Redox signaling in colonial hydroids: many pathways for peroxide
Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115 USA
* Author for correspndence (e-mail: neilb{at}niu.edu)
Accepted 15 November 2004
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Summary |
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Key words: anti-oxidant, clonal, cnidarian, colony development, Eirene, evolutionary morphology, hydroid, Podocoryna, Podocoryne, reactive oxygen species, redox signalling
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Introduction |
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In the mitochondrial ETC, there are two sites of ROS formation, site 1 on
complex I and site 2 at the interface between coenzyme Q and complex III
(Nishikawa et al., 2000;
Armstrong et al., 2003
).
Experimental manipulations of mitochondrial function in hydractiniid hydroids
suggest that it is site 2 that produces the ROS that affect colony growth and
development (Blackstone, 2003
).
At comparable physiological doses (determined by measures of oxygen uptake),
blocking the mitochondrial electron transport chain at complex III with
antimycin A1 produces the same phenotypic effects as blocking at
complex IV with azide. This phenotypic effect is similar to that observed in
areas of colonies that are only indirectly supplied with food from polyps
elsewhere in the colony (Blackstone,
2001
). In each case, ROS are increased, and the resulting
phenotype consists of `runner-like' growth with widely spaced polyps and
stolon branches. Conversely, at appropriate physiological doses the uncoupler
of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone, has
the same phenotypic effect as another uncoupler, 2,4-dinitrophenol. This
effect is similar to that observed in areas of colonies that are well fed -
`sheet-like growth', with closely spaced polyps and stolon branches - and
correlates with low levels of mitochondrial ROS. Rotenone was used to inhibit
electron transport `downstream' of site 1 of ROS formation and `upstream' of
site 2. The resulting phenotypic effects were very similar to those produced
by uncouplers and strikingly different from those produced by antimycin or
azide. This suggests that signal transduction is initiated at or near site 2,
and this role of site 2 has been found in other studies
(Nishikawa et al., 2000
;
Armstrong et al., 2003
). While
the effects of antimycin are sometimes difficult to interpret because of the
intricate interaction between coenzyme Q and complex III
(Armstrong et al., 2003
;
Osyczka et al., 2004
), in this
case the similarities between the effects of azide and antimycin suggest that
blocking the ETC anywhere downstream of site 2 will produce similar effects. A
lesser role for site 1 may be due to differences between sites 1 and 2 in
electron flux in colonies subject to a fat-rich diet.
At least in hydractiniid hydroids, the bulk of the mitochondria in a colony
are concentrated in narrow regions of contractile epitheliomuscular cells
(EMCs) located in polyp-stolon junctions
(Blackstone et al., 2004).
Within a colony, polyp-stolon junctions tend to be more centrally located as
compared with peripheral stolon tips. Both are similar in basic structure, for
instance, exhibiting a layer of endoderm and ectoderm covered by a protective
perisarc. Both are also connected by a continuous lumen through which
gastrovascular fluid circulates at a high rate. Nevertheless, peripheral
stolons are devoid of these muscular, mitochondrion-rich cells
(Schierwater et al., 1992
;
Blackstone et al., 2004
).
Mitochondrion-rich EMCs may be the locus of colony-wide redox signaling
(Blackstone et al., 2005a
).
Since the outward growth of a colony and its form are ultimately determined by
the behavior of peripheral stolon tips, signals from mitochondrion-rich EMCs
at polyp-stolon junctions may be conveyed to these peripheral tips. ROS from
mitochondrion-rich contractile regions can be considered a potential candidate
to provide stolons with signals influencing elongation, branching and
regression, leading to the emergence of colony growth form. To investigate
further the possible role of ROS in such redox signaling, perturbations of
colony growth and development were carried out using the hydroid
Podocoryna carnea. Some experiments also used Eirene
viridula. Chemical (vitamin C) and enzymatic (catalase) antioxidants were
used to attempt to diminish ROS, and these results were compared with those
that have been obtained previously using uncouplers of oxidative
phosphorylation to diminish mitochondrial ROS. ROS were also manipulated using
exogenous peroxide. Using fluorescent microscopy of both stolon tips and
mitochondrion-rich contractile regions, assays of ROS were carried out with
2',7'-dichlorofluorescein diacetate. The data obtained from these
experiments suggest that ROS in general and hydrogen peroxide in particular
are involved in a number of as-yet-uncharacterized signaling pathways in
colonial hydroids.
