Institut für Genetik, Abteilung Molekulargenetik;
Römerstraße 164, 53117 Bonn, Germany
* Present address: DLR-Projektträger, Gesundheitsforschung, Südstr.
125, 53175 Bonn, Germany
Author for correspondence (e-mail:
genetik{at}uni-bonn.de)
Accepted 28 April 2003
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Summary |
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Key words: Connexin, cx, Connexin-deficient mice, Gap junction, Epidermis, Wound healing
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Introduction |
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The epidermis, a stratified surface epithelium, protects the organism against external influences like mechanical, chemical or thermal stress and serves as a barrier to water. Keratinocytes pass through a program of differentiation which is accompanied by differential expression of several proteins like keratins and cadherins. The basal layer, the undermost layer of the epidermis, contains nondifferentiated, proliferating keratinocytes. When keratinocytes migrate into the spinous layer they loose their ability to proliferate and start the program of terminal differentiation. When they reach the cornified layer, keratinocytes peel off the epidermis as scurf (for a review, see Fuchs et al., 1994).
To date, nine different connexins have been described in the murine
epidermis (reviewed by Richard,
2000a). The connexin expression pattern in rodents is altered
during embryogenesis and after birth
(Risek et al., 1992
; Choudry
et al., 1997). In the epidermis of adult mice, Cx43 and Cx40 are expressed in
the basal layer and Cx43 and Cx31 are expressed in the spinous layer. In the
granulous layer, Cx43 expression no longer occurs, so that besides weak
expression of Cx26, only Cx31 appears in this layer
(Kamibayashi et al., 1993
;
Butterweck et al., 1994
)
(reviewed by Richard, 2000a
).
Transcripts of Cx30, Cx30.3, Cx31.1 and Cx57 have been found in mouse
epidermis but the epidermal expression pattern of the corresponding proteins
has not yet been analyzed (Manthey et al.,
1999
; Hennemann et al.,
1992
; Dahl et al.,
1996
). In the epidermis of adult rats, expression of Cx43 and
Cx31.1 has been detected in the basal and spinous layers, Cx37 is localized in
all epidermal layers besides the stratum corneum, and Cx26 has been found in
the granulous and at low concentrations in the spinous layer
(Risek et al., 1992
;
Goliger and Paul, 1994
).
Mutations in human connexin genes are known to cause several skin diseases.
For example, erythrokeratodermia variabilis, an autosomal dominant
genodermatosis, is characterized by the appearance of transient erythema and
localized or generalized hyperkeratosis that can be due to mutations in the
Cx31 or Cx30.3 gene
(Richard et al., 1998;
Richard, 2000a
;
Richard et al., 2000b
;
Macari et al., 2000
;
Gottfried et al., 2002
).
Different mutations in the Cx26 gene can lead to Vohwinkel's syndrome
which implies hearing loss in conjunction with palmoplantar keratoderma
(Maestrini et al., 1999
; for
reviews see White, 2000
, and
Kelsell et al., 2001
).
Mutations in the Cx26 gene can also cause the
Keratitis-ichthyosis-deafness syndrome
(Richard et al., 2002
).
Normal skin can regenerate after wounding or damaging. Wound healing
involves proliferation, migration and differentiation of keratinocytes
(Martin, 1997;
Jacinto et al., 2001
). The
expression of connexins in rat skin was shown to be altered in response to the
repair process (Goliger and Paul,
1995
). The healing process of an incision wound in rat tail skin
takes about five days (Goliger and Paul,
1995
). To study the effects of connexin loss on residual connexin
expression in the mouse epidermis, we have analyzed the expression of Cx43,
Cx31, Cx30 and Cx26 protein in intact and wounded tail skin of the transgenic
mouse mutants Cx43Cre-ER(T)/fl, Cx31/,
Cx30/ and wild-type control mice.
