| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Harvard Skin Disease Research Center,*
Brigham and Womens Hospital, Boston, Massachusetts; the Centre for
Cutaneous Research,
Queen Mary and Westfield
College, University of London, London, England; the Department of
Dermatology,
University Hospital Eppendorf,
University of Hamburg, Hamburg, Germany; and the Department of
Dermatology,§
Charité Hospital, Humboldt
University, Berlin, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Bcl-2 is the founding member of a large family of highly homologous apoptosis regulatory proteins.2-5 The members most closely related to Bcl-2, such as Bcl-xL, and Bcl-2 itself, promote cell survival, whereas more distant relatives, such as Bax, Bcl-xS, or Bak, induce apoptosis.2-5 Pro- and antiapoptotis members of this family can heterodimerize with each other by virtue of conserved sequence motifs, thereby regulating each other’s activity.2-5 One possible mechanism of action of the antiapoptotic proteins may involve their ability to inhibit the activation of caspases such as ICE (interleukin 1ß converting enzyme, caspase 1), the proteases that function in the execution phases of apoptosis (reviewed in refs. 2 and 11).
Bcl-2 expression in HF is strictly hair
cycle-dependent,10,12
being more strongly expressed during
phases of intense proliferative activity and down-regulated during HF
regression. Although functional evidence has not been available to
date, Bcl-2 has been suspected to play an important role in follicular
keratinocyte regression and growth during the hair
cycle.10,12
Disruption of the tight control of Bcl-2
expression might thus be expected to have a profound effect on hair
follicle morphogenesis and cycling. Indeed, Bcl-2
- and
Bcl-2ß-deficient mice reportedly show a substantial retardation of
HF cycling compared to wild-type (WT) mice13-15,
and
Bcl-2ß null mutants display retarded anagen development after
depilation.13
We describe here a new transgenic mouse line that overexpresses human Bcl-2 in the basal layer of epidermis and various components of the hair follicle, under the control of the human keratin-14 promoter. We have used this mouse model to investigate the effect of high-level expression of Bcl-2 on neonatal HF morphogenesis and adolescent HF cycling in mice, with particular emphasis on apoptosis-driven catagen development.10 Because we had previously shown that chemotherapy-induced alopecia in mice is associated with a massive increase in intrafollicular apoptosis,10 cyclophosphamide-induced alopecia16 was also compared between K14/Bcl-2 and WT mice. The effects of Bcl-2 overexpression on ultraviolet B (UVB)-induced apoptosis of murine epidermal keratinocytes in situ ("sunburn cells") and in vitro were examined to ensure that the transgenic Bcl-2 was functioning as expected. Taken together, these assays reveal surprisingly complex consequences of Bcl-2 overexpression for apoptosis-related phenomena in different epithelial tissue compartments of murine skin.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
FVB/N and Swiss Webster mice (Taconic Farms, Germantown, NY) were used for the construction of the transgenic lines. These mice were housed in community cages, with 12-hour light periods, at the Harvard Skin Disease Research Center, Animal Facilities (Boston, MA), and were fed water and mouse chow ad libitum.
Reagents
The Cell Death Detection ELISA was purchased from Boehringer-Mannheim (Indianapolis, IN). All enzymes were purchased from Boehringer-Mannheim. Hamster anti-human-Bcl-2, clone 6C8, and anti-mouse-Bcl-2, clone 3F11, were purchased from Pharmingen (San Diego, CA). Polyclonal rabbit anti-K14 was a kind gift of Dennis Roop (Baylor College of Medicine, Houston, TX), and a mouse monoclonal anti-human CK14, which cross-reacts with mouse cytokeratin 14, was purchased from Neomarker (Fremont, CA). Horseradish peroxidase-conjugated goat anti-hamster IgG (for Western blotting) was from Southern Biotechnology (Birmingham, AL), and the appropriate secondary biotinylated reagents for mouse and hamster primary antibodies (for immunohistochemistry) were from Amersham (Little Chalfont, UK).
