1 Department of Medicine, Phorbol esters
increase glucose (Glc) uptake and utilization in a variety of cell
types, and, in some cells, these changes have been attributed to
increased Glc phosphorylation and better functional coupling of
hexokinases (HKs) to facilitative Glc transporters. Phorbol esters are
potent mesangial cell mitogens, but their effects on HK-catalyzed Glc
phosphorylation and metabolism are unknown. When examined in murine
mesangial cells, active, but not inactive, phorbol esters increased HK
activity in a time- and dose-dependent manner. Maximal induction of HK
activity at 12-24 h was accompanied by parallel increases in both
Glc utilization and lactate production and was blocked by the specific
MEK1/2 inhibitor PD-98059 (IC50 ~3 µM). This effect involved early activation of protein kinase C
(PKC), MEK1/2, and ERK1/2, and the prolonged time course of subsequent
HK induction was attributable, in part, to requirements for ongoing
gene transcription and de novo protein synthesis. Mesangial cell HK
activity thus exhibits novel regulatory behavior involving both PKC and
classic MAPK pathway activation, suggesting specific mechanisms whereby
PKC activation may influence Glc metabolism.
glomerular mesangial cells; hexokinase; protein kinase C; MEK1/2; ERK1/2; mitogen-activated protein kinase
GLUCOSE (Glc) uptake and metabolism are of fundamental
importance to all mammalian cells, and hexokinases (HKs) play a
central role in these processes by catalyzing the
phosphorylation of Glc to yield Glc 6-phosphate
(Glc-6-P). By this mechanism, HKs
maintain the favorable downhill concentration gradient that permits
facilitated Glc entry into cells. In addition, they initiate all
subsequent pathways of Glc utilization, including the glycolytic
pathway, the pentose phosphate pathway, and the uronic acid pathway.
Thus primary changes in total HK activity can, in principle, have
profound effects on Glc uptake and utilization, as well as on the
ultimate metabolic fate(s) of Glc.
Total HK activity in the kidney increases throughout development (6,
21, 29) and declines slightly after weaning to adult levels (21, 33).
In the adult kidney, total HK activity increases along the axial
nephron, with the highest specific activities observed distally,
particularly in the thick ascending limb of Henle's loop (23). Thus
the specific activities observed in the adult renal medulla typically
exceed those observed in the cortex (3, 6, 32, 33). HK activity in the
adult kidney is specifically increased in genetically obese mice (27),
in experimental diabetes (2, 24-28), in compensatory unilateral renal hypertrophy (28), and in puromycin aminonucleoside-induced nephrosis (10). Increases in cortical activity have been described in
experimental diabetes, and increased glomerular activity has been
demonstrated both during development and in the setting of experimental
nephrosis. However, the contributions of individual cell types and the
molecular mechanisms underlying these changes have been largely
unexplored. Thus a description of the regulatory characteristics of HK
activity in an individual renal cell type such as the glomerular
mesangial cell should be of great interest as well as great
physiological, developmental, and pathophysiological relevance.
Phorbol esters act as diacylglycerol mimetics to activate protein
kinase C (PKC) and have been shown to increase Glc uptake by a variety
of cell types (7, 9, 13). In the case of thymocytes, they appear to do
so primarily via increased Glc phosphorylation and better functional
coupling of HKs to transport (17). The mitogenic capacity of phorbol
esters has also been linked to their ability to stimulate Glc uptake
(7). Although these compounds are known mitogens for mesangial cells
(11), their effects on Glc metabolism, and on Glc phosphorylation in
particular, in this cell type have been largely unexplored. In a
preliminary report published over a decade ago, Kreisberg et al. (12)
reported both contraction and increased glycolysis by cultured
mesangial cells treated with phorbol 12-myristate 13-acetate (PMA).
