Lysophosphatidic acid: a novel growth
and survival factor for renal proximal tubular cells
JERROLD S. LEVINE, JASON S. KOH, VERONICA TRIACA, AND WILFRED LIEBERTHAL
Renal Section, Department of Medicine, Boston Medical Center, Boston, Massachusetts 02118
apoptosis; phosphatidylinositol 3-kinase; phospholipids
LYSOPHOSPHATIDIC ACID (LPA; 1-acyl-sn-glycerol-3-phosphate), the smallest and structurally simplest of all
glycerol-based phospholipids, has long been recognized
as a precursor in phospholipid biosynthesis. Recently,
LPA has also emerged as an important intercellular
signaling molecule with a specific cell surface receptor
of its own (9, 14). Although LPA is not present in plasma
or freshly isolated blood, it is produced and released by
platelets during blood clotting and is therefore a normal constituent of serum (2, 28). The normal concentration of LPA in serum is in the range of 5–20 µM (2). LPA
binds with high affinity to serum albumin while retaining its biological activity (27, 28). Like most growth
factors or cytokines, LPA has a pleiotropic range of
effects (9, 14). These include stimulation or inhibition of
cell proliferation (depending upon the cell type), focal
adhesion formation, stress fiber formation, smooth
muscle cell contraction, and stimulation of tumor cell
invasion (9, 14, 20).
In this report, we show that LPA can act as a
‘‘survival factor’’ to inhibit apoptosis of primary cultures of mouse renal proximal tubular (MPT) cells
induced by removal of growth factors. Apoptosis refers
to an energy-requiring, gene-directed cellular process,
which, if activated, results in cell ‘‘suicide’’ (11, 19). The
morphological and biochemical characteristics of cells
dying by apoptosis differ from those of cells dying by
necrosis. Necrotic cells swell and lyse, with cellular
material spilling into the extracellular space and inducing an inflammatory response. In apoptotic cells, on the
other hand, the nucleus and cytoplasm shrink and
fragment into plasma membrane-bound vesicles called
apoptotic bodies. Recognition and ingestion by phagocytes of intact membrane-bound apoptotic bodies or
even entire apoptotic cells serves to protect tissues from
an otherwise harmful exposure to the contents of dying
cells (11, 21).
The viability of most if not all cells depends upon
so-called ‘‘survival factors,’’ which induce intracellular
signals that prevent the cell from committing suicide by
apoptosis (19). Competition for survival signals provides a simple means to ensure that a balance exists
between cell division and cell death. If a given level of a
survival factor supports a certain number of cells, then
any increase in the number of cells would tend to
increase competition and result in increased cell death,
thereby returning cell number to its original value.
Correspondingly, a decrease in cell number would
permit division of cells back to the original number.
We examined the effect of LPA on the survival of MPT
cells based on the following observations. We have
recently shown that MPT cells cultured in the absence
of growth factors undergo apoptosis (unpublished observations). Apoptosis may be inhibited by renal growth
factors such as epidermal growth factor (EGF) and
insulin-like growth factor-I (IGF-I). However, neither of
these factors is as effective in inhibiting apoptosis as
calf serum. Since albumin-bound LPA may be responsible for much of the non-cytokine-mediated activity of
whole serum (14), we tested the role of LPA on the
survival and proliferation of MPT cells. We show, for
the first time in any cell type, that LPA alone can act as
a ‘‘survival factor’’ to inhibit apoptosis and that LPA is
as effective as serum in prolonging the survival of MPT
cell monolayers. We also provide novel evidence that
LPA is a potent growth factor for MPT cells.
In addition, on the basis of the results of a recent
study in which the survival activity of EGF, insulin,
nerve growth factor (NGF), and fetal bovine serum was
inhibited by wortmannin and LY-294002 (32), two
structurally dissimilar inhibitors of phosphatidylinositol 3-kinase (PI3K) (29, 30), we examined the role of
PI3K in mediating the effects of LPA. Our data indicate
that stimulation of proliferation and inhibition of apoptosis by LPA may be mediated through the enzyme
PI3K.
METHODS
Reagents
Bisbenzamide (Hoechst dye or H-33342) was obtained from
Calbiochem (San Diego, CA). Sodium oleoyl-L-a-lysophosphatidic acid (LPA) was obtained from Avanti Polar Lipids
(Alabaster, AL). Trypsin-EDTA was obtained from GIBCOBRL (Grand Island, NY). LY-294002 [2-(4-morpholinyl)-8-
0363-6127/97 $5.00 Copyright r 1997 the American Physiological Society
F575
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Levine, Jerrold S., Jason S. Koh, Veronica Triaca,
and Wilfred Lieberthal. Lysophosphatidic acid: a novel
growth and survival factor for renal proximal tubular cells.
