©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calbindin Expression in Renal Tubular Epithelial Cells
ALTERED SODIUM PHOSPHATE CO-TRANSPORT IN ASSOCIATION WITH CYTOSKELETAL REARRANGEMENT (*)

Allan S. Pollock (§) , Hector L. Santiesteban (¶)

From the (1)Department of Medicine, Division of Nephrology, University of California, San Francisco, California 94143 and Department of Veterans Affairs Medical Center, San Francisco, California 94121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Sodium-phosphate transport in the opossum kidney (OK) cell line was studied in an OK clonal cell line that was transfected with an episomal vector expressing high levels of rat calbindin (28 kDa). High level expression of calbindin buffered the influx of calcium induced by ionomycin by 53% and raised the basal intracellular calcium from 100 ± 6 to 150 ± 8 nM. The decrement in sodium phosphate uptake induced by parathyroid hormone or forskolin was identical in the two cell lines. However, phorbol esters (10-10M), which decreased sodium phosphate uptake in the parental OK line, increased it in the calbindin-expressing line. Similarly, the parental clone did not respond to phosphate deprivation, while the calbindin-expressing clone did increase phosphate uptake in response to phosphate deprivation. In the calbindin-expressing cells, phorbol 12-myristate 13-acetate or low phosphate medium, which increased phosphate transport, produced actin filament aggregation, dissociation of the myristoylated alanine-rich C kinase substrate protein from sub-apical actin, and membrane-associated tyrosine phosphate staining. Agonists that reduced sodium phosphate uptake (cAMP, parathyroid hormone) did not affect these cellular features. The cytoskeletal rearrangement, redistribution of the myristoylated alanine-rich C kinase substrate protein, and membrane tyrosine phosphorylation are suggested to be involved in the events by which phosphate transport is increased in this cell line.


INTRODUCTION

Sodium phosphate co-transport is found in a range of epithelial cells where it serves to transport the essential charged nutrient phosphate. In the kidney, this transporter is involved in external phosphate homeostasis through its reabsorbtive function. The proximal tubule makes the quantitatively greatest contribution to phosphate reabsorbtion, affecting overall phosphate homeostasis (Dennis, 1992). The primary modulators of phosphate transport in the renal tubule are parathyroid hormone and subtle variations in dietary phosphate intake. A decrease in phosphate intake, even of brief duration, increases phosphate transport in the kidney (Dennis, 1992; Muff et al., 1992; Biber and Murer, 1993). PTH()as well as other hormones that raise intracellular cAMP decrease sodium phosphate co-transport. The activation of protein kinase C, provoked by both PTH and other hormones, also decreases sodium phosphate co-transport in the proximal tubule. The adaptation to phosphate depletion is the most physiologically relevant adjustment determining day-to-day phosphate homeostasis. The opossum kidney (OK) cell has been a widely studied cellular model of modulation of proximal renal tubule renal sodium phosphate transport (Caverzasio et al., 1986; Biber et al., 1988). In this cell, PTH, cAMP, and PKC activators decrease phosphate transport (Segal and Pollock, 1990), while some OK cell lines increase sodium phosphate co-transport with phosphate depletion. Physiologic and hormonal alterations in phosphate transport seen with the agonists mentioned may reflect movement of transporters into and out of the plasma membrane rather than direct allosteric alterations in transporter's properties. This is based on the prolonged time course (hours) required to change phosphate transport and a sensitivity to cytoskeletally active agents (Reshkin and Murer, 1992; Hansch et al. 1993). In the present study, we expressed calbindin (28 kDa), a calcium-binding protein normally found in distal renal tubule and intestine, in an OK cell line that did not normally respond to low phosphate medium. We found that expression of calbindin did indeed buffer intracellular calcium as well as restoring responsiveness to low phosphate media. These changes were also associated with alterations in the actin cytoskeleton and in cytoskeletal tyrosine phosphorylation. These findings suggest that cytoskeletal reorganization may be involved in the modulation of phosphate transport in response to both PKC and phosphate depletion.


MATERIALS AND METHODS

Preparation of Calbindin cDNA

Rat renal cortical homogenates were enriched in distal convoluted tubules as described by Vinay et al.(1981). Five micrograms of poly(A) RNA was prepared from these tubules, denatured with methyl mercury hydroxide and reverse transcribed using modified murine leukemia virus reverse transcriptase (Superscript H, Life Technologies, Inc.) primed with 1 µg of random hexamers. The first strand cDNA was amplified by the polymerase chain reaction using two primers annealing at the 5` and 3` ends of the coding sequence of the 28-kDa rat calbindin. The 5` primer introduced a NotI site followed by a Kozak consensus sequence (GCCACCATG), while the 3` polymerase chain reaction primer added an SfiI site following the termination codon. The polymerase chain reaction product was ligated into the vector pREP4 (Invitrogen). Recombinants were selected and verified by limited sequencing. Plasmid was prepared by CsCl gradient centrifugation.

Cell Culture, Transfection, and Selection of OK Cells

A single-cell OK cell clone was used. OK cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin, 2 mM glutamine. OK cells were transfected by electroporation exactly as described previously (Segal and Pollock, 1990) and selected in hygromycin B, 200 µg/ml. After initial selection, the hygromycin concentration was raised to 1000 µg/ml in a stepwise fashion and maintained at that concentration. Single-cell clones were obtained by repeated limiting dilution in 96-well round-bottom plates (0.3 cells/well), and single-cell colonies were expanded.

