(Received for publication, July 17, 1995; and in revised form, September 15, 1995)
From the
Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits both basolateral to apical and apical to basolateral transcytosis of ricin in Fisher rat thyroid (FRT) cells by 50% at 100 nM in a continuous transcytosis assay. In MDCK cells, a similar effect of wortmannin on basolateral to apical transcytosis of ricin was found, whereas apical to basolateral transcytosis was inhibited to a lesser degree. Transcytosis of dimeric IgA in MDCK cells expressing the polymeric immunoglobulin receptor was also reduced to 50% of controls, suggesting that wortmannin inhibits membrane translocation rather than sorting of specific proteins in the transcytotic pathway. This effect of wortmannin is selective, however, in that endocytosis at the basolateral domain and recycling at both the basolateral and apical membrane domains are unaffected, and apical endocytosis and apical secretion are only moderately reduced. We have shown previously that cAMP stimulates a late stage in basolateral to apical transcytosis in MDCK cells through activation of protein kinase A (Hansen, S. H., and Casanova, J. E.(1994) J. Cell Biol. 126, 677-687). Elevation of cellular cAMP still induced a 100% increase in transcytosis in wortmannin-treated cells, but transcytosis was no longer increased when compared to cells which received no drugs. In contrast, in experiments using a 17 °C block to accumulate ricin internalized from the basolateral surface in the apical compartment of MDCK cells, wortmannin had little effect on the stimulation of transcytosis by activators of protein kinase A observed under these conditions. The data thus suggest the existence of a wortmannin-sensitive step in the transcytotic pathway, positioned after endocytosis but prior to translocation into the protein kinase A-sensitive apical compartment, implying a role for phosphoinositide 3-kinase in an intermediate step in transcytosis in polarized epithelial cells.
Membrane homeostasis in eukaryotic cells is dependent on an interplay of membrane budding, fusion, and transport events, which must be regulated individually and collectively to maintain cell function. The generation of polarity in epithelial cells adds further complexity in that additional vesicle-mediated transport processes are required to allow selective traffic to and from the apical and basolateral membrane domains and between these domains(1, 2) . The latter process, transcytosis, i.e. vesicle-mediated transepithelial transport, has been shown to be a brefeldin A-sensitive process(3, 4) which in the case of basolateral to apical transcytosis is inhibited by calmodulin antagonists (5, 6) and requires intact microtubules(7, 8, 9) . However, little is known about how constitutive transcytosis is actually regulated.
Mammalian PI3K ()(p85/110), a heterodimer consisting of
an 85-kDa and a 110-kDa subunit, has been implicated in a wide array of
processes involving signaling through transmembrane receptors such as
mitogenesis, cell transformation, cellular differentiation, apoptosis,
membrane ruffling, and histamine secretion (for reviews, see (10, 11, 12) ). The 85-kDa regulatory subunit
contains one SH3 domain and two SH2 domains which mediate interactions
of PI3K with tyrosine-phosphorylated
proteins(10, 11, 12) . The 110-kDa catalytic
subunit phosphorylates PtdIns, PtdIns-4-P, and PtdIns-4,5-P
in the 3-position (13) and has also been shown to posses
protein kinase activity in that it phosphorylates the 85-kDa subunit
and the insulin receptor substrate 1 on serine (14, 15, 16) . Phosphorylation of the 85-kDa
subunit inhibits the lipid kinase activity of the 110-kDa subunit of
PI3K, a possible mechanism for regulating PI3K
activity(14, 15) . Both the phosphoinositide and the
protein kinase activities of PI3K are inhibited by nanomolar
concentrations of the microbial product
wortmannin(16, 17, 18) , which has proven to
be a useful tool in defining the role of PI3K in biological
processes(10, 11) .
