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Am J Physiol Cell Physiol
2017 May 01;3125:C550-C561. doi: 10.1152/ajpcell.00219.2015.
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Identification of a mammalian silicon transporter.
Ratcliffe S
,
Jugdaohsingh R
,
Vivancos J
,
Marron A
,
Deshmukh R
,
Ma JF
,
Mitani-Ueno N
,
Robertson J
,
Wills J
,
Boekschoten MV
,
Müller M
,
Mawhinney RC
,
Kinrade SD
,
Isenring P
,
Bélanger RR
,
Powell JJ
.
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Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.
Fig. 1. Identifying RnSlc34a2 as a candidate for Si transport. A: relative expression of solute-like carriers in the kidney of Rattus norvegicus from Si deplete (n = 4) compared with Si replete (n = 4) animals. Data were analyzed by gene array. Red indicates upregulation and blue indicates downregulation of expression; Si replete vs. Si deplete group. Multiple probe sets per gene can be present as was the case for Slc13a1. B: quantitative PCR analysis of Slc34a2 and of family members (inset) in the kidneys of Si-high reference, Si replete and Si deplete rats. Overall, the relative expression of RnSlc34a2 was inversely related to dietary Si exposure (P < 0.05), but there was no relationship with Slc34a1 or Slc34a3 (P = 0.5 and 0.4, respectively). Gene expression values are relative to the Si-high reference group. C: sequence alignment of the Rattus norvegicus Slc34 gene family. Slc34a2 is characterized by a ~30-residue stretch (highlighted in yellow) that is not present in Scl34a1 and Slc34a3. Asterisks (*) below sequence indicate identical amino acids, colons (:) indicate functionally similar amino acids, and dashes (–) indicate gaps in the alignment.
Fig. 2. Correlation between renal RnSlc34a2 expression (by quantitative RT-PCR analysis) and fasting urinary Si excretion. Urinary Si excretion in the rats (■, Si deplete; ●, Si replete) and laboratory chow reference group (▲, Si-high reference) showed an inverse relationship with Slc34a2 expression in the kidneys; r = 0.47.
Fig. 3. Immunohistochemistry analysis of Slc34a2 in freshly harvested rat kidney cortex. Sections of freshly harvested kidneys from a healthy wild-type rat were analyzed by immunohistochemistry with anti-Slc34a2 (green) antibody (this figure) or the appropriate isotype control (data not shown). Cell nuclei were counterstained (blue) with Hoescht 33342 and cell cytoskeleton (f-actin, red) with phalloidin CF633. Antibody-stained sections and isotype controls were collected under identical settings, as stated in materials
and
methods. A threshold removing all Slc34a2 attributable signal was defined on the isotype controls and uniformly applied to all images (i.e., antibody-stained images). Staining for Slc34a2 within the tubular epithelial cells was distinctly punctate so, as well as the signal above the isotype control being presented in an as-collected “intensity” format (i.e., the more secondary antibody that is bound, the brighter the signal) (A and B), it is also displayed as a binary format (i.e., all signal that is brighter than isotype threshold is given the maximum intensity value) as this aids visualization (C and D). All images are of the kidney cortex and scale bars are 50 µm. B: as-collected “intensity” format without actin staining. D: a high-power image (×63 magnification) of the area within the quadrant in image (C).
Fig. 4. Fasting urinary phosphorus excretion. Urinary P excretion was measured in the laboratory chow reference group (Si-high reference; n = 6), Si replete (n = 5), and Si deplete (n = 8) rats by ICP-OES and corrected for creatinine concentration (A). The higher P excretion in the laboratory chow reference group is due to the higher P content of the diet (see B). However, the difference in urinary P excretion between the Si replete and Si deplete rats cannot be explained by a difference in dietary P content, but rather due to the upregulation of Slc34a2 in the latter group mediated by Si deficiency in the diet and drinking water (B).