Analysis of Episomal Plasmid DNA

Low molecular weight DNA was prepared, as described by Hirt(1967), from approximately 10 cells that had been maintained under hygromycin selection for 10 months. The low molecular weight DNA was precipitated, and 10% of this DNA was used to transform Escherichia coli XL1-blue (transformation efficiency-efficiency of 5 10 colonies/µg of supercoiled plasmid DNA) by electroporation and plated on ampicillin agar. Resulting ampicillin-resistant colonies were grown in small scale culture, and plasmid DNA was prepared. Rescued plasmids were digested with BamHI and NotI and analyzed on agarose gels. For quantitave comparison, a known amount of supercoiled plasmid DNA was similarly treated and electroporated into bacteria.

Immunofluorescent Staining

Cells were grown on glass coverslips that had been etched with 7% nitric acid. For screening for calbindin, coverslips were rinsed with phosphate-buffered saline, fixed in 4% paraformaldehyde for 10 min, and permeabilized with ice-cold acetone for 30 s. After blocking with 5% goat serum, the primary antibody, monoclonal mouse anti-calbindin (Sigma), was applied at a 1:2000 dilution for 2 h. This was detected using fluoresceinated, affinity-purified F(ab`) fragments of a goat anti-mouse IgG (Zymed, 1:250). In order to visualize cytoskeletal and cytoskeleton-associated components, cells grown on coverslips were carefully maintained at 37 °C during the following steps. Cells were rinsed with calcium and magnesium-free phosphate-buffered saline, treated for 90 s with a cytoskeletal stabilization solution consisting of 80 mM K-PIPES, pH 6.6; 1 mM MgCl; 2 mM EGTA; 0.5% (v/v) Nonidet P-40; 1 µg/ml each pepstatin, leupeptin, and aprotinin; 1 mM phenylmethylsulfonyl fluoride followed by the same solution without Nonidet P-40 for 5 min. Finally, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (calcium and magnesium-free) for 30 min at 37 °C. Cells were blocked as described above. Immunostaining for calbindin was performed as described using a mouse monoclonal anti-calbindin (Sigma, 1:1000) followed by affinity-purified F(ab`) fragments of a goat anti-mouse IgG (Zymed, 1:250). Tubulin was stained with a monoclonal anti-tubulin antibody (Biogenex, 1:40) followed by the second antibody as indicated above. Actin was stained with either rhodamine-phallacidin or BIODIPY-FL-phallacidin (Molecular Probes, 20 units/ml). MARCKS protein was stained with a polyclonal rabbit anti bovine MARCKS antibody (a kind gift of Dr. Perry Blackshear) at 1:200 followed by lissamine-B/rhodamine-coupled, affinity-purified F(ab`) fragments of a goat anti-rabbit IgG (Accurate Chemical, 1:400). Cells were stained for phosphotyrosine after cytoskeletal fixation as described above, with the inclusion of 1 mM orthovanadate in the initial cell permeabilization solution. An anti-phosphotyrosine monoclonal antibody (UBI) was used at 10 µg/ml followed by a fluoresceinated goat anti-mouse F(ab`) IgG as described above. Cells were mounted in an aqueous mounting medium with N-propyl-gallate as an antifade reagent. Cells were examined under epifluorescence illumination with a Zeiss Axiophot microscope and photographed with Ektachrome EPH film. Confocal microscopy was performed with a Bio-Rad MRC600 confocal microscopy system with Nikon optics. Specimens for confocal microscopy were placed in mounting medium strain-free and examined with a 60 oil-immersion objective. For dual-wavelength studies, BIODIPY-FL phallacidin and lissamine/rhodamine second antibodies were used to minimize cross-channel signals.

Western Blotting

In order to detect calbindin, cells were homogenized using a Dounce homogenizer in 10 mM Tris, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, 1 mM phenanthroline and centrifuged at 15,000 g. Protein content of the cleared supernatant was determined by the BCA method (Pierce) and stored at -80 °C. Ten micrograms of homogenate protein were electrophoresed in a 12.5% SDS-PAGE gel and electroblotted onto nitrocellulose. Calbindin was detected using standard techniques using a primary monoclonal anti-calbindin IgG (Sigma) at 1:1000 and a secondary, peroxidase-coupled goat anti-mouse antibody (Zymed). As a positive control for Western blotting, superficial slices of renal cortex (containing calbindin-expressing distal tubules) were homogenized and treated as described by Christakos et al. (1987). Calbindin is heat-stable, and the heat treatment raises the relative calbindin concentration 3-4-fold over a crude homogenate (Christakos et al., 1987). Microtubule precipitation was accomplished by extraction and polymerization with taxol exactly as described by Collins(1991). Microtubule pellets were resuspended in PAGE loading buffer, heated to 95 °C, and electrophoresed in a 12.5% polyacrylamide gel, and Western blots for calbindin were performed.

Detergent-insoluble Phosphotyrosine Analysis

Cells in 150-cm culture dishes were treated with cytoskeletal stabilization solution containing 1 mM sodium orthovanadate followed by the same solution without Nonidet P-40 for 5 min. The remaining material was scraped and centrifuged at 18,000 g at room temperature for 3 min and dissolved in SDS-PAGE buffer, heated to 90 °C, resolved on 10% PAGE gels, and blotted to nitrocellulose. Western blots were probed with 1 µg/ml anti-phosphotyrosine antibody followed by a biotinylated goat anti-mouse IgG. Bands were detected with I-streptavidin, and autoradograms were obtained on Eastman Kodak XAR-2 film.

Transport Measurement

Cells were grown to confluence in multiwell clusters (Costar). Sodium phosphate uptake was performed exactly as described previously (Segal and Pollock, 1990). Amiloride-sensitive Na uptake was measured exactly as described previously (Pollock et al., 1986; Miller and Pollock, 1987).

