High capacity Na+/H+ exchange activity in mineralizing osteoblasts

Abstract

Osteoblasts synthesize bone in polarized groups of cells sealed by tight junctions. Large amounts of acid are produced as bone mineral is precipitated. We addressed the mechanism by which cells manage this acid load by measuring intracellular pH (pHi) in non-transformed osteoblasts in response to weak acid or bicarbonate loading. Basal pHi in mineralizing osteoblasts was ∼ 7.3 and decreased by ∼ 1.4 units upon replacing extracellular Na(+) with N-methyl-D-glucamine. Loading with 40 mM acetic or propionic acids, in normal extracellular Na(+), caused only mild cytosolic acidification. In contrast, in Na(+) -free solutions, weak acids reduced pHi dramatically. After Na(+) reintroduction, pHi recovered rapidly, in keeping with Na(+) /H(+) exchanger (NHE) activity. Sodium-dependent pHi recovery from weak acid loading was inhibited by amiloride with the Ki consistent with NHEs. NHE1 and NHE6 were expressed strongly, and expression was upregulated highly, by mineralization, in human osteoblasts. Antibody labeling of mouse bone showed NHE1 on basolateral surfaces of all osteoblasts. NHE6 occurred on basolateral surfaces of osteoblasts mainly in areas of mineralization. Conversely, elevated HCO 3- alkalinized osteoblasts, and pH recovered in medium containing Cl(-), with or without Na(+), in keeping with Na(+) -independent Cl(-) /HCO 3- exchange. The exchanger AE2 also occurred on the basolateral surface of osteoblasts, consistent with Cl(-) /HCO 3- exchange for elimination of metabolic carbonate. Overexpression of NHE6 or knockdown of NHE1 in MG63 human osteosarcoma cells confirmed roles of NHE1 and NHE6 in maintaining pHi. We conclude that in mineralizing osteoblasts, slightly basic basal pHi is maintained, and external acid load is dissipated, by high-capacity Na(+) /H(+) exchange via NHE1 and NHE6.

Fig. 1

Intracellular pH measurement using 2′, 7′-bis-(2-carboxyethyl)-5, 6-carboxyfluorescein (BCECF) in non-transformed mineralizing human osteoblasts. A: Confluent normal human osteoblasts. A bright field picture without phase shows mineralization, which reflects light, as focal dark nodules. The field is 500 μm across. B: For intracellular pH measurement, osteoblasts grown and differentiated on glass chips were loaded with 5 μM BCECF-AM at 37°C for 1 h. Green fluorescence shows the intracellular loading of the dye. The field is 200 μm across. C: BCECF loaded mineralizing human osteoblasts on glass were transferred to cuvette containing 130 mM KCl, 20 mM NaCl, 5 mM Hepes, and 10 μg/ml nigericin to equilibrate intracellular and extracellular pH. Solutions were adjusted to pH 5.0, 5.5, 6.0, 6.5, 7.0, and 8.0 using NH₄OH or acetic acid. Excitation scans with excitation from 400 to 520 nm and a fixed emission at 535 nm was performed using calibration solutions at each pH level as indicated. D: A standard curves relating fluorescence ratios with background fluorescence subtracted [(F500 nm F400 nm)/(F455 nm F400 nm)] to intracellular pH, from the excitation scans (C). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 2

