Regulation of Gene Expression by β-Glucans

Carolyn M. Klinge Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY 40292, USA Tell: 502-852-3668 Email: Carolyn.klinge@louisville.edu Abstract: β-D-glucans are diverse polysaccharides derived from plant cell walls, fungi and bacteria. β-D-glucans are composed of D-glucose monomers linked by (1-3) β-glycosidic bonds with varying amounts of (16) or (1-4) linked side chains that bind cell surface receptors ultimately triggering changes in intracellular pathways and gene transcription. β-Dglucans have anti-viral, immunomodulatory and anti-cancer activities. This article reviews the current identity and putative function of genes regulated by β-glucans purified from various sources in human cancer and immune cells and in murine dendritic, macrophage cells and lungs. β-D-glucans increase the expression of numerous cytokines and immunomodulatory factors and their receptors in dendritic cells. Pathways and transcription factors involved in the cellular responses to β-glucans are summarized.

There are many studies of β-glucans purified from the cell walls of yeast, fungi and plants as immunomodulators that increase the levels of proinflammatory cytokines, chemokines and cell adhesion molecules (Novak and Vetvicka, 2008). We will review immunomodulatory genes regulated by β-glucans in human and mouse cells. In addition, we reviewed the literature to identify additional genes increased or inhibited by glucan treatment of cells in vitro. We summarize the downstream effects of these transcriptional changes and how they inform the mechanisms by which glucans act in various cell types.

Glucans Increase Gene Transcription in Human Cancer Cells and Immortalized Normal Cells
A number of reports have examined the effect of βglucan on transcription in human and murine immune cells with a few studies examining the activity of glucans in human cancer cells. Table 1 summarizes those genes that were transcriptionally induced by βglucan in the cells examined. First, we will review the results in human cells.
More recent studies reported that two preparations of fungal β-glucans inhibited MCF-7 cell viability, stimulated apoptosis and ROS production and increased necrosis (Queiroz et al., 2015). BOT GLC and BOT FRU (100 µg mL −1 , Table 1) increased the mRNA and protein expression of p53 (TP53) and p27 (CDKN1B) after 48 h of treatment (Queiroz et al., 2015). The authors reported that BOT GLC activated AMPK, increased FOXO3a protein and reduced phospho-FOXO3a, suggesting that stimulation of FOXO3a transcriptional activity is the mechanism by which cell arrest is achieved. This was supported with the observation that treatment with these β-glucans resulted in an increase in p53 and p27 transcription (Queiroz et al., 2015).

β-Glucan Regulates Protein and Gene Expression in Gastric Cancer Cells and other Human Cell Lines
Treatment of human gastric carcinoma cells with βglucan increased the expression of HSP70 protein (HSPA1A gene) and MUC1 transcript levels (mucin-1, a transmembrane protein) -both of which are protective factors in the gastric mucosa (Tanaka et al., 2011). We note that the primary focus of this study was to examine the protective effects of β-glucans on gastric lesions in mice with confirmation of HSP70 and MUC1 in gastric cancer cells.
Synthetic glucans increased expression of PKC (protein kinase C), CXCL8 (IL-8), CDC-42 and PUMA (BBC3) transcripts in human Detroit-573 fibroblasts and HaCaT keratinocytes as measured by non-quantitative PCR . These genes are regulators of cell growth and PUMA is a BCL-2 pro-apoptotic protein.

