Fatostatin reverses progesterone resistance by inhibiting the SREBP1-NF-κB pathway in endometrial carcinoma
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Abstract Progesterone resistance can significantly restrict the efficacy of conservative treatment for patients with endometrial cancer who wish to preserve their fertility or those who suffer from advanced and recurrent cancer. SREBP1 is known to be involved in the occurrence and progression of endometrial cancer, although the precise mechanism involved remains unclear. In the present study, we carried out microarray analysis in progesterone-sensitive and progesterone-resistant cell lines and demonstrated that SREBP1 is related to progesterone resistance. Furthermore, we verified that SREBP1 is over-expressed in both drug-resistant tissues and cells. Functional studies further demonstrated that the inhibition of SREBP1 restored the sensitivity of endometrial cancer to progesterone both in vitro and in vivo, and that the over-expression of SREBP1 promoted resistance to progesterone. With regards to the mechanism involved, we found that SREBP1 promoted the proliferation of endometrial cancer cells and inhibited their apoptosis by activating the NF-κB pathway. To solve the problem of clinical application, we found that Fatostatin, an inhibitor of SREBP1, could increase the sensitivity of endometrial cancer to progesterone and reverse progesterone resistance by inhibiting SREBP1 both in vitro and in vivo. Our results highlight the important role of SREBP1 in progesterone resistance and suggest that the use of Fatostatin to target SREBP1 may represent a new method to solve progesterone resistance in patients with endometrial cancer.Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipogenesis. Release of membrane-bound SREBP requires SREBP cleavage-activating protein (SCAP) to escort SREBP from the endoplasmic reticulum (ER) to the Golgi for cleavage by site-1 and site-2 proteases. SCAP then recycles to the ER for additional rounds of SREBP binding and transport. Mechanisms regulating ER-to-Golgi transport of SCAP-SREBP are understood in molecular detail, but little is known about SCAP recycling. Here, we have demonstrated that SCAP Golgi-to-ER transport requires cleavage of SREBP at site-1. Reductions in SREBP cleavage lead to SCAP degradation in lysosomes, providing additional negative feedback control to the SREBP pathway. Current models suggest that SREBP plays a passive role prior to cleavage. However, we show that SREBP actively prevents premature recycling of SCAP-SREBP until initiation of SREBP cleavage. SREBP regulates SCAP in human cells and yeast, indicating that this is an ancient regulatory mechanism.
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SARS-CoV-2 induces major cellular lipid rearrangements, exploiting the host's metabolic pathways to replicate. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that control lipid metabolism. SREBP1 is associated with the regulation of fatty acids, whereas SREBP2 controls cholesterol metabolism, and both isoforms are associated with lipid droplet (LD) biogenesis. Here, we evaluated the effect of SREBP in a SARS-CoV-2-infected lung epithelial cell line (Calu-3). We showed that SARS-CoV-2 infection induced the activation of SREBP1 and SREBP2 and LD accumulation. Genetic knockdown of both SREBPs and pharmacological inhibition with the dual SREBP activation inhibitor fatostatin promote the inhibition of SARS-CoV-2 replication, cell death, and LD formation in Calu-3 cells. In addition, we demonstrated that SARS-CoV-2 induced inflammasome-dependent cell death by pyroptosis and release of IL-1β and IL-18, with activation of caspase-1, cleavage of gasdermin D1, was also reduced by SREBP inhibition. Collectively, our findings help to elucidate that SREBPs are crucial host factors required for viral replication and pathogenesis. These results indicate that SREBP is a host target for the development of antiviral strategies.
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皮膚血管は冷却刺激に応答して収縮し，体表面からの熱放散を制限する．この反応は，交感神経の興奮を介する全身性メカニズムと，局所的に皮膚血管の収縮反応性が増大する局所性メカニズムとが相乗的に機能して引き起こされる．局所性メカニズムの存在は，摘出血管を用いたin vitro実験によって証明されてきた．我々は，ラットやマウスを使ってin vivoで皮膚血流調節を解析する実験方法を確立した．皮膚循環は，様々な因子に起因する神経性の影響を強く受けるため，in vivoで皮膚血流を定量的に測定することは難しい．そこで，電位依存性Na+チャネル阻害薬であるテトロドトキシン（TTX）処置により神経伝導を遮断した条件下で皮膚血流を測定することを試みた．この条件下においても，ラットおよびマウスの後肢を局所冷却することにより，足底部の皮膚血流量が減少することを証明し，局所性メカニズムによる反応をin vivoで定量的に評価できることを示した．興味深いことに，マウスとラットでは，この反応の主要なメカニズムが異なっていた．すなわち，ラットでは，冷却刺激により血管あるいは周囲の細胞からATPが遊離され，これが交感神経終末のP2受容体に作用することでノルアドレナリン遊離を誘発し，平滑筋細胞の主にα1受容体の活性化を介して皮膚血管が収縮するのに対し，マウスでは，冷却刺激はRhoキナーゼの活性化を介して血管平滑筋のα2C受容体を介した収縮反応性を増大させることで皮膚血管を収縮させることが示唆された．In vivoでの解析は，レイノー病はもちろん，糖尿病やホルモン異常など末梢循環障害を来たす病態と皮膚循環調節との関係を解析する際に不可欠であり，この解析方法はこうした病態の治療薬や予防薬の開発にも役立つであろう．
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Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis. Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.