These authors speculated that increased Glc metabolism could provide the requisite energy necessary for phorbol ester-stimulated contraction by these cells. Although the effect of phorbol esters on mesangial cell
contraction has subsequently been characterized in greater detail (31),
the molecular mechanisms underlying the associated increase in Glc
metabolism have not yet been reported. Glc uptake and metabolism by
these cells have been extensively investigated, but little is known
about the relative contributions of HKs to these processes, and the
regulation of Glc phosphorylating capacity in this cell type has not
been addressed. Thus, to better understand this important family of
enzymes in the kidney, we sought to characterize the general regulatory
behavior of HK activity in SV40 MES 13 (murine mesangial) cells, which
exhibit morphological and biochemical characteristics of normal
mesangial cells in culture (16). More specifically, we sought to
characterize the effects of phorbol esters on HK activity and to
directly test the hypothesis that phorbol esters could affect mesangial
cell Glc metabolism via an effect on total Glc phosphorylating capacity.
Materials.
Dichloro-1- Cell culture. Mycoplasma-free SV40 MES
13 (murine mesangial) cells were obtained from the American Type
Culture Collection (Rockville, MD) at passage 27 and were routinely
maintained in DME-F12 (3:1) medium containing 6 mM Glc and supplemented
with both 5% fetal bovine serum and 14 mM HEPES. Cell monolayers were routinely grown to confluence at 37°C in 5%
CO2 before testing, and all
experiments were performed between passages 30 and 40 to minimize the
effects of phenotypic variation in continuous culture. Cultured rat
mesangial cells prepared from glomerular explants of male
Sprague-Dawley rats (kindly provided by Ashok Singh of the Hektoen
Institute, Chicago, IL) were also tested in parallel
during preliminary experiments. The preparation, characterization, and
maintenance of these cells has been described previously (14). When
inhibitors were employed, cells were routinely preincubated with
inhibitor alone for 30 min prior to the addition of phorbol esters.
Cell lysate preparation. Cell lysates
for HK activity assays were routinely prepared by a modification of the
methods of O'Doherty et al. (19) and Braithwaite et al. (5). In brief,
cells harvested in cold homogenization buffer (900 mM KCl, 20 mM
MgCl2, 10 mM EDTA, 11.1 mM
monothioglycerol, 0.25% Triton X-100, 10 mM Glc, and 20 mM
Tris · HCl, pH 8.1) were sonicated briefly
(30-60 J at 4°C) with a sonic Dismembrator (model
60; Fisher Scientific, Pittsburgh, PA) before centrifugation at 11,900 g and at 4°C for 10 min. Aliquots
of the resulting supernatants were then assayed for total HK activity
(see below). Cell lysates for PKC activity assays were similarly
prepared in 5 mM EDTA, 10 mM EGTA, 0.3% (vol/vol) Hexokinase assays. Total HK activity
was measured as the Glc-phosphorylating capacity of whole cell extracts
using a standard G6PDH-coupled assay (5, 34). In brief, the Glc- and
ATP-dependent reduction of Protein kinase C assays. PKC
activation was evaluated in cell extracts using a commercially
available protein kinase assay kit according to the manufacturer's
recommendations (Calbiochem). In brief, cell extracts corresponding to
the particulate and cytosolic cell fractions of whole cell lysates were
evaluated in parallel for the ability to specifically phosphorylate an
immobilized PKC pseudosubstrate. Specific
phosphotransferase activity was estimated by an
enzyme-linked immunosorbent assay employing a monoclonal antibody
specific for the phosphorylated form of the peptide substrate.
Glc utilization and lactate production
assays. Glc utilization and lactate production were
assayed as net Glc disappearance and net lactate accumulation in the
culture medium, respectively. For these experiments, cells were
routinely tested in defined growth medium containing 6 mM Glc and
lacking phenol red. At appropriate time points, medium aliquots were
assayed spectrophotometrically for both Glc and lactate content via
standard enzymatic coupled reactions using commercially available kits
(Sigma Diagnostics, St. Louis, MO). Glc was assayed by a modification
of the Trinder reaction (30), where chromagen formation coupled to the
oxidation of Glc by glucose oxidase was monitored
spectrophotometrically. The lactate assay was based on similar
chromagen formation coupled to lactate oxidation by lactate oxidase.
