* The National Institutes of Health, Bethesda, MD, USA
Food and Drug Administration, Washington, DC, USA
We read, with considerable dismay, a recent Research Article on
cholera toxin (CT) internalization
(Torgersen et al., 2001), in
which the authors extensively challenged methods, results and conclusions that
we had published four years ago (Orlandi
and Fishman, 1998
). As space limits a point-by-point rebuttal of
their comments and critique of the many deficiencies in their study, we
encourage readers to evaluate our response by comparing both papers. As a
preface to our reply, we state that most aspects of CT intoxication are
generally accepted, such as its structure and receptor, its mechanism of
retrograde trafficking through the Golgi and ER, its mechanism of activating
adenylyl cyclase and its pathophysiological effects on human enterocytes.
However, we believe that the crux of the dispute is our differing views on the
relationship between CT internalization and intoxication. Whereas most of the
cell-surface-bound CT is internalized, only a small percentage is activated on
release of the enzymatic A1 peptide
(Kassis et al., 1982
;
Orlandi and Fishman, 1998
).
Thus, to understand the mechanism of CT action, one must determine not only
the pathway(s) for CT internalization but also whether the uptake leads to
intoxication of the cell.
Our paper focused on whether both CT internalization and activation are
mediated by caveolae or by detergent-insoluble glycolipid-enriched complexes
(DIGs) (also known as lipid rafts) in cells deficient in caveolin and
caveolae. Torgersen et al. primarily were interested in showing
caveolae-independent endocytosis of CT. Our approach was to compare CT uptake
and action in three cells that have no, low or high levels of caveolin and
caveolae, and to use the cholesterol modifiers filipin and ß-cyclodextrin
(ßCD) to selectively inhibit caveolae/DIG-mediated endocytosis.
Chlorpromazine (CPZ) and diphtheria toxin (DT) served as inhibitor and probe
for clathrin-mediated uptake. One of our cell lines, human intestinal CaCo-2,
played a major role in their study and appears to be the source of many of
their repetitious complaints. By using anti-CT-A1 antibodies to
quantify CT uptake, we found 58% inhibition by filipin. As Torgersen et al.
found only 17% inhibition by using a different method, they speculated that
our assay may have overestimated CT uptake if the antibodies could not reach
CT clustered in the narrow necks connecting caveolae to the cell surface, and
if filipin could somehow alter the necks and increase antibody binding. They
ignored our second assay in which cells were labeled with rhodamine-conjugated
CT-B at 15°C. When warmed at 37°C, there is an extensive
redistribution of fluorescence from the plasma membrane to the perinuclear
region that is blocked by filipin but not CPZ. Even when Torgersen et al.
found filipin to be ineffective on two other cell lines, they failed to show
that the filipin was active. Filipin is known to be unstable in solution. This
led them to a circular argument: as CT uptake is only slightly inhibited by
filipin, it must not be via caveolae/DIGs. Thus when they found that ßCD
inhibits CT internalization in CaCo-2 cells by 43% (similar to our 39%), they
concluded that the uptake is clathrin-dependent based on the weak effect of
filipin and cited studies showing that ßCD also blocks the latter
pathway. Surprisingly, two of the four references cited were not relevant. We
found that both ßCD- and filipin-treated, but not CPZ-treated, CaCo-2
cells remain sensitive to DT. Others have shown that these agents selectively
inhibit caveolae/DIG-mediated, but not clathrin-mediated, endocytosis in a
variety of cells (Puri et al.,
2001; Wolf et al.,
2002
).
We also assayed CT activation and activity by A1 and cAMP
formation, respectively. Filipin totally blocks both CT activation and
activity in CaCo-2 cells, and ßCD inhibits CT activity by 98%. Thus, both
filipin and ßCD are more effective in inhibiting CT activity than
endocytosis. Although filipin- and ßCD-treated cells still internalize
substantial amounts of the bound CT by other pathways, CT remains inactive. In
this regard, filipin blocks CT-B trafficking from plasma membranes to Golgi,
but not clathrin-mediated endocytosis of CT-B and transferrin in COS-7 cells
(Nichols et al., 2001). We
found that filipin also inhibits CT activity in A431 and Jurkat cells that are
rich in and lacking caveolin and caveolae, respectively. Thus, the disruption
of CT intoxication of cells independent of the presence of caveolin and
caveolae led us to conclude that CT internalization and activation are
mediated through cholesterol- and glycolipid-rich microdomains rather than a
specific morphological structure. Torgersen et al. challenged our thesis by
asserting, "DIGs have been proposed to act as the vehicle for CT entry
in Jurkat T lymphoma cells (Orlandi and
Fishman, 1998
), but there are no data indicating how DIGs might be
internalized." We refer them to a review
(Simons and Ikonen, 1997
) and
an article on endocytosis of a GPI-anchored protein through DIGs in Jurkat
cells (Deckert et al.,
1996
).