DNA Constructs and Transgenic Mice
The full-length cDNA for human Bcl-2 (1.9 kb) contained in the Bluescript-KS vector was a kind gift of Stanley Korsmeyer (Harvard, Boston, MA). To specifically direct expression of Bcl-2 to the basal layer of the epidermis, the insert was excised from this vector by EcoRI digestion, modified with BglII linkers (New England Biolabs, Beverly, MA) and ligated into the single BamHI site of the K14/human growth hormone (hGH) expression vector,17 kindly provided by Elaine Fuchs (University of Chicago, Chicago, IL). The original vector had been modified for these experiments so that it no longer supported transcription of active growth hormone.18 The completed construct was electroporated into the competent DH5a strain of Escherichia coli; colonies bearing the vector in the correct orientation were identified and amplified; and the 6.1-kb insert was recovered with a single EcoRI digestion, purified, and used for microinjection into the pro-nuclei of fertilized FVB/N ova.19 Surviving two-cell embryos were transferred into preudopregnant Swiss Webster recipients, and the offspring were screened by polymerase chain reaction (PCR) amplification of genomic DNA obtained from ear punch biopsies, using primers directed at the hGH segment of the transgene.20 Two founder mice were identified, and the presence of the transgene was confirmed by Southern blotting, using the complete cDNA excised with EcoRI from the Bluescript-KS plasmid as a radioactively labeled probe.
Western Blotting
Human Bcl-2
Ears from transgenic and wild-type mice were mechanically scraped with a scalpel, and the resulting crude cell suspension was immediately lysed in isotonic buffer with protease inhibitors. The lysates were cleared of cell debris by high-speed centrifugation, and 15 mg of protein was separated on a 15% polyacrylamide gel, under denaturing conditions, transferred electrophoretically to nitrocellulose membranes in ice-cold Towbin’s buffer,21 probed with the hamster anti-human Bcl-2 monoclonal antibody, and developed with horseradish peroxidase-conjugated goat anti-hamster secondary reagent (1:2000) and an enhanced chemiluminescence (ECL) detection system (Amersham).
Murine Bcl-X(L)
Sixteen-day-old female Bcl-2 and wild-type mice were shaved, and full-thickness skin samples from one longitudinal half of the whole dorsal area were snap-frozen in liquid nitrogen and treated as described above. The lysates were probed with 0.5 µg/ml polyclonal rabbit anti-Bcl-x (Transduction Laboratories) and developed with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Pierce) secondary reagent and the ECL detection system (Amersham). To quantitate the amount of loaded protein more accurately, the membranes were reblocked for an hour after the ECL reaction was completed and probed with a monoclonal anti-actin antibody (clone C4, ICN, 1:20,000). Actin antibody binding was detected with a sheep anti-mouse immunoglobulin horseradish peroxidase-conjugated secondary reagent and ECL as before. All membrane blocking, washing, and antibody dilutions were performed using the buffer systems developed by Niland et al.22
Skin Harvesting and Skin Specimen Preparation
Sex- and age-matched, 1–20-day-old neonatal transgenic and wild-type littermates were used for the analysis of neonatal HF morphogenesis, postnatal HF regression (catagen), and the first anagen phase,23,24 and 6–9-week-old adolescent transgenic and wild-type mice were used for the analysis of cyclophosphamide-induced alopecia. These mice were housed in community cages, with 12-hour light periods, at the Harvard Skin Disease Research Center and were fed water and mouse chow ad libitum.
Using a special skin harvesting and processing technique for obtaining longitudinal cryosections through murine HF,24,25 we covered skin samples with embedding medium (GSV; SLEE Technik GmbH, Main, Germany). They were then snap-frozen in liquid nitrogen and stored at -80°C until cryosectioning (86 µm). Sections were placed on poly-L-lysine-covered glass slides, air-dried for 1 hour, and fixed at -20°C in acetone for 10 minutes to be stored at -20°C until immunohistology was performed.
