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From the ¶ Renal Division, Department of Medicine, Emory
University School of Medicine, Atlanta, Georgia and the
Received for publication, February 26, 2001
Growth factors suppress the degradation of
cellular proteins in lysosomes in renal epithelial
cells. Whether this process also involves specific classes of
proteins that influence growth processes is unknown. We investigated
chaperone-mediated autophagy, a lysosomal import pathway that depends
on the 73-kDa heat shock cognate protein and allows the degradation of
proteins containing a specific lysosomal import consensus sequence
(KFERQ motif). Epidermal growth factor (EGF) or ammonia, but not
transforming growth factor A major response of cells to growth factors is a generalized
increase in protein synthesis including the synthesis of specific classes of proteins (1). In addition to controlling synthesis, growth
factors can suppress the bulk degradation of proteins (2). For example,
in renal tubular epithelial cells we found that
EGF1 suppresses the breakdown
of the mass of intracellular proteins (3). The suppression of
proteolysis in response to growth factors involved decreased lysosomal
degradation rather than decreased proteasomal or calcium-sensitive
proteases (3). Despite reports that proteolysis is regulated, no one
has determined if specific classes of proteins are being regulated by
growth factors.
Lysosomes degrade extracellular proteins (via endocytosis), membrane
proteins, and organelles (via autophagy) and can degrade cytosolic proteins via direct import through the lysosomal membrane (4,
5). Dice and Terlecky (6) showed that there is a specific import
pathway involving the 73-kDa heat shock cognate protein (hsc73) called
chaperone-mediated autophagy. Hsc73 binds to a penta-peptide motif
(consensus sequence, KFERQ) on the target protein and, acting as a
chaperone, unfolds the target protein (7). Hsc73 bound to the substrate
protein then interacts with an intrinsic lysosomal membrane protein,
the 96-kDa lysosomal glycoprotein (lgp96, also called lysosomal
membrane protein 2a) (8). After recruiting other accessory proteins,
the target protein is transported through the lysosomal membrane and
degraded (9). Dice and co-workers (10) also showed that
chaperone-mediated autophagy can be regulated by calorie deprivation,
which accelerates the proteolysis of proteins with KFERQ motifs in the
lysosomes from liver. In kidney and liver, up to 30% of proteins
contains the KFERQ motif, including many of the proteins involved in
glycolysis. Because most glycolytic proteins have long half-lives, an
increase in degradation could function to down-regulate their abundance.
Because we found that growth factors suppress lysosomal proteolysis in
renal cells, we wanted to determine whether growth factors regulate the
half-life of proteins that are substrates for chaperone-mediated
autophagy. In pursuing this question, we uncovered a novel mechanism
that leads to the accumulation of specific proteins involved in the
regulation of cellular growth.
All chemicals or reagents were purchased from Sigma, except
Dulbecco's modified Eagle's medium, newborn calf serum,
Trypsin-EDTA, and penicillin-streptomycin, which were obtained from
Life Technologies, Inc. Recombinant human TGF- NRK-52E cells (a rat kidney epithelial cell line (11), passage 15) were
obtained from ATCC (Manassas, VA), subcultured, and grown in high
glucose Dulbecco's modified Eagle's medium supplemented with 25 mM HEPES, 25 mM glutamine, and 5% calf serum.
Studies were performed on cells from passages 19-29. Cells in 6-well
plates were grown to confluence and rendered quiescent by serum removal 48 h prior to experimental treatment. The cell culture medium was
refreshed every 24 h to maintain a constant pH; it did not differ
between control and treatment groups.