Calcium Measurements

Cells were grown to 50% confluence on 31-mm diameter glass coverslips (size 00, Biophysica Technologies). Coverslips were pretreated by etching in nitric acid. Coverslips were then placed in a tissue chamber (Biophysica Technologies) and studied on a Nikon Diaphot microscope with an inverted lens with a 40 oil-immersion fluorescent objective. A dual wavelength excitation spectroflourometer, (Photon Technology, Princeton New Jersey), with excitation at 340 and 380 nm while measuring fluorescence with a photomultiplier with a 510 nm bandpass filter, was used to determine fluorescence of Fura-2/AM loaded cells as described by Tsien.


RESULTS

After transfection, hundreds of hygromycin-resistant colonies were initially selected. The pooled colonies were screened by Western blotting, which demonstrated a calbindin protein band. However, immunofluorescence staining demonstrated that only a small fraction of cells expressed calbindin. Because the vector we used is capable of episomal replication in some cells and might respond to increasing concentrations of selective agents with an increase in episomal copy number, the hygromycin concentration was raised stepwise over several weeks to 1000 µg/ml, and single cell clones were isolated. Thirty-four clones were screened by both Western blotting and immunofluorescence. Three of these clones were found to express some calbindin by both criteria, and one, designated CB12, expressed considerably more than the others. A Northern blot of poly(A) RNA from clone CB12, demonstrated a hybridizing band at 1000 nucleotides, corresponding to the predicted size of the expression-vector transcript. (Fig. 1) (Note: The native rat calbindin mRNA is 2.1 kilobases. However, the predicted size of the calbindin transcript from this vector 800-nucleotide calbindin coding sequence and the heterologous SV40 3` sequences is 1 kilobase). We also selected another hygromycin-resistant clone, designated CB17, which had no calbindin mRNA, a negative Western blot, and negative immunofluorescence. This clone was used as a control throughout. However we note that the sodium phosphate uptake studies in this clone were no different from the wild-type cells, and the starting single-cell clone used in this lab (Segal and Pollock, 1990). Equal amounts of cell homogenate protein from calbindin-expressing clones were analyzed side-by-side with homogenates from renal cortex by Western blotting with anti-calbindin antibody (Fig. 1). Inspection of these blots indicated that the CB12 clone expressed approximately the same amount of calbindin as did rat renal cortex. Based on the published data regarding calbindin (Sonnenberg et al., 1984) content of various tissues, we can estimate that the CB12 clone contained on the order of 15-30 µg of calbindin/mg of cellular protein.()We have previously determined the intracellular volume of OK cells to be 5 µl/mg of protein (Pollock et al., 1986). Therefore, the calculated calbindin concentration in total cell water is on the order of 100-200 µM. This is similar to the level of calbindin expression obtained in other cell types using this vector system.()


Figure 1: Expression of calbindin in OK cells. An OK cell clone was transfected with pREP4-CB28, and single-cell clones were selected in hygromycin-B. Two clones were selected for analysis, OK CB17, which did not express calbindin, and OK CB12, which did. Upper left, 10 µg of cytoplasmic extract of rat renal cortex (A) and OK-CB12 cell clone (B) were separated by SDS-PAGE electrophoresis and detected by Western blotting using a monoclonal anti-calbindin antibody. Nontransfected OK cells and OK CB17 cells had no detectable calbindin. Upper right, 5 µg of poly(A) RNA from OK CB17 cells (A) and OK CB12 cells (B) were separated on a 1.2% agarose-formaldehyde gel, blotted to nylon, and probed with a P-labeled calbindin coding-sequence cDNA probe. Lower panel, OK CB17 cells (A) and OK CB12 cells (B), grown on coverslips, were fixed in ice-cold acetone followed by 4% paraformaldehyde and stained for calbindin using a monoclonal anti-calbindin antibody followed by a rhodamine-coupled goat anti-mouse antibody. Cells were photographed using epifluorescent illumination at the same exposure times.



After 10 months in continuous culture with hygromycin-B selection (200 µg/ml), we identified episomally-replicating plasmid DNA from both CB12 and CB17 cells by the Hirt rescue procedure (Hirt, 1967). Transformation of E. coli with the Hirt DNA from 10 CB12 cells yielded several hundred ampicillin-resistant colonies while only 2 colonies were obtained from extracts from an equivalent number of calbindin-negative CB17 cells. Based on simultaneous transformation with known amounts of supercoiled vector, we estimated 50-100 ampicillin-resistant episomal copies/cell. Restriction analysis of plasmid minipreps from these rescued plasmids was performed by digestion with NotI and BamHI, which are expected to release the 800-base pair calbindin cDNA insert from the vector polylinker. The plasmids rescued from the calbindin nonexpressing CB17 cells demonstrated an altered restriction pattern, while most of the CB12 plasmids rescued had a normal restriction pattern. Therefore, this EBNA-based plasmid replicated episomally over a prolonged period in this opossum cell line. The expression of calbindin after prolonged selection in the CB12 clone represented persistence of a high copy number of intact plasmids, while both the low copy number and the deletions found in the plasmids rescued from the calbindin-negative CB17 cells presumably affected the calbindin transcription unit while leaving a functional hygromycin resistance gene. We have used the CB17 cell line as a control in subsequent studies. Neither of these cells were substantially different in appearance than the wild-type OK cell starting material.

