Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H+-ATPase pump and in vitrovacuolar acidification in hepatoma HepG2 cells.. This cat
Trang 1acidification and cathepsin D in cholera toxin cytotoxicity Tatiana El Hage1,2,*, Cle´mence Merlen1,2*, Sylvie Fabrega1,2and Franc¸ois Authier1,2
1 INSERM, U756, Chaˆtenay-Malabry, France
2 Universite´ Paris-Sud, Faculte´ de Pharmacie, Chaˆtenay-Malabry, France
Cholera toxin (CT) is the causative agent of the
diarr-heal disease cholera, and mediates its effects by
increasing cAMP levels [1] The resulting increase in
intracellular cAMP causes net intestinal salt and water secretion, resulting in massive secretory diarrhea and changes in cell morphology, presumably due to
Keywords
acidification; cathepsin D; cholera toxin;
endosome; G protein
Correspondence
F Authier, INSERM U756, Universite´
Paris-Sud, Faculte´ de Pharmacie, 5 rue
Jean-Baptiste Cle´ment, 92296 Chaˆtenay-Malabry,
France
Fax: +33 1 46835844
Tel: +33 1 46835528
E-mail: francois.authier@u-psud.fr
*These authors contributed equally to this
work
(Received 19 December 2006, revised 7
March 2007, accepted 20 March 2007)
doi:10.1111/j.1742-4658.2007.05797.x
Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteoly-sis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi
D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J 272, 4385– 4397] Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and
47 kDa Gsa proteins These events coincided with the endosomal recruit-ment of ADP-ribosylation factor proteins, especially ADP-ribosylation fac-tor-6, with a time course identical to that of toxin and the A subunit of the stimulatory G protein (Gsa) translocation After an initial lag phase of
30 min, these constituents were linked to NAD-dependent ADP-ribosyla-tion of endogenous Gsa, with maximum accumulaADP-ribosyla-tion observed at 30–60 min postinjection Assessment of the subsequent postendosomal fate
of internalized Gsa revealed sustained endolysosomal transfer of the two Gsa isoforms Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H+-ATPase pump and
in vitrovacuolar acidification in hepatoma HepG2 cells The vacuolar H+ -ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A partially inhibited, both in vivo and in vitro, the cAMP response to cholera toxin This cathepsin D-dependent action of cholera toxin under the con-trol of endosomal acidity was confirmed using cellular systems in which modification of the expression levels of cathepsin D, either by transfection
of the cathepsin D gene or small interfering RNA, was followed by parallel changes in the cytotoxic response to cholera toxin Thus, in hepatic cells, a unique endocytic pathway was revealed following cholera toxin administra-tion, with regulation specificity most probably occurring at the locus of the endosome and implicating endosomal proteases, such as cathepsin D, as well as organelle acidification
Abbreviations
ARF, ADP-ribosylation factor; CT, cholera toxin; CT-A, cholera toxin A subunit; CT-B, cholera toxin B subunit; ER, endoplasmic reticulum; GSa, A subunit of the stimulatory G protein; LPS, postmitochondrial supernatant; si, small interfering.
Trang 2activation of cAMP-dependent protein kinase A.