Torgersen et al. chide us for not investigating the role of dynamin in
caveolae-mediated uptake and finally for suggesting that CaCo-2 cells have
caveolae based on small amounts of caveolin. The papers that link dynamin with
caveolae-mediated uptake were published while ours was in press
(Oh et al., 1998;
Henley et al., 1998
). Although
dynamin is now known to be involved in both clathrin- and caveolae-mediated
endocytosis, we are not aware of any role in DIG-mediated uptake. Regarding
the presence of caveolin and caveolae in CaCo-2 cells, others agree with us
(Mayor et al., 1994
;
Field et al., 1998
).
Regardless, our major thesis is that of the role of DIGs and not caveolae per
se in CT internalization and intoxication. Finally, we are not dogmatic about
our conclusions as some cell types may use a different pathway for CT
activation. Neurons, although lacking caveolin/caveolae, have DIGs to which CT
binds, but the internalization and activation of the toxin is
clathrin-mediated (Shogomori and Futerman,
2001
).
References
Deckert, M., Ticchioni, M. and Bernard, A. (1996). Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases. J. Cell. Biol. 133,791 -799.[Abstract]
Field, F. J., Born, E., Murthy, S. and Mathur, S. N.
(1998). Caveolin is present in intestinal cells: role in
cholesterol trafficking? J. Lipid Res.
39,1938
-1950.
Henley, J. R., Krueger, E. W., Oswald, B. J. and McNiven, M.
A. (1998). Dynamin-mediated internalization of caveolae.
J. Cell Biol. 141,85
-99.
Kassis, S., Hagmann, J., Fishman, P. H., Chang, P. P. and Moss,
J. (1982). Mechanism of action of cholera toxin on intact
cells. Generation of A1 peptide and activation of adenylate cyclase.
J. Biol. Chem. 257,12148
-12152.
Mayor, S., Rothberg, K. G. and Maxfield, F. R. (1994). Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264,1948 -1951.[Medline]
Nichols, B. J., Kenworthy, A. K., Polishchuk, R. S., Lodge, R.,
Roberts, T. H., Hirschberg, K., Phair, R. D. and Lippincott-Schwartz, J.
(2001). Rapid cycling of lipid raft markers between the cell
surface and Golgi complex. J. Cell Biol.
153,529
-541.
Oh, P., McIntosh, D. P. and Schnitzer, J. E.
(1998). Dynamin at the neck of caveolae mediates their budding to
form transport vesicles by GTP-driven fission from the plasma membrane of
endothelium. J. Cell Biol.
141,101
-114.
Orlandi, P. A. and Fishman, P. H. (1998).
Filipin-dependent inhibition of cholera toxin: evidence for toxin
internalization and activation through caveolae-like domains. J.
Cell Biol. 141,905
-915.
Puri, V., Watanabe, R., Singh, R. D., Dominguez, M., Brown, J.
C., Wheatley, C. L., Marks, D. L. and Pagano, R. E. (2001).
Clathrin-dependent and -independent internalization of plasma membrane
sphingolipids initiates two Golgi targeting pathways. J. Cell
Biol. 154,535
-547.
Simons, K. and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387,569 -572.[CrossRef][Medline]
Shogomori, H. and Futerman, A. H. (2001).
Cholera toxin is found in detergent-insoluble rafts/domains at the cell
surface of hippocampal neurons but is internalized via a raft-independent
mechanism. J. Biol. Chem.
276,9182
-9188.
Torgersen, M. L., Skretting, G., van Deurs, B. and Sandvig,
K. (2001). Internalization of cholera toxin by different
endocytic mechanisms. J. Cell Sci.
114,3737
-3747.
Wolf, A. A., Fujinaga, Y. and Lencer, W. I.
(2002). Uncoupling of the cholera toxin GM1 ganglioside-receptor
complex from endocytosis, retrograde Golgi trafficking, and downstream signal
transduction by depletion of membrane cholesterol. J. Biol.
Chem. 277,16249
-16256.
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