At least 20 HFs per mouse were examined: five mice on days 1 and 12 (neonatal morphogenesis) and days 18 and 19 after birth (first postnatal catagen and anagen), respectively. Thus, at least 60 to 100 different HFs obtained from three to five mice per stage of HF morphogenesis or cycling were analyzed by quantitative histomorphometry, as described.26-28
Immunohistochemistry
Human and murine Bcl-2 immunoreactivity (IR) and murine Bax and Bcl-X(L) IR were detected immunohistologically by the avidin-biotin complex and alkaline phosphatase technique (Vecta-Stain Kit, Vector, Burlingame, CA). The specific monoclonal antibody against human Bcl-2 was used in a dilution of 1:50; staining for murine ICE, Bcl-2, Bax, and NT-3 and double-labeling of murine Bcl-2/Bax were performed as described.10,27
Induction of Apoptosis by UVB
For the induction of apoptosis by UVB in vivo, groups of four or five mice aged 8 to 9 weeks were depilated on their dorsal surface with a commercial cream 1 to 2 days before irradiation. The mice were then anesthetized with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg) to immobilize them and irradiated in a Stratalinker 2400 (Stratagene, La Jolla, CA) fitted with lamps capable of delivering a chosen energy level of UVB at a wavelength of 312 nm. Either a single dose of 1250 J/m2 or five consecutive doses of 500 J/m2/day were given. Twenty-four hours after the last irradiation, the mice were sacrificed, and the dorsal skin was harvested and immersed in 10% buffered formalin. After appropriate fixation and deparaffinization, 5-µm sections were stained with hematoxylin and eosin; sunburn cells, defined as cells with a shrunken, highly eosinophilic cytoplasm, and condensed nuclei, were counted in both the basal and suprabasal layers of interfollicular epidermis.29
For the in vitro induction of apoptosis by UVB irradiation, 1 x 105 freshly prepared epidermal cells from mouse ears in 1 ml of culture medium (Dulbecco’s minimum essential medium without CaCl2, supplemented with glutamine and antibiotics (all Gibco, Paisley, Scotland), 5% chelex fetal calf serum, 0.1 mmol/L CaCl2, 2.5 ng/ml murine epidermal growth factor (EGF; Sigma)) were dispensed into Costar 24 wells and were allowed to adhere overnight. The medium was removed, the cells were washed with warm phosphate-buffered saline (PBS) containing 0.03 mmol/L CaCl2 and 0.8 mmol/L MgCL2 (PBS/Ca,Mg) and irradiated with a metal halide lamp (Mutzhas, Munich, Germany) filtered for emission of UVB light in the range of 290–330 nm. Power output was measured with an IL1700 International Light Research Radiometer (Newbury Port, MA). After irradiation, the PBS/Ca,Mg was replaced with culture medium, and the cells were incubated for a further 24 hours at 37°C. The degree of apoptosis was then quantitated using the Cell Death Detection ELISA, according to the manufacturer’s instructions.
Cyclophosphamide-Induced Alopecia
For the study of chemotherapy-induced apoptosis we used the well-studied model of cyclophosphamide-induced alopecia.10,16 Briefly, 7-week-old TG and WT mice with all back skin HF in telogen were depilated to induce anagen VI HF.24,30 At day 9 after depilation, 120 mg/kg cyclophosphamide was injected intraperitoneally, and 36 hours later, namely when the highest rate of apoptotic activity is found,10 the first group of TG and WT mice was harvested, and the second group was harvested after 5 days, when massive alopecia is visible macroscopically.10
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Pronuclear injection of fertilized ova with the construct described above yielded two founder mice, which were bred to establish hemizygous lines, designated bcl1 and bcl7. The founder mouse for the bcl7 line had only intermittent Bcl-2-positive cells in the epidermal basal layer, indicative of a mosaic pattern of expression (not shown). Both mice bred normally, and the bc17 founder was bred to give progeny with a complete expression pattern. The two transgenic lines appeared to have about equal gene copy numbers by Southern blot analysis (not shown); however, the probe used did not detect sufficient endogenous Bcl-2 to allow quantitative estimation of the absolute copy number.
