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• W2025968452 abstract "A characteristic feature of many vertebrate axons is their wrapping by a lamellar stack of glially derived membranes known as the myelin sheath. Myelin is a cholesterol-rich membrane that allows for rapid saltatory nerve impulse conduction. Axonal neuregulins instruct glial cells on when and how much myelin they should produce. However, how neuregulin regulates myelin sheath development and thickness is unknown. Here we show that neuregulin receptors are activated by drops in plasma membrane cholesterol, suggesting that they can sense sterol levels. In Schwann cells neuregulin-1 increases the transcription of the 3-hydroxy-3-methylglutarylcoenzyme A reductase, the rate-limiting enzyme for cholesterol biosynthesis. Neuregulin activity is mediated by the phosphatidylinositol 3-kinase pathway and a cAMP-response element located on the reductase promoter. We propose that by activating neuregulin receptors, neurons exploit a cholesterol homeostatic mechanism forcing Schwann cells to produce new membranes for the myelin sheath. We also show that a strong phylogenetic correlation exists between myelination and cholesterol biosynthesis, and we propose that the absence of the sterol branch of the mevalonate pathway in invertebrates precluded the myelination of their nervous system. A characteristic feature of many vertebrate axons is their wrapping by a lamellar stack of glially derived membranes known as the myelin sheath. Myelin is a cholesterol-rich membrane that allows for rapid saltatory nerve impulse conduction. Axonal neuregulins instruct glial cells on when and how much myelin they should produce. However, how neuregulin regulates myelin sheath development and thickness is unknown. Here we show that neuregulin receptors are activated by drops in plasma membrane cholesterol, suggesting that they can sense sterol levels. In Schwann cells neuregulin-1 increases the transcription of the 3-hydroxy-3-methylglutarylcoenzyme A reductase, the rate-limiting enzyme for cholesterol biosynthesis. Neuregulin activity is mediated by the phosphatidylinositol 3-kinase pathway and a cAMP-response element located on the reductase promoter. We propose that by activating neuregulin receptors, neurons exploit a cholesterol homeostatic mechanism forcing Schwann cells to produce new membranes for the myelin sheath. We also show that a strong phylogenetic correlation exists between myelination and cholesterol biosynthesis, and we propose that the absence of the sterol branch of the mevalonate pathway in invertebrates precluded the myelination of their nervous system. Myelination heavily influenced the evolution and structure of vertebrate brains, augmenting the reliability and speed of signal propagation in nervous pathways. Glial cells (oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system) extend plasma membrane processes, wrapping axons with specialized membrane named myelin. It is well established that myelin thickness depends largely on axon size (1Dietschy J.M. Turley S.D. J. Lipid Res. 2004; 45: 1375-1397Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 2Michailov G.V. Sereda M.W. Brinkmann B.G. Fischer T.M. Haug B. Birchmeier C. Role L. Lai C. Schwab M.H. Nave K.A. Science. 2004; 304: 700-703Crossref PubMed Scopus (752) Google Scholar). How axonal size signals the adequate myelin thickness produced by glial cells has remained largely unknown. However, it has been recently suggested that a product of the Nrg1 (neuregulin 1 gene) signals to myelinating cells about the axon diameter (2Michailov G.V. Sereda M.W. Brinkmann B.G. Fischer T.M. Haug B. Birchmeier C. Role L. Lai C. Schwab M.H. Nave K.A. Science. 2004; 304: 700-703Crossref PubMed Scopus (752) Google Scholar) regulating the myelin thickness. In addition, the artificial expression of this protein in unmyelinated axons converts them to a myelinated phenotype, suggesting that threshold levels of expressed neuregulin, and not strictly axon size, determines the myelination status of the neuron (3Taveggia C. Zanazzi G. Petrylak A. Yano H. Rosenbluth J. Einheber S. Xu X. Esper R.M. Loeb J.A. Shrager P. Chao M.V. Falls D.L. Role L. Salzer J.L. Neuron. 