The PI3K has also been implicated in vesicular trafficking. Mutagenesis studies have demonstrated that, upon ligand binding, the autophosphorylated PDGF receptor tail recruits PI3K to the membrane through binding of the SH2 domains in the 85-kDa subunit of PI3K to phosphotyrosine residues 740 and 751 of the PDGF receptor(19) . It has been shown that the these two tyrosine residues are required for efficient lysosomal transport of activated PDGF receptors (20) and that this process is inhibited by 25-50 nM wortmannin (21) . A similar mechanism may be operating in the postendocytic sorting of the receptor for colony-stimulating factor(22, 23) . Furthermore, insulin signaling through PI3K has been shown to recruit glucose transporters to the cell surface(24) , which is also a vesicle-mediated process(25, 26) .
The most compelling evidence for
the significance of phosphoinositide derivatives in membrane traffic
has come from studies by Emr and co-workers (27) on vacuolar
protein sorting in the yeast, Saccharomyces cerevisiae. These
studies have shown that the Vps34 gene encodes a
phosphatidylinositol-specific 3-kinase (PtdIns 3-kinase) with a
catalytic domain homologous to that of the 110-kDa subunit of mammalian
PI3K(27) . Through interaction with Vps15p, a serine-threonine
kinase, Vps34p mediates sorting of soluble hydrolytic enzymes to the
vacuole(28) . Extensive analysis of Vps15 and Vps34 mutants
have linked efficient sorting of soluble hydrolytic enzymes to the
actual generation of PtdIns 3-phosphate(29) . In addition to
its substrate specificity, this PtdIns 3-kinase differs from PI3K
further in that it is less sensitive to wortmannin, requiring low
micromolar concentrations for inhibition(30) . Furthermore,
yeast deficient in Vps34 fail to complement End12, a mutant defective
in postendocytic sorting of internalized -factor to the vacuole,
suggesting a role for Vps34p in this process(31) . Very
recently, the sorting and transport of lysosomal enzymes in mammalian
cells has also been shown to require PI3K
activity(32, 33) .
The importance and diversity of PI3K activity in cell biology, including its significance in the previously described aspects of vesicular trafficking, led us to investigate a possible role for the PI3K in transcytosis in polarized epithelial cells, using wortmannin as a tool. As demonstrated in this work, we found that treatment of FRT cells with 100 nM wortmannin resulted in a 50% reduction in basolateral to apical as well as apical to basolateral transcytosis of ricin, a toxic lectin which binds to terminal galactose residues on glycoproteins and glycolipids and thus serves as an effective marker of bulk membrane transport(4, 34) . Wortmannin had no effect on endocytosis from the basolateral membrane domain of FRT cells, while some reduction in apical endocytosis was detected. In MDCK cells expressing the polymeric immunoglobulin receptor (pIgR), basolateral to apical transcytosis of both ricin and dimeric IgA (dIgA) was reduced to 50% of controls in the presence of 100 nM wortmannin, showing that bulk membrane and receptor-mediated transcytosis are affected similarly. It has previously been shown that transcytosis in MDCK cells is stimulated by activators of protein kinase A (PKA)(35, 36) , and that the stimulation occurs at a late stage in the basolateral to apical transcytotic pathway(36) . Using a 17 °C block to accumulate ricin internalized from the basolateral domain in the apical compartment of MDCK cells, we provide evidence that wortmannin exerts its inhibitory effect on postendocytic transport earlier in the transcytotic pathway than the late stage where PKA stimulates. These data thus implicate a role for PI3K at an intermediate step in transcytosis in polarized epithelial cells.
