Regulation of cell proliferation and cell density by the inorganic phosphate transporter PiT1
© Byskov et al; licensee BioMed Central Ltd. 2012
Received: 11 December 2011
Accepted: 6 March 2012
Published: 6 March 2012
The inorganic phosphate (Pi) transporter, PiT1 (SLC20A1), is ubiquitously expressed in mammalian cells. It has previously been shown that down-regulation of PiT1 severely impaired the proliferation of two transformed human cells lines, HepG2 and HeLa, and the tumorigenicity of HeLa cells in nude mice. Moreover, PiT1 knock-out mice do not survive past E12.5 and from E10.5, the embryos were found to be growth-retarded and showed reduced proliferation of liver cells. Isolated mouse embryonic fibroblasts with knocked out as well as reduced PiT1 expression levels also exhibited impaired proliferation. Together these results suggest that a certain level of PiT1 is important for proliferation. We have here investigated the role of PiT1 in regulation of cell proliferation using two strictly density-inhibited cells lines, the murine MC3T3-E1 and NIH3T3 cells.
We found that knock-down of PiT1 in MC3T3-E1 cells led to impaired proliferation supporting that at least a certain level of PiT1 is important for wildtype level of proliferation. We, however, also observed that MC3T3-E1 and NIH3T3 cells themselves regulate their endogenous PiT1 mRNA levels with lower levels in general correlating with decreased proliferation/increased cell density. Moreover, over-expression of human PiT1 led to increased proliferation of both MC3T3-E1 and NIH3T3 cultures and resulted in higher cell densities in cultures of these two strictly density-inhibited cell lines. In addition, when we transformed NIH3T3 cells by cultivation in fetal bovine serum, cells over-expressing human PiT1 formed more colonies in soft agar than control cells.
We conclude that not only is a certain level of PiT1 necessary for normal cell division as suggested by previously published studies, rather the cellular PiT1 level is involved in regulating cell proliferation and cell density and an increased PiT1 expression can indeed make NIH3T3 cells more sensitive to transformation. We have thus provided the first evidence for that expression of the type III Pi transporter, PiT1, above the endogenous level can drive cell proliferation and overrule cell density constraints, and the results bridge previous observations showing that a certain PiT1 level is important for regulating normal embryonic growth/development and for tumorigenicity of HeLa cells.
KeywordsPiT1 SLC20A1 inorganic phosphate transporter cell proliferation cell density transformation
The mammalian type III sodium-dependent inorganic phosphate (NaPi) symporters, PiT1 (SLC20A1; formerly GLVR1) and PiT2 (SLC20A2; formerly GLVR2 and Ram1) [1–6] belong to the Pi transport (PiT) family (SLC20; TC #2.A.20 ), which members are present in all kingdoms of life . The mammalian PiT proteins were originally identified as receptors for gammaretroviruses [2, 3, 5]. Thus, e.g., human PiT1 (hPiT1) is receptor for gibbon ape leukemia virus (GALV)  and feline leukemia virus subgroup B (FeLV-B)  and human PiT2 (hPiT2) and murine PiT2 (mPiT2) are receptors for amphotropic murine leukemia virus (A-MLV) [1, 5]. There are, however, differences between the receptor functions of PiT proteins from different species, e.g., unlike hPiT1, murine PiT1 (mPiT1) does not support infection by GALV and FeLV-B [3, 10–14].
Analysis of the Pi transport functions of the two human PiT paralogs shows comparable abilities to transport Pi . Moreover, both paralogs are ubiquitously expressed in mammalian cells and have been suggested to be housekeeping Pi transporters supplying cells with their general Pi needs [1, 16–18] and thus to have overlapping functions in cellular Pi import. This notion is supported by results obtained by Beck and co-workers on an allelic series of mutant mice in which PiT1 was expressed from 0 to 100% . In agreement with results from Festing and co-workers, they found that PiT1 knock-out mice did not survive past E12.5 [19, 20]. Beck and co-workers, however, also found that E11.5 embryos showing PiT1 mRNA levels below 15% of that observed in wildtype embryos, had up-regulated PiT2 expression. And the authors could indeed not observe differences in the Pi uptake abilities of mouse embryonic fibroblasts (MEFs) derived from wildtype and PiT1-knock-out embryos . These results suggest that PiT2 might be compensating for the loss of PiT1 as a supplier, at least in part, of Pi to the embryonic cells up to E11.5 to E12.5. However, despite these suggested overlapping functions of PiT1 and PiT2 in cellular Pi import, Beck and co-workers also showed that PiT1 possesses a cellular function, which cannot be replaced by PiT2. Thus compared to wildtype embryos, at E10.5 PiT1-knock-out embryos started to show retarded growth and when investigated at E11.5 and/or E12.5, the PiT1 knock-out embryos also showed reduced proliferation of liver cells . Moreover, although MEF cells derived from PiT1-knock-out embryos and from embryos showing PiT1 mRNA levels at 50% of wildtype levels had unimpaired Pi uptake abilities, they did show severely impaired proliferation . These observations suggest a function of PiT1 in proliferation, which is not related to cellular Pi uptake. In line with this, the same group has also shown that knock-down of the expression of PiT1 in the two transformed human cell lines, HeLa and HepG2, severely impairs their proliferation . In addition, HeLa cells with knocked down PiT1 expression showed severely reduced tumor growth after injection into nude mice compared to control HeLa cells . The impaired proliferation of HeLa cells could not be rescued by over-expression of PiT2 although the latter restored cellular Pi uptake to nearly wildtype levels. However, expression of a PiT1 mutant unable to transport Pi did reestablish the proliferative potential of the PiT1-knock-down cells . Thus, the results from embryos and MEF and HeLa cells suggest that the role of PiT1 in cell proliferation is not related to PiT1 Pi uptake. Interestingly, studying MEF cells with either knocked out PiT1 expression or PiT1 mRNA levels at 50% of wildtype levels, Beck and co-workers found that the proliferative potentials of these cells correlated with the levels of PiT1 expression . Thus, the PiT1-knock-out MEFs proliferated very slowly (2.1-fold decrease in doubling time compared to wildtype), while the proliferative potential of the MEFs with a 50% reduction in PiT1 mRNA levels was between those of the knock-out and wildtype MEFs . The observations that lowering of the cellular PiT1 level led to reduced proliferation of different cell types, and of murine as well as human cells, [19, 21] suggest that mPiT1 and hPiT1 have the same function in cell proliferation and that a certain level of PiT1 in general is necessary for normal cell proliferation. In addition, these observations also open for the possibility that the PiT1 level per se might be involved in controlling cell proliferation as hypothesized by Beck and co-workers . In agreement with this hypothesis, upon establishing and cultivating murine MC3T3-E1 cells over-expressing hPiT1, we noticed that the hPiT1 expressing cells grew to higher densities than control cells albeit that the MC3T3-E1 cell line exhibits strictly density-inhibited proliferation (unpubl. observation).