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Materials and methods |
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Even though genetically identical stocks were used, colony growth may
differ between experiments because of environmental and perhaps epigenetic
effects (Ponczek and Blackstone,
2001). Seasonal effects are particularly common with more
sheet-like and slow-growing colonies occurring in the winter
(Ponczek and Blackstone,
2001
). Hence, control colonies were part of each experiment, and
all control and treated explants for an experiment were always made from the
same source colony. Nevertheless, some variation can be found even within a
group of explants made from the same colony. Typically, the slowest growing
explants (which are assigned the highest numbers in the figures) are also the
more sheet-like.
Treatment with vitamin C, catalase and peroxide
To investigate the pathways by which lithium ions affect development,
Jantzen et al. (1998), treated
Hydra vulgaris and Hydra magnipapillata with vitamin C,
vitamin E, and catalase. We have largely adopted their protocols. Vitamin E
(
-tocopherol), however, requires a solvent for treatment in aqueous
media, and in this regard ethanol is unsatisfactory for treatments of these
hydroids (Blackstone, 2003
).
While dimethyl sulfoxide (DMSO) can be used, experiments suggest that DMSO may
stimulate oxygen uptake (data not shown), perhaps because it is permeabilizing
the mitochondrial inner membrane. To simplify the interpretation of the
experiments, only vitamin C (ascorbic acid) was used to investigate the
effects of chemical antioxidants. For all experiments, vitamin C was prepared
in a 10 mmol l-1 stock solution and adjusted to a pH 8 with NaOH.
This stock was prepared afresh each day, immediately prior to use. Treatment
of hydroid colonies was carried out at 100 µmol l-1. While
vitamin C is generally considered an anti-oxidant, under some conditions it
can interact with catalytically active metals such as iron or copper and
produce ROS (Carr and Frei,
1999
). Hydroids are highly sensitive to such metals in their
culture medium (Lenhoff,
1983
). The seawater medium used (Reef Crystals, Aquarium Systems,
Mentor, Ohio, USA) contains chelators that probably keep concentrations of
such metals very low, particularly when reverse osmosis (RO) water is used to
mix up the medium. Catalase treatments were carried out at 0.1 mg
ml-1. Follow-up experiments show that similar effects are obtained
using 0.033 mg ml-1 (data not shown). Hydrogen peroxide treatments
were carried out at nominal concentrations of
20-50 µmol
l-1. Because peroxide is reactive, the actual concentrations may
have been somewhat less than this. Nevertheless, combining catalase and
peroxide at considerably more dilute solutions than those used in the
experiments quickly saturated an oxygen electrode, so considerable activity of
both the catalase and peroxide is thus assured.
Comparisons of colony growth and development
Experiments were conducted separately over a period of several years. For
each experiment, 14 replicates were explanted on 18 mm cover slips, with seven
each assigned to control and to the appropriate treatment. Occasionally,
broken cover slips resulted in smaller sample sizes. Each group was treated
with the appropriate solution in finger bowls for 6 h day-1.
As previously (e.g. Blackstone,
2003
), intermittent treatments seemed to be best tolerated by
colonies. As each colony covered the surface of the cover slip, that colony
was imaged. A colony was considered to be covering the surface when stolons
were contacting the edge of the cover slip throughout
60% of its
circumference. Images were processed to facilitate automatic measurement in
Image-Pro Plus software (Media Cybernetics, Silver Spring, Maryland, USA). The
gray level of some image objects (i.e. background, stolons or polyps) was
adjusted using Corel Photo-Paint software (Corel, Ottawa, Canada; background
gray level = 10, stolon = 201, polyp = 255). Processed images were checked
against the original images to insure accuracy. Processed images were measured
in Image-Pro for total colony area, total polyp area, and empty, unencrusted
areas within the colony (`inner' areas). Analyses focused on the mean size of
these inner areas, which largely depends on stolon branching and anastomosis
(i.e. as stolon development increases, mean inner area decreases). These data
were natural logarithm transformed before analysis of variance (ANOVA) with
PC-SAS software (SAS Institute, Carey, North Carolina, USA). Total polyp area
adjusted for total colony area was analyzed in the same way. Other parameters
(e.g. total colony area and number of days for a colony to cover the surface)
are also reported and compared as mean ± S.E.M. (twice the
S.E.M. provides a 95% confidence interval).