Recombinant human TGF- Measurements of Growth and Protein Turnover--
After exposure
to an experimental variable, cells were washed with PBS, incubated with
0.05% trypsin/0.5 mM EDTA for 5 min, centrifuged at
1500 × g for 5 min, and washed with PBS. The final pellet was resuspended in 1 ml of 50 mM
Na2PO4 (pH 7.4) and lysed on ice by repeated
passage though a 27-gauge needle. The lysate was divided and stored at
Protein degradation was measured as the release of
L-[U-14C]phenylalanine from cells prelabeled
as described (3-13). Briefly, 5 mM unlabeled phenylalanine
was added to the medium to minimize reuse of the phenylalanine released
by protein breakdown, and an initial 4-h washout period was used to
eliminate short lived proteins and unincorporated
L-[14C]phenylalanine. Aliquots of the medium
were removed at intervals and treated with trichloroacetic acid
to remove protein, and the radioactivity was determined. At the end of
the experiment, cell protein was solubilized in 1 ml/well of 1% SDS,
and the remaining radioactivity was measured. The protein degradation
rate was calculated as the slope of the logarithm of the
[14C]phenylalanine remaining in cell protein
versus time.
Turnover of Specific Proteins--
Confluent cells in 100-mm
dishes were incubated with 100 µCi of
L-[35S]cystine/methionine (ICN, Costa Mesa,
CA). For GAPDH, the labeling was performed in serum-free Dulbecco's
modified Eagle's medium with cold cystine/methionine present for
72 h. For Pax2, cells were treated with EGF in
cystine/methionine-free medium for 20 h to increase the labeling
of Pax2 because its abundance is very low in quiescent cells. After two
washes in serum-free medium, a 4-h washout in serum- and growth
factor-free cystine/methionine-containing medium was performed prior to
the addition of growth factors. Subsequently, cells were washed twice
with serum-free medium before adding the experimental variable in the
medium containing an excess of cold cystine and methionine. The culture
medium was always changed daily with an additional wash. At time 0 and
at various times up to 72 h for GAPDH and up to 24 h for
Pax2, cells were lysed in a 1% Nonidet P-40 lysis buffer containing
100 µg/ml phenylmethylsulfonyl fluoride, 2 mM
sodium EDTA, 4 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml
pepstatin. One µg/ml anti-GAPDH or Pax2 anti-serum was added to equal
amounts of cellular protein that was precipitated with protein
G-Sepharose beads. After three washes with lysis buffer, the
immunoprecipitate was separated by SDS-polyacrylamide gel
electrophoresis, underwent autoradiography, and was quantitated by use
of the Signmagel program. The protein half-life was calculated from the
slope of the logarithmic transformation of the densitometry data
plotted against time. We documented the completeness of recovery by
performing Western blots on the supernatants after immunoprecipitation (data not shown).
Western Blotting--
Cells in 60-mm tissue culture
dishes were washed twice in ice-cold PBS and lysed in a buffer
containing 100 µg/ml phenylmethylsulfonyl fluoride, 2 mM
sodium EDTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml
pepstatin. After centrifugation, the proteins in the supernatant
were determined and boiled in buffer containing 1% SDS and 0.5%
Lysosome Isolation--
Lysosomes were isolated as described by
Cuervo et al. (14). Briefly, cells in two 25-cm2
plates/group were washed in ice-cold PBS and then homogenized after
scraping in ice-cold buffer (2.5 mM Tris (pH 7.2), 0.25 M sucrose) by 20 strokes of a Teflon Polytron homogenizer
at 4 °C. Then 1 g of protein/7 ml of 0.25 M sucrose
was centrifuged at 2500 × g for 10 min and the
post-nuclear supernatant was placed on a discontinuous gradient of 35 and 17% metrizamide (pH 7.0) and 6% Percoll and centrifuged at
6800 × g for 25 min. The lysosome/mitochondrial fraction at the metrizamide/Percoll interface was resuspended to a
final concentration of 57% metrizamide. On top of this fraction, there
was a discontinuous metrizamide gradient of equal volumes of 35, 17, and 5%, metrizamide with a final 0.25% sucrose layer. This gradient
was centrifuged for 1 h at 95,000 × g. The
lysosomes sediment to the interface of 5-17% metrizamide and
mitochondria at the 35-57% interface. Purity of the lysosomal or
mitochondrial fractions was determined by the activity of
Statistics--
Results are expressed as mean ± S.E.