We demonstrated two effects of calbindin on intracellular calcium. First, the basal intracellular calcium concentration as measured by Fura-2 ratio fluorescence was 100 ± 6 nM (S.E., n = 5 experiments) in the CB17 clone, while it was 150 ± 8 nM in the CB12 clone. Thus the basal intracellular calcium was elevated in the calbindin-expressing cells. Secondly, since the presumed physiological role of calbindin is the binding and buffering of intracellular calcium in cells in which calcium concentrations or fluxes may vary (Chard et al., 1993; Batini et al., 1993; Muir et al., 1993; Lledo et al., 1992), we demonstrated buffering of calcium influx. In order to demonstrate calcium buffering, we exposed CB12 and CB17 cells to ionomycin while measuring the rate of rise of intracellular calcium. As expected, ionomycin introduced a dose-dependent calcium influx pathway, which raised intracellular calcium. The initial rate or rise in intracellular calcium was proportional to the initial concentration of ionomycin. There was a consistent difference between CB12 and CB17 cells. Fig. 2illustrates the arithmetic average of the initial rates of rise of intracellular calcium after the addition of 1 µM ionomycin in five sets of experiments. At this fixed ionomycin concentration, the initial rate of rise of intracellular calcium concentration in the CB12 line was consistently decreased to 53% of that found in the control CB17 line. Thus, the expression of the calcium-binding protein increased the basal intracellular calcium yet provided significant buffering of the influx of calcium when a fixed calcium entry pathway is introduced.


Figure 2: Demonstration of calcium buffering by calbindin. OK CB17 and CB12 cells were grown on large coverslips, loaded with Fura-2, and intracellular calcium concentration was continuously measured using a PTI-Deltascan fluorometer and Nikon Diaphot inverted microscope in the ratiometric mode. The arrow indicates the time of addition of 1 µM ionomycin. The data presented are the mathematical average of five experiments on each cell line indexed to the time of addition of the ionomycin.



The effect of calbindin expression on the PTH sensitivity of sodium phosphate co-transport was measured. In the wild-type OK cell, PTH produces a dose-dependent decreases in sodium phosphate co-transport. This decrease is evident at 3-4 h after PTH is added. cAMP generation seems to be the major proximate signaling agent of this effect. We found that CB17 control cells did display the predicted dose-dependent decrease in sodium phosphate uptake. The calbindin-expressing CB12 cells displayed an identical dose-dependent decrease in sodium phosphate co-transport in response to PTH (Fig. 3) For comparison, we included the response to 100 µM forskolin, which inhibited sodium phosphate transport maximally and equally in both cell lines. The absolute basal sodium-dependent phosphate uptake was lower in the CB12 cells, 234 pmol of POµg of protein5 minversus 410 pmol of POµg of protein5 min in the CB17 cells. Thus the expression of calbindin with its attendant increase in basal intracellular calcium and buffering of the intracellular calcium transients does not affect the PTH inhibition of sodium phosphate transport in any way.


Figure 3: Inhibition of sodium phosphate co-transport by PTH and forskolin in CB12 and CB17 cells. Calbindin-expressing CB12 cells and calbindin negative CB17 cells were grown in 6-well clusters, and sodium phosphate uptake was measured. Cells were exposed to increasing concentrations of bPTH(1-34) for 3 h prior to uptake measurements. Phosphate uptake is represented as a percent of control (no PTH). Errorbars are S.E. (n = 4).



PKC-associated pathways have been strongly implicated in the modulation of proximal tubule transport by hormones including PTH, and this same association has been made in the OK cell. It has been suggested that the PKC-mediated effect of PTH on these cells is responsible for the decrement in sodium phosphate transport seen at levels of PTH, which are insufficient to measurable raise cell cAMP, although data from this laboratory indicate that a functional protein kinase A is an absolute requirement for PKC effect (Segal and Pollock, 1990). We were therefore surprised to find that expression of calbindin markedly altered the response of sodium phosphate transport to direct activation of PKC with phorbol esters. As previously noted in several studies, phorbol esters decreased sodium phosphate transport in wild-type OK cells. The CB17 cell line, which does not express calbindin, displayed this inhibitory response. The calbindin-expressing CB12 cell line demonstrated a reproducible increase in the rate of sodium phosphate transport on the addition of PMA. This increase was on the order of 50% above basal. This is summarized in Fig. 4. The effects of PMA, both inhibition of sodium phosphate transport in the OK CB17 cells and the stimulation in the calbindin-expressing CB12 cells, was evident at as low as 10 nM PMA. The onset of this effect was detectable within 20 min and complete within 1 h, in contrast to the inhibition of sodium phosphate transport by PTH, which took 3-4 h as reported previously. Kinetic studies, varying the phosphate concentration (from 10 µM to 1.2 mM) at a saturating sodium concentration of 154 mM demonstrated that the PMA effect to increase sodium phosphate transport appeared to be an increase in the V. In addition, the PMA effects on phosphate transport in both the CB12 (increase) and CB17 (decrease) was prevented by 1 µM staurosporine, indicating that was a protein kinase-mediated effect.


Figure 4: Effect of PMA on sodium phosphate transport. CB12 and CB17 cells were exposed to PMA in the concentrations shown for 3 h, and phosphate uptake was measured. Errorbars are S.E. (n = 4).



Although the decrease in sodium phosphate co-transport induced by PTH and cAMP in the OK cell has been used as a representative model of the modulation of renal proximal tubule phosphate transport, a physiologically more important modulation of phosphate transport is to be found in the adaptive increase in phosphate transport associated with phosphate depletion in vivo (Dennis, 1992). Some OK cell lines have been reported to demonstrate an adaptive increase in sodium phosphate transport in response to conditioning in low phosphate media. In order to determine whether the calbindin-expressing CB12 clone displayed phosphate adaptation, we incubated both the CB12 and CB17 clones in either 1200 µM phosphate control medium or reduced phosphate medium (50 µM phosphate) for 3-16 h. In the control CB17 cells we noted no difference in sodium phosphate transport induced by low phosphate medium, and phosphate transport was not adaptively induced. This was characteristic of the starting clone of OK cells we used. After prolonged phosphate depletion, PMA still inhibited sodium phosphate transport by almost 50%. In contrast, phosphate depletion in the calbindin-expressing CB12 line increased sodium phosphate transport by up to 40% (Fig. 5). We addressed the interrelatedness of PKC activation and phosphate depletion. After overnight phosphate depeltion, PMA only increased sodium phosphate uptake in CB12 cells by 8%. Conversely, after overnight exposure to 10M PMA, lowered phosphate concentration did not affect sodium phosphate uptake. This data is consistent with both effects demonstrating a shared component of PKC mediation.


