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

Providing cholesterol for proliferation

Although cholesterol has received a lot of bad press, this lipid molecule is actually an essential component of cellular membranes. Growing or dividing cells need more cholesterol than quiescent cells. The activity of epidermal growth factor receptor (EGFR) family members stimulates cell proliferation in physiological and pathophysiological contexts, such as cancer. Haskins et al. found that neuregulin 1 (NRG1)–mediated activation of the EGFR member ERBB4 stimulated the transcription factor SREBP-2, which enhanced the expression of the receptor needed to uptake cholesterol-rich low-density lipoproteins and genes involved in cholesterol biosynthesis in cultured breast epithelial cells. Pharmacological inhibition of EGFR activity or SREBP-2 activity suppressed the NRG1-mediated induction of cholesterol synthesis–related genes. Thus, EGFR signaling alters cellular lipid metabolism, enabling cells to acquire or synthesize molecules necessary for proliferation.


Cholesterol is a lipid that is critical for steroid hormone production and the integrity of cellular membranes, and, as such, it is essential for cell growth. The epidermal growth factor receptor (EGFR) family member ERBB4, which forms signaling complexes with other EGFR family members, can undergo ligand-induced proteolytic cleavage to release a soluble intracellular domain (ICD) that enters the nucleus to modify transcription. We found that ERBB4 activates sterol regulatory element binding protein-2 (SREBP-2) to enhance low-density lipoprotein (LDL) uptake and cholesterol biosynthesis. Expression of the ERBB4 ICD in mammary epithelial cells or activation of ERBB4 with the ligand neuregulin 1 (NRG1) induced the expression of SREBP target genes involved in cholesterol biosynthesis, including HMGCR and HMGCS1, and lipid uptake, LDLR, which encodes the LDL receptor. Addition of NRG1 increased the abundance of the cleaved, mature form of SREBP-2 through a pathway that was blocked by addition of inhibitors of PI3K (phosphatidylinositol 3-kinase) or dual inhibition of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, but not by inhibition of AKT or mTORC1. Pharmacological inhibition of the activity of SREBP site 1 protease or of all EGFR family members (with lapatinib), but not EGFR alone (with erlotinib), impaired NRG1-induced expression of cholesterol biosynthesis genes. Collectively, our findings indicated that activation of ERBB4 promotes SREBP-2–regulated cholesterol metabolism. The connections of EGFR and ERBB4 signaling with SREBP-2–regulated cholesterol metabolism are likely to be important in ERBB-regulated developmental processes and may contribute to metabolic remodeling in ERBB-driven cancers.


The epidermal growth factor receptor (EGFR or ERBB) family member ERBB4 is essential for normal cardiac, neuronal, and mammary development and is activated by mutation in several cancers (14). As for other receptor kinases, ligand-induced phosphorylation of Tyr sites in ERBB4 recruits downstream signaling proteins. In addition, ERBB4 is unique in the ERBB family of receptor tyrosine kinases (RTKs) in that juxtamembrane-a (JM-a) spliced isoforms undergo ligand-induced ecto-domain and intramembrane proteolysis to release a soluble intracellular fragment that enters the nucleus and modifies transcription (57). In this context, ERBB4 can act as both a nuclear chaperone [such as for the transcription factor STAT5 (signal transducer and activator of transcription 5)] and a transcriptional modulator [such as for interactions with estrogen receptor, yes-associated protein (YAP), the transcription factor AP-2, the transcriptional coregulator TRIM28/KAP1, and TAB2 in complex with the transcriptional coregulator/N-CoR corepressor] (6, 811).

ERBB4 is alternatively spliced at two sites, a juxtamembrane region and a cytoplasmic (CYT) region, to generate four receptor isoforms. The JM-a isoform contains a proteolytic cleavage site for tumor necrosis factor–α converting enzyme (TACE, also known as ADAM17) that is absent in the JM-b isoform (12). Activation of TACE by ERBB4 ligands [heparin-binding EGF-like growth factor (HB-EGF), betacellulin, and neuregulins (NRG1–4)], phorbol esters, or other agonists induces shedding of the extracellular domain (ECD) of the receptor, leaving a membrane-embedded 80-kD isoform (m80) (13, 14). ECD shedding of JM-a (but not JM-b) isoforms enables intramembrane proteolysis at a γ-secretase cleavage site, which releases the soluble s80 form of the intracellular domain (ICD) (13). Tissue-specific alternative splicing to produce the ERBB4 JM-a isoform endows cells with the ability to signal through the ICD and couples the ERBBs to new avenues of signaling in addition to traditional RTK signaling at the membrane. Because the ERBBs promiscuously cross-activate when coexpressed, all ERBB receptors have the potential to signal through the ERBB4 JM-a ICD.

Alternative splicing of the ICD produces isoforms that differ by only 16 amino acids included in CYT-1, but absent in CYT-2. This region contains a predicted binding site for the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and a PPxY motif that overlap (12). The small amino acid sequence difference between CYT-1 and CYT-2 potentiates divergent biological properties: in tissue culture and mouse models, CYT-1 promotes mammary differentiation and survival phenotypes, whereas CYT-2 induces proliferation (1518). Expression of the CYT-2 isoform is enriched in the ER+HER2 subtype of breast cancer (19).

In a transcriptional analysis of pathways activated by full-length ERBB4 and the ICD of ERBB4 JM-a isoforms, we found that both full-length ERBB4 and ICD isoforms CYT-1 and CYT-2 increase expression of several genes associated with cholesterol metabolism, including those encoding 3-hydroxy-3-methylglutaryl (HMG)–coenzyme A (CoA) reductase (HMGCR), HMG-CoA synthase 1 (HMGCS1), and low-density lipoprotein receptor (LDLR) (18).