Am. J. Physiol. 273 (Renal Physiol. 42): F575–F585, 1997.—
Lysophosphatidic acid (LPA) is the smallest and structurally
simplest of all glycerophospholipids. LPA is a normal constituent of serum and binds with high affinity to albumin while
retaining its biological activity. The effects of LPA are pleiotropic and range from mitogenesis to stress fiber formation. In
this report, we demonstrate two novel functions for LPA. LPA
acts as a survival factor to inhibit apoptosis of primary
cultures of mouse renal proximal tubular (MPT) cells. LPA
also acts as a potent mitogen for MPT cells. The ability of LPA
to act as both a survival factor and a mitogen is mediated by
the lipid kinase phosphatidylinositol 3-kinase (PI3K), since
these activities were completely blocked by wortmannin or
LY-294002, two structurally dissimilar inhibitors of PI3K.
The identification of LPA as a proliferative and anti-apoptotic
factor suggests a potential role for this lipid mediator during
the injury and/or recovery phases following tubular damage.
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LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
phenyl-4H-1-benzopyran-4-one] was obtained from Biomol
Research Laboratories (Plymouth Meeting, PA). All other
culture supplies and reagents, including trypan blue, wortmannin, and delipidated fraction V bovine serum albumin
(BSA), were obtained from Sigma.
Primary Culture of MPT Cells
Preparation of LPA
Fatty acid-free BSA was dissolved at 10 mg/ml in calciumand magnesium-free phosphate-buffered saline (PBS), through
which oxygen-free nitrogen had been bubbled for 20 min. LPA
was added to achieve a final concentration of 0.5 mg/ml (1.1
mM) and stored under argon at 270°C.
Induction of Apoptosis
Upon reaching confluence, MPT cell monolayers were
washed with PBS and incubated in full or growth factor-free
medium (experimental wells). Calf serum (10%) or varying
concentrations of LPA in the presence or absence of wortmannin or LY-294002 were added to experimental wells. The
effects of LPA on MPT cell survival were determined using
growth factor-free medium, because we have previously shown
that withdrawal of insulin and hydrocortisone induces apoptosis of MPT cells (unpublished observations).
Cell Viability Assay
Cell viability was assessed 10 days after addition of calf
serum or LPA. Cell viability was quantitated by counting the
number of viable cells, defined as cells that both remained
adherent to the culture dish and excluded trypan blue.
Nonadherent cells were removed by two washes with ice-cold
PBS. Adherent cells were harvested by incubation with 0.05%
trypsin/0.53 M tetrasodium EDTA for 10 min at 37°C. Trypsin
was neutralized by addition of DMEM containing 10% calf
serum. Cells were centrifuged for 5 min at 100 g and
resuspended in DMEM. Trypan blue (0.04 g/dl) was added for
10 min, and the number of viable cells excluding trypan blue
was counted in a hemocytometer. Cell viability was expressed
as the percentage of viable cells in experimental wells compared with that in freshly confluent monolayers.
Immunofluorescent Staining of Cells
The morphology of nuclear chromatin was assessed by
staining with H-33342, a supravital DNA dye with an excitation wavelength of 348 nm and emission wavelength of 479
nm (12). H-33342 enters live cells and therefore stains the
Flow Cytometry
MPT cells were stained with H-33342 using the same
technique described for immunofluorescent microscopy and
then placed immediately on ice. Flow cytometry was performed on an Epics ESP Flow Cytometer (Coulter Electronics,
Hialeah, FLA) with an ultraviolet (UV)-enhanced argon laser.
Hoechst fluorescence was accomplished by excitation with ,5
mW of UV laser light (351–364 nm multiline) and detected
with a 525-nm bandpass optical filter. Data were analyzed by
Epic Elite software (Coulter Electronics). A constant number
of events was analyzed for each sample (10,000 events/
sample).
We used two methods of flow cytometric analysis to distinguish normal cells from apoptotic cells, as follows.
Light scatter measurements. Normal cells are characterized by relatively high forward angle light scatter (a measure
of cell size or volume) and relatively low side angle light
scatter (a measure of cell granularity). Apoptotic cells, in
contrast, are smaller and more granular than normal cells
and are characterized by relatively low forward scatter and
high side scatter (25).
Intensity of H-33342 fluorescence. A number of investigators have used the difference in intensity of Hoechst fluorescence between normal (faint nuclear fluorescence) and apoptotic cells (bright nuclear fluorescence) to distinguish these
populations on flow cytometry (17, 18, 24).
We have analyzed our cells using both methods, i.e., by
comparing forward scatter (x-axis) with side scatter (y-axis)
as well as by comparing forward scatter (x-axis) versus
intensity of H-33342 fluorescence (y-axis). In both cases,
cellular debris was gated out on the basis of size (forward
scatter) and therefore not included among the 10,000 events/
sample.
