Stimulated Pi Transport Research Articles

Enhanced Expression of the Inorganic Phosphate Transporter Pit-1 Is Involved in BMP-2–Induced Matrix Mineralization in Osteoblast-Like Cells

Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP-2-induced matrix calcification was studied. BMP-2 enhances Pit-1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification. Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP-2-induced matrix mineralization. Confluent MC3T3-E1 osteoblast-like cells were exposed to BMP-2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit-1 and Pit-2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport in MC3T3-E1 cells by increasing the V(max) of the transport system. This effect was preceded by an increase in mRNA encoding Pit-1 but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineralization induced by BMP-2 were blunted by a specific inhibitor of the c-Jun-N-terminal kinase (JNK) pathway. Results presented in this study indicate that, in addition to its well-known effect on several markers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1 cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP-2.

Stimulation of Na-dependent phosphate transport by platelet-derived growth factor in rat aortic smooth muscle cells

We investigated the effect of platelet-derived growth factor B homodimer (PDGF-BB) on inorganic phosphate (Pi) transport activity, which has been reported to be involved in the mechanism of atherosclerosis, in A-10 rat aortic vascular smooth muscle cells (VSMCs). PDGF-BB time- and dose-dependently stimulated Pi transport in A-10 cells. Using northern blot analysis, the PDGF-BB-enhanced Pi transporter (PiT) in A-10 cells was identified as Pit-1 (Glvr-1), a member of the type III Na-dependent PiT. An inhibitor of PDGF β-receptor tyrosine kinase suppressed PDGF-BB-induced Pi transport. Both a protein kinase C (PKC) inhibitor calphostin C and PKC down regulation suppressed the stimulatory effect of PDGF-BB on Pi transport. On the other hand, inhibition of mitogen-activated protein (MAP) kinases by selective inhibitors did not affect Pi transport. Ly294002, a phosphatidylinositol (PI) 3-kinase inhibitor, partially attenuated PDGF-BB-induced Pi transport. A selective inhibitor of S 6 kinase, rapamycin, reduced this effect of PDGF-BB, while Akt kinase inhibitor did not. In summary, these results indicated that PDGF-BB is a potent and selective stimulator of Pi transport in VSMCs. The mechanism responsible for this effect is not mediated by MAP kinase, but involves activation of PKC, PI 3-kinase and S 6 kinase.

Stimulatory effect of prostaglandin F 2α on Na-dependent phosphate transport in osteoblast-like cells

Prostaglandin F 2α (PGF 2α) has been reported to activate protein kinase C (PKC) through both phospholipase (PL) C and D, resulting in the proliferation of osteoblast-like cells. In addition, it has also been reported that Erk mitogen-activated protein kinase is also involved in the mechanism of PGF 2α-induced proliferation of these cells. Recently, we have reported that several growth factors stimulate Na-dependent phosphate transport (Pi transport) activity of osteoblast-like cells, which has been recognized to play an important role in their mineralization. In the present study, we investigated the effect of PGF 2α on Pi transport in MC3T3-E1 osteoblast-like cells. PGF 2α stimulated Na-dependent Pi transport dose dependently in the range between 1 nM and 10 μM in MC3T3-E1 cells. The effect was time dependent up to 24 h. Kinetic analysis revealed that PGF 2α induces newly synthesized Pi transporter. Pretreatment with actinomycin D and cycloheximide suppressed PGF 2α-induced enhancement of Pi transport. Combined effect of PMA and PGF 2α was not additive in Pi transport. Calphostin C, a PKC inhibitor, dose-dependently suppressed Pi transport induced by PGF 2α. On the contrary, U0126, which inhibits an upstream kinase of Erk (MEK), did not affect PGF 2α-induced enhancement of Pi transport. In conclusion, PGF 2α stimulates Pi transport through activation of PKC in osteoblast-like cells.

Transforming growth factor-beta stimulates inorganic phosphate transport and expression of the type III phosphate transporter Glvr-1 in chondrogenic ATDC5 cells.

Members of the transforming growth factor (TGF)-beta family are important regulators of skeletal development. In this study, we investigated the effect of TGF-beta1 on inorganic phosphate (Pi) transport and on expression of the type III Pi carriers Glvr-1 and Ram-1 in murine ATDC5 chondrocytes. TGF-beta1 induced a selective, dose- and time-dependent increase in sodium-dependent Pi transport in ATDC5 cells. This response was dependent on RNA and protein synthesis and reflected a change in the maximal rate of the transport system, suggesting that TGF-beta1 induces the synthesis of new Pi carriers and their insertion into the plasma membrane. Consistently, Northern blotting analysis showed a dose-dependent increase in Glvr-1 messenger RNA expression in response to TGF-beta1, which preceded the maximal stimulation of Pi transport by several hours. Glvr-1 thus likely mediates at least part of the increase in Pi uptake induced by TGF-beta1. Ram-1 messenger RNA expression was not affected by TGF-beta1. TGF-beta1 activated the Smad signaling pathway and the mitogen-activated protein kinases ERK and p38 in ATDC5 cells. Unlike the regulation of Pi transport by receptor tyrosine kinase agonists in osteoblasts, the effect of TGF-beta1 on Pi uptake in ATDC5 cells did not involve protein kinase C or mitogen-activated protein kinases, suggesting that a specific, possibly Smad-dependent, signal mediates this response. In conclusion, TGF-beta1 stimulates Pi transport and Glvr-1 expression in chondrocytes, suggesting that, like proliferation, differentiation, and matrix synthesis, Pi handling is subject to regulation by TGF-beta3 family members in bone-forming cells.

