Inhibition of cell differentiation by
G
q in the renal epithelial
cell line LLC-PK1
Lihyun
Sun1,
Debora J.
Weaver2,
Kurt
Amsler3, and
Ellen R.
Weiss1
1 Department of Cell Biology
and Anatomy, The University of North Carolina at Chapel Hill,
Chapel Hill 27599; 2 Department of
Biological Sciences, Campbell University, Buies Creek, North
Carolina 27506; and 3 Department
of Physiology and Biophysics, University of Medicine and Dentistry
of New Jersey-Robert Wood Johnson Medical School, Piscataway, New
Jersey 08854
 |
ABSTRACT |
LLC-PK1, an epithelial cell
line derived from the kidney proximal tubule, was used to study the
ability of the G protein
-subunit, G
q, to regulate cell
differentiation. A constitutively active mutant protein,
qQ209L, was expressed using the
LacSwitch-inducible mammalian expression system. Induction of
qQ209L expression with isopropyl-
-D-thiogalactopyranoside
(IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold.
Increasing concentrations of IPTG progressively inhibited the activity
of two differentiation markers,
Na+-dependent hexose transport and
alkaline phosphatase activity. Induction of
qQ209L expression also caused a
change from an epithelial to a spindle-shaped morphology. The effects
of
qQ209L expression on cell
differentiation were similar to those observed with
12-O-tetradecanoylphorbol 13-acetate
(TPA) treatment. However, protein kinase C (PKC) levels were
downregulated in TPA-treated cells but not in
qQ209L-expressing cells,
suggesting that the regulation of PKC by
G
q may be different from
regulation by TPA. Interestingly, the PKC inhibitor GF-109203X did not
inhibit the effect of IPTG on the development of
Na+-dependent hexose transport in
qQ209L-expressing cells. These data implicate PKC
and PKC
in the pathway used by
G
q to block the development of
Na+-dependent hexose transport in
IPTG-treated cells.
phospholipase C; kidney; proximal tubule; protein kinase C; G
protein
 |
INTRODUCTION |
THE RENAL EPITHELIAL cell line
LLC-PK1 has been used extensively
as a model for investigating the regulation of kidney differentiation in the proximal tubule (2-4, 28, 32, 43). These cells display the
characteristic morphology of a polarized renal epithelium on reaching
confluence (8, 9). They progressively acquire several properties
typical of the brush border of the kidney proximal tubule, such as
expression of Na+-dependent hexose
transport,
-glutamyltranspeptidase, and alkaline phosphatase
activities (7, 20, 43). The development of Na+-dependent hexose transport
activity is accelerated in response to compounds that raise cAMP levels
and is significantly decreased in
LLC-PK1 mutants deficient in
cAMP-dependent protein kinase (3, 4). Phorbol esters, such as
12-O-tetradecanoylphorbol 13-acetate
(TPA), which activate protein kinase C (PKC), can prevent or
drastically delay the expression of several of these differentiation markers, including the
Na+-dependent hexose transporter
(3, 8, 37),
-glutamyltranspeptidase, and alkaline phosphatase (5,
7). The conversion from undifferentiated, actively growing cells to a
culture expressing proximal tubule-specific traits and the ability to
manipulate the expression of these differentiation markers make these
cells particularly useful for studying the regulation of epithelial
cell differentiation. The inhibitory influence of TPA on the expression
of differentiation markers in this cell line has suggested the
participation of PKC in their regulation. However, the effect of
physiological activation of PKC through the stimulation of
phospholipase C (PLC) has not been studied. Members of the
G
q family of G proteins are
known to mediate the activation of phosphoinositide (PI)-specific
PLC
in response to stimulation of a variety of G protein-coupled
receptors (31). PI-PLC
hydrolyzes phosphatidylinositol bisphosphate
(PIP2), generating
diacylglycerol (DAG), and inositol trisphosphate
(IP3). Both of these metabolic
products play critical roles in cell signaling; DAG is known to be a
physiological activator of PKC, and
IP3 stimulates the release of
Ca2+ from intracellular stores
(34). Therefore these pathways may be important in the regulation of
cell differentiation.
The introduction of constitutively active mutants of G protein
-subunits into cells has been used by many laboratories to define
the signaling pathways regulated by a particular G protein without the
necessity of activating the appropriate G protein-coupled receptor
(17). The mutations that generate constitutive activation are in the
GTP binding domain of the
-subunit, resulting in loss of the ability
to hydrolyze GTP. These mutants have been found to constitutively
activate their downstream effectors. In fibroblasts, expression of
constitutively active mutants of members of the G
q family has profound effects,
inducing mitogenesis and transformation in NIH/3T3 cells (10, 11, 18)
and inhibiting the proliferation of Swiss-3T3 cells (29). The present
study represents the first investigation of the regulation of growth
control and differentiation in epithelial cells by a member of the
G
q family.
The GTPase-deficient constitutively active mutant protein
qQ209L (containing a mutation
of Gln-209 to Leu) was stably expressed in
LLC-PK1 cells using the
LacSwitch-inducible system, which allows for the stable integration of
this gene into cells without disturbing their normal pattern of growth
and differentiation. The use of an inducible promoter circumvents the
problem of lethality (29) or possible compensation in cells due to
constitutive expression of GTPase-deficient mutants (22). In the
present study, we show that induction of
qQ209L expression enhances PLC
activity and results in inhibition of cell differentiation in
LLC-PK1 cells. Although
qQ209L expression exerts
effects on cell differentiation similar to treatment with TPA, total
PKC activity is downregulated in TPA-treated cells but not in induced
qQ209L-expressing cells. Therefore the activation of the PLC pathway by its physiological regulator, G
q, may have effects
that are distinct from those observed with TPA.
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MATERIALS AND METHODS |
Cell culture.
The LLC-PK1 cell line was received
from the American Type Culture Collection. This cell line is maintained
in
-MEM supplemented with 10% fetal bovine serum in 5%
CO2-95% air at 37°C. Medium is replenished every 3-4 days. When cells reach confluence,
routine passage is carried out by washing cell monolayers in
Ca2+-,
Mg2+-free PBS (in mM: 137 NaCl,
2.7 KCl, 4.3 Na2HPO4,
and 1.4 KH2PO4, pH 7.4), followed by trypsin digestion [0.25% trypsin and 1 mM EDTA in Hanks' balanced salt solution (HBSS)].
Stable transfection of LLC-PK1 cells.