Since each method was dependent upon the formation of peroxide
intermediates, major findings were confirmed using commercially
available enzymatic assays based on the coupled reduction of NAD to
NADH by lactate dehydrogenase and G6PDH, respectively
(Sigma Diagnostics). Identical results in each case suggested that
endogenous
H2O2
did not contribute to either measure. Serial measures of medium Glc and
lactate content were uniformly performed in the presence of nonlimiting
concentrations of Glc and under conditions of linear net Glc
utilization and lactate accumulation.
Analysis of ERK1/2 phosphorylation and
activity. Specific extracellular signal-regulated
kinase 1 and 2 (ERK1/2) phosphorylation was assessed in cell lysates by
quantitative immunoblot analysis as described previously (8). In brief,
rabbit polyclonal IgGs specific for ERK1/2 were used to detect and
quantitate total ERK1/2 content in SV40 MES 13 cell lysates independent
of phosphorylation status. In parallel, phospho-specific rabbit
polyclonal IgGs directed against the characteristic dual-phosphorylated
tripeptide motifs of activated ERK1/2 were used to assess
phospho-ERK1/2 content. Following electrophoretic separation by
SDS-PAGE and transfer to nitrocellulose, cell lysates were probed with
primary antibodies at a 1:1,000 dilution. Specific protein bands were
then visualized by chemiluminescent detection of horseradish
peroxidase-conjugated anti-rabbit secondary antibodies using the
Phototope-HRP Western Detection System (New England BioLabs) per the
manufacturer's recommendations. Specific ERK1/2 phosphotransferase
activity was also assayed by an IP/kinase activity assay using a
commercially available kit (New England BioLabs) according to the
manufacturer's recommendations. In brief, activated ERK1/2
immunoprecipitates were prepared from cell lysates using immobilized
phospho-specific ERK1/2 monoclonal antibodies directed against
dual-phosphorylated (i.e., activated) ERK1/2. These immunoprecipitates
were then assayed for the ability to specifically Ser-phosphorylate
an Elk-1 fusion protein in vitro. Quantitative
immunoblotting was performed using rabbit polyclonal IgG specific for
phospho-Elk-1 and chemiluminescent detection as described above.
Control IP/kinase assays were routinely performed in parallel using
unstimulated cell lysates with and without the addition of functional
MEK-activated recombinant ERK2. Quantitative comparisons were made by
scanning transmission densitometry of the resulting autoradiograms
using an Eagle-Eye II still videoimaging system (Stratagene, La Jolla,
CA). Analysis of digital images was routinely performed using NIH Image
v1.61 software for Macintosh computers (NIH, Bethesda, MD).
MEK1/2 activity assays.
Mitogen/extracellular signal-regulated kinase kinase 1 and 2 (MEK1/2)
phosphotransferase activity was assayed by an IP/kinase activity assay
similar to that employed to measure ERK1/2 kinase activity. In brief,
rabbit polyclonal IgGs specific for phospho-MEK1/2 (New England
BioLabs) and protein A-agarose beads (Santa Cruz
Biotechnology, Santa Cruz, CA) were used to selectively
immunoprecipitate activated MEK1/2 from cell lysates. The resulting
immunoprecipitates were then assayed for the ability to specifically
phosphorylate inactive recombinant murine ERK2 (New England BioLabs) in
vitro. Phospho-ERK2 detection and quantitation was accomplished by
immunoblotting with mouse monoclonal IgG specific for
dual-phosphorylated ERK1/2 followed by chemiluminescent detection and
analysis as described above.
Statistical analysis. Unless otherwise
noted, data are presented as experimental means ± SE for at least
three independent measures. Statistical comparisons were performed by
paired t-testing where appropriate
using a significance level of 95% and StatView 5.0 software for
Macintosh computers (SAS Institute, Cary, NC).