Although the human small intestine mucosal cell is the
normal target of the toxin, CT is a ubiquitous
activa-tor of adenylate cyclase in most eukaryotic cells [2]
CT belongs to the AB family of bacterial exotoxins,
and consists of a pentameric B subunit (CT-B) and an
A subunit (CT-A) comprising two polypeptides, A1
and A2, linked by a disulfide bond CT-B binds with
high affinity to GM1, a ganglioside present in apical
membranes of all intestinal epithelial cells A1 has
ADP-ribosyl transferase activity, whereas A2 contains
a C-terminal KDEL endoplasmic reticulum (ER)
retrieval signal [2]
The intervening steps between CT binding and
adenylate cyclase activation are not fully understood
There is a characteristic lag period after CT binds to
the cell surface and before an increase in adenylate
cyclase activity is observed It is generally proposed
that this lag period corresponds to sequential steps of
CT uptake, CT activation and CT translocation to its
protein target, theA subunit of the stimulatory G
pro-tein (Gsa) Two major models have been proposed to
explain the events during this lag time The first model,
supported by in vivo and in vitro studies on the
intoxi-cation of rat hepatocytes, suggests that CT cytotoxicity
may be related, at least in part, to proteolytic events
within endocytic vesicles [3–7] Following CT binding
to the plasma membrane of hepatocytes, CT
accumu-lated in a low-density endosomal compartment, with
maximum accumulation observed by 15–30 min [4,7]
Following ATP-dependent endosomal acidification,
internalized CT was rapidly proteolyzed within hepatic
endosomes by aspartic acid protease cathepsin D [7]
In vivo studies showed that the acidotropic agent
chloroquine, as well as the carboxylic ionophore
monensin, inhibited CT activation of adenylate cyclase
and increased the lag period for this process [5,6]
In vitro experiments revealed that hydrolysates of CT
generated by cathepsin D displayed
ADP-ribosyltrans-ferase activity towards exogenous Gsa [7] However,
the mechanisms by which the endosome-activated
CT-A gains access to Gsa, which is mainly localized to the
inner face of the plasma membrane, remain undefined
A second activating pathway has been proposed to
operate within the ER, which CT accesses by retrograde
vesicular traffic via the trans-Golgi network In the
ER, the disulfide bond linking CT-A1 to CT-A2⁄
CT-B5 is reduced by protein disulfide isomerase, and
CT-A1 is then translocated to the cytosol in a process
involving ER-associated degradation The cytosolic
pool of CT-A1 escapes ubiquitin-mediated protein
deg-radation, due to its very limited number of internal
lysine residues [8,9], and subsequently ADP-ribosylates
Gsa However, mutagenesis studies have indicated that although the ER retrieval signal of CT-A2 and the ER localization of the toxin enhance the efficiency of CT cytotoxicity, they are not absolutely required for toxin action, suggesting the existence of alternative compart-ment(s) for CT activation [10,11]
At present, no experimental data exist to support a mechanism of interaction between the active frag-ment(s) of CT-A generated at the endosomal locus and its target, Gsa The object of the present study was
to investigate endosomally located mechanisms that regulate the activation and cytotoxic effect of CT in hepatocytes Using a subcellular fractionation approach to address the compartmentalization, activa-tion and acactiva-tion of CT in vivo, we demonstrate the existence of a complex of activated CT, Gsa and ADP-ribosylation factor (ARF) protein in the endo-somal membrane This coincided with ADP-ribosy-lation of Gsa in the endosomal compartment In addition, the aspartic acid protease inhibitor pep-statin A reduced, both in vivo and in vitro, the CT-stimulated cAMP response in hepatic cells, as did transfection of MCF-7 cells with cathepsin D small interfering (si)RNA In contrast, cathepsin D over-expression in rat tumor cells increased the cAMP response to CT Finally, we report on the endosomal acidification step, which was specifically increased by
CT and was required for its efficient action in rat liver and hepatoma cells
Results
CT-induced translocation and ADP-ribosylation
of Gsa within the endolysosomal apparatus
To determine whether the activated form of endosomal
CT remained functional within hepatic endosomes
in vivo, we first evaluated the subcellular content of Gsa (CT substrate) in endosomal fractions prepared from control and CT-injected rats (Fig 1) In agree-ment with our previous work [7], a time-dependent increase in CT-A and CT-B was observed in endoso-mal fractions 10–20 min after native CT injection (Fig 1, upper left blot) or 20–90 min after CT-B injec-tion (Fig 1, upper right blot) In control rats, immunoreactive Gsa was detected as a doublet of
47 kDa and 45 kDa (Fig 1, lanes 1 of lower blots)
In vivoinjection of native CT or CT-B effected a rapid increase of both the 47 kDa and 45 kDa Gsa isoforms, with maximal accumulation 20 min (native CT; 32% increase) or 30 min (CT-B; 77% increase) postinjec-tion By 90 min postinjection, both Gsa isoforms had returned to basal levels (Fig 1, lane 6 of lower blots)