Thymidine Incorporation
MPT cells were plated at 50,000 cells/well and incubated
overnight in growth factor-free medium. The medium was
then replaced with fresh growth factor-free medium alone or
growth factor-free medium supplemented with 10% calf serum or various concentrations of LPA. After an additional 24
h, 2 µCi of [3H]thymidine (2 Ci/mmol; New England Nuclear,
Boston, MA) were added to all wells. After 2 h, the cells were
washed three times with DMEM, then incubated with 2.0 ml
of ice-cold 5% trichloroacetic acid (TCA) for 1 h at 4°C. The
TCA was removed, and MPT cells were washed once with
fresh TCA. A quantity of 2.0 ml ice-cold ethanol containing
200 µM potassium acetate was added to each well for 5 min,
following which the cells were incubated twice in 2.0 ml of 3:1
mixture of ethanol:ether, for 15 min per incubation. After
allowing the monolayers to air dry, cells were solubilized in
1.0 ml of 0.1 N sodium hydroxide. [3H]thymidine counts per
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Cells were cultured from collagenase-digested fragments of
proximal tubules obtained from the cortices of kidneys of
C57Bl/6 mice by a modification of previously described methods (12). Cortical tubules were plated in serum-free, defined
culture medium [1:1 mixture of Dulbecco’s modified Eagle’s
medium (DMEM) and Ham’s F-12, containing 2 mM glutamine, 15 mM N-2-hydroxyethylpiperazine-N8-2-ethanesulfonic acid, 5 µg/ml transferrin, 5 µg/ml insulin, 50 nM
hydrocortisone, 500 U/ml penicillin, and 50 µg/ml streptomycin], hereafter denoted as ‘‘full medium.’’ Growth factor-free
medium is defined as full medium minus insulin and hydrocortisone. MPT cells grew to confluence from tubules over 4–5
days and were studied within 2 wk of achieving confluence.
Cell monolayers were previously shown to be of proximal
tubular origin by a combination of morphological, biochemical, and transport characteristics (12).
nuclei of viable cells, as well as cells that have died by
apoptosis or necrosis. Apoptotic cells may be distinguished
from viable and necrotic cells on the basis of nuclear condensation and fragmentation as well as increased fluorescent
intensity of nuclei stained with H-33342.
Adherent MPT cells (harvested as above) and cells that had
detached spontaneously from the monolayer were washed
once in PBS before staining with H-33342 (1.0 µg/ml) for 10
min at 37°C. Wet preparations of adherent and detached cells
were made on glass slides, and each field of cells was
photographed twice, under phase-contrast as well as fluorescence microscopy, for visualizing cell morphology and H-33342
nuclear staining in the same cells.
LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
F577
minute (cpm) were measured by adding samples to scintillation fluid and counting cpm using a Tri-Carb Liquid Scintillation Analyzer beta counter (model 1600TR; Packard Instrument, Meriden, CT).
more than one comparison was necessary, the Bonferroni
correction was used.
RESULTS
Statistics
Prolongation of MPT Monolayer Confluence by LPA
In each experiment, duplicate wells were examined, and
the results of duplicate wells were averaged. All data are
expressed as means 6 SE. Comparisons between the multiple
different graphs were made using a Student’s t-test. When
Freshly confluent MPT monolayers cultured in full
medium demonstrate the typical cobblestone appearance of epithelial cells and maintain confluence over 2
wk (Fig. 1A). In contrast, monolayers maintained in
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Fig. 1. Phase-contrast microscopy of mouse renal proximal tubular
(MPT) cells cultured in growth factor-free medium alone or growth
factor-free medium supplemented with calf serum, lysophosphatidic acid (LPA), or LPA plus LY-294002. A: freshly confluent cells
show the typical ‘‘cobblestone’’ appearance of normal epithelial
cells. B: most of the MPT cells cultured in growth factor-free
medium for 10 days have detached from the monolayer. Of the
remaining cells, many are smaller and more rounded, both characteristic features of apoptotic cells. C and D: in contrast, MPT cell
monolayers cultured for 10 days in growth factor-free medium
supplemented with either 10% calf serum (C) or 12 µM LPA (D)
remain fully viable and confluent. E: addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 (20 µM) blocked the
ability of LPA (12 µM) to maintain confluence of MPT monolayers
and resulted in cells having the morphological appearance of
apoptosis.
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LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
Fig. 2. Effect of LPA on cell viability. Mouse proximal tubular (MPT)
cell monolayers were cultured for 10 days in growth factor-free
medium alone or supplemented with 10% calf serum or 12 µM LPA.