Because there was experiment-to-experiment variation in the magnitude
of responses, results are presented as a percentage of the control
value determined simultaneously. The differences between two groups
were analyzed by the Student's t test, but multiple
comparisons were analyzed by analysis of variance. Comparisons of
slopes of lines representing the release of
L-[U-14C]phenylalanine were done by analysis
of co-variance.
We treated NRK-52E cells with growth factors and found different
growth properties (Fig. 1): EGF causes
hyperplasia (increased DNA content) and increases (~30%) the
half-life of long lived proteins. TGF- GAPDH has a KFERQ consensus sequence (17) and is a classic substrate
for chaperone-mediated autophagy (15,
18). As shown in Fig. 2 and Table I, the
half-life of GAPDH measured in a pulse-chase experiment increased
by ~ 90% in cells treated with either EGF or ammonia but did
not significantly change in cells treated with TGF-
A Mechanism Regulating Proteolysis of Specific Proteins during
Renal Tubular Cell Growth*
§¶,With the technical assistance of Li Ling Shen, and Atlanta Veterans Affairs Medical Center,
Decatur, Georgia 30033
ABSTRACT
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1, suppresses total protein breakdown in
cultured NRK-52E renal epithelial cells. EGF or ammonia prolonged the
half-life of glyceraldehyde-3-phosphate dehydrogenase, a classic
substrate for chaperone-mediated autophagy, by more than 90%, whereas
transforming growth factor 1 did not. EGF caused a similar increase
in the half-life of the KFERQ-containing paired box-related
transcription factor, Pax2. The increase in half-life was accompanied
by an increased accumulation of proteins with a KFERQ motif including glyceraldehyde-3-phosphate dehydrogenase and Pax2. Ammonia also increased the level of the Pax2 protein. Lysosomal import of KFERQ proteins depends on the abundance of the 96-kDa lysosomal glycoprotein protein (lgp96), and we found that EGF caused a significant decrease in
lgp96 in cellular homogenates and associated with lysosomes. We
conclude that EGF in cultured renal cells regulates the breakdown of
proteins targeted for destruction by chaperone-mediated
autophagy. Because suppression of this pathway results in an
increase in Pax2, these results suggest a novel mechanism for the
regulation of cell growth.
INTRODUCTION
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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1 and EGF were
obtained from R&D Systems (Minneapolis, MN), and
L-[U-14C]phenylalanine was obtained from
PerkinElmer Life Sciences. Anti-hsc73 antiserum was purchased
from Maine Biotechnology (Portland, ME), anti-M2 pyruvate kinase was
purchased from Scebo-Tech, A.G. (Wettenburg, Germany), and
anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased
from Biodesign International (Kennebunk, ME). Anti-Pax2 serum was
purchased from Zymed Laboratories Inc..
Affinity-purified anti-sera to the penta-peptide KFERQ and to lgp96
were a generous gift of J. F. Dice (Tufts University). The
anti-hexokinase serum was a gift of E. Knecht (Universidad de
Vallencia, Spain).
1 was reconstituted in 4 mM HCl
containing 0.1% heat-treated bovine serum albumin. Recombinant human EGF was reconstituted in PBS containing 0.1% heat-treated bovine serum
albumin. In all studies, concentrations of 10
10
M (TGF-1), 108 M (EGF), and 10 mM (NH4Cl) were used (12); the appropriate vehicle was added to control cells.
70 °C for protein and DNA determination as described (12).
-mercaptoethanol, separated by SDS-polyacrylamide gel
electrophoresis, and transferred to nitrocellulose filters, and 5%
fat-free milk protein or 3% bovine serum albumin was used as a
blocking reagent. Antibodies were detected using the ECL system
(Amersham Pharmacia Biotech) and Kodak BCL film.
