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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:978-982, May 2005

Biochemical and Molecular Actions of Nutrients

Riboflavin Deficiency Impairs Oxidative Folding and Secretion of Apolipoprotein B-100 in HepG2 Cells, Triggering Stress Response Systems1,2

Karoline C. Manthey, Yap Ching Chew and Janos Zempleni*,3

Department of Nutrition and Health Sciences and * Departments of Biochemistry and Animal Science, University of Nebraska at Lincoln, Lincoln, NE 68583

3To whom correspondence should be addressed. E-mail: jzempleni2{at}unl.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Secretory proteins such as apolipoprotein B-100 (apoB) undergo oxidative folding (formation of disulfide bonds) in the endoplasmic reticulum (ER) before secretion. Oxidative folding depends on flavoproteins in eukaryotes. Here, human liver (HepG2) cells were used to model effects of riboflavin concentrations in culture media on folding and secretion of apoB. Cells were cultured in media containing 3.1, 12.6, and 300 nmol/L of riboflavin, representing moderately deficient, physiological, and pharmacological plasma concentrations in humans, respectively. When cells were cultured in riboflavin-deficient medium, secretion of apoB decreased by >80% compared with controls cultured in physiological medium. The nuclear translocation of the transcription factor ATF-6 increased by >180% in riboflavin-deficient cells compared with physiological controls; this is consistent with ER stress. Nuclear translocation of ATF-6 was associated with activation of the unfolded protein response. Expression of stress-response genes coding for ubiquitin-activating enzyme 1, growth arrest and DNA damage inducible gene, and glucose regulated protein of 78 kDa was greater in riboflavin-deficient cells compared with other treatment groups. Finally, phosphorylation of the eukaryotic initiation factor (eukaryotic initiation factor 2{alpha}) increased in riboflavin-deficient cells, consistent with decreased translational activity. We conclude 1) that riboflavin deficiency causes ER stress and activation of unfolded protein response in HepG2 cells, and 2) that riboflavin deficiency decreases protein secretion in HepG2 cells. Decreased secretion of apoB in riboflavin-deficient cells might interfere with lipid homeostasis in vivo.

KEY WORDS: • riboflavin • human • apolipoprotein B-100 • oxidative folding • unfolded protein response

Riboflavin is a precursor of flavin mononucleotide and of flavin adenine dinucleotide (FAD),4 which serve as coenzymes for numerous oxidases and dehydrogenases in eukaryotic cells (1). Recently, it has been shown that flavoproteins play a role in the oxidative folding (formation of disulfide bonds) of secretory proteins in the endoplasmic reticulum (ER) (2,3). Proteins destined for secretion into the extracellular space enter the ER, where oxidative folding is mediated by 2 distinct FAD-dependent pathways catalyzed by protein disulfide isomerase and sulfhydryl oxidases. Protein disulfide isomerase is reduced during the folding of proteins and is subsequently re-oxidized by FAD-dependent Ero1p (2,4,5) and Ero1-L (GenBank accession number AF081886) in yeast and in humans, respectively. Sulfhydryl oxidases use FAD as a coenzyme (6).

Oxidative folding of secretory proteins is critical for their subsequent secretion (2). Accumulation of unfolded proteins in the ER causes cell stress, triggering the unfolded protein response, which has the following characteristics (7,8). First, translational activity decreases, caused by phosphorylation of the eukaryotic initiation factor 2{alpha} (eIF-2{alpha}). Phosphorylated eIF-2{alpha} loses its ability to recruit charged initiator methionyl tRNA to the 40S ribosomal subunit (9,10). Proteins that help to decrease the abundance of unfolded proteins are spared from this translational downregulation. Second, transcriptional activities of ER stress-response genes, such as ubiquitin-activating enzyme 1, glucose regulated protein of 78 kDa (BiP/Grp78), and the growth arrest and DNA damage inducible gene 153 (GADD153/CHOP), are upregulated (9,11,12). Ubiquitin-activating enzyme 1 catalyzes the first step in the ubiquitin-dependent degradation of unfolded proteins (11). BiP/Grp78 is a chaperone that facilitates protein folding in the ER (9). The transcription factor GADD153/CHOP is involved in cell-growth arrest and apoptosis (12), decreasing the proliferation of stressed cells. Increased expression of genes coding for BiP/Grp78 and GADD153/CHOP is mediated by ER stress elements (ERSE) located in regulatory regions of these genes (13). Transcription factors such as ATF-4, ATF-6, and X-box binding protein 1 bind to ERSE, mediating transcriptional activation (14,15).