Hormonal Regulation of Sodium-Dependent Phosphate Transport in Opossum Kidney Cells

There are multiple regulators of renal proximal tubule sodium-dependent phosphate (Na⁺-Pi) transport, including 1,25-dihydroxyvitamin D (1,25-Vit. D), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), and arachidonic acid (AA) and/or its metabolites. The purpose of our studies was to determine whether the effect of these factors on Pi transport is synergistic or antagonistic. The control solution or the substances were added independently or coincidentally to opossum kidney (OK) cells before incubation for 4 h. 1,25-Vit. D (10^–8<i>M</i>) had no significant effect on Pi transport (↑6.8%; p = 0.8). PTH (10^–7<i>M</i>) significantly inhibited Pi transport by 39.6% (p < 0.0001). IGF-1 (10^–8<i>M</i>) stimulated Pi transport by 19.6% (p < 0.0001). The AA metabolite 20-HETE (10^–7<i>M</i>) had no significant impact on Pi transport (⇓6.4; p = 0.3). The combined effect of 1,25-Vit. D and PTH was no different from PTH alone (p = 0.2). Likewise, addition of either 1,25-Vit. D or 20-HETE to IGF-1 failed to affect the magnitude of the increase on Pi transport induced by IGF-1 alone (p = 0.4, p = 0.6, respectively). The combination of 20-HETE and PTH was not different from that observed with PTH alone (p = 0.9). We conclude that in OK cells, PTH inhibits whereas IGF-1 stimulates Pi transport into OK cells. The effects of each of these hormones are independent and unaffected by either 1,25-Vit. D or 20-HETE.

Stimulation of sodium-dependent phosphate transport and signaling mechanisms induced by basic fibroblast growth factor in MC3T3-E1 osteoblast-like cells.

Physiological and pathological observations indicate that basic fibroblast growth factor (bFGF) is an important regulator of osteoblastic cell differentiation and in particular of cranial ossification. Experimental evidence suggests that inorganic phosphate (Pi) transport could be an important function of bone matrix calcification. In the present study, we address the influence of bFGF on Pi transport activity in MC3T3-E1 osteoblast-like cells derived from mouse calvaria. The results indicate that bFGF is a potent and selective stimulator of sodium-dependent Pi transport in these cells. The change in Pi transport activity induced by bFGF depends on transcription and translation and corresponds to a change in the maximum velocity of the Pi transport system (Vmax). These observations suggest that enhanced Pi transport activity in response to bFGF may result from insertion of newly synthesized Pi transporters into the plasma membrane. A selective inhibitor of fibroblast growth factor receptor (FGFR) tyrosine kinase, SU5402, blunted the stimulation of Pi transport induced by bFGF. It also prevented the increase in protein tyrosine phosphorylation induced by bFGF, including phosphorylation of FGFR-1, FGFR-2, phospholipase C-gamma (PLC-gamma), and Shc as well as the recruitment of the Grb2/Sos signaling complex. In addition, bFGF-induced the activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and p38, effects that were prevented by SU5402. Both the protein kinase C (PKC) inhibitor calphostin C and PKC down-regulation suppressed the stimulatory effect of bFGF on Pi transport. Selective inhibitors of ERK and p38 MAP kinases slightly reduced this cellular response with a significant effect observed with the highest concentration of the p38 MAP kinase inhibitor. In conclusion, the results of this study indicate that bFGF selectively stimulates Pi transport in calvaria-derived osteoblastic cells. The main signaling mechanism responsible for this effect involves tyrosine phosphorylation of PLC-gamma and activation of PKC, with a possible contribution of the p38 MAP kinase pathway.

Phosphate transport in Ehrlich ascites tumor cells: Inhibition by H+

The effect of changes in extracellular pH (pHo) and intracellular pH (pHi) on Na+-dependent and Na+-independent inorganic phosphate (Pi) transport in Ehrlich cells was investigated. In the presence of Na+, acutely reducing pHo from 7.30 to 5.50 results first in a transient (approximately 7 min) stimulation of Pi transport. The enhanced rate of transport is a saturable function of the extracellular [H+]; the Ks equals 2.3 X 10(-6) M (pHo 6.68). However, Pi transport is progressively inhibited as pHi falls below 6.50. The effect of pHi on Pi transport measured at various intracellular [Na+] suggests that inhibition develops as a consequence of H+ interaction with an intracellular Na+ site(s) on the Na+-dependent carrier. At pHo 7.4, about 15% of the steady state Pi flux persists in the absence of Na+. However, when pHo is reduced, transport is stimulated to the same extent and with the same time course and kinetic characteristics as in the presence of Na+. Thus, H+ stimulated Pi transport does not require Na+, raising the possibility that the Na+-independent component is mediated by the anion (Cl-) exchanger.