Figure 4:
Wortmannin inhibits an intermediate step
in the transcytotic pathway. A-D, continuous
transcytosis experiments. Monolayers of FRT cells (A and B) or MDCK cells (C) were treated with or without 100
nM wortmannin for 15 min at 37 °C and then further
preincubated with or without 20 µM Ro 20-1724 and 1
µM FSK for 15 min at 37 °C before addition of I-ricin to the basal or apical side of the filters. The
cells were then incubated for 60 min at 37 °C, and counts
representing transcytosed, intracellular, and bound
I-ricin were recovered and the data were expressed as
explained in Fig. 1. A, basolateral to apical
transcytosis in FRT cells. B, apical to basolateral
transcytosis in FRT cells. C, basolateral to apical
transcytosis in MDCK cells. D, summary of the protocol for the
experiments in A-C. E and F,
transcytosis experiments utilizing a 17 °C block. E,
monolayers of FRT cells were incubated with
I-ricin (400
ng/ml; 1
10
cpm/ng) in the basal chamber for 4 h at
17 °C. The basal side was then rinsed six times, and the cells were
incubated with or without 100 nM wortmannin for 15 min at 17
°C and then further incubated in the presence or absence of 20
µM Ro 20-1724 and 1 µM FSK for another
15 min at 17 °C. The cells were then chased for 15 min at 37 °C
with or without drugs in the presence of 0.1 M lactose in both
chambers. Finally, bound, internalized, and transcytosed
I-ricin were recovered and counted, and the data were
expressed as in continuous transcytosis experiments (see Fig. 1). F, summary of the protocol for the experiments
in E. Shown are the means ± S.D. from experiments, each
representative experiment of at least three independent
experiments.
Figure 1:
Wortmannin inhibits transcytosis of
ricin in FRT cells. Monolayers of FRT cells grown 5 days on Transwell
filters were preincubated with 0-250 nM wortmannin in
both basal and apical chambers for 15 min at 37 °C before addition
of I-ricin (250 ng/ml; 1
10
cpm/ng)
to the basolateral or apical side of the cells and further incubated
for 60 min at 37 °C. The medium containing soluble transcytosed
I-ricin was harvested directly, while membrane-associated
I-ricin was recovered by incubation with 0.1 M lactose for 5 min at 37 °C. Finally, the filters containing
the cells with intracellular
I-ricin were excised, and
all samples were counted. In a basolateral to apical transcytosis
experiment, total cell-associated counts were calculated as counts
removed with 0.1 M lactose from the basolateral side of the
filter plus counts remaining in the cells. Intracellular ricin was
expressed as the ratio of counts in the cells to total cell-associated
counts. Transcytosed ricin was expressed as the ratio of soluble
transcytosed counts plus transcytosed counts recovered with 0.1 M lactose to total cell-associated counts. Data were expressed
similarly in apical to basolateral transcytosis experiments except for
the reversed polarity. A, basolateral to apical transcytosis
of ricin in 60 min at 37 °C. B, apical to basolateral
transcytosis of ricin in 60 min at 37 °C. C-F,
intracellular accumulation of ricin administered for 60 min at 37
°C from the basolateral side (C), 60 min at 37 °C from
the apical side (D), 15 min at 37 °C from the basolateral
side (E), and 15 min at 37 °C from the apical side (F). A and C and B and D,
respectively, are derived from the same experiment. Negligible amounts
of
I-ricin were transcytosed in 15 min in either
direction. The data shown are mean ± S.D. (n =
3) from experiments, each representative of at least three independent
experiments.
Figure 2:
Transcytosis and recycling of dIgA. MDCK
cells expressing the pIgR were pretreated for 15 min with 100 nM wortmannin (open symbols) or with vehicle alone (closed symbols), then allowed to internalize I-dIgA from either the basolateral (A) or apical (B) surface for 10 min. After washing, cells were placed in
fresh medium with or without wortmannin and chased for the times
indicated. A, basolateral internalization: closed
circles, transcytosis (control); open circles,
transcytosis (+wortmannin); closed squares, basolateral
recycling (control); open squares, basolateral recycling
(+wortmannin). B, apical internalization: closed
circles, transcytosis (control); open circles,
transcytosis (+wortmannin); closed squares, apical
recycling (control); open squares, apical recycling
(+wortmannin). Data are the mean ± S.D. of triplicate
determinations and are representative of four
experiments.