-N-hexosaminidase (lysosome) and mitochondrial succinic
dehydrogenase and by the presence of lgp96 (15).
RESULTS
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1 increases in the protein/DNA
ratio (hypertrophy) but does not significantly suppress proteolysis.
The combination of EGF plus TGF-1 causes hypertrophy with the
suppression of proteolysis, whereas ammonia causes less hypertrophy
even though there is an even greater suppression of proteolysis. We
have shown previously that EGF, TGF-1, and EGF plus TGF-1
increase protein synthesis, whereas ammonia does not affect synthesis
(12, 13, 16).
View larger version (16K):
[in a new window]
Fig. 1.
Growth factors exert different effects on
growth and proteolysis. NRK-52E cells were treated with EGF
(10 8 M), TGF-1 (1010
M), EGF + TGF-1, and NH4Cl (10 mM). A, protein, DNA, and protein to DNA levels
after 72 h of treatment are expressed as a percentage of the
increase over the value measured in cells treated with the vehicle
only. n = 14-18. B, protein
degradation expressed as the slope of the log of the percentage of the
counts remaining in cells at different times. The figure is a
representative experiment of three repeats, (n = 6/group). The slopes of all lines are significantly different from each
other (p < 0.05) except for control compared with
TGF-1 and EGF compared with EGF + TGF-1.
1 alone.
View larger version (17K):
[in a new window]
Fig. 2.
Growth factors change GAPDH protein
half-life. A, autoradiogram of NRK-52E cell lysates
after immunoprecipitation with anti-GAPDH in pulse-chase experiments.
Cells were treated as in Fig. 1 except they were radiolabeled when
quiescent. Lanes are labeled according to treatment
(control, EGF, or TGF- ) and
duration of treatment (hours) after removal of
L-[35S]cystine/methionine. 0 represents initial labeling. The autoradiogram shown is representative
of five repeats. B, densitometry of all repeats are plotted
as in Fig. 1B (n = 5).
Growth factors change GAPDH protein half-life
We also tested whether these agents change the abundance of specific
proteins with the KFERQ lysosomal import sequence. GAPDH abundance
increased with treatments that suppress proteolysis, but TGF-1,
which does not affect proteolysis, did not increase GAPDH. We also
examined the M2 isoform of pyruvate kinase because it is a glycolytic
enzyme with KFERQ consensus sequences (19), it binds hsc73, and it is
imported into lysosomes (6). In contrast, hexokinase is a glycolytic
enzyme that lacks a KFERQ sequence (15). Conditions that stimulate cell
growth also increase the abundance of the M2 isoform of pyruvate kinase
as well as other proteins recognized by anti-KFERQ affinity-purified
sera (Fig. 3). Hexokinase abundance did
not increase with any of the growth factors. Although the pattern of
changes was similar between proteins recognized by anti-KFERQ serum and
the M2 isoform of pyruvate kinase, the magnitude of increase in the M2
isoform of pyruvate kinase was much greater, suggesting that its
synthesis is also stimulated (Fig. 3, B and
C).
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Glycolytic enzymes are not the only proteins containing KFERQ
sequences, which may be important intermediates influencing renal cell
growth. For example, the renal paired box-related transcription factor,
Pax2, contains a conserved KFERQ sequence at amino acids 38-42 (20).
We found that the half-life of Pax2 (Fig.
4) also increased with EGF treatment and
that the abundance of Pax2 was increased by growth factors; the
smallest rise occurred with TGF-1. Interestingly, the Pax2 abundance
also increased when lysosomal proteolysis was inhibited with
NH4Cl (Fig. 3D). Thus different stimuli causing
growth also increase Pax2 abundance.