The LacSwitch-inducible mammalian expression system (Stratagene) was
used. The Lac repressor vector (p3'SS) was transfected into the
LLC-PK1 cells by calcium phosphate
precipitation (16). Cells
(106/10-cm dish) were transfected
with 10 µg of the plasmid DNA. Stable clones were selected for
resistance to 1 mg/ml hygromycin (LC Laboratories) and examined for the
expression of the Lac repressor by indirect immunofluorescence using a
polyclonal antibody to the Lac repressor (Stratagene). A clone that
showed the highest expression of the Lac repressor based on the
brightness of nuclear staining was selected. This clone was stably
transfected with the pOPRSVI operator vector alone or pOPRSVI
containing the cDNA for mutant
qQ209L (a gift from Dr. Gary L. Johnson, National Jewish Medical and Research Center, Denver, CO) using
calcium phosphate precipitation. For the second transfection, 2 × 106 cells were transfected with 1 µg of the specific DNA and 10 µg of pSP72 carrier DNA per 10-cm
dish. G418 (GIBCO BRL) at 1 mg/ml was added into the media to select
for clones containing the lac operator
vector.
Screening of clones by RT-PCR.
To measure the expression of
qQ209L in the presence of
isopropyl-
-D-thiogalactopyranoside
(IPTG, Stratagene), hygromycin- and G418-resistant clones were screened
by RT-PCR. For each clone, 5 mM IPTG was added to a 10-cm dish of cells
for 12 h. Total RNA was isolated from each clone. After reverse
transcription with Moloney murine leukemia virus RT (Boehringer
Mannheim), cDNA was amplified by PCR with
Taq DNA polymerase (Promega) using a
31-base 5'-primer corresponding to nucleotides 739-769 in
the
q cDNA and a 31-base
3'-primer corresponding to the 5'-end of the thymidine kinase poly(A) consensus sequence in pOPRSVI. The predicted size of the
PCR product is 351 bp.
PLC assay.
Cells were plated at a density of 2 × 105/35-mm dish in hygromycin- and
G418-free media and allowed to grow for the indicated times in culture.
One day before measurement, cells were washed with PBS and labeled with
myo-[2-3H]inositol
(Amersham) at 1 µCi/ml for 16-24 h in serum-free
-MEM containing 0.1% BSA at 37°C. After labeling, cells were washed twice with serum-free
-MEM-0.1% BSA and once with serum-free
-MEM-0.1% BSA containing 20 mM LiCl. Cells were then incubated for
30 min in
-MEM-0.1% BSA containing 20 mM LiCl to prevent degradation of inositol phosphates (IPs). Different concentrations of
IPTG were added as described in the text. At the end of the incubation,
cells were fixed in 1 ml of ice-cold acidified methanol (methanol-HCl,
100:1), scraped, and transferred into a tube containing 2 ml of
chloroform, 1 ml of H2O, and 1 ml
of acidified methanol. After mixing and centrifugation, the aqueous
phase containing IPs was passed through an AG1-X8 (200-400 mesh,
formate form, Bio-Rad) anion-exchange column. Total IPs were eluted
with 0.1 M formic acid-1.0 M ammonium formate (6). Aliquots were
assayed for radioactivity by liquid scintillation spectroscopy. Total [3H]inositol
incorporation into lipid was determined by measuring the radioactivity
in the organic phase. The IPs produced were calculated as a percentage
of the total labeled lipids.
Na+-dependent
hexose transport activity.
To measure the activity of the
Na+-dependent hexose transporter,
uptake of the nonmetabolizable glucose analog,
methyl-
-D-glucopyranoside (
-MeG) was performed as described by Amsler and Cook (3). Cells were
plated at a density of 2 × 105/35-mm dish in hygromycin- and
G418-free media. Different concentrations of IPTG were added as
described in the figure legends. Where indicated, cells were also
incubated with 2 µM GF-109203X (GFX), 50 µg/ml of
oleyl-2-acetylglycerol (OAG), or 100 nM A-23187, a
Ca2+ ionophore. At the desired
time points, the medium was aspirated, and cells were incubated in HBSS
without glucose (in mM: 137 NaCl, 5 KCl, 1.3 CaCl2, 0.4 MgSO4 · 7H2O,
0.5 MgCl2 · 6H2O,
0.3 Na2HPO4, 0.3 KH2PO4,
and 20 mM HEPES, pH 7.2) at 37°C for 2 min. The uptake assay was
initiated by adding 1 ml of HBSS containing 100 µM
-[14C]MeG (0.2 µCi/ml;
-[14C]MeG
was from New England Nuclear;
-MeG was from Sigma), and dishes were
incubated at 37°C for 60 min. Uptake was terminated by aspiration
of the solution, and dishes were rinsed three times in ice-cold
Tris-buffered saline [15 mM Tris · HCl (pH
7.4)-0.15 M NaCl]. Cell monolayers were solubilized in 0.2% SDS,
and radioactivity was determined by liquid scintillation spectroscopy.
Protein was measured using a modified Lowry assay (Bio-Rad) at 750 nm,
with BSA as a standard.
Alkaline phosphatase activity.
Alkaline phosphatase activity was measured using a modification of the
method described by Amsler (2). Cells were plated at a density of 2 × 105/35-mm dish in
hygromycin- and G418-free media. IPTG at different final concentrations
(0.005, 0.05, 0.5, and 5 mM) or TPA (0.1 µM) was added the following
day and replaced every other day. At the desired time points, cells
were rinsed three times with ice-cold HBSS and solubilized in the assay
buffer (in mM: 150 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, and 10 Tris · HCl, pH 10) containing 1% Triton X-100.
Approximately 200-300 µg of cell protein per sample were
incubated in the assay buffer containing 10 mM
p-nitrophenyl phosphate (Sigma) for 60 min at 37°C. The reaction was stopped by the addition of 0.2 N
NaOH. Activity was determined by measurement of optical density at
405 nm using
p-nitrophenol (Sigma) as a standard.
Protein was determined from the same dishes using the Bradford assay.
Counting cells.
Cells were plated at a density of 2 × 105/35-mm dish, and IPTG at a
final concentration of 5 mM was added the following day. Cells were
trypsinized on various days indicated in the legend to Fig. 2 and
diluted into
-MEM. Trypan blue was added to a final concentration of
0.04%, and the number of viable cells was determined using a
hemocytometer.
Microscopy.