2005; 47: 681-694Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). Myelin chemical composition differs greatly from other cellular membranes. Thus, high cellular levels of cholesterol are necessary for myelin membrane growth (4Saher G. Brugger B. Lappe-Siefke C. Mobius W. Tozawa R. Wehr M.C. Wieland F. Ishibashi S. Nave K.A. Nat. Neurosci. 2005; 8: 468-475Crossref PubMed Scopus (491) Google Scholar). In contrast to other tissues, the brain is unable to obtain cholesterol from circulating plasma lipoproteins and depends entirely on de novo cholesterol biosynthesis, mostly performed by glial cells (1Dietschy J.M. Turley S.D. J. Lipid Res. 2004; 45: 1375-1397Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 4Saher G. Brugger B. Lappe-Siefke C. Mobius W. Tozawa R. Wehr M.C. Wieland F. Ishibashi S. Nave K.A. Nat. Neurosci. 2005; 8: 468-475Crossref PubMed Scopus (491) Google Scholar). The rate-limiting step in vertebrate cellular cholesterol production is the synthesis of mevalonate performed by the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) 5The abbreviations used are: HMGR3-hydroxy-3-methylglutaryl-CoA reductaseLPDSlipoprotein-deficient serumFBSfetal bovine serumDMEMDulbecco's modified Eagle's mediumCREcAMP-response elementCREBCRE-binding proteinSREsterol-response elementPI3Kphosphatidylinositol 3-kinaseMAPKmitogen-activated protein kinaseSMDFsensory and motor-neuron derived factorMβCDmethyl-β-cyclodextrinGSTglutathione S-transferaseERendoplasmic reticulumqPCRquantitative PCRTBPTATA-binding proteinEGFepidermal growth factorSREBPsterol regulatory element-binding protein. (5Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4566) Google Scholar). Studies in vivo and in vitro have shown that HMGR is highly regulated at the transcriptional level (6Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). When cholesterol concentration drops in the endoplasmic reticulum (ER), the SREBP-2 transcription factor is released and binds to a sterol-response element (SRE) located on the HMGR promoter. This leads to increased transcription of the HMGR gene, stimulating the cholesterol biosynthesis and safeguarding the adequate cholesterol concentration within the cell (6Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). Other regulatory elements on this promoter have been described, suggesting that additional transcription factors regulate HMGR expression (7Di Croce L. Vicent G.P. Pecci A. Bruscalupi G. Trentalance A. Beato M. Mol. Endocrinol. 1999; 13: 1225-1236Crossref PubMed Scopus (34) Google Scholar). 3-hydroxy-3-methylglutaryl-CoA reductase lipoprotein-deficient serum fetal bovine serum Dulbecco's modified Eagle's medium cAMP-response element CRE-binding protein sterol-response element phosphatidylinositol 3-kinase mitogen-activated protein kinase sensory and motor-neuron derived factor methyl-β-cyclodextrin glutathione S-transferase endoplasmic reticulum quantitative PCR TATA-binding protein epidermal growth factor sterol regulatory element-binding protein. Most of the free cellular cholesterol is located within the plasma membrane (1Dietschy J.M. Turley S.D. J. Lipid Res. 2004; 45: 1375-1397Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar) where levels are tightly regulated. Despite this, no cholesterol sensor has been proposed for this cell compartment. However, it has been shown that the epidermal growth factor receptor (also known as ErbB1), which resides in the plasma membrane, is phosphorylated after acute cholesterol depletion (8Chen X. Resh M.D. J. Biol. Chem. 2002; 277: 49631-49637Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In turn, ErbB1 phosphorylation causes hyperactivation of the PI3K and the mitogen-activated protein kinase (MAPK) pathways, showing that membrane cholesterol depletion can elicit intracellular signaling cascades (9Furuchi T. Anderson R.G. J. Biol. Chem. 1998; 273: 21099-21104Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) and suggesting that some ErbB receptors could be part of a mechanism for sensing plasma membrane cholesterol concentration. Here we show that neuregulin receptors (ErbB2, ErbB3, and ErbB4) are transactivated by ErbB1 after acute drops in