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Open Access Publications from the University of California

Scholarly Works (41 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2016)
Zhang et al. found that plasma concentrations of the thiamine antimetabolite oxythiamine are significantly increased in patients with end-stage renal disease. These investigators discuss the potential sources of oxythiamine and the consequences of its plasma elevation. This commentary addresses the significance of these findings and expands on the potential role of gut microbiome in the generation of this antithiamine metabolite.
    Cover page: Functional thiamine deficiency in end-stage renal disease: malnutrition despite ample nutrients
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2014)
    The human SLC52A1 gene encodes the riboflavin transporter-1 (RFVT-1), a plasma membrane protein that transports vitamin B2 (riboflavin, RF) into cells, and thus, plays a role in controlling cellular homeostasis of RF in those tissues that express the carrier protein (e.g. placenta and intestine). Currently, there is nothing known about transcriptional regulation of the SLC52A1 gene, therefore, we aimed to clone and characterize its 5'-flanking region. Using rapid amplification of the cDNA ends (5'-RACE), we identified one transcription start site (TSS). A 579 bp segment of the 5'-flanking region of this gene was cloned which exhibited robust promoter activity upon transfection in human intestinal epithelial cells. Deletion analysis revealed that the core promoter activity to be embedded in a region between -234 and -23 that lacked TATA element, was GC-rich, and harbored several putative cis-regulatory sites including KLFs, AP-2, EGRF and Sp-1. Mutating each of these sites led to a significant decrease in promoter activity (which was highest for the Sp-1 site), suggesting their possible involvement in regulating SLC52A1 transcription. Focusing on the Sp-1 site, EMSA, super-shift and ChIP analysis was performed that established the interaction of the Sp-1 transcription factor with the SLC52A1 promoter; also, co-transfection of the minimal SLC52A1 promoter with an Sp-1 containing vector in Drosophila SL-2 cells led to significant promoter activation. These results are the first to reveal the identity of the minimal SLC52A1 promoter and to establish an important role for Sp-1 in its activity.
      Cover page: Identification and characterization of 5′-flanking region of the human riboflavin transporter 1 gene (SLC52A1)
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2016)
      The water-soluble biotin (vitamin B7) is indispensable for normal human health. The vitamin acts as a cofactor for five carboxylases that are critical for fatty acid, glucose, and amino acid metabolism. Biotin deficiency is associated with various diseases, and mice deficient in this vitamin display enhanced inflammation. Previous studies have shown that biotin affects the functions of adaptive immune T and NK cells, but its effect(s) on innate immune cells is not known. Because of that and because vitamins such as vitamins A and D have a profound effect on dendritic cell (DC) function, we investigated the effect of biotin levels on the functions of human monocyte-derived DCs. Culture of DCs in a biotin-deficient medium (BDM) and subsequent activation with LPS resulted in enhanced secretion of the proinflammatory cytokines TNF-α, IL-12p40, IL-23, and IL-1β compared with LPS-activated DCs cultured in biotin-sufficient (control) and biotin-oversupplemented media. Furthermore, LPS-activated DCs cultured in BDM displayed a significantly higher induction of IFN-γ and IL-17 indicating Th1/Th17 bias in T cells compared with cells maintained in biotin control or biotin-oversupplemented media. Investigations into the mechanisms suggested that impaired activation of AMP kinase in DCs cultured in BDM may be responsible for the observed increase in inflammatory responses. In summary, these results demonstrate for the first time that biotin deficiency enhances the inflammatory responses of DCs. This may therefore be one of the mechanism(s) that mediates the observed inflammation that occurs in biotin deficiency.
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2005)

        In mammalian cells (including those of the ocular system), the water-soluble vitamin B-2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study was to address this issue using the hRPE ARPE-19 cells as the retinal epithelial model. Our results show RF uptake in the hRPE to be: (1) energy and temperature dependent and occurring without metabolic alteration in the transported substrate, (2) pH but not Na+ dependent, (3) saturable as a function of concentration with an apparent K-m of 80 +/- 14 nM, (4) trans-stimulated by unlabelled RF and its structural analogue lumiflavine, (5) cis-inhibited by the RF structural analogues lumiflavine and lumichrome but not by unrelated compounds, and (6) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) as well as by the Na+-H+ exchange inhibitor amiloride and the sulfhydryl group inhibitor p-chloromercuriphenylsulphonate (p-CMPS). Maintaining the hRPE cells in a RF-deficient medium led to a specific and significant up-regulation in RF uptake which was mediated via changes in the number and affinity of the RF uptake carriers. While modulating the activities of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, protein tyrosine kinase (PTK)-, and nitric oxide (NO)-mediated pathways were found to have no role in regulating RF uptake, a role for the Ca2+-calmodulin-mediated pathway was observed. These studies demonstrate for the first time the involvement of a specialized carrier-mediated mechanism for RF uptake by hRPE cells and show that the process is adaptively regulated in RF deficiency, and also appears to be under the regulation of an intracellular Ca2+-calmodulin-mediated pathway.