Cells were plated at the density of 5 × 104/35-mm dish, and IPTG at a
final concentration of 5 mM was added the next day. Morphological changes induced by IPTG were viewed with a Zeiss IM35 phase-contrast light microscope containing a ×16 Zeiss lens (0.35 NA).
Photographs were taken with an Olympus OM-2 camera.
PKC assay.
Cells were plated at the density of 1.4 × 106/10-cm plate in hygromycin- and
G418-free media. IPTG at different final concentrations (0.005, 0.05, 0.5, and 5 mM) or TPA (0.1 µM) was added the following day
(day 0) and replaced every 2 days
throughout the experiments. The kinase assay was performed on
day 7 using the PKC Assay System (GIBCO BRL) according to the manufacturer's protocols. Briefly, total
cellular PKC was extracted and partially purified by DEAE-cellulose ion-exchange column chromatography. Kinase reactions were performed by
measuring the phosphorylation of an acetylated synthetic peptide from
myelin basic protein in the presence of 20 µM
[
-32P]ATP (5 µCi/ml, Amersham) at 30°C for 5 min. TPA at a final concentration of 10 µM was included in the reaction to maximally activate PKC. The
specificity of the reaction for PKC was confirmed using the PKC
pseudosubstrate inhibitor peptide. Aliquots of the reaction mixtures
were spotted onto phosphocellulose disks, washed twice in 1%
phosphoric acid and twice in water. The radioactivity was determined by
liquid scintillation spectroscopy. Proteins were precipitated with 10%
TCA and dissolved in 0.5 M NaOH for quantification using the modified
Lowry assay as described above.
Statistical analysis.
Statistics were calculated using the computer programs Statview and
SuperANOVA from Abacus Concepts.
 |
RESULTS |
Expression of constitutively active
qQ209L increases PLC
activity.
To investigate the effect of G
q
expression on cell differentiation, clones stably expressing the
GTPase-deficient mutant protein,
qQ209L, were established.
Although the expression of mRNA for
qQ209L could be detected by
RT-PCR in cells treated with IPTG, protein could not be detected in
whole cell homogenates (data not shown). This may be due to the
inability of the antibody to detect small increases in protein
expression against a background of endogenous
G
q in these cells. Because
qQ209L is constitutively active, high levels of expression may not be necessary to cause a
significant increase in
G
q activity. PLC activity was
assayed to determine the induction of
qQ209L expression. A time
course (Fig.
1A)
demonstrated a maximal increase in IP accumulation after a 12-h
incubation in the presence of IPTG in one of the clones,
qQ2. No IP accumulation was
observed in cultures incubated in the absence of IPTG. In contrast, a
clone transfected with the vector alone (v1) demonstrated no increase
in IP accumulation in the presence or absence of IPTG. PLC activity
measured after a 12-h incubation with IPTG was selected to screen for
IPTG inducibility in all clones found to express
qQ209L mRNA by RT-PCR. Among
different
qQ209L-expressing
clones, functional inducibility by IPTG varied from no induction to 6- to 7.5-fold induction (data not shown). Two clones,
qQ1 and
qQ2, demonstrated approximately
two- and sixfold increases in IP accumulation, respectively (Fig.
1B). These clones were selected for
further study because, in the absence of IPTG, both of them
demonstrated PLC activity comparable to that of the vector control
clone (v1), suggesting that no expression of
qQ209L occurred in uninduced
cells. In addition, they represent two different levels of
qQ209L activity.

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Fig. 1.
Phospholipase C (PLC) activity in pOPRSVI and
pOPRSVI- qQ209L transfected
LLC-PK1 cells.
A: time course for
isopropyl- -D-thiogalactopyranoside
(IPTG) induction. Cells of v1, a control clone transfected with vector
pOPRSVI ( , ), and qQ2, a
pOPRSVI- qQ209L-transfected
clone ( , ), were plated at 2 × 105 cells/35-mm dish and allowed
to grow for 2 days before being labeled overnight with
myo-[2-3H]inositol
as described in MATERIALS AND METHODS.
IPTG at a final concentration of 5 mM was added to some of dishes for
0, 6, 12, and 24 h during labeling with
myo-[2-3H]inositol.
PLC activity assayed in absence (open symbols) or presence (filled
symbols) of IPTG was measured at each time point as accumulation of
total inositol phosphates (IPs) for 30 min as described in
MATERIALS AND METHODS. IPs produced
were calculated as a percentage of total labeled lipids. Data are
expressed as means ± SD of triplicate measurements.
B: PLC activity in v1 cells and
qQ209L-expressing
qQ1 and
qQ2 cells. Procedures for cell
plating and labeling were the same as those described for
A. Cells were uninduced ( IPTG,
filled bars) or induced (+IPTG, hatched bars) with 5 mM IPTG for 12 h
in presence of
myo-[2-3H]inositol.
IP formation over a 30-min incubation period was measured. Total IPs
produced were calculated as a percentage of total labeled lipids. Bars
represent means ± SD of triplicate measurements.
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qQ209L expression
inhibits
Na+-dependent
hexose transport activity.
Na+-dependent hexose transport
occurs at the apical membrane of proximal tubule cells and is a
biochemical marker for the differentiation of
LLC-PK1 cells (8, 9, 21). To study
the effect of
qQ209L expression
on differentiation in this cell line, the development of
Na+-dependent hexose transport
activity in the absence or presence of IPTG was compared over a 14-day
culture period (Fig.
2A). In the absence of IPTG, both v1 and the
qQ209L-expressing clone,
qQ2, acquired transport
activity progressively over time. Incubation with IPTG blocked the
development of this activity in
qQ2, but not in v1. Transport
activity was also plotted as a function of protein amount, as an
indicator of cell density (Fig. 2B).
In clones v1 (in either absence or presence of IPTG) and
qQ2 (in the absence of IPTG),
the ability to transport
-MeG was apparent when the cells reached
400-500 µg of protein/dish. In contrast, IPTG-treated
qQ2 cells exhibited almost no
acquisition of transport activity even at cell densities far above
those of untreated
qQ2 and v1
cells. Furthermore, the
qQ2
clone was shown to reach higher cell densities in the presence of IPTG
compared with uninduced cells (Fig.
2C).
Na+-dependent hexose transport
activity was also examined in
qQ1, the
G
q-expressing clone that showed
lower inducibility of IP accumulation by IPTG (Fig.
