SREBP1) pathway; however, the details are still unknown. Here, the regulation and mechanism of sodium acetate (SA) in milk fat synthesis in bovine mammary epithelial cells (BMECs) were assessed. BMECs were treated with SA supplementation (SA+) or without SA supplementation (SA-), and milk fat synthesis and activation of the SREBP1 pathway were increased (P=0.0045; P=0.0042) by SA+ and decreased (P=0.0068; P=0.0031) by SA-, respectively. Overexpression or inhibition of SREBP1 demonstrated that SA promoted milk fat synthesis (P=0.0045) via the SREBP1 pathway. Overexpression or inhibition of TATA element modulatory factor 1 (TMF1) demonstrated that TMF1 suppressed activation of the SREBP1 pathway (P=0.0001) and milk fat synthesis (P=0.0022) activated by SA+. Overexpression or inhibition of TMF1 and SREBP1 showed that TMF1 suppressed milk fat synthesis (P=0.0073) through the SREBP1 pathway. Coimmunoprecipitation analysis revealed that TMF1 interacted with SREBP1 in the cytoplasm and suppressed the nuclear localization of SREBP1 (P=0.0066). The absence or presence of SA demonstrated that SA inhibited the expression of TMF1 (P=0.0002) and the interaction between TMF1 and SREBP1 (P=0.0001). Collectively, our research suggested that TMF1 was a new negative regulator of milk fat synthesis. In BMECs, SA promoted the SREBP1 pathway and milk fat synthesis by suppressing TMF1. This study enhances the current understanding of the regulation of milk fat synthesis and provides new scientific data for the regulation of milk fat synthesis.

Sterol regulatory element binding transcription factor 1 promotes proliferation and migration in head and neck squamous cell carcinoma.

Sterol-regulatory element-binding protein 1 (SREBP1) is a transcription factor involved in lipid metabolism that is encoded by sterol regulatory element binding transcription factor 1(SREBF1). SREBP1 overexpression is associated with the progression of several human tumors; however, the role of SREBP1 in head and neck squamous cell carcinoma (HNSC) remains unclear. SREBF1 expression in pan-cancer was analyzed using the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data, and the association between SREBF1 expression and clinical characteristics of HNSC patients was examined using the UALCAN database. Enrichment analysis of SREBF1-related genes was performed using the Cluster Profiler R package. TCGA database was used to investigate the relationship between immune cell infiltration and SREBF1 expression. CCK-8, flow cytometry, and wound healing assays were performed to investigate the effect of SREBF1 knockdown on the proliferation and migration of HNSC cells. SREBF1 was significantly upregulated in several tumor tissues, including HNSC, and SREBF1 overexpression was positively correlated with sample type, cancer stage, tumor grade, and lymph node stage in HNSC patients. Gene enrichment analysis revealed that SREBF1 is associated with DNA replication and homologous recombination. SREBF1 upregulation was positively correlated with the infiltration of cytotoxic cells, B cells, T cells, T helper cells, and NK CD56 bright cells in HNSC. Knockdown of SREBF1 inhibited the proliferation and migration of HNSC cells (Hep2 and TU212) and induced apoptosis by downregulating the expression of steroidogenic acute regulatory protein-related lipid transfer 4 (STARD4). SREBF1 may promote HNSC proliferation, migration and inhibit apoptosis by upregulating STARD4 and affecting the level of immune cell infiltration.

Goat FADS2 controlling fatty acid metabolism is directly regulated by SREBP1 in mammary epithelial cells.