Cholesterol is an essential component of cell membranes and acts as a signaling molecule, as a precursor to steroid hormones, and as a major nutritional source for neonates in milk (20, 21). Cholesterol homeostasis is coordinated by sterol regulatory element binding proteins (SREBPs), which promote expression of genes encoding enzymes for de novo cholesterol production via the mevalonate pathway and expression of the LDLR to enhance extracellular lipoprotein uptake (22). When cholesterol abundance is high, SREBPs bind to SREBP cleavage-activating protein (SCAP) in the endoplasmic reticulum (ER), which forms a complex with insulin-induced gene (INSIG) proteins that sequester SREBPs in the ER and prevent cleavage. When intracellular cholesterol accumulation is low, SCAP transports SREBPs from the ER to the Golgi apparatus, where they are subsequently cleaved to release N-terminal fragments that bind to promoters and activate gene transcription (23).

The ERBBs have been linked to SREBP signaling. EGF activates fatty acid synthase through SREBP-1 in prostate and breast carcinoma cells and in glioblastoma (GBM) to enhance fatty acid synthesis (2426). In GBM, activated EGFR induces cleavage of SREBP-1 after 6 hours of EGF treatment and increases over 24 hours. EGFR activation of SREBP-1 occurs through PI3K-AKT pathway signaling (26) and is independent of mammalian target of rapamycin (mTOR) complex 1 (mTORC1) in GBM cell lines (25). Targeting fatty acid synthesis reduces tumor growth in a xenograft mouse model of EGFR-activated GBM (25). EGFRvIII activates a PI3K- and SREBP-1–dependent tumor survival pathway by increasing LDLR in GBM through SREBP-1 but not SREBP-2 (27). Targeting LDLR with a liver X receptor agonist causes LDLR degradation and tumor cell death in an in vivo GBM model (27). EGFR has not been reported to activate SREBP-2, and there is little information on the intersection of ERBB signaling with cholesterogenic pathways.

NRG1-activated ERBB4 regulation of cholesterogenic genes that we identified by transcriptional profiling (10, 18) could occur through an SREBP-dependent or SREBP-independent mechanism. Because SREBP-2 is the major regulator of genes promoting cholesterol synthesis, NRG1-induced increases in expression of these genes may work through enhanced SREBP-2 cleavage. Alternatively, ERBB4 might directly bind SREBP-2 and enhance its nuclear translocation or the transcriptional activation of SREBP-2 target genes. To address these possibilities, we investigated the ability of the ERBB signaling network to activate the SREBP-2–regulated cholesterol biosynthetic pathway through ERBB4. The results indicate that NRG1 binding to ERBB4 activates SREBP-2 by inducing cleavage and that this is accompanied by a corresponding increase in the expression of cholesterol metabolic enzymes, LDL uptake, and cholesterol biosynthesis.


ERBB4 ICD enriches for SREBP-regulated cholesterol biosynthesis genes

Our earlier transcriptional analysis revealed that ERBB4 induces genes involved in cholesterol metabolism (18). Both NRG1-activated full-length ERBB4 and the constitutively active ERBB4 ICD CYT-1 and CYT-2 isoforms enhance expression of genes associated with cholesterol metabolism, including HMGCR, HMGCS1, DHCR7, DHCR24, LDLR, FDFT1, FDPS, IDI1, MVD, SQLE, LSS, NSDHL, SC5DL, INSIG1, ACLY, and ACSS2 (fig. S1).

To further analyze SREBP-dependent cholesterol pathway activation, we used gene set enrichment analysis (GSEA) to test for overlap of ERBB4 ICD–induced genes and genes associated with cholesterol metabolism. Engineered expression of ERBB4 ICD CYT-1 or CYT-2 in MCF10A cells, which do not express endogenous ERBB4, induced enrichment of transcripts associated with the REACTOME_CHOLESTEROL_BIOSYNTHESIS gene signature [normalized enrichment score (NES) = 2.33, P = 1.46 × 10−4 (CYT-1), or NES = 2.42, P < 0.0001 (CYT-2)] and the HORTON_SREBF_TARGET gene signature [NES = 2.40, P < 0.0001 (CYT-1), or NES = 2.32, P < 0.0001 (CYT-2)] (Fig. 1), and the core enriched genes in each data set were identified (fig. S1, A and B).

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.

SREBPs [also denoted sterol regulatory element–binding transcription factors (SREBFs)] are master regulators of genes involved in cholesterol metabolism and fatty acid synthesis. The SREBP isoform SREBP-1c activates genes involved in fatty acid synthesis, including FASN (encoding fatty acid synthase), ACLY [encoding ATP (adenosine 5′-triphosphate) citrate lyase], and ACC (encoding acetyl-CoA carboxylase), whereas SREBP-2 predominantly activates genes involved in cholesterol metabolism (including HMGCR, HMGCS1, and LDLR) (20). SREBP-1a is capable of activating both fatty acid synthesis and cholesterol metabolism (28). To test whether ERBB4 broadly activates multiple SREBPs, we analyzed the ERBB4 ICD transcriptional data for increases in the expression of genes encoding proteins involved in fatty acid synthesis. We found no significant enrichment for the fatty acid synthesis gene sets KEGG_FATTY_ACID_METABOLISM or KEGG_BIOSYNTHESIS_OF_UNSATURATED_FA. Only a few fatty acid synthesis genes were significantly altered upon expression of ERBB4 CYT-1 or CYT-2 ICD (fig. S2, A and B). Unlike cholesterol metabolism genes, fatty acid synthesis genes were both up- and down-regulated (fig. S2, C and D).