cholesterol, suggesting that they could also form part of a plasma membrane cholesterol-sensing mechanism. In addition, we show that the activation of the neuregulin 1-ErbB pathway in Schwann cells up-regulates the expression of HMGR, the rate-limiting enzyme in cholesterol biosynthesis. We propose that neurons, by activating the NRG-ErbB pathway, simulate a drop of cholesterol to which Schwann cells respond augmenting cholesterol biosynthesis. Cholesterol up-regulation will help to increase plasma membrane size, which will wrap around the axon to form the myelin sheath. Constructs, Promoter Cloning, and Site-directed Mutagenesis—The HMGR promoter (nucleotides -309, +77) was amplified from mouse genomic DNA (using primers sense 5′-CTGAGTTCGGGGTACTCCAC and antisense 5′-CTCACCTCCGGATCTCAATG), cloned in pCRII, and subcloned in pGL3 basic (Promega). Deletions of SRE and CRE were produced by inverse PCR with a high fidelity DNA polymerase. Briefly, SRE was mutated by inverse PCR using the primers sense 5′-TGGTGCGACGTCCGTTCTCCGCCCGGGTGC and antisense 5′-CGGGCAGACGTCCATCTCTCACCACGCAGA; CRE site was mutated using the primers sense 5′-TTCGTGGACGTCGCCGTCAGGCTGAGCAGC and antisense 5′-CGGCCTGACGTCCGAACGGTCTCCCTAACA. Amplicons were digested with DpnI to remove the template and with AatII to create compatible ends before ligation. Digested products were ligated and transformed in Escherichia coli DH5α. All constructs were verified by automatic sequencing. The pcDNA3-ErbB2, pcDNA3-ErbB3, and pcDNA3-ErbB4 vectors were kindly provided by Professor Yossef Yarden (The Weizmann Institute of Science, Rehovot, Israel). pECF1-myr-Akt was kindly provided by Professor F. Mayor (Centro de Biologia Molecular Severo Ochoa, Madrid, Spain). mRNA Detection and Quantification by Reverse Transcription-PCR and qPCR—To detect and quantify gene expression, Schwann cell total RNA was isolated and retro-transcribed to cDNA with SuperScript II Reverse transcriptase (Invitrogen). Control reactions were performed by omitting retrotranscriptase. First strand cDNA was PCR-amplified with specific primers for SREBP-2 (sense 5′-AAGTCTGGCGTTCTGAGGAA and antisense 5′-CCAGGAAGGTGAGGACACAT), SCAP (sense 5′-CGATGTACTAACAGGCAGCCG and antisense 5′-GCCGGTCACCAGAAGGTTA), INSIG1 (sense 5′-CTTGTGGTGGACGTTTGATCG and antisense 5′-CACTGTGACACCTCCTGAGA), INSIG-2 (sense 5′-CGGTGCTCTTCTTCATTGGCG and antisense 5′-GTGGCTCTCCTAGATGCCTGTC), site 1 protease (5′-GTTTGAAGACAACATCGCCCG and antisense 5′-AGCTCCCGCTTCTGTACTG); and site 2 protease (sense 5′-TGAAGTCGCAGAGGACTCAC and antisense 5′-GCCATTCAGTAGAACCATCTAGTCG). Real time PCR analysis was performed using Platinum® SYBR®Green qPCR Supermix UDG (Invitrogen) with 400 nm of gene-specific primers for rat HMGR (sense 5′-CCAAGGTGGTGAGAGAAGTATT and antisense 5′-TCTCTATAGACGGCATGGTACA). Reactions were performed in duplicate, and threshold cycle values were normalized to the housekeeping rat TBP mRNA (sense 5′-GAGAGCCACGAACAACTGCG and antisense 5′-AGCTTCTGCACAACTCTAGC). The specificity of the products was determined by melting curve analysis and gel electrophoresis. The ratio of the relative expression of HMGR to TBP was calculated by using the 2ΔCT formula. Cell Culture and Transfection—COS-7 and MCF-7 were obtained from the ATCC and were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). The Oligo-neu cell line was kindly provided by Prof. J. Trotter (University of Heidelberg, Germany) and cultured in SATO medium (10Jung M. Kramer E. Grzenkowski M. Tang K. Blakemore W. Aguzzi A. Khazaie K. Chlichlia K. von Blankenfeld G. Kettenmann H. Eur. J. Neurosci. 