High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24 h and had shifted more cells from G0/G1 to S+G2/M within 12 h, suggesting that an early event may play a role. We here show that expression of human PiT1 in NIH3T3 cells led to faster cell adhesion; this effect was not cell type specific in that it was also observed when expressing human PiT1 in MC3T3-E1 cells. We also show for NIH3T3 that PiT1 overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby contribute to their proliferative advantage within the first 24 h after seeding.     

Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium-dependent phosphate transport function of human PiT2

The mammalian members of the inorganic phosphate (P(i)) transporter (PiT) family, the type III sodium-dependent phosphate (NaP(i)) transporters PiT1 and PiT2, have been assigned housekeeping P(i) transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na+) or proton (H+) gradients to transport P(i). Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N- and C-terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N- and C-terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed P(i) uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaP(i) transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild-type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na+-dependent P(i) transport function of human PiT2. The conservation of the aspartates among proteins using either Na+- or H+-gradients for P(i) transport suggests that they are involved in H+-dependent P(i) transport as well. Current results favor a membrane topology model in which the N- and C-terminal PiT family signature sequences are positioned in intra- and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.     

Regulation of nebramycin biosynthesis by inorganic phosphate

The effect of inorganic phosphate on the biosynthesis of nebramycin factors2, 4 and5′ was studied inStreptomyces tenebrarius strain A (forming2, 4 and5′ in natural ratios) and its mutants B (forming predominantly2), C (forming2 as the only major product) and D (forming predominantly5′). In phosphate-supplemented complex media, the production of2 in A, B and C was reduced by 20–70%, while the yields of5′ remained unchanged in A and decreased by 30–60% in B. The production of4 increased by 50–90% in A and was fully suppressed in B. In D the biosynthesis of the three factors was inhibited completely.     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Regulation of 5-phosphoribosyl-1-pyrophosphate synthesis in human fibroblasts by the concentration of inorganic phosphate

During the growth cycle of normal fibroblasts and of fibroblasts deficient in glucose-6-phosphate dehydrogenase activity, the concentration of 5-phosphoribosyl-1-pyrophosphate and of P_i, as well as the activity of 5-phosphoribosyl-1-pyrophosphate synthetase, decreased to stable values in confluent cultures. A high degree of correlation (0.89 and 0.91 for two normal and 0.69 for one glucose-6-phosphate dehydrogenase-deficient cell strain, respectively) was shown between intracellular P_i and 5-phosphoribosyl-1-pyrophosphate concentrations under varying culture and incubation conditions. 5-phosphoribosyl-1-pyrophosphate concentrations were elevated in normal fibroblasts incubated with methylene blue only if intracellular P_i levels were high. Neither methylene blue nor 6-aminonicotinamide, singly, affected intracellular P_i concentrations. However, when normal cells were pretreated with 6-aminonicotinamide and then with methylene blue, intracellular P_i decreased, 5-phosphoribosyl-1-pyrophosphate was depleted, and its rate of generation decreased. Under similar conditions, glucose-6-phosphate dehydrogenase-deficient fibroblasts maintained unaltered P_i levels, and 5-phosphoribosyl-1-pyrophosphate concentration and generation were slightly increased. The decrease in intracellular P_i in normal cells after the combined treatment was commensurate with an accumulation of 6-phosphogluconate, which did not take place in mutant cells. The changes in 5-phosphoribosyl-1-pyrophosphate synthesis, whether due to the stage of growth or various experimental manipulations, were always concordant with changes in intracellular P_i level. The regulatory role of P_i is consistent with the known enzymic properties of 5-phosphoribosyl-1-pyrophosphate synthetase.     