Comparisons of reactive oxygen species
Hydrogen peroxide represents a major component of ROS under physiological
conditions (Chance et al.,
1979), and 2',7'-dichlorofluorescein diacetate
(H2DCFDA; Molecular Probes, Eugene, Oregon, USA) is usually used to
assay H2O2 (Jantzen
et al., 1998
; Nishikawa et
al., 2000
; Pei et al.,
2000
). This non-fluorescent dye is freely permeable to living
cells. Once inside a cell, the acetate groups are removed by intracellular
esterases. In turn, H2DCF is usually oxidized by peroxides in the
presence of peroxidase, cytochrome c, or Fe2+ to form
2',7'-dichlorofluorescein which can then be visualized with
fluorescent microscopy. There is some debate as to whether the activation of
H2DCF is specific for the detection of H2O2
(Finkel, 2001
).
Conservatively, this assay should be regarded as a semi-quantitative measure
of general ROS activity. A 10 mmol l-1 stock solution of
H2DCFDA was prepared in anhydrous DMSO. Twenty-four hours after
feeding, 5-7 naïve colonies (i.e. colonies previously untreated) were
incubated in the appropriate treatment with an equivalent number of control
colonies. After 1 h, H2DCFDA was added to a concentration of 10
µmol l-1, and colonies were incubated an additional hour in the
dark prior to measurement. Colonies were imaged in a RC-16 chamber (Warner
Instruments, Hamden, USA) in plain seawater immediately after being removed
from the treatment solution. Using a Orca-100 cooled-CCD camera (Hamamatsu
Photonics, Hamamatsu City, Japan) and a Axiovert 135 (Carl Zeiss, Jena,
Germany), peroxide (as indicated by H2DCFDA-derived
2',7'-dichlorofluorescein) was imaged for a
50 x150
µm region at the base of three polyps per colony (excitation 450-490 nm,
emission 515-565 nm). At these wavelengths, negative controls show that there
is little native fluorescence. Images with 12-bit depth (4096 gray levels)
were thus obtained and were analyzed using Image-Pro Plus software. In such
images, fluorescence is visible from many
10 mm2-sized
clusters of mitochondria from EMCs at polyp-stolon junctions
(Blackstone et al., 2004
). The
luminance and area for each of these fluorescent objects was measured in Image
Pro Plus software by: (1) selecting the object and an equivalent area of its
immediate surroundings (background) as a circular region of interest; (2)
allowing the software to identify the area and luminance of the foreground
`bright' region (i.e. the area of fluorescent signal); (3) exporting these
measures to file; (4) automatically identifying the area and luminance of the
complementary background `dark' region and exporting these measures to file.
The area of each cluster was thus calculated, and the luminance of the cluster
was adjusted for the background luminance by subtraction. These measures were
analyzed by a nested ANOVA, clusters nested within polyps, polyps nested
within clonal replicates and replicates within treatments. In separate
experiments with similarly treated naïve colonies, three peripheral
stolon tips were measured per colony. Images of stolon tips were analyzed
similarly, except the entire stolon tip was measured and compared with an
equivalent area of the background fluorescence outside the colony.
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Results |
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Catalase, conversely, triggers rapid growth of peripheral stolons away from
the center of the colony in P. carnea, and the result is a
fast-growing and extremely runner-like colony with few, widely spaced polyps
and long stolonal connections (Fig.