Goat milk provides benefits to human health due to its richness in bioactive components, such as polyunsaturated fatty acids (PUFAs). The fatty acid desaturase 2 (FADS2) is the first rate-limiting enzyme in PUFAs biosynthesis. However, its role and transcriptional regulation mechanisms in fatty acid metabolism in dairy goat remain unclear. Here, our study revealed that the FADS2 gene was highly expressed during the peak lactation compared with the dry period, early lactation, and late lactation. The content of triacylglycerol (TAG) was enhanced with the increasing mRNA expression of TAG synthesis genes (diacylglycerol acyltransferase 1/2, DGAT1/2) in FADS2-overexpressed goat mammary epithelial cells (GMECs). Overexpression of FADS2 was positively correlated with the elevated concentrations of dihomo-gamma-linolenic acid (DGLA) and docosahexaenoic acid (DHA) in GMECs. BODIPY staining showed that FADS2 promoted lipid droplet accumulation in GMECs. To clarify the transcriptional regulatory mechanisms of FADS2, 2,226 bp length of FADS2 promoter was obtained. Deletion mutation assays revealed that the core region of FADS2 promoter was located between the -375 and -26 region, which contained SRE1 (-361/-351) and SRE2 (-191/-181) cis-acting elements of transcription factor sterol regulatory element-binding protein 1 (SREBP1). Overexpression of SREBP1 enhanced relative luciferase activity of the single mutant of SRE1 or SRE2, vice versa, and failed to alter the relative luciferase activity of the joint mutant of SRE1 and SRE2. Chromatin immunoprecipitation (ChIP) and site-directed mutation assays further demonstrated that SREBP1 regulated the transcription of the FADS2 gene by binding to SRE sites in vivo and in vitro. In addition, the mRNA levels of FADS2 were significantly decreased by targeting SRE1 and SRE2 sites in the genome via the CRISPR interference (CRISPRi) system. These findings establish a direct role for FADS2 regulating TAG and fatty acid synthesis by SREBP1 transcriptional regulation in dairy goat, providing new insights into fatty acid metabolism in mammary gland of ruminants.

Oncolytic avian reovirus σA‐modulated fatty acid metabolism through the PSMB6/Akt/SREBP1/acetyl-CoA carboxylase pathway to increase energy production for virus replication

We have demonstrated previously that the σA protein of avian reovirus (ARV) functions as an activator of cellular energy, which upregulates glycolysis and the TCA cycle for virus replication. To date, there is no report with respect to σA-modulated regulation of cellular fatty acid metabolism. This study reveals that the σA protein of ARV inhibits fatty acids synthesis and enhance fatty acid oxidation by upregulating PSMB6, which suppresses Akt, sterol regulatory element-binding protein 1 (SREBP1), acetyl-coA carboxylase α (ACC1), and acetyl-coA carboxylase β (ACC2). SREBP1 is a transcription factor involved in fatty acid and cholesterol biosynthesis. Overexpression of SREBP1 reversed σA-modulated suppression of ACC1 and ACC2. In this work, a fluorescence resonance energy transfer‐based genetically encoded indicator, Ateams, was used to study σA-modulated inhibition of fatty acids synthesis which enhances cellular ATP levels in Vero cells and human cancer cell lines (A549 and HeLa). By using Ateams, we demonstrated that σA-modulated inhibition of Akt, SREBP1, ACC1, and ACC2 leads to increased levels of ATP in mammalian and human cancer cells. Furthermore, knockdown of PSMB6 or overexpression of SREBP1 reversed σA-modulated increased levels of ATP in cells, indicating that PSMB6 and SREBP1 play important roles in ARV σA-modulated cellular fatty acid metabolism. Furthermore, we found that σA R155/273A mutant protein loses its ability to enter the nucleolus, which impairs its ability to regulate fatty acid metabolism and does not increase ATP formation, suggesting that nucleolus entry of σA is critical for regulating cellular fatty acid metabolism to generate more energy for virus replication. Collectively, this study provides novel insights into σA-modulated inhibition of fatty acid synthesis and enhancement of fatty acid oxidation to produce more energy for virus replication through the PSMB6/Akt/SREBP1/ACC pathway.

Leptin Silencing Attenuates Lipid Accumulation through Sterol Regulatory Element-Binding Protein 1 Inhibition in Nasopharyngeal Carcinoma.