Figure 5: Effect of low phosphate media on phosphate uptake. Cells were placed in either 1.2 mM phosphate media or 50 µM phosphate medium for 6 h prior to the measurement of phosphate uptake. Errorbars are S.E. (n = 3).



Since the obvious diference between the control and calbindin-expressing cells was the basally-elevated intracellular calcium, we asked whether simply raising the intracellular calcium in control cells with ionomycin could alter the PMA effect on sodium phosphate transport from an inhibition to a stimulation. CB17 cells were incubated in 1 or 10 µM ionomycin in 1 mM external calcium for 2 h, and phosphate uptake, with or without PMA for an additional hour, was measured. The basal sodium phosphate uptake was not affected by ionomycin. This is similar to the findings of Quamme et al. (1989a, 1989b). Whether or not ionomycin pretreatment was performed, PMA decreased sodium phosphate transport by 10-21%. Thus, increments in intracellular calcium do not reproduce the effect of calbindin expression.

Finally, in order to demonstrate that the effect on sodium phosphate transport was not a generalized effect on transport, we report the effect of PMA on amiloride-sensitive Na uptake, representing sodium/hydrogen exchange, in these cells. We treated the CB17 line and the calbindin-expressing CB12 line to 10 and 10M PMA for 1 h and measured Na uptake in the presence of 10 mM external sodium at pH 7.5. We found that 0.1 and 1 µM PMA reduced amiloride-sensitive Na uptake by 19 and 23%, respectively, in the CB17 control cells and to 21 and 28%, respectively, in the CB12 cells. The basal Na uptake was 32% lower in the CB12 cells than in the CB17 control cells. It is well known that PMA inhibits sodium/hydrogen exchange in this cell line. Thus it appears that PMA has the same qualitative and quantitative effects on sodium/hydrogen exchange in the CB12 cells as it does in the control cells. This also indicates that the calbindin effects on phosphate transport are not a reflection of generalized or overall effects on all other membrane transport systems.

Cytoskeletal rearrangement is known to be central to trafficking of proteins within cells and has been implicated in the normal economy of several membrane proteins including transport proteins (Luna, 1991; Luna and Hitt, 1992; Rodriguez-Boulan and Powell, 1992; Marja and Matlin, 1990; Ojakian and Schwimmer, 1992; Nelson, 1991), and there is evidence in intestinal cells that calbindin may be associated with the microtubular cytoskeleton (Nemere and Norman, 1990; Nemere et al., 1991). Additionally, there is a report that in the OK cell that microtubule disruption with nocodozole alters the response of some OK cell clones to low phosphate medium (Hansch et al., 1993). We therefore determined whether calbindin was associated with microtubules in these cells ectopically expressing calbindin and evaluated the state of microtubular organization. CB12 cells were homogenized, and microtubules were polymerized in the presence of GTP and taxol. The precipitated microtubules were analyzed by Western blotting for the presence of calbindin. Calbindin was readily detected in the cell homogenate but not in the microtubule fraction, indicating that in the CB12 cell, calbindin does not co-precipitate with polymerized microtubules. These findings were confirmed by staining cytoskeletally stabilized, Nonidet P-40-treated cells for calbindin. Although a striking calbindin fluorescence was evident in the acetone-fixed cells, the permeabilized cells in which only cytoskeletal components remain contained no calbindin staining.

OK cells demonstrated a well organized microtubular structure that was more developed in the CB12 cells than in the control cells. The tubulin structure was not markedly reorganized by forskolin, PTH, or phorbol esters in either control OK CB17 cells or the calbindin-expressing CB12 line. (Fig. 6) Low phosphate medium incubation, a perturbation that increased sodium phosphate co-transport only in the CB12 cells, had a significant effect on the tubulin organization of both CB17 and CB12 cells where it markedly disrupted the microtubular staining pattern (Fig. 6). Since this occurred in both control and CB12 cells, it seems unlikely that there is a direct microtubular participation in these processes responsible for an increased phosphate transport only in the CB12 cells. These findings suggest that microtubular rearrangement perse is not a direct mediator of these alterations in transport and that the calbindin effect is not mediated through a direct microtubule binding of calbindin.


Figure 6: Organization of the tubulin microtubules in OK cells. Control CB17 cells (A-D) and CB12 cells (E-H) were grown on glass coverslips, fixed as described, and stained for tubulin with a monoclonal anti-tubulin antibody. This was detected with a fluorescein-coupled goat anti-mouse antibody. A, E, control; B, F, cells exposed to 100 nM bPTH(1-34); C, G, cells exposed to 50 µM phosphate medium overnight; D, H, cells exposed to 10 nM PMA for 3 h. Cells were photographed using epifluorescent illumination at an original magnification of 1000.



The actin cytoskeleton is involved with the placement of membrane proteins in many cells, and agents acting to alter the disposition of membrane proteins may achieve this via rearrangement of the actin cytoskeleton. In particular, activation of PKC and tyrosine phosphorylation, which produces disparate effects on phosphate transport in the calbindin-expressing cells, may act to disrupt the actin cytoskeleton via phosphorylation of the MARCKS protein (Aderem, 1992; Downward, 1992; Ridley and Hall, 1992). In addition, actin polymerization/depolymerization is a target of a range of rho family G-protein-mediated events originating in a range of cellular signals (Hall, 1992; Reshkin and Murer, 1992). The actin organization, probed with rhodamine-phallacidin, demonstrated a greater frequency of stress fibers in the CB12 cells under resting conditions (Fig. 7A). PTH and cAMP, both agents that decrease phosphate transport, somewhat disrupted the actin networks in the control CB17 cells as well as the CB12 cells. Fragmentation of actin after PTH exposure has been reported in proximal tubular cells (Goligorsky et al., 1986). However, the two agonists that did increase phosphate transport, PMA and low PO media, produced focal actin aggregation only in the CB12 cells. Optical sectioning of these cells by confocal microscopy indicated that the actin aggregates produced by either low PO medium or by PMA were present in the apical 1-2 µm of the cells (which ranged from 5 to 6 mm in height, Fig. 7B).