NRG1 increases expression of SREBP-regulated genes

Because the ERBB4 ICD used for these experiments is encoded by an artificial construct, we next determined whether stimulation of full-length ERBB4 with its ligand NRG1 also activates these cholesterol metabolism genes in the T47D mammary carcinoma cell line, which endogenously expresses ERBB4. Full-length ERBB4 might stimulate cholesterogenic genes either by activation of SREBPs or through SREBP-independent mechanisms, so we compared the ability of NRG1 to increase SREBP target genes under conditions where SREBP is active or inhibited.

Use of lipoprotein-deficient serum (LPDS) reduces the bioavailability of exogenous cholesterol and therefore activates SREBPs. The increased mRNA expression of the SREBP-regulated genes HMGCR, HMGCS1, and LDLR observed in cells grown in LPDS was reduced when cells were supplemented with exogenous cholesterol in the form of low-density lipoproteins (LDLs) (fig. S3A). LDLs inhibit the processing of SREBPs by inducing the formation of a complex containing SCAP, SREBP, and INSIG1 that sequesters SREBPs in the ER (23).

Under lipoprotein-deficient (LPDS) conditions in which SREBPs are activated, NRG1 further increased HMGCR, HMGCS1, and LDLR mRNA an additional two- to threefold (fig. S3A). This induction was more apparent with 2 hours than with 4 hours of NRG1 treatment. Under conditions inhibiting SREBP activation (+LDL), LDL reduced but did not completely abolish NRG1 induction of HMGCR, HMGCS1, and LDLR mRNA (fig. S3A). NRG1-stimulated SREBP target gene expression was slightly reduced after 4 hours of LDL incubation (2 hours before treatment and 2 hours with LDL and NRG1 co-incubation) compared with LPDS controls, and was reduced by ~50% after 6 hours of LDL treatment (2-hour LDL and 4-hour LDL plus NRG1 co-incubation) (fig. S3A).

Because LDL and NRG1 regulate SREBP target genes with different kinetics, we conducted experiments during periods of maximal NRG1 gene expression (2 hours) and maximal LDL-induced inhibition of SREBP activity (6 hours). Two hours of NRG1 treatment increased HMGCR expression twofold, HMGCS1 expression twofold, and LDLR expression threefold (Fig. 2A). NRG1 weakly increased HMGCR and HMCS1 expression in the presence of LDL. However, in the presence of exogenous LDL, NRG1 still induced a substantial threefold increase in LDLR expression despite lower absolute amounts of LDLR expression (Fig. 2A).

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.

NRG1 stimulates SREBP-2 cleavage

Because NRG1 induces multiple SREBP-2 target genes, we determined whether NRG1 activates SREBP-2 cleavage. Indeed, in LPDS, incubation with NRG1 for 2 hours induced a marked increase in the amount of mature, cleaved SREBP-2 (Fig. 2B). However, 4 hours of NRG1 treatment did not strongly affect SREBP-2 cleavage (fig. S3B). Addition of LDL reduced the absolute amount of cleaved SREBP-2 in the absence of NRG1. NRG1 still induced a twofold increase in cleaved SREBP-2 in the presence of LDL with 2 hours in NRG1, but not 4 hours (Fig. 2B and fig. S3B). Together, these data indicate that NRG1 activates SREBP-2 to induce SREBP-2–regulated genes and that NRG1 may partially override sterol-mediated inhibition of SREBP-2 cleavage in the presence of cholesterol in the media in the form of LDL.

NRG1 induces acute activation of SREBP-2 followed by increases in protein expression of cholesterogenic genes

To determine whether NRG1-dependent increases in expression of SREBP-2 target genes are recapitulated at the protein level, we analyzed HMGCR and LDLR protein expression. T47D cells were incubated with NRG1 for up to 6 hours after 24 hours of lipoprotein depletion. Incubation in serum (negative control) strongly inhibited HMGCR protein expression. NRG1 induced an increase in both HMGCR and LDLR protein abundance within as little as 4 hours (Fig. 2C). The HMGCR inhibitor simvastatin was used as a positive control for HMGCR expression because inhibition of cholesterol biosynthesis with simvastatin activates the SREBP pathway, thereby increasing HMGCR protein abundance. The increases in expression elicited by NRG1 augmented that which was induced by lipoprotein depletion, suggesting that ERBB4 can activate cholesterol biosynthesis genes beyond the amounts induced by lipoprotein depletion.

We next compared the timing of SREBP-2 cleavage and changes in HMGCR expression in NRG1-stimulated T47D cells (Fig. 2D). NRG1 induced SREBP-2 cleavage within as little as 30 min, and the mature form of SREBP-2 continued to increase in abundance through 2 hours of stimulation. Abundance of mature SREBP-2 returned to baseline amounts induced by lipoprotein depletion after 4 hours of NRG1 treatment. HMGCR expression increased through 6 hours of NRG1 treatment and returned to baseline within 24 hours. ERBB4 cleavage (Fig. 2D, arrowhead) was detected 30 min after NRG1 treatment, was sustained through 1 hour, and returned to baseline at 2 hours (Fig. 2D). The presence of the ERBB4 ICD temporally preceded the increase in the mature form of SREBP-2, which was followed by increased abundance of HMGCR. Therefore, transient activation of ERBB4 was sufficient to activate SREBP-2 and induce persistent expression of cholesterogenic genes.

Inhibition of SREBP cleavage reduces NRG1-induced expression of HMGCR

Having determined that NRG1 enhances SREBP-2 cleavage, we next investigated whether SREBPs mediate the induction of cholesterol metabolism genes in response to NRG1. We used a serine protease inhibitor (PF-429242) that is selective for SREBP site 1 protease (S1P) to block SREBP cleavage. Cotreatment of T47D cells with LPDS and PF-429242 for 24 hours reduced the abundance of both precursor and mature SREBP-2 protein (Fig. 2E). Because SREBP-2 activates its own promoter, leading to feed-forward induction, inhibition of SREBP-2 cleavage was expected to reduce total SREBP-2 protein (29). PF-429242 reduced the amount of HMGCR protein abundance induced by 6 hours of NRG1 stimulation in a dose-dependent fashion (Fig. 2E). NRG1 increased HMGCR relative to cells cultured in PF-429242 alone, so NRG1 can still induce SREBP-2 cleavage despite reduced expression of SREBP-2. NRG1 did not affect SREBP-2 cleavage at the 6-hour time point, which is consistent with the data presented above.