Regulation of murine T cell proliferation by B cell stimulatory factor-1

The proliferation of mitogen-activated primary T cells, antigen-activated memory T cells from mixed leukocyte culture, and antigen-dependent alloreactive T cell clones in response to purified murine recombinant B cell stimulatory factor-1 (also known as interleukin 4) was examined. We found that B cell stimulatory factor-1 (BSF-1) stimulated optimal proliferation of these T cells only after their recent activation by antigen or mitogen. Analysis of cell surface BSF-1 receptor expression indicated that although T cell activation is accompanied by a small increase in BSF-1 receptor expression, the cells also express BSF-1 receptors prior to activation at a time when they do not proliferate in response to BSF-1. BSF-1 was as effective a stimulus as interleukin 2 for inducing proliferation of the Lyt-2+ subpopulation of concanavalin A-activated murine spleen cells and an alloreactive cytolytic T cell clone. However, the L3T4+ subpopulation of concanavalin A-activated spleen and an alloreactive helper T cell clone were less responsive to BSF-1 than to interleukin 2. Taken together, the data indicate an important role for BSF-1 in the regulation of normal T cell proliferation.     

Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillus ficuum

Two types of extracellular acid phosphatases are synthesized by Aspergillus ficuum NRRL 3135: a nonspecific orthophosphoric monoester phosphohydrolase (EC 3.1.3.2) with an optimum pH of 2.0, and an enzyme with restricted specificity, a mesoinositol-hexaphosphate phosphohydrolase (EC 3.1.3.8; phytase) with an optimum pH of 5.5. Although the pH 5.5 enzyme is termed a phytase, both enzymes hydrolyze phytin. Synthesis of the enzymes is repressed by high orthophosphate concentrations in the fermentation medium. The highest total level for each enzyme is synthesized in low orthophosphate medium. In high orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme. In low orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme during the early stages of growth, but the reverse occurs after 5 days. The enzymes are differentiated by heat denaturation at acid and alkaline pH levels. They are separated into two distinct fractions on Sephadex G-100 followed by carboxymethylcellulose column chromatography. This indicates that the two enzymes are structurally different. The K(m) for both enzymes is 1.25 mm when calcium phytate is the substrate. Orthophosphate competitively inhibits the pH 2.0 (K(i) = 1.1 x 10(-2)m) but not the pH 5.5 phosphatase. Neither enzyme is denatured by 50% (w/v) urea or inhibited by 0.01 m tartrate. Thus, they differ from human prostatic phosphatase.     

Regulation of muscle phosphorylase b kinase activity by inorganic phosphate and calcium ions

The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P(i) and Ca(2+) was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P(i) increased activity severalfold. The effect was concentration-dependent, with an apparent K(m) of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg(2+) in addition to P(i). The apparent K(m) for Mg(2+) was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P(i), and was strongly inhibited by K(+), Na(+) and Li(+). At physiological concentrations, Ca(2+) activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca(2+) of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca(2+), and showed a large increase in activity over a narrow range of Ca(2+) concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca(2+), and had a smaller increase over a wide range of Ca(2+) concentration. The concentrations of Ca(2+) required for half-activation were 0.1 and 1mum for the blowfly and rabbit enzymes respectively. The pH-activity profiles of the non-activated, phosphate- and Ca(2+)-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.     

Regulation of glutamate transporter GLT-1 by MAGI-1

The sodium-dependent glutamate transporter, glutamate transporter subtype 1 (GLT-1) is one of the main glutamate transporters in the brain. GLT-1 contains a COOH-terminal sequence similar to one in an isoform of Slo1 K(+) channel protein previously shown to bind MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1). MAGI-1 is a scaffold protein which allows the formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. The glutathione S-transferase pull-down assay demonstrated that MAGI-1 was a binding partner of GLT-1. The interaction between MAGI-1 and GLT-1 was confirmed by co-immunoprecipitation. Immunofluorescence of MAGI-1 and GLT-1 demonstrated that the distribution of MAGI-1 and GLT-1 overlapped in astrocytes. Co-expression of MAGI-1 with GLT-1 in C6 Glioma cells resulted in a significant reduction in the surface expression of GLT-1, as assessed by cell-surface biotinylation. On the other hand, partial knockdown of endogenous MAGI-1 expression by small interfering RNA in differentiated cultured astrocytes increased glutamate uptake and the surface expression of endogenous GLT-1. Knockdown of MAGI-1 increased dihydrokainate-sensitive, Na(+) -dependent glutamate uptake, indicating that MAGI-1 regulates GLT-1 mediated glutamate uptake. These data suggest that MAGI-1 regulates surface expression of GLT-1 and the level of glutamate in the hippocampus.     