We have here investigated the role of PiT1 in governing proliferation and cell density of two strictly density-inhibited cell lines, the murine MC3T3-E1  and NIH3T3  cells. In order to investigate whether an increased PiT1 level could influence the proliferation of these cells, we exploited, as elaborated above, that previous results suggest that mPiT1 and hPiT1 have the same function in cell proliferation and used MC3T3-E1 and NIH3T3 cells stably expressing hPiT1 for our experiments. This approach also allowed for verification of the presence of functional transgenic hPiT1 protein at the cell surface by exploiting the differences in retroviral receptor functions of mPiT1 and hPiT1. Furthermore, it allowed us to discriminate between exogenously and endogenously expressed hPiT1 and mPiT1 mRNAs, respectively, and thus to follow the mRNA levels of the endogenous mPiT1 in murine cells stably expressing hPiT1. Using a combination of PiT1 knock-down and hPiT1 over-expression studies, we found that the level of PiT1 in cells exhibiting density-inhibited growth, can determine their proliferative potential and the density to which these cells can grow. Specifically for both cell lines, over-expression of hPiT1 led to increased proliferation and cell density showing that a PiT1 level above the endogenous level can drive cell proliferation and to some degree overrule the cell density constraints of these strictly density-inhibited cell lines. Moreover, upon investigating their ability to form colonies in soft agar, we also found that over-expression of hPiT1 made NIH3T3 cells more sensitive to transformation with fetal bovine serum (FBS). We, furthermore, found that the endogenous PiT1 expression is regulated in a manner which indeed is in agreement with a role of PiT1 in controlling cell proliferation in density-inhibited cells.
Knock-down of PiT1 impairs overall proliferation and cell density in cultures of MC3T3-E1 cells
The endogenous PiT1 mRNA level is decreased in dense MC3T3-E1 cell cultures
Characterization of MC3T3-E1 cells over-expressing hPiT1 or hPiT2
The observation that knock-down of the PiT1 mRNA level in human  and murine (Figure 1D and ref. ) cells led to impaired proliferation and cell density strongly suggest that PiT1 has the same function in cell proliferation in mouse and man. In order to further address the role of the PiT1 level in cell proliferation and cell density, we therefore employed MC3T3-E1 cells stably expressing hPiT1. The use of hPiT1 allowed us both to investigate whether the transgenic hPiT1 protein was present in the cell membrane and to discriminate between endogenous and exogenous PiT1 mRNA levels in the cells.
Transduction study analyzing the presence of functional hPiT1, hPiT2, and mPiT2 on the cell surface of MC3T3-E1-LXSN, -LPiT1SN, -LPiT2SN, NIH3T3-LXSN, and - LPiT1SN cells
Pseudotype of vectors encoding LacZ
4.7 × 104 c)
4.5 × 104
4.3 × 103
6.5 × 104
6.4 × 105
5.4 × 105
3.6 × 104
The endogenous mPiT1 and mPiT2 mRNA levels were also followed over time in culture in MC3T3-E1-LXSN, -LPiT1SN, and -LPiT2SN cells. In the experiment shown in Figures 3C and 3D, the cells were seeded at 500 cells/cm2, thus the cells grew in colonies. In agreement with the observed regulation of endogenous mPiT1 in wildtype MC3T3-E1 cells, the mPiT1 mRNA levels decreased when the cells started to touch each others within the colonies, i.e., approximately around 3-5 days in culture in the experiment shown (Figure 3C). No significant difference in mPiT1 mRNA levels was observed between MC3T3-E1-LXSN, -LPiT1SN or -LPiT2SN cells (Figure 3C) indicating that the expression of the endogenous mPiT1 was not per se influenced by the exogenously expressed hPiT1 or hPiT2. The endogenous mPiT2 mRNA levels decreased from day 1 to 3 in culture and remained thereafter at the same level (Figure 3D). At day 5 in culture, MC3T3-E1-LPiT1SN cells showed lower mPiT2 mRNA levels than the other cultures, but otherwise, no significant difference in mPiT2 mRNA levels was observed between MC3T3-E1-LXSN, -LPiT1SN or -LPiT2SN cells (Figure 3D) indicating that the expression of the endogenous mPiT2 was not per se influenced by the exogenously expressed hPiT1 or hPiT2.