3). While catalase-treated colonies were imaged at slightly
smaller total areas than controls (controls, 135.86±7.2 mm2;
treated, 107.15±10.82 mm2), this likely reflects their
extremely runner-like growth form. In other words, when covering the surface
the long, unbranched stolons of the treated colonies enclosed a smaller area
than the more branched stolons of the controls. Catalase-treated colonies
covered the surface more quickly than controls (controls, 30.4±4 days;
treated, 18.1±2.3 days). Treated colonies exhibited less branching and
anastomosis of stolons as indicated by the mean size of unencrusted areas
within the colony (Fig. 4;
F=46.8, d.f.=1, 12, Px0.001). Treated colonies also
exhibited a smaller percent of the total area devoted to polyp growth
(F=13.9, d.f.=1, 12, P<0.01). In E. viridula,
catalase had similar effects. Treated colonies covered the surface more
quickly than controls (controls, 19.4±1 days;treated, 16±0.7
days). Since the time of covering is sometimes difficult to judge in E.
viridula, the time that the first stolon touched the cover slip edge was
also measured, with similar results (controls, 12.1±1.2 days; treated,
7.2±0.9 days). While treated colonies did not exhibit significantly
greater branching of stolons as indicated by the mean size of unencrusted
areas within the colony (F=2.8, d.f.=1, 12, P>0.1), this
is a less than ideal measure for the catalase-treated colonies of E.
viridula because they branched so little that they did not form many
inner areas. Other measures of growth form such as total colony perimeter
divided by the square root of total colony area did show significant
differences between catalase-treated and control colonies of E.
viridula (F=19, d.f.=1, 12, P<0.001), indicating
that the treated colonies exhibited a more irregular, runner-like growth form
(Blackstone and Buss,
1991).
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Peroxide experiments were conducted in the winter; hence colonies of P. carnea were relatively slow-growing and sheet-like. Nevertheless, colonies treated with exogenous peroxide covered the surface faster than untreated colonies (56±1.9 days versus 64±2.6 days). No significant effect was found of peroxide treatment on branching and anastomosis of stolons as indicated by the mean size of unencrusted areas within the colony (Fig. 5; F=2.1, d.f.=1, 12, P>0.15), nor did other measures of growth form show significant differences. Perhaps notably, the slowest growing treated and control colonies (replicates 6 and 7) did show a large difference in mean inner area and other measures. It may be that peroxide treatment has an effect under some circumstances, e.g. perhaps when endogenous levels of peroxide are low.
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Comparisons of reactive oxygen species
In mitochondrion-rich polyp-stolon junctions in naïve colonies of
P. carnea, vitamin C diminished levels of peroxide 2 h after
initiating treatment, as indicated by H2DCFDA-derived
2',7'-dichlorofluorescein (Fig.
6), and this different is statistically significant
(F=19, d.f.=1, 12, P<0.001). In other naïve colonies
after
2 h, however, peripheral stolon tips in five colonies treated with
vitamin C showed greatly increased ROS levels compared with five controls
(Fig. 7; F=140,
d.f.=1, 8, Px0.001). This dramatic flux in peroxide occurs as
these stolon tips are regressing
(Blackstone et al., 2005a
). In
colonies treated repeatedly over many days, such stolon death does not occur;
rather, stolons grow out very slowly with high rates of branching, i.e. there
are not really any `peripheral' stolons (e.g.
Fig. 1). Conversely, in
mitochondrion-rich polyp-stolon junctions in naïve colonies of P.
carnea, catalase has no detectable effect on peroxide
2 h after
initiating treatment (F=2, d.f.=1, 8, P>0.2). In other
naïve colonies after
2 h, peripheral stolon tips in colonies treated
with catalase were again no different from those in the controls
(Fig. 8; for the foreground -
background difference; F=1.5, d.f.=1, 12, P>0.2). While
the naïve catalase-treated colonies showed no difference in relative
luminance (foreground - background), they nevertheless did show an absolute
difference such that treated stolon tips exhibit greater absolute levels of
ROS when compared with controls (Fig.