Figure 5:
Effect of wortmannin on apical secretion
of gp 80 in MDCK cells. Monolayers of MDCK cells were starved for 20
min at 37 °C, pulse-labeled with EXPRES
S
(1 mCi/ml) for 15 min at 37 °C and chased for 2 h at 19.5 °C,
to accumulate proteins in the TGN, in the presence or absence of 100
nM wortmannin for the last 30 min (A-F) and
with or without 20 µM Ro 20-1724 and 1 µM FSK for the last 15 min at 19.5 °C (D-F). Next,
the cells were warmed to 37 °C for 15 (A-F) or 30 (A-C) min in the presence or absence of drugs. The
apical medium was sampled, and aliquots thereof were subjected to
SDS-polyacrylamide gel electrophoresis and fluorography as detailed
under ``Materials and Methods.'' gp 80 is the major secretory
product which under reducing conditions is easily identifiable as three
polypeptides of 35-45 kDa. A-C, effect of
wortmannin on the constitutive level of apical delivery of gp 80. D-F, effect of wortmannin on apical transport of gp 80
in the presence of PKA activators. The data shown are from experiments
representative of at least three separate experiments of each kind. The
data in B are mean ± S.D. (n = 3). C and F, summary of the protocols for the experiments
in A and B and D and E,
respectively.
Figure 3:
Activators of PKA stimulate transcytosis
of ricin in FRT cells. Monolayers of FRT cells were preincubated with
or without 30 µM H-89 for 30 min at 37 °C and then
further with 20 µM Ro 20-1724 or 1 µM FSK alone, or with 20 µM Ro 20-1724 in the
presence of 1 µM FSK or 1 µM dideoxyforskolin (DDF) or 500 µM 8-Br-cAMP for 15 min at 37 °C
before addition of I-ricin to the basal or apical side of
the filters. The cells were then incubated for 60 min at 37 °C, and
counts representing transcytosed, intracellular, and bound
I-ricin were obtained as described in Fig. 1. A, basolateral to apical transcytosis. B, apical to
basolateral transcytosis. The data were first expressed as the ratio of
transcytosed to total cell-associated (bound + internalized)
counts and then normalized to controls (100%) from the same
experiments. The data are representative of at least two separate
experiments, each performed in triplicate, with a coefficient of
variation less than 15% in the data shown. C, summary of the
protocol for these experiments.
Wortmannin did not affect the generation of cAMP by Ro 20-1724 and FSK (data not shown). In the presence of 100 nM wortmannin, the combination of forskolin and Ro 20-1724 still increased transcytosis by 100% above the level of wortmannin-treated cells in a continuous 1-h transcytosis assay in both FRT and MDCK cells (Fig. 4, A-C). However, transcytosis was no longer increased when compared to cells which received no drugs (Fig. 4, A-C). In contrast, in pulse-chase experiments using a 17 °C block to accumulate ricin internalized from the basolateral surface in the apical compartment of MDCK cells, the presence of wortmannin had little effect on the stimulation in transcytosis by forskolin and Ro 20-1724 observed under these conditions (Fig. 4D). These data suggest that the effects of wortmannin and agents which increase cellular levels of cAMP are separable and implicate a role for PI3K in an intermediate step in transcytosis after internalization but before translocation to the PKA-sensitive compartment.
In an attempt to identify mechanisms regulating vesicular transport in polarized epithelial cells, we have investigated a possible role for PI3K in transcytosis, using the fungal metabolite wortmannin as a tool. At nanomolar concentrations, wortmannin is an irreversible, noncompetitive, and specific inhibitor of this kinase (17, 18) . Indeed, we found that as little as 10 nM wortmannin gave a significant (25%) reduction in bidirectional transcytosis of ricin in FRT cells, while, at a concentration of 100 nM, transcytosis was reduced to 50% of control values. In MDCK cells, both receptor-mediated (dIgA) and bulk-membrane (ricin) transcytosis was inhibited by 50% in the basolateral to apical direction, whereas apical to basolateral transport of ricin was inhibited by 25%. Recycling of dIgA was unimpaired at either the basolateral or apical surface. These data suggest a role for PI3K in transcytosis, a postendocytic vesicular trafficking pathway that is unique to epithelial cells.