Leptin is a crucial regulator of metabolism and energy homeostasis in mammals. Many studies have investigated the impacts of leptin on human cancers, such as proliferation and metastasis. However, the mechanisms underlying leptin-mediated regulation of lipid metabolism in nasopharyngeal carcinoma (NPC) remain incompletely understood. In the current study, leptin downregulation ameliorated lipid accumulation, triglyceride, and cholesterol levels. Mechanistically, diminished leptin by siRNA not only inhibited sterol regulatory element-binding protein 1 (SREBP1), a master regulator of lipid metabolism, at the mRNA and protein levels, but also reduced SREBP1 downstream target expressions, such as fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1), in NPC cells. In addition, leptin expression could modulate the promoter activity of SREBP1. We also found that pharmacological inhibition of poly-ADP ribose polymerase-γ (PPAR-γ) resulted in increased SREBP1 expression in leptin-depleted NPC cells. Functionally, SREBP1 overexpression overcame the effects of leptin-silencing attenuated triglyceride level, cholesterol level and cell survival in NPC cells. Taken together, our results demonstrate that leptin is an important regulator of lipid metabolism in NPC cells and might could be a potential therapeutic target for treatment of NPC patients.

Regulation of Expression of Sterol Regulatory Element-binding Protein 1 in Thyroid Cancer Cells.

Expression of sterol regulatory element-binding protein 1 (SREBP1) is upregulated in thyroid cancer and associated with shorter disease-specific survival. The molecular regulatory mechanisms governing SREBP1 over-expression in thyroid cancer are still unclear. Thyroid cancer cell lines BHT-101 (with the BRAF V600E mutation) and FTC-131 (wild-type for BRAF) were treated with specific inhibitors. The expression of SREBP1 was determined at the mRNA level using quantitative real-time PCR and at the protein level using immunoblotting. Lenvatinib and a MEK inhibitor, selumetinib, suppressed SREBP1 expression in BHT-101 but not FTC-133 cells. Olitigaltin, a galectin-3 inhibitor, decreased SREBP1 expression in a time- and dose-dependent manner in both cells. MK2206, an allosteric AKT inhibitor, did not change SREBP1 expression in either cell line. The galectin-3 inhibitor attenuates SREBP1 expression in thyroid cancer cells, likely independent of AKT phosphorylation. Lenvatinib and selumetinib decreases SREBP1 expression in the BRAF-mutant cell line BHT-101.

SREBP1 promotes invasive phenotypes by upregulating CYR61/CTGF via the Hippo-YAP pathway.

Aberrant lipid metabolism provides bioenergetic, biosynthetic, and redox supplies to cancer cells. Previous studies have reported differential lipid profiling in thyroid malignancies. Sterol regulatory element-binding protein 1 (SREBP1), encoded by the SREBF1 gene, is a master regulator of cellular lipid homeostasis. The clinical and functional significance of SREBP1 in thyroid cancer is not well understood. Here, we showed that SREBP1 expression is significantly upregulated in invasive thyroid cancer than in normal thyroid tissue or benign thyroid nodules. High tumoral SREBP1 expression was associated with extrathyroidal extension, advanced disease stage, and shorter disease-specific survival in patients with differentiated thyroid cancer. SREBP1 overexpression significantly increased the oxygen consumption rate, filopodia formation, and migratory and invasive capacities of thyroid cancer cells. Knockdown of SREBF1 or treatment with an SREBP1 activation inhibitor fatostatin had the opposite effect. RNA-Seq analysis showed that modulation of SREBP1 expression was accompanied by corresponding changes in the expression of epithelial-mesenchymal transition markers and CYR61/CTGF. SREBP1-facilitated cell invasion could be abrogated by treatment with a YAP inhibitor such as verteporfin or genetic silencing of CYR61 or CTGF. In summary, SREBP1 upregulation can be used as a prognostic indicator for thyroid cancer and SREBP1 overexpression is involved in cancer invasiveness, at least partly, through upregulation of CYR61/CTGF via the Hippo-YAP pathway.

SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

Renal cell carcinoma (RCC) cells have increased lipogenesis and cholesterol synthesis. Sterol regulatory element-binding protein-1 (SREBP1) is cleaved by site 1 protease (S1P) to release the transcriptionally active amino-terminal domain. PF-429242 is a potent and competitive S1P inhibitor. We here tested its activity in RCC cells. In established and primary human RCC cells, PF-429242 potently inhibited cell proliferation, migration, and invasion. The S1P inhibitor provoked apoptosis activation in RCC cells. Furthermore, shRNA-mediated S1P silencing or CRISPR/Cas9-induced S1P knockout led to RCC cell growth inhibition and apoptosis activation. Conversely, ectopic overexpression of SREBP1 or S1P augmented RCC cell proliferation and migration. Daily i.v. injection of a single dose of PF-429242 robustly inhibited RCC xenograft growth in severe combined immunodeficiency mice. Additionally, intratumoral injection of S1P shRNA lentivirus inhibited RCC xenograft growth in mice. SREBP1, S1P, and its target gene low density lipoprotein receptor (LDLR) were significantly elevated in human RCC tissues. These results suggest that targeting S1P by PF-429242 inhibited RCC cell growth in vitro and in vivo.

ACSL4 reprograms fatty acid metabolism in hepatocellular carcinoma via c-Myc/SREBP1 pathway.

Lipid metabolic reprogramming plays a pivotal role in hepatocellular carcinoma (HCC) development, but the underlying mechanisms are incompletely characterized. Long chain acyl CoA synthetase 4 (ACSL4), a member of acyl-CoA synthetases (ACS) family, has been identified as a novel marker of alpha-fetoprotein-high subtype HCC and as an oncogene. Here, we identified a new function of ACSL4 in HCC lipid metabolism. ACSL4 can modulate de novo lipogenesis by accumulating intracellular triglycerides, cholesterols, and lipid droplets in HCC. Mechanistically, ACSL4 upregulates the master lipogenesis regulator sterol regulatory element binding protein 1 (SREBP1) and its downstream lipogenic enzymes in HCC cells via c-Myc. Moreover, SREBP1 is crucial for ACSL4-mediated regulation of lipogenesis as well as HCC cell proliferation and metastasis, as SREBP1 overexpression rescues lipogenic deficiency and decreased oncogenic capabilities associated with ACSL4 suppression in vitro and in vivo. Clinically, our data showed that the expression of ACSL4 was positively correlated with that of SREBP1 in HCC patients, and the combinational biomarkers showed strong predictive value for HCC. Together, our findings uncover a new mechanism by which ACSL4 modulates aberrant lipid metabolism and promotes the progression of HCC.

Novel mechanisms underlying anti-polycystic ovary like syndrome effects of electroacupuncture in rats: suppressing SREBP1 to mitigate insulin resistance, mitochondrial dysfunction and oxidative stress

BackgroundAcupuncture, a therapy of traditional Chinese medicine, is confirmed to exert the therapeutic action on polycystic ovary syndrome (PCOS). However, the detailed therapeutic mechanisms of acupuncture in PCOS remain ambiguous. In this study, we further investigated whether electroacupuncture (EA) alleviated PCOS-like symptoms in rats via regulating a metabolic regulator, sterol regulatory element binding protein-1 (SREBP1).MethodsThe PCOS-like rat model was built by hypodermic injection with dehydroepiandrosterone (DHEA). The rats were subjected to EA intervention (ST29 and SP6 acupuncture points) for 5 weeks. Primary granulosa cells were isolated from control and PCOS-like rats for evaluating insulin resistance, mitochondrial dysfunction and oxidative stress in vitro.ResultsThe expression of SREBP1 was increased in PCOS-like rats, which was suppressed by EA treatment. In addition, lentivirus-mediated overexpression of SREBP1 restrained EA treatment-induced improvement in pathological changes, serum hormone levels and insulin resistance in rats. In addition, overexpression of SREBP1 repressed insulin-stimulated phosphorylation of insulin receptor β (IR) and AKT in primary granulosa cells. Moreover, upregulation of SREBP1 further exacerbated mitochondrial dysfunction and oxidative stress in granulosa cells isolated from PCOS-like rats. Mechanically, EA treatment suppressed SREBP1 expression through inducing the activation of AMP-activated protein kinase (AMPK) signaling pathway in PCOS-like rats.ConclusionEA intervention alleviated PCOS-like symptoms in rats via improving IR, mitochondrial dysfunction and oxidative stress through regulating SREBP1, a lipid metabolism regulator. Our findings illuminate the novel protective mechanisms of EA in the treatment of PCOS.

