The Whittier Institute for Diabetes and Endocrinology, Department of Pediatrics, University of California at San Diego, School of Medicine, La Jolla, California 92037
Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor-induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.
The identification of mechanisms involved in the formation and function of the mammalian endocrine
system is one of the most important issues in developmental biology. The differentiation and growth of endocrine organs can be regulated at several levels. One level is
the regulation of reactions controlled by intracellular signal messengers. This type of regulation provides a different gene expression response to various external stimuli,
which is critical for rates of hormone biosynthesis and release.
Phosphatidylinositol 3-kinase (PI3K)1 is a ubiquitous
enzyme that has been shown to be an important mediator
of intracellular signaling in mammalian cells. To date, the
expanding family of mammalian PI3K consists of three
members, each containing a different p110 catalytic subunit (Hiles et al., 1992 While the process of endocrine cell differentiation has
been extensively studied, no specific intracellular signaling
pathway directly involved in regulating expression of endocrine-specific genes has been identified. Because of the
role of PI3K in mitogenesis, differentiation, and stimulussecretion pathways, we have investigated the possibility
that this enzyme regulates endocrine differentiation in
mammalian cells. Until recently, most of the studies addressing the role of PI3K in cellular proliferation and differentiation were undertaken using a variety of cell lines
and transfection methodologies. Such transformed cells
are capable of indefinite replication in culture and express
only some of the differentiated properties of their cell of
origin. Thus, these approaches provide only limited information about the potential link between PI3K activity and
development. With the identification of the drugs wortmannin (Powis et al., 1994 We now report that wortmannin or Ly294002 blockade
of PI3K activity significantly increased the number of hormone-producing cells growing in ICCs. These unexpected
results indicate that PI3K plays a role as a negative regulator of cellular differentiation during fetal neogenesis of endocrine system.
Tissue Culture
The use of human fetal tissue for these studies was reviewed and approved
by the Institutional Review Board at our university. Human fetal pancreases at 18-24 gestational wk were obtained with appropriate permissions
and patient consent through nonprofit organ procurement programs (Advanced Bioscence Resources, Oakland, CA; Anatomic Gift Foundation,
Laurel, MD). Experiments were started by the enzymatic digestion with
collagenase P (Boehringer Mannheim Corp., Indianapolis, IN) of the human fetal pancreases followed by tissue culture for 5 d, which led to the
formation in vitro of ICCs, as previously described in detail (Otonkoski et al., 1993 Isolation of Fetal Pancreatic Islets
Effects of PI3K inhibitors on endocrine function were tested not only in
ICCs, but also in purified islets. Undifferentiated epithelial cells account
for ~75-80% of the total cell mass in ICCs. By contrast, purified fetal islets contain about 10-fold fewer undifferentiated epithelial cells (Beattie
et al., 1996b RNA Isolation and Analysis
Transcriptional analyses on total RNA (0.5 µg) were performed using a
multiprobe ribonuclease protection assay, as previously described (Otonkoski et al., 1993 Insulin Content, Insulin Secretion, and DNA Synthesis
After incubation with various factors in culture, the ICCs were harvested,
and measurements of insulin content, insulin release in response to glucose plus theophylline, and [3H]thymidine incorporation into DNA were
performed as described previously (Otonkoski et al., 1993 PI3K Assay
Aliquots of cell lysates normalized for protein content were incubated for
3 h with anti-PI3K antibodies directed against the 85-kD regulatory subunit or with antiphosphotyrosine antibodies (Upstate Biotechnology, Inc.,
Lake Placid, NY). The immune complexes were absorbed onto protein
A-Sepharose and washed as described (Ptasznik et al., 1995 Determination of Total Cellular PIP3
The ICCs, which were pretreated for 5 d with PI3K inhibitors, NIC, HGF/
SF, or control buffer, were subsequently harvested and suspended at a
concentration of 2 × 105 cells/ml in buffer A (30 mM Hepes, pH 7.2, 110 mM
NaCl, 10 mM KCl, 1 mM MgCl2, 10 mM glucose), and 1 mCi/ml [32P]orthophosphate (HCl-free; DuPont/NEN) was added. The cells were incubated at 37°C for 2 h and then washed 3× with buffer A. Depending on
the type of experiment, the labeled islet cells were either directly lysed by
addition of 3 ml chloroform/methanol (1:2, vol/vol), followed by 4 ml chloroform/2.4 M HCl (1:1, vol/vol) (to measure basal levels of PIP3), or labeled cells were first stimulated with 25 ng/ml HGF/SF for the indicated
times, and subsequently the reaction was stopped as above (to measure
the inducible levels of PIP3). Phospholipids were extracted and analyzed
by thin-layer chromatography, and the total cellular PIP3 was quantified,
as we described previously in detail (Ptasznik et al., 1996 Triple Immunofluorescence and Confocal Microscopy
ICCs cultured for 5 d in either control medium or medium containing 10 µM Ly294002 were paraffin embedded, and 5-µm sections were stained
for hormone immunoreactivity. To simultaneously identify cells producing
insulin, glucagon, somatostatin, and pancreatic polypeptide (pp), we followed a modification of our previous protocol for multiple labeling (Otonkoski et al., 1996 Morphometric Analysis and Statistics
Sections were prepared from control and Ly294002-treated ICCs from
three independent experiments. After immunostaining, confocal images
were acquired from 57 control and 61 Ly294002-treated ICC sections. All
images collected (one image per section) were then analyzed for total surface area and insulin-, glucagon-, somatostatin-, and pp-positive cell surface area by using measurement tools in the software NIH Image 1.60 (National Institutes of Health, Bethesda, MD). Data were analyzed in Stat
View 4.01 (Abacus Concepts, Inc., Berkeley, CA) for calculation of mean,
standard deviation, and parametric statistic (t test).