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Materials and Methods |
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Dye transfer in acute thick sections of mouse ear skin
After sacrificing the mice, the ears were immediately dissected and
transferred into Dulbecco's modified Eagle's Medium (Gibco, Karlsruhe,
Germany), warmed to 35°C. Tissue slices (250 µm) of mouse ear were
produced using a microtome (VT1000S, Leica, Wetzlar, Germany) with commercial
razor blades. Sections were microinjected, submerged in a chamber at 35°C
under constant flow of oxygenated Dulbecco's Modified Eagle's Medium, after an
initial incubation period of 15 minutes to 2 hours at 35°C in carbogen
(95% O2 and 5% CO2). Microinjection of the fluorescent
dye Alexa Fluor 488 (Molecular Probes Europe BV, Leiden, The Netherlands) was
performed by iontophoresis into single cells under visual inspection. The dye
was injected according to Romualdi et al.
(Romualdi et al., 2002) with 2
nA for 2 minutes. Electrodes were filled with 200 mM KCl. These conditions
resulted in the expected membrane potential of 25 mV when starting the
injection of Alexa Fluor 488. Injections at lower membrane potentials were
excluded from the final analysis. The intercellular spreading of the
microinjected dye was followed by visual inspection under UV light and phase
contrast. Finally, gap junctional coupling was evaluated 10 minutes after
injection by counting fluorescent cells under UV light, and micrographs were
taken with a SONY CCD camera. Dye spreading experiments were carried out on
two to three skin sections per mouse with the following numbers:
Cx43Cre-ER(T)/fl induced: 6 mice, 28 injections;
Cx43Cre-ER(T)/fl uninduced: 4 mice, 16 injections;
Cx43fl/fl uninduced: 3 mice, 16 injections; Cx43fl/+
induced: 2 mice, 17 injections; wild-type: 13 mice; 44 injections;
Cx31/: 5 mice, 31 injections; Cx31+/+: 3
mice, 26 injections. The statistically significant decrease of dye transfer in
skin sections from Cx43Cre-ER(T)/fl mice (P<0.001) has
been evaluated by the paired t-test.
Immunoblot analysis
Wounded or uninjured pieces of tail skin were dissected on ice and
immediately frozen in liquid nitrogen. Homogenized tissue was taken up in
protein lysis buffer [60 mM Tris HCl, pH 7.4 and 3% sodium dodecyl sulphate
(SDS)], supplemented with proteinase inhibitor `Complete' (Roche, Mannheim,
Germany) and sonicated three times for 20 seconds on ice. Protein
concentration was determined using the bichinchoninic acid protein assay
(Sigma, Taufkirchen, Germany). For electrophoresis, 50 µg of protein were
separated on 12.5% SDS-polyacrylamide gels. Proteins were electroblotted on
nitrocellulose membranes (Hybond ECL, Biosciences, Bucks, UK) for 2 hours at
100 V. Membranes were blocked for 1 hour with blocking solution [(20 mM Tris,
HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20 (TBS-Tween)] and 5% skim milk powder
(w/v) and afterwards incubated with primary antibodies over night at 4°C.
The antibodies were diluted in blocking solution (anti-Cx43: 1:500
(Traub et al., 1994);
anti-Cx31: 1:250 (Butterweck et al.,
1994
); anti-Cx30: 1:250 (Zytomed, Berlin, Germany); anti-Cx26:
1:500 (Zytomed)). After washing for 30 minutes in blocking solution, membranes
were incubated with anti-rabbit horseradish peroxidase-conjugated secondary
antibodies at room temperature for 1 hour (Dianova, Hamburg, Germany), diluted
1:20000 for anti-Cx43 and 1:5000 for the other antibodies in blocking
solution. Afterwards, membranes were washed for 1 hour in TBS-Tween and
incubated with an ECL chemiluminescence detection system (Amersham
Biosciences, Freiburg, Germany). In order to check equal loading in all lanes
of immunoblot, we performed Ponceau or Coomassie staining of the blotting
membranes, followed by densitometric analyses of the stained protein. Lysates
of HeLa cells after stable transfection with the respective connexin and
lysates of untransfected HeLa cells were used as positive and negative
controls.