SREBP1 silencing inhibits the proliferation and motility of human esophageal squamous carcinoma cells via the Wnt/β-catenin signaling pathway.

Sterol regulatory element-binding protein 1 (SREBP1) is dysregulated in a variety of types of human cancer. However, the functional roles of SREBP1 in esophageal squamous cell carcinoma (ESCC) remain poorly understood. The present study investigated the function of SREBP1 in cell proliferation and motility. Microarray datasets in Oncomine, reverse transcription-quantitative PCR and western blot analysis revealed that SREBP1 was overexpressed in ESCC tumors when compared with normal tissues. In addition, SREBP1 overexpression was significantly associated with tumor differentiation, lymphatic metastasis and Ki67 expression. Results suggested that silencing SREBP1 inhibited the proliferation, migration and invasion of ESCC cells, whereas overexpression of SREBP1 had opposite effects on proliferation and metastasis. In addition, loss of SREBP1 significantly increased E-cadherin and decreased N-cadherin, Vimentin, Snail, matrix metalloproteinase 9 and vascular endothelial growth factor C expression levels, which were restored via SREBP1-overexpression. Mechanistically, loss of SREBP1 suppressed T-cell factor 1/lymphoid enhancer factor 1 (TCF1/LEF1) activity and downregulated TCF1/LEF1 target proteins, including CD44 and cyclin D1. Moreover, knockdown of SREBP1 downregulated the expression levels of stearoyl-CoA desaturase 1 (SCD1), phosphorylated glycogen synthase kinase-3β and nuclear β-catenin. Furthermore, the inhibitors of SREBP1 and/or SCD1 and small interfering RNA-SCD1 efficiently inhibited the activation of the Wnt/β-catenin pathway driven by constitutively active SREBP1. Finally, in vivo results indicated that SREBP1-knockdown suppressed the proliferation and metastasis of ESCC. Taken together, these findings demonstrated that SREBP1 exerts oncogenic effects in ESCC by promoting proliferation and inducing epithelial-mesenchymal transition via the SCD1-induced activation of the Wnt/β-catenin signaling pathway.

MTOR Signaling and SREBP Activity Increase FADS2 Expression and Can Activate Sapienate Biosynthesis

SummaryCancer cells display an increased plasticity in their lipid metabolism, which includes the conversion of palmitate to sapienate via the enzyme fatty acid desaturase 2 (FADS2). We find that FADS2 expression correlates with mammalian target of rapamycin (mTOR) signaling and sterol regulatory element-binding protein 1 (SREBP-1) activity across multiple cancer types and is prognostic in some cancer types. Accordingly, activating mTOR signaling by deleting tuberous sclerosis complex 2 (Tsc2) or overexpression of SREBP-1/2 is sufficient to increase FADS2 mRNA expression and sapienate metabolism in mouse embryonic fibroblasts (MEFs) and U87 glioblastoma cells, respectively. Conversely, inhibiting mTOR signaling decreases FADS2 expression and sapienate biosynthesis in MEFs with Tsc2 deletion, HUH7 hepatocellular carcinoma cells, and orthotopic HUH7 liver xenografts. In conclusion, we show that mTOR signaling and SREBP activity are sufficient to activate sapienate metabolism by increasing FADS2 expression. Consequently, targeting mTOR signaling can reduce sapienate metabolism in vivo.

Leelamine Is a Novel Lipogenesis Inhibitor in Prostate Cancer Cells In Vitro and In Vivo.