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Sterol regulation-defective (SRD) 4 cells expressing a mutant sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP D443N) and Chinese hamster ovary (CHO) cells stably expressing SCAP (CHO-SCAP) and SCAP D443N (CHO-SCAP-D443N) have increased cholesterol and fatty acid synthesis because of constitutive processing of SREBPs. We assessed whether constitutive activation of SREBPs also influenced the CDP-choline pathway for phosphatidylcholine (PtdCho) biosynthesis. Relative to control CHO 7 cells, SRD 4 cells displayed increased PtdCho synthesis and degradation as indicated by a 4–6-fold increase in [3H]choline incorporation into PtdCho and 10–15-fold increase in intracellular [3H]glycerophosphocholine. [3H]Phosphocholine levels in SRD 4 cells were reduced by over 10-fold, suggesting enhanced activity of CTP:phosphocholine cytidylyltransferase α (CCTα). CHO-SCAP and CHO-SCAP D443N cells displayed modest increases in [3H]choline incorporation into PtdCho (2-fold) and only a 2-fold reduction in [3H]phosphocholine. Elevated PtdCho metabolism in SRD 4, compared with SCAP-overexpressing cells, was correlated with fatty acid synthesis. Inhibition of fatty acid synthesis by cerulenin resulted in almost complete normalization of PtdCho synthesis and choline metabolite profiles in SRD 4 cells, indicating that fatty acids or a fatty acid-derived metabolite was responsible for up-regulation of PtdCho synthesis. In contrast to apparent activation in vivo, CCTα protein, mRNA, and in vitro activity were reduced in SRD 4 cells and unchanged in SCAP transfected cells. Unlike control and SCAP transfected cells, CCTα in SRD 4 cells was localized by immunofluorescence to the nuclear envelope, suggesting that residual enzyme activity in these cells was in an active membrane-associated form. Translocation of CCTα to the nuclear envelope was reproduced by treatment of CHO 7 cells with exogenous oleate. We conclude that the SREBP/SCAP pathway regulates PtdCho synthesis via post-transcriptional activation of nuclear CCTα by fatty acids or a fatty acid-derived signal. Sterol regulation-defective (SRD) 4 cells expressing a mutant sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP D443N) and Chinese hamster ovary (CHO) cells stably expressing SCAP (CHO-SCAP) and SCAP D443N (CHO-SCAP-D443N) have increased cholesterol and fatty acid synthesis because of constitutive processing of SREBPs. We assessed whether constitutive activation of SREBPs also influenced the CDP-choline pathway for phosphatidylcholine (PtdCho) biosynthesis. Relative to control CHO 7 cells, SRD 4 cells displayed increased PtdCho synthesis and degradation as indicated by a 4–6-fold increase in [3H]choline incorporation into PtdCho and 10–15-fold increase in intracellular [3H]glycerophosphocholine. [3H]Phosphocholine levels in SRD 4 cells were reduced by over 10-fold, suggesting enhanced activity of CTP:phosphocholine cytidylyltransferase α (CCTα). CHO-SCAP and CHO-SCAP D443N cells displayed modest increases in [3H]choline incorporation into PtdCho (2-fold) and only a 2-fold reduction in [3H]phosphocholine. Elevated PtdCho metabolism in SRD 4, compared with SCAP-overexpressing cells, was correlated with fatty acid synthesis. Inhibition of fatty acid synthesis by cerulenin resulted in almost complete normalization of PtdCho synthesis and choline metabolite profiles in SRD 4 cells, indicating that fatty acids or a fatty acid-derived metabolite was responsible for up-regulation of PtdCho synthesis. In contrast to apparent activation in vivo, CCTα protein, mRNA, and in vitro activity were reduced in SRD 4 cells and unchanged in SCAP transfected cells. Unlike control and SCAP transfected cells, CCTα in SRD 4 cells was localized by immunofluorescence to the nuclear envelope, suggesting that residual enzyme activity in these cells was in an active membrane-associated form. Translocation of CCTα to the nuclear envelope was reproduced by treatment of CHO 7 cells with exogenous oleate. We conclude that the SREBP/SCAP pathway regulates PtdCho synthesis via post-transcriptional activation of nuclear CCTα by fatty acids or a fatty acid-derived signal. phosphatidylcholine acyl-CoA:cholesterol acyltransferase Chinese hamster ovary cholinephosphotransferase CTP:phosphocholine cytidylyltransferase glycerophosphocholine sterol regulation-defective sterol regulatory element-binding protein SREBP cleavage-activating protein phosphate-buffered saline polyacrylamide gel electrophoresis calcium-independent phospholipase A2 Eukaryotic cell membranes are composed of a complex array of proteins, phospholipids, sphingolipids, and cholesterol. The relative proportions and fatty acyl composition of these components dictate the physical properties of membranes, such as fluidity, surface potential, microdomain structure, and permeability (1.Cullis P.R. Fenske D.B. Hope M.J. Vance D.E. Vance J.E. New Comprehensive Biochemistry: Biochemistry of Lipids, Lipoproteins and Membranes. 