We confirmed that PF-429242 treatment blocked SREBP-2 cleavage and did not inhibit ERBB4 phosphorylation or cleavage after 2 hours of NRG1 stimulation (fig. S4, A and B). The highest dose of PF-429242 used enhanced the accumulation of the ERBB4 cleavage product (fig. S4B), which was nearly undetectable in the absence of inhibitor after 2 hours of NRG1 treatment (Fig. 2D). Overall, these data confirm that NRG1 activation of SREBP target genes is at least partially dependent on SREBPs. We cannot rule out the possibility that SREBP-1 is also involved because S1P also cleaves the SREBP-1 isoform.

Collectively, these data support the conclusion that NRG1 activates SREBP-2 target genes involved in the biosynthesis and uptake of cholesterol, and does so beyond amounts induced by lipoprotein depletion.

SREBPs mediate NRG1-induced activation of cholesterogenic genes through ERBB kinases and PI3K, independent of AKT and mTORC1

The mechanism by which SREBP activation is regulated by growth factor receptors (such as the insulin receptor and EGFR) is not completely understood. In prostate carcinoma and GBM cell lines and tumors, EGFR activates SREBP-1 to increase fatty acid synthesis through a PI3K- and AKT-dependent mechanism that in GBM is mTORC1-independent (25, 26). In keratinocytes, EGF and TGFα (transforming growth factor α) induce HMGCR through mitogen-activated protein kinase (MAPK) pathways, but the involvement of SREBPs in this process was not determined (30).

To test whether ERBB kinase activity is required for NRG1 activation of SREBP-2 cleavage, we used a pan-ERBB kinase inhibitor, lapatinib, and an EGFR kinase inhibitor, erlotinib. In control cells treated with dimethyl sulfoxide (DMSO), NRG1 increased cleavage of SREBP-2 and phosphorylation of ERBB4, EGFR, AKT, and p70 S6 kinase (Fig. 3A). Inhibition of ERBB kinases with lapatinib reduced the phosphorylation of ERBB4 and EGFR and diminished NRG1-induced cleavage of SREBP-2. Lapatinib also lowered baseline and NRG1-induced phosphorylation of AKT and p70 S6 kinase. Treatment with the EGFR inhibitor erlotinib did not block NRG1-induced phosphorylation of EGFR, presumably through cross-phosphorylation of EGFR by ERBB4. However, erlotinib did reduce baseline EGFR phosphorylation. EGFR inhibition did not greatly affect NRG1 activation of SREBP-2 cleavage. Erlotinib lowered baseline and NRG1-induced phosphorylation of p70 S6 kinase but had no effect on AKT or ERBB4 phosphorylation.

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.

We next examined whether NRG1 activates SREBP-2 through the PI3K pathway, because inhibitors of mediators of this pathway (PI3K, AKT, and mTORC1) can reduce SREBP cleavage in various cellular contexts. PI3K inhibition with GDC-0941 reduced baseline and NRG1-induced phosphorylation of AKT and p70 S6 kinase, raised baseline SREBP-2 cleavage, and prevented any NRG1-induced increase in mature SREBP-2 (Fig. 3A). AKT inhibition with MK-2206 strongly reduced baseline and NRG1-induced AKT phosphorylation, but—unlike PI3K inhibition—did not affect NRG1-induced activation of SREBP-2 cleavage (Fig. 3A). Likewise, mTORC1 inhibition with rapamycin caused a dose-dependent reduction in p70 S6 kinase phosphorylation, but did not affect NRG1-induced SREBP-2 cleavage (Fig. 3B).

Having ruled out contributions from AKT and mTORC1, we tested a more extensive panel of PI3K and mTOR inhibitors for effects on ERBB4 phosphorylation and NRG-dependent HMGCR production (Fig. 3C) and SREBP-2 cleavage (Fig. 3D). Four PI3K inhibitors [wortmannin (PIKK superfamily), GDC-0941, BYL-719 (PI3Kα), and a dual PI3K and mTOR inhibitor] reduced HMGCR abundance in the presence of NRG1. Of these, wortmannin, BYL-719, and the dual PI3K and mTOR inhibitor also blocked NRG1-induced cleavage of SREBP-2. Rapamycin (mTORC1), AZD-8055 (mTORC1 and mTORC2), MK-2206 (AKT inhibitor), and SB-203580 (p38 MAPK) all partially reduced HMGCR, and only AZD-8055 (mTORC1 and mTORC2) also consistently blocked NRG1-induced cleavage of SREBP-2. The PI3K inhibitors wortmannin and BYL-719 and the dual mTORC1 and mTORC2 inhibitor AZD-8055 were the most effective agents at blocking both NRG1-induced cleavage of SREBP-2 and up-regulation of HMGCR abundance. Each of these drugs reduced NRG1-dependent phosphorylation of p70 S6 kinase without substantially affecting NRG1-dependent ERBB4 phosphorylation (Fig. 3E). Overall, these results indicate that NRG1 activates SREBP-2 through a mechanism requiring ERBB4 kinase activity and PI3K, independently of AKT or mTORC1.