Increased de novo synthesis of fatty acids is implicated in the pathogenesis of human prostate cancer, but a safe and effective clinical inhibitor of this metabolic pathway is still lacking. We have shown previously that leelamine (LLM) suppresses transcriptional activity of androgen receptor, which is known to regulate fatty acid synthesis. Therefore, the current study was designed to investigate the effect of LLM on fatty acid synthesis. Exposure of 22Rv1, LNCaP, and PC-3 prostate cancer cells, but not RWPE-1 normal prostate epithelial cell line, to LLM resulted in a decrease in intracellular levels of neutral lipids or total free fatty acids. LLM was superior to another fatty acid synthesis inhibitor (cerulenin) for suppression of total free fatty acid levels. LLM treatment downregulated protein and/or mRNA expression of key fatty acid synthesis enzymes, including ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase, and sterol regulatory element-binding protein 1 (SREBP1) in each cell line. Consistent with these in vitro findings, we also observed a significant decrease in ATP citrate lyase and SREBP1 protein expression as well as number of neutral lipid droplets in vivo in 22Rv1 tumor sections of LLM-treated mice when compared with that of controls. LLM-mediated suppression of intracellular levels of total free fatty acids and neutral lipids was partly attenuated by overexpression of SREBP1. In conclusion, these results indicate that LLM is a novel inhibitor of SREBP1-regulated fatty acid/lipid synthesis in prostate cancer cells that is not affected by androgen receptor status.

Abstract 5081: Leelamine is a novel inhibitor of fatty acid synthesis in prostate cancer

Abstract Increased beta-oxidation of fatty acids to meet energy demand is a rather unique characteristic of a subset of human prostate cancers. A safe and effective intervention for inhibition of fatty acid synthesis is still a clinically unmet need. We have shown previously that leelamine (LLM), a phytochemical derived from pine tree bark, suppresses expression and activity of full-length androgen receptor (AR) and its splice variants in vitro and in vivo in preclinical models of prostate cancer. Because AR is implicated in regulation of fatty acid metabolism, the present study was undertaken to determine the effect of LLM on this metabolic pathway. Treatment of a castration-resistant (22Rv1) as well as an androgen-responsive (LNCaP) human prostate cancer cell line with LLM (2.5 and 5 µmol/L) resulted in downregulation of key fatty acid synthesis enzyme proteins including ATP citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBP1). LLM treatment also decreased intracellular levels of total free fatty acids and neutral lipid droplets in LNCaP and 22Rv1 cells. Consistent with the in vitro results, we also observed a significant decrease in ACLY and SREBP1 protein expression and number of neutral lipid droplets in vivo in tumor tissue sections of 22Rv1 xenografts after intraperitoneal administration of LLM (9.1 mg/kg bw/ day, 5 times/week) compared to controls. Studies are in progress to determine if overexpression of AR and/or SREBP1 confers protection against fatty acid synthesis inhibition by LLM. In conclusion, it is reasonable to postulate that suppression of AR-SREBP1 regulated fatty acid metabolism is an important mechanism in prostate cancer inhibition by LLM. This study was supported by the grant RO1 CA101753 awarded by the National Cancer Institute. Citation Format: Krishna B. Singh, Shivendra V. Singh. Leelamine is a novel inhibitor of fatty acid synthesis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5081.

Interplay of PKD3 with SREBP1 Promotes Cell Growth via Upregulating Lipogenesis in Prostate Cancer Cells.

Protein kinase D (PKD) has been implicated in cancer cell survival, proliferation, migration and angiogenesis. However, it is still unknown whether PKD regulates cell proliferation through lipid metabolism in cancer cells. Here we report a novel function of PKD3, a member of PKD family, in regulating of prostate cancer cell proliferation by modulation of SREBP1-mediated de novo lipogenesis. We show that silencing of PKD3 significantly reduces lipid content and expression of the lipogenic genes encoding FASN and ATP-citrate lyase (ACLY). Moreover, endogenous PKD3 interacts with sterol regulatory element binding protein 1(SREBP1) in DU145 cells. Interestingly, PKD3 silencing decreases not only the level of matured-SREBP1 (68KD) but also the binding of SREBP1 to the promoter of fasn gene. In addition, overexpression of SREBP1 reverses the suppression of cell growth caused by PKD3 depletion. Finally, immune-histochemical staining indicate that PKD3 expression is positively correlated with expression of FASN and SREBP1 in prostate cancers. Taken together, these data suggest that targeting PKD3-mediated de novo lipogenesis may be a potential therapeutic approach to block prostate cancer progression.