Trang 3Next, we used the in situ liver model system for
endosome–lysosome transfer analysis to determine the
endosomal fate of the internalized CT and Gsa
(Fig 2) Transfer of CT and Gsa from the endosomal
compartment to the lysosomal compartment was
examined by Nycodenz density gradient analysis of the
postmitochondrial supernatant (LPS) fractions
pre-pared 20 min after CT administration (Fig 2A) When
LPS fractions were incubated at 4C, most of the
CT-B and Gsa appeared in a single broad region with a
density of 1.077–1.119 gÆmL)1 (Fig 2A, left blots),
which mainly coincided with the Golgi marker
galacto-syltransferase (Fig 2A, upper left panel) and the
endo-somal marker EEA1 or procathepsin D precursor
(Fig 2B) When the LPS fraction was incubated at
37C, there were only minor changes in the
distribu-tion of CT-B (Fig 2A, upper right blot), with a slight
shift to the right that partially coincided with the
lyso-somal marker acid phosphatase (Fig 2A, upper right
panel) and the mature 45 kDa cathepsin D enzyme
(Fig 2B, lower blot) This was accompanied by a
par-tial loss in CT-B immunoreactivity at the endosomal
position (Fig 2A, upper right blot) However, a major
transfer of both Gsa proteins from the endosomal to the lysosomal position was clearly detectable, along with a partial decrease in the total amount of immuno-reactive Gsa throughout the gradient (Fig 2A, lower right blot)
The cofactor ARF, and especially ARF-6, is required for full ADP-ribosylation of Gsa by activated
CT [12] Therefore, we evaluated the subcellular con-tent of ARF proteins in hepatic fractions prepared from CT and CT-B-injected rats (Fig 3A) An increase
in ARF content was observed in endosomal fractions isolated 5 min postinjection of CT-B, and this increase was maintained for up to 60 min (Fig 3A, upper panel) CT administration led to a low and brief recruitment of ARF-6 to the endosomal membrane 15–30 min postinjection (Fig 3A, lower panel EN),
a decrease in plasma membrane ARF-6 content 5–15 min postinjection (Fig 3A, panel PM), and a sus-tained association of ARF-6 with the cytosolic fraction 5–60 min postinjection (Fig 3A, panel S)
Finally, we performed an in vivo CT substrate labeling experiment using [32P]NAD and endocytic vesicles that contained in vivo internalized native
Fig 1 CT-mediated internalization of Gsa in the endosomal apparatus Rat liver endosomal fractions were isolated at the indicated times after the in vivo administration of native CT or CT-B, and evaluated by western blotting for their content of both CT subunits and Gsa Fifty micrograms of protein was applied to each lane Molecular mass markers are indicated on the left of the upper panels Arrows to the right indicate the mobility of CT-A ( 28 kDa), CT-B ( 12 kDa) and Gsa ( 47 and 45 kDa) Lower panels: quantification of Gsa signals by scan-ning densitometry, with results expressed as percentage of signal intensity in the endosomal fraction prepared from control (noninjected) rats.
Trang 4CT (Fig 3B) Radiolabeling of endosomal Gsa
was observed with endosomal fractions prepared 30
and 60 min postinjection of native CT Thus, CT is
active in vivo towards endosomal Gsa following
a 30 min lag period, which probably
corres-ponds to the time required for its internalization
into endocytic structures and subsequent proteolytic
activation
Effect of CT on ATP-dependent endosomal acidification
It has been previously reported that 18 h after the intraperitoneal injection of CT into rats, hepatic endo-somes displayed increased rates of acidification and a more acidic steady-state intravesicular pH [13] There-fore, we investigated whether CT altered endosomal
A
B
Fig 2 Transfer of CT-B and Gsa from the endosomal to the lysosomal position on Nycodenz gradients (A) The LPS fraction was isolated
20 min after CT administration, and immediately subfractionated on linear Nycodenz density gradients (left panels, 4 C), or incubated with ATP and an ATP-regenerating system at 37 C for 60–90 min prior to subfractionation on linear Nycodenz density gradients (right panels,
37 C) Galactosyltransferase (circles) and acid phosphatase (squares) activities were determined, and results expressed as a percentage of total enzymatic activity recovered CT subunits and Gsa content were evaluated for each subfraction by immunoblotting Thirty microliters of each subfraction was loaded onto each lane Arrowheads indicate the median densities of galactosyltransferase (closed arrowhead) and acid phosphatase (open arrowhead) Arrows on the right indicate the mobilities of immunodetected CT-B ( 12 kDa) and Gsa ( 47 and 45 kDa) CT-A was below the limits of detection (results not shown) (B) The content of early endosome antigen 1 (EEA1) and cathepsin D (CD) was evaluated by immunoblotting for each subfraction isolated from the LPS fraction incubated at 37 C Components appearing at densities 1.075–1.105 and 1.11–1.14 gÆmL)1were scored, respectively, as truly endosomal and lysosomal.