Over-expression of hPiT1 in MC3T3-E1 cells increases cell proliferation and cell density
Effect of regulation of PiT1 and PiT2 expressions on the ability of MC3T3-E1 cells to import Pi
The impaired proliferation of MC3T3-E1 (Figure 1D) and the previously reported impaired proliferation of MEF cells , as consequences of knock-down of PiT1 expression, were both associated with upregulated PiT2 expression and neither could be related to decreased cellular Pi import. Moreover, the previously reported down-regulatory effect of knock-down of PiT1 on proliferation of HeLa cells was shown to be independent on the ability of PiT1 to transport Pi . We, moreover, find that when seeded at a density of 50,000 cells/cm2, or 20,000 cells/cm2 as in the experiment shown in Figure 4, analyses of the Pi uptake as in the experiment shown in Figure 1C show that the ability of MC3T3-E1-LPiT1SN cells to import Pi is indistinguishable from that of MC3T3-E1-LXSN cells, while that of MC3T3-E1-LPiT2SN cells is several fold higher (unpubl. results). The Km's of hPiT1 and hPiT2 are in the range of 100 to 300 μM Pi , and the Pi uptake was also analyzed in the presence of 100 μM and 300 μM Pi. However, no increase in Pi uptake by the MC3T3-E1-LPiT1SN cells compared to control MC3T3-E1-LXSN cells was observed, neither at 100 μM Pi (Figure 3E) nor at 300 μM Pi (not shown); MC3T3-E1-LPiT2SN cells showed 4-fold (100 μM Pi) (Figure 3E) and 3.3-fold (300 μM Pi) (not shown) increased Pi uptake compared to control MC3T3-E1-LXSN cells. The lack of increased Pi import in the PiT1 over-expressing MC3T3-E1 cells are in support of that the effects of the PiT1 level on the proliferation and density of MC3T3-E1 cells could be independent of its Pi-transport function. Further supporting that the cellular Pi import per se is not the cause of the effect of the increased PiT1 level on the proliferative potential of MC3T3-E1 cells is that MC3T3-E1-LPiT2SN cells, which exhibited increased Pi uptake ability, showed the opposite pattern of distribution of cells in G0/G1 and S + G2/M than MC3T3-E1-LPiT1SN cells (Figure 4C).
Characterization of NIH3T3 cells over-expressing hPiT1
Over-expression of hPiT1 in NIH3T3 cells increases cell proliferation and cell density
Effect of over-expression of hPiT1 on the ability of NIH3T3 cells to import Pi
The effect of over-expressing hPiT1 on the ability of NIH3T3 cells to import Pi was also investigated. Unlike MC3T3-E1 cells, which did not support increased Pi import when hPiT1 was over-expressed, the hPiT1 expressing NIH3T3-LPiT1SN cells were found to support increased (approximately 20-fold) Pi import compared to control NIH3T3-LXSN cells (Figure 7C).
Effect of over-expression of hPiT1 on the ability of NIH3T3 cells to form colonies in soft agar
Colony formation by NIH3T3-LXSN and NIH3T3-LPiT1SN cells in soft agar
0.5 ± 0.5c)
69.0 ± 8.3
2.5 ± 1.8
97.5 ± 9.8d)
In the present work, we show that the type III NaPi transporter, PiT1, can regulate proliferation and cell density of two different non-transformed cell lines, MC3T3-E1 and NIH3T3, which both exhibit strictly density-inhibited cell proliferation, and that over-expression of PiT1 indeed made NIH3T3 cells more sensitive to transformation with FBS. Working with MC3T3-E1 cells, we found in agreement with the in vitro work of Beck and co-workers in MEF, HeLa, and HepG2 cells [19, 21] and the reported effect of PiT1 knock-out on proliferation of liver cells in the developing embryo , that lowering of the PiT1 level led to impaired cell proliferation; interestingly, so far we have not identified a PiT1-knock-down level, which the NIH3T3 cells could survive (unpubl. observations). Thus, a certain PiT1 level is necessary for maintaining the wildtype proliferative potentials of a number of different cell lines/cell types. We, however, also found that over-expression of PiT1 increased the proliferation and culture densities of MC3T3-E1 and NIH3T3 cells and made NIH3T3 cells more sensitive to transformation with FBS. This is the first time that it is shown that an increase in the PiT1 expression above the endogenous levels of cells is sufficient to increase cell proliferation and that it indeed also can increase the density to which strictly density-inhibited cell lines can grow as well as increase anchorage independent growth/transformation. Thus, not only is a certain level of PiT1 necessary for normal cell proliferation (Figure 1 and refs. [19, 21]), an increased PiT1 level above the endogenous level of the cell can impel proliferation and to some degree overrule the constraints on cell density. Hence, the results suggest that PiT1 is not only necessary for normal cell proliferation, but can regulate the proliferative potential of cells, cell density, and anchorage-independent growth.
Investigations of the endogenous PiT1 mRNA level in control MC3T3-E1 and NIH3T3 cells showed that it seemed to follow the proliferative potential of the cells, i.e., the lowest PiT1 mRNA levels were found in cultures showing a low degree of proliferation, in agreement with a role of the endogenous PiT1 level in proliferation. Our observations also suggest a role of the endogenous PiT1 level in regulating cell density. Thus we found that the endogenous PiT1 mRNA level was down-regulated in dense cultures. Moreover, while increasing the PiT1 levels in NIH3T3 and MC3T3-E1 cells by over-expressing hPiT1 did not lead to an obvious abrogation of the density-inhibited cell proliferation per se, the cultures did grow to higher cell densities than control cell cultures before they stopped proliferating. Furthermore, while the NIH3T3 cells over-expressing hPiT1 showed similar endogenous PiT1 mRNA levels as the control cells at day 1 in culture, at day 2 in culture, these cells had reached their maximal density and had down-regulated the endogenous PiT1 mRNA levels, which stayed down. It is possible that the down-regulation of endogenous PiT1 could have occurred at a lesser cell density than the maximal density of the hPiT1 over-expressing cultures, since over-expression of hPiT1 led to higher cell densities, than observed in control cultures, and we only had one analysis-point per day. Nevertheless, the observed down-regulation of the endogenous PiT1 expression in response to cell density is interesting in that the studies of MC3T3-E1 (Figure 1) and MEF  cells showed that down-regulation of their PiT1 mRNA levels was sufficient to down-regulate the proliferation of these cells and that they ceased to proliferate although they had not reached the density of the control cells. It is in this respect also interesting, that Beck and co-workers reported on a less dense packaging of cells in the liver of PiT1 knock-out embryos . Together these data make us hypothesize that the cells themselves utilize their PiT1 levels to control their proliferation in response to cell density, i.e., when the cells attain a certain density, they down-regulate the endogenous PiT1 level, which again leads to down-regulation of the proliferation.
