Borate Transport and Cell Growth and Proliferation: Not Only in Plants

Boron is an abundant mineral essential for the life cycle of plants and may play a role in animal development and growth. Very little is known about boron homeostasis in plant and animal cells and the physiological roles of boron in animals. The recent identification of boron transporters, BOR1 in plants and NaBC1 in mammals, and that NaBC1 functions as an electrogenic Na+-coupled borate transporter essential for cell growth and proliferation open the way to probe the roles of boron in cellular function and physiology.     

Soil bacteria confer plant salt tolerance by tissue-specific regulation of the sodium transporter HKT1

Elevated sodium (Na(+)) decreases plant growth and, thereby, agricultural productivity. The ion transporter high-affinity K(+) transporter (HKT)1 controls Na(+) import in roots, yet dysfunction or overexpression of HKT1 fails to increase salt tolerance, raising questions as to HKT1's role in regulating Na(+) homeostasis. Here, we report that tissue-specific regulation of HKT1 by the soil bacterium Bacillus subtilis GB03 confers salt tolerance in Arabidopsis thaliana. Under salt stress (100 mM NaCl), GB03 concurrently down- and upregulates HKT1 expression in roots and shoots, respectively, resulting in lower Na(+) accumulation throughout the plant compared with controls. Consistent with HKT1 participation in GB03-induced salt tolerance, GB03 fails to rescue salt-stressed athkt1 mutants from stunted foliar growth and elevated total Na(+) whereas salt-stressed Na(+) export mutants sos3 show GB03-induced salt tolerance with enhanced shoot and root growth as well as reduced total Na(+). These results demonstrate that tissue-specific regulation of HKT1 is critical for managing Na(+) homeostasis in salt-stressed plants, as well as underscore the breadth and sophistication of plant-microbe interactions.     

Effect of extracellular calcium,pH and borate on growth oscillations in Lilium formosanum pollen tubes

Calcium ions (Ca(2+)), protons (H(+)), and borate (B(OH)(4)(-)) are essential ions in the control of tip growth of pollen tubes. All three ions may interact with pectins, a major component of the expanding pollen tube cell wall. Ca(2+ )is thought to bind acidic residues, and cross-link adjacent pectin chains, thereby strengthening the cell wall. Protons are loosening agents; in pollen tube walls they may act through the enzyme pectin methylesterase (PME), and either reduce demethylation or stimulate hydrolysis of pectin. Finally, borate cross-links monomers of rhamnogalacturonan II (RG-II), and thus stiffens the cell wall. It is demonstrated here that changing the extracellular concentrations of Ca(2+), H(+) and borate affect not only the average growth rate of lily pollen tubes, but also influence the period of growth rate oscillations. The most dramatic effects are observed with increasing concentrations of Ca(2+) and borate, both of which markedly reduce the rate of growth of oscillating pollen tubes. Protons are less active, except at pH 7.0 where growth is inhibited. It is noteworthy, especially with borate, that the faster growing tubes exhibit the shorter periods of oscillation. The results are consistent with the idea that binding of Ca(2+) and borate to the cell wall may act at a similar level to alter the mechanical properties of the apical cell wall, with optimal concentrations being high enough to impart sufficient rigidity to the wall so as to prevent bursting in the face of cell turgor, but low enough to allow the wall to stretch quickly during periods of accelerating growth.     

Expression of the Arabidopsis borate efflux transporter gene, AtBOR4, in rice affects the xylem loading of boron and tolerance to excess boron

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.     

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter

Regulation of intra- and extracellular ion activities (e.g. H(+), Cl(-), Na(+)) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na(+)/HCO(3)(-) cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO(3)(-) transporters are known: a neutral Na(+)/ HCO(3)(-) cotransporter, a K(+)/ HCO(3)(-) cotransporter, and a Na(+)-dependent Cl(-)-HCO(3)(-) exchanger. We report the cloning and characterization of a Na(+)-coupled Cl(-)-HCO(3)(-) exchanger and a physiologically unique bicarbonate transporter superfamily member. This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughout Drosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl(-), Na(+), H(+), and HCO(3)(-) but does not require HCO(3)(-). Transport is blocked by the stilbene 4,4'-diisothiocyanodihydrostilbene- 2, 2'-disulfonates and may not be strictly electroneutral. Our functional data suggest this Na(+) driven anion exchanger (NDAE1) is responsible for the Na(+)-dependent Cl(-)-HCO(3)(-) exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.     

Plasmalemmal V-H(+)-ATPases regulate intracellular pH in human lung microvascular endothelial cells

The lung endothelium layer is exposed to continuous CO(2) transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na(+)/H(+) exchanger and HCO(3)(-)-dependent H(+)-transporting mechanisms regulate intracellular pH (pH(cyt)) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H(+)-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na(+)/H(+) exchanger and HCO(3)(-)-based H(+)-transporting mechanisms, to maintain pH(cyt) homeostasis. Immunocytochemical studies revealed V-H(+)-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na(+) and HCO(3)(-) that were similar to those observed in the presence of either Na(+), or Na(+) and HCO(3)(-). The Na(+)- and HCO(3)(-)-independent pH(cyt) recovery was inhibited by bafilomycin A(1), a V-H(+)-ATPase inhibitor. These studies show a Na(+)- and HCO(3)(-)-independent pH(cyt) regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases.     

AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.     

Molecular expression of SLC4-derived Na+-dependent anion transporters in selected human tissues

NaHCO(3) transporters are involved in maintenance of intracellular pH and transepithelial HCO(3)(-) movement in many rodent tissues. To establish the human relevance of the many investigations on rodents, this study aimed to map these transporters and a related polypeptide, NaBC1 [solute carrier 4 (SLC4)A11], to several human tissues by using PCR on reverse transcribed human mRNA and immunoperoxidase histochemistry. The mRNA encoding the electroneutral Na(+):HCO(3)(-) cotransporter (NBCe1; SLC4A4), was expressed in renal cortex, renal medulla, stomach, duodenum, jejunum, ileum, colon, pancreas, choroid plexus, cerebellum, cerebrum, and hippocampus. NBCe2 (SLC4A5) and NBCn1 (SLC4A7) mRNAs were mainly found in kidney and brain tissues, as was mRNA encoding the Na(+)-dependent anion exchangers NCBE (SLC4A10) and NDCBE1 (SLC4A8). In addition to previous findings, NBCn1 protein was localized to human renal medullary thick ascending limbs and duodenal epithelial villus cells and NBCe2 protein to renal collecting ducts. Finally, the message encoding NaBC1 was found in kidney, stomach, duodenum, pancreas, and brain, and the corresponding protein in the anterior and posterior corneal epithelia, renal corpuscules, proximal tubules, collecting ducts, pancreatic ducts, and the choroid plexus epithelium. In conclusion, the selected human tissues display distinct expression patterns of HCO(3)(-) transporters, which closely resemble that of rodent tissues.     

Genes essential to sodium-dependent bicarbonate transport in cyanobacteria: function and phylogenetic analysis

The cyanobacterium Synechocystis sp. strain PCC 6803 possesses two CO(2) uptake systems and two HCO(3)(-) transporters. We transformed a mutant impaired in CO(2) uptake and in cmpA-D encoding a HCO(3)(-)transporter with a transposon inactivation library, and we recovered mutants unable to take up HCO(3)(-) and grow in low CO(2) at pH 9.0. They are all tagged within slr1512 (designated sbtA). We show that SbtA-mediated transport is induced by low CO(2), requires Na(+), and plays the major role in HCO(3)(-) uptake in Synechocystis. Inactivation of slr1509 (homologous to ntpJ encoding a Na(+)/K(+)-translocating protein) abolished the ability of cells to grow at [Na(+)] higher than 100 mm and severely depressed the activity of the SbtA-mediated HCO(3)(-) transport. We propose that the SbtA-mediated HCO(3)(-) transport is driven by DeltamuNa(+) across the plasma membrane, which is disrupted by inactivating ntpJ. Phylogenetic analyses indicated that two types of sbtA exist in various cyanobacterial strains, all of which possess ntpJ. The sbtA gene is the first one identified as essential to Na(+)-dependent HCO(3)(-) transport in photosynthetic organisms and may play a crucial role in carbon acquisition when CO(2) supply is limited, or in Prochlorococcus strains that do not possess CO(2) uptake systems or Cmp-dependent HCO(3)(-) transport.     

Syntheses of (±)-Epoformin (Desoxyepoxydon) and (±)-Epiepoformin (Desoxyepiepoxydon)

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.     

Pharmacological modulation of ion channels and transporters

Ion channels and transporter proteins are prerequisites for formation and conduction of cardiac electrical impulses. Acting in concert, these proteins maintain cellular Na(+) and Ca(2+) homeostasis. Since intracellular Ca(2+) concentration determines contractile activation, we expect the majority of agents that modulate activity of ion channels and transporters not only to influence cellular action potentials but also contractile force. Drugs which block ion channels usually possess antiarrhythmic properties, those inhibiting the Na(+) pump have predominantly inotropic effects and those affecting Na(+),Ca(2+)- or Na(+),H(+)-exchanger protect against ischaemic cell damage. However, irrespective of their primary indication, all compounds targeted against ion channels and transporter proteins possess potential proarrhythmic activity.     

cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1

Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.     

Intracellular Na and K distribution in Debaryomyces hansenii. Cloning and expression in Saccharomyces cerevisiae of DhNHX1

Debaryomyces hansenii is a salt-tolerant yeast that contains high amounts of internal Na(+). Debaryomyces hansenii kept more sodium than Saccharomyces cerevisiae in both the cytoplasm and vacuole when grown under a variety of NaCl concentrations. These results indicate a higher tolerance of Debaryomyces to high internal Na(+), and, in addition, suggest the existence of a transporter driving Na(+) into the vacuole. Moreover, a gene encoding a Na(+) (K(+))/H(+) antiporter from D. hansenii was cloned and sequenced. The gene, designated DhNHX1, exhibited significant homology with genes of the NHE/NHX family. DhNHX1 expression was induced neither at low pH nor by extracellular NaCl. A mutant of S. cerevisiae lacking its own Na(+) transporters (ena1-4Delta nha1 Delta nhx1 Delta), when transformed with DhNHX1, partially recovered cation tolerance as well as the ability to accumulate Na(+) and K(+) into the vacuole. Our analysis provides evidence that DhNhx1p transports Na(+) (and K(+)) into the vacuole and that it can play an important role in ion homeostasis and salt tolerance.     