Microarray Analysis in Human Monocyte-Derived Dendritic Cells
Gene array profiling of human monocyte-derived Dendritic Cells (DCs) treated with a purified extract of Polysaccharide (PS-G, a branched (1→6)-β-d-glucan) from Ganoderma lucidum, a Chinese medicinal mushroom, identified 3477 (17%) probe sets upregulated (2-fold) and 4418 (19%) probe sets downregulated (2fold) after 16 h of treatment (Lin et al., 2006). The identity of genes included in the text of that manuscript (Lin et al., 2006) are shown on Table 1 and 2. Significant increases in transcript levels were observed for a number of cytokines (IL-12A, IL-12B, IL-23A, IL-27 and EBI2; also known as IL-27B), chemokines and chemokine receptors (CCL20, CCL19, CCL5, CXCL10, CXCL11 and CCR7) and cell surface proteins (CD80, CD83 and CD86). The authors also noted that an increase in transcripts for NFKB1, NBKB2, RELA, RELB and MAPK11which support the model that β-glucans activate human dendritic cells through the NFκB and p38 MAPK pathways. Further, the authors suggested that the increase in CCL20, IL-27, IL-23A, IL-12A and IL-12B transcripts after PS-G treatment of human DCs may play a role in the anti-tumor activity of β-glucans (Lin et al., 2006).
A more recent microarray profiling of human monocyte-derived DCs treated with β-D glucan (10 µg mL −1 ) for 4 or 12 h identified ~1500 genes that were either inhibited (38%) or induced (62%) (Cardone et al., 2014). The β-D glucan-regulated genes were divided into 6 groups: (1) Early-inhibited genes (4-6 h after activation); (2) genes inhibited similarly at both early and late times (early-late-inhibited genes); (3) lateinhibited genes; (4) early-induced genes; (5) genes induced similarly at both early and late times (earlylate-induced genes; and (6) late-induced genes (12 h after activation). Gene ontology analysis indicated that the early induced genes were likely involved in apoptosis or stress and antiviral responses, e.g., IFNB1, NFKBID, NFATC1, TNF, proIL1β, NFKBIZ and that these genes were potentially regulated by Interferon Regulatory Factors (IRFs). The early-induced genes were identified to be involved in chemotaxis, IL-1 production and NFκB or STAT signaling and were predicted to be regulated by NFκB and IRFs. The lateinduced genes included chemokines, cytokines and other factors involved in DC activation and proliferation, e.g., IL6, I10, IL12B, IL20, IL23A, IL1F9, CSF7, CSF3; and were predicted to be regulated by NFκB, IRFs, AP1, STATs and CEBPs. The authors demonstrated that β-glucan stimulates IL-1 expression which once processed by the caspase/inflammasome pathway acts in an autocrine fashion to stimulate the expression of the late-induced cytokines at the transcriptional level via IκB-ζ modulation (Cardone et al., 2014). Further, the authors reported that β-glucan also promoted an IFN-I gene signature consistent with the reported induction of IFN-I in response to fungi via Dectin-1 signaling. These data demonstrated that IL-1 and IFN-γ differentially regulate the β-glucan-induced Th responses in human DCs.

β-Glucan Stimulates IL23A and IL-10 Transcription But not IL-12 in Human Dendritic Cells
In a study designed to determine the effect of βglucan on the production of IL12 and IL23 in human dendritic cells, high and low concentrations of purified β-glucan (10 versus 200 µg mL −1 zymosan) were shown to induce transcription of IL-23 and IL-10, which was enhanced when cells were simultaneously treated with R848 (a Toll-like receptor 7 and 8 agonist) or with Pam2C (TLR2 ligand) . IFN-γ priming inhibited zymosan-stimulated IL-23 transcription . In contrast, transcription of IL-12 was not induced by zymosan alone. Instead, expression of IL12 was observed only when cells were first primed with IFN-γ and treated with low concentrations of zymosan and R848. Based on these observations, the authors concluded that the differential expression of IL-23 and IL-12 is dependent on the specific PPRs (pattern recognition receptors) that are activated by different microorganisms. Specifically, TLR2 is able to differentially regulate the expression of these two cytokines which allows DCs to respond appropriately to different pathogens .
In a more recent study, Rodríguez et al. (2014) showed that selective upregulation of IL-23 by zymosan (1 mg mL −1 ) involves activating transcription Factor 2 (ATF2) in addition to NFκB which stimulates both IL-23 and IL-10. In these studies, the authors suggest that zymosan interaction with dectin-1 activates PKA-MEK-ERK and PKC signaling leading to activation of ATF2 and coordinates with TLR2 signaling to activate NFκB. ATF2 and NFκB stimulate IL23A transcription (Rodríguez et al., 2014). While zymogen and two other insoluble β-glucans (curdlan-and pustulancontaining latex beads) resulted in the disappearance of the Endoplasmic Reticulum Stress Response (ERS) transcription factors CHOP and XBP-1 from the nucleus of human DCs and macrophages, there was no evidence that CHOP is involved directly in IL-23 induced expression (Rodríguez et al., 2014).

β-Glucans Induce Cytokine Production in Human B Lymphocytes
While most studies have focused on the activation of gene expression in DCs, a recent report demonstrated that purified β-glucan (curdlan) induces expression of several cytokines including TNFα, IL-6 andIL-8 in human B lymphocytes at both the mRNA and protein level (Ali et al., 2015). Activation of IL-8 was shown to be mediated through the Dectin-1 receptor and involved SYK, ERK and JNK and activation of the transcription factors AP-1 and NFκB. Interestingly, β-glucan had no effect on antibody production or proliferation suggesting that β-glucans induce a different response than TLR-9 agonist CpG oligodeoxynucleotide (CpG) that is a bacterial and viral DNA component known to induce cytokine production in B lymphocytes (Ali et al., 2015).