Previous studies suggested that riboflavin supply affects the oxidative folding and secretion of IL-2 in Jurkat (lymphoma) cells (3). Notwithstanding the roles of flavoproteins in oxidative folding, the secretion of IL-2 was impaired in severely riboflavin-deficient Jurkat cells but not in moderately deficient cells (3). We propose that the following characteristics of protein secretion contributed to the relative resistance of Jurkat cells to moderate riboflavin deficiency: 1) IL-2 is the only known disulfide-containing protein secreted by Jurkat cells (16); 2) IL-2 is secreted in small quantities [<100 pg/(106 cells x h)] by Jurkat cells (17); and 3) IL-2 contains only one disulfide bond (18).

In the present study, we tested the hypothesis that moderate riboflavin deficiency impairs oxidative folding in cells that secrete large quantities of protein. HepG2 cells (human hepatocarcinoma cells) were used as a model, given that these cells secrete at least 15 distinct proteins in large quantities (16). For example, HepG2 cells secrete ~500 µg of apolipoprotein B-100 (apoB)/(h x mg of cell protein) (19). Specifically, we sought to determine whether riboflavin deficiency decreases oxidative folding and secretion of apoB, triggering the unfolded protein response in HepG2 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Cell culture. HepG2 cells were purchased from ATCC. Cells were cultured in customized RPMI-1640; riboflavin concentrations in culture media were adjusted to 3.1 nmol/L (denoted "deficient"), 12.6 nmol/L (denoted "physiological"), and 300 nmol/L (denoted "pharmacological"), as described in our previous studies, taking into account the residual concentrations of flavins in dialyzed bovine growth serum (3). Cells were cultured in riboflavin-defined media for ≥8 d. For the assays described below, samples were collected at 60–70% confluence. Cells cultured in physiological medium were considered the control group.

Riboflavin concentrations in media were chosen based on the following: 300 nmol/L represents the riboflavin concentration in plasma from riboflavin-supplemented adults (20), 12.6 nmol/L represents the riboflavin concentration in normal human plasma (20), and 3.1 nmol/L represents the riboflavin concentration observed in plasma from moderately deficient pregnant women (21).

    Riboflavin transport. Rates of riboflavin transport into HepG2 cells were quantified using a physiological concentration of [3H]riboflavin (10 nmol/L) for all treatment groups (22).

    Glutathione metabolism. Both cellular activities of FAD-dependent glutathione reductase and concentrations of reduced glutathione are markers for flavin status (1); both variables were quantified in lysed HepG2 cells as described (23,24), with minor modifications (3).

    ApoB secretion. After 8 d of culturing in riboflavin-defined media, 3 x 106 cells were seeded in 250-mL culture flasks in a total volume of 15 mL (t = 0 h); the medium was replaced with fresh medium at t = 48 h; cell-free medium and cell pellets were collected at t = 72 h. ApoB secretion into media was determined by using ELISA ("CardioCHEK," ALerCHEK) according to the manufacturer’s instructions. ApoB secretion was normalized by cell protein as determined by bicinchoninic acid assay (Pierce).

    Immunocytochemistry. Intracellular apoB was visualized by standard procedures of immunocytochemistry (25). Cells were stained with mouse antihuman apoB antibody (Santa Cruz Biotechnology) and CyTM5-conjugated donkey antimouse IgG (Jackson ImmunoResearch). ß-actin (control) was stained with rhodamine phalloidin (Molecular Probes). Cells were viewed using an Olympus FV500 confocal microscope equipped with a X40 oil immersion lens.