The proportion of viable cells (adherent and excluding trypan blue) is
expressed as a percent of the absolute number of cells in freshly
confluent control monolayers (274 6 12 3 103 cells/well; n 5 6). * P ,
0.0001 compared with growth factor-free medium. The survival
effects of LPA and 10% calf serum are comparable.
Fig. 3. Dose response for effect of LPA on cell viability. MPT cell
monolayers were cultured for 10 days in growth factor-free medium
alone or supplemented with 12, 6, or 1 µM LPA. The proportion of
viable cells (adherent and excluding trypan blue) is expressed as a
percent of the absolute number of cells in freshly confluent monolayers (305 6 12 3 103 cells/well; n 5 6). * P , 0.001 compared with
growth factor-free medium.
of LPA (P , 0.005, for 12 and 6 µM LPA; P 5 0.25 for 1
µM LPA).
Inhibition of Apoptosis by LPA
The increased numbers of viable cells seen with
addition of LPA to growth factor-free medium may
potentially be the result of two factors, namely, increased production of MPT cells from stimulation of
proliferation and/or decreased loss of MPT cells from
inhibition of apoptosis. LPA has been shown to stimulate proliferation in a variety of cells (9, 14), but to our
knowledge, no studies have yet addressed a role for LPA
as a survival factor. As will be shown, both increased
proliferation and decreased apoptotic cell death seem to
contribute to the protective effect of LPA on MPT cells
in culture.
Fig. 4. Phase-contrast and fluorescence microscopy of H-33342stained MPT cells from freshly confluent MPT cells (A and B) and
MPT cells after culture in growth factor-free medium alone (C and D)
or supplemented with calf serum (E and F) or LPA (G and H). After
pooling adherent MPT cells with those that had detached from the
monolayer, cells were stained with H-33342, mounted onto glass
slides as wet preparations, and photographed under phase-contrast
(A, C, E, G) and fluorescence (B, D, F, and H) microscopy for
visualizing cell morphology and H-33342 nuclear staining in the
same cells. H-33342 staining of cells from freshly confluent monolayers (B) shows faint fluorescence of nuclei with a delicate chromatin
pattern. This is a normal nuclear morphology. These cells appear
normal under phase (A). In contrast, H-33342 staining of cells
cultured in growth factor-free medium for 10 days (D) shows nuclei
with varying morphology. The majority of nuclei are abnormal and
appear as brightly staining, homogeneous masses of varying size.
These morphological features are characteristic of apoptosis and
result from chromatin condensation and nuclear fragmentation.
Cells displaying apoptotic nuclear morphology may also be identified
as apoptotic by phase-contrast microscopy on the basis of decreased
cell size (C). Supplementation of growth-factor-free medium with
either 10% calf serum (F) or 12 mM LPA (H) prevents apoptosis and
results in a nuclear H-33342 staining pattern comparable to that
seen with freshly confluent cells.
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growth factor-free medium (full medium without insulin and hydrocortisone) fail to maintain confluence and
display typical features of apoptosis. Individual cells
become small and rounded before detaching from the
monolayer. The detachment of apoptotic cells occurs
over many days, resulting in a gradual but progressive
loss of the cell monolayer (Fig. 1B).
Apoptosis may be inhibited by the addition of calf
serum to growth factor-free medium (Fig. 1C). In
support of the idea that albumin-bound LPA accounts
for much of the non-cytokine-mediated biological activity of whole serum, the addition of LPA was as effective
as serum in maintaining confluence of MPT monolayers (Fig. 1D). The concentration of LPA used in these
experiments (12 µM) is within the range of concentrations normally found in serum (5–20 µM).
We next compared the protective effect of LPA to that
of calf serum by counting the number of viable MPT
cells remaining in the monolayer after 10 days of
culture (Fig. 2). In comparison with freshly cultured
MPT cells, only 27 6 4% of MPT cells were still viable
after 8–10 days of incubation in growth factor-free
medium. The addition of 10% calf serum or 12 µM LPA
to growth factor-free medium increased viability to
86 6 4% and 77 6 4%, respectively (n 5 6, P , 0.001 in
comparison to growth factor-free medium for each
condition). The survival effect of LPA was not different
from that of calf serum (P . 0.1).
A clear dose-response relationship existed between
LPA concentration and MPT cell survival (Fig. 3). Cell
survival, after 7–10 days of culture in growth factorfree medium containing LPA (12, 6, or 1 µM; n 5 6) was
77 6 4%, 55 6 4%, and 35 6 8%, respectively, compared
with only 27 6 6% for MPT cells cultured in the absence
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LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
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LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
Stimulation of Proliferation by LPA
We assessed directly the contribution of LPA-induced
proliferation of MPT cells to maintenance of confluence
by measuring [3H]thymidine incorporation as an index
of DNA synthesis (Fig. 8). MPT cells were plated at a
density of 50,000 cells/well and were cultured for 24 h
in growth factor-free medium alone or in growth factorfree medium to which 10% calf serum or LPA had been
added. [3H]thymidine was added for the final 2 h.