2D). After a 7-day exposure to IPTG, Na+-dependent hexose transport
activity was reduced to a lesser extent than observed in the
qQ2 clone. Because TPA has been
shown to drastically delay the development of
Na+-dependent hexose transport
activity in wild-type LLC-PK1
cells (3), its effect was also examined in the
qQ1 cells. As shown in Fig.
2D, 0.1 µM TPA caused a greater
inhibition of the development of transport activity in
qQ1 compared with IPTG
induction, ruling out the possibility that transport activity is less
sensitive to PKC activation in this clone. These results are consistent with the lower level of IP accumulation when this clone is treated with
IPTG (Fig. 1B). The data suggest
that
qQ209L expression inhibits
the development of Na+-dependent
hexose transport activity and that different levels of expression
induced by IPTG, demonstrated by the differences in IP accumulation in
the two G
q-expressing clones,
result in different extents of inhibition of
Na+-dependent hexose transport
activity.

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Fig. 2.
Na+-dependent hexose transport
activity in v1 and
qQ209L-expressing
LLC-PK1 cells.
A: acquisition of
methyl- -D-glucopyranoside
( -MeG) uptake capacity in v1 cells ( , ) and
qQ209L-expressing
qQ2 cells ( , ). Cells
were plated at 2 × 105/35-mm
dish 1 day before IPTG was added and maintained in absence (open
symbols) or presence (filled symbols) of 5 mM IPTG from
day 0 through day
13. Medium was replenished every other day. -MeG
uptake was measured at 37°C in HBSS containing 100 µM
-[14C]MeG (0.2 µCi/ml) for 60 min. Level of uptake was normalized to the amount of
protein determined from the same dishes. Data are expressed as means ± SD of triplicate determinations in a single experiment and are
representative of 2 independent experiments.
B: analysis of -MeG uptake capacity
in v1 ( , ) and qQ2 ( ,
) cells as a function of protein amount/dish. Data from the time
course shown in A were replotted as a
function of protein level in cultures.
C: comparison of number of cells per
culture for uninduced qQ2 cells
( ) and qQ2 cells induced
with IPTG ( ). Cells were cultured as described in
MATERIALS AND METHODS. IPTG was added
on day 0. Cells were trypsinized on
days 3,
7, and
13 and counted as described in
MATERIALS AND METHODS. Error bars
represent SD of 6 determinations from a single experiment.
D: -MeG uptake capacity in
qQ1 cells. Cells were plated as
described above. On day 0, cells were
either untreated (control) or treated with IPTG (5 mM; +IPTG) or
12-O-tetradecanoylphorbol 13-acetate
(0.1 µM; +TPA). Medium containing these reagents was replenished
every other day up to day 7 when
-MeG uptake was measured as described in MATERIALS
AND METHODS. Bars represent means ± SD of
triplicate measurements.
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On the basis of the results shown in Fig. 2, we tested the hypothesis
that varying IPTG concentrations would induce different levels of
qQ209L expression, resulting in
modulation of the development of
Na+-dependent hexose transport
activity. As shown in Fig.
3A, there is a progressive increase in IP accumulation with increasing
concentrations of IPTG. IP accumulation reached a maximum of
approximately fivefold over basal activity at 2 mM IPTG. The
half-maximal increase was at 0.03 mM IPTG. A corresponding decrease in
Na+-dependent hexose transport
activity to ~50% of maximum at an IPTG concentration of 0.01 mM was
observed (Fig. 3B). At 0.05 mM IPTG,
transport activity declined to ~13% of maximal levels and reached
its lowest level at 2 mM IPTG. These results suggest that cell
differentiation is modulated in an IPTG concentration-dependent manner,
presumably by regulating the level of
qQ209L expression in a manner
directly proportional to the induction of PLC activity.

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Fig. 3.
IPTG-dependent changes in PLC and
Na+-dependent hexose transport
activities in qQ2.
A:
qQ2 cells were plated at a
density of 2 × 105/35-mm dish. IPTG was added the
following day (day 0) at 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, and 5 mM and replaced every other
day throughout experiment. Cells were labeled with
myo-[2-3H]inositol
on day 6, and PLC activity was
measured on day 7 as described in
MATERIALS AND METHODS. Total IPs
accumulated over a 30-min period were calculated as a percentage of
total labeled lipids. Symbols represent means ± SD of triplicate
determinations. B:
qQ2 cells were plated at a
density of 2 × 105/35-mm
dish. IPTG at different final concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, and 5 mM was added the following day
(day 0) and replaced every other
day. -MeG uptake was measured on day
7 over a 60-min incubation period at 37°C and
normalized to protein amount determined from the same dishes, as
described in MATERIALS AND METHODS.
Symbols represent means ± SD of triplicate determinations.
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Expression of
qQ209L
inhibits alkaline phosphatase activity.
To examine whether IPTG-dependent expression of
qQ209L can inhibit other
differentiation markers, the activity of alkaline phosphatase in
qQ2-expressing cells was
assayed. Alkaline phosphatase is another differentiation marker in
LLC-PK1 cells that demonstrates increased expression at the apical surface over time in culture (20,
30, 43). As shown in Fig. 4, alkaline
phosphatase activity was higher on day
7 than on day 1 in
both the control cell line v1 and
qQ2 cells in the absence of
IPTG. TPA treatment reduced acquisition of this differentiated function
in both v1 and
qQ2 cells on
day 7. Similarly, increasing
concentrations of IPTG reduced alkaline phosphatase activity in
qQ2 cells. There was also a gradual decrease in alkaline phosphatase activity with increasing concentrations of IPTG. Therefore regulated expression of
qQ209L also inhibited alkaline
phosphatase activity.

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Fig. 4.
Alkaline phosphatase activity in v1 and
qQ2 cells. Cells were plated at
a density of 2 × 105 in
35-mm dishes and allowed to grow for 1 day. IPTG at different final
concentrations (0.005, 0.05, 0.5, and 5 mM) or TPA (0.1 µM) was added
the following day (day 0). Alkaline
phosphatase activities were measured as described in
MATERIALS AND METHODS. On
day 1 (filled bars), when cells were
still actively growing and subconfluent, alkaline phosphatase activity
was determined from cells treated without or with 5 mM IPTG or with 0.1 µM TPA. On day 7 (hatched bars),
when cells reached confluence and were differentiated, alkaline
phosphatase activity was determined in cells incubated in 0.005, 0.05, 0.5, and 5 mM IPTG. Amounts of
p-nitrophenol produced from ~300
µg of protein in day 1 cultures and
from 200 µg protein in day 7 cultures were measured for 60 min at 37°C. Results were normalized
to amount of protein determined from the same dishes. Data are
expressed as means ± SD of triplicate measurements and are
representative of 2 independent experiments. Ability of IPTG to reduce
the level of alkaline phosphatase activity in
qQ2 cells treated for 7 days was analyzed using a Dunnett ANOVA test. * Concentrations of
IPTG caused a statistically significant reduction
(P < 0.01) in the level of alkaline
phosphatase activity compared with cells without IPTG.