    Western blot analyses. Whole-cell proteins for analysis of ubiquitin-activating enzyme 1 and GADD153/CHOP were extracted as described (26). Extranuclear proteins for analysis of apoB, eIF-2{alpha}, and phosphorylated eIF-2{alpha} (eIF-2{alpha}-p) were extracted as described above. Proteins were resolved by electrophoresis, using 3–8% Tris acetate gels and 4–12% BisTris gels (Invitrogen) (26). The following antibodies were used to probe proteins: mouse monoclonal IgG1 antihuman ubiquitin-activating enzyme 1 (Upstate); mouse monoclonal IgG1 antihuman GADD153/CHOP (Santa Cruz); mouse monoclonal IgG1 antihuman apoB (Santa Cruz); goat polyclonal IgG antihuman eIF-2{alpha} (Santa Cruz); and rabbit IgG antihuman eIF-2{alpha}-p (Upstate). The following secondary antibodies were used: goat antimouse IgG peroxidase conjugate, mouse monoclonal antigoat/sheep IgG peroxidase conjugate, and mouse monoclonal antirabbit IgG peroxidase conjugate (Sigma). ß-actin (control) was probed using a goat polyclonal antihuman ß-actin antibody and a mouse monoclonal antigoat/sheep IgG peroxidase conjugate (Sigma). Bands were visualized by chemiluminescence (26).

    Reporter-gene experiments. A construct of the luciferase reporter gene driven by 5 repeats of an ATF-6 binding site (denoted p5xATF6GL3) and a promoter-free control (pOFluc-GL3) were provided by R. Prywes, Columbia University (27). A construct of the luciferase reporter gene driven by the 5'-flanking region of the Grp78/BiP gene (denoted Grp78GL3) was provided by A. Lee, University of Southern California Keck School of Medicine (15). A promoter-free plasmid containing the luciferase gene (pGL3-Basic; Promega) was used to quantify baseline luciferase expression. Constructs of the luciferase reporter gene driven by the wild-type or mutated ERSE from the human GADD153/CHOP gene (denoted CHOP-ERSE-Luc and CHOP-M-ERSE-Luc, respectively) were provided by C. Glembotski, San Diego University (28). A construct of the SV promoter linked to the ß-galactosidase reporter gene (pSV ß-Gal, Promega) was used as a control for transfection efficiency. Reporter-gene experiments were conducted in analogy to our previous studies (17).

    Proliferation rates. Proliferation rates of HepG2 cells were quantified by measuring the cellular uptake of [3H]thymidine as described (29).

    Statistics. Homogeneity of variances among groups was confirmed using Bartlett’s test (30). Significance of differences among groups was tested by one-way ANOVA. Fisher’s protected least significant difference multiple comparison test was used for posthoc testing (30). StatView 5.0.1 (SAS Institute) was used to perform all calculations. Differences were considered significant if P < 0.05. Data are expressed as means ± SD.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Flavin homeostasis. If cells were cultured in riboflavin-deficient medium, the activity of glutathione reductase decreased to 44 ± 24% of physiological controls (Fig. 1). Likewise, if cells were cultured in riboflavin-deficient medium, the intracellular concentration of reduced glutathione decreased to 79 ± 12% of controls (Fig. 1). Concentrations of reduced glutathione were significantly greater in cells cultured in medium containing a pharmacological riboflavin concentration compared with physiological controls. Transport rates of riboflavin were significantly lower in riboflavin-deficient cells compared with other treatment groups [units = pmol riboflavin/(µg protein x 10 min); n = 5; P < 0.05]: 0.6 ± 0.2 (deficient medium); 7.5 ± 2.8 (physiological medium); and 8.9 ± 4.9 (pharmacological medium). Collectively, these findings suggest that the concentration of riboflavin in culture media affected the flavin homeostasis in HepG2 cells.



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  		FIGURE 1 Riboflavin concentrations in culture media affect activities of glutathione reductase and concentrations of reduced glutathione in HepG2 cells. Cells were cultured in riboflavin-defined media for 8 d. Values are means ± SD, n = 4. Means for a variable without a common letter differ, P < 0.05.

 
    ApoB metabolism. Secretion of apoB into culture media was lower in riboflavin-deficient cells compared with other treatment groups. If HepG2 cells were cultured in riboflavin-deficient medium, secretion of apoB decreased to 14 ± 29% compared with cells cultured in physiological medium (Fig. 2A). Secretion of apoB was not significantly different between cells cultured in media containing physiological and pharmacological concentrations of riboflavin.



