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Cited by 24Cholesterol and Lipid MetabolismLipoproteins and Cardiovascular HealthCancer, Lipids, and Metabolism

The DOI matches its Crossref record; 32 of the 32 references it cites were found in a registry or an index; none of the 16 sources its claims cite is checked for support yet.

Transmembrane Protein 55B Is a Novel Regulator of Cellular Cholesterol Metabolism

Open access
Medina, Marisa W., Bauzon, Frederick, Naidoo, Devesh, Theusch, Elizabeth, Stevens, Kristen, Schilde, Jessica, Schubert, Christian, Mangravite, Lara M., Rudel, Lawrence L., Temel, Ryan E., Runz, Heiko, Krauss, Ronald M.·2014·Arteriosclerosis, Thrombosis and Vascular Biology
DOI 10.1161/atvbaha.113.302806PMC PMC4141484
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Abstract

Objective—: Interindividual variation in pathways affecting cellular cholesterol metabolism can influence levels of plasma cholesterol, a well-established risk factor for cardiovascular disease. Inherent variation among immortalized lymphoblastoid cell lines from different donors can be leveraged to discover novel genes that modulate cellular cholesterol metabolism. The objective of this study was to identify novel genes that regulate cholesterol metabolism by testing for evidence of correlated gene expression with cellular levels of 3-hydroxy-3-methylglutaryl-CoA reductase ( HMGCR ) mRNA, a marker for cellular cholesterol homeostasis, in a large panel of lymphoblastoid cell lines. Approach and Results—: Expression array profiling was performed on 480 lymphoblastoid cell lines established from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, and transcripts were tested for evidence of correlated expression with HMGCR as a marker of intracellular cholesterol homeostasis. Of these, transmembrane protein 55b ( TMEM55B ) showed the strongest correlation ( r =0.29; P =4.0E−08) of all genes not previously implicated in cholesterol metabolism and was found to be sterol regulated. TMEM55B knockdown in human hepatoma cell lines promoted the decay rate of the low-density lipoprotein receptor, reduced cell surface low-density lipoprotein receptor protein, impaired low-density lipoprotein uptake, and reduced intracellular cholesterol. Conclusions—: Here, we report identification of TMEM55B as a novel regulator of cellular cholesterol metabolism through the combination of gene expression profiling and functional studies. The findings highlight the value of an integrated genomic approach for identifying genes that influence cholesterol homeostasis.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

Genome-wide expression profiling in 480 lymphoblastoid cell lines identified TMEM55B as the top novel gene correlated with HMGCR transcript levels, a marker of cellular cholesterol homeostasis. Functional studies in hepatoma cells showed that TMEM55B is sterol-regulated and modulates LDLR decay, cell-surface LDLR, LDL uptake, and intracellular cholesterol.

Key findings

  • In 480 LCLs from Cholesterol and Pharmacogenetics statin trial participants, 110 genes had correlated expression with HMGCR at Bonferroni adjusted p<0.05; TMEM55B was the strongest novel candidate not previously implicated in cholesterol metabolism (r=0.29, Bonferroni adjusted p=4.0E-08).
  • TMEM55B was sterol-regulated: statin + LPDS induced TMEM55B transcript 1.7- to 2.8-fold in HepG2, Hep3B, and Huh7 cells, with sterol add-back reversing this; simvastatin exposure increased TMEM55B in primary human hepatocytes; cholesterol feeding reduced hepatic TMEM55B ∼40% in African Green monkeys (n=5, p=0.01) and dose-dependently in an independent monkey study.
  • TMEM55B knockdown in HepG2 reduced total cholesterol (0.64±0.1 fold, n=8, p=0.01), free cholesterol (0.70±0.07 fold, n=8, p=0.008), and cholesterol esters (0.39±0.1 fold, n=8, p=0.05); it accelerated LDLR protein decay, reduced cell-surface LDLR (e.g., HepG2 TMEM-1 0.87±0.05 fold, n=12, p=0.02; TMEM-2 0.79±0.05 fold, n=12, p=0.001), and reduced LDL uptake (e.g., HepG2 TMEM-1 0.92±0.02, n=20, p=0.004; TMEM-2 0.92±0.02, n=20, p=0.001). Overexpression produced opposite effects.
  • SREBF2 binding motifs were found in the TMEM55B promoter region, and SREBF1 or SREBF2 knockdown reduced TMEM55B transcript levels ∼30% (p=0.03 and p=0.04, respectively).