31. Elsevier Science Publishers B.V., Amsterdam1996: 1-33Google Scholar). This in turn regulates the localization and activity of membrane-associated proteins (2.Yeagle P.L. Biochimie (Paris). 1991; 73: 1303-1310Crossref PubMed Scopus (290) Google Scholar). Assembly of membranes necessitates the coordinate synthesis and catabolism of phospholipids, sterols, and sphingolipids to create the unique properties of a given cellular membrane. This must be an extremely complex process that requires coordination of multiple biosynthetic and degradative enzymes and lipid transport activities. There is evidence from different experimental models showing coordinate synthesis of sphingomyelin, phosphatidylcholine (PtdCho),1 and cholesterol (reviewed in Refs. 3.Kolesnick R.N. Prog. Lipid Res. 1991; 30: 138-150Crossref Scopus (256) Google Scholar and 4.Ridgway N.D. Byers D.M. Cook H.W. Storey M.K. Prog. Lipid Res. 2000; 38: 337-360Crossref Scopus (31) Google Scholar). In several cases the affected enzymes in each pathway have been identified, but the mediators and mechanism for co-regulation remain unknown. A potentially important pathway for coordinate regulation of membrane composition involves the sterol regulatory element-binding proteins (SREBPs); transcription factors with a dual role in controlling fatty acid and cholesterol biosynthesis (5.Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar, 6.Bennett M.K. Lopez J.M. Sanchez H.B. Osborne T.F. J. Biol. Chem. 1995; 270: 25578-25583Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 7.Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (534) Google Scholar). SREBPs are tethered to nuclear/endoplasmic reticulum membranes by two transmembrane segments and undergo two proteolytic reactions that release the soluble, nuclear-localized N-terminal transcription factor domain. The first proteolysis step is sterol-regulated, requires SREBP cleavage-activating protein (SCAP), and is catalyzed by a unique membrane-bound protease (5.Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). The second proteolytic step is constitutive and occurs at a site within the first transmembrane domain (5.Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). Results from transgenic and cell models demonstrated that SREBP-2 and -1a stimulated transcription of numerous cholesterol biosynthetic enzymes and the low density lipoprotein receptor, whereas SREBP-1c/ADD-1 (adipocyte determination anddifferentation factor-1) isoforms are exclusively involved in the transcription of several key genes in fatty acid biosynthesis and desaturation (8.Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar, 9.Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (678) Google Scholar). This specificity is not exclusive because both SREBP-1a and -2 appear to stimulate expression of both cholesterol and fatty acid biosynthetic genes. SREBPs also regulate expression of a series of genes involved in the production of NADPH and acetyl-CoA required for fatty acid biosynthesis (10.Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. J. Biol. Chem. 1998; 273: 35299-35306Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Recently, SREBP-1c/ADD-1 expression was demonstrated to be insulin-dependent (11.Shimomura I. Bashmakov Y. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (622) Google Scholar) and required for insulin regulation of gene expression (12.Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (590) Google Scholar, 13.Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3786Crossref PubMed Scopus (452) Google Scholar). In addition to regulation of cholesterol and fatty acid biosynthesis, SREBP/SCAP could potentially regulate the synthesis of phospholipids and sphingolipids by direct or indirect mechanisms. For example, SREBP 1 was shown to regulate the transcription of glycerol-3-phosphate acyltransferase in cultured cells (14.Ericsson J. Jackson S.M. Kim J.B. Spiegalman B.M. Edwards P.A. J. Biol. Chem. 1997; 272: 7298-7305Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) and transgenic mouse models (10.Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. J. Biol. Chem. 1998; 273: 35299-35306Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Glycerol-3-phosphate acyltransferase is the initial enzyme in the synthesis of phosphatidic acid and diglyceride, two important precursors of phospholipids. Other evidence suggests that SREBPs influence phospholipid synthesis by indirect mechanisms. PtdCho synthesis was reduced in sterol-regulatory-defective (SRD) cells 6 (15.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. J. Lipid. Res. 1997; 38: 711-722Abstract Full Text PDF PubMed Google Scholar) that have reduced cholesterol and fatty acid synthesis as a consequence of defective SREBP processing (5.Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar, 16.Evans M.J. Metherall J.E. Mol. Cell. Biol. 1993; 13: 5175-5185Crossref PubMed Scopus (28) Google Scholar). The absence of active SREBPs in SRD 6 cells did not affect the activity of the first and last enzymes in the CDP-choline pathway, choline phosphotransferase (CPT) and choline kinase. Instead, the activity of CCTα, the rate-limiting enzyme in the CDP-choline pathway (17.Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (190) Google Scholar), was reduced as the result of insufficient activation by fatty acids or a related derivative (15.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. J. Lipid. Res. 1997; 38: 711-722Abstract Full Text PDF PubMed Google Scholar). Sphingomyelin synthesis was also decreased in SRD 6 cells, but again the in vitro activity of biosynthetic enzymes in the pathway was unaffected (18.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1998; 336: 247-256Crossref PubMed Scopus (75) Google Scholar). Reduced sphingomyelin synthesis in SRD 6 cells could have been secondary to a relative deficiency in PtdCho, which provides the phosphocholine headgroup to sphingomyelin. Collectively, these results suggest that SREBPs regulate PtdCho and sphingomyelin synthesis indirectly by modifying the supply of precursors or lipid activators for CCT. We have now examined PtdCho synthesis in SRD 4 cells (19.Metherall J.E. Ridgway N.D. Dawson P.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1991; 266: 12734-12740Abstract Full Text PDF PubMed Google Scholar, 20.Nohturfft A. Hua X. Brown M.S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13709-13714Crossref PubMed Scopus (57) Google Scholar), and CHO cell lines expressing wild-type or a sterol-resistant SCAP mutant (D443N). SRD 4 cells express a SCAP allele with a point mutation (D443N) in the putative "sterol sensing" domain that renders it insensitive to suppression by sterols, resulting in constitutive proteolysis of SREBP 1 and SREBP-2 to the mature transcription factors (20.Nohturfft A. Hua X. Brown M.S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13709-13714Crossref PubMed Scopus (57) Google Scholar, 23.Hua X. Nofturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). As expected, these cell lines had elevated cholesterol and fatty acid synthesis but also displayed a 2–6-fold increase in PtdCho synthesis because of increased CCTα activity in vivo. In SRD 4 cells, activation of PtdCho synthesis (6-fold) was correlated with increased fatty acid synthesis and CCTα localization to the nuclear envelope. The modest increase in PtdCho and fatty acid synthesis in SCAP transfected cells (2-fold) was not accompanied by changes in CCTα expression or localization. Nuclear envelope localization of CCTα in control cells was reproduced by exogenous oleate, suggesting that elevated synthesis of this CCTα activator (or a derivative thereof) stimulated PtdCho synthesis in sterol regulation-defective cells. [methyl-3H]Choline, CDP-[methyl-3H]choline, [1-14C]acetate, [α-32P]dATP, [32P]phosphate, and [methyl-3H]phosphocholine were purchased from NEN Life Science Products. Choline, CDP-choline, phosphocholine, and cerulenin were from Sigma. Lovastatin was provided by Merck. Silica gel 60 TLC plates were from BDH. Monoclonal antibodies against HSV and T7 epitope tags were purchased from Novagen. The SCAP polyclonal antibody R-139 was kindly provided by Dr. Axel Nohturfft (University of Texas Southwestern Medical Center, Dallas TX; Ref. 21.Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Lipoprotein-deficient serum was prepared from fetal calf serum by centrifugation at 150,000 × g for 32 h, followed by extensive dialysis against 10 mm phosphate (pH 7.4) and 150 mm NaCl (PBS) (22.Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1278) Google Scholar). Cell culture medium, fetal calf serum, S1 nuclease (from Aspergillus oryzae), and G418 were from Life Technologies, Inc. SRD 4 cells were cultured in Dulbecco's modified Eagle's medium with 5% lipoprotein-deficient serum, 33 μg/ml proline (medium A), and 0.3 μg/ml 25-hydroxycholesterol at 37 °C in CO2/air (1:19). CHO 7 cells were maintained in medium A without 25-hydroxycholesterol. For experiments, SRD 4 and CHO 7 cells were subcultured in 60-mm dishes in medium A without 25-hydroxycholesterol (refer to figure legends for specific details). CHO 7 cells overexpressing SCAP and SCAP D443N were prepared by calcium phosphate transfection with pTK-HSV-SCAP-T7, pTK-HSV-SCAP-T7 (D443N), or empty vector (23.Hua X. Nofturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Cells were grown in medium A with 600 μg/ml G418 until individual colonies were evident at 12–14 days. SCAP transfected cells were trypsinized and cultured in medium A containing 600 μg/ml G418 and 0.5 μg/ml 25-hydroxycholesterol. After 10 days, thirty G418 and 25-hydroxycholesterol resistant colonies were harvested, expanded in culture for 10–14 days, and screened for expression of SCAP and SCAP D443N by immunoblotting of total cell extracts with monoclonal antibodies specific for T7 or HSV epitope tags. Mock transfected cells were selected after growth in G418. Four transfected cell lines expressing epitope-tagged SCAP, SCAP D443N, or empty vector were selected for further characterization. Stock cultures of SCAP transfected cells were maintained in medium A with 300 μg/ml G418 and 0.3 μg/ml 25-hydroxycholesterol and were subcultured for experiments in medium A without G418 or 25-hydroxycholesterol. After labeling with [3H]choline (see figure legends for specific conditions), cells were rinsed once with cold PBS and scraped in 1 ml of methanol-water (5:4, v/v). The culture dish was rinsed with 1 ml of methanol-water, and the extracts were combined in a glass screw cap tube. [3H]PtdCho and aqueous [3H]choline metabolites were separated by extraction with chloroform as described previously (15.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. J. Lipid. Res. 1997; 38: 711-722Abstract Full Text PDF PubMed Google Scholar). Labeled PtdCho was resolved by TLC in chloroform-methanol-water (65:25:4, v/v/v), whereas aqueous metabolites were separated in a solvent system of ethanol-water-ammonia (48:95:6, v/v/v). In some experiments, [3H]PtdCho was measured by scintillation counting of an aliquot of the chloroform phase (>98% of the radioactivity was in PtdCho). Sterol and fatty acid synthesis was measured by [14C]acetate labeling (24.Brown M.S. Faust J.R. Goldstein J.L. Kandeko I. Endo A. J. Biol. Chem. 1978; 253: 1121-1128Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were incubated with 5 μCi/ml [14C]acetate for 2 h, cell monolayers were dissolved in 1 ml of 0.5 n NaOH, transferred to screwcap tubes, and saponified in 3 ml of ethanol and 0.5 ml of 50% (w/v) KOH for 1 h at 60 °C. The sterol fraction was extracted with 4 ml of hexane and resolved by TLC in petroleum ether-diethyl ether-acetic acid (60:40:1, v/v/v). The zone corresponding to cholesterol was scraped into vials, and radioactivity was measured by scintillation counting. Fatty acids were extracted from the hydrolysate with 4 ml of hexane after acidification (pH <3) with HCl. Radioactivity in an aliquot was measured by scintillation counting (>99% of the radioactivity in this fraction were in fatty acids). Total cellular phosphocholine levels were determined by thin layer chromatography and phosphate analysis (15.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. J. Lipid. Res. 1997; 38: 711-722Abstract Full Text PDF PubMed Google Scholar). Cells were harvested by scraping in cold PBS, sedimented at 2,000 × g for 5 min, and homogenized in 20 mm Tris-HCl (pH 7.4), 10 mm EDTA, 5 mm dithiothreitol, and 0.1 mmphenylmethylsulfonyl fluoride by 20 passages through a 23-gauge needle. The homogenates were centrifuged for 60 min at 100,000 ×g, the soluble fraction was collected, and the particulate (total membrane) fraction was resuspended in the same buffer containing 250 mm sucrose. CCT activity in the membrane and soluble fractions was measured by conversion of [3H]phosphocholine to CDP-[3H]choline in the presence or absence of PtdCho-oleate (1:1, mol/mol) vesicles as described previously (15.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. J. Lipid. Res. 1997; 38: 711-722Abstract Full Text PDF PubMed Google Scholar, 25.Cornell R.B. J. Biol. Chem. 1989; 264: 9077-9082Abstract Full Text PDF PubMed Google Scholar). CPT activity in membranes was measured by conversion of CDP-[3H]choline to [3H]PtdCho in the presence of 1 mmdioleoylglycerol/0.015% Tween-20 (26.Cornell R.B. Methods Enzymol. 