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  		FIGURE 2 Riboflavin deficiency decreases synthesis and secretion of apoB in HepG2 cells. Cells were cultured in riboflavin-defined media for 8 d. (A) Secretion of apoB into culture media, as quantified by enzyme-linked immunosorbent assay. Values are means ± SD, n = 4. Means without a common letter differ, P < 0.05. (B) ApoB in cell extracts and ß-actin (control) were visualized by using immunocytochemistry. Merged images are depicted in the right column. (C) Intracellular apoB was quantified by Western blot analysis.

 
Immunocytochemical analysis suggested that intracellular concentrations of apoB paralleled riboflavin concentrations in culture media (Fig. 2B). Riboflavin did not affect the cellular abundance of ß-actin (control). Western blot analysis of cell extracts yielded similar data: the abundance of apoB correlated with riboflavin concentrations in culture media (Fig. 2C), whereas the abundance of ß-actin did not depend on riboflavin (data not shown). These data suggest that decreased synthesis of apoB accounted for some of the decreased secretion of apoB by riboflavin-deficient cells.

    Cellular stress response. The nuclear abundance of ATF-6 increased in response to riboflavin deficiency. The following transcriptional activities of p5xATF6GL3 were observed (units = ratio promoter-driven plasmid/promoter-free plasmid; n = 4; P < 0.05 for deficient cells vs. other treatment groups): 799 ± 72, 284 ± 51, and 372 ± 34 in cells cultured in deficient, physiological and pharmacological media, respectively. These findings suggest that riboflavin deficiency increases the transcription of ATF-6 dependent genes.

Consistent with this notion, the transcriptional activity of the BiP/Grp78 promoter increased in response to riboflavin deficiency. The following transcriptional activities were observed for Grp78GL3 (units = ratio promoter-driven plasmid/promoter-free plasmid; n = 4; P < 0.05 among all treatment groups): 423 ± 32, 114 ± 5, and 248 ± 68 in cells cultured in deficient, physiological, and pharmacological media, respectively. The differences among all treatment groups were significant.

The abundance of proteins involved in ER stress response correlated negatively with riboflavin concentrations in culture media, as indicated by Western blot analysis (Fig. 3). Increased abundance of ubiquitin-activating enzyme 1 in riboflavin-deficient HepG2 cells is consistent with increased ubiquitin-dependent degradation of unfolded proteins. Increased abundance of eIF-2{alpha}-p in riboflavin-deficient cells is consistent with impaired translation. The abundance of nonphosphorylated eIF-2{alpha} was not affected by riboflavin (data not shown). Increased abundance of GADD153/CHOP in riboflavin-deficient HepG2 cells is consistent with decreased rates of cell proliferation and increased apoptotic activities. The abundance of ß-actin (control) was not affected by riboflavin.



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  		FIGURE 3 Riboflavin deficiency causes ER stress in HepG2 cells. Cells were cultured in riboflavin-defined media for 8 d. Protein abundance was quantified by Western blot analysis.

 
Riboflavin deficiency was associated with a decreased proliferation rate; the following rates of thymidine uptake were observed [units = pmol thymidine/(µg protein x h); n = 4; P < 0.01 for deficient cells vs. other treatment groups]: 306 ± 21, 769 ± 70, and 836 ± 65 in cells cultured in media containing deficient, physiological, and pharmacological concentrations of riboflavin, respectively. Cells cultured in physiological and pharmacological media did not differ.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
This study is consistent with the following notions. 1) Riboflavin deficiency causes ER stress, triggering both nuclear translocation of stress-related transcription factors and unfolded protein response. Increased binding of transcription factors to ERSE increases the expression of proteins that help to reduce ER stress. 2) Unfolded protein response is associated with decreased secretion of apoB and proliferation rates in HepG2 cells. Likely, these effects are caused by the following sequence of events. Riboflavin deficiency depletes cellular FAD, decreasing the activity of enzymes, such as Ero1-L and sulfhydryl oxidases. A decreased activity of these enzymes impairs the folding of secretory proteins; unfolded proteins accumulate in the ER. This triggers nuclear translocation of transcription factors, such as ATF-6 and XBP1. These transcription factors bind to ERSE in the promoter regions of genes that mediate unfolded protein response, e.g., BiP/GRP78 and GADD153/CHOP. In addition, increased phosphorylation of eIF-2{alpha} reduces the global translational activity in riboflavin-deficient HepG2 cells. It remains to be determined whether riboflavin deficiency also impairs protein folding in normal human hepatocytes.