1995; 7: 1245-1265Crossref PubMed Scopus (210) Google Scholar). Schwann cells were cultured from sciatic nerves of neonatal rats as described previously by Brockes et al. (11Brockes J.P. Fields K.L. Raff M.C. Brain Res. 1979; 165: 105-118Crossref PubMed Scopus (916) Google Scholar). All the procedures were performed following European Union and institutional guidelines. Cells cultures were expanded in DMEM supplemented with 3% FBS, 5 μm forskolin, and 50 nm GST-NRG1 and used up to eighth passage except where indicated. Cells were transfected with plasmid DNA using Lipofectamine™ 2000 (Invitrogen) following the manufacturer's recommendations. Purification of Recombinant Neuregulins and Tyrosine Phosphorylation Assay—Cloning of pGEX-SMDF was already described elsewhere (12Cabedo H. Luna C. Fernandez A.M. Gallar J. Ferrer-Montiel A. J. Biol. Chem. 2002; 277: 19905-19912Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). cDNA encoding EGF-like domain of NRG1 was amplified by PCR and cloned into pGEX-4T-1. Bacterial cells were grown until reaching 0.6-0.8 mOD. Thereafter, cells were induced with isopropyl 1-thio-β-d-galactopyranoside at 0.3 mm for 4 h and pelleted. The pellet was resuspended in phosphate-buffered saline, 5 mm dithiothreitol, and sonicated. Triton X-100 was added to reach 1% and centrifuged at 10,000 × g for 10 min. Protein was purified from the supernatant with GSH-agarose beads. After extensive washing, neuregulin was eluted from the beads with 10 mm GSH in 5 mm dithiothreitol, 50 mm Tris-HCl, pH 8.8. The concentration of protein was determined using the method of Bradford et al. (13Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Neuregulin-induced tyrosine phosphorylation of ErbB receptors was carried out as described by Ho et al. (14Ho W.H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Briefly, MCF-7 cells were grown until ≥80% confluence in 24-well plates. Thereafter, cells were serum-starved for 5 h and incubated with recombinant SMDF for 3 min. Medium was removed, and cells were harvested with 100 μl of β-mercaptoethanol containing SDS sample buffer. Whole cell extracts were heat-denatured, separated by SDS-PAGE, and analyzed by immunoblotting with a monoclonal anti-phosphotyrosine antibody from Sigma (1:1,000). Cholesterol Depletion Assays—To ensure comparable levels of the receptor per well, pcDNA3-ErbB transiently transfected COS-7 cells were trypsinized, reseeded, and grown to ≈80% confluence in 24-well plates. Thereafter, cells were serum-starved for 18 h and incubated with methyl-β-cyclodextrin (MβCD) at the concentration indicated for 1 h at 37 °C and 5% CO2. Where indicated, AG1478 (in Me2SO) was preincubated for 1 h before MβCD treatment. Controls were treated with vehicle (Me2SO). ErbB phosphorylation analysis in transfected cells was performed by SDS-PAGE and immunoblot with epitope-specific antibodies (anti-p-ErbB2-Tyr-1248 and anti-ErbB2 from Upstate and anti-phosphotyrosine from Sigma). Reporter Activity Assays—Schwann cells, MCF-7, Oligo-neu, or COS-7 cells were growth in 60-mm culture dishes and transfected with the pHMGR-Luc construct (or the promoter deletions). 6 h later cells were trypsinized, replated into 48-well dishes (100,000 cells/well), and incubated with the indicated treatment in DMEM. 48 h later cells were lysed, and luciferase activity was determined with the luciferase assay system (Promega) using the manufacturer's recommendations. The β-galactosidase activity (Beta-Glo Assay System, Promega) of a pCMV-LacZ reporter co-transfected at 1:100 was used to normalize variations in cell number, viability, and transfection efficiency. No major changes in β-galactosidase activity or cell morphology were observed. Neuregulin Receptors Are Activated by Transphosphorylation after Drops in Plasma Membrane Cholesterol—To test whether, similar to ErbB1 (8Chen X. Resh M.D. J. Biol. Chem. 2002; 277: 49631-49637Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), other members of the ErbB receptor family can also sense the plasma membrane cholesterol level, the cDNA encoding for ErbB2 was transfected into the fibroblast cell line COS-7. Plasma membrane cholesterol was acutely depleted with increasing concentrations of MβCD and receptor phosphorylation status monitored with an antiphospho-ErbB2 (Tyr-1248)-specific antibody. As shown in Fig. 1a, ErbB2 was strongly phosphorylated in an MβCD dose-dependent manner, suggesting that cholesterol levels modulate the phosphorylation status of ErbB2. Additionally, we found that all the other members of this receptor family, ErbB3 and ErbB4, are also phosphorylated during cholesterol depletion (Fig. 1b). Because COS-7 cells naturally express ErbB1, which heterodimerizes with other ErbB receptors, the possibility exists that cholesterol depletion-induced ErbB1 activation