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Morphological changes in
qQ209L-expressing
LLC-PK1 cells.
The morphology of uninduced and induced
qQ2 cells was examined at the
light-microscopic level (Fig. 5). Uninduced
subconfluent cells exhibit the islandlike clusters of closely apposed
cells typical of cultured epithelia, which are indistinguishable from the control v1 clone. In the presence of IPTG, these cells acquire a
spindle-shaped morphology, extending long, spiny processes with ruffled
edges, and appear more loosely associated. In larger islands, uninduced
cells form a monolayer of rounded cells, whereas IPTG-treated cells are
more disorganized and appear to overgrow each other in multicellular
aggregates. Interestingly, Mullin et al. (27) observed that PKC
is
expressed preferentially in
LLC-PK1 cells in areas that are
multilayered. Untreated confluent monolayers of these cells were able
to form domes, whereas IPTG-treated cells did not. Similar
morphological changes were observed in five different clones that
demonstrated at least a twofold induction in IP accumulation by IPTG.
These morphological changes could be observed within 12 h of IPTG
incubation (data not shown).

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Fig. 5.
Morphological changes are induced by IPTG in subconfluent
qQ209L-expressing
LLC-PK1 cells. The
qQ209L-expressing clone,
qQ2, was plated at a density of
5 × 104 cells/35-mm dish and
5 mM IPTG was added the following day. After treatment with (+IPTG) or
without ( IPTG) IPTG for 1 day, the morphology of these cells at
lower (L) and higher (H) densities was recorded as described in
MATERIALS AND METHODS.
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qQ209L expression does
not downregulate PKC activity.
The activation of G
q stimulates
PLC-mediated hydrolysis of PIP2,
leading to the release of DAG and
IP3. Because DAG stimulates PKC,
the effect of IPTG-induced expression of
qQ209L on the levels of PKC was
examined. Prolonged treatment with phorbol esters has been shown to
downregulate PKC, a process attributed to the proteolytic degradation
of the activated enzymes (39). We were therefore interested in the
levels of total PKC activity in cells incubated with IPTG compared with
cells chronically exposed to TPA. As illustrated in Fig.
6, chronic TPA treatment caused
downregulation of PKC activity in both control (v1) and
qQ209L-expressing cells
(
qQ2), as reported previously
for LLC-PK1 cells (26). However,
qQ209L expression induced by
IPTG did not downregulate PKC. The total PKC activity in
qQ2 cells treated with IPTG was
comparable to that of untreated
qQ2 cells and of untreated and
treated v1 cells.

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|
Fig. 6.
Protein kinase C (PKC) activity in v1 and
qQ2 cells. Cells were plated at
a density of 1.4 × 106 in
10-cm plates. IPTG at different final concentrations (0.005, 0.05, 0.5, and 5 mM) or TPA (0.1 µM) was added the following day
(day 0). PKC assays were performed
on day 7 as described in
MATERIALS AND METHODS. PKC activities
were calculated as picomoles of phosphate per minute per milligram of
protein determined from the same dishes and normalized as a percentage
to the level of activity in v1 cells in absence of IPTG in the same
experiment, which was regarded as 100%. Data represent average of 2 independent experiments performed in triplicate. Error bars represent
means ± SE of 6 measurements.
|
|
Effect of PKC activators and inhibitors on
Na+-dependent
hexose transport activity.
The activation of PLC by G
q
results in the generation of DAG and
IP3. Both DAG and
IP3 can stimulate downstream
signaling pathways; DAG activates members of the PKC family of kinases
and IP3 induces the release of
Ca2+ from intracellular stores
(34). To determine the ability of these pathways acting separately or
together to influence the development of
Na+-dependent hexose transport,
qQ2 cells were incubated with
the DAG analog OAG and the Ca2+
ionophore A-23187 (Fig. 7). After a 7-day
incubation, cells treated with OAG showed ~50% decrease in the
development of Na+-dependent
hexose transport, whereas cells treated with A-23187 were not affected,
suggesting that PKC activation, but not increased cytosolic
Ca2+, is important in the
regulation of Na+-dependent hexose
transport. The addition of A-23187 together with OAG had no greater
effect than OAG alone. The role of PKC was further investigated using
the inhibitor GFX. At 2 µM, this inhibitor blocks the morphological
changes observed in these cells (Sun and Weiss, unpublished
observations). However, GFX had no effect on the ability of cells to
develop Na+-dependent hexose
transport activity in the presence or absence of IPTG in either v1 or
qQ2 cells (Fig.
8).

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Fig. 7.
Effect of oleyl-2-acetylglycerol (OAG) and the
Ca2+ ionophore A-23187 on
development of Na+-dependent
hexose transport. qQ2 cells
were plated at a density of 2 × 105/35-mm dish on
day 0 and incubated as described in
MATERIALS AND METHODS. Cells were
refed every day with medium containing 50 µg/ml OAG and every other
day with 100 nM A-23187 and IPTG where indicated. Cultures were assayed
on day 5 for
Na+-dependent hexose transport
activity as described in MATERIALS AND
METHODS and in legend to Fig. 2. Error bars represent
means ± SE of a single experiment representative of 2 independent
experiments.
|
|
 |
DISCUSSION |
We have described for the first time the inducible expression of a
constitutively active mutant of
G
q in a renal epithelial cell
to investigate its participation in the regulation of proximal tubule-specific markers. Our results demonstrate that the expression of
qQ209L inhibits the acquisition
of Na+-dependent hexose transport
and alkaline phosphatase activities, both markers for the kidney
proximal tubule. The inhibition of differentiation was regulated in a
concentration-dependent manner by IPTG, suggesting that the level of
active G
q directly affects the
extent of inhibition. In addition, expression of constitutively active
G
q induced a conversion from an
epithelial to a spindle-shaped morphology. The cells became more
disorganized and reached a higher cell density than those not treated
with IPTG. The morphological changes observed in
qQ209L-expressing cells are
similar to those reported previously on exposure to TPA (3, 26). OAG
was also found to inhibit the development of
Na+-dependent hexose transport
activity, further implicating PKC in this regulatory pathway.