Inhibition of PI3K Increases Islet-specific Hormone
Biosynthesis and Hormone Secretion in Developing
Fetal Pancreatic Cells
To investigate whether PI3K activation is important for
endocrine differentiation of human fetal pancreatic cells,
we continuously treated ICCs for 5 d with 100 nM wortmannin or 10 µM Ly294002, concentrations that block over
90% of total PI3K activity in intact fetal islet cells (data not
shown; see Fig. 7). We established that these concentrations of wortmannin and Ly294002 almost completely inhibited the rise in PIP3 formation stimulated by growth
factors in intact [32P]orthophosphate-labeled islet cells.
By contrast, these concentrations of inhibitors did not affect significantly the ratio of [32P]PIP2 to [32P]PIP and
[32P]PIP to [32P]PI in phospholipid labeling experiments
where PIP3 levels were measured, implying that other kinases (PI5K and PI4K) were not inhibited under these
conditions. Wortmannin, a fungal metabolite, functions as
a covalent inhibitor of the catalytic p110 subunits of PI3Ks
at nanomolar concentrations, whereas Ly294002, a structurally and mechanistically distinct compound, functions
as a noncovalent, competitive inhibitor of PI3Ks at 100fold higher concentrations than wortmannin (Okada et al.,
1994
We measured the transcriptional expression of islet-specific hormone genes in ICCs growing for 5 d in the presence of PI3K inhibitors. As shown in Fig. 1, wortmannin
and Ly294002 increased the transcriptional levels of insulin, glucagon, and somatostatin in cells within the ICCs.
The pattern of alterations of mRNA levels was strikingly
similar to that of insulin protein (see below). Therefore,
these data indicate that two structurally distinct compounds have similar effects on hormone transcription as a
consequence of their shared ability to function as specific
inhibitors of PI3K. The inhibitors had no effects on cyclophilin mRNA, which was used as an internal control. The
quantitative analysis of islet hormone transcription levels,
after normalization to cyclophilin expression, is shown in
Fig. 1 C. To better understand the effect of these PI3K inhibitors on islet hormone gene expression, we have compared the effect of PI3K inhibitors alone to the NIC-induced increase in expression of islet-specific hormone genes
(Otonkoski et al., 1993
We also measured the insulin content and insulin secretion per cellular DNA in fetal ICCs cultured for 5 d in
the presence of the PI3K inhibitors. As shown in Fig. 2, A
and B, we found that both insulin content and secretion
were significantly increased in Ly294002-treated cells, as
compared to untreated control cells. Wortmannin also induced increases in these parameters, but to a lesser extent.
(Wortmannin appears to be a much less stable agent than
Ly294002 in culture medium [Kimura at al. 1994] [data not
shown].) The pattern of alterations of insulin secretion was almost identical to that of insulin content, indicating a close functional association between these two parameters. This
would suggest that a continuous blockade of PI3K activity
could secondarily increase insulin secretion through potent stimulation of insulin biosynthesis in developing islet
cells. Consistent with this, we observed no direct effect of
PI3K inhibitors on insulin secretion when islet cells were
treated with these inhibitors for a short time (0.5 and 2 h;
data not shown). No significant differences were observed
in insulin content under these conditions. The observation that short-term inhibition of PI3K does not significantly
affect insulin secretion was also recently shown in rat islets
and insulin-secreting
To determine whether PI3K inhibitors increase the proportion of islet cells expressing hormones, confocal immunofluorescent detection of all islet-specific hormones, followed by morphometric analysis of the ICCs, was carried
out. Fig. 3 shows representative fields of a microscopic
analysis performed on sections from human fetal ICCs,
cultured with or without Ly294002 for 5 d. Only a few hormone-positive cells were visible in ICCs cultured in control medium. By contrast, hormone-positive cells were more
common in ICCs cultured with the PI3K inhibitor (4.4-fold
increase in the total percentage of endocrine-positive cells
in Ly294002-treated ICCs, as compared to control ICCs).