In the present study, the abundance of ubiquitin-activating enzyme 1, GADD153/CHOP, and eIF-2{alpha}-p was greater in cells cultured in physiological medium (12.6 nmol/L riboflavin) compared with cells cultured in pharmacological medium (300 nmol riboflavin). This finding suggests that 12.6 nmol/L of extracellular riboflavin may not be sufficient to maintain riboflavin homeostasis in HepG2 cells. The selection of 12.6 nmol/L riboflavin as a physiological control in the present study was based on riboflavin concentrations observed in peripheral plasma. Of note, liver cells receive some of their vitamins through the portal vein in vivo. Studies in rats provided evidence that flavin concentrations in portal blood are substantially higher than in peripheral blood (31). We propose that cells cultured in medium containing 12.6 nmol/L riboflavin are moderately riboflavin deficient, causing early signs of the unfolded protein response.

In the present study, transport rates of riboflavin were lower in severely riboflavin-deficient HepG2 cells compared with riboflavin-sufficient controls. We propose that translation and translocation of riboflavin transporters to the cell membrane decrease in response to severe riboflavin-deficiency in HepG2 cells. Consistent with this notion, transport rates of riboflavin increased in response to moderate riboflavin deficiency in HepG2 cells, caused by riboflavin-deficient medium for only 4 d (K. C. Manthey and J. Zempleni, unpublished results). Likewise, moderate riboflavin deficiency in Jurkat cells is associated with increased riboflavin transport rates (3). The cellular uptake of riboflavin is a transporter-mediated process but the identity of the riboflavin transporter is not yet known (32,33). Hence, we could not test the hypothesis that severe riboflavin deficiency in HepG2 cells decreases the translocation of riboflavin transporters to the cell membrane. Note that riboflavin deficiency is associated with DNA damage and activation of apoptotic pathways (K. C. Manthey and J. Zempleni, unpublished results). We cannot formally exclude the possibility that this mediates some of the effects observed in the present study.

Decreased secretion of proteins in response to riboflavin deficiency is physiologically important, given the essential roles of secretory proteins in intermediary metabolism. Riboflavin deficiency has been observed in preterm newborns treated with phototherapy (34), in patients with cystic fibrosis (35), and in pregnant women (21). The moderate riboflavin deficiency observed in these risk groups is likely to affect hepatic protein secretion, given that liver cells deplete rapidly of flavins (36). Note that effects of riboflavin deficiency on oxidative folding in HepG2 cells are not limited to apoB but extend to other secretory proteins such as plasminogen (K. C. Manthey and J. Zempleni, unpublished results).


   ACKNOWLEDGMENTS
 
We thank C. Glembotsky (San Diego University), A. Lee (University of Southern California), and R. Prywes (Columbia University) for generously providing plasmids for this study.


   FOOTNOTES
 
1 Presented in part at Experimental Biology 04, April 17–21, 2004, Washington, DC [Manthey, K. C. & Zempleni, J. (2004) Oxidative folding of secretory proteins is impaired in riboflavin-deficient HepG2 cells. Abstract #939]. Back

2 Supported by NIH grants DK 60447 and DK 063945. This paper is a contribution of the University of Nebraska Agricultural Research Division, Lincoln NE 68583 (Journal series number 14847). Back

4 Abbreviations used: apoB, apolipoprotein B-100; BiP/Grp78, glucose regulated protein of 78 kDa; eIF-2{alpha}, {alpha}-subunit of eukaryotic initiation factor 2; eIF-2{alpha}-p, phosphorylated eIF-2{alpha}; ER, endoplasmic reticulum; ERSE, endoplasmic reticulum stress element; FAD, flavin adenine dinucleotide; GADD153/CHOP, growth arrest and DNA damage inducible gene. Back

Manuscript received 15 November 2004. Initial review completed 30 December 2004. Revision accepted 23 January 2005.


   LITERATURE CITED
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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