[3H]thymidine incorporation by confluent MPT monolayers incubated in growth factor-free medium was 563 6
56 disintegrations per minute (dpm) per well. In comparison, calf serum increased [3H]thymidine incorporation to 3,002 6 544 dpm/well (n 5 5, P , 0.02). LPA
increased [3H]thymidine incorporation in a dosedependent fashion (Fig. 8). Stimulation was maximal
at a concentration of 96 µM (1,346 6 164 dpm/well, P ,
0.01 vs. growth factor-free medium) and was increased
at a dose as low as 3 µM (n 5 5, P , 0.05 vs. growth
factor-free medium).
Thus, like calf serum, LPA inhibits apoptosis and
stimulates proliferation of MPT cells in culture. Therefore, maintenance of MPT cell monolayers by LPA is
likely the result of both increased proliferation and
decreased apoptotic cell death. It should be noted that
both effects of LPA were seen at concentrations as low
as 3 µM. As the normal serum concentration of LPA is
,5–20 µM, these data suggest that a significant portion of the effect of 10% serum may be attributable to
albumin-bound LPA.
Dependence of LPA-Induced Proliferation and
Inhibition of Apoptosis on PI3K
Finally, we determined the role of PI3K in mediating
the effects of LPA using wortmannin and LY-294002,
two structurally dissimilar inhibitors of PI3K (29, 30).
Addition of wortmannin (10 nM) blocked the ability of
LPA (12 µM) to maintain confluence of MPT monolayers (Fig. 1E). We confirmed this observation by counting the number of viable MPT cells remaining in the
monolayer after 8–10 days of culture (Fig. 9, A and B).
Both wortmannin (10 nM) (n 5 5) and LY-294002 (20
µM) (n 5 6) blocked the ability of LPA (12 µM) to
maintain MPT cell survival (Fig. 9, A and B). Since we
have shown that increased cell survival is attributable
both to increased proliferation and to inhibition of
apoptosis, we examined the effects of wortmannin and
LY-294002 on each of these processes separately. Inhibition of PI3K by either wortmannin or LY-294002 completely blocked the ability of LPA both to inhibit
apoptosis (Fig. 7, d and e) and to stimulate proliferation
(Fig. 10, A and B). Thus both LPA-induced proliferation
and inhibition of apoptosis appear to be mediated by a
PI3K-dependent signaling pathway.
DISCUSSION
In this report, we demonstrate that the glycerolbased phospholipid LPA can act as a survival factor to
inhibit apoptosis of MPT cells. This represents the first
report showing in any cell type that LPA alone, in the
absence of any other growth factor, is capable of inhibiting apoptosis. Thus inhibition of apoptosis should be
added to the growing list of biological activities attributable to LPA. These activities include such diverse
effects as focal adhesion assembly, stress fiber formation, platelet aggregation, smooth muscle cell contraction, and stimulation of tumor cell invasion (9, 14, 20).
We also demonstrate that LPA stimulates the proliferation of MPT cells. Although the proliferative effects
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We first determined the effect of LPA on apoptotic
loss of MPT cells. We have previously shown that
apoptotic MPT cells can be readily distinguished from
viable and necrotic cells by morphological criteria (12).
In comparison with viable cells, apoptotic cells are
smaller and show nuclear condensation and fragmentation upon staining with the cell-permeant supravital
DNA dye H-33342. Adherent MPT cells obtained from
freshly confluent monolayers (Fig. 4, A and B) demonstrated the faint chromatin staining pattern of normal
nuclei. In marked contrast, the majority of nuclei of
MPT cells cultured in growth factor-free medium (Fig.
4, C and D) were abnormal. Nuclei from cells at a
relatively early stage of apoptosis showed bright
H-33342 staining indicative of nuclear condensation
but maintained a relatively normal overall morphology.
Nuclei from cells at a later stage of apoptosis also
stained brightly but had undergone nuclear fragmentation as well. The addition of either calf serum (Fig. 4, E
and F) or LPA (Fig. 4, G and H) led to a dramatic
decrease in the number of MPT cells demonstrating
apoptotic morphology.
We used flow cytometry to confirm the inhibitory
effect of LPA on apoptosis (Fig. 7). MPT cells were
separated into viable and apoptotic populations on the
basis of size (forward scatter), granularity (side scatter), and intensity of H-33342 nuclear staining. Viable
cells were defined in left of Fig. 7 as those having
normal size and low granularity and in right of Fig. 7 as
those having normal size and faint H-33342 nuclear
staining. Apoptotic cells were defined in left of Fig. 7 as
those having decreased size and increased granularity
and in right of Fig. 7 as those having decreased size and
bright H-33342 staining. Electron microscopic examination of sorted populations based upon size and intensity
of H-33342 nuclear staining validated these definitions
(Figs. 5 and 6). Cells of normal size and faint H-33342
staining were uniformly viable (Figs. 5A and 6A),
whereas cells of decreased size and bright H-33342
staining showed predominantly apoptotic morphology
(Figs. 5B and 6, B and C). Comparable micrographs
were obtained when cells were sorted on the basis of
size and granularity.