SV40 MES 13 cells exhibited morphological and
biochemical characteristics of normal mesangial cells in
culture. SV40 MES 13 cells, originally derived from
glomerular explants of mice transgenic for the SV40 early region, have
been shown to exhibit morphological and biochemical characteristics of
normal mesangial cells in culture (16). As previously reported, we
found that these cells stained for SV40 large T antigen, exhibited the
typical stellate morphology of mesangial cells in culture, and, unlike
fibroblasts which lack D-amino
acid oxidase, were capable of normal growth following equimolar
substitution of D-valine for its
natural L-stereoisomer in normal
growth medium. In addition, they uniformly expressed mesangial cell
markers such as the intermediate filaments vimentin and desmin, but
failed to express detectable levels of epithelial cell markers such as
cytokeratins by immunoblot analysis (data not shown). In preliminary
experiments, we observed similar levels of basal HK activity in both
SV40 MES 13 cells and cultured rat mesangial cells maintained in normal
growth medium (27 ± 3 vs. 26 ± 1 U/g protein; not significant).
Serum deprivation of SV40 MES 13 cells for 48 h decreased basal HK
activity slightly but significantly by ~20% (28 ± 3 vs. 22 ± 3 U/g protein; P < 0.001), and
similar results were observed for cultured rat mesangial cells. Since,
in preliminary studies, phorbol esters increased total HK activity
similarly in both cell types (22), we sought to further characterize
this response in SV40 MES 13 cells.
Active, but not inactive, phorbol esters induced total
HK activity in a time- and dose-dependent manner. As
shown in Fig. 1A,
exposure to 1 µM PMA increased total HK activity in SV40 MES 13 cells
by over 20% within 5 h (P = 0.07),
and a significant increase of almost 40% was observed within 12 h
(P < 0.002). Maximal activity was
observed within 24 h and was over 50% greater than in unstimulated
controls (P < 0.02). Thereafter,
total HK activity began to decrease but was still over 40% higher than
controls at 36 h. In Fig. 1B, the dose
dependence of PMA-induced HK activity at 24 h in these cells can also
be appreciated. PMA significantly increased total HK activity at
concentrations as low as 10 nM (P < 0.05), and maximal activation was observed within 24 h of exposure to
PMA concentrations
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-ribofuranosylbenzimidazole
(DRB) was obtained from Fluka (Milwaukee, WI), PD-98059 was from
Calbiochem (La Jolla, CA), and Leuconostoc
mesenteroides Glc-6-P
dehydrogenase (G6PDH) was from Boehringer-Mannheim (Indianapolis, IN).
Electrophoretic and immunoblotting reagents were routinely obtained
from Bio-Rad (Hercules, CA), and all cell culture reagents were
supplied by Life Technologies (Gaithersburg, MD). All
antibodies, substrates, and the chemiluminescent detection system
employed for both immunoblot analysis and immunoprecipitation/kinase
(IP/kinase) activity assays were obtained from New England BioLabs
(Beverly, MA). All other chemicals, including PMA,
phorbol-12,13-didecanoate (PDD), 4
-phorbol-12,13-didecanoate (4
-PDD),
-NADP, ATP, cycloheximide, and actinomycin D, were obtained from Sigma (St. Louis, MO) unless otherwise noted and were the
finest quality available.
-mercaptoethanol,
1 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, and 20 mM
Tris · HCl, pH 7.5. The particulate fraction was
routinely separated from the soluble, or cytosolic, fraction by
differential centrifugation (100,000 g
for 1 h at 4°C). After solubilization in lysis buffer supplemented
with 1% (vol/vol) Triton X-100, cell extracts corresponding to the
particulate fraction were diluted to yield final Triton X-100
concentrations <0.02% (vol/vol) in PKC phosphotransferase assays,
which were routinely performed in parallel with corresponding cytosolic
extracts (see below). Cell lysates for both immunoblot analysis and
other protein kinase activity assays were routinely prepared in 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM
Na3VO4,
1 g/ml leupeptin, and 20 mM Tris · HCl, pH 7.5. Cells
were typically washed once with ice-cold phosphate-buffered saline, pH ~ 7.2, before harvesting in cold lysis buffer. After brief sonication
(30-60 J at 4°C), samples were centrifuged at
11,900 g and at 4°C for 10 min,
and the resulting supernatants were used for direct immunoblot analysis or for immunoprecipitation of protein kinases prior to kinase activity
assays. Protein content in cell lysates was routinely evaluated by the
Bradford method (4) using bovine
-globulin (Bio-Rad) as a reference standard.