As expected, the K14-driven expression of the transgene in the
epidermis was restricted to the basal layer (Figure 1A)
. No transgenic Bcl-2 protein could be
detected at all in control mice (Figure 1B)
, whereas K14 was equally
expressed in interfollicular epidermis in both transgenic and wild-type
mice (not shown). Western blotting of lysates prepared from epidermal
ear scrapings demonstrated that the Bcl-2 protein detected by this
antibody had an expected size of about 26 kd (Figure 2)
.
|
|
Intrafollicular Bcl-2 Transgene Expression Is Strictly Dependent on Hair Follicle Development and Cycling and Corresponds to K14 Immunoreactivity
The immunohistological analysis of K14-driven transgenic Bcl-2 IR
in murine skin revealed that the human Bcl-2 protein in murine skin was
not constitutively expressed in the hair follicle, in contrast to the
expected expression patterns,17
but that the transgene
expression was strictly dependent on the respective stage of hair
follicle development and cycling. During early skin and HF development,
transgenic Bcl-2 IR was found strongly on all keratinocytes of the
basal epidermal layer and the developing hair germ (Figure 1C)
. Later,
the developing inner root sheath (IRS) showed decreasing K14/Bcl-2 IR
(Figure 1C)
, and the entire outer root sheath (ORS) displayed strong
K14/Bcl-2 IR (Figure 1C)
. In contrast, the entire proximal part of
fully developed HF displayed only very faint K14/Bcl-2 IR (Figure 1D)
.
During catagen, on the other hand, strong transgene IR was found on all
follicular keratinocytes and was particularly strong on the
keratinocytes of the regressing epithelial strand (Figure 1E)
. Telogen
HF displayed only some K14/Bcl-2 IR on keratinocytes of the proximal
ORS and the secondary hair germ close to the dermal papilla (DP)
(Figure 1F)
. During the first anagen after birth, the central ORS and
the developing IRS displayed virtually no transgene IR, whereas the
proximal ORS was strongly K14/Bcl-2-positive (Figure 1G)
. Thus this
developmentally controlled and hair cycle-dependent transgene IR was
found exactly in those regions that have been defined as "apoptosis
hot spots" during murine HF morphogenesis and cycling (central IRS,
distal ORS, epithelial strand),10
and a substantial
decline of transgene IR was found on the entire proximal part of fully
developed HF during morphogenesis (Figure 1E)
and anagen (not shown).
To determine whether the hair cycle-dependent expression of the
transgene was dependent on a hair growth-dependent expression of K14 or
was the result of Bcl-2 function in the HF, we have analyzed the IR
patterns of K14 during neonatal HF morphogenesis in wild-type mice.
Similar to the transgene IR patterns shown in Figure 1, C–G
, strong
K14 IR was first found on all keratinocytes of the epidermis and stage
2–3 hair buds (Figure 1, H and I)
, as well as on the ORS of stage 4 HF
(Figure 1, I and J)
, but not on their developing IRS (Figure 1, I and J)
. Fully developed stage 8 HFs display strong K14 IR on the distal and
central ORS (Figure 1K
, arrowhead), but not on the proximal parts
(Figure 1K
, arrows).