Interestingly, in Ly294002-treated ICCs, we detected more
cells positive for more than one protein, indicating that the
PI3K inhibitor can trigger activation of multiple hormonespecific genes in a single cell. It was previously shown that
coexpression of multiple hormones represents an early step in the endocrine differentiation program of islet cell progenitors (Alpert et al., 1988
Inhibition of PI3K Decreases DNA Synthesis in Fetal
Pancreatic Cells
We measured DNA synthesis in fetal ICCs cultured in the
presence of the PI3K inhibitors. As shown in Fig. 4 A, wortmannin and Ly294002 significantly decreased the [3H]thymidine incorporation into DNA in cells within the ICCs.
Treatment with the combination of the PI3K inhibitor and
NIC resulted in a synergistic decrease in DNA synthesis.
To make certain that the observed increase in islet-specific
hormone expression in PI3K inhibitor-treated cells is not
secondary to nonspecific blockade of the cell cycle, we measured insulin protein expression in mitomycin C-treated
or serum-starved cells. As shown in Fig. 4, B and C, neither mitomycin C-induced blockade of DNA synthesis nor
serum starvation affected insulin protein expression in
ICCs. Consistent with this observation, neither mitomycin
C, which is known to interact directly with DNA (Tomasz
et al., 1987
HGF/SF or NIC-dependent Regulation of Endocrine
Differentiation Is Inversely Related to PI3K Activity
To clarify the correlation between PI3K activity and endocrine-specific gene expression, we examined whether any
of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in islet cells. Mesenchyme-derived HGF/SF is a physiological
modulator of endocrine differentiation in human fetal islet
cells. When fetal ICCs were induced to proliferate by the
addition of HGF/SF, a marked downregulation of both insulin and glucagon gene transcription, as well as insulin
protein biosynthesis, was observed (Beattie et al., 1996a
In contrast to HGF/SF, NIC is known to increase both
insulin content and insulin release in ICCs (Sandler et al.,
1989 The cellular signaling pathways that are required for endocrine differentiation are unknown. The present studies
clearly indicate that lipid products of PI3K are an important part of the regulatory network that links differentiation signals at the cell surface of endocrine precursors to
transcriptional responses in the nucleus. We demonstrate
a previously unrecognized function for PI3K The basis for the interaction of cytoplasmic phospholipid messengers with transcriptional factors in the nucleus
is, at present, unknown. Only a few potential biochemical
targets of phosphoinositides have been found in mammalian cells (for review see Carpenter and Cantley, 1996 The data presented here strongly suggest that the functional association between PI3K activity and islet-specific
gene expression is part of a more general developmental
program that coordinates cell differentiation and cell division. It has previously been suggested that the general
function of HGF/SF is to allow various epithelial cells to
rearrange during embryogenesis by promoting their proliferation, scattering, and invasiveness (Brinkmann et al.,
1995 It is known that external stimulation of islet cells with
insulin inhibits insulin gene expression in these cells (Koranyi et al., 1992 It has been shown that during development, multipotential epithelial stem cells give rise to all islet cell phenotypes
(Teitelman and Lee, 1987 In conclusion, our results describe a role of PI3K in regulating development of the human endocrine system. Interestingly, it was shown just recently that inhibition of the
PI3K displays a stimulatory effect on melanogenesis and
dendrite outgrowth in B16 murine melanoma cell line
(Busca et al., 1996; Hu et al., 1993
; Stoyanov et al., 1995
).
Upon activation, PI3K phosphorylates inositides at the D-3
position of the inositol ring to generate such lipid messengers as: PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (see
abbreviations footnote for explanation of nomenclature). The exact role and downstream molecular targets of these
lipid products are unknown. However, it is known that
overall increases in the levels of these messengers correlates with mitogenic signaling by growth factors (Cantley
et al., 1991
), secretion, and vesicle trafficking (Brown et al.,
1995
), as well as chemotaxis, cell shape changes, and membrane ruffling (Traynor-Kaplan et al., 1988
; Eberle et al.,
1990
; Wennstrom et al., 1994
). PI3K was reported to be
important for the regulation of insulin receptor-induced intracellular pathways, including glucose transport (Backer
et al., 1993
). Similarly, members of the seven transmembrane-spanning receptor family, hormone and sensory receptor system in mammalian cells, were recently shown to
use PI3K to transduce signals to the interior of the cell and
to assemble the ras activation complex (Ptasznik et al.,
1995
, 1996; Touhara et al., 1995
). Several studies have
shown that the PI3K lipid products are signaling intermediates in the induction of cellular differentiation of PC12
pheochromocytoma cells (Hempstead et al., 1992
; Kimura
et al., 1994
) as well as of adipocytic 3T3-F442A cells (Saad
et al., 1994
), suggesting that this enzyme may function as a
positive regulator of cellular differentiation in these cell
lines.