Figure 7: A, organization of the actin cytoskeleton in OK cells. Control CB17 cells (A-D) and CB12 cells (E-H) were grown on glass coverslips, fixed as described, and stained for actin using rhodamine-phallacidin. A, E, control; B, F, cells exposed to 100 nM bPTH(1-34); C, G, cells exposed to 50 µM phosphate medium overnight; D, H, cells exposed to 10 nM PMA for 3 h. Arrows indicate actin aggregates. Cells were photographed using epifluorescent illumination at an original magnification of 1000. B, localization of actin aggregates in OK cells by confocal microscopy. OK-CB12 cells, treated with PMA as above, were stained with rhodamine phallacidin and examined by confocal microscopy. Sections at 1-2 µm of the basal aspect of the cell are seen on the leftpanel, while sectioning at 1 µm from tha apical membrane were seen on the rightpanel.



The MARCKS protein is a major mediator of protein kinase-mediated events affecting the actin cytoskeleton. In order to determine whether MARCKS-related rearrangement of the actin cytoskeleton was altered in the CB12 cells, cells were stained for both actin and the MARCKS protein, using a rabbit anti-MARCKS antibody (Fig. 8). Cells were examined by confocal microscopy at a regions within 1-2 µm of the base of the cell and within 1-2 µm of the apical membrane. In control CB17 and CB12 cells, actin and MARCKS staining was present both apically and basolaterally. On the addition of PMA, no change in MARCKS protein distribution was noted in the CB17 cells, but the CB12 cells demonstrated disappearance of staining from the apical regions of the CB12 cell (Fig. 9). Thus, increase in sodium phosphate co-transport in the CB12 cells only correlated with the disappearance of the MARCKS protein from the subapical region of the cell. This is consistent with activation of the MARCKS protein by PKC, upon which it is known to dissociate MARCKS from the actin cytoskeleton. In addition it is consistent with the more apical location of the actin aggregates found.


Figure 8: Co-localization of actin and MARCKS protein. OK CB12 cells were permeabilized in a cytoskeletal stabilization buffer and stained for actin using fluorescein phallacidin and for the MARCKS protein using a polyclonal rabbit antibody followed by rhodamine-coupled goat anti rabbit IgG. A, phallacidin staining of actin; B, staining for the MARCKS antigen. Original magnification 640.




Figure 9: Co-distribution of MARCKS protein and actin in CB12 and CB17 cells after PMA evaluated by confocal microscopy. Cells grown on coverslips were treated with control media or 10 nM PMA for 30 min and permeabilized in cytoskeletal stabilization buffer and fixed. Actin and MARCKS protein were stained as in Fig. 8. Cells were photographed using confocal microscopy at a basal region (about 1 µm from the basal aspect of the cell) and at an apical region (about 1 µm from the apical aspect of the cell). Images were simultaneously obtained using a Bio-Rad MRC600 confocal microscope with Nikon 60 oil-immersion objective and 4 electronic magnification. Images of actin, pseudocolored green, and of MARCKS, red, were superimposed. A-D, OK CB17 cells; E-H, OK CB12 cells. A and B represent the apex and base of the cells in control conditions, while C and D represent the apex and base of the cells after PMA. E and F represent the apex and base of the cells in control conditions, while G and H represent the apex and base of the cells after PMA.



Caverzasio et al.(1986) observed that orthovanadate, which increased net membrane tyrosine phosphorylation in OK cells, increased sodium phosphate transport activity (Caverzasio and Bonjour, 1992). This suggests that membrane-associated tyrosine phosphorylation might be involved in the alterations in transporter activity. We therefore stained CB17 and CB12 cells for cytoskeletally-associated phosphotyrosine after exposure to agonists that alter transport. The control cells did not demonstrate significant membrane staining for phosphotyrosine either in control conditions nor on exposure to low phosphate media or phorbol esters. In contrast, the CB12 line demonstrated membrane-like staining for phosphotyrosine when exposed to either low phosphate medium or phorbol esters, both situations that increase sodium phosphate transport (Fig. 10). Detergent-insoluble fractions from cells were prepared using the same protocol used to prepare them for phosphotyrosine immunohistology, and they were analyzed by Western blotting using an anti-phosphotyrosine antibody. A number of major tyrosine-phosphorylated proteins were noted (data not shown). However, there was no difference between control cells and those treated with PMA, suggesting that it was possible that there was a redistribution of phosphorylated proteins. Indeed, we noted a marked decrease in the perinuclear localization of phosphotyrosine with its appearance in the membrane.


Figure 10: Membrane-associated phosphoytrosine. Cells were grown on coverslips and permeabilized in cytoskeletal permeabilization buffer containing 1 mM orthovanadate and fixed. Phosphotyrosine was detected with a monoclonal anti-phosphotyrosine antibody and in turn with rhodamine-labeled goat anti-mouse IgG. A-C, CB17 cells; D-F, CB12 cells. A and D, control; B and E, 50 µM phosphate medium overnight; C and F, 10 nM PMA for 1 h. The arrows indicate prominent membrane staining. Cells were photographed under epifluorescent illumination. Photos of A-C were overexposed relative to D-F because of lesser degrees of staining.