The Bcl-2 Transgene Protects against Sunburn Cell Formation by Epidermal Keratinocytes in Vivo and Prevents UVB-Induced Apoptosis in Vitro
Because the transgenic animals demonstrated no overt skin
abnormalities, we first undertook to verify that the Bcl-2 transgene
was active. One of the well-characterized apoptotic stimuli against
which Bcl-2 protein protects is UVB radiation, which was first
demonstrated for lymphoid cells.31
Skin irradiation with
UVB induces the appearance of apoptotic ("sunburn") cells, mainly
in the basal layer of the epidermis.32
Indeed, irradiation
of both transgenic lines bcl1 and bcl7 with UVB resulted in a
significant (P < 0.02) reduction in the numbers
of sunburn cells in the interfollicular basal layer of epidermis of the
transgenic strains compared to controls, 24 hours after a single dose
of 1250 J/m2, confirming the normal functioning
of the transgene (Figure 3A)
. Similar
results were obtained in mice up to 12 months of age (not shown) and in
two further experiments in which mice from each of the other strains
were irradiated on 5 consecutive days with 500
J/m2
and sunburn cells were counted after 24
hours. The numbers of sunburn cells in the suprabasal layers were much
lower than in the basal layers (not shown), and no significant
difference was observed between transgenic and nontransgenic animals,
as expected, because the Bcl-2 transgene was not expressed in this
location.
|
shows that cultured primary
keratinocytes from normal FVB/N mice undergo UVB-induced apoptosis in a
dose-dependent manner, allowing their histones to be detected in an
ELISA-based assay. At the same doses, virtually no histones can be
detected in keratinocytes derived from the transgenic strains. Histones
were nevertheless detectable by ELISA in cultures of cells of both
transgenic and nontransgenic origin when, in some experiments, there
was some degree of spontaneous histone release by nonirradiated cells
(not shown). Bcl-2 Transgenic Mice Display No Alteration of Hair Follicle Morphogenesis and Exhibit Normal Onset but Accelerated Progression of Hair Follicle Regression (Catagen) and Premature Anagen Development
To precisely analyze the velocity of HF morphogenesis, the
percentage of HFs in distinct stages of HF development was assessed by
quantitative histomorphometry in TG and WT mice.26
Compared to WT mice, the transgenic mice displayed no statistically
significant difference in the velocity of early HF development (day 1
after birth) (Figure 4A)
and—surprisingly—no significant difference in the onset of the first
phase of HF regression (catagen) after birth (Figure 4B)
.
|
, and a statistically significant
(P < 0.05) acceleration of the first anagen
development at day 19 in TG mice compared to WT littermates (Figure 5, D–F)
. In addition, catagen development in the transgenic mice was
significantly (P < 0,01) less well-synchronized
than in age-matched WT controls, ie, the K14/Bcl-2 mice still showed
catagen VIII alongside anagen III HF, whereas WT mice displayed only
telogen and anagen I/II HF (Figure 5F)
. Furthermore, at day 18 after
birth the transgenic mice showed a significant increase in
TUNEL+ hair bulb keratinocytes
(P < 0.05) compared to WT controls (Figure 6, A–C)
, also supporting an acceleration
of catagen development.10
Thus, in contrast to our
expectations, the Bcl-2 transgene seems to promote apoptosis of HF
keratinocytes.
|
|
Bcl-2 Transgenic Mice Displayed Increased Alopecia, Hair Follicle Dystrophy, and Apoptosis after Cyclophosphamide Injection
Consistent with the acceleration of spontaneous catagen
development, the transgenic mice also showed a significant increase in
TUNEL+ hair bulb keratinocytes
(P < 0.05) compared to WT controls 36 hours
after cyclophosphamide injection (Figure 6, D–F)
. Compared to WT mice,
the K14/Bcl-2 mice displayed macroscopically visible hair loss 2 days
earlier (not shown), and the wave of alopecia progressing from neck to
tail was substantially accelerated (Figure 7A)
. In addition, 5 days after
cyclophosphamide treatment, K14/Bcl-2 mice displayed a statistically
significant acceleration of dystrophic catagen, ie, a significantly
higher rate of shorter and deformed hair follicles, as well as more
abnormally widened hair canals (P < 0.05)
(Figure 7B)
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß and
Bcl-2-deficient mice reportedly show a substantial retardation of
the first anagen development and depilation-induced
anagen.13-15,35
Although these observations were not
supported by quantitative histomorphometry, our data complement in part
the findings reported in these studies: our Bcl-2-overexpressing mice
paradoxically showed a marked accelerated spontaneous and
cyclophosphamide-induced catagen progression, as well as an accelerated
first anagen development. Bcl-2-deficient mice should thus be tested by
quantitative histomorphometry to determine whether they also
consistently display a retardation of the first catagen progression.