) and Ly294002 (Vlahos et al.,
1994
) as potent PI3K inhibitors, it became possible to directly inhibit the endogenous PI3K activity in cultured primary cells. In the present experiments, we have used, as a
model for endocrine differentiation, human fetal-derived
pancreatic cells, growing in vitro as islet-like cell clusters
(ICCs) (Sandler et al., 1989
). The cellular composition of
ICCs consists mostly of undifferentiated epithelial cells
(~80%) containing putative precursors of the hormoneproducing cells (Sandler et al., 1989
; Otonkoski et al.,
1993
; Beattie et al., 1994
). Endocrine cells developing in
vitro within ICCs originate from undifferentiated, pluripotent epithelial cells and contain insulin-producing
cells and the three other cell types,
,
, and pp, secreting glucagon, somatostatin, and pancreatic polypeptide, respectively. An advantage of this model system is the ability to
mimic steps of the differentiation process in cell culture, as
evidenced by the fact that after being transplanted into
athymic nude mice, ICCs develop into morphologically and functionally mature endocrine tissue (Sandler et al.,
1985
; Beattie et al., 1994
).
Materials and Methods
). The cells, cultured for 5 d, were treated continuously with 10 µM
Ly294002 (Calbiochem Corp., La Jolla, CA), 100 nM wortmannin (Sigma
Chemical Co., St. Louis, MO), 10 mM nicotinamide (NIC) (Sigma Chemical Co.), or 25 ng/ml hepatocyte growth factor/scatter factor (HGF/SF) (a
kind gift of J.S. Rubin, National Cancer Institute, Bethesda, MD). These
concentrations of wortmannin and Ly294002 were found to be effective in
inhibiting PI3K activity in our preliminary, dose-response experiments.
The effective doses for NIC and HGF/SF as modulators of endocrine differentiation in fetal islet cells were established in our laboratory previously (Otonkoski et al., 1993
; Beattie et al., 1996a
). PI3K inhibitors, NIC,
or HGF/SF was added to the culture medium from the very beginning. Medium plus factor was changed every day for 5 d. For direct comparison, portions of the same pancreases were grown, treated with factors, and
used for transcriptional analyses, insulin content and secretion, PI3K activities, and DNA synthesis.
). Purification of human fetal pancreatic islets was performed
as recently described (Beattie et al., 1996b
). Fetal islets, identified as homogeneous differentiated clusters of dithizone-positive cells, were incubated with factors as above.
). The housekeeping gene cyclophilin was used as an internal control, and yeast tRNA (10 µg) was included as a negative control.
Probes used were of human origin and were described previously (Otonkoski et al., 1993
). Target RNAs were quantitated in autoradiographs by
scanning densitometry (LKB UltroScan XL Laser) and integrated using
Gel Scan XL software (Pharmacia LKB Biotechnology, Inc., Piscataway,
NJ). The probe-specific mRNA signals were normalized to the cyclophilin
signal in each sample to account for differences in sample loading between
lanes.
).
). PI3K assays
were performed directly on beads. Briefly, the reaction was carried out for
10 min in a buffer containing 40 mM Hepes, pH 7.2, 6 mM MgCl2, 1 mM
EDTA, 10 µg of PI (Avanti Polar Lipids, Alabaster, AL), 10 µM ATP,
and 10 µCi [
-32P]ATP (6,000 Ci/mmol; DuPont/NEN, Wilmington, DE).
Adenosine (0.2 mM) was added to the reaction mixture to inhibit residual
PI4-kinase activity. After the incubation, the reaction was stopped with
methanol plus 2.4 N HCl (1:1, vol/vol), and lipids were extracted, analyzed
by thin-layer chromatography, and quantified as described previously
(Ptasznik et al., 1995
). In some experiments, the direct binding of PI3K to
p190Met after HGF/SF stimulation of islet cells was determined, as previously described in detail (Graziani et al., 1991
).
).
). Briefly, sections were incubated for 1 h at room temperature with a mixture of primary antibodies: IgG fraction of a sheep
anti-human insulin antiserum (The Binding Site, Birmingham, England)
(5 µg/ml), mouse monoclonal anti-human glucagon (Sigma Chemical Co.)
(1 µg/ml), rabbit anti-human somatostatin antiserum (Dako Corporation,
Carpinteria, CA) (used at 1:100 dilution), and rabbit anti-human pp antiserum (Chemicon International, Inc., Temecula, CA) (used at 1:100 dilution). In separate sections, a mixture of normal sheep, rabbit, and mouse IgGs was used as control reference for specificity of primary antibodies.
After washings in PBS-DS (5 mM glycine, 0.2% donkey serum, 0.1% BSA),
sections were incubated for 1 h at room temperature with a cocktail of
F(ab
)2 fractions from secondary antibodies: lissamine-rhodamine-conjugated affinity-purified donkey anti-sheep IgGs (5 µg/ml); FITC-conjugated affinity-purified donkey anti-rabbit IgGs (5 µg/ml); and indodicarbocyanine-conjugated affinity-purified donkey anti-mouse IgGs (5 µg/ml).
All F(ab
)2 were preadsorbed on appropriate multiple species to eliminate
the possibility of cross-reactivity in multiple labeling protocols (Jackson
ImmunoResearch Labs, Inc., West Grove, PA). The sections were processed as previously described (Otonkoski et al., 1996
) and viewed on a laser scanning confocal microscope (model MRC-1024; BioRad Laboratories, Hercules, CA).