-NADP was monitored
spectrophotometrically at 340 nm in the presence of excess G6PDH. All
assays (final assay mixture composition: 1 U/ml G6PDH, 0.5 mg/ml
-NADP, 6.7 mM ATP, 7.7 mM MgCl2, 3.8 mM Glc, 45 mM KCl,
1 mM NaH2PO4, 10.6 mM monothioglycerol, 0.01%
Triton X-100, 0.5 mM EDTA, and 42 mM Tris · HCl, pH
8.5) were performed at 25°C under conditions of linear HK-limited
NADPH formation (34). Total HK activity was routinely normalized for cellular protein content and was expressed in enzyme activity units
corresponding to the Glc phosphorylation rate in micromoles per
minute. Where appropriate, changes in activity relative to paired unstimulated controls were calculated to facilitate direct comparisons.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
100 nM. To ensure that induction of HK activity by
phorbol esters was specific for PKC activation, we also tested both PDD
and its inactive 4
-analog, 4
-PDD, for the ability to induce total
HK activity. As shown in Fig. 2, the active
phorbol ester PDD increased HK activity at 24 h in a dose-dependent fashion and mimicked the effects of PMA. At concentrations
10 nM, PDD
significantly increased HK activity by ~60%
(P < 0.05 vs. unstimulated control
cells). In contrast, 4
-PDD, which is incapable of activating PKC
(1), or vehicle alone (DMSO) failed to alter total HK activity when
tested in parallel. Consistent with these observations, the ability of
particulate cell extracts to specifically phosphorylate a PKC
pseudosubstrate increased over 10-fold within 1 min of 1 µM PMA
stimulation, an effect that persisted for at least 30 min. In contrast,
the phosphotransferase activities of corresponding soluble (cytosolic)
extracts decreased as much as ~86% within this time period,
suggesting activation and membrane translocation of PKC. At 24 h, both
particulate and cytosolic activities were decreased by ~72% and
~97%, respectively, consistent with downregulation of PKC activity
in response to prolonged phorbol ester exposure.
View larger version (16K):
[in a new window]
Fig. 1.
Phorbol ester induction of mesangial cell hexokinase (HK) activity.
A: time course of induction.
Cells were exposed to 1 µM phorbol 12-myristate 13-acetate (PMA) for
the indicated times before assay of total HK activity. Significant
induction was observed within 12 h, and maximal activity was observed
within 24 h. Vehicle (DMSO) alone did not mimic this effect (data not
shown). Individual data points represent the mean ± SE for at least
3 independent measures. Statistical comparisons were performed by
paired t-testing using a significance
level of 95%. * P < 0.02 vs.
unstimulated controls. B: dose
dependence of phorbol ester-induced HK activity. Cells were exposed to
PMA at the indicated concentrations for 24 h before assay of total HK
activity. Significant induction was observed at PMA concentration 10
nM. * P < 0.05.
View larger version (22K):
[in a new window]
Fig. 2.
Specificity of HK induction for phorbol esters capable of protein
kinase C (PKC) activation. Twenty-four-hour exposure to
phorbol-12,13-didecanoate (PDD) mimicked the effect of PMA and
significantly increased total HK activity at concentrations 10 nM.
* P < 0.05. In contrast, the
inactive 4
-analog of PDD, 4
-PDD, had no effect, suggesting that
this effect is specific for phorbol esters capable of PKC activation.
Each individual data point represents the mean ± SE for 3-5
independent experiments.
Sustained exposure to phorbol esters was not required
for maximal induction of HK activity at 24 h. Although
the findings above suggested short-term activation and long-term
depletion of PKC activity, long-acting phorbol esters (e.g., PMA) are
capable of eliciting cellular responses attributable to sustained PKC activation (18). We therefore also tested the ability of brief PMA
exposure (1 h) to mimic the effect of continuous 24-h exposure. To
this end, we exposed cells to 1 µM PMA for varying time periods before thorough washing and replacement with phorbol ester-free medium.