1992; 209: 267-272Crossref PubMed Scopus (29) Google Scholar). Protein was measured by the method of Lowry et al. (27.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Soluble and total membrane fractions from cells were prepared as described above. Membranes were treated with 1% (v/v) Nonidet P-40 on ice for 15 min, and the detergent-soluble fraction was isolated by centrifugation at 15,000 × g for 15 min. A total cell Nonidet P-40 soluble fraction was prepared in a similar manner. Equivalent amounts of protein from Nonidet P-40-solubilized cells, Nonidet P-40 solublilized membranes, and cytosol were resolved by SDS-10% PAGE and transferred to nitrocellulose. The filter was incubated with a polyclonal antibody against 45 amino acids of the C-terminal phosphorylation domain of CCTα (provided by Martin Post, Hospital for Sick Children, Toronto, Canada; Ref. 28.Yang J. Wang J. Tseu I. Kuliszewski M. Lee W. Post M. Biochem. J. 1997; 325: 29-38Crossref PubMed Scopus (24) Google Scholar), followed by a goat anti-rabbit antibody conjugated to horseradish peroxidase, and developed by the chemiluminescence method according to the manufacturer's instructions (Amersham Pharmacia Biotech). All antibody incubations were in 20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5% (w/v) skim milk powder, and 0.1% (v/v) Tween-20. This antibody does not cross-react with the CCTβ isoforms because of sequence divergence in the C-terminal phosphorylation domain (29.Lykidis A. Murtis K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 30.Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Phosphorylation of CCTα was measured by labeling cells for 15 h in phosphate-free medium A containing 25 μCi/ml [32P]phosphate. Soluble and total membrane fractions were prepared in buffer A as described previously (15.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. J. Lipid. Res. 1997; 38: 711-722Abstract Full Text PDF PubMed Google Scholar). The membrane fraction was treated with 0.3% Triton X-100 on ice for 15 min, and the detergent-soluble fraction was recovered after centrifugation at 400,000 × g for 20 min at 4 °C. [32P]CCTα was immunoprecipitated from cytosol and solubilized membranes (50 μg) in buffer A containing 1% Triton X-100 with a 1:400 dilution of an antibody against the membrane binding region of CCTα (kindly provided by Rosemary Cornell, Simon Fraser University, Vancouver, Canada; Ref. 31.Johnson J.E. Aebersold R. Cornell R.B. Biochim. Biophys. Acta. 1997; 1324 (184): 273Crossref PubMed Scopus (39) Google Scholar) for 1 h at 4 °C. Protein A-Sepharose was added for 45 min at 20 °C, followed by 6–8 washes with 0.5 ml of PBS containing 1% Triton X-100. Samples were boiled in SDS-PAGE sample buffer and separated by SDS-PAGE in 10% gels. Dried gels were exposed to film at −70 °C. CCTα mRNA was quantitated by S1 nuclease protection assays using a [α32P]dATP-labeled antisense probe corresponding to a 86-base pairHindIII-EcoRI fragment of the rat cDNA as described previously (18.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1998; 336: 247-256Crossref PubMed Scopus (75) Google Scholar). The CCTα S1 probe is against the 5′ end of the mRNA and will not hybridize to CCTβ mRNA because of limited sequence similarity in that region (29.Lykidis A. Murtis K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 30.Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an internal load control. Our previous results showing reduced PtdCho synthesis in cholesterol auxotrophic SRD 6 cells prompted an analysis of PtdCho synthesis in SRD 4 cells, which display elevated cholesterol synthesis that is resistant to down-regulation by 25-hydroxycholesterol (19.Metherall J.E. Ridgway N.D. Dawson P.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1991; 266: 12734-12740Abstract Full Text PDF PubMed Google Scholar, 20.Nohturfft A. Hua X. Brown M.S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13709-13714Crossref PubMed Scopus (57) Google Scholar, 23.Hua X. Nofturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). PtdCho synthesis and turnover was measured in SRD 4 cells, as well as parental CHO 7 cells, by pulse labeling with [3H]choline for 1 h followed by 4 h of chase in medium containing unlabeled choline (Fig. 1). Following a 1-h pulse, choline incorporation into PtdCho was 4-fold higher in SRD 4 cells relative to controls. The increase in PtdCho synthesis in SRD 4 cells after the 1-h choline pulse was accompanied by a 12-fold decrease in [3H]phosphocholine and 3-fold increase in [3H]CDP-choline, indicative of increased CCT activity. [3H]Phosphocholine was gradually lost from CHO 7 cells, because