The data presented in the present study imply that Bcl-2 per
se cannot protect hair follicle keratinocytes against
catagen-associated apoptosis. Furthermore, the induction and the
progression of catagen seem to be two distinctly controlled processes,
because the onset of catagen displayed no statistically significant
differences between wild-type and transgenic mice, whereas subsequent
catagen progression was markedly different (Figures 4B and 5)
.
In contrast to the expected expression patterns,17
we show
here that the K14-driven transgene is not expressed constitutively in
follicular keratinocytes, but that its expression is developmentally
controlled and is strictly associated with distinct stages of HF
morphogenesis and cycling (Figure 1)
.
These stage-dependent transgene expression patterns are consistent with
the immunoreactivity patterns of K14 during murine HF morphogenesis and
cycling (Figure 1, H–K)
. However, these findings have to be confirmed
by additional methods, such as K14-driven ß-galactosidase or green
fluorescent protein expression, to exclude the possibility that cells
are altered when they express K14-linked Bcl-2, leading indirectly to a
hair growth-dependent expression pattern caused by Bcl-2 function. In
addition, the stage-dependent transgene expression is also
age-dependent, because skin and HF morphogenesis and cycling follow a
precisely controlled timetable.36
This finding has
important implications for all studies performed with transgenes under
the control of the K14 promoter. In particular, in the case of forced
expression of secreted molecules, in light of our data, it would seem
critical to analyze the stage-/age-dependent level of the K14-driven
transgene in various tissues and in the circulation, and to ensure that
experimental comparisons between test and control animals are only
performed between age-matched mice that are in precisely the same stage
of the hair cycle, so that artifacts arising from hair cycle-associated
differences in transgene expression are avoided.
It is intriguing that Bcl-2, a potent apoptosis inhibitory protein, when it is overexpressed in follicular keratinocytes, promotes accelerated spontaneous and cyclophosphamide-induced HF regression (catagen), processes that are driven mainly by apoptosis of HF keratinocytes.10 In interfollicular epidermal keratinocytes, the transgene itself was active as expected, because transgenic mice had a very marked reduction in UVB-induced sunburn cells, specifically in the basal keratinocytes, in which the transgene was expressed. A similar reduction in UVB-induced sunburn cells in mouse skin has been described for mice transgenic for another Bcl-2 family member, Bcl-xL, which also protects against UVB-induced apoptosis.37 That the reduction of sunburn cells implies protection against UVB-induced apoptosis is supported by the finding that cultured keratinocytes from transgenic mice produce no histones, a hallmark of apoptotic cells, when irradiated with the same doses of UVB that elicited high histone release from nontransgenic cells. This suggests that Bcl-2 exerts different apoptosis-modulating functions in epidermal versus follicular keratinocytes.
In contrast to Rodriguez-Villanueva et al,38 who overexpressed Bcl-2 ectopically in suprabasal epidermal layers by using a keratin-1 promoter, we have expressed Bcl-2 orthotopically in the basal epidermal layer and in the ORS of the hair follicle. Surprisingly, we did not find any of the changes reported by Rodriguez-Villanueva et al,38 such as multifocal hyperplasia without associated hyperkeratosis. These data suggest that ectopic Bcl-2 expression and orthotopic Bcl-2 expression substantially differ in their effects on keratinocyte biology, depending on their location (differences in the local signaling milieu) and state of differentiation (proliferative versus terminally differentiating cell pool).