It is currently not known how the increased and decreased PiT1 levels lead to increased and decreased, respectively, cell proliferation. Beck and co-workers found that impairment of proliferation of HeLa cells by knock-down of endogenous PiT1 could be rescued by a PiT1 mutant (S128A mutation), which is unable to transport Pi, but not by over-expression of PiT2 albeit the latter nearly restored the Pi uptake ability of the cells to wildtype level . Furthermore, Beck and co-workers also found that the impaired proliferation of MEF cells with down-regulated PiT1 expression was not due to impaired cellular Pi uptake . In the present work, did over-expression of hPiT1 not lead to increased Pi uptake in MC3T3-E1 cells. However, over-expression of hPiT2 in these cells did lead to increased Pi uptake ability, but not to an increased proportion of cells in the S + G2/M phases of the cell cycle as over-expression of hPiT1 did. Thus, we have no evidence supporting that the here shown function of PiT1 in cell proliferation in MC3T3-E1 cells is related to Pi translocation by PiT1. However, since hPiT1 over-expression did increase the Pi-uptake ability of NIH3T3 cells, we can presently not rule out that increased Pi import play a role in NIH3T3 cells. Given the ubiquitous expression of PiT1 in mammalian cells , we do however hypothesize that the mechanism by which the PiT1 level exerts its function in the regulation of cell proliferation/cell density is the same in the different cell types.
In PiT1-deficient MEF cells, Beck and co-workers investigated the activation of S6K1, S6, Akt1, and 4EBP1, which are proteins involved in coupling signals from growth factors and nutrients to coordinate cell growth and cell division, but found no defects in their regulation . It should, however, be stressed that numerous studies show that PiT1 is a genuine cellular Pi transporter, i.e., that it is able to import Pi into cells, hereunder studies in Xenopus laevis oocytes [1, 4, 15, 18, 26], as we also find when we over-express hPiT1 in NIH3T3 cells. Thus, although the role of PiT1 in regulating cell proliferation may not be directly linked to its Pi transport function, we nevertheless hypothesize that the role of the PiT1 level in regulating cell proliferation, relates to PiT1's suggested role in cellular Pi homeostasis albeit in a yet unknown manner. It is in this respect, however, interesting that the yeast Pi transporter, Pho84, can function as a nutrient sensor, a transceptor . Results obtained by Popova and co-workers suggest that binding of phosphate/phosphate-containing compounds to Pho84 can elicit a signal transduction. The Pho84-mediated signal transduction did require a specific conformational change as in a Pi-transport-process, but not a complete transport cycle, thus Pi translocation was not involved in the signal transduction . Albeit, it is not known whether PiT1 has transceptor function, it is important to notice that if there is analogy to the transceptor function of Pho84, a PiT1 Pi transport knock-out mutant, as, e.g., the PiT1 S128A mutation shown to be able to rescue the impairment of proliferation of HeLa cells caused by knock-down of endogenous PiT1 , could be captured in a constitutive signaling conformation.
Another question arises from the present and published results on the role of PiT1 as a regulator of cell proliferation/cell density, namely, whether the abilities of PiT1 to transport Pi and function as a regulator of cell proliferation/cell density are functions that are performed by the same PiT1 molecule? It is in this respect interesting, that PiT1 has been published to be able to bind another ubiquitously expressed protein, the multimembrane-spanning protein, progressive ankylosis protein (ANK), which exports pyrophosphate out of the cell . How this binding affects the function of PiT1, is, however, not yet clear.
We conclude that not only is a certain level of PiT1 necessary for normal cell division as suggested by previous published results, rather the cellular PiT1 level is involved in regulating cell proliferation and cell density, and expression of PiT1 above the endogenous level can drive cell proliferation, overrule cell density constraints, and indeed make NIH3T3 cells more sensitive to transformation. We have thus provided the first evidence for a role of the type III Pi transporter, PiT1, as a protein, which can increase normal cell proliferation and cell density.
Expression plasmids and transfer vectors
The expression plasmids pOJ74 and pOJ75 (Wyeth-Ayerst Research, Pearl River N.Y., USA) encoding hPiT2 and hPiT1, respectively, have been described . The pLXSN  derivatives, pLPiT1SN and pLPiT2SN carrying the open reading frames from pOJ75 and pOJ74, respectively, were constructed as follows. HindIII-NotI fragments harboring the coding sequences of pOJ74 and pOJ75 were purified, blunt ended using Klenow Fragment, and inserted into the HpaI site of the pLXSN plasmid , encoding the retroviral transfer vector LXSN; the resulting plasmids, pLPiT1SN and pLPiT2SN, respectively, were verified by sequence analysis (Kjærgaard, M. M. and Pedersen, L., unpublished work). The LMP  (Open Biosystems, France) derived transfer vector, LMP-MmPiT1shRNAmiR, was made by insertion of a shRNA encoding sequence targeting murine PiT1: 5'TGCTGTTGAC AGTGAGCGAA CCCATTGTAT TGTCGGTGC A TAGTGAAGCC ACAGATGTAT GCACCGACAA TACAATGGG T CTGCCTACTG CCTCGGA 3' (bold being the target and complementary sequences for mouse PiT1). The authenticity of the LMP-derived vector was confirmed by sequence analysis. His-tagged FeLV-B RBD was produced from a pCDNA3 vector encoding the first 212 N-terminal amino acids of the SU of FeLV-B (G/A) fused to a C-terminal His-tag via a short linker (Dreyer, K. and Pedersen, L., unpublished work). C-terminally His-tagged A-MLV RBD encoded by a FBASALF-derived vector has been described elsewhere .