Both 0.5-h and 1.0-h exposures resulted in over 60% increases in total
HK activity at 24 h (P < 0.02 vs.
unstimulated controls) that were indistinguishable from that observed
in cells continuously exposed to PMA over the same time period.
Increased total HK activity was accompanied by
parallel increases in both Glc utilization and lactate
production. As demonstrated in Fig.
3, net Glc utilization and net lactate
production were both increased within 18-24 h of phorbol ester
stimulation, and these changes temporally corresponded to maximal
induction of HK activity. Regression analysis revealed a linear
relationship between net Glc utilization and net lactate accumulation
that was the same for both stimulated and unstimulated cells. Moreover, the slope of the line generated using all data was not different from
that generated from data obtained from unstimulated or PMA-stimulated cells alone or from that defined by the individual means (see Fig. 3,
inset). In both stimulated and
unstimulated cells, the disappearance of three molecules of Glc from
the medium was associated with a net accumulation of approximately five
molecules of lactate (slope ~ 1.7 lactate/Glc;
r2 = 0.82, where
r = correlation coefficient).
|
HK induction by phorbol esters was
PD-98059-inhibitable and was associated with the early activation of
MEK1/2. Since the downstream effects of PKC activation
in mesangial cells may be mediated, in part, by classic
mitogen-activated protein kinase (MAPK) pathway activation (15), we
tested the ability of the specific MEK1/2 inhibitor PD-98059 to block
induction of HK activity by phorbol esters. As shown in Fig.
4, this compound inhibited PMA-stimulated
HK activity in a dose-dependent manner with an apparent
IC50 of ~3 µM and at
concentrations considered specific for the inhibition of MEK1/2
activity. Basal HK activity was not similarly affected, and vehicle
alone (DMSO) failed to mimic this effect, suggesting that the effect
was specific for PD-98059 and was not attributable to direct inhibition
of HK activity by this compound. Consistent with this interpretation, 1 µM PMA markedly increased MEK1/2 activity within 1 min of PMA
exposure, and activity rapidly declined thereafter toward normal levels
(see Fig.
5A). As
expected, PD-98059 also inhibited the activation of MEK1/2 by PMA (data
not shown).
|
|
Phorbol esters increased the phosphorylation and
activity of ERK1/2 in a time- and dose-dependent manner, and these
effects were also blocked by PD-98059. To
further evaluate the involvement of the classic MAPK pathway, we tested
the ability of PMA to induce the specific phosphorylation and
activation of ERK1/2. As depicted in Fig.
5B, specific dual-phosphorylated
ERK1/2 content increased markedly within 1 min of PMA exposure, was
maximal within 5 min, and declined thereafter, albeit never to basal
levels over the 60-min period monitored. As shown in Fig.
6A, ERK1/2
activity increased approximately 10-fold within 1 min of PMA
stimulation, and the time course of activation paralleled increases in
dual-phosphorylated ERK1/2 content (see Fig.
5B). The effect of PMA was dose
dependent, and only those concentrations capable of inducing total HK
activity at 24 h were found to be capable of ERK1/2 activation at 5 min (see Fig. 6B). As shown in Fig.
6C, pretreatment of cells with 1 µM
PMA for 24 h to deplete active PKC completely blocked the subsequent
ability of phorbol esters to induce ERK1/2 activity. In Fig.
