Departments of 1 Pediatrics, 2 Ophthalmology and Visual Sciences, and 3 Pharmacology, University of Wisconsin-Madison, Madison, Wisconsin 53792
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ABSTRACT |
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Cell proliferation, survival, and
differentiation are carefully orchestrated processes during
nephrogenesis that become aberrant during renal cyst formation.
Signaling through focal adhesion kinase (FAK) impacts these processes,
although its role during nephrogenesis requires further delineation. We
previously demonstrated that phosphorylation of FAK and paxillin is not
downregulated in cystic kidneys from B cell lymphoma/leukemia-2 (bcl-2)
/
mice. Here we examine whether FAK downstream signaling pathways are affected in these cystic kidneys. Cystic kidneys from bcl-2
/
mice exhibited sustained phosphorylation of Src and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK, ERK1).
However, similar levels of expression were noted for phosphorylated c-Jun NH2-terminal kinase, phosphatidylinositol-3-kinase,
and its target protein kinase B/ATP-dependent tyrosine kinase in
kidneys from postnatal day 20 bcl-2 +/+ and bcl-2
/
mice. We also examined expression of the adapter protein Shc,
implicated in growth and apoptosis. Expression of
p66Shc decreases to low levels in postnatal kidneys,
whereas p52/p46Shc was constitutively expressed during
nephrogenesis. Shc expression was similar in normal and cystic kidneys.
Therefore, sustained activation of MAPK/ERKs through the Src/FAK
pathway may contribute to the hyperproliferation observed in cystic
kidneys from bcl-2
/
mice.
mitogen-activated protein kinases; renal cysts; signal transduction
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INTRODUCTION |
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B CELL LYMPHOMA/LEUKEMIA-2 (bcl-2) is a death repressor
that plays an important role during nephrogenesis. Mice deficient
in bcl-2 (bcl-2 /
) develop renal hypoplasia/cystic dysplasia (16). Renal cystic disease occurs in these mice by
postnatal day 20 (P20) and is characterized by
hyperproliferation in the cortex and medulla, increased amounts of
apoptosis, and epithelial cells that have not fully
differentiated (15). Cystic kidneys from these mice
display altered signaling through focal adhesion kinase (FAK). We have
observed sustained phosphorylation of FAK and paxillin and altered
distribution and decreased expression and activity of the tyrosine
phosphatases Src homology-2 domain phosphatase (SHP-2) and protein
tyrosine phosphatase (PTP) 1B in cystic kidneys from these mice
(17, 18). Bcl-2 forms a complex with paxillin/FAK,
potentially influencing cell adhesion-mediated signaling
(18). Thus the early loss of bcl-2 during nephrogenesis may result in an inability to complete renal differentiation/maturation as a consequence of not receiving the appropriate signals or not forming appropriate signaling complexes.
The role of the FAK/paxillin signal transduction pathway(s) in nephrogenesis and in the pathogenesis of kidney disease has not been elucidated. Signaling through FAK can prevent apoptosis and promote cell proliferation and migration. Our studies indicate that proper regulation of FAK/paxillin during renal maturation is important (18). FAK plays a pivotal role in integrin and growth factor signaling pathways. It interacts with signaling and cytoskeletal proteins such as paxillin, Src kinase, phosphatidylinositol-3 (PI3)-kinase, and p130cas. Src's association may protect FAK from dephosphorylation by phosphatases and allow Src to phosphorylate FAK on additional tyrosine residues, keeping it in an active state. However, continued signaling through FAK could result in the aberrant activation of downstream signaling pathways, including mitogen-activated protein kinase (MAPK), PI3-kinase, and protein kinase B (PKB)/ATP-dependent tyrosine kinase (Akt) (3).
MAPKs, including extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal kinase (JNK), and p38, play important roles in the cell by transmitting extracellular signals from the cell membrane to the nucleus (9). MAPKs are activated by various stimuli, influencing cell proliferation, differentiation, and apoptosis. Altered regulation of MAPKs can have profound cellular effects. Constitutive activation of MAPK/ERKs is observed in an organ-specific manner in primary tumors and tumor cell lines, with kidney, colon, and lung having the highest frequencies (4). We recently demonstrated that sustained activation of ERKs results in dedifferentiation of Madin-Darby canine kidney epithelial cells, affecting cell adhesive mechanisms (13). Activation of FAK can also result in signal transduction through PI3-kinase. PI3-kinase contributes to cell growth and survival. PI3-kinase can influence cell survival by activation of PKB/Akt. PKB/Akt, a serine-threonine kinase, inhibits apoptosis by a variety of stimuli (2). Thus, altered regulation of cell proliferation and differentiation observed in renal cystic disease would be consistent with aberrant regulation of MAPKs and/or PI3-kinase signaling pathways.