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To determine the mechanisms that control suppression of proteolysis, we
examined if the regulatory proteins of chaperone-mediated autophagy
change in response to growth factors. Hsc73 did not change in abundance
(Fig. 3A). We also examined the lysosomal membrane receptor
for protein translocation, lgp96, in lysates and in association with
isolated lysosomes using sera directed against the 12-amino acid
cytoplasmic portion of lgp96 that binds to hsc73 (8). The quality of
lysosomes isolated did not vary between the control and EGF-treated
cells as assayed by hexosaminidase activity (Table
II). Isolated lysosomes exhibited
immunostaining for lgp96 (Fig.
5A); the level was 7-fold
higher than in whole cell lysates (Fig. 5B). Lgp96 was not
detected in the mitochondrial fractions. In whole cell lysates, lgp96
abundance decreased by 30-40% after 24 or 96 h of treatment with
stimuli that suppress proteolysis (Fig. 5, C and
E). In contrast, TGF-1, which did not affect proteolysis,
did not affect lgp96 levels. Because the lysosomal-associated lgp96
correlates more closely with the activity of chaperone-mediated
autophagy than total cellular lgp96 (21), we examined
lysosomal-associated lgp96 with EGF treatment and found a 48 ± 10% decrease (p < 0.05, n = 3)
compared with lysosomes isolated from control cells (Fig. 5,
D and E).
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In the early 1980s, it was recognized that specific growth factors and activated oncogenes could suppress protein degradation in certain cell types including epithelial cells (22). It was not known, however, which classes of proteins develop longer half-lives during growth or how this response was regulated. We found that EGF suppresses the breakdown of the bulk of proteins in NRK-52E cells by a mechanism that involved the suppression of lysosomal function but not proteolysis by proteasomal or calcium-activated proteases (3).
Physiologic conditions can regulate specific pathways of lysosomal proteolysis. For example, calorie deprivation increases the degradation of proteins with a KFERQ motif in liver and kidney lysosomes (10). How does this finding bear on growth factor-induced renal cell growth? Conditions stimulating renal cell growth increase glycolysis, and many glycolytic enzymes contain KFERQ motifs (17, 23-25). Thus, by acting in the opposite fashion as calorie deprivation, growth factors could suppress the degradation of glycolytic enzymes and contribute to the increase in glycolysis that accompanies renal growth. Our results confirm that EGF acts to prolong the half-life of the classic substrate for chaperone-mediated autophagy, GAPDH, and increase the abundance of KFERQ-containing proteins.
Our results provide additional insights into the relationship among
growth factors, cell growth, and lysosomal protein degradation. First,
only specific growth factors influence lysosomal function. For example,
EGF clearly stimulates cell growth and suppresses total proteolysis and
the proteolysis of substrates of chaperone-mediated autophagy. In
contrast, TGF-1 caused the smallest increase in growth and has
almost no effect on proteolysis. We do not conclude that suppression of
proteolysis is the sole mechanism causing KFERQ-containing protein
accumulation, because the accumulation of KFERQ proteins that occurred
with TGF- treatment almost certainly reflects increased synthesis
(Figs. 3, B and C).
Second, our results show that regulation of this lysosomal pathway by growth factors leads to prolongation of the half-life of Pax2, which has been implicated in renal cell growth in development, cyst formation, and renal cell carcinoma (20, 26). Because there is also an increase in the abundance of Pax2 in cells treated with EGF and because EGF causes only trivial increases in Pax2 mRNA in renal tubular cells (27), the increase in half-life we found could be physiologically relevant. Because Pax2 acts as a transcription factor, these responses suggest a new mechanism by which growth factors regulate cell growth; not only do they suppress the degradation of the bulk of cytoplasmic proteins (3), but they increase the availability of at least one critical transcription factor.