          Cover page: Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2020)
          SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.
            Cover page: pH-dependent pyridoxine transport by SLC19A2 and SLC19A3: Implications for absorption in acidic microclimates
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2016)
            Riboflavin (RF) is indispensable for normal cell metabolism, proliferation, and growth. The RFVT-3 protein (product of the Slc52a3 gene) is expressed in the gut with the expression being restricted to the apical membrane domain of the polarized intestinal epithelial cells. The relative contribution of RFVT-3 to total carrier-mediated RF uptake in the native intestine, however, is not clear. We addressed this issue in the current investigation using a conditional (intestinal-specific) RFVT-3 knockout (cKO) mouse model developed by the Cre/Lox approach. All RFVT-3 cKO mice were found to be RF deficient and showed a significant growth and development retardation; also, nearly two-thirds of them died prematurely between the age of 6 and 12 wk. In vivo (intestinal and colonic loops) and in vitro (native isolated intestinal epithelial cells) uptake studies showed a severe inhibition in carrier-mediated RF uptake in the cKO mice compared with control littermates. We also observed a significant increase in the level of expression of oxidative stress-responsive genes in the intestine of the cKO mice compared with control littermates. Supplementation of the RFVT-3 cKO mice with pharmacological doses of RF led to a complete correction of the growth retardation and to normalization in the level of expression of the oxidative stress-responsive genes in the gut. These results show, for the first time, that the RFVT-3 system is the main transporter involved in carrier-mediated RF uptake in the native mouse small and large intestine, and that its dysfunction impairs normal RF body homeostasis.
              Cover page: Conditional (intestinal-specific) knockout of the riboflavin transporter-3 (RFVT-3) impairs riboflavin absorption.
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2011)
              Interleukin-1β (IL-1β) is a prototypical multifunctional cytokine that plays an important role in intestinal inflammation of Crohn's disease and other inflammatory conditions of the gut. Previous studies have shown that IL-1β causes an increase in intestinal epithelial tight junction (TJ) permeability both in in vivo animal and in vitro cell culture model systems. The IL-1β-induced increase in intestinal epithelial TJ permeability has been postulated to be an important pathogenic mechanism contributing to intestinal inflammation. However, the signalling pathways and the molecular processes that mediate the IL-1β modulation of intestinal epithelial TJ barrier remain unclear. Here, we show that the IL-1β-induced increase in Caco-2 monolayer TJ permeability was mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2) signalling pathway and that inhibition of ERK1/2 activity inhibits the IL-1β-induced increase in Caco-2 TJ permeability. The activation of ERK1/2 pathway caused a downstream activation of nuclear transcription factor Elk-1. The activated Elk-1 translocated to the nucleus and binds to the cis-binding motif on myosin light chain kinase (MLCK) promoter region, triggering MLCK gene activation, MLCK mRNA transcription and MLCK protein synthesis and MLCK catalysed opening of the intestinal epithelial TJ barrier. These studies provide novel insight into the cellular and molecular processes that mediate the IL-1β-induced increase in intestinal epithelial TJ permeability.
                Cover page: Cellular and molecular mechanism of interleukin‐1β modulation of Caco‐2 intestinal epithelial tight junction barrierCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2016)
                The recently identified human thiamine pyrophosphate transporter (hTPPT; product of the SLC44A4 gene) is responsible for absorption of the microbiota-generated TPP in the large intestine. The hTPPT is highly expressed in the colon, but not in other regions of the intestinal tract and is localized exclusively at the apical membrane domain of epithelia. The hTPPT protein is predicted to have multiple TM domains with a number of putative N-glycosylation sites, but it is not known if the protein is actually glycosylated, and if so at which site, and their role in the functionality of the transporter. Using several approaches including inhibiting de novo N-glycosylation in human colonic epithelial NCM460 cells with tunicamycin as well as enzymatic de-glycosylation, we show that the hTPPT protein is, indeed, a glycoprotein. Glycosylation of hTPPT was shown, by mean of site-directed mutagenesis, to occur at Asn(69), Asn(155), Asn(197), Asn(393), and Asn(416). However, only N-glycosylation at Asn(69), Asn(155), and Asn(393) appeared to be important for transporter functionality possibly through an effect on protein conformation and/or interaction with its ligand (but not through changes in expression at the cell membrane as determined by live cell confocal imaging). Results of this study showed, for the first time, that the hTPPT is glycosylated and that N-linked glycosylation occurs at multiple sites with some of them being important for function. The results also provide an indirect support for a membrane topology for hTPPT with 10 transmembrane domains as predicted by the TMHMM transmembrane helixes prediction program.
                  Cover page: The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2014)

                  Background

                  The human thiamine transporter-2 (hTHTR-2) is involved in the intestinal absorption of thiamine. Recent studies with membrane transporters of other nutrients/substrates have shown that they have associated proteins that affect different aspects of their physiology and cell biology. Nothing is known about protein(s) that interact with hTHTR-2 in intestinal epithelial cells and influence its physiological function and/or its cell biology.

                  Aims

                  The aim of this study was to identify protein partner(s) that interact with hTHTR-2 in human intestinal cells and determine the physiological/biological consequence of that interaction.

                  Methods

                  The yeast split-ubiquitin two-hybrid approach was used to screen a human intestinal cDNA library. GST-pull-down and cellular co-localization approaches were used to confirm the interaction between hTHTR-2 and the associated protein(s). The effect of such an interaction on hTHTR-2 function was examined by (3)H-thiamine uptake assays.

                  Results

                  Our screening results identified the human TransMembrane 4 SuperFamily 4 (TM4SF4) as a potential interactor with hTHTR-2. This interaction was confirmed by an in vitro GST-pull-down assay, and by live-cell confocal imaging of HuTu-80 cells co-expressing hTHTR-2-GFP and mCherry-TM4SF4 (the latter displayed a significant overlap of these two proteins in intracellular vesicles and at the cell membrane). Co-expression of hTHTR-2 with TM4SF4 in HuTu-80 cells led to a significant induction in thiamine uptake. In contrast, silencing TM4SF4 with gene-specific siRNA led to a significant decrease in thiamine uptake.

                  Conclusions

                  These results show for the first time that the accessory protein TM4SF4 interacts with hTHTR-2 and influences the physiological function of the thiamine transporter.
                    Cover page: Association of TM4SF4 with the Human Thiamine Transporter-2 in Intestinal Epithelial Cells
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2017)
                    [This corrects the article DOI: 10.1371/journal.pone.0149255.].
                      Cover page: Correction: Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter
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