Using these definitions, we found the percentage of
viable cells was only 16% when cells were cultured in
growth factor-free medium (Fig. 7a). The addition of
either calf serum (Fig. 7b) or LPA (Fig. 7c) markedly
increased the percentage of viable cells to 60% and 59%,
respectively. Combined with Fig. 4, these data demonstrate that the increased survival of MPT monolayers
in the presence of LPA is at least in part attributable to
inhibition of apoptosis.
LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
F581
of LPA are well documented in other cell types (9, 14),
including vascular smooth muscle cells, fibroblasts,
Jurkat T cells, and keratinocytes, ours is the first
report to demonstrate that LPA is a trophic factor for
renal tubular cells. Although most biological effects of
LPA are mediated by nanomolar concentrations of the
phospholipid, LPA-stimulated proliferation has been
found to require micromolar concentrations (9). This
was true in our study as well, in which stimulation of
proliferation, as well as inhibition of apoptosis, required micromolar concentrations of LPA. It is unclear
why these two biological actions should be seen only
with higher concentrations of LPA, but it has been
suggested that the reason may relate to loss of activity
from degradation or oxidation given the prolonged
incubation times needed to study proliferation and
apoptosis (9).
On the basis of inhibition studies with wortmannin
and LY-294002, we suggest that both stimulation of
proliferation and inhibition of apoptosis by LPA are
mediated via PI3K activation. We base this conclusion
on the following lines of evidence. At the nanomolar
concentrations used in this study, wortmannin is an
irreversible and noncompetitive inhibitor of PI3K (29).
Inhibition appears to be highly specific for PI3K, as
wortmannin does not inhibit the function of a wide
variety of protein and lipid kinases, including protein
kinases A, C, and G and the structurally related
phosphatidylinositol 4-kinase (15, 29, 31). Nonspecific
effects of wortmannin, such as inhibition of myosin
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Fig. 5. Electron microscopy (low-power
view) of MPT cells sorted by flow cytometry on the basis of forward light scatter (size) and intensity of H-33342
nuclear staining. On low-power view,
cells characterized on flow cytometry
by normal size and faint H-33342
nuclear staining all demonstrate normal, viable morphology (A, 34,200 magnification). In contrast, cells characterized on flow cytometry by decreased
size and bright H-33342 nuclear staining are predominantly apoptotic (B,
34,200 magnification).
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LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
light chain kinase, occur only at 100-fold higher concentrations than those used in this study (26, 29, 31).
Moreover, we obtained identical results using LY294002, a recently developed alternative inhibitor of
PI3K. As for wortmannin, the effective concentration of
LY-294002 (20 µM) is consistent with its activity as a
PI3K inhibitor (30). Since wortmannin and LY-294002
are structurally dissimilar (29, 30), it is unlikely that
their effects in our study can be attributed to any
nonspecific interactions. It remains possible, however,
that wortmannin and LY-294002 also inhibit an as yet
uncharacterized member of the PI3K family and that
inhibition of this family member, rather than inhibition
of PI3K itself, is responsible for the effects of these
inhibitors (cf. below). Moreover, our data do not address
the issue of whether signaling events downstream of
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Fig. 6. Electron microscopy (high-power
views) of MPT cells sorted by flow cytometry on the basis of forward light
scatter (size) and intensity of H-33342
nuclear staining. The MPT cell shown
in A (36,900 magnification) is a normal, viable MPT cell sorted with the
population of cells characterized by normal size and faint H-33342 staining on
flow cytometry. Representative cells
from the population sorted by flow cytometry on the basis of decreased size
and bright H-33342 nuclear staining
(B, 36,900 magnification; and C,
311,800 magnification) are both apoptotic. The apoptotic cell shown in B is
slightly smaller than the normal cell
(A), whereas the apoptotic cell (C) is
about one-half the size of the normal
cell (magnification for C is almost 2-fold
greater than for A). In addition to being
smaller than normal, both apoptotic
cells (B and C) demonstrate chromatin
condensation and nuclear fragmentation (both are indicated by arrowheads)
that are classic morphological features
of apoptosis. Other features of apoptosis demonstrated by B and C include
morphologically intact plasma membranes that have lost their microvilli
and preservation of the structure of
mitochondria and other subcellular organelles.
PI3K affect cell proliferation and apoptosis by the same
or separate pathways.