Glutamate-induced glioma cell proliferation is prevented by functional expression of the glutamate transporter GLT-1

A tetracycline-dependent inducible system was used to achieve controlled expression of the glutamate transporter 1 (GLT-1) in C6 glioma cells. Non-induced cells show modest glutamate uptake and, in the presence of L-cystine, these cells tend to release substantial amounts of glutamate. Overnight exposure to doxycycline increased D-[3H]-aspartate uptake, reaching similar capacity as observed in cultured astrocytes. Efficient clearance of exogenously applied glutamate was evidenced in these cells, even in the presence of l-cystine. The addition of glutamate (100 microM) to the medium of non-induced cells significantly increased their proliferation rate, an effect that was blocked when the expression of GLT-1 was induced. This suggests that impaired glutamate uptake capacity in glioma cells indirectly contributes to their proliferation.     

Regulation of tomato HMG1 during cell proliferation and growth

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is encoded by a small multigene family in tomato (Lycopersiconesculentum Mill.) and catalyzes the synthesis of mevalonic acid (MVA), a committed step in the biosynthesis of sterols and isoprenoids. A chimeric HMG1::GUS reporter gene fusion was used to analyze the regulation of HMG1 gene expression in detail. HMG1 promoter 5′ deletion mutants established the boundary of a fully inducible promoter. In HMG1::GUS transgenic tomato plants, histochemical staining with 5-bromo-3-indolyl-glucuronide demonstrated that HMG1 was primarily expressed in shoot and root meristems, and in young tomato fruit. This result was confirmed by both HMG1 in-situ hybridization and RNA gel blot analysis. Tomato suspension cell experiments showed that steady-state HMG1 mRNA accumulated during lag and exponential growth phases, but not during the stationary phase. Transient expression of the HMG1::GUS in tissue culture cells treated with mevinolin indicated that HMG1 expression was subject to feedback regulation by a biosynthetic product derived from MVA. These results suggest that a primary, although not exclusive, role of HMG1 is to supply the MVA demand associated with cell division and growth. Received: 26 October 1998 / Accepted: 16 December 1998     

Regulation of T cell proliferation by IL-7

The regulation of murine T cell proliferation by IL-7 was investigated. Highly purified resting splenic T cells were induced to proliferate in a short term assay by IL-7 in the presence of the comitogen, Con A. The proliferation of these resting T cells showed both IL-2-dependent and -independent components as determined by the susceptibility of the response to the blocking effects of anti-IL-2 mAb. Furthermore, IL-7 was found to augment the Con A-induced production of IL-2 and expression of IL-2R by resting splenic T cells. In contrast, Con A blasts and long term, Ag-dependent cloned T cells proliferated in response to IL-7 independently of any involvement of IL-2. Finally, differences were observed between IL-7 and IL-6 with regard to the regulation of T cell growth and activation. As with IL-7, IL-6 stimulated resting splenic T cells to proliferate in the presence of comitogen. However, in contrast to IL-7, IL-6 failed to stimulate the proliferation of Con A blasts or T cell clones and did not augment the Con A-induced expression of IL-2R on resting T cells.     

