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The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development.

Beck L, Leroy C, Beck-Cormier S, Forand A, Salaün C, Paris N, Bernier A, Ureña-Torres P, Prié D, Ollero M, Coulombel L, Friedlander G - PLoS ONE (2010)

Bottom Line: In contrast, mutant fetal livers display decreased proliferation and massive apoptosis.Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic.This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U845, Centre de Recherche Croissance et Signalisation, Paris, France. laurent.beck@inserm.fr

ABSTRACT

Background: PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na(+)-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.

Methodology/principal findings: To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and alleles in heterozygous compounds results in late embryonic lethality (E14.5-E16.5) with phenotypic features intermediate between and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.

Conclusion/significance: This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

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Disruption of PiT1 in MEFs does not affect Na+-Pi cotransport but leads to reduced proliferation.(A) MEFs proliferation curve. PiT1Δ5/Δ5 MEFs display a mean doubling time of 38 h, whereas PiT1+/+ and PiT1Δ5/+ MEFs had a growth rate of 18 h and 20 h, respectively. (B) Na+-Pi uptake in MEFs. Disruption of PiT1 in MEFs does not modify the overall Pi uptake. (C) Quantification of the expression of PiT1 and PiT2 mRNAs in PiT1+/+ and PiT1Δ5/Δ5 MEFs by real-time RT-PCR. Note the 1.8-fold overexpression of PiT2 mRNA in PiT1- MEFs. * and # indicate significant differences as compared to wild-type controls with P<0.05 and P<0.001, respectively (Student's t test).
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pone-0009148-g008: Disruption of PiT1 in MEFs does not affect Na+-Pi cotransport but leads to reduced proliferation.(A) MEFs proliferation curve. PiT1Δ5/Δ5 MEFs display a mean doubling time of 38 h, whereas PiT1+/+ and PiT1Δ5/+ MEFs had a growth rate of 18 h and 20 h, respectively. (B) Na+-Pi uptake in MEFs. Disruption of PiT1 in MEFs does not modify the overall Pi uptake. (C) Quantification of the expression of PiT1 and PiT2 mRNAs in PiT1+/+ and PiT1Δ5/Δ5 MEFs by real-time RT-PCR. Note the 1.8-fold overexpression of PiT2 mRNA in PiT1- MEFs. * and # indicate significant differences as compared to wild-type controls with P<0.05 and P<0.001, respectively (Student's t test).

Mentions: As genetic inactivation of PiT1 resulted in embryonic lethality, together with decreased cell proliferation, we evaluated whether the absence of PiT1 could also affect MEFs proliferation in vitro. Results show that PiT1Δ5/Δ5 MEFs proliferated very slowly, displaying a mean doubling time of 38 h, a 2.1-fold increase compared with the doubling time of 18 h observed for PiT1+/+ MEFs (Fig. 8A). PiT1Δ5/+ MEFs had an intermediate growth rate (mean doubling time of 20 h). As the main reported function of PiT1 is to couple inward Pi uptake to the Na+ gradient across the cell plasma membrane, we characterized the Na+-dependent Pi uptake in PiT1-deficient MEFs. Interestingly, our results showed that Na+-Pi uptake in PiT1- MEFs was unaffected (Fig. 8B), with no change in its kinetic properties (Vmax  = 19.2±0.7 and 19.1±1.7 nmol.mg prot−1 for PiT1+/+ and PiT1Δ5/Δ5 MEFs, respectively; Km  = 124.5±38.5 and 134.5±17.9 µM for PiT1+/+ and PiT1Δ5/Δ5 MEFs, respectively). While real-time RT-PCR confirmed that PiT1 expression in PiT1Δ5/Δ5 MEFs was abolished, this was associated with a 1.8-fold overexpression of PiT2 mRNA (Fig. 8C), which could account for the maintenance of normal Na+-Pi transport in PiT1- MEFs. Maintenance of a normal Pi uptake in PiT1- MEFs supports the idea that the defect in MEF proliferation was not a consequence of Pi deprivation but resulted in a transport-independent function of PiT1, consistent with our recent data obtained in HepG2 and HeLa cells [9]. However, additional in vivo studies are necessary to assign the observed phenotype to a specific loss of either function of PiT1.


The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development.