transphosphorylates other ErbB partners. To test this hypothesis, cells were preincubated with 30 nm of the ErbB1-specific inhibitor tyrphostin AG1478 (ErbB1 IC50 = 3 nm) (15Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1628) Google Scholar). As is shown in Fig. 1c, AG1478 completely inhibited the MβCD-induced phosphorylation of ErbB2, ErbB3, and ErbB2/ErbB3 in transfected COS-7 cells. Thus, our results indicate that ErbB1 can sense drops in plasma membrane cholesterol concentration and transactivate other ErbB receptors. Activation of the Neuregulin-ErbB Pathway Stimulates HMGR Gene Transcription in Cell Lines of Different Lineages—It has been shown that the epidermal growth factor (a ligand for ErbB1) up-regulates the cholesterol biosynthetic pathway in adenocarcinoma cells (16Asslan R. Pradines A. Pratx C. Allal C. Favre G. Le Gaillard F. Biochem. Biophys. Res. Commun. 1999; 260: 699-706Crossref PubMed Scopus (31) Google Scholar). To further understand the role of ErbB signaling in cholesterol biosynthesis, we investigated the effects produced by activation of this pathway in different ways on the transcription of the HMGR gene. To this goal, we cloned the mouse HMGR promoter (nucleotides -309 to +77) into a luciferase reporter vector (pGL3-basic). The resulting construct (pHMGR-Luc) and the pcDNA3-ErbB2 and pcDNA-ErbB3 vectors were co-transfected into COS-7 cells, and the resulting luciferase activity was determined. As shown in Fig. 2a, the ErbB2-ErbB3 complex nearly doubled the transcriptional activity of the HMGR promoter (1.87 ± 0.09-fold (n = 6)). This result suggests that neuregulins, by activating the ErbB2-ErbB3 complex are involved in the control of cholesterol biosynthesis in cultured mammalian cells. To test this hypothesis further, we took advantage of MCF-7 cells, a human adenocarcinoma cell line that naturally expresses the neuregulin receptors ErbB2 and ErbB3 (17Chan S.D. Antoniucci D.M. Fok K.S. Alajoki M.L. Harkins R.N. Thompson S.A. Wada H.G. J. Biol. Chem. 1995; 270: 22608-22613Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). MCF-7 cells transfected with pHMGR-Luc were incubated with recombinant sensory and motor-neuron derived factor (SMDF), a human type III neuregulin 1 highly expressed in the peripheral nervous system (14Ho W.H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). As shown in Fig. 2b, recombinant SMDF (GST-SMDF) promotes the phosphorylation of the ErbB2-ErbB3 complex (inset) and induces the activity of the HMGR promoter in MCF-7 cells (1.9 ± 0.3-fold increase (n = 6)). Neuregulin-ErbB signaling pathway plays an essential role in glial cell development and myelination (18Garratt A.N. Britsch S. Birchmeier C. BioEssays. 2002; 22: 987-996Crossref Scopus (253) Google Scholar). To unveil whether it also has a role in cholesterol biosynthesis in glial cells, we turned to Oligo-neu cells, a murine oligodendrocyte cell line immortalized by the expression of a constitutive active form of ErbB2 (10Jung M. Kramer E. Grzenkowski M. Tang K. Blakemore W. Aguzzi A. Khazaie K. Chlichlia K. von Blankenfeld G. Kettenmann H. Eur. J. Neurosci. 1995; 7: 1245-1265Crossref PubMed Scopus (210) Google Scholar). As is shown in Fig. 2c, two ErbB inhibitors (AG1478 and AG825) decreased significantly the activity of the HMGR promoter in these cells, suggesting that neuregulin-ErbB pathway controls cholesterol biosynthesis also in glial cells. Taken together, our results show that activity of the neuregulin-ErbB pathway controls the HMGR gene expression at the transcriptional level in mammalian cell lines of different lineage, including glial cells. Neuregulin 1 Increases the Steady-state Levels for HMGR mRNA in Schwann Cells—It has been shown that the thickness of the myelin sheath in the peripheral nervous system is graded to the amount of neuregulin expressed on the surface of the neuronal axon (2Michailov G.V. Sereda M.W. Brinkmann B.G. Fischer T.M. Haug B. Birchmeier C. Role L. Lai C. Schwab M.H. Nave K.A. Science. 2004; 304: 700-703Crossref PubMed Scopus (752) Google Scholar, 3Taveggia C. Zanazzi G. Petrylak A. Yano H. Rosenbluth J. Einheber S. Xu X. Esper R.M. Loeb J.A. Shrager P. Chao M.V. Falls D.L. Role L. Salzer J.L. Neuron. 