In the studies described here, we examined FAK downstream signal
transduction in kidneys from P20 bcl-2 +/+ and bcl-2 /
mice. Cystic kidneys from P20 bcl-2
/
mice display
sustained activation of Src and MAPK/ERK (ERK1), although their levels
of expression are similar. In contrast, JNK, PI3-kinase, and its target
PKB/Akt were not affected. The adapter protein Shc has been implicated
in growth and apoptosis. Consistent with their described roles,
expression of p66Shc decreases to low levels in postnatal
kidneys, whereas p52/p46Shc was constitutively expressed
during nephrogenesis. Shc expression was similar in normal and cystic
kidneys. Thus sustained activation of MAPK/ERKs (ERK1) through the
Src/FAK pathway may contribute to the hyperproliferation observed in
cystic kidneys from bcl-2
/
mice.
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MATERIALS AND METHODS |
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Animal breeding. Bcl-2 heterozygote animals were interbred, and the genotypes were determined as previously described (16). For expression studies, mice were interbred and the kidneys were surgically dissected at the times noted. Embryos were removed from anesthetized pregnant female mice on day 15 of pregnancy. The protocols were performed with approval from the University of Wisconsin Animal Care and Use Committee.
Protein lysate preparation, immunoprecipitation, and Western blot
analysis.
The protein lysates were prepared in a modified RIPA buffer [(in mM)
142.5 KCl, 5 MgCl2, 10 HEPES, pH 7.4, 2 orthovanadate, and
2 sodium difluoride, as well as 1% Nonidet P-40 and a complete protease inhibitor cocktail (Boehringer Mannheim)], and 20 µg of
total protein were electrophoresed in a 4-20% polyacrylamide gel,
transferred to a Hybond ECL nitrocellulose membrane (Amersham, Arlington Heights, IL), blocked, and incubated with anti-PKB-/Akt (Transduction Laboratories; 1:1,000), phosphorylated Akt (New England
Biolabs; 1:1,000) Src (Santa Cruz Biotechnology; 1:1,000), ERK (Santa
Cruz Biotechnology; 1:1,000), phosphorylated ERK (Santa Cruz
Biotechnology; 1:1,000), JNK (Promega; 1:2,000), phosphorylated JNK
(Santa Cruz Biotechnology; 1:1,000), or Shc (Transduction Laboratories;
1:2,000) overnight at 4°C. The membranes were then washed, incubated
with the appropriate secondary antibody (Pierce, Rockford, IL), washed
again, and developed with ECL (Amersham). For immunoprecipitations, 600 µg of kidney lysate were incubated with 3 µg of antiphosphotyrosine
(PY20, Transduction Laboratories) or mouse IgG (control), incubated
with GammaBind Plus Sepharose (Pharmacia Biotech, Piscataway, NJ), and
washed as previously described (18). Western blot
quantitation was performed using a Molecular Dynamics PhosphorImager.
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RESULTS |
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Sustained Src phosphorylation in kidneys from P20 bcl-2 /
mice.
Renal cystic disease in bcl-2
/
mice is characterized by
hyperproliferation in the cortex and medulla, increased
apoptosis, and sustained phosphorylation of FAK and paxillin
(15, 18). Src phosphorylates FAK, and signal transduction
through this pathway impacts proliferation. To determine whether Src
could potentially contribute to sustained phosphorylation of FAK in
P20 bcl-2
/
mice, we examined Src expression and
phosphorylation. Kidney protein lysates prepared from postnatal
day 10 (P10) mice (before renal maturation) and
P20 mice (after renal maturation in normal mice; cystic
kidneys from bcl-2
/
mice) were immunoprecipitated with mouse IgG
(negative control) or PY20 (antiphosphotyrosine) and blotted with
anti-Src to determine Src tyrosine phosphorylation. Levels of
phosphorylated Src were similar before renal maturation (P10) in bcl-2 +/+ and bcl-2
/
mice (Fig.