Surprisingly, the PKC inhibitor GFX, which blocks the morphological
changes observed in our cells and is reported to inhibit the
TPA-induced disruption of tight junctions (23), had no effect on the
inhibition of Na+-dependent hexose
transport by IPTG. It has been shown that
LLC-PK1 cells possess the PKC
isoforms
,
,
,
, and
(5, 25). PKC
,
PKC
, and PKC
are most sensitive to GFX, with
IC50 values ranging from 0.01 to
0.02 µM. In contrast, the IC50
values are an order of magnitude higher (0.21 and 0.13 µM,
respectively) for PKC
and PKC
and 5.8 µM for PKC
(24, 36).
Therefore GFX is unlikely to be an effective inhibitor of
,
, and
in our experiments. Because PKC
is not regulated by DAG (12), PKC
and PKC
are the most likely candidates for the regulation of
Na+-dependent hexose transport
activity by
qQ209L in
LLC-PK1 cells.

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Fig. 8.
Development of Na+-dependent
hexose transport activity in cultures treated with GF-109203X (GFX). v1
and qQ2 cells were plated as
described in legend to Fig. 2, and a time course was performed in
presence or absence of 5 mM IPTG and 2 µM GFX. Cells were refed every
other day. Na+-dependent hexose
transport activity was assayed as described in
MATERIALS AND METHODS. ,
, IPTG; , , +IPTG; open symbols, GFX; filled
symbols, +GFX. Error bars represent means ± SE of a
single experiment representative of at least 2 independent
experiments.
|
|
The most significant difference between TPA treatment and expression of
constitutively active
q was on
the levels of cellular PKC. Chronic TPA treatment downregulated PKC
levels in both v1 and
qQ2
cells, consistent with previous reports in both
LLC-PK1 and other cell types (13,
26). In contrast, long-term treatment of
qQ2 cells with IPTG did not
significantly alter PKC levels, suggesting that physiological
activation of PLC by
qQ209L
expression does not result in downregulation of PKC. It should be noted
that our experiments do not measure the activity of PKC in TPA-treated or G
q-expressing cells.
Designing an assay that preserves and measures actual PKC activity in a
cell under a given set of experimental conditions is difficult, since
breaking open the cells changes compartmentalization,
Ca2+ levels, and probably the
state of activation of PKC. The reason for the difference in PKC levels
observed for TPA treatment and IPTG induction is not clear but may be
due to differences in the metabolism of DAG and TPA. DAG, the
endogenous activator, is rapidly metabolized and appears to cause only
transient activation of PKC. On the other hand, TPA is degraded very
slowly in cells, causing persistent activation of PKC (39) and
demonstrating greater potency than DAG in inducing the differentiation
of macrophages (1, 40). Incubation with TPA has generally been shown to cause the complete loss of PKC, whereas this has been reported not to
occur with DAG in the monoblastoid cell line U937 (19). In previous
studies, phorbol ester and DAG displayed differences in phosphorylation
substrates, the activation of specific enhancer elements, and the
stimulation of cell differentiation (35, 38, 40). These data suggest
that the downregulation of PKC observed in TPA-treated cells may not be
a critical step in preventing the expression of proximal
tubule-specific traits. Interestingly, TPA was previously shown to
reduce the expression of PKC
but enhanced the expression of PKC
and had no effect on PKC
(5). Therefore the levels of PKCs whose
activities are known to be regulated by TPA in vitro may be
differentially regulated in vivo. This may be due in part to
compartmentalization of PKC isoforms within the cell as well as
compartmentalization of the various factors that influence the levels
of expression of these proteins.
The mechanism for the progressive, concentration-dependent inhibition
of differentiation by G
q is
unknown. The expression of
Na+-dependent hexose transport
activity that begins at confluence has been correlated with an increase
in the synthesis of its mRNA and protein (41, 42). A 1-h treatment with
TPA induces a rapid loss of transporter mRNA in postconfluent,
differentiated LLC-PK1 cells, a
phenomenon attributed to posttranscriptional degradation of the mRNA
(33). These data are consistent with the observation that activation of
PKC by phorbol esters or DAG analogs inhibits Na+-dependent hexose transport
activity in renal proximal tubular primary cell culture through a
decrease in maximal enzyme reaction rate (Vmax)
(14), suggesting a change in the level rather than the activity of this
protein. Phorbol esters can also block the expression of
tissue-specific markers in the intestinal epithelial cell line, HT-29
(15). Therefore PKC is implicated in regulating the formation of the
differentiated phenotype in both kidney and intestinal epithelia.
The expression of
qQ209L has
been found to cause transformation in fibroblasts in a cell
type-specific manner (10, 18). The expression of constitutively active
mutants of the G
q family was
either growth stimulatory or inhibitory in different fibroblasts (11,
29). With the inducible expression of
qQ209L, we were able to show a
growth-stimulatory effect, indicated by an increase in the amount of
protein per dish and in the number of cells, in these epithelial cells.
Taken together with the data for cell differentiation, inducible
expression of constitutively active
qQ209L resulted in stimulation
of growth and inhibition of differentiation in
LLC-PK1 epithelial cells. Future
studies will include transformation assays to test whether these cells
are transformed by the expression of
qQ209L. The mechanism whereby
G
q-mediated pathways affect cell growth and differentiation remains to be elucidated. PKC phosphorylates a number of cellular substrates that could lead to
changes in expression of proximal tubule markers in the
LLC-PK1 cell line. Investigating
the downstream events regulated by PKC in this cell line is in progress
in the laboratory.
In summary, we have successfully expressed
qQ209L under the control of an
inducible expression system in
LLC-PK1 kidney epithelial cells.
In an IPTG concentration-dependent manner, the expression of
constitutively active G
q
inhibited differentiation of these cells, as indicated by measurement
of both Na+-dependent hexose
transport and alkaline phosphatase activity, and caused changes in the
epithelial morphology of these cells. From morphological changes
occurring in the
qQ209L-expressing cells,
cell-cell contacts seem to be altered in cells treated with IPTG (Sun
and Weiss, unpublished observations). The inhibition of differentiation
resulting from
qQ209L
expression could be due to impaired cell-cell contacts induced by IPTG.