Results
; Powis et al., 1994
; Vlahos et al., 1994
; Wymann et
al., 1996
). At nanomolar concentrations, wortmannin is
thought to be selective for PI3K. Ly294002, even at micromolar concentrations, is quite specific for PI3K and does not affect PI4K or a number of intracellular Ser/Thr and
Tyr kinases (Vlahos et al., 1994
). Finally, we have also
shown that continuous treatment for 5 d with 100 nM wortmannin or 10 µM Ly294002 does not cause notable cytotoxity nor induce apoptosis in fetal pancreatic cells growing as islet-like cell clusters (data not shown).
Fig. 7.
Effect of continuous treatment for 5 d with
NIC, wortmannin, or
Ly294002 on cellular PI3K
activity in fetal ICCs. Fetal
islet cells were incubated for
5 d with control medium
(Control), 10 mM nicotinamide (NIC), 100 nM wortmannin (W), 10 µM
Ly294002 (LY) or combined
NIC plus inhibitor. PI3K activity in p85 precipitates from
islet cells was analyzed as described in Materials and
Methods. Results shown are
representative of two independent experiments.
[View Larger Version of this Image (38K GIF file)]
). NIC is an inhibitor of the enzyme
poly(ADP-ribose) synthetase and can potently induce human fetal islet cell differentiation by influencing the transcription of DNA (Yonemura et al., 1984
; Sandler et al.,
1989
). Treatment with the combination of a PI3K inhibitor and NIC resulted in a synergistic increase in mRNA
levels of islet-specific hormones (maximum increase is
about 10-fold for insulin and somatostatin, and fourfold
for glucagon). Taken together, these data suggest that PI3K
is a negative regulator of islet-specific gene expression in
developing pancreatic cells.
Fig. 1.
Stimulatory effect of PI3K inhibitors on islet hormone
gene expression. Total RNA from ICCs cultured for 5 d was hybridized simultaneously to radiolabeled antisense riboprobes for
(A) glucagon (Glu), insulin (Ins), and cyclophilin (Cyclo), or (B)
somatostatin (Som) and cyclophilin (Cyclo). Protected fragments
for glucagon (389 nucleotides), insulin (262 nucleotides), somatostatin (206 nucleotides), and cyclophilin (135 nucleotides) are indicated. Lanes: CONT, control ICCs; NIC, W, and LY, ICCs cultured in the presence of nicotinamide, wortmannin, or Ly294002,
respectively; NIC+W or NIC+LY, ICCs cultured in the presence
of the combination of nicotinamide and either wortmannin or
Ly294002; tRNA, yeast tRNA (10 µg) used as a negative control;
P, mixture of undigested probes (from top: [A] glucagon, insulin,
cyclophilin; [B] somatostatin, cyclophilin); M, RNA molecular size
markers, in nucleotides (Ambion, Inc., Austin, Texas). (C) Quantitative analysis of hormone transcription levels. Densitometrically determined band intensities of target mRNAs are shown after normalization to the cyclophilin signal from the RNase
protection assays shown in A and B. Values are compared to the
control samples. Results shown are representative of two independent experiments.
[View Larger Versions of these Images (57 + 51 + 31K GIF file)]
-TC3 cells and has already been exhaustively discussed by other investigators (Gao et al.,
1996
). Taken together, these data suggest that the continuous blockade of PI3K activity triggered changes in mRNA
levels and that these changes were followed by a significant increase in hormone biosynthesis and a subsequent
increase in hormone secretion.
Fig. 2.
Stimulatory effects of PI3K inhibitors on insulin content and insulin secretion in fetal islet cells. Replicate groups of 50 human fetal ICCs were cultured for 5 d in the presence or absence of the inhibitors wortmannin (W) or Ly294002 (LY). Nicotinamide (NIC), a potent inducer of endocrine cell differentiation,
was also included in some cultures. Insulin content (A) and insulin release (B) were measured. *P < 0.05, **P < 0.005, ***P < 0.0005; n = 8.
[View Larger Version of this Image (41K GIF file)]
; Herrera et al., 1991
). The present
results thus suggest that PI3K inhibitors induce a process
of endocrine differentiation in fetal islet precursor cells.
Fig. 3.
Increased frequency of endocrine-positive cells and endocrine precursors in PI3K inhibitor-treated fetal islet cultures. Representative fields, collected by confocal microscopy, are shown for control and Ly294002-treated ICCs. Immunoreactivity for insulin is
shown in red, somatostatin and pp in green, and glucagon in blue. Bar graphs represent the morphometric analysis of endocrine cell frequency for each hormone and show the immunoreactive surface area expressed as percent of total surface area of ICCs. Notably, the endocrine surface area is significantly increased for all hormones in Ly294002-treated ICCs (insulin, P < 0.01; somatostatin and pp, P < 0.03; glucagon, P < 0.03; n = 3). When the three fluorescence spectra are merged (lower panels) to measure the total endocrine cell surface, hormone colocalization can also be appreciated. Insulin and glucagon coexpression is highlighted by the appearance of a purple
color (arrowheads) resulting from the overlap of red and blue, whereas colocalization of insulin and somatostatin or pp is shown by the
yellow color (arrow) resulting from the overlap of red and green fluorescences. Note that the frequency of cells coexpressing multiple
hormones is increased in Ly294002-treated ICCs: seven cells coexpressing insulin and glucagon (purple) and two cells coexpressing insulin and somatostatin or pp (yellow). Morphometric analysis demonstrates that the total endocrine cell surface area is significantly increased in Ly294002-treated ICCs (lower right bar graph). Control sections incubated with irrelevant primary antibodies did not show
any detectable immunoreactivity (left panels). Bar, 12 µm.