As mentioned in the introduction, a role for PI3K in postendocytic sorting of activated PDGF receptors has been described. The observation that mutant receptors lacking PI3K binding sites are efficiently internalized but not degraded has led to the assumption that PI3K is involved in the sorting of the internalized receptors into the lysosomal pathway(20) . This hypothesis has been supported by the demonstration that 25-50 nM wortmannin also inhibits degradation of receptors in the presence of PDGF(21) . Our results, however, using ricin and dIgA as markers of bulk membrane and receptor-mediated transcytosis, respectively, suggest that wortmannin at similar concentrations significantly inhibits the constitutive flow of membrane into the transcytotic pathway rather than sorting of individual proteins.
Another constitutive membrane transport process
that is known to require PI3K activity is the sorting of soluble
hydrolytic enzymes to the yeast vacuole. Vps34p, the PtdIns 3-kinase
which is essential to this process, has an IC of 4
µM and requires 10 µM wortmannin for complete
inhibition, a 100-fold higher concentration than mammalian
p110(30) . Vps34p appears to be required for a specific sorting
event since trafficking of vacuolar membrane proteins remains
unaffected following inactivation of Vps34p(29) . Yet, the
possibility remains that soluble and membrane vacuolar proteins are
cargoed in two separate vesicle populations, of which only the first
requires Vps34p for a budding or other membrane translocation event.
The organelle in the biosynthetic pathway where the action of Vps34p is
required is probably the TGN, although Munn and Riezman (31) have recently determined that Vps34p is also required for
transport of internalized
-factor to the vacuole, suggesting that
it may function at the exit from late endosomes as well. Very recently,
Brown et al.(33) have presented evidence in favor of
a similar role for a PI3K in transport of newly synthesized lysosomal
enzyme (cathepsin D) from the TGN to late endosomes/prelysosomes in the
mammalian cell lines NRK and CHO. As for Vps34p, high wortmannin
concentrations (1-3 µM) were required for maximal
inhibition of cathepsin D processing in late endosomes. Furthermore,
this mammalian enzyme appears to function in a M6PR-dependent sorting
event in the TGN, since M6PRs were detected at normal levels in the TGN
but were depleted from the late endosomes in wortmannin-treated cells (33) . These authors suggest that the most likely candidate is
a PtdIns 3-kinase characterized by Stephens et
al.(49) , which, like Vps34p, has a high IC
for wortmannin. Davidson (32) , however, has found that
in K562, NRK, and Cos 1 cells the sorting of procathepsin D is
inhibited by low nanomolar concentrations of wortmannin. Davidson
suggests that the transport of lysosomal enzymes in these cells is
regulated by a mammalian homolog of Vps34p, which very recently has
been cloned by Volinia et al.(50) and shown to
interact with a mammalian homolog of Vps15p. This PtdIns 3-kinase is
indeed inhibited by concentrations of wortmannin in the low nanomolar
range(50) . Thus, it is possible that homologs of Vps34p
function in transport of soluble hydrolytic enzymes to the lysosome in
mammalian cells.