Intracellular pH changes in response to acid load and Na⁺ withdrawal. BCECF loaded normal human osteoblasts or HeLacells (controls in B) were grown on glass, equilibrated in Hepes-buffered ${\text{HCO}}_3^{-}$ free saline (standard solution): 140 mMNaCl, 3 mMKCl, 1.5 mM CaCl₂, 5 mM Hepes, 5 mM glucose, pH 7.4, transferred into test solutions. A: Effect of mineralization on resting osteoblast intracellular pH. Mineralizing osteoblast resting intracellular pH is significantly higher than that of non-mineralizing osteoblasts. N = 4, mean ± SD, P < 0.01. B: Change in intracellular pH in HeLa cells, non-mineralizing human osteoblasts, and mineralizing human osteoblasts after a 40 mM acid load using propionate acid in standard solution. The change in pH in HeLa cells and nonmineralizing osteoblasts was similar, while mineralizing osteoblasts, which express much larger amounts of sodium-hydrogen exchangers (see Fig. 4), was significantly reduced. N = 4, mean ± SD, P<0.05. C: Real-time measurements of intracellular pH: effect of replacing extracellular sodium. In mineralizing normal osteoblasts (black trace): replacement of standard solution with Na+-free solution, 140 mMN-methyl-d-glucamine chloride (NMDGCl), 5 mM Hepes, 5 mM glucose, pH 7.4from 140 to 505 sec. At 505 sec, the cells were transferred back to standard solution. Growing, non-mineralizing normal osteoblasts (grey trace): replacement of standard solution with Na⁺-free solution from 150 to 580 sec. At 580 sec, the cells were transferred backto standard solution. D: Effect of acetic acid loading on mineralizing normal human osteoblasts with and without extracellular sodium. Cells were exposed to acetic acid, 40 mM, in the presence of Na⁺ (black trace), from 110 to 640 sec, then transferred back into standard solution. The same cells grown on another glass chip were exposed to acetic acid load in Na⁺-free solution (grey trace) from 145 to 625 sec, then transferred back into standard solution. Standard solution with 40 mM potassium acetate was used as acetic acid load. Na⁺-free acid load was the same except for the replacement of NaCl with NMDGCl. E: Effect of propionic acid loading on mineralizing normal human osteoblasts with and without extracellular sodium. Osteoblasts were exposed to propionate acid, 40 mM, in the presence of Na⁺ (black trace), from 190 to 695 sec, then transferred back into standard solution. The same cells grown on another glass chip were exposed to propionate acid load in Na⁺-free solution (grey trace) from 150 to 625 sec, and then transferred back into standard solution. Standard solution with 40 mM potassium propionate was used as propionate acid load. Na⁺-free acid load was the same except forthe replacement of NaCl with NMDGCl. Intracellular pHwasderived asdescribed under Materials and Methods Section. F: Comparison of initial pHi recovery rate of osteoblasts from acid load in presence or absence of Na⁺.

Fig. 3

Effect of amiloride on intracellular pH recovery after acid load. A: BCECF loaded mineralizing human osteoblasts on glass were first equilibrated in 140 mM NaCl, 3 mM KCl, 1.5 mM CaCl₂, 5mM Hepes, 5 mM glucose, pH 7.4, and transferred into acetate loading solution in the presence (grey trace) or absence of 10 μM amiloride (black trace). For comparison the two curves are overlaid. Loading solution is standard solution plus 40 mM potassium acetate, pH 7.4. B: Acid extrusion as rate of pH recovery (in pH units/min) calculated from the initial slope of the pH recovery curves (A). N = 2, mean ± range. C: Amiloride concentration in relation to the fractional inhibition of pH recovery (B). A second independent experiment showed similar results (not illustrated).

Fig. 4

Sodium–hydrogen exchangers 1 and 6 are highly expressed in human and mouse osteoblasts. A, B: Real-time RT-PCR analysis of NHE isoform 1 (A) and isoform 6 (B) in growing and mineralizing normal human osteoblasts and MG63 human osteosarcoma cells. Total RNA were isolated from growing normal human osteoblasts and MG63cells, mineralizing normal human osteoblasts and MG63 cells treated with osteoblast differentiation medium for 2weeks. Results were presented as fraction of control. N = 2, mean W ± range. C, D: Fluorescent immune labeling of NHE 1 (C) and NHE6 (D) in mouse vertebral bone frozen sections. Sections were labeled with goat anti-NHE6, goat anti-NHE1, and secondary donkey anti-goat cy3 (red) as described in Materials and Methods Section. Negative controls using specific blocking peptide to neutralize each primary antibody are also shown (second and fourth columns). Calcein labeling of bone formation at the mineralizing surface (green signal) was shown separately (middle row) and overlaid with NHE1 orNHE6 signals (red) (last row). Bright field picture with no phase of the same field was also taken to show the contour of the bone (first row). Fields are 300 μm across. E: Labeling for calcein and NHE6 at higher magnification. Fields are 100 μm across. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 5