Methods

  • Expression array profiling was performed on 480 LCLs established from participants of the Cholesterol and Pharmacogenetics statin clinical trial; transcripts were tested transcriptome-wide for correlated expression with HMGCR, with 110 genes meeting Bonferroni adjusted p<0.05.
  • Functional validation used human hepatoma cell lines (HepG2, Hep3B, Huh7), HeLa cells, human fibroblasts, primary human hepatocytes, and African Green monkeys; assays included siRNA knockdown and overexpression, sterol depletion/add-back, cycloheximide chase for LDLR decay, cell-surface LDLR measurement, LDL uptake, and cholesterol quantification.
  • Materials and Methods are available in the online-only Data Supplement; the main analyses were correlation of gene expression with HMGCR and functional assays of TMEM55B manipulation.

Limitations

  • The paper states that further studies are necessary to determine the precise mechanism by which TMEM55B regulates LDLR, and notes that the larger reduction in intracellular cholesterol (30-60%) versus the smaller reduction in LDLR surface protein and LDL uptake (closer to 10%) suggests TMEM55B may modify other pathways such as cholesterol efflux, synthesis, or VLDL metabolism.
  • The paper notes that TMEM55B appears to be the only sterol-responsive gene in the phosphatidylinositol pathway based on personal communication, and that the mechanism may involve non-clathrin-mediated effects.
  • Evident from the text: the initial LCL correlation is modest (r=0.29), some supporting cell-model results were not statistically significant (HeLa 2.4±0.7 fold, n=4, p=0.13; fibroblasts 3.4±0.6 fold, n=3, p=0.05), and the functional work relies on cell lines and animal models rather than direct human causal evidence.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1161/atvbaha.113.302806

Cites

32 of 32 references verified0 citation errors · 0 not checkable

Statistics

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Verification

Claims the paper makes

0 of 16 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Overexpression of TMEM55B in HepG2 cells increased cell surface LDLR protein and DiI-LDL uptake relative to control transfected cells.

    “Overexpression of TMEM55B in HepG2 cells produced the opposite effects, with increases of both cell surface LDLR protein and DiI-LDL uptake relative to control transfected cells (Figures 2E and 2F).”
    Quote in the paperSubstantial claim
  • Findingown data

    There was no relationship between variation in hepatic LDLR transcript levels and hepatic cholesterol (r2=0.01 for total cholesterol).

    “whereas there was no relationship between variation in hepatic LDLR transcript levels with hepatic cholesterol (r2=0.01 for total cholesterol).”
    Quote in the paperSubstantial claim
  • Findingown data

    Inter-individual variation in hepatic TMEM55B expression levels was directly correlated with variation in hepatic total cholesterol, free cholesterol, and cholesterol esters in African Green monkeys.

    “Furthermore, inter-individual variation in hepatic TMEM55B expression levels was directly correlated to variation in hepatic total cholesterol, free cholesterol and cholesterol esters (Figures 1E, SIA and SIB)”
    Quote in the paperSubstantial claim
  • Findingown data

    TMEM55B knock-down in HepG2 reduced cholesterol esters (0.39±0.1 fold, n=8, p=0.05).

    “Importantly, TMEM55B knock-down in HepG2 reduced overall cellular total cholesterol (0.64±0.1 fold, n=8, p=0.01), free cholesterol (0.70±0.07 fold, n=8, p=0.008) as well as cholesterol esters (0.39±0.1 fold, n=8, p=0.05), Figure 2C.”
    Quote in the paperSubstantial claim
  • Findingown data

    Simvastatin exposure (0.5 and 2.0 μM) induced TMEM55B transcript levels in primary human hepatocytes derived from four unrelated donors.

    “Induction of TMEM55B transcript levels with statin treatment was also observed after in vitro simvastatin exposure (0.5 and 2.0μM) of primary human hepatocytes derived from four unrelated donors (Figure 1B).”
    Quote in the paperSubstantial claim
  • It may state two separate claims as one. 11
  • Probably not a substantial claim. 6

  • It may state two separate claims as one.