Trang 5acidification during the early stage of CT action, when
most of the internalized CT should be located within
hepatic endosomes We used a fluorescent weak base,
acridine orange, which concentrates within acidic
compartments and has been widely used to assess
vacuolar H+-ATPase activity [14,15] A time-dependent
decrease in fluorescence intensity was observed within
hepatic endosomes prepared from uninjected rats as
well as toxin-injected rats, both with (closed symbols)
and without (open symbols) addition of ATP
(Fig 4A) However, endosome acidification was
strongly ATP-dependent, and, in the presence of ATP,
the rate of acidification was markedly increased
follow-ing CT administration (closed squares) The initial rate
of ATP-dependent acidification of endosomes (which
was linearly related to incubation time for the first
5 min) increased two-fold in endosomes isolated from CT-injected rats (closed squares), but this was not observed for CT-B-injected (closed diamonds) or diptheria toxin-injected rats (closed circles)
Bafilomycin A1 neutralizes endosomal acidification
by inhibiting the vacuolar ATPases responsible for maintaining proton gradients [16] It was therefore of interest to determine whether bafilomycin A1 would similarly affect endosomal acidification in control and CT-treated cells (Fig 4B) Incubation of HepG2 cells for 30 min with bafilomycin A1 alone (0.2 lm) abol-ished the granular fluorescence of DAMP almost com-pletely (Fig 4B, lower left panel) However, a residual fluorescent staining reminiscent of vesicular acidifica-tion was clearly observed in cells pretreated with bafilomycin A1 and then incubated with CT for 2 h (Fig 4B, lower right panel) These data are consistent with our finding that CT increased endosomal acidifi-cation at the early stage of CT action
Role of endosomal acidification and cathepsin D
in CT action
To assess whether the aspartic acid protease cathep-sin D and endosomal acidity might be two major requirements for CT cytotoxicity in hepatic cells, we examined the in vivo and in vitro effects of agents that inhibit aspartic acid protease activity and⁄ or vesicle acidification (Fig 5) Animals were given an intraperi-toneal injection of either pepstatin A, an inhibitor of aspartic acid proteases [17], or a mixture of bafilo-mycin A1 and folibafilo-mycin, two inhibitors of the vacuolar ATPases [16], prior to CT administration (50 lg per
100 g body weight) Rats were then killed 50 min post-CT injection The cAMP content in rat liver homogenates isolated from control rats was increased
5-fold over basal levels after CT injection (Fig 5A,
cf Basal and CT-Control) Both pepstatin A and bafilomycin A1⁄ folimycin treatment caused a 3-fold decrease in hepatic cAMP content in CT-treated rats (Fig 5A, cf PA, Bafi⁄ Foli and Control)
Cellular cAMP content was next measured in vitro
in hepatoma HepG2 cells treated with CT in the presence or absence of pepstatin A or bafilomycin A1 (Fig 5B) Cellular cAMP content increased 45 min after the addition of CT, and reached a maximum 90–120 min (closed circles) Bafilomycin A1 (closed triangles) extended the lag phase by 15 min, and decreased the rate at which CT increased cellular cAMP content Pepstatin A (closed squares) was less effective at inhibiting the initial rate of cAMP pro-duction, but did reduce the maximal extent of cAMP production
A
B
Fig 3 CT-mediated recruitment of ARF-6 and ADP-ribosylation of
Gsa in hepatic endosomes (A) Rat liver endosomal (EN), plasma
membrane (PM) and cytosolic (S) fractions were isolated at the
indicated times after the in vivo administration of native CT or
CT-B, and evaluated by western blotting for their content of ARF
and ARF-6 using their respective polyclonal and monoclonal
anti-bodies Arrows to the right of each panel indicate the mobilities of
immunodetected ARF proteins ( 21 kDa) (B) Endosomal fractions
were isolated at the indicated times after the in vivo administration
of native CT, and immediately incubated with 0.54 l M [32P]NAD at
30 C in an ADP-ribosylation buffer; this was followed by
SDS ⁄ PAGE and autoradiography Molecular mass markers are
indi-cated to the left of the panel The arrow on the right indicates the
mobility of [ 32 P]-labeled Gsa ( 45 kDa).