[View Larger Versions of these Images (7 + 29K GIF file)]
), nor serum starvation significantly affected
basal PI3K activity in fetal islet cells (data not shown).
Thus, the stimulatory effect of PI3K inhibitors on endocrine differentiation is not secondary to blockade of cell proliferation but is due to the specific blockade of PI3K.
Fig. 4.
Inhibition of proliferation in fetal islet cells by PI3K inhibitors. (A) Effect of PI3K inhibitors on [3H]thymidine incorporation into DNA. ICCs were cultured as described in Fig. 2.
[3H]thymidine (1 µCi/ml) was added to the cultures 16 h before
the assay. Data are combined from three independent experiments and are presented as percentage of control ICCs from each
experiment. (Absolute control values were 2086 ± 172, 4552 ± 583, and 7625 ± 536 cpm/µg DNA). *P < 0.05 compared to control, **P < 0.05 compared to treatment with nicotinamide or
wortmannin. Inhibition of DNA synthesis by mitomycin C or serum starvation does not affect insulin protein expression in fetal
ICCs. ICCs were cultured in the presence of 2 µg/ml mitomycin C
for 5 d (B) or starved in 0.5% FBS for 2 d (C). Subsequently,
[3H]thymidine incorporation and insulin protein content were
measured as described in Materials and Methods.
[View Larger Version of this Image (51K GIF file)]
;
Otonkoski et al., 1996
). By contrast, addition of NIC under the same conditions resulted in an increase in insulin
and glucagon mRNA levels and insulin protein biosynthesis. These data indicate a role for HGF/SF in promoting
proliferation and inhibiting the endocrine function of fetal
islet cells. The HGF/SF receptor is known to function by
activating PI3K in a variety of cellular systems (Graziani et al., 1991
). Consistent with this, cellular PI3K is robustly activated in fetal islet cells after acute HGF/SF stimulation, and this activation results from direct binding of
PI3K to the HGF/SF receptor (Fig. 5 and data not shown).
Similarly, analysis of [32P]orthophosphate-labeled ICCs,
growing continuously in the presence of HGF/SF, showed
a threefold increase in levels of the major lipid product of
PI3K; PIP3 (Fig. 6). Thus, the HGF/SF-triggered downregulation of hormone-specific genes is associated with an increase in proliferation of islet cells and with an increase of
PI3K activity.
Fig. 5.
HGF/SF-induced increase of
PI3K activity in fetal ICCs. ICCs starved
for 24 h were stimulated for the indicated times with HGF/SF (25 ng/ml).
Equivalent amounts of cell lysates were
immunoprecipitated with antiphosphotyrosine antibody (Upstate Biotechnology, Inc.), and immune complexes were
assayed for PI3K activity as described in
Materials and Methods. The position of
migration of phosphatidylinositol 3 phosphate [PI(3)P] is indicated. Results
shown are representative of two independent experiments.
[View Larger Version of this Image (12K GIF file)]
Fig. 6.
Effect of continuous treatment for 5 d with
HGF/SF or NIC on PIP3 formation in fetal ICCs. (A) Autoradiogram of a thin-layer chromatographic separation
of cell phospholipids showing
levels of PIP3 in fetal islet
cells treated continuously
for 5 d in the absence (Cont)
or presence of 25 ng/ml
HGF/SF (HGF) or 10 mM
nicotinamide (NIC). (B) Radioactivity in PIP3 spots was
quantitated and is expressed
relative to control. Results
shown are representative of
three similar experiments.
PA, phosphatidic acid; PC,
phosphatidylcholine.
[View Larger Version of this Image (34K GIF file)]
), as well as to upregulate the expression of islet-specific hormone genes (Otonkoski et al., 1993
). Fig. 7 shows
the amount of PI3K activity measured in p85 subunit antibody immunoprecipitates from control, NIC-treated, and
PI3K inhibitor-treated fetal ICCs. A significantly lower
(about fivefold) amount of PI3K activity was present in
p85 immunoprecipitates from NIC-treated cells, as compared to untreated cells. We consistently observed that the
basal level of PIP3 was significantly lower in NIC-treated
ICCs than in control cells (Fig. 6). Treatment with wortmannin or Ly294002 eliminated >90% of control PI3K activity under these conditions (Fig. 7). Inducibility of cellular PI3K activity by growth factors, as well as the de novo
formation of PIP3, was significantly reduced in NIC-treated
cells. There was no change in the actual level of p85 protein during NIC treatment, as determined by Western
blotting, nor was there a direct inhibitory effect of various
concentrations of NIC on PI3K activity in vitro in p85 immunoprecipitates, indicating that NIC is not a direct inhibitor of PI3K (data not shown). The mechanism by which
NIC attenuates cellular PI3K activity in developing fetal
islet cells remains to be determined. The cell culture and treatment conditions used in Fig. 7 were identical to those
used in Fig. 1 and Fig. 2, indicating that an inverse correlation exists between the amount of PI3K activity and the
stage of endocrine maturity in islet cells during fetal neogenesis.