Immunofluorescence analyses
Cryosections (5 µm) of tail epidermis were stained with the following
antibody solutions: rabbit anti-Cx43 (1:2000)
(Traub et al., 1994), rabbit
anti-Cx31 (1:100) (Butterweck et al.,
1994
), rabbit anti-Cx30 (1:300; Zytomed) and rabbit anti-Cx26
(1:500; Zytomed). Primary antibodies were detected with Alexa594 conjugated
goat anti-rabbit immunoglobulin (1:2000; MoBiTech, Goettingen, Germany). For
immunofluorescence analyses, cryosections were fixed in 100% ethanol
(20°C) for 5 minutes, blocked with 4% bovine serum albumin (BSA,
PAA Laboratories GMBH, Linz, Austria) in phosphate buffered saline (PBS) and
incubated with antibodies diluted in 0.4% BSA in PBS over night at 4°C.
Afterwards, sections were washed with 0.4% BSA in PBS and incubated with Alexa
conjugated antibodies for 1 hour at room temperature. Nuclear staining was
performed by 15 minutes incubation with 0.2 µg/ml Hoechst 33258 fluorescent
dye in PBS (Sigma B-2883). Slices were mounted with fluorescent mounting
medium (Dako, Glostrup, Denmark).
Antibody-stained tissue slices were analyzed using the photomicroscope Axiophot (Zeiss, Jena, Germany). Scoring of sections to quantify the extent of immunostaining was carried out under the microscope to ensure that only immunofluorescent and not background signals were counted. The size of immunofluorescent signals was taken into account as follows: images were digitally recorded at the same magnification and time of exposure. Immunosignals were analyzed by counting immunopositive pixels in distinct areas (approx. 30 µm2). The unit `immunosignals/field' matched the number of pixels counted per 30 µm2 area. The mean values of the counted samples with the corresponding standard deviations were determined and Student's t-tests were performed.
Sections of the corresponding connexin deficient mice were used as negative controls. Stable transfected HeLa cells or sections of connexin expressing tissues were used as positive controls.
Injection of 4-hydroxytamoxifen in Cx43Cre-ER(T)/fl and
control mice
Fifty milligrams of 4-hydroxytamoxifen (Sigma, H-6278) were suspended in
200 µl ethanol with a sonicator. Peanut oil (4.8 ml) was added, and the
mixture was sonicated and mixed vigorously (final concentration: 10 mg/ml).
Injections were performed intraperitonally with a 26 gauge needle.
4-Hydroxytamoxifen (3.5 mg) was injected five times (once every 24 hours) and
wounds were cut 5 days after the last injection.
Preparation of skin sections and histochemistry
Animals were killed by cervical dislocation. The tail skin was dissected,
immediately frozen in liquid nitrogen and stored at 70°C. Tail
epidermis was cut lengthwise (5 µm sections) with a cryostat. For
5-bromo-4-chloro-3-indolyl-ß-galactoside staining, sections were
processed as described previously (Theis
et al., 2001). Tissue used for paraffin embedding was fixed with
100% ethanol overnight at 4°C. Tissue infiltration was performed in an
infiltration machine (TP1020; Leica, Bensheim, Germany) with the following
protocol: 30 minutes H2O; 1 hour 70% ethanol; 30 minutes 80%; 30
minutes 96%; 45 minutes 96%; 30 minutes 100%; 30 minutes 100% ethanol; 30
minutes xylol; 30 minutes xylol; paraplast 2 hours; paraplast 2 hours.
Embedding was carried out with a paraffin embedding station (EG 1140 H;
Leica). Slices of 5 µm were produced with a rotation microtome (HM 360;
Microm). Deparaffination was performed with the following protocol: Xylol 3
minutes; 100% EtOH 1 minute; 96% EtOH 1 minute; 80% EtOH 1 minute; 60% EtOH 1
minute. Afterwards slices were stained with haematoxylin and eosin and mounted
with mounting medium (DAKO).