Finally, our results provide unexpected information about a potential mechanism by which ammonia could increase cell growth. The growth of renal cells characteristically found in response to metabolic acidosis is attributed to ammonia, which can reach concentrations as high as 5 mM in the cortex of the kidney (28). Ammonia had been thought to act only by changing lysosomal pH and nonspecifically suppressing lysosomal proteolysis leading to the accumulation of cytosolic proteins (16, 29). However, our results suggest that ammonia also acts by suppressing the degradation of specific signaling proteins such as Pax2. The up-regulation of transcription factors could allow the expression of particular proteins important for growth without an increase in global protein synthesis.
Regarding the mechanism involved in changing lysosomal degradation, we and others find that the abundance of hsc73 does not change even when activity of this pathway changes (9, 14). Curiously, hsc73 contains KFERQ sequences but is resistant to degradation within hepatic lysosomes responding to starvation (9). On the other hand, we did observe a decrease in the abundance of lgp96 including a sharp decrease in the amount of lgp96 specifically associated with lysosomes (Fig. 5). This finding is consistent with the close correlation between the lgp96 associated with lysosomes and the activity of chaperone-mediated autophagy (14, 21, 30).
Although there are similarities between the effects of EGF and ammonia on the proteolysis of KFERQ-containing proteins and lgp96 levels, there are differences in their actions on lysosomes. We found that pharmacologic agents that specifically inhibit lysosomal proteolysis (ammonia, methylamine, bafilomycin A1, or leupeptin plus the protease inhibitor, E64) convert the cellular proliferation in response to EGF into hypertrophy (31). The change in lgp96 abundance may be a common pathway increasing growth-promoting proteins such as glycolytic enzymes and Pax2, and an additional influence of lysosomal inhibitors may account for the conversion of hyperplasia to hypertrophy.
Besides the regulation of chaperone-mediated autophagy, EGF could affect the function of other pathways of lysosomal proteolysis. Autophagy may also be regulated by growth factors (32), leading to slower degradation of organelles and membranes. EGF acts through phosphoinositide 3-kinase as it suppresses proteolysis in renal cells,2 and phosphoinositide 3-kinase has been reported to regulate autophagy in cultured liver cells (33).
One practical prediction of these results is that a KFERQ sequence may
be used to identify proteins that are up-regulated during renal cell
growth. Besides glycolytic enzymes, there are a large number of
proteins in the National Center for Biotechnology Information data base
that contain conserved KFERQ sequences and are important for renal
tubule cell growth. These proteins include enzymes involved in
phospholipid metabolism (choline kinase (GenBankTM
accession number 139962) and phosphorylcholine transferase (34)), ion
transporters ( subunit of the sodium/potassium ATPase (35)), and
signaling molecules such as Pax2 (20). The KFERQ sequence is present in
the Pax isoforms expressed in the urinary tract (Pax2, -5, and -8) but
not in other Pax isoforms, suggesting that the link between Pax
proteins and this proteolytic pathway may be specific to the urinary
tract (36, 37). Finally, the signaling proteins MARKS and I
B have
been shown to have their abundance regulated by this pathway (30,
38).
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ACKNOWLEDGEMENTS |
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We thank Drs. J. Fred Dice and Ana Maria Cuervo for help with reagents and techniques and Drs. William Mitch and Russ Price for advice and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health K08 DK02496 a Young Investigator Research Grant from the National Kidney Foundation, a Veterans Administration Merit Review Award (to H. F.), an American Heart Association Scientist Development Award (to J. D.), and a fellowship grant from the National Kidney Foundation of Georgia (to S. S.).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. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Renal Division, Emory University School of Medicine, W.M.B., Rm. 338, 1639 Pierce Dr., N. E., Atlanta, GA 30322. Tel.: 404-727-9217; Fax: 404-727-3425; E-mail: hfranch@emory.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101777200
2 H. A. Franch and J. Du, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
EGF, epidermal
growth factor;
hsc73, 73-kDa heat shock cognate protein;
lgp96, 96-kDa lysosomal glycoprotein;
TGF-1, transforming
growth factor 1;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PBS, phosphate-buffered saline.
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