Beck L, Leroy C, Beck-Cormier S, Forand A, Salaün C, Paris N, Bernier A, Ureña-Torres P, Prié D, Ollero M, Coulombel L, Friedlander G - PLoS ONE (2010)

Disruption of PiT1 in MEFs does not affect Na+-Pi cotransport but leads to reduced proliferation.(A) MEFs proliferation curve. PiT1Δ5/Δ5 MEFs display a mean doubling time of 38 h, whereas PiT1+/+ and PiT1Δ5/+ MEFs had a growth rate of 18 h and 20 h, respectively. (B) Na+-Pi uptake in MEFs. Disruption of PiT1 in MEFs does not modify the overall Pi uptake. (C) Quantification of the expression of PiT1 and PiT2 mRNAs in PiT1+/+ and PiT1Δ5/Δ5 MEFs by real-time RT-PCR. Note the 1.8-fold overexpression of PiT2 mRNA in PiT1- MEFs. * and # indicate significant differences as compared to wild-type controls with P<0.05 and P<0.001, respectively (Student's t test).
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Related In: Results  -  Collection

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pone-0009148-g008: Disruption of PiT1 in MEFs does not affect Na+-Pi cotransport but leads to reduced proliferation.(A) MEFs proliferation curve. PiT1Δ5/Δ5 MEFs display a mean doubling time of 38 h, whereas PiT1+/+ and PiT1Δ5/+ MEFs had a growth rate of 18 h and 20 h, respectively. (B) Na+-Pi uptake in MEFs. Disruption of PiT1 in MEFs does not modify the overall Pi uptake. (C) Quantification of the expression of PiT1 and PiT2 mRNAs in PiT1+/+ and PiT1Δ5/Δ5 MEFs by real-time RT-PCR. Note the 1.8-fold overexpression of PiT2 mRNA in PiT1- MEFs. * and # indicate significant differences as compared to wild-type controls with P<0.05 and P<0.001, respectively (Student's t test).
Mentions: As genetic inactivation of PiT1 resulted in embryonic lethality, together with decreased cell proliferation, we evaluated whether the absence of PiT1 could also affect MEFs proliferation in vitro. Results show that PiT1Δ5/Δ5 MEFs proliferated very slowly, displaying a mean doubling time of 38 h, a 2.1-fold increase compared with the doubling time of 18 h observed for PiT1+/+ MEFs (Fig. 8A). PiT1Δ5/+ MEFs had an intermediate growth rate (mean doubling time of 20 h). As the main reported function of PiT1 is to couple inward Pi uptake to the Na+ gradient across the cell plasma membrane, we characterized the Na+-dependent Pi uptake in PiT1-deficient MEFs. Interestingly, our results showed that Na+-Pi uptake in PiT1- MEFs was unaffected (Fig. 8B), with no change in its kinetic properties (Vmax  = 19.2±0.7 and 19.1±1.7 nmol.mg prot−1 for PiT1+/+ and PiT1Δ5/Δ5 MEFs, respectively; Km  = 124.5±38.5 and 134.5±17.9 µM for PiT1+/+ and PiT1Δ5/Δ5 MEFs, respectively). While real-time RT-PCR confirmed that PiT1 expression in PiT1Δ5/Δ5 MEFs was abolished, this was associated with a 1.8-fold overexpression of PiT2 mRNA (Fig. 8C), which could account for the maintenance of normal Na+-Pi transport in PiT1- MEFs. Maintenance of a normal Pi uptake in PiT1- MEFs supports the idea that the defect in MEF proliferation was not a consequence of Pi deprivation but resulted in a transport-independent function of PiT1, consistent with our recent data obtained in HepG2 and HeLa cells [9]. However, additional in vivo studies are necessary to assign the observed phenotype to a specific loss of either function of PiT1.

Bottom Line: In contrast, mutant fetal livers display decreased proliferation and massive apoptosis.Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic.This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U845, Centre de Recherche Croissance et Signalisation, Paris, France. laurent.beck@inserm.fr

ABSTRACT

Background: PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na(+)-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.

Methodology/principal findings: To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and alleles in heterozygous compounds results in late embryonic lethality (E14.5-E16.5) with phenotypic features intermediate between and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.

Conclusion/significance: This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

Show MeSH
Related in: MedlinePlus