ERBB4 cleavage is not required for NRG1 increases in HMGCR

The ERBB4 ICD produced by cleavage of JM-a isoforms has unique intracellular signaling properties. Because both the ERBB4 ICD and the full-length receptor may be capable of activating SREBP signaling, we investigated whether cleavage and release of the ERBB4 ICD are required for SREBP-2 signaling. We produced MCF10A pINDUCER20 cell lines expressing doxycycline (DOX)–inducible ERBB4 JM-a CYT-2 or the TACE-insensitive spliced isoform ERBB4 JM-b CYT-2. As expected, NRG1 or PMA (phorbol 12-myristate 13-acetate) treatment of JM-a, but not JM-b, ERBB4 induced production of the ~80-kD JM-a cleavage product (fig. S5). In pINDUCER20-ERBB4 MCF10A cells cultured without DOX, NRG1 slightly enhanced production of HMGCR (Fig. 4), likely either through leaky production of ERBB4 by the uninduced pINDUCER-regulated complementary DNAs (cDNAs) or through activity of endogenous ERBB3. DOX induction of ERBB4 enhanced NRG1-dependent accumulation of HMGCR, and this occurred comparably in cells expressing either JM-a or JM-b (Fig. 4). Despite inducing ERBB4 cleavage in JM-a ERBB4 cells, PMA did not consistently increase HMGCR abundance above baseline (Fig. 4). Hence, ERBB4 cleavage mediated exclusively through the JM-a isoform is neither necessary nor sufficient (in the absence of NRG1) for up-regulation of HMGCR.

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.

NRG1 activates SREBP-2 through ERBB4

EGF activates SREBP-1 cleavage in prostate carcinoma cells and GBM. We investigated the impact of EGF on SREBP-2 by directly comparing the ability of EGF and NRG1 to induce SREBP-2 cleavage and up-regulation of HMGCR in T47D cells. NRG1 increased mature SREBP-2 after 2 hours of treatment but not 24 hours (Fig. 5A). EGF did not alter the amount of mature SREBP-2 compared with baseline accumulation induced in LPDS at 2 or 24 hours (Fig. 5A). NRG1 increased HMGCR protein abundance, and EGF increased the abundance of HMGCR. NRG1 consistently induced a greater increase in HMGCR protein abundance than did EGF, and HMGCR abundance correlated with ERBB4 phosphorylation (Fig. 5B).

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.

After 2 or 6 hours of incubation with NRG1, NRG1-induced ERBB4 phosphorylation was strong, but EGF-induced EGFR phosphorylation was considerably weaker (Fig. 5, A and B). Because T47D cells express roughly similar amounts of EGFR and ERBB4, this may be explained by suboptimal activity of EGF or by differences in timing of EGFR and ERBB4 down-regulation and/or dephosphorylation. Hence, we compared NRG1- or EGF-induced phosphorylation of ERBB4 and EGFR after 10 min and 6 hours of stimulation (Fig. 5C). EGFR and ERBB4 were similarly phosphorylated with 10 min of incubation with their respective ligands, but EGFR phosphorylation at Tyr1068 decayed much more rapidly than did ERBB4 phosphorylation. Despite comparable initial activation of ERBB4 and EGFR by their respective ligands, NRG1 more robustly activated SREBP-2 than did EGF.

MCF10A cells express endogenous EGFR and ERBB3, but not endogenous ERBB4. As another approach to determining the ERBB4 dependence of the NRG1 response, we evaluated NRG1 activation of SREBP-2 in MCF10A cells engineered to express ERBB4 under control of DOX-inducible promoter. MCF10A pINDUCER20 ERBB4 JM-a CYT-2 cells were cultured in reduced serum media in the presence or absence of DOX for 24 hours and cocultured with NRG1 or EGF for the final 6 hours. DOX treatment induced ERBB4 CYT-2 protein abundance, which was undetectable in untreated cells (Fig. 5D and fig. S6, B and C). NRG1 increased the phosphorylation of ERBB4 and the abundance of HMGCR when ERBB4 was expressed (in the presence of DOX) but had no effect on HMGCR when ERBB4 was not expressed (in the absence of DOX). EGF, on the other hand, increased the phosphorylation of EGFR and the abundance of HMGCR regardless of the presence of ERBB4, indicating that EGF increases HMGCR abundance through EGFR in MCF10A cells.

To quantitatively compare the ability of NRG1 and EGF to activate SREBP target genes, we measured the ability of each ligand to induce LDLR, HMGCR, and HMGCS1 mRNA expression in MCF10A pINDUCER20 ERBB4 JM-a CYT-2 cells in the absence or presence of DOX (fig. S6, A and B). LDLR was induced comparably with EGF treatment regardless of whether ERBB4 expression was induced with DOX. Consistent with the HMGCR protein phenotype (Fig. 4D), NRG1 induced LDLR expression only when ERBB4 was present (+DOX). There were no changes in HMGCR or HMGCS1 mRNA expression in the presence of ERBB4. Collectively, these data demonstrate that NRG1 activates SREBP-2 through ERBB4.

NRG1 increases LDL binding to LDLR and LDL uptake

To determine whether NRG1-induced changes in cholesterol genes and SREBP-2 cleavage functionally affect cholesterol metabolism, we first examined the impact of NRG1 on binding of LDL to LDLR and on LDL uptake (Fig. 6, A and B). Incubation of T47D cells in LPDS for 24 hours increased LDL binding and uptake compared with cells grown in the presence of serum (FBS), demonstrating effective lipoprotein depletion (Fig. 6, A and B). Eight hours of incubation with NRG1 increased LDL binding and uptake compared with LPDS cultured cells. In the presence of serum, NRG1 had no effect on LDL binding, but there was a trend toward enhanced LDL uptake. These data are consistent with NRG1-induced SREBP-2 cleavage and activation of SREBP-2–regulated cholesterol genes and indicate that NRG1 has a functional impact on cholesterol metabolism.

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.