The effects of 1alpha,24(S)-dihydroxyvitamin D(2) analog on cancer cell proliferation and cytokine expression

It is well established that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, plays a role in regulating proliferation and differentiation of cells, in addition to its classic function in mineral homeostasis. Recent studies have also provided evidence for the involvement of 1alpha,25(OH)(2)D(3) in regulating the immune system. However, therapeutic application of 1alpha,25(OH)(2)D(3) to hyperproliferative diseases such as cancer, or for immunologic purposes, is thwarted by its hypercalcemic activity. In order to overcome this obstacle, analogs of 1alpha,25(OH)(2)D(3) have been produced that exhibit decreased hypercalcemic activity while retaining the growth and immunologic regulating properties. In the present study, the efficacy of 1alpha,24(S)-dihydroxyvitamin D(2) (1alpha,24(S)(OH)(2)D(2)), a vitamin D(2) analog, in restraining cell proliferation was compared to that of 1alpha,25(OH)(2)D(3). In parallel studies, cancer cell lines were grown in increased concentrations (10(-10)-10(-7) M) of each compound for various incubation periods (1-4 days). Growth was assessed by measuring [(3)H]thymidine incorporation. The results revealed that 1alpha,24(S)(OH)(2)D(2) significantly inhibits proliferation to an extent similar to that observed for 1alpha,25(OH)(2)D(3). Moreover, incubating the human leukemia cell line, HL-60, with 1alpha,24(S)(OH)(2)D(2) resulted in an induction of differentiation of these promyelomonocyte cells into monocyte-macrophage-like cells, in a manner similar to that observed with 1alpha,25(OH)(2)D(3). Using a Western procedure, it was also shown that 1alpha,24(S)(OH)(2)D(2) like 1alpha,25(OH)(2)D(3) enhances the expression of vitamin D receptors (VDR) in the rat osteosarcoma cell line, ROS 17/2.8. The expression of tumor necrosis factor (TNF) alpha (TNF-alpha) in human peritoneal macrophages (HPM) obtained from uremic patients treated with continuous ambulatory peritoneal dialysis (CAPD) was found to be regulated by 1alpha,25(OH)(2)D(3) as well as by 1alpha,24(S)(OH)(2)D(2). Incubations of HPM with 1alpha,25(OH)(2)D(3) or 1alpha,24(S)(OH)(2)D(2), have inhibited the expression of TNF-alpha on both mRNA and protein levels. These results suggest that 1alpha,25(OH)(2)D(3) has a role in controlling the rate of inflammation in the peritoneal cavity of CAPD treated patients. Since 1alpha,24(S)(OH)(2)D(2) does not cause hypercalcemia, the present results encourage the possible use of this vitamin D(2) analog in the treatment of cancer and hyper-inflammatory diseases.     

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia

The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO(3)(-) transporter family (Na(+)/HCO(3)(-) cotransporter NBC1 and Cl(-)/HCO(3)(-) exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO(2). Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na(+)/H(+) exchangers (NHE1A, NHE1B) during the first days (by 50-60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na(+)-K(+)-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO(2) exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na(+)-K(+)-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.     

The Arabidopsis kinase-associated protein phosphatase regulates adaptation to Na+ stress

The kinase-associated protein phosphatase (KAPP) is a regulator of the receptor-like kinase (RLK) signaling pathway. Loss-of-function mutations rag1-1 (root attenuated growth1-1) and rag1-2, in the locus encoding KAPP, cause NaCl hypersensitivity in Arabidopsis thaliana. The NaCl hypersensitive phenotype exhibited by rag1 seedlings includes reduced shoot and primary root growth, root tip swelling, and increased lateral root formation. The phenotype exhibited by rag1-1 seedlings is associated with a specific response to Na(+) toxicity. The sensitivity to Na(+) is Ca(2+) independent and is not due to altered intracellular K(+)/Na(+). Analysis of the genetic interaction between rag1-1 and salt overly sensitive1 (sos1-14) revealed that KAPP is not a component of the SOS signal transduction pathway, the only Na(+) homeostasis signaling pathway identified so far in plants. All together, these results implicate KAPP as a functional component of the RLK signaling pathway, which also mediates adaptation to Na(+) stress. RLK pathway components, known to be modulated by NaCl at the messenger RNA level, are constitutively down-regulated in rag1-1 mutant plants. The effect of NaCl on their expression is not altered by the rag1-1 mutation.     

Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)(2)D(3) levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)(2)D(3) on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)(2)D(3), although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1 alpha-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)(2)D(3) and/or activation. Importantly, 1,25(OH)(2)D(3) up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)(2)D(3) may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.     

Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.