The pre-osteoblastic murine cell line, MC3T3-E1  (a kind gift from Dr. H. Kodama) and derivatives of this were cultured in Minimum Essential Medium alpha (MEMα) (Gibco BRL) supplemented with 10% FBS (Gibco BRL) and 1% Penicillin and Streptomycin (PS) (Gibco BRL) (MEMα-FBS-PS). Only MC3T3-E1 cells and derivatives of this cell line below the 5th passage were used for experiments. The murine fibroblastic cell line, NIH3T3 (ATCC CRL-1658), and derivatives of this were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco BRL) supplemented with 10% Newborn Calf Serum (NCS) (Gibco BRL) and 1% PS (DMEM-NCS-PS). The 293T-derived retroviral packaging cell lines, Platinium Eco (PlatE)  and Phoenix™Eco (Orbigen) (http://www.stanford.edu/group/nolan/retroviral_systems/phx.html), and derivatives of the latter were cultivated in DMEM-FBS-PS. A-MLV (4070A isolate) and GALV (SEATO) pseudotypes of the β-galactosidase-encoding transfer vector G1BgSvN (GBN)  were obtained from the producer cell lines, PA317-GBN [29, 34] and PG13-GBN , respectively. Both producer cell lines were cultivated in DMEM-NCS-PS. Vectors were harvested as supernatants from confluent producer cells and vector containing supernatants were filtered (0.45-μm pore size) and stored at −80°C until use. Dog osteosarcoma D17 (ATCC CCL-183) cells were cultivated in MEMα-FBS-PS. All plastic ware used for cell culture were from NUNC.
PiT1-knock-down in MC3T3-E1 cells
Phoenix™-Eco cells were seeded at 1 × 104 cells/cm2 in 100-mm-diameter dishes and transfected with LMP or LMP-MmPiT1shRNAmiR transfer vectors using the calcium-phosphate DNA precipitation method . Two days after transfection, each 100-mm-diameter dish were transferred to two T175 flasks and selected with 1.25 μg/mL puromycin (Sigma-Aldrich) the first three days, and thereafter cultivated in 0.5 μg/mL puromycin. The Phoenix™-Eco-LMP and - LMP-MmPiT1shRNAmiR cells were grown to confluence, media were changed to MEMα-FBS-PS, and retroviral vector containing supernatants were harvested, filtrated (0.45-μm pore size), and stored at −80°C until use. MC3T3-E1 cells were seeded at 2.5 × 103 cells/cm2, and vector-containing supernatants supplemented with 2 μg/mL Polybrene (PB, Sigma-Aldrich) were added 3 times to the cells over 2 days. The cells were transferred to T175 flasks and selected with 4 μg/mL puromycin for 2 days before used in experiments.
Establishment of LPiT1SN, LPiT2SN, and LXSN carrying MC3T3-E1 and NIH3T3 cells
Retroviral transfer vectors for transduction of MC3T3-E1 and NIH3T3 cells were produced in PlatE cells. PlatE cells were seeded at 4 × 106 cells/dish in 100-mm-diameter dishes in standard growth medium. The next day, the media were changed to fresh media and the cells were transfected with 30 μg of pLXSN, pLPiT1SN, or pLPiT2SN using the calcium-phosphate DNA precipitation method. The day after transfection, the media were changed to harvest media, and 24 hours later, vector containing supernatants were harvested, filtrated (0.45-μm pore size), and either used directly or stored at −80°C until use. A second harvest was made after an additional 24 hours using the same procedure. MC3T3-E1 cell populations carrying LPiT1SN, LPiT2SN, or empty vector LXSN were established and referred to as MC3T3-E1-LPiT1SN, MC3T3-E1-LPiT2SN, and MC3T3-E1-LXSN, respectively. NIH3T3 cell populations carrying LPiT1SN or empty vector LXSN were established and referred to as NIH3T3-LPiT1SN or NIH3T3-LXSN, respectively. The establishment of these cells was done as follows. MC3T3-E1 and NIH3T3 cells seeded at 6 × 103 cells/cm2 in 60-mm-diameter dishes in standard growth media were transduced over a period of approximately 30 hours. Approximately two hours after seeding, media were changed to 3 mL per dish of filtered (0.45 μm) retroviral vector containing supernatants from Plat-E cells with 2 μg/mL PB. Additional transductions were carried out twice the following day. The transduced cells were selected with 400 μg active G418 per mL (Gibco BRL). The day after the last transduction, each 60-mm-dish was passaged into two T175 flasks in standard growth medium, and 48 hours later, 400 μg/mL active G418 was added, and G418 resistant cell populations established. The selected MC3T3-E1 cell populations were frozen at −80°C for at least 24 hours before their use in experiments.
Cells were seeded in 4-well plates in quadruplicates in standard growth medium. At indicated days, the cells were detached from the wells using Trypsin-EDTA (5 × T) diluted in PBS from a stock of 10× Trypsin-EDTA (0.5% Trypsin, Gibco BRL), and re-suspended in 500 μL standard growth medium. Aliquots were mixed 1:1 with Trypan Blue Stain (Gibco BRL) and counted using a cell counting chamber.