Trang 6Time of incubation (min)
control
control + ATP
cholera toxin
cholera toxin + ATP
diphtheria toxin
diphtheria toxin + ATP
A
30 15
200
400
Baf-A1
B
No drugs
CT-B subunit + ATP
Fig 4 Effect of cholera toxin on endosomal acidification (A) Rat liver endosomal fractions were isolated 2 h after the in vivo administration
of native CT, CT-B or diphtheria toxin (60 lg per 100 g body weight), and incubated in 0.15 M KCl containing 5 m M MgCl2, 5 l M acridine orange and, when indicated, 5 m M ATP The relative decrease in fluorescence intensity was immediately recorded at 37 C for 30 min using
a recording spectrofluorometer Results are expressed as arbitrary units of fluorescence intensity Baseline fluorescence at zero time was 284.63 ± 7.41 (– ATP) and 422.97 ± 1.79 (+ ATP) (B) HepG2 hepatoma cells were incubated at 37 C for 30 min with or without bafilomy-cin A1 (0.2 l M ), and this was followed by the addition of CT (1.3 l M ) or buffer alone for an additional 2 h The acidic compartments were visualized by immunofluorescence using the DAMP method.
Fig 5 Role of vesicular acidification and
cathepsin D in cellular cAMP production by
CT (A) Rats were injected with native CT
(50 lg per 100 g body weight) 1 h after an
intraperitoneal injection of either 12.5%
dimethylsulfoxide, 625 lg pepstatin A
methyl ester (PA) or a mixture of
bafilomy-cin A1⁄ folimybafilomy-cin (Bafi ⁄ Foli) (0.75 lg each).
Fifty minutes after CT injection, rats were
killed, hepatic homogenates were prepared,
and cAMP content was measured by
radio-immunoassay The data were expressed as
pmol cAMP per mG protein Each histogram
represents the mean ± SD of at least three
independent determinations (B) HepG2
hepatoma cells were treated with CT
(10 lgÆmL)1) and incubated at 37 C in the
absence (control) or presence of pepstatin A
(120 lgÆmL)1) or bafilomycin A1 (0.2 l M ) for
the indicated times Cellular cAMP content
was measured as described for (A), and
the data were expressed as pmolÆ(mG
pro-tein))1 Results are the mean ± SD of three
separate experiments.