1A). In contrast, Src
phosphorylation was sustained in lysates from P20 bcl-2
/
mice compared with P20 bcl-2 +/+ littermates (after
renal maturation; Fig. 1, A and C). Similar
results were obtained when lysates were immunoprecipitated with
anti-Src and blotted with 4G10 (antiphosphotyrosine; data not shown).
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Sustained activation of ERKs but not JNK or PKB/Akt is observed in
cystic kidneys.
FAK can activate downstream signaling pathways, including MAPK/ERKs and
PI3-kinase (3). We next examined the expression and
phosphorylation of MAPKs. The level of total ERK1 and ERK2 expression
was similar at all times (Fig.
2B). Levels of phosphorylated ERK1 were similar before renal maturation (P10) in bcl-2 +/+
and bcl-2 /
mice (Fig. 2A). After renal maturation
(P20) in normal mice, the level of ERK1 phosphorylation
declines significantly. In contrast, ERK1 phosphorylation lysates from
P20 bcl-2
/
mice remained at higher levels, similar to
those observed at P10. Expression of activated
(phosphorylated) ERK2 was similar at all times in bcl-2 +/+ and bcl-2
/
mice.
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Expression of adapter proteins Shc and Grb2.
Shc isoforms (p46, p52, and p66) are SH2-containing proteins implicated
in the regulation of apoptosis and life span (p66) and growth
(p52/p46) (8). The loss of p66Shc expression
confers resistance to H2O2-induced
apoptosis (11). In Fig.
3A, we examined Shc expression
in kidneys from mice at embryonic day 15, postnatal
day 0 (birth), P10, and P20 to
delineate expression during normal nephrogenesis and after renal
maturation (P20). In addition, we examined Shc expression in
cystic kidneys from P20 bcl-2 /
mice. The expression of
p66Shc decreased to low levels in kidneys from postnatal
mice. This suggests that decreased expression of p66Shc may
aid cell survival in the postnatal kidney. In contrast, expression of
p46Shc and p52Shc was similar throughout
nephrogenesis. Expression of Shc isoforms was similar in kidneys from
P20 bcl-2 +/+ and bcl-2
/
mice. We also observed
constitutive expression of the adapter protein Grb2 during
nephrogenesis and in cystic kidneys (Fig. 3B).
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DISCUSSION |
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Signal transduction through FAK impacts cell proliferation,
survival, and differentiation. Altered regulation of this pathway can
lead to abnormal growth and differentiation. We previously demonstrated
that sustained phosphorylation of FAK and paxillin in cystic kidneys
from bcl-2 /
mice correlated with an increased rate of cell
proliferation and an inability of renal epithelial cells to terminally
differentiate (15, 18). Here, we have further investigated
FAK signal transduction in cystic kidneys from bcl-2
/
mice. Our
major findings are as follows: 1) sustained phosphorylation
of Src is observed in cystic kidneys, 2) MAPK/ERKs (ERK1)
remains activated in cystic kidneys, and 3) PI3-kinase/Akt, JNK, and Shc expression is not affected in cystic kidneys from P20 bcl-2
/
mice. Thus our data indicate that sustained
phosphorylation of Src and FAK may initiate downstream signaling,
resulting in sustained activation of MAPK/ERKs in cystic kidneys from
P20 bcl-2
/
mice.
FAK binding to paxillin or p130cas can activate MAPK/ERKs
via Crk (3). FAK is required for transducing survival
signals from a complex extracellular matrix. The FAK survival signal is
mediated through PI3-kinase/Akt-dependent and -independent pathways.
Our data suggest that sustained phosphorylation of FAK leads to
sustained activation of MAPK/ERKs, disruption of renal epithelial cell
terminal differentiation, and formation of renal cysts. Activated ERKs are observed in samples from patients with autosomal-dominant polycystic kidney disease (1, 19). Sustained activation of ERKs in renal cystic disease is consistent with our in vitro data demonstrating an inability of kidney epithelial (Madin-Darby canine kidney) cells with activated ERKs to form stable adherens junctions (13). These cells also have downregulated adherens
junction proteins, including E-cadherin, -catenin, and
-catenin.
We have demonstrated that inhibition of MAPK/ERKs in these cells is
sufficient to reestablish a normal epithelial phenotype and formation
of adherens junctions (13). Sustained activation of
MAPK/ERKs in these cells also inhibits tube formation in collagen gels.