Cell cycle analysis
Cells were seeded in T25-flasks at 20,000 cells/cm2. After 24 hours, the cells were detached using 5 × T, pelleted by centrifugation, re-suspended in PBS, cooled on ice, mixed 1:1 with ice-cold 99.9% ethanol, incubated at least 10 minutes on ice and used for staining or stored at 4°C until staining. Staining was carried out by pelleting the cells by centrifugation, re-suspending them in 1 mL PBS containing 20 μg/mL RNase A followed by addition of 100 μL propidium iodide (1 mg/mL). The cells were then analyzed by flow cytometry (FL2 channel) using a FACSCalibur flow cytometer (Becton Dickinson). CellQuest software was used for acquisition. FlowJo software was used for subtraction of aggregates and doublets  and for data analysis employing the Watson-Pragmatic model.
RBD binding assay
His-tagged RBDs were produced in 293T cells. Shortly, 293T cells were seeded at 4 × 106 cells/dish in 100-mm-diameter dishes and transfected the following day with 15 μg His-tagged FeLV-B RBD or His-tagged A-MLV RBD encoding expression plasmids together with 15 μg carrier plasmid (pUC18) per dish using the calcium-phosphate DNA precipitation method. At days 2 and 3 after transfection, RBD containing supernatants were harvested in standard growth medium, filtrated (0.45-μm pore size), and stored in aliquots at −80°C until use. For analysis of RBD binding, cells were detached using 1 mM EDTA in PBS and counted. Aliquots of 106 cells/sample were pelleted by centrifugation for 5 minutes at 1200 rpm. The cell pellets were re-suspended in 1 mL RBD-containing supernatant or standard growth medium, as control, and incubated 20 minutes at 37°C. The cells were then washed 2× in 5% FBS in PBS and incubated on ice with primary antibody, Mouse-Anti-Penta-His (Qiagen), diluted 1:400 in 50 μL 5% FBS in PBS/sample for 45 minutes. Then the cells were washed twice in ice-cold 5% FBS in PBS and incubated for at least 45 minutes in the dark with 1 μg/sample of PE-conjugated goat anti-mouse Ig (BD Pharma) diluted in 300 μL 5% FBS in PBS. Finally, the cells were washed 1× in ice cold 5% FBS in PBS, fixed in 1% paraformaldehyde (Ampliqon) for 10 minutes at RT, washed 1× in PBS, and re-suspended in 600 - 1000 μL PBS. The samples were analyzed using the FL2-H channel on a FACSCalibur flow cytometer. For data analysis, CellquestPro software was used.
MC3T3-E1-LXSN, -LPiT1SN, -LPiT2SN, NIH3T3-LXSN, -LPiT1SN, and D17 cells were seeded in 24-well plates with a density of 104 cells/cm2 in standard growth media and analyzed for their susceptibility to transduction with GALV and A-MLV vector pseudotypes as previously described for titer determination using dilution series of vector-containing supernatants from PG13-GBN and PA317-GBN producer cells, respectively . The transduction efficiency was evaluated as blue (β-galactosidase-positive) colony forming units (CFUs).
Cells were lyzed on the plates and total RNA purified using RNAqueous®-4PCR kit (Applied Biosystems). The RNA samples were DNase1 treated and after inactivation of the DNase used directly for cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) as recommended by the supplier. The samples were either stored at −20°C until use or used directly for qRT-PCR analysis. For qRT-PCR analyses, the following TaqMan® Gene Expression Assays (Applied Biosystems) were used according to the protocol: Mouse PiT1 (Mm00489378_m1), mouse PiT2 (Mm00660197_m1), human PiT1 (Hs00965596_m1), human PiT2 (Hs01108472_m1) and, as endogenous control, mouse β-2-microglobulin (B2M)-VIC (Mm00437762_m1). The individual qRT-PCR reactions contained 10 μL TaqMan® Fast Universal PCR Master Mix ((Applied Biosystems), 1 μL TaqMan® Gene Expression Assay, 1 μL B2M, and 8 μL cDNA (approximately 10 ng). All samples were made in triplicates for all genes analyzed. The efficiencies of each set of primers were determined on dilution series of cDNA and were used in calculations of relative gene expression as described in ref. . The PCR cycles employed were: 95°C for 10 minutes, 40 cycles of 95°C for 1 second, and 60°C for 20 seconds.
Cells were seeded in 4-well plates, the next day, they were washed in Pi-free DMEM (Gibco BRL) and hereafter incubated 5 minutes in 37°C warm serum-free, Pi-free DMEM containing KH2 32PO4 (900-1100 mCi/mmol, Perkin Elmer) with or without added non-radioactive Pi. The endconcentrations of Pi in the uptake experiments were 5 μM, 100 μM, or 300 μM Pi. The cells were then washed 3× in ice cold PBS or serum-free, Pi-free DMEM, and lyzed in 0.5% (w/v) SDS in PBS or in 0.5% (v/v) Triton X-100, and 2/3 of the lysate was mixed with 5 mL Ultima Gold Liquid Scintillation Counter Cocktail (Perkin Elmer) or Optiphase "Hisafe" 3 (Perkin Elmer) and measured in liquid scintillation counters. The differences in washing and lysis procedures did not influence the results (unpubl. observation). The rest of the sample was stored at −20°C until used for protein determination using the BCA™ protein assay kit (Pierce).
Soft agar assay
NIH3T3-LXSN and NIH3T3-LPiT1SN cells were pre-incubated for two days in either standard growth medium or DMEM-FBS-PS. Base agar was made by melting 1% agar (SeaPlaque®GTG®Agarose, Lonza), cooling it to 40°C in a water bath and mixing it with an equal volume of 40°C warm 2× DMEM (Gibco BRL) supplemented with 3.7 g NaHCO3 (MERCK), 2% PS, and 20% FBS (2 × DMEM/FBS) or 20% NCS (2 × DMEM/NCS); 0.5 mL base agar was plated per well in 24-well plates and allowed to set at 4°C. NIH3T3-LXSN or NIH3T3-LPiT1SN cells grown in either DMEM-FBS-PS or DMEM-NCS-PS were loosened with 5 × T and diluted to a concentration of 105 cells/mL, and 50 μL of the cells were mixed with 1 mL melted 40°C 0.7% agar and 1 mL 40°C 2 × DMEM/FBS or 2 × DMEM/NCS, respectively, and 0.5 mL per well were seeded on top of the solidified base agar containing DMEM/FBS or DMEM/NCS, respectively. When the top agar had set, 0.25 mL DMEM-NCS-PS or DMEM-FBS-PS were added to each well. The dishes were incubated for 18 days at 37°C and 5% CO2. Colonies were stained by addition of 200 μL 0.005% Crystal Violet (MERCK) to each well followed by 1 hour incubation at room temperature before counting the colonies under a microscope.