Trang 7As pepstatin A treatment produced, both in vivo
and in vitro, a sustained reduction in the cAMP
response to CT, we next evaluated the role of the
pepstatin A-sensitive enzyme cathepsin D in CT
cyto-toxicity by using cathepsin D-deficient 3Y1-Ad12 cells
transfected with the human cathepsin D gene [18]
Immunoblot analysis of equal amounts of protein
from 3Y1-Ad12 cell lysates confirmed the absence of
cathepsin D in nontransfected cells, and the presence
of both the 31 kDa mature cathepsin D and the
45 kDa procathepsin D in cathepsin D-overexpressing
cells (Fig 6A, lower panel) Measurement of cellular
cAMP levels in 3Y1-Ad12 cells after CT treatment
revealed that cells expressing the transfected gene
were 3–5-fold more sensitive to CT treatment than
were control cells deficient in cathepsin D (Fig 6A,
upper panel) To strengthen the possibility of a direct action of cathepsin D in CT cytotoxicity, cAMP assays were performed with MCF-7 cells whose endogenous cathepsin D expression was inhibited
by small RNA-mediated gene silencing (cathepsin D siRNA) (Fig 6B) A progressive decrease in expres-sion of both procathepsin D and mature cathepsin D was observed in MCF-7 cells 48–72 h after transfec-tion with cathepsin D siRNA (Fig 6B, lower panel)
On the basis of cellular cAMP levels, transfected MCF-7 cells were 4-fold less effective in responding
to CT treatment as compared to nontransfected MCF-7 cells (Fig 6B, upper panel), supporting the notion that cathepsin D might play a crucial role in
CT activation and action in hepatic cells as well as in other cell types
B A
Fig 6 Relationship between cathepsin D expression and cellular cAMP response to CT (A) Rat embryonic 3Y1-Ad12 tumor cells expressing either no cathepsin D (control cells, open histograms) or overexpressing human wild-type cathepsin D (3Y1-Ad12-CD, closed histograms) were incubated with CT (1.3 l M ) for 2 h Cellular cAMP content was measured, and expressed as fold stimulation over basal (unstimulated) activity [6 pmolÆ(mG protein))1] (upper panel) Results are the mean ± SD of three separate experiments Whole cell lysates (60 lg of protein per lane) were evaluated by immunoblotting for their content of human cathepsin D (lower panel) Arrows on the right indicate the mobility
of procathepsin D ( 45 kDa) and mature cathepsin D ( 31 kDa) (B) MCF-7 cells, whose cathepsin D expression was inhibited by siRNA silencing for 48–72 h, were incubated with CT (1.3 l M ) for 2 h Cellular cAMP content was measured and expressed as fold stimulation over basal (unstimulated) activity [ 28 pmolÆ(mG protein))1] (upper panel) Results are the mean ± SD of three separate experiments Whole cell lysates (60 lg of protein per lane) were evaluated by immunoblotting for their content of human cathepsin D (lower panel) Arrows on the right indicate the mobility of procathepsin D ( 45 kDa) and mature cathepsin D ( 31 kDa).
Trang 8Assessment of the KDEL peptide integrity
of endosomal CT-A
The results presented thus far established that CT-A
represented a high-affinity substrate for endosomal
cathepsin D that assisted in the release of CT-A
frag-ment(s) that are active towards Gsa However, the
endosomal degradative CT-A fragment(s) remained
undefined, and it was unknown whether the processed
form(s) of internalized CT-A had lost part or all of its
C-terminal ER-retention KDEL motif
To investigate this, we characterized three polyclonal
antibodies to KDEL for their specificity towards CT-A
by western blot analysis of pure CT or CT-A
(Fig 7A) Each antibody revealed a specificity for
CT-A, with the antibody to KAVKKDEL revealing
the highest affinity Therefore, the antibody to
KAVKKDEL was used to assess the presence of the
KDEL peptide in the internalized CT-A No percep-tible KDEL immunoreactivity was detected in rat liver endosomal fractions isolated 5–90 min post-CT injec-tion (Fig 7B), suggesting that the integrity of the C-terminal KDEL peptide was rapidly lost during CT endocytosis
We have previously identified a 25 kDa endosomal CT-A fragment, which we postulated might be involved in the ADP-ribosylation of Gsa [7] The
25 kDa fragment was consistently observed in endo-somal hydrolysates of CT obtained at acidic pH, and was strictly cathepsin D-dependent; its detection coin-cided with 32P-labeling of Gsa by CT hydrolysates at acidic pH [7] Consequently, we examined whether, under conditions where a 25 kDa CT-A fragment was generated by endosomal cathepsin D, we would observe a corresponding loss of the C-terminal KDEL peptide (Fig 7C) Indeed, whereas rapid production of
CT-A
(28-kDa)
CT-B
(12-kDa)
A
α-KAVKKDEL
CT-A (28-kDa) CT-B (12-kDa)
0 5 15 30 60 90
CT
B
α-CT
0 5 15 30 60 90
CT
α-KAVKKDEL α-CT
CT-A (28-kDa)
CT-B (12-kDa)
C
Fig 7 Metabolic fate of the KDEL peptide during endosomal proteolysis of internalized CT (A) Polyclonal sera against CT or KDEL peptides were assessed by western blotting for their ability to bind specifically to CT-A Each lane contained 1 lg of CT-A or 5 lg of CT Arrows to the left indicate the mobilities of CT-A ( 28 kDa) and CT-B ( 12 kDa) Each antibody to KDEL showed specificity for CT-A Polyclonal anti-serum to CT bound to both subunits (B) Rat liver endosomal fractions were isolated at the indicated times after the in vivo administration of
CT, and evaluated by western blotting for their immunoreactivity using polyclonal antiserum to synthetic peptide KAVKKDEL (a-KAVKKDEL)
or polyclonal IgG against CT (a-CT) (incubation with the same membrane) Fifty micrograms of protein was applied to each lane Arrows to the left indicate the mobilities of CT-A ( 28 kDa) and CT-B ( 12 kDa) (C) Endosomal fractions were incubated with 10 lg of native CT at
37 C for the indicated times in 30 m M citrate ⁄ phosphate buffer at the indicated pH The incubation mixtures were then analyzed by western blotting using polyclonal antibody to CT (left panel) or polyclonal antibody to KAVKKDEL (right panel) The mobilities of each intact CT subunit are indicated on the left (CT-A, 28 kDa; CT-B, 12 kDa) A major 25 kDa CT-A fragment was evident after 30 min of incubation, but was not recognized by the antibody to KDEL.