Insulin stimulates Pi transport in brush border vesicles from proximal tubular segments.

Induction of hyperinsulinemia in dogs results in enhanced reabsorption of Pi from glomerular filtrate in the renal proximal tubule. To determine whether this may be a direct action of insulin mediated by altered transport characteristics of the proximal tubular brush border membrane, we measured Na+-dependent 32Pi transport in brush border membrane vesicles prepared from isolated proximal tubular segments originating from dog kidney that had been incubated with or without insulin. Specific high affinity binding sites for insulin were detected in proximal tubular segments. Increased initial rates (15 s) of Na+-dependent 32Pi transport were measured in brush border vesicles prepared from segments that had been incubated with insulin. This effect of insulin was concentration dependent over the range of 10(-10) to 10(-6) M insulin. These studies demonstrate the feasibility of using brush border vesicles prepared from proximal tubular segments to study solute transport. Our findings suggest that insulin-induced increased Pi reabsorption in the proximal tubule is mediated by a direct action of insulin on the proximal tubular cell, which results in increased Na+-Pi cotransport across the brush border membrane.

Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport.

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.

Effect of 1,25-dihydroxyvitamin D3 on phosphate homeostasis in the X-linked hypophosphatemic (Hyp) mouse.

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on homeostasis of inorganic phosphate (Pi) in murine hypophosphatemia (Hyp), a homologue of X-linked hypophosphatemia (XLH) in man. Normal mice given 1,25-(OH)2D3 (83 pg/g.d) by continuous subcut, infusion x 10 d) had a significant rise in plasma calcium (Ca) (p less than 0.001), plasma Pi (p less than 0.025) and fractional Ca excretion (p less than 0.001), without change in fractional Pi excretion or Na+-stimulated Pi transport in purified renal brush-border membrane vesicles (BBMV). Hyp littermates did not respond to this dose of 1,25-(OH)2D3. At 415 pg/g.d, Hyp mice had increased plasma Ca (p less than 0.001), fractional Ca excretion (p less than 0.001), and plasma Pi (p less than 0.001) but no change in either fractional Pi excretion or Na+-stimulated Pi transport in BBMV. At this dose, 1,25-(OH)2D3 also stimulated Pi transport by everted sacs of Hyp small intestine (p less than 0.001). No deficiency of Pi transport was found in untreated Hyp intestine. We conclude that 1,25-(OH)2D3 improves Pi homeostasis in the Hyp phenotype by its effect on intestine; the defect in renal Pi reabsorption remains unchanged.

1184 PERSISTANCE OF THE DEFECT IN RENAL BRUSH-BORDER MEMBRANE PHOSPHATE TRANSPORT IN 1α,25-(OH)2 VITAMIN D3 (l,25-(OH)2D3)-TREATED X-LINKED HYPOPHOSPHATEMIA

We studied the effect of 1,25(OH)2D3 on phosphate (Pi) homeostasis in the Hyp mouse, a homologue of X-linked hypophosphatemia in man. 1,25(OH)2D3 (83 pg/g·day, infused subcut. × 10 days) produced significant hypercalcemia in normal mice (11.34±0.07 vs 10.14±0.15 mg/dl, p<0.001) and a rise in fractional excretion index of Ca (FEICa) (0.037±0.007 vs 0.005±0.001, p <0.001), without significant alteration of serum Pi (8.94±0.46 vs 8.36±0.23 mg/dl, NS), FEIpi (0.21±0.02 vs 0.19±0.03, NS) or Na+-stimulated Pi transport by purified renal brush-border membrane vesicles (BBMV) (844±32 vs 764±41 pmoles/mg protein·15s, NS). At this dose, 1,25(OH)2D3 had no observable effect in Hyp littermates. At 415 pg/g·day, Hyp mice experienced an increase in serum Ca (11.25 ±0.44 vs 9.64±0.06 mg/dl, p <0.001), FEICa (0.013±0.003 vs 0.005±0.001, p <0.005) and serum Pi (7.70±0.29 vs 4.35±0.15 mg/dl, p <0.001) but no change in FEIpi (0.40±0.05 vs 0.45±0.05, NS) and no enhancement of Na+-stimulated Pi transport by renal BBMV (385±23 vs 359±50 pmoles/mg protein·15s, NS). We conclude that 1,25(OH)2D3 does not correct the renal BBM transport defect in Hyp mice, at the supraphysiologic dose that improves Pi homeostasis. On the other hand, 1,25(OH)2D3 enhances (p <0.02) intestinal absorption of Pi in the Hyp mouse, thus explaining its effect on Pi homeostasis in the mutant phenotype of mouse and man.