Although the mechanism by which receptor tyrosine
kinases (such as those for EGF or IGF-I) activate PI3K
is understood, the mechanism for G protein-coupled
receptors, such as that for LPA, remains unclear (9, 14,
29). PI3K is a heterodimer consisting of a 110-kDa
catalytic subunit (p110a and p110b) and an 85-kDa
regulatory subunit (p85a and p85b). Activation of PI3K
by receptor tyrosine kinases occurs following recruitment of PI3K to membrane-bound signaling complexes.
Recruitment is mediated by the binding of src-homology 2 domains within the p85 regulatory subunit to
specific phosphotyrosine residues. In contrast, induction of PI3K activity by LPA may involve the recently
characterized p100g isotype of the catalytic subunit
LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
(23). The p100g isotype does not interact with p85, and,
at least in vitro, is activated by directly associating
with G proteins.
PI3K has been implicated in a wide range of cellular
processes such as mitogenesis, differentiation, membrane ruffling, and vesicular trafficking (10). A role for
PI3K in regulation of apoptosis has emerged from a
recent study in which inhibition of PI3K activity by
wortmannin or LY-294002 blocked the ability of NGF,
EGF, insulin, and serum to prevent apoptosis of PC12
cells in culture (32). An attractive downstream target of
PI3K that may be responsible for stimulation of proliferation and inhibition of apoptosis is the 70-kDa S6
kinase (pp70S6k ) (1). Activation of pp70S6k is an established part of the mitogenic response and leads to the
phosphorylation of the S6 polypeptide of the 40S ribosomal subunit. Full activation of pp70S6k depends not
only on PI3K but also on phospholipase Cg and the
rapamycin-inhibitable enzyme, FK506 binding proteinrapamycin-associated protein. In this regard, it is
noteworthy that administration of rapamycin enhanced apoptosis in several different cell lines (22).
These data are consistent with the view that pp70S6k is
a critical target of PI3K in inhibiting apoptosis.
An alternative explanation for the effects of wortmannin and LY-294002 in this study relates to the fact that
the double-stranded DNA-dependent protein kinase
(DNA-PK) has a PI3K domain and is inhibitable by
wortmannin, albeit at concentrations about 100-fold
greater than those used in this study (8). Given the role
of DNA-PK in repair of double-stranded DNA breaks, it
is tempting to speculate whether there exist other
members of this family that are inhibitable by lower
concentrations of wortmannin and that participate in
regulation of apoptosis via recognition and/or repair of
damaged DNA.
Although our inhibition studies focus attention on
PI3K, stimulation of proliferation and inhibition of
apoptosis by LPA may also be viewed in a larger
context. Virtually all cell types depend upon so-called
‘‘survival factors,’’ which deliver a signal that prevents
the cell from committing suicide by apoptosis. Such
survival factors frequently derive from paracrine or
autocrine secreted growth factors, but in the case of
adhesive cells such as MPT cells, survival factors also
Fig. 8. Dose-response curve for effect of LPA on thymidine uptake.
Confluent MPT monolayers were cultured for 24 h in growth factorfree medium alone or supplemented with 10% calf serum and LPA (1
to 96 µM). [3H]thymidine was added for the final 2 h. Data are
presented as the percent increment above baseline [3H]thymidine
uptake seen for monolayers cultured in growth factor-free medium
(n 5 5). * P , 0.002 compared with growth factor-free medium. † P ,
0.05 compared with 3 µM LPA. ¶ P , 0.05 compared with 12 µM LPA.
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.5 on June 17, 2017
Fig. 7. Flow cytometric analysis of MPT cells cultured in growth
factor-free medium alone (a) and supplemented with serum (b), LPA
(c), or LPA plus inhibitors of PI3K (d and e). After pooling adherent
MPT cells with those that had detached from the monolayer, cells
were stained with H-33342 and sorted by flow cytometry on the basis
of size (forward scatter), granularity (side scatter), and log fluorescent intensity of H-33342 staining. Viable cells were defined on left as
those having normal size and granularity (region R1) and on right as
those having normal size and faint H-33342 staining (bottom right
quadrant). Apoptotic cells were defined on left as those having
decreased size and increased granularity and on right as those
having decreased size and bright H-33342 staining (top left quadrant). The percentage of viable cells after 10 days in growth factorfree medium is ,20% (a). Supplementation with either 10% calf
serum (b) or 12 µM LPA (c) improves viability to ,60%. Addition of
the PI3K inhibitors, wortmannin (10 nM) (d) or LY-294002 (20 µM)
(e), completely blocked the ability of LPA (12 µM) to maintain
viability of MPT cells. This flow cytometric analysis is representative
of 5 experiments.