NRG1 enhances de novo cholesterol biosynthesis

We next evaluated the impact of NRG1 on de novo cholesterol biosynthesis. T47D cells in LPDS were cultured with NRG1 for 6 or 24 hours in the presence of heavy-labeled [2-13C]acetate or nonlabeled acetate (Fig. 6, C and D, and table S1). Relative 13C incorporation into cholesterol isotopomers was evaluated using gas chromatograph–mass spectrometry (GC-MS). Twenty-four hours of incubation with NRG1 significantly increased incorporation of 13C into cholesterol at peaks M +3 through M +7 (Fig. 6D and table S1). A similar trend was observed with 6 hours of NRG1 treatment, but was not statistically significant. In contrast, simvastatin treatment significantly reduced [13C]acetate conversion into cholesterol, in accordance with inhibition of HMGCR by this agent (Fig. 6, C and D, and table S1).

We used isotopomer spectral analysis (ISA) to estimate the parameters of cholesterol synthesis (31, 32) from the isotopomer labeling patterns (Fig. 6D). After 24 hours of culture with NRG1, ISA revealed a 20% increase in the fraction of newly synthesized cholesterol compared with control cells, but no significant increase at 6 hours of treatment (table S1). According to ISA, acetate contributed about half of the lipogenic acetyl CoA pool, and this value was not affected by NRG1 (table S1). Thus, the increased labeling of NRG1 cultured cells is the result of increased de novo synthesis. Overall, these findings demonstrate that NRG1 stimulates the mevalonate pathway, leading to increased cholesterol biosynthesis.


The ERBBs are major regulators of cell growth, a process that requires sufficient nutrients and metabolites. We found that NRG1-activated ERBB4 enhances cholesterogenic gene expression through SREBP-2, a master regulator of cholesterol metabolism. Activated ERBB4 increased the abundance of both mevalonate pathway enzymes and the LDLR, and significantly increased the binding and uptake of LDL and de novo cholesterol biosynthesis. NRG1 enhanced SREBP-2 cleavage and target gene expression above amounts induced by lipoprotein depletion. The reduction in NRG1-dependent effects on SREBP cleavage and cholesterogenic genes by LDL and serum, along with the sensitivity to protease inhibitor PF-429242, indicates that ERBB4 activates cholesterol metabolism through SREBP-2.

Our results suggest the model in Fig. 7. Both the ICD and full-length ERBB4 activate SREBP-2 signaling, but release of the ICD does not appear to be required. Multiple PI3K inhibitors blocked both NRG-1–induced SREBP-2 cleavage and increases in HMGCR protein abundance. Despite inhibition of AKT activity by MK-2206 and mTORC1 activity by rapamycin, NRG1 still increased SREBP-2 cleavage. This sets ERBB4 to SREBP-2 signaling apart from mechanisms of SREBP activation that are proposed to require AKT, including EGFR and insulin receptor signaling, and suggests that ERBB4 activates SREBP-2 through AKT-independent PI3K signaling arms. Because a dual mTORC1 and mTORC2 inhibitor, but not rapamycin (targeting mTORC1), blocked SREBP-2 signaling, it is possible that mTORC2 facilitates ERBB4 activation of SREBP-2.

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.

The ERBB4 ICD activates SREBP-2, independent of the cytoplasmic isoform (CYT-1 or CYT-2) that is expressed. The similar abilities of JM-a and JM-b isoforms to activate SREBP-2 imply that formation of the ERBB4 ICD is not required. This interpretation is not definitive without further experimentation, because γ-secretase cleavage of ERBB4 in lung alveolar cells does not require TACE activity (33). Nonetheless, it is possible that expression of soluble ICD in the original transcription profiling experiments (17) activated PI3K signaling and subsequent SREBP signaling. Work is under way to evaluate this hypothesis and more speculative possibilities including ERBB4 directly enhancing SREBP-2 activation, through stabilization of the SCAP complex with SREBP, dissociation of the SCAP complex away from INSIG1 allowing for ER-to-Golgi transport and SREBP cleavage, and/or promotion of nuclear import of mature SREBP-2.

ERBB4 activation of SREBP-2 appears to occur though a mechanism distinct from that of EGFR regulation of SREBP-1 (Fig. 7). NRG1, but not EGF, activates SREBP-2 cleavage in T47D cells, leading to concomitant increases in HMGCR. ERBB4 is required for NRG1-induced increases in HMGCR. Unlike EGFR activation of SREBP-1 in GBM cells, ERBB4 induction of SREBP-2 cleavage does not depend on AKT or EGFR kinase activity. NRG1 activates SREBP-2 cleavage acutely within 0.5 to 2 hours of NRG1 treatment in breast cancer cell lines, whereas EGF induces SREBP-1 cleavage over 6 to 24 hours in GBM cell lines (25). These data suggest that NRG1 and ERBB4 activate SREBP-2 distinctly from EGFR but do not rule out the possibility that EGFR might activate SREBP-1a to regulate cholesterol biosynthesis genes in parallel with or in place of ERBB4 to SREBP-2 in some tissues.

ERBBs were linked to metabolic regulation as early as 1965, when Stanley Cohen found that injection of EGF into newborn rodents induces fatty liver (including some increase in cholesteryl esters) (34), and now reinforced with the finding that the Dsk5 gain-of-function mutation in the EGFR induces fatty liver with elevated HMGCR and FAS in mice (35).

Several ERBB4-regulated biological processes might rely on SREBP-2 regulation of cholesterol metabolism. In the mammary gland, EGFR, ERBB2, and ERBB3 are important for ductal tissue expansion at puberty and in pregnancy, whereas ERBB4 plays an essential role in tissue differentiation and the expression of milk proteins during lactation through ICD activation of STAT5 (1, 2, 16). The lactating mouse mammary gland secretes a milk lipid equivalent to its entire body weight over a 20-day lactation cycle (36). ERBB4 might help coordinate production of milk proteins with the anabolic shift that occurs at onset of lactation by increasing expression of mevalonate pathway enzymes and LDLR.