of conversion to PtdCho, and by 4 h was similar to SRD 4 cells. Phosphocholine pool size in CHO 7 and SRD 4 cells were similar (6.9 ± 1.2 versus 4.6 ± 0.7 nmol/mg protein (n = 3), respectively), indicating that diminished precursor pool size in SRD 4 cells cannot account for rapid synthesis and turnover of radiolabeled PtdCho. In SRD 4 cells, [3H]glycerophosphate (GPC), a product of PtdCho catabolism by phospholipase A, was elevated over 10-fold at the end of the pulse and increased during the chase period. Elevated [3H]GPC in SRD 4 cells during the chase period was accompanied by progressive decay of [3H]PtdCho. However, the loss of radioactivity from PtdCho in SRD 4 cells from 0 to 4 h was not entirely accounted for by increased intracellular GPC. The difference (approximately 500 dpm/mg protein × 10−3) was recovered in the cell culture medium as [3H]choline (results not shown). [3H]Choline pulse experiments showed that SRD 4 cells had elevated PtdCho synthesis and degradation (Fig. 1). Consistent with this observation, PtdCho mass in SRD 4 cells was not significantly different than CHO 7 controls (60.9 ± 4.7 nmol/mg protein in CHO 7 cells (n = 3) versus 53.4 ± 8.1 nmol/mg protein in SRD 4 cells (n = 3)). The mass of phosphatidylethanolamine, phosphatidylserine, and sphingomyelin in SRD 4 cells was also similar to controls. In addition to the SCAP D443N mutation, SRD 4 cells have a single point mutation in the ACAT gene that renders the enzyme inactive (32.Cao G. Goldstein J.L. Brown M.S. J. Biol. Chem. 1996; 271: 14642-14648Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Although chronic ACAT inhibition by 58–035 in CHO 7 or SCAP transfected CHO cells (see below) did not affect PtdCho synthesis (results not shown), we could not rule out the possibility that reduced ACAT activity or another mutation in the SRD 4 cells contributed to the results shown in Fig. 1. To address this question, CHO 7 cells were stably transfected with the cDNA for wild-type SCAP or the SCAP D443N mutant and PtdCho synthesis was examined. Initially, four clones resistant to killing by 0.5 μg of 25-hydroxycholesterol/ml and expressing epitope-tagged SCAP or SCAP D443N (hereafter referred to as CHO-SCAP or CHO-SCAP D443N) were isolated, and PtdCho synthesis was measured by a 1-h [3H]choline pulse. The average rate of PtdCho synthesis in four mock transfected cell lines was 122.5 ± 32.4 dpm/μg protein, compared with 221.1 ± 22.8 and 206.2 ± 22.0 dpm/μg protein in four CHO-SCAP and CHO-SCAP D443N cells, respectively. Increased PtdCho synthesis in SCAP transfected cells was also accompanied by a 2–3-fold reduction in [3H]phosphocholine. Because all four SCAP- and SCAP D443N-overexpressing cell lines appeared to have a similar phenotype with respect to increased PtdCho synthesis, one cell line from each group was examined in detail. The expression of epitope-tagged and endogenous SCAP and SCAP D443N in the two cell lines is shown in Fig. 2. Immunoblotting with a HSV monoclonal antibody detected a protein doublet of approximately 140–150 kDa in the Nonidet P-40 extracts of CHO-SCAP and CHO-SCAP D443N cell membranes but not in mock transfected controls (Fig. 2 A). It appeared that higher expression of wild-type SCAP compared with the D443N mutant was required to maintain 25-hydroxycholesterol resistance. The cell extracts from Fig.2 A, as well as those from CHO 7 and SRD 4, were also probed with antibody R-139 to detect both endogenous and overexpressed SCAP (21.Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). This antibody also detected a 140–150-kDa protein doublet in all cells. As expected, expression was highest in the CHO-SCAP and CHO-SCAP D443N cells relative to mock, CHO 7, and SRD 4 cells (Fig.2 B). Irrespective of differences in expression, both CHO-SCAP and CHO-SCAP D443N cells displayed a 2-fold increase in [14C]acetate incorporation into cholesterol relative to mock transfected controls (Fig. 3). Cholesterol and fatty acid synthesis in SRD 4 cells was increased 3-fold above CHO 7 cells. Unlike that of SRD 4 cells, [14C]acetate incorporation into fatty acids in the SCAP-overexpressing cell lines was not significantly increased. PtdCho synthesis was examined in the SCAP-overexpressing cells by [3H]choline labeling followed by 4-h chase in medium containing unlabeled choline (Fig. 4). At the end of the 1-h [3H]choline pulse (0 h) and throughout the 4-h chase, PtdCho synthesis was increased 1.5–2.5-fold in CHO-SCAP and CHO-SCAP D443N cells. Increased PtdCho synthesis in these cells was accompanied by a 2-fold reduction in [3H]phosphocholine levels, a 2–3-fold increase in [3H]CDP-choline, and a 2-fold elevation in [3H]GPC production. Our previous results in SRD 6 cells showed that decreased PtdCho synthesis was not correlated with changes in cellular cholesterol levels but rather with the availability of fatty acids (15.Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. J. Lipid. Res. 1997; 38: 711-722Abstract Full Text PDF PubMed Google Scholar). However, these studies did not rule out the possibilit
