From the
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
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
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)
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.
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
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.
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
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 (
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
-10
M), 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.
(
)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.
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.
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.
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 protein
5
min
versus 410 pmol of
PO
µg of protein
5
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.
Na
uptake, representing sodium/hydrogen exchange, in these cells. We
treated the CB17 line and the calbindin-expressing CB12 line to
10
and 10
M 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.
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.
transporters in rat proximal tubule, consistent with its
existence in vesicular structures.
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.