In the brain, NRG1 stimulates myelination through ERBB4, and cholesterol is a major component of myelin. SREBP-2 might mediate the effects of NRG1 and ERBB4 in the brain, including neuronal cell migration, NMDA (N-methyl-d-aspartate) receptor signaling, timing of astrogenesis, and differentiation of radial glia. NRG1 and ERBB4 are both schizophrenia-linked genes. ERBB4 to SREBP-2 signaling could be altered in schizophrenia, where disruption of myelination is believed to contribute to pathogenesis (37).

ERBB4 is activated by mutation in several cancers including melanoma (14%, COSMIC) and lung cancer (5%, COSMIC). CD74-NRG1 fusions are amplified in a subset of human lung cancers, and ligand-activated ERBB4 is enriched in chemotherapy-resistant mouse models of lung cancer (38, 39). ERBB4 to SREBP-2 signaling may be co-opted in cancerous cells.

Increased mevalonate pathway activity and cholesterol abundance are seen in several cancers. In prostate cancer, cholesterol is elevated due to enhanced activity of the mevalonate pathway and activation of SREBPs (40, 41). High mRNA expression of HMGCR and other genes of the mevalonate pathway correlates with poor prognosis in primary breast cancer (42). Furthermore, up-regulation of the mevalonate pathway by mutant p53 is necessary and sufficient for induction of spheroids by nonmalignant mammary epithelial cells (43). Introduction of activated PI3K or KRAS into mammary cells up-regulates lipid biosynthesis through mTORC1 and mTORC2, leading to activation of SREBP-1 and SREBP-2, and knockdown experiments indicate the importance of both SREBP-1 and SREBP-2 on lipogenesis and proliferation (44). Ectopic expression of HMGCR, the rate-limiting enzyme in the mevalonate pathway, promotes cell growth and cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts (42). Therefore, ERBB4 activation of SREBP-2 has the potential to contribute to cancer cell growth and survival.

The union of the ERBB receptors, major regulators of cell growth and development, and SREBPs, master regulators of cholesterol and fatty acid homeostasis, sheds light on potential biological roles for ERBB to SREBP signaling in development and disease. ERBB4 can couple all expressed ERBB receptors to cholesterol metabolism through receptor cross-activation and is unusual in its ability to undergo cleavage and act as a transcriptional coactivator. The discovery that ERBB4 activates cholesterol metabolism through SREBP-2 highlights an underappreciated connection between the ERBBs and cell metabolism. The network of signaling interactions through which the four ERBBs modulate cholesterol and fat biosynthesis has important implications for cellular signaling and metabolism. The importance of ERBBs including ERBB4 as cancer drivers, and of SREBP pathways and cholesterol in cancer cell maintenance (4446) means that further elucidation of this signaling axis will provide insights into cancer metabolic dysregulation.



The following reagents were used: NRG1 (Sigma), EGF (Sigma), lapatinib (Selleck), erlotinib (LC Laboratories), GDC-0941 (Selleck), MK-2206 (Selleck), rapamycin (LC Laboratories), PF-429242 (Tocris), and simvastatin (Selleck).

Cell culture

T47D human breast cancer cells [American Type Culture Collection (ATCC)] were maintained in RPMI (Life Technologies) with 10% FBS, 1% penicillin/streptomycin, and insulin (5 μg/ml; Gibco). LPDS was prepared as described previously (47). Briefly, fumed silica was added to FBS (heat-inactivated, 70 mg of silica per milliliter of FBS) and was mixed overnight at 4°C. The mixture was ultracentrifuged at 11,290g for 10 min at 4°C to pellet lipoproteins, and the LPDS layer was transferred to a fresh tube and filter-sterilized twice. LPDS medium (10% LPDS, RPMI) was tested for the ability to induce HMGCR after 24 hours of treatment to confirm depletion of lipoproteins. MCF10A human breast cancer cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium/F12 (Life Technologies) with 5% horse serum, 1% penicillin/streptomycin, insulin (10 μg/ml; Gibco), EGF (20 ng/ml; Sigma), hydrocortisone (0.5 μg/ml; Sigma), and cholera toxin (100 ng/ml; Sigma).

Quantitative RT-PCR

RNA was isolated with the RNeasy Mini Plus Kit using QIAshredder columns (Qiagen). cDNA was prepared using the iScript Kit (Bio-Rad). Real-time PCR was performed using Universal TaqMan Master Mix (Applied Biosystems) coupled with TaqMan FAM-labeled probes and ran on a ViiA 7 RT-PCR machine (Life Technologies). Relative mRNA expression was determined using the 2−ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. The following TaqMan probes were used: LDLR (Hs00181192_m1), HMGCR (Hs00168352_m1), HMGCS1 (Hs00940429_m1), and GAPDH (Hs02758991_g1).


Cells were lysed in buffers described below that were supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors 2 and 3 (Sigma). For analysis of SREBPs (48), cells were lysed in SREBP lysis buffer [20 mM tris-HCl (pH 8.0), 120 mM KCl, 1 mM DTT (dithiothreitol), 2 mM EGTA, 0.1% Triton X-100, 0.5% NP-40]. Samples were diluted in 2× Laemmli sample buffer and incubated at 100°C for 5 min. For detection of HMGCR (49), cells were lysed in lysis buffer A [10 mM tris-HCl, 1% (w/v) SDS, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA]. Then, an equal volume of lysis buffer B was added [62.5 mM tris-HCl, 15% (w/v) SDS, 8 M urea, 15% glycerol, 100 mM DTT]. Samples were diluted in 6× Laemmli sample buffer (without 2-mercaptoethanol) and incubated at 37°C for 20 min. For analysis of LDLR (50), cells were lysed in LDLR lysis buffer [50 mM tris-HCl (pH 6.8), 2.6 mM CaCl2, 1% Triton X-100].