Regulation of cell proliferation by epidermal growth factor

Epidermal Growth Factor (EGF) is a 6045 dalton polypeptide which stimulates the proliferation of various cell types in vitro and in vivo. EGF binds to diffusely distributed membrane receptors which rapidly cluster primarily on coated pits areas on the plasma membrane. Subsequently, the EGF-receptor complexes are endocytosed and degraded by lysosomal enzymes. The lateral diffusion coefficient (D) of EGF-receptor complexes on cultured cells increases gradually from D = 2.8 X 10(-10) cm2/sec at 5 degrees C to 8.5 X 10(-10) cm2/sec at 37 degrees C. In the same range of temperature the rotational correlation times change from 25 to 50 microseconds to approximately 350 microseconds. Hence, at 4 degrees C, the occupied EGF receptors translate and rotate rapidly in the plane of the membrane. At 37 degrees C, EGF receptors form microclusters composed of 10 to 50 molecules. Moreover, it is concluded that both at 4 degrees C and 37 degrees C lateral diffusion of the occupied receptors is not the rate determining step for either receptor clustering or internalization. EGF receptor is a 150,000 to 170,000 dalton glycoprotein. The receptor is in close proximity to an EGF-sensitive, cAMP-independent, tyrosine-specific protein kinase which also phosphorylates the receptor molecules itself. The EGF sensitive kinase is similar to the kinase activity which is associated with certain RNA tumor viruses. The fact that the non-mitogenic cyanogen-bromide cleaved EGF is as potent as native EGF in stimulating phosphorylation suggests that EGF-induced, protein phosphorylation is a necessary but insufficient signal for the induction of DNA synthesis by EGF. EGF receptor serves also as the binding site for Transforming Growth Factors (TGF) which compete with EGF and induce anchorage-independent growth of normal cells in soft agar. Tumor promoters such as phorbol ester effect the binding of EGF to its membrane receptors and its ability to stimulate DNA synthesis. EGF itself has also some tumor promoting activity. Hence, the membrane receptor for EGF seems to participate in the regulation of normal and neoplastic growth. Monoclonal antibodies against EGF receptor (IgM) induce various early and delayed effects of EGF, while their monovalent Fab' fragments are devoid of biological activity. These observations support the notions that EGF receptor rather than EGF itself is the active moiety and that the role of the hormone is to perturb the receptor in the appropriate way, probably by inducing the microaggregation of EGF receptors.     

Regulation of cell proliferation by a morphogen gradient

One model to explain the relationship between patterning and growth during development posits that growth is regulated by the slope of morphogen gradients. The Decapentaplegic (DPP) morphogen controls growth in the Drosophila wing, but the slope of the DPP activity gradient has not been shown to influence growth. By employing a method for spatial, temporal, and quantitative control over gene expression, we show that the juxtaposition of cells perceiving different levels of DPP signaling is essential for medial-wing-cell proliferation and can be sufficient to promote the proliferation of cells throughout the wing. Either activation or inhibition of the DPP pathway in clones at levels distinct from those in surrounding cells stimulates nonautonomous cell proliferation. Conversely, uniform activation of the DPP pathway inhibits cell proliferation in medial wing cells. Our observations provide a direct demonstration that the slope of a morphogen gradient regulates growth during development.     

Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae.

A kinetic study of Pi transport with 32Pi revealed that Saccharomyces cerevisiae has two systems of Pi transport, one with a low Km value (8.2 microM) for external Pi and the other with a high Km value (770 microM). The low-Km system was derepressed by Pi starvation, and the activity was expressed under the control of a genetic system which regulates the repressible acid and alkaline phosphatases. The function of the PHO2 gene, which is essential for the derepression of repressible acid phosphatase but not for the derepression of repressible alkaline phosphatase, was also indispensable for the derepression of the low-Km system.     

Expression of PiT1 and PiT2 retroviral receptors and transduction efficiency of tumor cells

Recombinant retroviral vectors are still the most common gene delivery vehicles for gene therapy purposes, especially for construction of genetically modified tumor vaccines (GMTV). However, these vehicles are characterized by relatively low titre and in the case of many tumor cell lines, low transduction efficiency. We constructed bicistronic retroviral vector pseudotypes of amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GaLV), encoding enhanced green fluorescent protein (EGFP) as a rapid and easily detectable reporter gene. Transduction of five different human melanoma and four renal carcinoma cell lines by these two virus pseudotypes revealed differences in transduction efficiency, which wase markedly lower for the renal carcinoma cell lines. Stimulation of retroviral receptor expression (PiT1 and PiT2) by phosphate depletion induced a limited increase of receptor mRNA levels, but did not improve the gene transfer efficiency. In contrast, simultaneous transduction with both vector pseudotypes markedly increased the transduction efficiency, compared to GaLV or A-MuLV alone. The same effect could be achieved by several repeated exposures of target cells to fresh vector preparation. Overexpression of GaLV receptor (PiT1) in target cells significantly increased the transduction rate and enabled retrovirus mediated gene transfer into the cells which normally are not transducible by GaLV pseudotypes. We demonstrated that, using different transduction strategies, the relatively inefficient, widely used retroviral vector systems could be significantly improved.