F583
F584
LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
Fig. 9. Effect of the PI3K inhibitors
wortmannin and LY-294002 on LPAmediated cell survival. MPT cell monolayers were cultured for 10 days in
growth factor-free medium alone or
supplemented with 10% calf serum, 12
µM LPA, PI3K inhibitor alone, or 12
µM LPA plus the PI3K inhibitor. Data
for wortmannin (W, 10 nM) are shown
in A (n 5 5), and data for LY-294002
(LY, 20 µM) are shown in B (n 5 6). The
proportion of viable cells (adherent and
excluding trypan blue) is expressed as a
percent of the absolute number of cells
in freshly confluent control monolayers
(A, 301 6 17 3 103 cells/well; B, 283 6 2
cells/well). * P , 0.01 compared with
growth factor-free medium. †P , 0.01
compared with LPA.
Fig. 10. Effect of the PI3K inhibitors
wortmannin and LY-294002 on LPAmediated thymidine uptake. MPT cell
monolayers were cultured for 10 days
in growth factor-free medium alone or
supplemented with 10% calf serum, 12
µM LPA, PI3K inhibitor alone, or 12
µM LPA plus the PI3K inhibitor.
[3H]thymidine was added for the final 2
h. Data are presented as the absolute
[3H]thymidine uptake (dpm/well). Data
for wortmannin (W, 20 nM) are shown
in A (n 5 8), and data for LY-294002
(LY, 20 µM) are shown in B (n 5 3).
* P , 0.01 compared with growth factorfree medium. †P , 0.05 compared with
LPA.
it is noteworthy that adhesion-dependent cell cycle
progression and inhibition of apoptosis show similar
signaling requirements (4, 13). Finally, in a fibroblast
model of c-Myc-induced apoptosis, only those cytokines
known to be important for traversal of G1 checkpoints
(i.e., PDGF and IGF-I) were able to inhibit apoptosis,
even when these cells were growth arrested in the S
phase of the cell cycle (6).
In summary, we have provided novel data showing
that the glycerol-based phospholipid LPA not only
stimulates the proliferation of MPT cells in culture but
can also itself act as a survival factor to inhibit apoptosis. Both effects of LPA appear to be mediated via the
intracellular lipid kinase PI3K. Given recent interest in
the contribution of apoptosis to the pathogenesis of
renal diseases resulting from injury to renal tubular
epithelial cells (11) and the protective effect of growth
factors following experimental acute renal failure, the
identification of LPA as a proliferative and antiapoptotic factor suggests a potential role for this lipid
mediator during the injury or recovery phases of tubular damage. Moreover, although activated platelets are
the best characterized source of LPA, injured cells may
also release LPA (9, 14) and thus provide an autocrine
or paracrine survival and proliferative factor following
renal injury.
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.5 on June 17, 2017
include adhesive signals from the extracellular matrix
or intercellular contact (3, 4, 7). In this regard, activation by LPA of the Ras-related cytoplasmic GTPbinding protein Rho assumes importance (5, 14, 20).
LPA-mediated activation of Rho potently induces the
assembly of actin stress fibers and the formation of
focal adhesion complexes, which are involved in cellmatrix and some cell-cell interactions (5, 20).
Combined with the data presented in this report,
several facts induce us to hypothesize that an intimate
connection exists between survival factors, cytoskeletal
rearrangement, and cell cycle progression. First, many
potent survival factors, such as platelet-derived growth
factor (PDGF), EGF, and IGF-I, have all been shown to
induce profound changes in the actin cytoskeleton,
mediated via various members of the Rho family of
GTP-binding proteins (5, 20). Second, as shown in a
recent study, activation of Rho may itself play a significant role in progression through the G1 phase of the cell
cycle (16). Microinjection of Rho into Swiss 3T3 fibroblasts stimulated cell progression through G1, whereas
microinjection of a dominant negative form of Rho
blocked traversal of G1 by serum-stimulated cells.
Third, not only can signals derived from the extracellular matrix act as survival signals in some cells (3), but
adhesion is necessary in many of these same anchoragedependent cells for progression through G1 (4). Indeed,
LPA IS A GROWTH AND SURVIVAL FACTOR FOR RENAL CELLS
We are grateful to John Daley for technical assistance in flow
cytometric analysis.
This work was supported by National Institutes of Health Grants
AR/AI-42732 (to J. S. Levine), DK-375105 (W. Lieberthal), and
HL-53031 (W. Lieberthal); by American Cancer Society Grant IN97-N
(to J. S. Levine); and by a Young Investigator Award from the
National Kidney Foundation (to J. S. Levine).
Address for reprint requests: J. S. Levine, Renal Section, E428,
Boston Medical Center, 88 East Newton St., Boston, MA 02118
(E-mail: [email protected]).
Received 2 January 1997; accepted in final form 26 June 1997.
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