    Findingown data

    TMEM55B knockdown with either TMEM-1 or TMEM-2 in both HepG2 and Huh7 cells did not reduce LDLR transcript levels, but rather caused a 20-50% increase under all conditions tested.

    “Lastly, we found that TMEM55B knockdown with either TMEM-1 or TMEM-2 in both HepG2 and Huh7 cells did not reduce LDLR transcript levels, but rather caused a 20-50% increase under all conditions tested, Figure SIV.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Extreme sterol depletion (simvastatin + LPDS) induced TMEM55B transcript levels 1.7 to 2.8-fold depending on cell type in HepG2, Hep3B, and Huh7 cells, and sterol add-back reversed this effect.

    “Similarly, extreme sterol depletion (statin + LPDS) induced TMEM55B transcript levels 1.7 to 2.8-fold depending on cell type, while sterol add-back reversed this effect (Figure 1A).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    TMEM55B knockdown reduced LDL uptake in HepG2 cells transfected with TMEM-1 (0.92±0.02, n=20, p=0.004) or TMEM-2 (0.92±0.02, n=20, p=0.001), and in Hep3B cells transfected with TMEM-1 (0.95±0.02, n=12, p=0.03) or TMEM-2 (0.94±0.02, n=12, p=0.01).

    “Consistent with the reduction in cell surface LDLR protein, we also found that TMEM55B knockdown reduced LDL uptake in HepG2 cells transfected with either TMEM-1 (0.92±0.02, n=20, p=0.004) or TMEM-2 (0.92±0.02, n=20, p=0.001), as well as in Hep3B cells transfected with either TMEM-1 (0.95±0.02, n=12, p=0.03) or TMEM-2 (0.94±0.02, n=12, p=0.01), Figure 2F.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In HeLa cells and human fibroblasts cultured in lipoprotein-depleted serum and exposed to 2-hydroxyl-β-cyclodextrin, TMEM55B transcript was up-regulated by 2.4±0.7 fold (n=4, p=0.13) and 3.4±0.6 fold (n=3, p=0.05), respectively.

    “For instance, in HeLa cells and human fibroblasts cultured in lipoprotein-depleted serum and exposed to the cholesterol-reducing agent 2-hydroxyl-β-cyclodextrin, TMEM55B transcript was up-regulated by 2.4±0.7 fold (n=4, p=0.13) and 3.4±0.6 fold (n=3, p=0.05), respectively.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    TMEM55B knockdown reduced cell surface LDLR protein in HepG2 cells transfected with TMEM-1 (0.87±0.05 fold, n=12, p=0.02) or TMEM-2 (0.79±0.05 fold, n=12, p=0.001), and in Huh7 cells transfected with TMEM-2 (0.75±0.08 fold, n=12, p=0.02).

    “As shown in Figure 2E, cell surface LDLR was reduced in HepG2 cells transfected with either TMEM-1 (0.87±0.05 fold, n=12, p=0.02) or TMEM-2 (0.79±0.05 fold, n=12, p=0.001), as well as in a second hepatoma cell line, Huh7, transfected with TMEM-2 (0.75±0.08 fold, n=12, p=0.02).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    TMEM55B knock-down in HepG2 caused a reduction in LDLR protein over three hours after cycloheximide treatment, consistent with an accelerated LDLR protein decay rate.

    “Although we did not observe detectable degradation of total cellular LDLR protein in the NTC treated cells, TMEM55B knock-down caused a reduction in LDLR protein over three hours, consistent with an accelerated LDLR protein decay rate (Figure 2D, SIIIB).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    TMEM55B knock-down did not influence levels of cell surface transferrin receptor or rate of transferrin-568 uptake.

    “We found no evidence that TMEM55B knock-down influenced either levels of cell surface transferrin receptor or rate or transferrin-568 uptake (Figures SIIIC and SIIID).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    SREBF1 and SREBF2 knock-down reduced TMEM55B transcript levels by approximately 30% in Huh7 cells (p=0.03 and p=0.04 respectively), with no additive effect of dual knock-down.