The hypothesis that two mean values were identical was tested by a two-tailed Student's t-test; a p-value <0.05 was considered statistically significant.
- Pi :
gibbon ape leukemia virus
feline leukemia virus subgroup B
amphotropic murine leukemia virus
mouse embryonic fibroblast
colony forming unit
receptor binding domain
We thank Merete Scheving for expert technical assistance. We thank Drs. Bryan O'Hara for pOJ74 and pOJ75, Maribeth V. Eiden for the PA317GBN and PG13GBN cell lines, and Dimitri Lavillette for A-MLV RBD encoding vector. This work was supported by the Lundbeck Foundation (Grant number 14/02), the Novo Nordisk Foundation, the Danish Medical Research Foundation (Grant numbers 09-061652 (271-06-0564), 09-063569 (271-07-0598), 09-066064 (271-08-1005), and the Intramural Budget at the Institute of Clinical Medicine at Aarhus University. CH was sponsored by Aarhus Graduate School of Science. KB was supported by a scholarship from the Danish Cancer Society and a fellowship from the Institute of Clinical Medicine at Aarhus University. IBK was supported by a scholarship from the Danish Cancer Society.
- Kavanaugh MP, Miller DG, Zhang W, Law W, Kozak SL, Kabat D, Miller AD: Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci USA 1994,91(15):7071–7075. 10.1073/pnas.91.15.7071PubMed CentralView ArticlePubMedGoogle Scholar
- Miller DG, Edwards RH, Miller AD: Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. Proc Natl Acad Sci USA 1994,91(1):78–82. 10.1073/pnas.91.1.78PubMed CentralView ArticlePubMedGoogle Scholar
- O'Hara B, Johann SV, Klinger HP, Blair DG, Rubinson H, Dunn KJ, Sass P, Vitek SM, Robins T: Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus. Cell Growth Differ 1990,1(3):119–127.PubMedGoogle Scholar
- Olah Z, Lehel C, Anderson WB, Eiden MV, Wilson CA: The cellular receptor for gibbon ape leukemia virus is a novel high affinity sodium-dependent phosphate transporter. J Biol Chem 1994,269(41):25426–25431.PubMedGoogle Scholar
- van Zeijl M, Johann SV, Closs E, Cunningham J, Eddy R, Shows TB, O'Hara B: A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family. Proc Natl Acad Sci USA 1994,91(3):1168–1172. 10.1073/pnas.91.3.1168PubMed CentralView ArticlePubMedGoogle Scholar
- Wilson CA, Eiden MV, Anderson WB, Lehel C, Olah Z: The dual-function hamster receptor for amphotropic murine leukemia virus (MuLV), 10A1 MuLV, and gibbon ape leukemia virus is a phosphate symporter. J Virol 1995,69(1):534–537.PubMed CentralPubMedGoogle Scholar
- Saier MH Jr: A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev 2000,64(2):354–411. 10.1128/MMBR.64.2.354-411.2000PubMed CentralView ArticlePubMedGoogle Scholar
- Bøttger P, Pedersen L: 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. FEBS J 2005,272(12):3060–3074. 10.1111/j.1742-4658.2005.04720.xView ArticlePubMedGoogle Scholar
- Takeuchi Y, Vile RG, Simpson G, O'Hara B, Collins MK, Weiss RA: Feline leukemia virus subgroup B uses the same cell surface receptor as gibbon ape leukemia virus. J Virol 1992,66(2):1219–1222.PubMed CentralPubMedGoogle Scholar
- Johann SV, van Zeijl M, Cekleniak J, O'Hara B: Definition of a domain of GLVR1 which is necessary for infection by gibbon ape leukemia virus and which is highly polymorphic between species. J Virol 1993,67(11):6733–6736.PubMed CentralPubMedGoogle Scholar
- Pedersen L, Johann SV, van Zeijl M, Pedersen FS, O'Hara B: Chimeras of receptors for gibbon ape leukemia virus/feline leukemia virus B and amphotropic murine leukemia virus reveal different modes of receptor recognition by retrovirus. J Virol 1995,69(4):2401–2405.PubMed CentralPubMedGoogle Scholar
- Tailor CS, Takeuchi Y, O'Hara B, Johann SV, Weiss RA, Collins MK: Mutation of amino acids within the gibbon ape leukemia virus (GALV) receptor differentially affects feline leukemia virus subgroup B, simian sarcoma-associated virus, and GALV infections. J Virol 1993,67(11):6737–6741.PubMed CentralPubMedGoogle Scholar
- Pedersen L, van Zeijl M, Johann SV, O'Hara B: Fungal phosphate transporter serves as a receptor backbone for gibbon ape leukemia virus. J Virol 1997,71(10):7619–7622.PubMed CentralPubMedGoogle Scholar
- Wilson CA, Farrell KB, Eiden MV: Comparison of cDNAs encoding the gibbon ape leukaemia virus receptor from susceptible and non-susceptible murine cells. J Gen Virol 1994,75(Pt 8):1901–1908.View ArticlePubMedGoogle Scholar
- Bøttger P, Hede SE, Grunnet M, Høyer B, Klærke DA, Pedersen L: Characterization of transport mechanisms and determinants critical for Na + −dependent Pi symport of the PiT-family paralogs, human PiT1 and PiT2. Am J Physiol Cell Physiol 2006,291(6):C1377-C1387. 10.1152/ajpcell.00015.2006View ArticlePubMedGoogle Scholar
- Johann SV, Gibbons JJ, O'Hara B: GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus. J Virol 1992,66(3):1635–1640.PubMed CentralPubMedGoogle Scholar