Changes in cell-cell contacts, such as the adherens junctions, between
these epithelial cells are currently under investigation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Gary L. Johnson for the gift of the
qQ209L cDNA, Dr. Benjamin Peng
for assistance with microscopy, and Dr. Shoji Osawa for review of the
manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of General Medical
Sciences Grant GM-43582 and a grant from the University Research Council, The University of North Carolina at Chapel Hill, and was
performed during the tenure of E. R. Weiss as an Established Investigator of the American Heart Association.
Present address of L. Sun: Dept. of Neurology, San Francisco General
Hospital, University of California, San Francisco, San Francisco, CA
94110.
Address for reprint requests: E. R. Weiss, Dept. of Cell Biology and
Anatomy, CB7090, 108 Taylor Hall, The University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599-7090.
Received 28 October 1996; accepted in final form 22 November 1997.
 |
REFERENCES |
1.
Aihara, H.,
Y. Asaoka,
K. Yoshida,
and
Y. Nishizuka.
Sustained activation of protein kinase C is essential to HL-60 cell differentiation to macrophage.
Proc. Natl. Acad. Sci. USA
88:
11062-11066,
1991[Abstract].
2.
Amsler, K.
Role of cell density/cell-cell contact and growth state in expression of differentiated properties by the LLC-PK1 cell.
J. Cell. Physiol.
159:
331-339,
1994[Medline].
3.
Amsler, K.,
and
J. S. Cook.
Development of Na+-dependent hexose transport in a cultured line of porcine kidney cells.
Am. J. Physiol.
242 (Cell Physiol. 11):
C94-C101,
1982[Abstract/Free Full Text].
4.
Amsler, K.,
S. Ghatani,
and
B. A. Hemmings.
cAMP-dependent protein kinase regulates renal epithelial cell properties.
Am. J. Physiol.
260 (Cell Physiol. 29):
C1290-C1299,
1991[Abstract/Free Full Text].
5.
Amsler, K.,
J. Murray,
R. Cruz,
and
J.-L. Chen.
Chronic TPA treatment inhibits expression of proximal tubule-specific properties by LLC-PK1 cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C322-C340,
1996.
6.
Berridge, M. J.,
R. M. C. Dawson,
C. P. Downes,
J. P. Heslop,
and
R. F. Irvine.
Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.
Biochem. J.
212:
473-482,
1983[Medline].
7.
Chen, J. L.,
and
K. Amsler.
Expression of
-glutamyl transpeptidase by renal epithelial cells occurs on a cell-by-cell basis and is inhibited by chronic TPA treatment.
J. Cell. Biochem.
58:
73-82,
1995[Medline].
8.
Cook, J. S.,
K. Amsler,
E. R. Weiss,
and
C. Shaffer.
Development of Na+-dependent hexose transport in vitro.
In: Membranes in Growth and Development. New York: Liss, 1982, p. 551-567.
9.
Cook, J. S.,
and
E. R. Weiss.
Cell kinetics of differentiation of Na+-dependent hexose transport in a cultured renal epithelial cell line.
In: INSERM Symposium on Brush Border Membrane and Sodium-Coupled Transport, edited by F. Alvarado,
and C. H. van Os. Amsterdam: Elsevier, 1986, p. 335-343.
10.
De Vivo, M.,
J. Chen,
J. Codina,
and
R. Iyengar.
Enhanced phopholipase C stimulation and transformation in NIH-3T3 cells expressing Q209LGq-
-subunits.
J. Biol. Chem.
267:
18263-18266,
1992[Abstract/Free Full Text].
11.
De Vivo, M.,
and
R. Iyengar.
Activated Gq-
potentiates platelet-derived growth factor-stimulated mitogenesis in confluent cell cultures.
J. Biol. Chem.
269:
19671-19674,
1994[Abstract/Free Full Text].
12.
Dekker, L. V.,
and
P. J. Parker.
Protein kinase C: a question of specificity.
Trends Biochem. Sci.
19:
73-77,
1994[Medline].
13.
Dong, L.,
J. L. Stevens,
D. Fabbro,
and
S. Jaken.
Protein kinase C isozyme expression and down-modulation in growing, quiescent, and transformed renal proximal tubule epithelial cells.
Cell Growth Differ.
5:
881-890,
1994[Abstract].
14.
Friedlander, G.,
and
C. Amiel.
Protein kinase C activation has dissimilar effects on sodium-coupled uptakes in renal proximal tubular cells in primary culture.
J. Biol. Chem.
264:
3935-3941,
1989[Abstract/Free Full Text].
15.
Garcia de Herreros, A.,
M. Fabre,
E. Batlle,
C. Balagué,
and
F. X. Real.
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate blocks differentiation of HT-29 human colon cancer cells.
J. Cell Sci.
105:
1165-1172,
1993[Abstract/Free Full Text].
16.
Graham, F. L.,
and
A. J. van der Eb.
A new technique for the assay of infectivity of human adenovirus 5 DNA.
Virology
52:
456-467,
1973[Medline].
17.
Gupta, S. K.,
C. Gallego,
and
G. L. Johnson.
Mitogenic pathways regulated by G protein oncogenes.
Mol. Biol. Cell
3:
123-128,
1992[Medline].
18.
Kalinec, G.,
A. J. Nazarali,
S. Hermouet,
N. Xu,
and
J. S. Gutkind.
Mutated
subunit of the Gq protein induces malignant transformation in NIH 3T3 cells.
Mol. Cell. Biol.
12:
4687-4693,
1992[Abstract].
19.
Kraft, A. S.,
V. V. Baker,
and
W. S. May.
Bryostatin induces changes in protein kinase C location and activity without altering c-myc gene expression in human promyelocytic leukemia cells (HL-60).
Oncogene
1:
111-118,
1987[Medline].
20.
Lasheras, C.,
J. A. Scott,
and
C. A. Rabito.
Na+-sugar cotransport system as a polarization marker during organization of epithelial membrane.
Am. J. Physiol.
255 (Cell Physiol. 24):
C745-C753,
1988[Abstract/Free Full Text].
21.
Lever, J. E.
Chemical inducers of differentiation in a long-term renal cell line.
Environ. Health Perspect.
80:
173-180,
1989[Medline].
22.