Wounding and isolation of mouse tail skin
Incision wounds into mouse tail were cut with a scalpel as previously
described for rat tail (Goliger and Paul,
1995). Six to eight transverse sections through the tail skin with
a length of 1 cm were performed per mouse. Two of them were processed for
immunofluorescence, two for histological analysis and four were used for
immunoblot analyses. At each time point after wounding, connexin-deficient and
control mice were analyzed.
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Results |
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Cx30 protein was barely detectable in the granulous layer of normal tail epidermis. In other epidermal layers, Cx30 protein was not found in nonirritated skin (Fig. 1B). In contrast to these results, ß-galactosidase staining of Cx30/ tail epidermis yielded signals in all epidermal layers (Fig. 2B). Thus, Cx30 transcripts were probably abundant in all epidermal layers but Cx30 protein was only barely detected in the granule layer of mouse tail epidermis, suggesting post-transcriptional control of Cx30 expression.
Cx43 expression pattern in mouse epidermis of the back was already found in
the stratum basale and lowest layers of the stratum spinosum
(Butterweck et al., 1994). This
corresponds to the expression found in wild-type tail epidermis
(Fig. 1C). In
Cx31/ mice, the level of Cx43 protein was
significantly reduced (Fig. 3A) compared with wild-type tail epidermis
(Fig. 1C). We observed by
immunoblot analyses of Cx31/ mice only about
one third of the Cx43 protein found in wild-type controls
(Fig. 3B), which is consistent
with the above finding.
|
Cx30 and Cx26 proteins were barely detectable in the granulous layer of wild-type tail epidermis (Fig. 1B,D). By contrast, Cx30-specific immunoreactivity was much stronger in the granulous and spinous layers in induced Cx43Cre-ER(T)/fl mice (Fig. 4A), compared with wild-type controls (Fig. 4B). Thus, our results show an altered expression of Cx43 protein in Cx31-deficient mice and of Cx30 protein in Cx43Cre-ER(T)/fl mice.
|
Expression of Cx43, Cx31, Cx30 and Cx26 during wound healing of
wild-type mouse tail epidermis
To study wound healing in mice we adapted the method described by Goliger
and Paul (Goliger and Paul,
1995) for wounding of rat tail skin.
Fig. 5 shows that the healing
process in mouse skin took 5 days, similar to that reported for rat skin
(Goliger and Paul, 1995
). In
the first 12 to 24 hours after wounding, the incision wound could be
recognized on micrographs as cut-through epidermis and dermis of the mouse
tail (cf. Fig. 5A). On day 1-2
of the healing process, the epidermis at the wound edges was thickened and
rounded keratinocytes began to migrate under the coagulum (cf.
Fig. 5B). Three days after
wounding, the migrating keratinocytes closed the epidermal wound under the
coagulum and began their process of terminal differentiation
(Fig. 5C). Four to five days
after wounding, the epidermis was again completely stratified
(Fig. 5D).
|
The expression pattern of Cx43 and Cx26, which had been studied in wounded
tail epidermis of 10-day-old rats (Goliger
and Paul 1995), was largely consistent with our results on Cx43
and Cx26 in mouse tail epidermis. Furthermore, we found that the expression
patterns of Cx26 and Cx30 after wounding were remarkably similar
(Fig. 6A,C). Both Cx26 and Cx30
were barely expressed in the granulous layer of uninjured tail epidermis.
After wounding, the expression of both connexins was upregulated in all
epidermal layers, reaching a maximum on day 3 after wounding and declining
afterwards to the expression level in uninjured epidermis.
|
Cx31 was strongly expressed in suprabasal layers and weakly in the stratum basale of uninjured tail epidermis. After wounding, Cx31 expression in suprabasal layers was drastically downregulated, whereas expression in the basal layer increased to a maximum on day 3 postwounding (Fig. 6D). Cx31 expression in suprabasal layers started again to increase on day 3 after wounding, in contrast to the basal layer, where it returned to the low expression level of uninjured epidermis.