    “Both SREBF1 and 2 knock-down reduced TMEM55B transcript levels ∼30% (p=0.03 and p=0.04 respectively) with no additive effect of the dual knock-down (Figure SIC).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    RHOA was identified as a novel candidate gene implicated in LDL-cholesterol lowering in response to statin treatment.

    “Through expression array analysis of 480 in vitro simvastatin and sham incubated LCLs derived from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, we recently reported the identification of RHOA as a novel candidate gene implicated in LDL-cholesterol lowering in response to statin treatment9.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Medina MW Theusch E Naidoo D Bauzon F Stevens K Mangravite LM Kuang YL Krauss RM RHOA is a modulator of the cholesterol-lowering effects of statin PLoS genetics 2012 8 e1003058 23166513 PMC3499361

  • Probably not a substantial claim.

    Premisecitation

    LDLR recycles between the endosome/lysosome compartment and the cell surface.

    “Since TMEM55B has been shown to localize to the endosome/lysosome13, and LDLR recycles between this compartment and the cell surface12, we hypothesized that TMEM55B could modulate LDLR turnover, activity or localization.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] Goldstein JL Brown MS The LDL receptor Arterioscler Thromb Vasc Biol 2009 29 431 438 19299327 PMC2740366

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisecitation

    HMGCR encodes the rate-limiting enzyme of the cholesterol biosynthesis pathway and is tightly regulated at the level of gene transcription by SREBF2 in response to changes in intracellular sterol content.

    “Since HMGCR encodes the rate-limiting enzyme of the cholesterol biosynthesis pathway and is tightly regulated at the level of gene transcription by sterol response element binding factor 2 (SREBF2) in response to changes in intracellular sterol content10, we identified genes whose statin-induced expression level changes were most highly correlated with statin-induced changes in HMGCR.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Goldstein JL Brown MS Regulation of the mevalonate pathway Nature 1990 343 425 430 1967820

  • It may state two separate claims as one.

    Interpretationown data

    TMEM55B regulates cellular levels of cholesterol and LDLR.

    “We have validated this finding by functional studies demonstrating that TMEM55B regulates cellular levels of cholesterol and LDLR.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Human hepatoma cell lines (HepG2, Hep3B, Huh7) are valid models for human hepatocyte cholesterol metabolism.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Immortalized lymphoblastoid cell lines (LCLs) have been used as a model system to study genetic variation affecting cholesterol metabolism and statin response.

    “Immortalized lymphoblastoid cell lines (LCLs), created by Epstein-Barr virus transformation of peripheral blood mononuclear cells1, have been used as a model system to study genetic variation affecting cholesterol metabolism and statin response.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Pressman S Rotter JI Epstein-barr virus transformation of cryopreserved lymphocytes: Prolonged experience with technique Am J Hum Genet 1991 49 467 1651051 PMC1683279

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    TMEM55B may modify other pathways that influence cellular cholesterol metabolism such as cholesterol efflux or synthesis.

    “This discrepancy suggests that TMEM55B may modify other pathways that influence cellular cholesterol metabolism such as cholesterol efflux or synthesis.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Human hepatoma cell lines (HepG2, Hep3B, Huh7) are valid models for human hepatocyte cholesterol metabolism.

  • It may state two separate claims as one.

    Interpretationown data

    TMEM55B modulates intracellular cholesterol levels and LDLR metabolism.

    “TMEM55B modulates intracellular cholesterol levels and LDLR metabolism”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Human hepatoma cell lines (HepG2, Hep3B, Huh7) are valid models for human hepatocyte cholesterol metabolism.

  • Probably not a substantial claim.

    Premisecitation

    PIP2 is required for clathrin-mediated endocytosis, with roles in clathrin-coated pit initiation, stabilization, and maturation.

    “It has been well established that PIP2, the predominant PI formed at the plasma membrane, is required for clathrin-mediated endocytosis, with roles in clathrin-coated pit initiation, stabilization, and maturation14-17.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [14] Jost M Simpson F Kavran JM Lemmon MA Schmid SL Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation Current biology:CB 1998 8 1399 1402 9889104
    • cites [15] Zoncu R Perera RM Sebastian R Nakatsu F Chen H Balla T Ayala G Toomre D De Camilli PV Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate Proc Natl Acad Sci U S A 2007 104 3793 3798 17360432 PMC1805489
    • cites [16] Antonescu CN Aguet F Danuser G Schmid SL Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size Molecular biology of the cell 2011 22 2588 2600 21613550 PMC3135483
    • cites [17] Abe N Inoue T Galvez T Klein L Meyer T Dissecting the role of ptdins(4,5)p2 in endocytosis and recycling of the transferrin receptor J Cell Sci 2008 121 1488 1494 18411250 PMC3579524
16 of 16

  • The paper relies on this without support.