2005; 47: 681-694Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). Because myelin is a cholesterol-enriched specialized plasma membrane, and cholesterol is critical for myelin biogenesis (4Saher G. Brugger B. Lappe-Siefke C. Mobius W. Tozawa R. Wehr M.C. Wieland F. Ishibashi S. Nave K.A. Nat. Neurosci. 2005; 8: 468-475Crossref PubMed Scopus (491) Google Scholar), we tested whether the neuregulin-ErbB pathway controls the cholesterol biosynthesis in primary cultures of Schwann cells, the cell type responsible for myelination in the peripheral nervous system. To this goal, Schwann cells were obtained from excised sciatic nerves of newborn rats and cultured (see “Experimental Procedures”). Schwann cells primary cultures (6-8 passes) were transfected with the pHMGR-Luc and pCMV-LacZ constructs described previously and incubated with recombinant SMDF. As is shown in Fig. 3a, GST-SMDF increased the HMGR promoter activity by 2.21 ± 0.28-fold (n = 9) in Schwann cells, supporting the tenet that axonally derived neuregulin controls cholesterol biosynthesis in myelinating cells. All neuregulin 1 products share an EGF-like domain that binds and activates ErbB receptors (15Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1628) Google Scholar). To test whether the effect of SMDF on HMGR transcriptional activity is mediated by the EGF-like domain, we cloned, expressed, and purified this domain in bacteria. As is shown in Fig. 3a, the recombinant EGF-like domain of neuregulin 1 (GST-NRG1) increased the transcription of the HMGR gene as well (3.47 ± 0.51-fold, (n = 6)), strongly suggesting that SMDF effect is mediated by direct ErbB3-ErbB2 complex activation. Because we regularly expand the Schwann cell cultures in medium containing serum, forskolin, and neuregulin, the possibility exists that the activation-induced down-regulation of neuregulin receptors masked partially the transcriptional effects of neuregulin. To explore the role of neuregulin on HMGR transcriptional activity in a more physiological model, we repeated the experiments with nonexpanded rat Schwann cell cultures. The presence of fibroblasts in these cultures should not affect the conclusions because they do not express neuregulin receptors and cannot contribute to the transcriptional effects of neuregulin (data not shown). In fact, a putative diluting effect caused by a higher reporter (β-galactosidase) activity is expected. Remarkably, as is shown in Fig. 3b, the responsiveness of the HMGR promoter to GST-NRG1 in nonexpanded Schwann cell cultures was much higher (14.3 ± 1.4-fold increase (n = 9)) than that found in expanded cultures. Therefore, our results show that the transcriptional effect of neuregulin decreases in expanded Schwann cell cultures, probably as a consequence of the down-regulation of ErbB receptors. Steady-state levels of mRNA depend on the balance between synthesis and degradation rates. To check whether the transcriptional effect of neuregulin is translated into an increase in the steady-state amount of the HMGR mRNA in Schwann cells, we determined, using real time qPCR, the changes in HMGR mRNA in Schwann cultures in response to neuregulin 1 application. To avoid any genomic DNA amplification, specific primers for rat HMGR were designed in separated exons that include a large intron interposed between them (see “Experimental Procedures”). To normalize gene expression levels, a housekeeping gene (the rat TATA-binding protein (TBP)) was used. As is shown in Fig. 3c, GST-NRG1 increased up to 181 ± 5% (n = 4) the amount of the HMGR transcript. Thus, our results so far show that the axonal product neuregulin 1 stimulates the transcription of the HMGR gene in cultured Schwann cells and produces an almost 2-fold increase in the total amount of the HMGR mRNA transcript. Intracellular Pathways Involved in Neuregulin 1 Signaling—The HMGR promoter contains at least two transcription factor-binding sites, shown schematically in Fig. 4a. The transcriptional control of HMGR in response to cholesterol depletion in hepatocytes and fibroblasts is mainly mediated by a sterol response element (SRE) located in the HMGR promoter (6Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar, 20Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). Because Schwann cells do express the mRNA for the transcription factor SREBP-2 (Fig. 6b), we decided to explore whether SRE mediates the neuregulin-induced HMGR transcriptional activation in these cells. To this goal, we deleted the SRE of the pHMGR-Luc construct (Fig. 4a). Schwann cells were transfected with the wild type construct or the mutant, and the responsiveness to neuregulin SMDF was compared with the luciferase/β-galactosidase assay. As is shown in Fig. 4b, we were unable to detect any modification in the neuregulin-induced transcriptional activation after SRE disruption (1.94 ± 0.19-fold increase in the mutant versus 2.19 ± 0.19 in wild type), suggesting that the neuregulin effect is mediated by a different transcription factor-binding site in this promoter. It has been shown previously that thyrotropin-mediated stimulation of the HMGR gene transcription in FRTL-5 rat thyroid cells is mediated by a CRE located downstream of the SRE (21Bifulco M. Perillo B. Saji M. Laezza C. Tedesco I. Kohn L.D. Aloj S.M. J. Biol. Chem. 1995; 270: 15231-15236Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) (see Fig. 4a). To test whether the CRE site mediates the transcriptional effect of neuregulin in Schwann cells, we introduced disruptive mutations in its core by inverse PCR. As is shown in Fig. 4b, mutations in CRE decreased (although not abrogated) the GST-SMDF-induced stimulation of the HMGR promoter (1.33 ± 0.15-fold (n = 6)), suggesting that neuregulin transcriptional control of HMGR is in part mediated by the cAMP-response element. To explore this tenet further, a CRE-luciferase construct (pCRE-Luc) was transfected into cultured rat Schwann cells, and the transcriptional effect of neuregulin was determined by the luciferase/β-galactosidase assay. As is shown in Fig. 4c, GST-SMDF was able to stimulate CRE promoter activity in Schwann cells by 2.37 ± 0.29-fold (n = 6). Even a larger increment (4.52 ± 0.22-fold (n = 3)) was obtained with GST-NRG1. Taken together our results suggest that the transcriptional effect of neuregulin 1 on the HMGR gene in Schwann cells is in part mediated by a CREB/ATF transcription factor. Indeed, when the cAMP-dependent pathway was stimulated with forskolin (Fig. 4d), the transcriptional effect of GST-NRG1 on HMGR gene doubled (6.34 ± 0.69-fold (n = 6) versus 3.24 ± 0.44-fold (n = 6) in nontreated cultures).FIGURE 6Cholesterol deprivation up-regulates the transcription of the HMGR gene in COS-7 but not in Schwann cells. a pHMGR-Luc/pCMV-LacZ-transfected cultures were incubated in culture with cholesterol-containing medium (10% FBS) or cholesterol-deprived medium (10% LPDS). After 48 h, luciferase and β-galactosidase activity of cell extracts were determined. No major changes in β-galactosidase activity were found. Data are given as mean ± S.E. The number of experiments (n) is indicated. b Schwann cells express SREBP-2 mRNA and the mRNA for the proteins involved in its processing. Total RNA from Schwann cells was extracted, retrotranscribed to cDNA, and PCR-amplified with specific primers for SREBP-2, INSIG-1, INSIG-2, Site 1 protease, and Site 2 protease. A fraction (10%) of the PCR product was submitted to agarose gel electrophoresis and detected with ethidium bromide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Neuregulin activates two major signal transduction cascades in mammalian cells, the PI3K and the MAPK pathways (19Falls D.L. Exp. Cell Res. 2003; 284: 14-30Crossref PubMed Scopus (858) Google Scholar, 21Bifulco M. Perillo B. Saji M. Laezza C. Tedesco I. Kohn L.D. Aloj S.M. J. Biol. Chem. 1995;" @default.
• W2025968452 created "2016-06-24" @default.
• W2025968452 creator A5014885640 @default.
• W2025968452 creator A5018676466 @default.
• W2025968452 creator A5060725086 @default.
• W2025968452 creator A5062124982 @default.
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• W2025968452 date "2007-09-01" @default.
• W2025968452 modified "2023-09-29" @default.
• W2025968452 title "Transcriptional Control of Cholesterol Biosynthesis in Schwann Cells by Axonal Neuregulin 1" @default.
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