Fig. 5. Transport activity in RnSlc34a2-expressing oocytes. A–C: influx transport activity of Rattus norvegicus Slc34a2 for arsenate, HAsO42− (P = 0.0001) (A), phosphate, HPO42− (P = 0.0008) (B), and silicic acid, H4SiO4 (P = 0.66) (C) . Rice transporter Lsi1 was used as a positive control for H4SiO4 influx (P < 0.0001). D: the concentrations of sodium and phosphate in the medium did not influence H4SiO4 influx by RnSlc34a2-expressing oocytes, nor that by OsLsi1-expressing oocytes (P < 0.0001 in both instances). Water-injected oocytes were used as a negative control. E: in H4SiO4 efflux studies, rice transporter Lsi2 was used as a positive control. Data were corrected against water-injected control oocytes. F: changes in sodium and phosphate concentration did not affect H4SiO4 efflux by Slc34a2 expressing oocytes. Data are shown as means ± SE (n = 15).
Fig. 6. Germanium transport activity in RnSlc34a2-expressing oocytes. Transport activity for H4GeO4 showing a lack of influx (P = 0.14) (A) but significant efflux (B) following a 2 h incubation (P = 0.004). Efflux was not significant at 30 min (P = 0.14) for Slc34a2-expressing oocytes. The rice Si transporters Lsi1 and Lsi2 were used as positive controls for influx and efflux activity, respectively (P < 0.0001 in both cases compared with negative control, water-injected oocytes).
Fig. 7. Pairwise alignment of the transmembrane domains of Si efflux transporters. Pairwise alignment of the transmembrane domains predicted in RnSlc34a2 rat protein (red) with the four Si efflux transporters in plants (green). Transmembrane domains were predicted by OCTOPUS (56), and subsequent alignment was performed by AlignMe tool (51). A: OsLsi2 (rice); B: ZmLsi2 (maize); C: HvLsi2 (barley); D: CmLsi2–1 (pumpkin).
Fig. 8. Phylogeny of Slc34a gene family member in vertebrates. A: the tree was produced using PhyML maximum likelihood analysis with the JTT+G+I model from an alignment of 880 positions. Numbers at nodes are a percentage of 100 bootstrap replicates, with nodes having <70% bootstrap support being collapsed. B: the tree was produced using Phylobayes Bayesian MCMC analysis under the CAT +G+I model (15 parallel chains with sampling every 100 cycles, burn-in one-fifth the total size of the chain) from an alignment of 880 positions. Numbers at nodes indicate posterior probabilities, with nodes having <0.95 support being collapsed. The scale bar indicates the average number of amino acid substitutions per site. The Slc34a1 clade is in green, the Slc34a2 clade is in blue, and the Slc34a3 clade is in red. The trees are rooted using the single Slc34a homolog identified from the lamprey genome. The Slc34a gene phylogeny largely agrees with the species phylogeny for vertebrates (33), with incongruent branches (e.g., the basal branches of the a2 clade) only having low statistical support. The maximum likelihood phylogenetic analyses resolve that the Slc34a clade evolved from a single ancestor in jawless vertebrates, and likely involved two main duplication events, initially producing the a3 and a1+2 clades, with a further divergence of the a1 and a2 clades. A teleost-specific duplication event resulted in the evolution of Slc34a2a and Slc34a2b, as found in stickleback and zebrafish. The Bayesian analysis had poor phylogenetic resolution at the base of the a2 clade, but still resolves the a1 and a3 groups as distinct monophyletic clades, and is not incongruous with the maximum likelihood analysis results. For full details of the species and sequences used see Supplemental Table S1.
Fig. 9. Alignment of vertebrate Slc34a protein sequences showing characteristic motif conserved across members of the Slc34a2 group. The alignment shows the region around the portion identified as unique to rat Slc34a2 in comparison to rat Slc34a1 or Slc34a3 (see Fig. 1C). Highlighted in yellow are the homologous regions in other vertebrate Slc34a2 proteins, and in the Slc34a-type lamprey sequence. The characteristic Slc34a2 motif identified within this region contains at least three positive residues uninterrupted by any negatively charged residues, with the positive residues regularly spaced apart by at least four small residues (primarily cysteines). A Slc34a sequence containing this motif was found in all vertebrate species investigated. The only members of the Slc34a2 clade (see Fig. 8) where this motif was incomplete was are in the zebrafish and stickleback SLC34a2b (highlighted in blue). Positively charged residues are shown in bold and small amino acids are in italics. Sequence names correspond to the species and gene identifiers given in Supplemental Table S1 and to the phylogeny shown in Fig. 8. The incomplete spiny shark and skate NaPi-IIb sequences are omitted due to this region being missing from the EMBL/GenBank data. The alignment was generated using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/).
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