${\text{HCO}}_3^{-}/{\text{CI}}^{-}$ exchange activity and anion exchanger 2(AE2) in osteoblasts. A: BCECF loaded mineralizing human osteoblasts grown on glass first equilibrated in standard solution, 140 mM NaCl, 3 mM KCl, 1.5 mM CaCl₂, 5 mMHepes, 5 mM glucose, pH 7.4, and transferred into bicarbonate loading solution: 40 mM NaHCO₃, 100 mM NaCl, 5 mM Hepes, 5 mM glucose, pH 7.4. The base loading was reversed by transferring the cells into standard solution (black curve) or Na⁺-free solution with Na⁺ replaced with N-methyl-d-glucamine (NMDG⁺) (grey curve). Intracellular pH was derived as described in Materials and Methods Section. B: Bicarbonate-loaded cells as in A were transferred into bicarbonate free medium but with chloride replaced with gluconate; pHi did not re-equilibrate until this Cl^- free solution was additionally replaced with standard chloride-containing medium. C: Indirect fluorescent labeling of antibody localization of AE2 in mouse vertebra frozen sections. Sections were cut and labeled with goat anti-AE2 and donkey anti-goat-Cy3 (red) as described in Materials and Methods Section. Negative controls using AE2 specific blocking peptide to neutralize primary AE2 antibody are shown (right column). Calcein labeling of bone formation at the mineralizing surface (green signal) was shown separately (middle row) and overlaid with AE2 signal (red signal) (last row). Note that the basolateral surface of bone lining cells, with or without calcein (green signal), is labeled with the antibody. Bright field of the same area is shown to indicate the contour of bone trabeculae (first row). All fields shown are 300 μm across. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 6

Effect on pHi of overexpression of NHE6 or NHE1 knockdown in MG63 cells. MG63 cells grown on glass were transfected with either control vector, NHE6-expressing plasmid, control siRNA, or NHE1 siRNA. Forty-eight hours later, a glass chip was loaded with BCECF and equilibrated in standard solution. Standard solution, Na⁺-free solution, and acetic acid load solution are the same as in previous figures. A: Effect of NHE6 over expression on Na⁺ withdrawal in MG63 cells. Black trace: control vector transfected MG63 cells were exposed to Na⁺-free solution from 90 to 355 sec then transferred back into standard solution. Grey trace: MG63 cells transfected with NHE6-expressing plasmids were exposed to Na⁺-free solution from 210 to 380 sec then transferred back in to standard solution. Horizontal bars indicate periods of Na⁺ withdrawal. B: Effect of NHE6 over expression on acetic acid load in MG63 cells. Black trace: control vector transfected MG63 cells were exposed to acetic acid loads olution from 210 to 610 sec then transferred back in to standard solution. Grey trace: MG63 cells transfected with NHE6-expressing plasmids were exposed to acetic acid load from 185to 835 sec then transferred back into standard solution. Horizontal bars indicate periods of acetic acid loading. C: Real-time PCR analysis of NHE6 expression in MG63 cells transfected with control vector or NHE6-expressing plasmid. Results are presented as percentage of GAPDH level. Note that the difference is several hundred fold; the Y-axis is log scale. D: Effect of NHE1 knockdown on acetic acid load in MG63 cells. Black trace: control siRNA transfected MG63 cells were exposed to acetic acid load solution from 105 to 380 sec then transferred back into standard solution. Grey trace: MG63 cells transfected with NHE6-expressing plasmids were exposed to acetic acid load from 105 to 565 sec then transferred back into standard solution. Horizontal bars indicate periods of acetic acid loading. E: Real-time PCR analysis of NHE1 expression in MG63 cells transfected with control siRNA or NHE1 siRNA. Results are presented as percentage of GAPDH level.

Fig. 7

Proposed sodium-hydrogen exchange mechanism to alkalinize the bone mineral formation compartment, which produces net acid. Bone is formed by gap-junction linked groups of osteoblasts, this unit referred to as an osteon. Its superficial (bone lining) cells isolate extracellular fluid from the bone matrix with tight junctions. Its deep surface is a cement line; mineralized bone is deposited from the cement line toward the surface at a narrow front. As cells are incorporated in matrix, new layers of osteoblasts differentiate at the basolateral surface and are added to the unit. Cells buried in matrix no longer synthesize bone, but have regulatory functions, and are called osteocytes. Mineralized bone is impervious to ions, so deposition of mineral is strictly vectorial. At the mineralization front, ${\mathrm{H}}_2{\text{PO}}_4^{-}$ and water are precipitated with Ca²⁺ as a ${\text{PO}}_4^{3-}$ and OH^- salt, with evolution of H⁺, ∼1.5 moles per mole of Ca²⁺ deposited. The osteoblasts maintain a slightly alkaline pH, which promotes mineral precipitation, and manage the acid produced, by greatly expanded expression and activity of sodium-hydrogen exchangers NHE1 and NHE6 at their basolateral surfaces. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]