- Uckert W, Willimsky G, Pedersen FS, Blankenstein T, Pedersen L: RNA levels of human retrovirus receptors Pit1 and Pit2 do not correlate with infectibility by three retroviral vector pseudotypes. Hum Gene Ther 1998,9(17):2619–2627. 10.1089/hum.1998.9.17-2619View ArticlePubMedGoogle Scholar
- Ravera S, Virkki LV, Murer H, Forster IC: Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements. Am J Physiol Cell Physiol 2007,293(2):C606-C620. 10.1152/ajpcell.00064.2007View ArticlePubMedGoogle Scholar
- Beck L, Leroy C, Beck-Cormier S, Forand A, Salaün C, Paris N, Bernier A, Ureña-Torres P, Prié D, Ollero M, et al.: The Phosphate Transporter PiT1 (Slc20a1) Revealed As a New Essential Gene for Mouse Liver Development. PLoS One 2010,5(2):e9148. 10.1371/journal.pone.0009148PubMed CentralView ArticlePubMedGoogle Scholar
- Festing MH, Speer MY, Yang H-Y, Giachelli CM: Generation of mouse conditional and null alleles of the type III sodium-dependent phosphate cotransporter PiT-1. Genesis 2009,47(12):858–863.PubMed CentralPubMedGoogle Scholar
- Beck L, Leroy C, Salaü C, Margall-Ducos G, Desdouets C, Friedlander G: Identification of a Novel Function of PiT1 Critical for Cell Proliferation and Independent of Its Phosphate Transport Activity. J Biol Chem 2009,284(45):31363–31374. 10.1074/jbc.M109.053132PubMed CentralView ArticlePubMedGoogle Scholar
- Kodama H, Amagai Y, Sudo H, Kasai S, Yamamoto S: Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Jpn J Oral Biol 1981, 23: 899–901. 10.2330/joralbiosci1965.23.899View ArticleGoogle Scholar
- Jainchill JL, Aaronson A, Todaro G: Murine sarcoma and leukemia viruses: Assays using clonal cell lines of contact-inhibited mouse cells. J Virol 1969,4(5):549–553.PubMed CentralPubMedGoogle Scholar
- Nielsen LB, Pedersen FS, Pedersen L: Expression of type III sodium-dependent phosphate transporters/retroviral receptors mRNAs during osteoblast differentiation. Bone 2001,28(2):160–166. 10.1016/S8756-3282(00)00418-XView ArticlePubMedGoogle Scholar
- Suzuki A, Ghayor C, Guicheux J, Magne D, Quillard S, Kakita A, Ono Y, Miura Y, Oiso Y, Itoh M, et al.: Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells. J Bone Miner Res 2006,21(5):674–683. 10.1359/jbmr.020603View ArticlePubMedGoogle Scholar
- Bøttger P, Pedersen L: Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life. BMC Biochem 2011, 12: 21. 10.1186/1471-2091-12-21PubMed CentralView ArticlePubMedGoogle Scholar
- Popova Y, Thayumanavan P, Lonati E, Agrochão M, Thevelein J: Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor. Proc Natl Acad Sci USA 2010,107(7):2890–2895. 10.1073/pnas.0906546107PubMed CentralView ArticlePubMedGoogle Scholar
- Wang J, Tsui HW, Beier F, Tsui FWL: The CPPDD-Associated ANKH M48T Mutation Interrupts the Interaction of ANKH with the Sodium/Phosphate Cotransporter PiT-1. J Rheumatol 2009,36(6):1265–1272. 10.3899/jrheum.081118View ArticlePubMedGoogle Scholar
- Miller AD, Rosman GJ: Improved retroviral vectors for gene transfer and expression. Biotechniques 1989,7(9):980–990.PubMed CentralPubMedGoogle Scholar
- Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ, Lowe SW: Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 2005,37(11):1289–1295.PubMedGoogle Scholar
- Lavillette D, Boson B, Russell SJ, Cosset FL: Activation of membrane fusion by murine leukemia viruses is controlled in cis or in trans by interactions between the receptor-binding domain and a conserved disulfide loop of the carboxy terminus of the surface glycoprotein. J Virol 2001,75(8):3685–3695. 10.1128/JVI.75.8.3685-3695.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Morita S, Kojima T, Kitamura T: Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 2000,7(12):1063–1066. 10.1038/sj.gt.3301206View ArticlePubMedGoogle Scholar
- McLachlin JR, Mittereder N, Daucher MB, Kadan M, Eglitis MA: Factors affecting retroviral vector function and structural integrity. Virology 1993,195(1):1–5. 10.1006/viro.1993.1340View ArticlePubMedGoogle Scholar
- Miller AD, Buttimore C: Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 1986,6(8):2895–2902.PubMed CentralPubMedView ArticleGoogle Scholar
- Miller AD, Garcia JV, von Suhr N, Lynch CM, Wilson C, Eiden MV: Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol 1991,65(5):2220–2224.PubMed CentralPubMedGoogle Scholar
- Gorman C, Glover E DM: High Efficiency Gene Transfer into Mammalian Cells. In DNA Cloning, Volume II: A Practical Approach. Oxford: IRL Press; 1985:143–190.Google Scholar
- Givan AL, Givan AL: Cells from Within: DNA in Life and Death. In Flow Cytometry: First Principles. 2nd edition. Wiley-Liss, Inc; 2001:123–158.View ArticleGoogle Scholar
- Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J: qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 2007,8(2):R19. 10.1186/gb-2007-8-2-r19PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.