Discussion
as a negative regulator of endocrine differentiation in developing mammalian cells. Blockade of PI3K activity in primary cultured fetal pancreatic cells resulted in a robust activation
of endocrine differentiation. Treatment of ICCs with PI3K
inhibitors increased transcription of islet-specific hormone
genes, expression of islet-specific hormone proteins, insulin content, insulin release in response to secretagogues,
the total number of endocrine-positive cells developing
in islets, and the number of precursor islet cells coexpressing multiple hormones. By contrast, DNA synthesis was significantly decreased in the PI3K inhibitor-treated islet
cells, as compared to untreated cells. However, inhibition of
DNA synthesis by serum starvation or by treatment with
cell cycle-blocking antibiotics had no effect on islet-specific hormone expression. This implies that the observed
effects are not secondary to nonspecific blockade of the
cell cycle, but they can be attributed directly to specific inhibition of PI3K. As a further indication of the involvement of PI3K in regulating endocrine differentiation, we
also observed that the activity of this enzyme was inversely
correlated with the HGF/SF-induced downregulation or
NIC-induced upregulation of islet-specific hormone gene
expression, providing support for the role of PI3K as a
negative regulator of endocrine differentiation.
). The
exact role and immediate downstream molecular targets of
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 have not been identified. This is accurate for all known cellular functions of
PI3K in various systems. Thus, the precise molecular
mechanisms that link the inhibition of the PI3K to the induction of endocrine differentiation remain to be elucidated. It has been reported that proteins of the jun family
can inhibit islet-specific hormone gene transcription both
in vivo (Inagaki et al., 1992
) and in vitro (Henderson and Stein, 1994
). The jun transcription factors block activity of these genes through the E-box elements, which are common for insulin, glucagon, and somatostatin (Kruse et al.,
1993
; Cordier-Bussat et al., 1995
). The known ability of
PI3K to activate ras and subsequently the MAPK-Jun cascade (Pulverer et al., 1991
; Thomas et al., 1992
; Hu et al.,
1995
) provides a potentially direct link between PI3K signaling and the inhibition of islet-specific hormone gene expression. This scenario would explain why treatment with
PI3K inhibitors can release the blockade of all islet-specific hormone genes, which is described in the present experiments. Alternatively, it is possible that the functional
association of PI3K with the islet-specific hormone genes
is mediated by protein kinase C (PKC)-dependent pathways. Several recent reports have indicated that PI3K
might activate PKC isoforms both in vitro and in vivo (Nakanishi et al., 1993
; Toker et al., 1994
). PKC-dependent
branches of signal transduction pathways are known as upstream regulators of several regulatory genes and transcription factors, including members of the c-rel family
(NF-kB), as well as members of the fos/jun family involving AP-1 sites (Leonardo and Baltimore, 1989
; De Tata et
al., 1993). PKC was previously suggested to be involved in
intracellular control of insulin anabolism and secretion
(for review see Newgard and McGarry, 1995
). Thus, the
PI3K could modify islet-specific gene expression in a PKCdependent manner. Another explanation for our results
would have to imply that PI3K can control biosynthesis
of transcriptional factors for hormone gene expression
during fetal neogenesis. We have recently shown that phosphoinositides may regulate the expression of islet/
duodenum homeobox-1 (IDX-1) transcriptional factor in
undifferentiated rat insulinoma cells growing in vitro at low
passages (Ptasznik, A., unpublished data). IDX-1 (PDX-1,
IPF-1, STF-1) is known to be important for activation of
islet-specific genes and development of endocrine pancreas (Josson et al., 1994
; Miller et al., 1994
; Watada et al.,
1996
). Experiments to test this alternative in primary
growing human fetal cells are in progress.
). The ability of PI3K to inhibit islet-specific hormone gene expression, which we show in our present experiments, provides an explanation for the link between the
activation of the HGF/SF receptor and the downregulation of islet-specific gene transcription. Since PI3K is activated in islet cells by HGF/SF (Figs. 5 and 6), it could
serve in these proliferating cells at the same time to block
insulin synthesis and secretion. We have clearly shown that
induction of endocrine differentiation by PI3K inhibitors is associated with a decrease in DNA synthesis in ICCs
(Figs. 1, 2, 3, 4) and, vice versa, that the transition of fetal
ICCs towards proliferation, by the addition of HGF/SF, is
associated with downregulation of islet-specific hormone
gene expression and a decrease in the hormone protein
content (Beattie et al., 1996a
; Otonkoski et al., 1996
). Thus,
according to our present results, it is possible to suggest
that PI3K may functionally convert activation of growth
factor receptors into downregulation of tissue-specific genes,
and in this way accommodate the rates of differentiation
versus proliferation in developing tissues. Thus, cell differentiation and cell division would be modulated in a coordinated way, by the common signaling transducer-PI3K. An
inverse relationship between proliferation and endocrine
differentiation in insulin-producing cells has already been
demonstrated previously (Philippe et al., 1987a
,b; Oberg et
al., 1994
). Our observation is also in agreement with the
general view that tissue-specific functions inversely correlate with cellular growth during embryogenesis.