Trang 9a 25 kDa product of CT-A was evident following
pro-teolysis of CT at acidic pH (Fig 7C, left panel), no
detectable KDEL immunoreactivity was associated
with this degradative CT-A product (Fig 7C, right
panel)
Discussion
In the present study, we show that rat hepatocytes
dis-play endosomally located mechanisms to regulate CT
activation and action Two of these regulatory
mecha-nisms may be at the level of intraendosomal toxin
pro-teolysis and endosome acidification (Fig 8) The
assessment of CT compartmentalization during its
endocytosis into rat liver has revealed specific in vivo
regulation during the early phase (0–60 min) of toxin
internalization First, a complex of activated CT-A,
cointernalized Gsa and recruited ARF proteins was
observed in endosomes 5–30 min postinjection
Sec-ond, efficient ADP-ribosylation of the cointernalized
Gsa proteins occurred at the endosomal locus after a
lag phase of 30 min Third, CT-mediated
hyperacidifi-cation of endosomes increased over a time course sim-ilar to that of endosomal activation of CT Therefore,
we propose that endosome regulation would serve as
an effective amplification mechanism for promoting
CT and cathepsin D interaction to induce a maximal cytotoxic effect (Fig 8) Finally, using in vitro cellular systems, we have obtained evidence that the cAMP response in CT-treated cells was, at least in part, rela-ted to the proteolytic activity and expression level of the aspartic acid protease cathepsin D (Fig 8) How-ever, internalized CT that has been localized within the
ER in murine hepatocyte BNL CL.2 cells [19] may also follow another activating pathway operating at a late stage of endocytosis and requiring retrograde transport
Although G proteins are widely accepted as media-tors of signal transduction by cell surface recepmedia-tors, several lines of evidence now indicate that trimeric
G proteins are located in the endosomal compartment
of various cells and are involved in vesicular transport events through the endocytic pathway [20] Consistent with previous studies [21,22], similar amounts of two
Fig 8 Endosomal regulation of CT activation and action in rat liver.