Overexpression of SREBF chaperone (SCAP) enhances nuclear SREBP1 translocation to upregulate fatty acid synthase (FASN) gene expression in bovine mammary epithelial cells

Fatty acid synthase is a key enzyme for the synthesis of milk fat in the ruminant mammary gland. In nonruminants, sterol regulatory element binding protein 1 (SREBP1) is a regulator of FASN gene expression, and SREBF chaperone (SCAP) is essential for SREBP1 maturation and activity. However, the role of SCAP on the regulation of FASN gene expression in ruminants is unknown. The objective of this study was to investigate the transcriptional regulation of FASN by overexpressing SCAP in bovine mammary epithelial cells. A bovine SCAP expression vector, SREBP1 expression vector, and the promoter of FASN were cloned. The transcription factor binding sites of FASN promoter were predicted using bioinformatics analysis. After transfection with FASN promoter vectors in the immortalized bovine mammary epithelial cell line MAC-T, we co-overexpressed the SCAP + SREBP1 expression vector with pcDNA3.1 vector as control. The effect of SCAP + SREBP1 overexpression on the regulation of FASN was investigated using luciferase assay, immunofluorescence, Western blot, real-time PCR, and lipid droplet staining. We observed that co-overexpression of SCAP + SREBP1 significantly increased activity of the FASN promoter containing a sterol response element binding site. The FASN mRNA abundance and lipid droplet formation increased due to co-overexpression of SCAP + SREBP1. Compared with overexpression of SREBP1 alone, co-overexpression of SCAP + SREBP1 enhanced the nuclear translocation and nuclear SREBP1 protein abundance. Overall, as in nonruminants cells, results indicate that SCAP is essential for promoting nuclear translocation of SREBP1 and activation of FASN gene transcription, leading to lipid droplet formation in bovine mammary epithelial cells.

Nutrient sensor O-GlcNAc transferase controls cancer lipid metabolism via SREBP-1 regulation.

Elevated O-GlcNAcylation is associated with disease states such as diabetes and cancer. O-GlcNAc transferase (OGT) is elevated in multiple cancers and inhibition of this enzyme genetically or pharmacologically inhibits oncogenesis. Here we show that O-GlcNAcylation modulates lipid metabolism in cancer cells. OGT regulates expression of the master lipid regulator the transcription factor sterol regulatory element binding protein 1 (SREBP-1) and its transcriptional targets both in cancer and lipogenic tissue. OGT regulates SREBP-1 protein expression via AMP Activated protein kinase (AMPK). SREBP-1 is critical for OGT-mediated regulation of cell survival and of lipid synthesis, as overexpression of SREBP-1 rescues lipogenic defects associated with OGT suppression, and tumor growth in vitro and in vivo. These results unravel a previously unidentified link between O-GlcNAcylation, lipid metabolism and the regulation of SREBP-1 in cancer and suggests a crucial role for O-GlcNAc signaling in transducing nutritional state to regulate lipid metabolism.

Overexpression of SREBP1 (sterol regulatory element binding protein 1) promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells

Sterol regulatory element binding protein 1 (SREBP1; gene name SREBF1) is known to be the master regulator of lipid homeostasis in mammals, including milk fat synthesis. The major role of SREBP1 in controlling milk fat synthesis has been demonstrated in bovine mammary epithelial cells. Except for a demonstrated role in controlling the expression of FASN, a regulatory role of SREBP1 on milk fat synthesis is very likely, but has not yet been demonstrated in goat mammary epithelial cells (GMEC). To explore the regulatory function of SREBP1 on de novo fatty acids and triacylglycerol synthesis in GMEC, we overexpressed the mature form of SREBP1 (active NH2-terminal fragment) in GMEC using a recombinant adenovirus vector (Ad-nSREBP1), with Ad-GFP (recombinant adenovirus of green fluorescent protein) as control, and infected the GMEC for 48 h. In infected cells, we assessed the expression of 20 genes related to milk fat synthesis using real time-quantitative PCR, the protein abundance of SREBP1 and FASN by Western blot, the production of triacylglycerol, and the fatty acid profile. Expression of SREBF1 was modest in mammary compared with the other tissues in dairy goats but its expression increased approximately 30-fold from pregnancy to lactation. The overexpression of the mature form of SREBP1 was confirmed by >200-fold higher expression of SREBF1 in Ad-nSREBP1 compared with Ad-GFP. We observed no changes in amount of the precursor form of SREBP1 protein but a >10-fold increase of the mature form of SREBP1 protein with Ad-nSREBP1. Compared with Ad-GFP cells (control), Ad-nSREBP1 cells had a significant increase in expression of genes related to long-chain fatty acid activation (ACSL1), transport (FABP3), desaturation (SCD1), de novo synthesis of fatty acids (ACSS2, ACLY, IDH1, ACACA, FASN, and ELOVL6), and transcriptional factors (NR1H3 and PPARG). We observed a >10-fold increase in expression of INSIG1 but SCAP was downregulated by Ad-nSREBP1. Among genes related to milk fat synthesis and lipid droplet formation, only LPIN1 and DGAT1 were upregulated by Ad-nSREBP1. Compared with the Ad-GFP, the cellular triacylglycerol content was higher and the percentage of C16:0 and C18:1 increased, whereas that of C16:1, C18:0, and C18:2 decreased in Ad-nSREBP1 cells. Overall, the data provide strong support for a central role of SREBP1 in the regulation of milk fat synthesis in goat mammary cells.

Sfrp2 is a transcriptional target of SREBP-1 in mouse chondrogenic cells

Secreted frizzled-related protein 2 (Sfrp2) is highly expressed in developing limbs and is associated with skeletal malformation such as Brachydactyly and Syndactyly. However, the mechanism by which Sfrp2 gene was transcriptionally regulated in chondrogenic cells is largely unknown. Here, we found that sterol regulatory element binding protein-1 (SREBP-1) regulated transcriptional activation of the Sfrp2 gene in chondrogenic cells. Overexpression of SREBP-1 led to stimulation of Sfrp2 promoter activity and increase of Sfrp2 mRNA in chondrogenic ATDC5 cells. Reporter gene assays using deleted Sfrp2 promoter constructs showed that the position between -1150 and -840 is required for SREBP-1-mediated Sfrp2 promoter activity. Mutagenesis analysis showed that the SREBP-1-binding site at nucleotide -935 to -926 is a functional motif for Sfrp2 transcription. Promoter enzyme immunoassay and chromatin immunoprecipitation assay demonstrated that SREBP-1 binds directly to the SREBP-1-binding motif in the mouse Sfrp2 promoter. Thus, these results demonstrated that SREBP-1 acts as a positive regulator of Sfrp2 transcription in chondrogenic cells.

Fatty acid synthase promoter: Characterization, and transcriptional regulation by sterol regulatory element binding protein-1 in goat mammary epithelial cells

Fatty acid synthase (FASN) is the central enzyme of the de novo fatty acid biosynthesis pathway. Although the FASN transcriptional regulatory mechanism has been elucidated clearly in many tumor cells, its mechanism is still not clear in the ruminant mammary gland. In this study, we cloned and sequenced a 1.8-kb fragment of the FASN 5′ flanking region from goat genomic DNA. Multiple alignment analysis demonstrated that the entire 1.8-kb fragment has little homology but that the sub-section nearest the transcriptional start site (−203 to +1) is more conserved across species, in particular the binding motifs for transcriptional regulation. Deletion analysis revealed a putative core promoter region located in −297/−14bp upstream of the transcription site within the high homology domain. Mutations of sterol response elements (SRE1 and SRE2) and the nuclear factor Y (NF-Y) binding site appeared to significantly down-regulate the FASN promoter activity in goat mammary epithelial cells (P<0.05). Further analysis showed that both SRE sites responded to sterol regulatory element-binding protein 1 (SREBP-1). SREBP-1 overexpression and knockdown by small interference RNA influenced the abundance of endogenous FASN. These data suggested that SREBP-1 may regulate FASN expression at the transcriptional level in the lactating goat mammary gland. Hence, the current work will contribute valuable information to understanding the molecular regulatory mechanisms of FASN during lactation.