8; for absolute foreground luminance; F=20.7, d.f.=1, 12,
P<0.001). For the latter measures, identical camera settings were
used for all images to ensure that absolute measures of luminance were
comparable.
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In naïve colonies of P. carnea, those treated with exogenous
peroxide for 2 h show increased levels of ROS in stolon tips as compared
with controls (Fig. 9;
F=29.5, d.f.=1, 8, P<0.001). In untreated colonies of
P. carnea, peripheral and central stolon tips were examined for ROS,
and a gradient was found such that central stolon tips exhibit greater amounts
of ROS (Fig. 10; paired
comparison t-test, t=4, P<0.01). Finally,
colonies of E. viridula exhibit higher levels of ROS in stolon tips
than colonies of P. carnea (Fig.
11; F=44.5, d.f.=1, 10, P<<0.001).
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Discussion |
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Catalase, conversely, is a large (350 kDa) tetramer and is likely not taken
up by a colony, nor can its enzymatic function be transferred across a plasma
membrane. The lack of an effect on ROS levels of mitochondria-rich EMCs
supports this hypothesis. Nevertheless, catalase probably does rapidly convert
any peroxide released by the colony into water and oxygen. ROS emitted by
colonies may serve a function (perhaps anti-bacterial, but see
Bolm et al., 2004), hence the
diminished amounts of ROS outside the colony may lead to compensatory
formation and emission from within the colony. In treated colonies, stolon
tissues thus have absolutely greater amounts of ROS, as suggested by the data.
Such elevated levels of ROS are still considerably less than levels needed to
provoke an acute response leading to cell death. Nevertheless, these elevated
levels are sufficient to mimic mitochondrial redox signaling and result in
rapid, runner-like growth. In support of this hypothesis, colonies treated
with exogenous peroxide cover the surface of an 18 mm diameter cover slip
faster than untreated colonies. Furthermore, there is a gradient in colonies
of P. carnea with central stolon tips, which are closer to the
majority of the mitochondrion-rich EMCs, exhibiting higher levels of ROS than
peripheral stolon tips. Finally, colonies of E. viridula exhibit
higher levels of ROS in stolon tips than colonies of P. carnea, and
this correlates with their extremely rapid, runner-like growth. It is not
known what molecules may be the targets of such ROS. Nevertheless,
cysteine-rich proteins involved in vascular development (e.g. vascular
endothelial growth factors; Seipel et al.,
2004
) provide plausible candidates.
On the basis of these data, we hypothesize that in hydroid colonies ROS
participate in a number of putative signaling pathways. High levels of ROS may
be a factor in the cell and tissue death that seem to affect peripheral stolon
tips when the environment is rapidly changing. Such a process would seem
adaptive - if the colony becomes `overextended,' stolons can retreat and the
nutrients in the cells and tissues of the stolon may be taken up by the
remainder of the colony. ROS emitted from the colony also seem to have an
extra-colony function, perhaps in suppressing the growth of bacteria or other
parasites. Hydractiniid hydroid colonies grow on snail shells that are crowded
with epifauna and probably some of these can be rebuffed by peroxide. Notably,
the foot region of Hydra is characterized by the activity of a
peroxidase (Hoffmeister-Ullerich et al.,
2002). Hydra may also emit peroxide and may use this
peroxidase to protect its own tissue at the point of attachment to the
substratum. More moderate levels of ROS in stolon tips seem to act as a growth
factor, triggering outward growth, inhibiting branching and, possibly,
mediating the redox signaling emanating from mitochondrion-rich EMCs.
Treatment with exogenous peroxide suggests that stolon tips are capable of
concentrating peroxide. Peroxide emitted from polyp-stolon junctions could be
carried by gastrovascular flow to stolon tips. Nevertheless, because of the
multiple pathways for peroxide, the particular phenotypic effects may depend
on the spatial and temporal patterns of ROS formation within the colony. While
the work reported here serves to outline the broad possibilities for signaling
using ROS in colonial hydroids, considerable amounts of future research will
be required to elucidate these spatial and temporal patterns, as well as the
molecular targets of ROS.
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Acknowledgments |
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