Lowndes, J. M.,
S. K. Gupta,
S. Osawa,
and
G. L. Johnson.
GTPase-deficient G
i2 oncogene gip2 inhibits adenylylcyclase and attenuates receptor-stimulated phospholipase A2 activity.
J. Biol. Chem.
266:
14193-14197,
1991[Abstract/Free Full Text].
23.
Marano, C. W.,
K. V. Laughlin,
L. M. Russo,
and
J. M. Mullin.
The protein kinase C inhibitor, bisindolylmaleimide, inhibits the TPA-induced but not the TNF-induced increase in LLC-PK1 transepithelial permeability.
Biochem. Biophys. Res. Commun.
209:
669-676,
1995[Medline].
24.
Martiny-Baron, G.,
M. G. Kazanietz,
H. Mischak,
P. M. Blumberg,
G. Kochs,
H. Hug,
D. Marme,
and
C. Schachtele.
Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976.
J. Biol. Chem.
268:
9194-9197,
1993[Abstract/Free Full Text].
25.
Middleton, J. P.,
W. A. Khan,
G. Collinsworth,
Y. A. Hannun,
and
R. M. Medford.
Heterogeneity of protein kinase C-mediated rapid regulation of Na/K-ATPase in kidney epithelial cells.
J. Biol. Chem.
268:
15958-15964,
1993[Abstract/Free Full Text].
26.
Mullin, J. M.,
K. V. Snock,
R. D. Shurina,
J. Noe,
K. George,
L. Misner,
S. Imaizumi,
and
T. G. O'Brien.
Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology.
J. Cell. Physiol.
152:
35-47,
1992[Medline].
27.
Mullin, J. M.,
A. P. Soler,
K. V. Laughlin,
J. A. Kampherstein,
L. M. Russo,
D. T. Saladik,
K. George,
R. D. Shurina,
and
T. G. O'Brien.
Chronic exposure of LLC-PK1 epithelia to the phorbol ester TPA produces polyp-like foci with leaky tight junctions and altered protein kinase C-
expression and localization.
Exp. Cell Res.
227:
12-22,
1996[Medline].
28.
Mullin, J. M.,
J. Weibel,
L. Diamond,
and
A. Kleinzeller.
Sugar transport in the LLC-PK1 renal epithelial cell line: similarity to mammalian kidney and the influence of cell density.
J. Cell. Physiol.
104:
375-389,
1980[Medline].
29.
Qian, N.-X.,
M. Russell,
A. M. Buhl,
and
G. L. Johnson.
Expression of GTPase-deficient G
16 inhibits Swiss 3T3 cell growth.
J. Biol. Chem.
269:
17417-17423,
1994[Abstract/Free Full Text].
30.
Rabito, C. A.,
J. I. Kreisberg,
and
D. Wight.
Alkaline phosphatase and
-glutamyl transpeptidase as polarization markers during the organization of LLC-PK1 cells into an epithelial membrane.
J. Biol. Chem.
259:
574-582,
1984[Abstract/Free Full Text].
31.
Rhee, S. G.,
and
Y. S. Bae.
Regulation of phosphoinositide-specific phospholipase C isozymes.
J. Biol. Chem.
272:
15045-15048,
1997[Free Full Text].
32.
Sepulveda, F. V.,
K. A. Burton,
and
J. D. Pearson.
The development of
-glutamyltransferase in a pig renal-epithelial-cell line in vitro.
Biochem. J.
208:
509-512,
1982[Medline].
33.
Shioda, T.,
T. Ohta,
K. J. Isselbacher,
and
D. B. Rhoads.
Differentiation-dependent expression of the Na+/glucose cotransporter (SGLT1) in LLC-PK1 cells: role of protein kinase C activation and ongoing transcription.
Proc. Natl. Acad. Sci. USA
91:
11919-11923,
1994[Abstract/Free Full Text].
34.
Sternweis, P. C.,
and
A. V. Smrcka.
Regulation of phospholipase C by G proteins.
Trends Biochem. Sci.
17:
502-506,
1992[Medline].
35.
Strulovici, B.,
S. Daniel-Issakani,
E. Oto,
J. Nestor,
H. Chan,
and
A. P. Tsou.
Activation of distinct protein kinase C isozymes by phorbol esters: Correlation with induction of interleukin 1
gene expression.
Biochemistry
28:
3569-3576,
1989[Medline].
36.
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakane,
V. Baudet,
P. Boissin,
E. Boursier,
F. Loriolle,
L. Duhamel,
D. Charon,
and
J. Kirilovsky.
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781,
1991[Abstract/Free Full Text].
37.
Van Den Bosch, L.,
H. De Smedt,
and
R. Borghgraef.
Influence of PMA and a low extracellular Ca2+ concentration on the development of the Na+-dependent hexose carrier in LLC-PK1 cells.
Biochim. Biophys. Acta
1092:
244-250,
1991[Medline].
38.
Ways, D. K.,
R. C. Dodd,
and
H. S. Earp.
Dissimilar effects of phorbol ester and diacylglycerol derivative on protein kinase activity in the monoblastoid U937 cell.
Cancer Res.
47:
3344-3350,
1987[Abstract].
39.
Wilkinson, S. E.,
and
T. J. Hallam.
Protein kinase C: is its pivotal role in cellular activation over-stated.
Trends Pharmacol. Sci.
15:
53-57,
1994[Medline].
40.
William, F.,
F. Wagner,
M. Karin,
and
A. S. Kraft.
Multiple dose of diacylglycerol and calcium inonophore are necessary to activate AP-1 enhancer activity and induce markers of macrophage differentiation.
J. Biol. Chem.
265:
18166-18171,
1990[Abstract/Free Full Text].
41.
Wu, J. R.,
and
J. E. Lever.
Developmentally regulated 75-kilodalton protein expressed in LLC-PK1 cultures is a component of the renal Na+/glucose cotransport system.
J. Cell. Biochem.
40:
83-89,
1989[Medline].
42.
Yet, S.-F.,
C.-T. Kong,
H. Peng,
and
J. E. Lever.
Regulation of Na+/glucose cotransporter (SGLT1) mRNA in LLC-PK1 cells.
J. Cell. Physiol.
158:
506-512,
1994[Medline].
43.
Yoneyama, Y.,
and
J. E. Lever.
Induction of microvillar hydrolase activities by cell density and exogenous differentiation inducers in an established kidney epithelial cell line (LLC-PK1).
J. Cell. Physiol.
121:
64-73,
1984[Medline].
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