DISCUSSION

The regulation of sodium phosphate transport in the OK cell model has revealed that both activation of protein kinase A and of PKC reduce this transport activity, as does PTH. Other renal cell lines, such as the LLC-PK1, demonstrate different responses to these agonists. The small intracellular calcium transient reported in some OK cell lines after PTH had been suggested to mediate part of the PTH effect. Recent evidence suggests, however, that the inhibition calcium transients can be dissociated from inhibition of transport (Quamme et al., 1994). Our initial rationale for the expression of calbindin, a naturally occurring calcium buffer, was to probe the relationship between calcium and the inhibition of sodium phosphate transport in a homogenous single-cell OK cell clone. Calbindin over-expression both acted as a buffer to diminish the rate of rise of calcium while raising basal intracellular calcium. Both of these patterns have been reported in cells in which calbindin is expressed (Lledo et al., 1992; Muir et al., 1993). The major findings of this study were that over-expression of the calcium binding protein calbindin (28 kDa), produced a significant alteration of several features of the sodium phosphate co-transport in these cells. These results were unanticipated.

A novel finding was the dissociation of the effects of the PKC activator PMA and the cAMP-mediated effect of PTH and cAMP-raising agents such as forskolin. Whereas both of these agents decrease sodium phosphate transport in the native OK cell line, the expression of calbindin allows PMA to increases in sodium phosphate transport. This response to PMA has been noted in two other epithelial cell lines (Raymond et al., 1991; Bringhurst et al., 1993). In addition, calbindin expression allows expression of an adaptive increase in sodium phosphate transport in response to low phosphate medium. This effect, previously reported in several OK cell clones, was clearly restored to an otherwise unresponsive OK by calbindin expression. Because both the restoration of the low phosphate response and the increase in phosphate transport with direct PKC treatment were both induced in the calbindin-expressing cells, they may operate through related mechanisms. Although an elevation of intracellular calcium is the most obvious difference between the control and calbindin-expressing cells, our studies raising intracellular calcium with ionomycin suggest that this alone is not the cause.

In the renal proximal tubule epithelia, PTH and cAMP decreases, and phosphate depletion increases this transport activity. The OK cell retains many of these properties. PTH and cyclic AMP both reduce phosphate transport. Experiments from this laboratory have indicated that activation of protein kinase A is an essential feature of this process (Segal and Pollock, 1990). The OK cell additionally displays a reduction in phosphate transport with direct PKC activation. This PKC-associated reduction in transport has been used to explain the response of phosphate transport to low concentrations of PTH, below the level required for measurable production of cAMP. In this setting it has been assumed that PTH activates PKC in order to effect this change. Phosphate transport has also been studied in the nonrenal HeLa cell where it is inhibited by cAMP (Raymond et al., 1991). However in this line, direct activation of PKC increases sodium phosphate transport in a manner similar to that we observed. The LLC-PK1 cell line, normally lacking PTH receptors, displays an increase in phosphate transport when calcitonin or transfected PTH receptors are activated. This effect is cAMP-insensitive (Bringhurst et al., 1993). The effect of calbindin expression in OK cells appears to simulate the features of transport modulation found in the PTH receptor-transfected LLC-PK1 cell and in the HeLa cell.

Several sodium phosphate transporters have recently been isolated by expression cloning from rat and human kidney and by homology from the OK cell (Biber and Murer, 1993; Sorribas et al., 1994). Its amino acid structure predicts a cytoplasmic PKC consensus phosphorylation site but no protein kinase A consensus site. This structure, in addition to the relatively slow responses to protein kinase-activating agonists (cAMP, PTH, and phorbol esters), suggest that agonists that induce alterations in transport might do so by mechanisms other than direct protein modification by phosphorylation. These might include alterations in its placement in the apical membrane. Recent studies (Cluster et al., 1994) demonstrate a subapical pool of immunoreactive NaP transporters in rat proximal tubule, consistent with its existence in vesicular structures.

Calbindin 28 kDa is the major vitamin D-responsive calcium binding protein and is primarily expressed in the distal convoluted tubule, intestinal mucosa, and nervous tissue (Christakos et al., 1992). In kidney and intestine (epithelia responsible for significant net trans-epithelial calcium movement, as well as in neuronal tissues) the calbindins are thought to buffer intracellular calcium and allow large transcellular or intracellular fluxes of calcium without a large increase in free intracellular calcium concentration (Chard et al., 1993; Batini et al., 1993; Muir et al., 1993; Lledo et al., 1992; Dowd et al., 1992). Indeed, mathematical modeling of epithelial calcium absorption essentially requires the presence of a significant intracellular calcium buffer in order to maintain thermodynamically favorable calcium gradients (Stein, 1992). Recent reports of transfection-mediated expression of calbindin in cell lines suggest that calbindin can buffer or limit the rate of rise in intracellular calcium resulting from calcium influx (Lledo et al., 1992; Muir et al., 1993) and raise the basal intracellular calcium to levels similar to those observed in these studies. However, in addition to intracellular calcium buffering, there is in vitro evidence that calbindin (28 kDa) may modulate the activities of calmodulin-sensitive enzymes such as phosphodiesterase and calcium-ATPase (Resiner et al., 1992). In this setting, calbindin apparently substitutes for Ca-calmodulin in the activation of these calmodulin-dependent enzymes. An additional effect of calbindin is found in insulin-producing rat insulinoma cells, which normally do express some calbindin. Overexpression of calbindin in these cells to levels similar to those attained in these studies (30-fold basal) increased insulin secretion and synthesis rated to over 30-fold control without affecting basal intracellular calcium.()Thus calbindin appears to have effects independent of changes in overall intracellular calcium concentration.

