Research ArticleCholesterol Metabolism

Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake

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Science Signaling  03 Nov 2015:
Vol. 8, Issue 401, pp. ra111
DOI: 10.1126/scisignal.aac5124
  • Fig. 1 ERBB4 ICD expression enriches for SREBP target genes and cholesterol biosynthesis.

    (A) GSEA in MCF10A cells expressing ERBB4 ICD CYT-1. Upper plot: Enrichment for REACTOME_CHOLESTEROL_BIOSYNTHESIS gene set (NES = 2.33, P = 1.47 × 10−4). Lower plot: Enrichment for HORTON_SREBF_TARGETS gene set (NES = 2.40, P < 0.0001). (B) GSEA in MCF10A cells expressing ERBB4 ICD CYT-2. Upper plot: Enrichment for REACTOME_CHOLESTEROL_BIOSYNTHESIS gene set (NES = 2.42, P < 0.0001). Lower plot: Enrichment for HORTON_SREBF_TARGETS gene set (NES = 2.32, P < 0.0001). FDR, false discovery rate.

  • Fig. 2 NRG1 activates SREBP-2 cleavage and enhances expression of cholesterogenic genes.

    (A) Reverse transcription polymerase chain reaction (RT-PCR) of HMGCR, HMGCS1, or LDLR in T47D cells incubated in LPDS for 24 hours followed by concomitant NRG1 (50 ng/ml) treatment for the final 2 hours. +LDL, cells were pretreated with LDL (50 μg/ml) for 4 hours before addition of NRG1 to the media. Data are means ± SD from three experiments. (B) Immunoblot of SREBP-2 cleavage in T47D cells treated as in (A). p, uncleaved SREBP-2 precursor; m, cleaved SREBP-2 mature form. Blots are representative of three experiments. (C) Immunoblot of HMGCR and LDLR in T47D cells incubated in LPDS for 24 hours followed by concomitant NRG1 (50 ng/ml) treatment for the final 0 to 6 hours. +serum, cells were incubated in the presence of fetal bovine serum (FBS). Positive control cells (+simv) were incubated in LPDS and 1 μM simvastatin for 24 hours. Blots are representative of three experiments. (D) Immunoblot of SREBP-2 cleavage and HMGCR abundance in T47D cells incubated in LPDS for 48 hours with concomitant NRG1 (50 ng/ml) treatment for the final 0.5 to 24 hours. Positive control cells (+simv) were incubated in LPDS along with 1 μM simvastatin for 24 hours. long, longer exposure. Arrowhead marks ICD; FL marks full-length ERBB4. (E) Immunoblot of HMGCR abundance and SREBP-2 cleavage in T47D cells incubated in LPDS along with PF-429242 for 24 hours and then concomitant NRG1 (50 ng/ml) treatment for the final 6 hours. Blots are representative of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 NRG1 induces SREBP-2 cleavage through ERBB kinases, independent of AKT and mTORC1.

    (A) SREBP-2 cleavage in T47D cells incubated in LPDS for 24 hours followed by concomitant NRG1 (50 ng/ml) treatment for the final 2 hours. Cells were pretreated for 30 min with DMSO control or agents indicated (1 μM) before the addition of NRG1 and immunoblotted for SREBP-2 and signaling molecules as marked. Y, Tyr residue. (B) Immunoblot of SREBP-2 cleavage and phosphorylation of p70 S6 kinase (P-S6K) and ERBB4 in T47D cells incubated in LPDS for 24 hours followed by concomitant NRG1 (50 ng/ml) treatment for the final 2 hours. Cells were pretreated for 30 min before the addition of NRG1 with DMSO or rapamycin. p, precursor; m, mature form of SREPB-2. (C) Immunoblot of HMGCR protein abundance and phosphorylation of ERBB4 Tyr1056 in T47D cells incubated in LPDS for 24 hours followed by NRG1 (50 ng/ml) treatment for the final 6 hours. Cells were pretreated for 30 min before the addition of NRG1 with DMSO (control), rapamycin (100 nM), or other agents indicated at a concentration of 1 μM. (D) Immunoblot of SREBP-2 cleavage and phosphorylation of ERBB4 Tyr1056 in T47D cells incubated in LPDS for 24 hours followed by NRG1 (50 ng/ml) treatment for the final 2 hours. Cells were treated as in (C). (E) Immunoblot of SREBP-2 cleavage, phosphorylation of ERBB4 Tyr1056, and phosphorylation of p70 S6 kinase in T47D cells incubated in LPDS for 24 hours followed by NRG1 (50 ng/ml) treatment for the final 2 hours. Cells were treated as in (C) in the presence or absence of NRG1. Blots are representative of two (A) or three (B to E) experiments.

  • Fig. 4 NRG1-induced HMGCR is similar for ERBB4 juxtamembrane domain isoforms JM-a and JM-b.

    Immunoblot of ERBB4 cleavage and HMGCR abundance in MCF10A cells expressing pINDUCER20 encoding DOX-inducible JM-a or JM-b CYT-2 isoforms of ERBB4. Cells were incubated in Opti-MEM reduced serum media in the presence or absence of DOX (5 ng/ml) for 24 hours followed by concomitant treatment with DMSO, NRG1 (50 ng/ml), or PMA (100 ng/ml) for the final 3 hours. Arrowheads mark ICD; FL marks full-length ERBB4; long indicates long exposure. Blots are representative of three experiments.

  • Fig. 5 NRG1 activates SREBP-2 through ERBB4.