Glucans Increase gene Transcription in Murine (Mouse) Immune Cells
Treatment of murine Myeloid-Derived Suppressor Cells (MDSCs) with whole β-glucan particles was shown to increase the expression of Runx1, a transcription factor that induces differentiation in Myeloid-Derived Suppressor Cells (MDSC) and mmu-miR-181d in M-MDSCs (downregulated in G-MDSCs) (Tian et al., 2015). These authors did not examine the function of miR-181d and a search in PubMed revealed no studies examining miR-181d in MDSCs. miR-181d is a stress-responsive miRNA in thymocytes with expression decreasing in response to LPS (Belkaya and van Oers, 2014). A search of microRNA.org revealed 8084 targets for has-miR-181d, but the role of increased miR-181d in response to β-glucan in MDSCs remains to be defined.
Treatment of murine bone marrow derived DCs with Whole β-Glucan Particles (WGPs) rapidly activated the dectin-1 receptor, increased SYK phosphorylation (10-20 min) and increased cell surface GITRL protein (RNFSF18) levels measured 48 h later (Tian et al., 2012). GIRTL (also called TL6) is a cytokine member of the TNF ligand family. GIRTL primes cytotoxic T lymphocyte responses and downregulates the suppressive activity of regulatory T cells, thus inhibiting tumor progression in the mouse Lewis Lung Carcinoma (LLC) cell model (Tian et al., 2012). The authors also showed that treatment of mice implanted with LLC tumor cells with WBPs resulted in an increase in GITRL on DCs in vivo and slower tumor development.
Time course experiments in murine macrophagederived RAW264.7 cells showed that β (1->3),(1->6)-Dglucan, purified from Aureobasidium pullulan, strongly upregulated the inflammatory cytokines IL-1β and IL-6 and weakly upregulated TNFα. Increases in GM-CSF (CSF2) and G-CSF (CSF3), cytokines important for differentiation and proliferation, were also observed by 3 h with increased Ddx58 (RIG-1-retinoic acid inducible gene-1) and Mda5 (melanoma differentiation-associated protein 5) seen 6 h after treatment (Muramatsu et al., 2012). RIG-1 is necessary for Type I interferon production when cells are infected with a virus and MDA5 is an intracellular pattern recognition molecule for virus-derived RNAs which is necessary for Type I interferon production when cells are virus infected. These results correlate with the anti-viral activity of βglucan observed in mice (Muramatsu et al., 2012).
Treatment of RAW264.7 mouse macrophage cells with β-glucan increased TNFα and GM-CSF (granulocyte macrophage colony stimulating factor) after 3 h. CSF2 is involved in the development and differentiation of lymphocytes from stem cells increased at 3 h and remained upregulated through 24 h.

Genes Inhibited by β-Glucan
A number of studies have identified genes whose expression is decreased by β-glucan treatment in human and mouse cells. Table 2 summarizes the information on these genes that have been identified. First, the results in human cells will be reviewed followed by studies in mouse cells.

β-Glucan Inhibits Gene Transcription in Breast Cancer Cells
Early studies in MCF-7 cells showed that a 48 h treatment with 400 µg mL −1 PCM3-II, a water-soluble β-glucan, inhibited cell viability and coordinately reduced Cyclin D1 and Cyclin E protein expression (Zhang et al., 2006). However, that study did not examine the impact of PCM3-II on gene transcription. We examined the effect of two concentrations of β-Dglucan on the expression of a set of genes implicated in breast cancer in MCF-7 and LCC9 cells using a PCR array. We reported that β-D-glucan inhibited the expression of cell proliferation and differentiation markers in MCF-7 cells, e.g., CDKN1C, CTNNB1, ERBB2, MUC1 and RARB (Jafaar et al., 2014). These changes in gene expression correspond to the observation that β-D-glucan dissolved in DMSO, but not water, inhibits MCF-7 cell proliferation (Jafaar et al., 2014). β-D-glucan also inhibited the expression of CDKN1C and CTNNB1in LCC9 cells (Jafaar et al., 2014). The mechanism(s) for these effects in MCF-7 and LCC9 cells has not been examined.