Trang 10forms of Gsa, with apparent molecular masses of
47 kDa (large form, Gsa-L) and 45 kDa (small form,
Gsa-S), have been identified in our endosomal
frac-tions isolated from noninjected rats These isoforms
are produced by alternative splicing of a single
precur-sor mRNA [23]
The translocation of G proteins from the plasma
membrane to the endosomal apparatus has been
demon-strated biochemically and morphologically in various
cells stimulated by agonists such as glucagon [21],
iso-proterenol [24], carbachol [25], thyrotropin-releasing
hormone [26] and bradykinin [27] Our studies extend
these observations to CT, for which maximal endosomal
association of Gsa was observed 20 min (native CT) or
30 min (CT-B) postinjection The underlying
mecha-nisms involved in CT-induced endosomal translocation
of Gsa may well originate in target lipid rafts at the cell
surface, which show significant enrichment of
stimula-tory and inhibistimula-tory G protein and predominant
localiza-tion of the endogenous CT receptor ganglioside GM1
[28,29] This would facilitate interactions between CT,
GM1 and Gsa and, potentially, their subsequent
coin-ternalization Alternatively CT, which increases the
endosomal content of fluid-phase endocytosis probes in
rat liver [30], might induce the uptake of various plasma
membrane molecules, such as its own cellular protein
target Gsa, into the endocytic pathway
Our data suggest a direct interaction between
activa-ted CT-A and Gsa in the endosomal membrane It has
clearly been shown that the endosomal acidic pH
faci-litates the membrane insertion and penetration of
intact CT-A and⁄ or activated CT-A fragment(s) across
the endosomal membrane Using the lipid bilayer
mat-rix containing ganglioside GM1, fluorescence and
phosphorescence spectroscopy studies have shown that
upon CT binding to GM1, CT-A faces the membrane
surface but does not significantly penetrate into the
hydrophobic core of the bilayer at neutral pH [31,32]
However, CT-A1 peptide released from CT-A2 peptide
exhibits hydrophobic behavior in aqueous solution and
when membrane-bound, suggesting that free CT-A1
peptide or CT-A fragment may partition
spontane-ously into the hydrophobic core of the endosomal
membrane Fluorescence resonance energy transfer,
used to monitor pH-dependent structural changes in
CT-B, has revealed that the low endosomal pH is
cap-able of inducing structural changes in CT, which, in
turn, exerts its effect on the structure of the membrane
to which CT-B is bound [33] The role of endosomal
acidity in facilitating CT-A translocation across the
endosomal membrane has also been demonstrated
using hepatic endosomes isolated after injection of
native CT, and then examined for their ability to bind
antibodies to CT-A and to stimulate exogenous plasma membrane-associated adenylate cyclase [6] Time- and acid-dependent exteriorization of CT-A was observed with no translocation of CT-B [6] These findings would be consistent with a model in which CT mark-edly increases endosomal acidification rates (this study) [13], to allow maximal insertion of activated CT-A into the endosomal membrane, leading to efficient ADP-ribosylation of cointernalized Gsa
The enzymatic activity of CT-A1 is allosterically sti-mulated by ARFs, which are host-cell small GTP-binding proteins active in the GTP-bound form [34]
On the basis of the reconstitution of a signal transduc-tion pathway in a bacterial two-hybrid system, a direct interaction between human ARF-6 (belonging to the class III ARFs) and CT-A1 was demonstrated [34] Recently, the cocrystallization of CT and ARF-6 has defined the structural basis for activation of CT by human ARF-6 [35] However, inhibition of the ARF-6 pathway had minimal effects on CT entry, intracellular
CT transport, and CT-induced activation of adenylate cyclase [36] Although the CT–ARF interaction has been extensively characterized in vitro, little is known about their in vivo interaction, and the subcellular binding site(s) between CT and ARF-6 remain(s) unde-fined Originally, ARF-6 was thought to be an uncon-ventional member of the ARF family that was found exclusively in the plasma membrane of Chinese ham-ster ovary cells [37] However, assessment of the sub-cellular distribution of endogenous ARF-6 in various other tissues and cells has established both a cytosolic and membrane-bound localization [38], and over-expressed ARF-6 has been localized to the plasma membrane and endosomes [39] In the present study,
we demonstrate the existence of an endosomal pool of ARFs whose amount was strongly increased after CT treatment ARF-6 was undetectable in endosomes pre-pared from untreated rats, but its endosomal recruit-ment was rapidly observed after CT injection Recently, it was shown that V-ATPase-dependent endosomal acidification stimulates the recruitment of ARF-6 from proximal tubule cytosol to endosomal membranes, implicating this process in endosomal function in situ [40] However, the precise functional role of endosomal ARF-6 in the full activation of internalized CT-A remains to be determined
Subcellular fractionation techniques used to assess the in vivo localization of [125I]CT uptake into rat liver have previously shown that some radioactivity (30 min
to 2 h postinjection) is intrinsic to acid-phosphatase-containing structures, presumably lysosomes [4] Using the in situ rat liver model system for endosome– lysosome fusion, we have confirmed a low lysosomal