    (A) Immunoblot of SREBP-2 cleavage ERBB phosphorylation, and HMGCR in T47D cells incubated in LPDS for 24 hours followed by concomitant NRG1 (N; 50 ng/ml) or EGF (E; 50 ng/ml) treatment for the times indicated. +simv, control cells incubated in LPDS plus 1 μM simvastatin for 24 hours; p, uncleaved SREBP-2 precursor; m, cleaved SREBP-2 mature form. (B) Immunoblot of ERBB phosphorylation and HMGCR in T47D cells incubated in LPDS for 24 hours followed by concomitant NRG1 (N; 50 ng/ml) or EGF (E; 50 ng/ml) treatment for the final 6 hours. Negative and positive control cells were incubated, respectively, in the presence of FBS (+serum) or in LPDS plus 1 μM simvastatin (+simv) for 24 hours. (C) Immunoblot of ERBB phosphorylation and HMGCR in T47D cells incubated in LPDS for 24 hours followed by concomitant NRG1 (N; 50 ng/ml) or EGF (E; 50 ng/ml) treatment for the times indicated. (D) Immunoblot of ERBB phosphorylation and HMGCR in MCF10A cells harboring pINDUCER20 ERBB4 JM-a CYT-2 incubated in Opti-MEM reduced serum medium in the absence or presence of DOX (50 ng/ml) for 24 hours followed by concomitant NRG1 (N; 50 ng/ml) or EGF (E; 50 ng/ml) treatment for the final 6 hours. Immunoblots are representative of two (C) or three (A, B, and D) experiments.

  • Fig. 6 NRG1 increases LDL binding and uptake, and enhances biosynthesis of cholesterol from [2-13C]acetate precursor.

    (A and B) Flow cytometry analysis of diI-LDL binding (A) and uptake (B) in T47D cells incubated in LPDS or in the presence of FBS for 24 hours. During the final 6 hours, cells were treated with or without NRG1 (50 ng/ml). Data are representative of three experiments. Data are means ± SD with data points from each trial shown (filled circles, squares, and triangles represent data from trials 1, 2, and 3, respectively). n.s., not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001. (C and D) Cholesterol-trimethylsilane isotopomer spectra from T47D cells incubated without (-) or with NRG1 (NRG) for 6 hours (C) or 24 hours (D), or in the presence of simvastatin (Simv) to inhibit cholesterol synthesis. NRG promoted cholesterol synthesis from [2-13C]acetate as evidenced by the higher fractional abundance of [13C]cholesterol (M +3→M +7), whereas simvastatin inhibited cholesterol synthesis from [1-13C]acetate resulting in less 13C incorporation. Shown are representative data from the two replicates in one of three experiments described in table S1.

  • Fig. 7 Model of ERBB4 activation of SREBP-2/cholesterol signaling.

    NRG1 activates ERBB4 and induces proteolytic cleavage and release of membrane-anchored and soluble forms of the ICD. FL and ICD ERBB4 contribute to SREBP-2 activation through PI3K signaling pathways and, hypothetically, through direct interactions. ERBB4 enhances SREBP-2 cleavage through ERBB kinase activity but independent of AKT and mTORC1, resulting in increased expression of cholesterogenic genes (including HMGCR, HMGCS1, and LDLR) and increased LDL uptake and cholesterol synthesis. EGFR regulates fatty acid synthesis and increases LDLR expression through SREBP-1 (2426), so activated EGFR might also regulate cholesterogenesis in parallel with ERBB4.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/401/ra111/DC1

    Fig. S1. Gene lists for cholesterol/SREBP enrichment in the ERBB4 ICD data set.

    Fig. S2. Fatty acid synthesis genes in the ERBB4 ICD data set.

    Fig. S3. NRG1 transcriptionally activates expression of SREBP-regulated genes and induces SREBP-2 cleavage.

    Fig. S4. PF-429242 reduces SREBP-2 cleavage and does not inhibit ERBB4 phosphorylation.

    Fig. S5. Impact of NRG1 and PMA on cleavage of ERBB4 juxtamembrane isoforms JM-a and JM-b.

    Fig. S6. Regulation of cholesterogenic gene mRNA and HMGCR protein by EGF and NRG1 in MCF10A cells.

    Table S1. Determination of cholesterol biosynthesis by ISA.

  • Supplementary Materials for:

    Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake

    Jonathan W. Haskins, Shannon Zhang, Robert E. Means, Joanne K. Kelleher, Gary W. Cline, Alberto Canfrán-Duque, Yajaira Suárez, David F. Stern*

    *Corresponding author. E-mail: df.stern{at}yale.edu

    This PDF file includes:

    • Fig. S1. Gene lists for cholesterol/SREBP enrichment in the ERBB4 ICD data set.
    • Fig. S2. Fatty acid synthesis genes in the ERBB4 ICD data set.
    • Fig. S3. NRG1 transcriptionally activates expression of SREBP-regulated genes and induces SREBP-2 cleavage.
    • Fig. S4. PF-429242 reduces SREBP-2 cleavage and does not inhibit ERBB4 phosphorylation.
    • Fig. S5. Impact of NRG1 and PMA on cleavage of ERBB4 juxtamembrane isoforms JM-a and JM-b.
    • Fig. S6. Regulation of cholesterogenic gene mRNA and HMGCR protein by EGF and NRG1 in MCF10A cells.
    • Table S1. Determination of cholesterol biosynthesis by ISA.

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    Citation: J. W. Haskins, S. Zhang, R. E. Means, J. K. Kelleher, G.W. Cline, A. Canfrán-Duque, Y. Suárez, D. F. Stern, Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake. Sci. Signal. 8, ra111 (2015).

    © 2015 American Association for the Advancement of Science

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