6D, we similarly confirmed the ability
of 50 µM PD-98059 to completely block the downstream activation of
ERK1/2 by 1 µM PMA at 5 min.
|
PMA induction of HK activity required both ongoing
gene transcription and de novo protein synthesis. The
prolonged time course of HK induction following PMA treatment also
prompted us to examine the effects of general inhibitors of gene
transcription and protein translation on this effect. As depicted in
Fig. 7, the general transcriptional
inhibitor DRB also inhibited PMA-stimulated HK activity at 24 h in a
dose-dependent manner with an apparent
IC50 of ~10 µM. At this
concentration, DRB significantly inhibited PMA-stimulated activity
(P = 0.0005) without a corresponding
effect on basal activity. At 20 µM, however, DRB inhibited
PMA-stimulated activity by 81 ± 5%
(P < 0.005) while decreasing basal
activity by 19 ± 5% (P < 0.005). These effects were mimicked by both 1 µg/ml actinomycin D and
10 µg/ml cycloheximide, which completely blocked the induction of HK
activity by PMA at 24 h (100%; P = 0.05 and P < 0.02, respectively) and
resulted in corresponding decreases in basal activity of 16 ± 14%
(not significant) and 22 ± 6% (P < 0.02), respectively.
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DISCUSSION |
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The specific HK activities reported herein for cultured mesangial cells were comparable in magnitude to those previously reported for both isolated rat glomeruli (6, 10, 23) and whole rat renal cortex (3, 32, 33), typically in the 10-30 U/g protein range. Although much lower than the specific activities observed in the distal nephron and the whole medulla (23), they are comparable in magnitude to those reported for normal murine muscle and adipose (5), tissues responsible for mediating the bulk of peripheral Glc utilization. Thus mesangial cells clearly exhibit a substantial capacity for Glc phosphorylation.
The present demonstration of regulated HK activity in cultured mesangial cells is, to our knowledge, the first such description in this cell type. In addition, the specific regulation of total Glc phosphorylating capacity by phorbol esters has not heretofore been described for any cell type that we are aware of. Thus the ability of phorbol esters to increase mesangial cell HK activity is relevant not only to the understanding of mesangial cell Glc metabolism but also to HK regulation in general. To better understand the role and regulation of HK activity in mesangial cells, we have attempted to characterize this regulatory response and some of its underlying molecular mechanisms.
We have shown that phorbol esters increase total HK activity in both a time- and dose-dependent manner. Maximal induction of HK activity occurred within 12-24 h of phorbol ester stimulation, and activity remained persistently elevated for at least an additional 12-24 h. The prolonged time course of HK induction, coupled with the observation that 30-min PMA exposure was sufficient for maximal HK induction at 24 h, suggests 1) that PKC activation initiates an early chain of signaling events culminating 12-24 h later in increased HK activity and 2) that sustained PKC activation is not required for this effect. Our findings of early activation and subsequent depletion of PKC with continued exposure to PMA are consistent with these interpretations. Since PKC is the only known intracellular target for diacylglycerol-mimetic phorbol esters, the demonstration that active, but not inactive, phorbol esters were capable of increasing HK activity supports a causal relationship between PKC activation and subsequent HK induction.
We have also shown that increases in HK activity were accompanied by corresponding increases in both net Glc utilization and net lactate accumulation. Regression analysis of the data suggested that both stimulated and unstimulated cells generated at least five molecules of lactate for every three molecules of Glc consumed (~1.7 lactate/Glc) and did not suggest metabolic uncoupling by phorbol esters. Thus glycolysis probably accounted for at least 85% of the Glc utilization by these cells, with the difference reflecting, in large part, the contributions of nonglycolytic Glc utilization (e.g., via the pentose phosphate or uronic acid pathways) and lactate reutilization. These findings are consistent with those reported previously by Kreisberg et al. (12) and suggest that changes in the total Glc phosphorylating capacity of cultured mesangial cells are associated with parallel changes in Glc metabolism.
In an effort to identify downstream effectors of PKC activation that
contribute to the induction of mesangial cell HK activity, we also
investigated the classic MAPK pathway (Raf MEK
ERK). This pathway has been shown to mediate a number of downstream effects
of PKC activation in a variety of cell types. Although PKC activation
by phorbol esters has already been shown to result in ERK1/2 activation
in glomerular mesangial cells (15), the physiological consequences of
such activation have not been described. The ability of the specific
MEK1/2 inhibitor PD-98059 to completely block PMA induction of HK
activity at 24 h suggests the involvement of classic MAPK pathway
signaling downstream of PKC. The demonstration of rapid PMA-stimulated
ERK1/2 phosphorylation and activation is consistent with such a
hypothesis, and the corresponding time course of MEK1/2 activation is
temporally consistent with a role for these dual-specificity kinases in
activating ERK1/2 and mediating this effect. Taken together, these
findings suggest that the signaling events initiated by phorbol esters,
and ultimately contributing to increased HK activity, in mesangial
cells include the immediate activation of PKC, MEK1/2, and ERK1/2.