The effect of wortmannin on transcytosis was further characterized through experiments that included activators of PKA. These experiments were prompted by the finding that wortmannin appeared to inhibit vesicular transport processes that are stimulated by PKA activators. In 37 °C continuous transcytosis experiments, the inhibition by wortmannin in percent was the same in cells treated with PKA activators as in controls. In contrast, in experiments with MDCK cells employing a 17 °C temperature block where marker internalized from the basolateral domain is accumulated in the apical compartment(8) , the presence of wortmannin had little effect on the stimulation by PKA activators on transport of ricin to the apical membrane. These results show that wortmannin exerts its inhibitory effect on transcytosis prior to the late stage in the transcytotic pathway where activators of PKA stimulate. In addition, it was found that wortmannin has a small inhibitory effect on apical secretion of gp 80 which also is stimulated strongly by activators of PKA. A simple hypothesis compatible with these data is that wortmannin inhibits vesicle budding from a postendocytic compartment whereas activators of PKA exert their effect by stimulating fusion of transcytosing and secretory vesicles with the apical membrane in MDCK cells (and possibly with both apical and basolateral membranes in FRT cells).
Very recently, an isoform of the 110-kDa subunit of PI3K,
p110, has been cloned which has no binding site for the 85-kDa
regulatory subunit and is activated by G protein subunits(51) .
Although it is tempting to speculate that this isoform is involved in
the regulation of transcytosis, there are several discrepancies which
are difficult to reconcile with this possibility. First, as discussed
above, the data obtained in this work implicate PKA (activated by
G
, adenylyl cyclase, and cAMP) and PI3K at different
stages in the transcytotic pathway. Second, the p110
is less
sensitive to wortmannin requiring higher concentrations for complete
inhibition than those significantly inhibiting transcytosis. Third, in
addition to
subunits of G
proteins(51, 52) , the activity of p110
appears
to be stimulated strongly by the G
group of G
subunits(51) , whereas pertussis toxin, an inhibitor of this
group, has no effect on transcytosis(36) .
Serine/threonine kinases clearly serve important roles in regulating transcytosis. In addition to PKA, it has been shown that transcytosis of the pIgR is also stimulated by phorbol esters which activate protein kinase C(53) . It remains unknown, however, if this effect of phorbol esters is specific for the pIgR or, like PKA, affects bulk membrane transport as well. Furthermore, it has been demonstrated that sorting of the pIgR into the transcytotic pathway is stimulated by phosphorylation of the receptor itself by an as yet unidentified kinase (38) . The catalytic subunit of PI3K is also a protein kinase (see introduction), and it cannot be excluded that it is the protein and not the lipid kinase activity of PI3K which is involved in the regulation of constitutive transcytosis, as both activities are inhibited by wortmannin. However, there is to our knowledge no precedence for a role for the serine kinase activity of PI3K in membrane traffic.
How the PI3K functions in membrane traffic is not known in detail, but it has been suggested that the negative charge of the phosphoinositide products of the kinase may be important in membrane invagination and/or recruitment of adaptor proteins(12) . The negative charge of phospholipid products have also been implicated in the possible function of other lipid kinases in membrane traffic(54) , like phosphatidylinositol 4-kinase and phosphatidylinositol 4-phosphate 5-kinase, which may operate through a mechanism that involves ADP-ribosylation factor(55, 56) . These suggestions fit our data well in that the effect of wortmannin does not appear to distinguish between receptor-mediated and bulk membrane transport, arguing against a role for PI3K in sorting but in favor of a possible function in formation of vesicles that mediate transcytosis. This could also explain the differential effect of wortmannin on the transcytotic pathways in MDCK cells, since the apical and basolateral membranes of these cells have been shown to exhibit distinct differences in lipid composition(57) . It seems likely that either the extent to which the charged phospholipids are generated and/or their effect on vesicle formation would differ depending on whether the membrane derives from the apical or basolateral domain of MDCK cells. FRT cells, in contrast to most other polarized epithelial cells studied (including MDCK), have been shown to sort most of their glycosylphosphatidylinositol-anchored proteins to the basolateral domain(58) . This suggests that the lipid compositions of the apical and basolateral membrane domains in FRT cells differ substantially from those of MDCK cells and could provide an explanation for the lack of polarity in the effect of wortmannin on transcytosis in FRT cells. However, whether this is actually the case remains speculative until more is known about the role of lipids and their kinases in membrane traffic.