β-Glucan Inhibits ERCC5 Expression in HepG2 Cells
Treatment of HepG2 human hepatoma cells with a β-glucan-containing polysaccharide complex purified from Agaricusblazei (Murill mushroom of Brazilian origin) by aqueous extraction (Gonzaga et al., 2005) had no effect on cell viability (MTT assay), CYP1A1 or CASP9 gene transcript levels, but inhibited ERCC5 expression (da Silva et al., 2013). ERCC5 is involved in excision repair following DNA damage, but the authors did not pursue this finding. Interestingly, βglucan treatment of HepG2 cells increased metabolites involved in bioenergetic metabolism, including alanine, glutamate and creatine. From these studies, the authors concluded that β-glucan increases metabolites needed for high energy metabolism and stimulates bioenergetics (da Silva et al., 2013). Interestingly, injection of yellow croaker (Pseudosciaenacrocea) fish with β-glucan (5 mg kg −1 body weight, from Sigma) 6 h prior to hypoxic stress inhibited reactive oxygen formation in the liver and increased transcription of Pyruvate Kinase (PK) while reducing F-ATPase, Succinate Dehydrogenase (SDH) and Malate Dehydrogenase (MDH) expression (Zeng et al., 2016). Which subunit of SDH or form of MDH was examined by PCR was not indicated. These results suggest that β-glucan inhibits the TCA cycle and enhances glycolysis in vivo in the liver of yellow croaker fish, although direct examination of TCA cycle metabolites, oxygen consumption or ATP production was not performed.

β-Glucan Inhibits miR-9 Expression in Mouse Macrophage Cells
A microRNA microarray in Whole β-Glucan Particles (WGP)-treated MDSCs isolated from the spleen of Lewis lung carcinoma bearing mice identified 61 miRNAs that were downregulated whereas 40 miRNAs were upregulated (Tian et al., 2015). Specifically, the authors identified miR-9 as an important regulator of Runx1 (runt-related transcription factor) as a direct, bona fide target of miR-9 (verified by 3'UTR luciferase reporter assay in HEK-293T cells). They also reported a decrease in Creb transcript levels in WGP-treated MDSCs, identified a binding site for CREB in the miR-9 promoter region and verified CREB suppression by a promoter luciferase reporter assay. Both CREB and mi-RNA suppression by WGP were shown to be mediated by the Dectin-1 pathway. These results support the conclusion that through the Dectin-1 receptor, CREB directly regulates miR-9 expression in response to β-glucan. The downregulation of miR-9 results in an increase in Runx1 allows for differentiation, decreased immunosuppressive function and reduced tumor growth (Tian et al., 2015).

β-Glucan Inhibits Proteins that Regulate Nucleic Acid Synthesis in a Human Tumor Cell Xenograft
As reviewed above, treatment of female athymic mice with lentinan at a dose of 1 mg/kg/day 2X/week for 3 weeks reduced xenograft human KB cell tumors (Harada et al., 2010). The authors reported reduced thymidylate synthase and dihydropyrimidine dehydrogenase proteins in the KB xenografts, results that correlated with inhibition of KB xenograft growth in vivo (Harada et al., 2010).
As discussed above, gene array profiling of human monocyte-derived DCs treated with a PS-G identified 4418 (19%) probe sets downregulated (2-fold) after 16 h of treatment (Lin et al., 2006). The identity of downregulated genes included in the text of that manuscript (Lin et al., 2006) is shown in Table 2. The authors suggested that the decrease in ARHGDIB, ATM, CEBPA, CSFIR, CST, F13A1, ITGAM and ITGB2 transcripts after PS-G treatment may play a role in the anti-tumor activity of β-glucans (Lin et al., 2006). Another microarray profiling of human monocytederived DCs treated with β-D glucan (10 µg mL −1 ) for 4 or 12 h identified ~ 1500 genes that were either inhibited (38%) or induced (62%) (Cardone et al., 2014). The authors focused on upregulated transcripts and none of the downregulated genes were described; thus, these are not included in Table 2.

Conclusion
From the genes identified as regulated by β-glucan in this review, it is clear that β-glucan regulates the transcription of numerous immunomodulatory genes in human and mouse dendritic and macrophage cells. In addition, gene expression changes in response to βglucans have been identified in human breast, gastric and liver cancer cells as well as a few other cell lines as summarized in Table 1 and 2. Figure 1 is a model of the generalized pathway by which β-glucan regulates gene transcription by interaction with a plasma membrane receptor which then activates an intracellular pathways leading to activation or inhibition of transcription factor activity. The activated transcription factors then regulate the transcription of cell-specific target genes.