    Cholesterol-fed African Green monkeys are a suitable model for human hepatic cholesterol metabolism.

    “quantification of hepatic TMEM55B in African Green monkeys before and after a 19-week 0.6mg/kcal cholesterol supplemented diet found that cholesterol loading reduced TMEM55B transcript levels ∼40% (n=5 animals, p=0.01, Figure 1C).”
    “We observed similar results in an independent study of African Green monkeys fed a diet containing 0.002, 0.2 or 0.4 mg/kcal cholesterol for 10 weeks (n=5 per diet, Figure 1D), with cholesterol loading down-regulatingTMEM55B transcript levels in a dose-dependent manner.”

  • The paper relies on this without support.

    Human hepatoma cell lines (HepG2, Hep3B, Huh7) are valid models for human hepatocyte cholesterol metabolism.

    “To test if endogenous TMEM55B transcript levels were changed in response to intracellular sterol content, we treated three human hepatoma cell lines, HepG2, Hep3B and Huh7, to conditions of extreme sterol depletion using media containing 2 μM simvastatin + 10% lipoprotein deficient serum (LPDS), thus reducing both cholesterol synthesis and uptake.”
    “We transfected human hepatoma cell lines with TMEM55B-siRNAs (aka TMEM-1 and TMEM-2) that reduced TMEM55B transcript levels and protein levels ∼80% and 60% respectively (Figures 2A and 2B, SII).”
2 of 2

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  • SREBF2 dataset is cited to an SREBP1 study

    Minor

    The text identifies reference 11 as a source of SREBF2 ChIP-Seq data, but the cited study's title identifies its genome-wide occupancy analysis as SREBP1.

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Metadata

Authors
Medina, M. W., Bauzon, F., Naidoo, D., Theusch, E., Stevens, K., Schilde, J., Schubert, C., Mangravite, L. M., Rudel, L. L., Temel, R. E., Runz, H., & Krauss, R. M.
Affiliations
Sage Bionetworks (US)
ORCID
Venue
Arteriosclerosis, Thrombosis and Vascular Biology
Publisher
Ovid Technologies (Wolters Kluwer Health)
Published
2014-09
Volume
34
Issue
9
Pages
1917-1923
ISSN
  • 1079-5642 (ISSN-L)
  • 1524-4636
DOI
10.1161/atvbaha.113.302806
PMC
PMC4141484
OpenAlex
W2002837940
Cited by
24
References
32
Citations by year
2026: 3 · 2024: 1 · 2023: 3 · 2022: 2 · 2021: 3 · 2020: 2 · 2019: 3 · 2018: 1 · 2017: 2 · 2016: 3
FWCI
0.71
Type
journal article
Language
en
Open access
Locations
  • Arteriosclerosis Thrombosis and Vascular Biology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Institutes of Health (5r00hl088528-05)
  • National Heart, Lung, and Blood Institute (R00-HL088528, U19HL069757, R01HL104133, P01HL049373)
  • National Institute of Diabetes and Digestive and Kidney Diseases (P30DK026743)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Lipoproteins and Cardiovascular Health
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Lipids, and Metabolism
Keywords
cholesterol homeostasis, HMGCR, LDL receptor, LDL uptake, sterol regulation, lymphoblastoid cell lines, hepatoma cells, gene expression correlation, serum cholesterol, cardiovascular risk
MeSH terms
Biological Transport, Cell Membrane, Cholesterol, Homeostasis, Humans, Hydroxymethylglutaryl CoA Reductases, Intracellular Fluid, Lymphocytes, Receptors, LDL, RNA, Messenger, Gene Expression Profiling, Hepatocytes, Lipid Metabolism, Sterol Regulatory Element Binding Protein 1, Sterol Regulatory Element Binding Protein 2, Hep G2 Cells

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