In uninjured tail epidermis, immunofluorescence analysis of Cx43 protein revealed its expression in basal and spinous layers. Between days 1 and 2 after wounding, the amount of Cx43 protein was clearly reduced at the wound edges and in the periphery of the wounds, a region where proliferation has been described (Fig. 6B). At day 3 postwounding, Cx43 expression started to increase in basal and suprabasal layers to the expression level in uninjured epidermis. Thus, localization of Cx43 protein did not change during wound-healing process.
Expression of Cx43, Cx31, Cx30 and Cx26 during epidermal wound
healing in connexin-deficient mice
Mice with reduced levels of Cx43 protein showed premature fusion of tail
epidermis on day 2 after wounding in seven out of eight cases. Thus, in
contrast to wild-type mice, where the epidermis under the coagulum fused on
day 3 after wounding in 14 of 16 cases (Figs
7,
8), Cx30 and Cx31 showed an
earlier peak of expression in tail epidermis of mice with decreased amounts of
Cx43 (Fig. 6E,F). Immunofluorescence analyses revealed a peak in Cx30 protein expression in the
tail epidermis of mice, with reduced Cx43 levels already at day 2
postwounding, whereas wild-type controls displayed maximal Cx30 protein on day
3 (Fig. 7). In addition, the
total amount of Cx30 protein on days 2 and 3 of the wound-healing process was
clearly higher in tail epidermis of Cx43-deficient mice than in wild-type
controls. Cx30 expression decreased in the basal layer of mice with reduced
levels of Cx43 one day earlier (on day 3) compared with wild-type
epidermis.
|
|
Immunostained Cx31 protein was clearly more abundant on days 1 and 2 postwounding in tail epidermis of mice with decreased amounts of Cx43 protein than in wild-type controls (Fig. 8). Cx31 expression already increased on day 2 in the spinous and granulous layers, whereas it occurred only on day 3-4 in wild-type epidermis. On day 3 there was a reduction of the Cx31 protein in the basal layer. By contrast, Cx31 protein did not decrease until day 4 after wounding of wild-type tail epidermis.
In Cx31-deficient mice, only traces of immunoreactive Cx30 protein were found in the epidermis on days 1 and 2 after wounding (data not shown). By contrast, Cx30 protein in wild-type epidermis was increased on day 1 and 2 postwounding compared with intact epidermis. In wounded wild-type tail epidermis, the highest amount of Cx26 and Cx30 protein was detected on day 3 postwounding (Fig. 9). By contrast, Cx30/ and Cx31/ mice showed maximal expression of Cx26 on day 4 after wounding, together with maximal expression of Cx30 in Cx31/ mice (data not presented).
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Dye transfer in acute thick sections of mouse ear epidermis
Injection of the fluorescent dye Alexa Fluor 488 was performed in thick
sections of the ear and not in tail epidermis because the stratum corneum of
the tail was too thick to allow correct positioning of the injection needle in
a cell of the basal layer. The microinjected, fluorescent dye spread only
between epidermal cells, i.e. no dye spreading across the basal lamina into
dermal cells was noticed (Fig.
10A,B). The epidermis of Cx31-deficient mice showed no
statistically significant difference in dye transfer compared with wild-type
skin from Cx31+/+ litter mates of
Cx31/ mice or C57BL/6 mice for comparison
(Fig. 10C). By contrast,
epidermal sections from Cx43Cre-ER(T)/fl-mice after
induction with 4-hydroxytamoxifen yielded a 40% reduction in coupling compared
with uninduced Cx43Cre-ER(T)/fl-mice and wild-type mice
(Fig. 10A-C).