). Consistent with our present results, it
is possible to suggest that PI3K, which is known to be a
downstream target for the insulin receptor (Backer et al.,
1993
), might functionally link activation of this receptor to
downregulation of insulin and other hormone-specific genes
in fetal islet cells. This is also consistent with the recent
observation that islet
-cells express the insulin receptor
mRNA and insulin receptor substrate 1, i.e., the same signal transducers that are known to mediate insulin action in
peripheral insulin target tissues (Harbeck et al., 1996
).
Since PI3K is activated by insulin in target tissues, it could
serve in
-cells at the same time to block insulin biosynthesis and secretion. An autocrine feedback loop acting
through PI3K would be part of the signaling mechanism
maintaining homeostatic control within developing fetal
cells.
; Alpert et al., 1988
). The double
control mechanism was suggested to be necessary during
islet development (Alpert et al., 1988
; Herrera et al., 1991
;
Cordier-Bussat et al., 1995
). The first step occurs when all
the islet-specific hormone genes are activated, and the
cells are able to coexpress multiple hormone genes. The
second step occurs when the differentiating cells become restricted to express only one hormone gene. The data presented here strongly point to the early islet progenitor
cells as a target, which responded to our treatment with
PI3K inhibitors. Thus, we observed that treatment with
these inhibitors released the blockade of all hormone
genes in undifferentiated pancreatic cells and significantly
increased the number of cells coexpressing multiple hormone proteins, which are known to represent precursors
of terminally differentiated islet cells. As a further indication of the involvement of progenitor islet cells, we also
observed that the PI3K inhibitors combined with NIC
caused the synergistic increase of the mRNA levels of islet-specific hormone genes. NIC alone was previously shown
to induce these mRNA levels only in precursor cells, without any effect on mature endocrine islet cells (Otonkoski
et al., 1993
). Finally, we observed no effect of PI3K inhibitors or NIC on endocrine function in purified fetal islets
(data not shown). These results would suggest, again, that
the effects that we found in primary cultured ICCs, rich in
precursor cells, were developmentally dependent. For this
reason, these effects cannot be detected in purified islets,
which contain mostly terminally differentiated endocrine
cells and few precursor cells (see Materials and Methods).
). Thus, the negative regulation of cellular differentiation by PI3K, which we independently discovered in primary growing human cells during fetal neogenesis, may be a general phenomenon. Nevertheless,
other authors have previously shown a positive involvement of PI3K in PC12 pheochromocytoma and 3T3F442A adipocytic cell lines differentiation (Hempstead et
al., 1992
; Kimura et al., 1994
; Saad et al., 1994
). Taken together, all available results suggest that the PI3K may play
a dual role as both a positive and negative regulator of cellular differentiation in mammalian cells. Future studies directed at the downstream signaling elements coupled to
PI3K should prove informative, as will further investigation of the transcriptional factors by which PI3K links to
tissue-specific gene regulation.
Received for publication 20 January 1997 and in revised form 21 March 1997.
1. Abbreviations used in this paper: HGF/SF, hepatocyte growth factor/ scatter factor; ICC, islet-like cell cluster; NIC, nicotinamide; PI3K, phosphatidylinositol 3-kinase; PI, phosphatidylinositol; PIP, phosphatidylinositol 4 phosphate; PIP2, phosphatidylinositol 4,5 bisphosphate; PIP3 or Pt-dIns(3,4,5)P3, phosphatidylinositol 3,4,5 trisphosphate; PKC, protein kinase C; PtdIns(3)P, phosphatidylinositol 3 phosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4 bisphosphate.We are grateful to Drs. Gordon Gill, Alexis Traynor-Kaplan, and Fred Levine for their valuable comments. We thank Drs. P. Miettinen, D. Drucker, and D. Bergsma for kindly providing cDNA probes, Dr. J.S. Rubin for HGF/SF, Dr. J.F. Habener for IDX-1 antiserum, and Dr. M. Ellisman for his advice on confocal microscopy performed at The National Center for Microscopy and Imaging Research, University of California San Diego (supported by National Institutes of Health grant RR 04050).
This work was supported by research grant No. 196097 from the Juvenile Diabetes Foundation International (to A. Ptasznik) and the Herbert O. Perry Research Fund (to A. Hayek). M. Mally is a recipient of a research grant, and V. Cirulli is a recipient of a Career Development Award from the Juvenile Diabetes Foundation.