We noted phorbol ester-induced disappearance of the MARCKS protein only in the calbindin-expressing CB12 cells in subapical area of the cell. In this setting, PKC activation in the wild-type cell, which lowers sodium phosphate transport activity, is not associated with MARCKS dissociation from the membrane. In contrast, the calbindin-expressing CB12 cells increase phosphate transport and demonstrate MARCKS dissociation from the membrane as well as actin filament reorganization. Both of these occur beneath the apical membrane, which is the location of the sodium phosphate transporter. This was also the site of actin aggregation both upon PMA treatment and upon exposure to low phosphate media; both are situations that increased phosphate transport. Dissociation of MARCKS from membrane structures after PKC phosphorylation varies between cells (Byers et al., 1993). Actin aggregation is a frequently described result of signal transduction by mitogens, cytokines, and extracellular matrix (Yamamoto et al., 1992; Aharoni et al., 1993; McWhirter and Wang, 1993). The mildly elevated basal intracellular calcium level in the CB12 cells might increase the activity of calcium-calmodulin that acts along with PKC on the MARCKS protein.

The cytoskeleton is involved with many aspects of membrane protein economy, and several pathways including PKC-induced reorganization and tyrosine phosphorylation of actin-associated proteins appear to be related to these activities. Several membrane transport properties, including Cl channels (Suzuki et al., 1993), the Na/K-ATPase (Marrs et al., 1993) and volume-regulatory adaptation (Cantiello et al., 1993), have been linked to the state of the actin cytoskeleton or to properties of actin binding proteins. The MARCKS protein, a major protein kinase C substrate, is an integrator of extracellular signals acting on membrane structures. Normally bound to polymerized, sub-membrane actin filaments, PKC acts to phosphorylate this protein in the presence of calcium/calmodulin. This subsequently allows dissociation from the cytoskeleton where it exerted a stabilizing role on the polymerized form of actin. Cytoskeletal reorganization has also been associated with the discrete activation of tyrosine kinases in nonepithelial cells such as platelets and in cells in which focal adhesions are reorganized. Over-expression of the insulin receptor in rat-1 cells is associated with co-localization of both actin reorganization and protein tyrosine phosphorylation. Epithelial cells, including renal epithelial brush-border membranes, both protein tyrosine kinases, as as well as tyrosine-phosphorylated substrates have been noted (Tremblay et al., 1992). Brush-border and membrane tyrosine phosphorylation, modulated directly either by the by the inhibition of tyrosine kinases or the inhibition of tyrosine phosphatases, have been demonstrated to alter epithelial transport of sodium in intestine (Donowitz et al., 1994) or the A6 distal nephron cell line (Matsumoto et al., 1993). In the OK cell, tyrosine phosphorylation induced by vanadate also was associated with an increase in sodium phosphate transport (Caverzasio and Bonjour, 1992) as was found here. The studies of Quamme et al. (Quamme et al., 1989a, 1989b) indicate that the level of intracellular calcium may not affect the downward regulation of phosphate transport by cAMP and PTH. Acute elevation of intracellular calcium was not found responsible for the calbindin effects on up-regulation of transport. Alternatively, albindin in vitro can directly affect the activity of several enzymes (Resiner et al., 1992). These authors found that calbindin could activate several calmodulin-dependent enzymes by substituting for calcium-calmodulin, albeit requiring 10-75-fold more calbindin. A dissociation between PKC activation and MARCKS dissociation from actin has been noted previously in the C6 and N1E-115 neuroblastoma cell lines (Byers et al., 1993). In the C6 line, PKC activation by TPA was followed by phosphorylation and prompt release of cytoskeltally-associated MARCKS, while in the N1E-115 line, MARCKS phosphorylation was not associated with its cytoskeletal dissociation. This is analogous to our observations in the OK-CB17 cell and the calbindin-expressing CB12 cell. It is conceivable that the over-expressed calbindin substituted for calcium/calmodulin and allowed a MARCKS activation that was not present in the control cells; calcium/calmodulin must interact with MARCKS before PKC activation will allow it to dissociate from actin (Aderem, 1992). Although the effect of PKC activation to increase phosphate transport in the OK-CB12 cell is not characteristic of the OK cell, it is found in both HeLa cells and in LLC-PK1 cells. Taken together, these findings suggest that calbindin expression has effects on PKC responsiveness unrelated to elevation of intracellular calcium and that cytoskeletal reorganization may be associated with an apparent increase in OK cell sodium phosphate transport. These changes are associated with a complex of PKC activation and membrane-associated tyrosine phosphorylation and may underlie the response to phosphate deprivation seen in renal cells.


FOOTNOTES

*
This work was supported by the Research Service of the Department of Veterans Affairs Medical Center, San Francisco and National Institutes of Health Grants R01DK31398 and DK37423. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: VA Medical Center, 111J, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2032; Fax: 415-750-6905.

Supported by National Institutes of Health Training Grant 5T32 DK07219.

The abbreviations used are: PTH, parathyroid hormone; OK, opossum kidney; PKC, protein kinase C; MARCKS, myristoylated alanine-rich C kinase substrate; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate.

Rat cortical homogenate was made according to Christakos et al. (1987) and heat-treated, and the heat-stable protein was retained. The heat-stable homogenate is 4-fold enriched in calbindin relative to total cellular protein as described (Christakos et al., 1987). The calbindin content of rat renal cortex is 7.3 µg/mg protein (Sonnenberg et al., 1984), and therefore, the content of the heat-treated cortex is 29 µg/mg of protein. As judged by Western blotting, the calbindin content of the CB12 line is approximately equal to that of the heat-treated renal cortical homogenate.

Rat insulinoma cells transfected with this vector and selected as described expressed 14.3 µg of calbindin/mg of cell protein as determined by specific calbindin radioimmunoassay (S. Christakos, personal communication).

S. Christakos, personal communication.


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