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Discussion |
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Our results suggested that the reduction in Cx43 and Cx31 protein may be associated with dedifferentiation and mobilization of keratinocytes at the wound edge during the first two days of wound healing. This notion was supported by the observation that mice with decreased Cx43 protein showed premature closure of the tail epidermis after wounding. Furthermore, in thick sections of the ear skin from Cx43-deficient mice, we found 40% decrease of dye transfer, in contrast to Cx31-deficient mice. Thus, the lack of Cx43 protein led to decreased gap junctional intercellular communication in epidermal cells of the skin. Therefore, the Cx43 protein seems to be important for maintaining epidermal gap junctional communication. Possibly, lack of Cx43 protein may accelerate the wound closure, as the amount of Cx43 did not have to be decreased before the keratinocytes started to migrate. For technical reasons dye transfer experiments were carried out with thick sections from ear. Connexin expression pattern in mouse ear epidermis was compared using immunofluorescence analyses with connexin expression in tail skin. Although, no obvious differences in connexin expression pattern were found between tail and ear epidermis, we cannot rule out the possibility that the intercellular coupling may be different.
Keratinocyte proliferation after wounding takes place in the periphery of
the wound and not at the wound edge (Garlick et al., 1994). Because Cx43 was
also decreased in the periphery on days 1 and 2 after wounding, Cx43 might
have to be downregulated before extensive proliferation of keratinocytes could
occur in the periphery of the wound. Because Cx43 protein was essential for
maintaining the normal level of gap junctional communication, we conclude that
decreased coupling between epidermal cells may be necessary for mobilization
and proliferation of keratinocytes. Keratinocytes of induced
Cx43CreER(T)/flox mice showed reduced coupling, and
therefore cell migration and proliferation might have started earlier.
Downregulation of Cx43 was also described after treatment of mouse back skin
with tumor promoters or oncogenes
(Brissette et al., 1991;
Budonova, 1994
).
Ca2+-mediated differentiation of cultured mouse keratinocytes also
resulted in a decrease of Cx43 protein
(Brissette et al., 1994
). Thus,
Cx43-deficient mice might show an earlier onset of keratinocyte migration
and/or proliferation compared with wild-type mice. This suggested function of
Cx43 (i.e. downregulation of Cx43 appears to be necessary for mobilization and
proliferation of keratinocytes) is a new feature that may be restricted to the
skin. It was previously shown that the lack of Cx43 resulted in decreased
migration of neural crest cells during development
(Huang et al., 1998
). Thus,
Cx43-containing gap junctions, or at least the presence of functional Cx43
protein, might support the migration of neural crest cells. This could be
explained by the finding that neural crest cells in mouse embryos appear to
express Cx43 and Cx46 (Bannerman et al.,
2000
), in contrast to keratinocytes, which were shown to express
nine different connexin isoforms.
The expression level of Cx31 in suprabasal epidermal layers appeared to depend on the state of keratinocyte differentiation: outer epidermal layers expressed more Cx31 protein. This notion was supported by the finding that the amount of Cx31 was strongly reduced in suprabasal layers of the wound edges during the first two days after wounding, when barely differentiated keratinocytes migrated under the coagulum to close the wounded epidermis.
In contrast to the Cx43 expression pattern, Cx31 expression was totally altered during wound healing. Cx31 protein, which was found in suprabasal layers of uninjured epidermis, was mainly expressed during the first two days after wounding in the stratum basale and only in small amounts in the suprabasal layers.
The function of Cx31 in the basal layer during the first three days of
wound healing is not known. When Cx43 was decreased to a low level, Cx31
protein seemed to partially adopt the site of Cx43 expression during the first
two days of wound healing. The correlation of Cx43 and Cx31 expression could
be due to the observed downregulation of Cx43 in Cx31-deficient mice.