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Sterol regulatory element binding proteins(SREBPs) are membrane-bound transcription factors that activate genes encoding enzymes of cholesterol and fatty acid biosynthesis.SREBPs are synthesized as precursor proteins in the endoplasmic reticulum(ER),where they form a complex with another protein,SREBP cleavage activating protein(SCAP).SCAP escorts SREBP to activation in the Golgi apparatus.There,SREBP undergoes two proteolytic cleavage steps to release the mature,biologically active transcription factor,nuclear SREBP(nSREBP).The process is induced by two proteins,SCAP and Insig.This review is to summarize the role of SCAP and Insig in the procession and maturation of SREBP and the lipid metabolism.
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The objective of this study was to investigate the molecular mechanisms by which nutrients regulate sterol regulatory element binding protein-1 (SREBP1) in bovine mammary epithelial cells (Mac-T). Three models were tested. First, the relationship between SREBP1 and the mechanistic target of rapamycin (mTOR) signaling was tested through mTOR activation/inhibition as well as SREBP1 knockdown by siRNA. Second, the relationship between AMPK and SREBP1 was tested in t10,c12-CLA-treated Mac-T cells. Third, the activation of SREBP1 was tested by glucose supplementation. Results showed that mTOR activation increased SREBP1 protein as well as the lipogenic gene expression by over 50%. While inhibition on mTOR failed to increase SREBP1, siRNA-directed SREBP1 knockdown confirmed that insulin enhanced lipogenic gene transcription through SREBP1. Further examination found that mTOR signaling regulates SREBP1 by preventing its proteosomal degradation. t10,c12-CLA decreased SREBP1 protein and lipogenic gene expression through phosphorylation of AMPK, while inhibition of AMPK phosphorylation partially rescued the SREBP1 reduction. Lastly, low glucose (1 mmol/L) was able to increase mature SREBP1 level by 2.2-fold. Increasing glucose concentration increased SREBP-cleavage activating protein, a key regulator of SREBP1 activation, in a dosage- and time-dependent manner. In conclusion, these results showed that major cellular metabolic regulators play roles in SREBP1 activation and degradation thus regulating lipogenesis in response to the nutrients provided.
Lipogenesis
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ABSTRACT Sterol regulatory element binding proteins (SREBPs) are essential transcriptional factors that control expression of lipogenic genes and adipocyte differentiation. Human cytomegalovirus (HCMV) infection has been shown to require the induction of lipogenesis. Here we show that the induction of lipogenesis and expression of key lipogenic enzymes in human fibroblasts occurs by 24 h post-HCMV infection. This activation correlates with increased cleavage of the SREBP1 precursors to form the mature active transcription factors that enter the nucleus to transcriptionally activate lipogenic genes. SREBP1 cleavage is normally inhibited by increased sterol levels; however, our data show that this level of control is overridden in infected cells to allow constitutive activation of lipogenesis. This process requires viral protein synthesis, since UV-irradiated HCMV cannot activate SREBP cleavage. The cleavage of SREBP1 requires it to be in complex with SREBP cleavage activation protein (SCAP). Depleting SCAP using short hairpin RNA (shRNA) showed that SREBP1 cleavage and the induction of lipogenic genes and lipid synthesis are all inhibited in HCMV-infected cells. As a result, production of infectious virions is reduced in SCAP-depleted cells. Thus, the SCAP-mediated mechanism for SREBP cleavage is utilized by HCMV during infection. Our studies suggest that HCMV induces adipocyte-like lipogenesis and overrides normal sterol feedback controls in order to maintain high levels of constitutive lipid synthesis during infection.
Lipogenesis
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Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis.
Lipogenesis
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