The ability of general inhibitors of both gene transcription and protein translation to block the effect of phorbol esters on HK activity also suggests that maximal induction at 24 h requires both ongoing gene transcription and de novo protein synthesis. The much smaller, albeit statistically significant, effects on basal activity suggest a more limited dependence upon continuing gene expression for maintenance of basal HK activity and may be suggestive of limited enzyme turnover. The persistence of elevated HK activity following induction provides indirect support for such a hypothesis. In addition, requirements for both ongoing gene transcription and de novo protein synthesis may explain, at least in part, the prolonged time course of HK induction following PMA stimulation.
We have previously reported in preliminary form that 24- to 48-h exposure to elevated ambient Glc at concentrations capable of PKC activation in mesangial cells failed to mimic the effect of phorbol esters on HK activity (22). It is therefore of additional interest that other known activators of PKC exhibit a variable capacity to induce HK activity in this cell type. For example, other preliminary work in our laboratory indicates that the effect of PMA can be mimicked by thrombin (20), but not by angiotensin II or endothelin-1 (R. B. Robey, unpublished observations). The reasons underlying these discrepant observations are not presently clear, but they may reflect, in part, the diversity and complexity of PKC signaling in this cell type. The inability of some known activators of PKC to increase mesangial cell HK activity also suggests that this is a specific, rather than a general, response to PKC activation.
In summary, phorbol esters are clearly capable of initiating an early chain of signaling events in mesangial cells that ultimately leads to increased HK activity. This effect involves the immediate activation of PKC and the classic MAPK pathway, and the prolonged lag time between these early signaling events and subsequent increases in Glc phosphorylating capacity may reflect specific requirements for ongoing gene expression. Although the specific gene expression requirements for induction have not yet been identified and the possibility of altered regulatory gene product expression cannot be excluded, it would be attractive to speculate that phorbol esters ultimately exert their effect on HK activity by increasing HK gene expression. We conclude that mesangial cell HK activity exhibits novel regulatory behavior that requires both PKC and classic MAPK pathway activation, as well as ongoing gene expression. In addition to suggesting specific mechanisms whereby PKC activation may influence Glc metabolism, these findings may have physiological or pathophysiological implications for certain conditions associated with altered mesangial cell PKC activation.
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ACKNOWLEDGEMENTS |
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We thank Jose A. L. Arruda and Subhash C. Pandey for critical reading of the manuscript. We also thank Richard L. Printz for helpful discussions during both the execution of this work and the preparation of the manuscript. In addition, we gratefully acknowledge the excellent technical assistance of Katie Tinich, Yuan Cai, Sohail Usman, Navin Taneja, and Badal Raval.
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FOOTNOTES |
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Portions of this work were presented in preliminary form at the 30th Annual Meeting of the American Society of Nephrology in San Antonio, TX, November 4, 1997, and at the 10th International Conference on Second Messengers and Phosphoproteins in Jerusalem, Israel, November 9, 1998.
This work was supported, in part, by Grants-in-Aid from the National Kidney Foundation of Illinois (to R. B. Robey) and the American Heart Association of Metropolitan Chicago (to R. B. Robey), as well as by a Department of Veterans Affairs Merit Review Award (to R. B. Robey).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. B. Robey, Dept. of Medicine, Section of Nephrology (M/C 793), 820 South Wood St., Rm. 418W CSN, Chicago, IL 60612-7315 (E-mail: RBRobey{at}uic.edu).
Received 16 February 1999; accepted in final form 22 June 1999.
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