Obviously, the expression level of Cx31 seemed to influence Cx43-containing
gap junctions. Cx31 channels show only homotypic coupling in HeLa cells
(Elfgang et al., 1995). Hence,
the coupling of Cx43 hemichannels with those of Cx31 is unlikely. However,
heteromeric channels, including Cx43 and Cx31 in the epidermal layers with
overlapping expression, i.e. basal and innermost spinous layers, may form in
wild-type mice. This might explain the decreased amounts of Cx43 protein in
Cx31-deficient mice.
In contrast to the above-mentioned hypothesis, however, Cx43 and Cx31 showed expression patterns that were more or less separated from each other. In the overlapping expression areas, Cx31 was only present in small amounts. Thus, extensive formation of heteromeric channels is unlikely.
Taken together, Cx43 expression seemed to be dependent on the occurrence of Cx31 protein, because lack of Cx31 protein was associated with a reduced amount of Cx43 in the epidermis. There appears to be an inverse relationship between both connexins that led to the complementary distribution pattern in the mouse epidermis. This was supported by the observation that an increased amount of Cx31 in 60% of all observed cases was found in epidermis with a reduced Cx43 protein level. This could mean that expression of Cx43 affected the amount and site of Cx31 expression and vice versa. The mechanism of regulatory interactions between epidermal connexins remains to be clarified.
In contrast to Cx43 and Cx31 expression, Cx30 and Cx26 proteins could
barely be detected in intact epidermis. They responded to wounding with a
slight increase in protein on days 1 and 2 postwounding and reached their peak
of expression on day 3. At this time the epidermis fused under the coagulum
and then the protein level decreased until the expression level of normal
epidermis was reached again. Thus, Cx26 and Cx30 both showed the same
immediate response to epidermal irritation. Cx26 upregulation has already been
reported under hyperproliferative skin conditions
(Labarthe et al., 1998).
Epidermal wounding results in a loss of the epidermal Ca2+.
Premature regeneration of this gradient would complicate the restoration of
the epidermal permeability barrier (Menon
et al., 1992
). The immediate increase of Cx26 and Cx30 expression
in all epidermal layers could prevent the premature regeneration of the
Ca2+ gradient (for Cx26 see
Goliger and Paul, 1995
). The
hypothesis that Cx26 and Cx30 channels are responsible for fast gap junctional
intercellular communication at the site of skin irritations is supported by
the existence of an alternative, faster trafficking pathway for Cx26
(Martin et al., 2001
; Ahmad et
al., 2002). Cx26- and Cx30-containing channels could therefore be responsible
for the transient formation of a network of cells for exchanging metabolites
or ions. Cx30 and Cx26 protein in the epidermis seem to be partially
redundant, as Cx30-deficient mice do not show obvious abnormalities in normal
and wounded tail epidermis.
Overall, the expression level of Cx43, Cx31, Cx30 and Cx26 proteins was clearly increased on day 3 postwounding when the epidermis of wild-type mice had fused under the coagulum. After this, keratinocyte migration ended and the stratification of the epidermis under the coagulum began. Cx26 and Cx30, which were associated with a fast irritation response, were strongly increased in concentration at day 3. They could provide the required level of coupling for initiating restratification under the coagulum until the normal expression pattern of Cx43 and Cx31 was re-established and could contribute to normal epidermal function. Currently, we cannot explain the drop of Cx26 and Cx30 protein expression on day 4 after wounding. It might be due to a general change of expression in epidermis and dermis. Owing to the fibrin clot, the epidermis could not be dissected from the dermis by dispase treatment. Thus, for immunoblot analyses we used total skin lysates including dermis and epidermis. By contrast, our immunofluorescence analyses represent the epidermal changes of expression in the direct wound region.
There appears to be an extensive functional redundancy between connexins in the mouse epidermis. Analysis of double connexin-deficient mice or simultaneous ablation of several connexins using conditional gene targeting approaches or the use of transdominant connexin mutants might help to dissect this redundancy for the proper function of the skin. Primary cultures of keratinocytes which lack one or more connexins could be useful to analyse the function of gap junction channels between keratinocytes in response to various growth factors.
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Acknowledgments |
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References |
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