These in vitro data are consistent with our observation that cell-cell adhesive interactions are aberrant in kidneys from bcl-2
/
mice (14, 18).
We believe that, in the absence of bcl-2, focal adhesion complexes do
not form properly in early development and are not properly regulated
at maturation. Apoptosis, proliferation, and differentiation are affected in cystic kidneys. Continued signaling through FAK (phosphorylated FAK) may lead to signaling through MAPK/ERKs and/or PI3-kinase, affecting apoptosis, proliferation, and
differentiation. However, the data presented here suggest that
sustained activation of Src, FAK, and MAPK/ERKs (ERK1) plays a causal
role in the hyperproliferation we previously reported in the cortex and
medulla of cystic kidneys from bcl-2 /
mice (15).
Figure 4 summarizes the potential cellular effects of signaling through MAPK/ERKs. Aberrant signal transduction through this pathway is consistent with described renal
cystic phenotype.
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Protein tyrosine phosphorylation is a dynamic reversible process in
which the level of phosphorylation, at any time, is the result of
kinase and/or phosphatase activity. Altered regulation of kinases
and/or phosphatases can lead to abnormal growth and differentiation.
Here we show that sustained activation of Src and MAPK/ERKs (ERK1) may
contribute to the aberrant phosphorylation/activation of signaling
proteins in cystic kidneys from bcl-2 /
mice. However, the
important role of tyrosine phosphatases in this process should not be
neglected. We recently showed that cystic kidneys from P20
bcl-2
/
mice have reduced expression, a sixfold decrease in
activity, and altered distribution of SHP-2 and PTP 1B, whereas PTP-proline, glutamate, serine, and threonine (PEST) sequence expression and distribution were similar in bcl-2 +/+ and bcl-2
/
mice. Therefore, our data indicate that the hyperproliferation observed
in cystic kidneys from bcl-2
/
mice may be due to an inability to
complete the final stages of terminal differentiation, aberrantly
keeping signal transduction pathways active that are normally downregulated.
Signal transduction through Src/FAK is one way to affect
proliferation. However, growth and apoptosis can also be
influenced by Shc isoforms. Shc isoforms regulate apoptosis and
life span (p66Shc) as well as growth
(p52/p46Shc). p52/p46Shc isoforms are
ubiquitously expressed. However, p66Shc expression varies
in different cell types and is lacking in some cells, including
hematopoietic cells (12). In the kidney,
p66Shc is highly expressed embryonically, and its
expression decreases to low levels postnatally, whereas
p52/p46Shc is constitutively expressed. Similarly,
p66Shc is highly expressed in the embryonic lung, becomes
restricted late in gestation, and is nearly absent in the postnatal
lung (6). The decline in p66Shc expression
postnatally may give us an insight into its function. Mice lacking
p66Shc have increased resistance to paraquat and increased
life span, whereas fibroblasts lacking p66Shc have enhanced
resistance to apoptosis due to H2O2 or
ultraviolet light (10). Thus, reducing
p66Shc expression in the postnatal kidney or lung may
provide additional protection from apoptosis in a
stressful/harsh environment. We observed similar expression of
p66Shc in kidneys from P20 bcl-2 +/+ and bcl-2
/
mice, perhaps suggesting redundancy of this pathway.
In summary, the appropriate regulation of FAK and its downstream signal
transduction pathway(s) plays an important role during terminal
differentiation of renal epithelial cells. Aberrant activation of these
pathways may contribute to sustained activation of MAKP/ERKs and
hyperproliferation in cystic kidneys from bcl-2 /
mice.
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ACKNOWLEDGEMENTS |
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The authors thank R. Gordon and J. Oswald for preparation of the figures.
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FOOTNOTES |
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This research and C. M. Sorenson were supported in part by the Solomon Papper, M.D., Young Investigator Grant of the National Kidney Foundation and the Polycystic Kidney Foundation. N. Sheibani was funded by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-45599.
Address for reprint requests and other correspondence: C. M. Sorenson, Dept. of Pediatrics, University of Wisconsin-Madison, H4/444 CSC, 600 Highland Ave., Madison, WI 53792-4108 (E-mail: cmsorenson{at}facstaff.wisc.edu).
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.
June 26, 2002;10.1152/ajprenal.00380.2001
Received 27 December 2001; accepted in final form 19 June 2002.
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