Samples were loaded onto 4 to 12% bis-tris gradient gels (NuPAGE, Life Technologies) and run in Mops buffer. Protein was transferred to polyvinylidene difluoride membranes at constant amperage at 500 mA for 1 hour. Membranes were blocked in 5% milk/TBST (HMGCR) or 5% bovine serum albumin/TBST (SREBP, LDLR). Membranes were incubated with horseradish peroxidase–conjugated secondary antibodies and developed by chemiluminescence (Pierce). The following antibodies were used for immunoblotting: SREBP-2 (BD Pharmingen, catalog no. 557037, mouse), HMGCR (from mouse hybridoma), LDLR (Cayman Chemical, catalog no. 10007665, rabbit), phosphorylated ERBB4 at Tyr1056 (Santa Cruz Biotechnology, sc-33040, rabbit), phosphorylated EGFR at Tyr1068 (Cell Signaling, catalog no. 3777, rabbit), EGFR (Santa Cruz Biotechnology, sc-03, rabbit), phosphorylated S6 kinase at Thr389 (Cell Signaling, catalog no. 9205, rabbit), p70 S6 kinase (Cell Signaling, catalog no. 9202, rabbit), phosphorylated AKT at Ser473 (Cell Signaling, catalog no. 4060, rabbit), AKT (Cell Signaling, catalog no. 9272, rabbit), and GAPDH (Santa Cruz Biotechnology, sc-25778, rabbit).

LDL uptake and binding assays

T47D cells were incubated for 24 hours in 10% LPDS medium or grown in the presence of 10% FBS. During the final 6 hours, cells were incubated with or without NRG1 (50 ng/ml). For LDL uptake (50), diI-LDL (30 μg/ml) alone or diI-LDL along with unlabeled LDL (600 μg/ml), to account for unspecific uptake, was added to the medium for 2 hours. Cells were washed with phosphate-buffered saline (PBS), trypsinized, and fixed in 1% paraformaldehyde (PFA)/PBS and stored at 4°C. For LDL binding, cells were treated with NRG1 for 6 hours and then incubated on ice for 30 min to stop LDLR endocytosis. Then, diI-LDL alone or diI-LDL along with unlabeled LDL, as indicated above, was added for 1.5 hours on ice. Cells were washed with PBS, trypsinized, and fixed in 1% PFA/PBS and stored at 4°C. LDL binding and uptake were analyzed by flow cytometry using the FACSCalibur (BD Biosciences) system. Specific diI-LDL uptake or binding was calculated using geometric mean after subtracting nonspecific LDL uptake or binding. Samples were normalized to LPDS-treated cells (51).

Cholesterol synthesis by ISA

Fractional amounts of newly synthesized cholesterol were determined from the mass isotopomer distribution of cholesterol from cells incubated for 6 and 24 hours with [2-13C]acetate (Cambridge Isotope Laboratories) (31, 32). T47D cells were grown in LPDS medium for 24 hours. Simvastatin (1 μM) was added to control wells simultaneously with LPDS. Medium was aspirated, and fresh LPDS was added along with NRG1 (50 ng/ml) and 2 mM acetate or [2-13C]acetate. After 6 or 24 hours of incubation with NRG1 and labeled acetate, cells were scraped into PBS, pelleted, and flash-frozen in liquid nitrogen. Cells were resuspended in 500 μl of water and disrupted by pulse sonication, and lipids were extracted in 3 ml of CH3Cl/CH3OH (2:1). The organic phase was dried under N2 gas and silylated with 150 μl of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) at 60°C for 30 min. Cholesterol isotopomer distribution was determined at the Yale Mouse Metabolic Phenotyping Center from GC-MS (HP6890-5975C MSD) in the EI mode monitoring masses from M−1 to M+18 (367–368). ISA was performed as previously described (31, 32).

Gene set enrichment analysis

GSEA was performed on transcription profiles from MCF10A cells overexpressing ERBB4 ICD CYT-1 or CYT-2. Our identification of genes significantly altered by ERBB4 ICD (adjusted P < 0.05) compared with empty vector controls was previously reported [(18); GEO GSE57339)]. The REACTOME_CHOLESTEROL_BIOSYNTHESIS and HORTON_SREBF_TARGETS (52) gene sets were manually curated from the MSigDB_v4.0 (Broad Institute). ERBB4 CYT-1 genes (n = 6865) or CYT-2 genes (n = 5965) were first rank-ordered by fold change in expression over vector-transfected cells. This list was evaluated with GSEA under default settings [GSEA preranked, 10,000 permutations; (53),].

Statistical analysis

Two-tailed Student’s t tests were performed where appropriate. Error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


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.


Acknowledgments: We thank anonymous Reviewer 2 for informing us about (34). Funding: This work was supported by U.S. Public Health Service RO1 CA80065 (D.F.S.), pilot funding to D.F.S. and Y.S. from the Yale Cancer Center (P30 CA16359), and NIH training grant T32GM07223 (J.W.H.). The Yale Mouse Metabolic Phenotyping Center is supported by NIH/National Institute of Diabetes and Digestive and Kidney Diseases (U24 DK-059635). Author contributions: J.W.H. performed the conceptual design, acquisition, and analysis of experimental data and assisted in the preparation of the manuscript. S.Z., R.E.M., and A.C.-D. obtained and analyzed experimental data. J.K.K. performed the isotopomer data analysis with experimental assistance from G.W.C. Y.S. and D.F.S. contributed to the conceptual design and analysis of the experiments, and prepared the manuscript. Competing interests: The authors declare that they have no competing interests.
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