The suppression of UVB-induced apoptosis in our K14/bcl-2 mice reveals for the first time that orthotopic Bcl-2 overexpression in vivo indeed results in reduced UVB-induced apoptosis. This is suggested by previous evidence from in vitro experiments on lymphoid cells31 and ectopically Bcl-2-expressing transgenic mice.38
The promotion of catagen- and chemotherapy-associated apoptosis in K14/bcl-2 transgenic mice is a very unexpected finding because the overexpression of an apoptosis inhibitor such as Bcl-2 should, in theory, reduce follicular apoptosis and should delay spontaneous or dystrophic catagen development. Arriola et al39 demonstrated in another system (testicular germ cell tumors) that the overexpression of Bcl-2 reciprocally down-regulates Bcl-X(L), another antiapoptotic member of the Bcl-2 family, and leads to a higher susceptibility of these cells to chemotherapy-induced apoptosis. In addition, compared to Bcl-2, Bcl-X(L) appears to play a far more important role in chemotherapy-induced apoptosis in these cells. However, we did not detect any marked difference in the ratio of Bcl-2 and Bcl-X(L) in K14/bcl-2 mouse skin compared to controls, by immunohistology or Western blotting of full dorsal skin homogenates (not shown).
Because the overexpression of Bcl-2 might have been counterregulated by up-regulation of proapoptotic Bcl-2 family members such as Bax or counterregulatory activity of caspases that are directly upstream of DNA fragmentation, we have analyzed in the transgenic versus WT mice, for example, the ratio of endogenous Bcl-2/Bax IR as well as caspase 1 (ICE) IR as one key member of the large caspase family. However, no significant alteration between the two mouse lines was found.
Furthermore, K14/Bcl-2 may prolong the survival of the HF keratinocyte subpopulation in the ORS otherwise destined to undergo apoptosis, which now continue to secrete catagen-promoting factors such as NT-3. We have recently demonstrated that NT-3 promotes catagen development and that many NT-3+ keratinocytes can be found in the ORS of late anagen VI HF.26 Thus K14/bcl-2 overexpression of ORS keratinocytes in late anagen may retain such NT-3+ ORS keratinocytes inside the HF for a longer time than normal and consequently accelerate catagen progression. However, additional immunohistological analyses of NT-3 expression in transgenic versus control mice did not reveal any substantial differences in any HF compartment between the two strains (not shown).
Interestingly, during the revision of this paper, Pena et al40 presented an explanation for a skin phenotype similar to the one observed by us in a transgenic mouse line overexpressing Bcl-X(L) under control of the K14 promoter (K14/BclX(L) mice). As in K14/Bcl-2 mice, the transgene was expressed on basal layer keratinocytes of the epidermis and the ORS, leading to accelerated catagen development. These authors have suggested the plausible argument that accelerated catagen development in K14/Bcl-X(L) mice might be explained, at least in part, by prolonged FGF-5 production of ORS keratinocytes surviving longer than normal, because the skin phenotype was substantially reversed in FGF-5-deficient mice, given that FGF-5 is an important catagen-promoting factor41 and that FGF-5 mRNA steady-state levels are maximally high during catagen in mice.42
Therefore, K14/Bcl-2 overexpression in the ORS of transgenic mice may well lead to the survival of ORS keratinocytes that elaborate catagen-promoting factors such as FGF-5 or NT-3. Unfortunately, however, with the methodology we have used we have failed to detect significant expression differences in this respect between transgenic and WT mice (our attempts to obtain reliable FGF-5 IR patterns well above background were frustrated).
Nevertheless, in concert with the publication by Pena et al,40 our findings in K14/Bcl-2 mice suggest that Bcl-2 plays a crucial role in regulating death and survival of ORS keratinocytes and invite one to exploit in future studies the novel transgenic mouse strain reported here as an instructive model for further exploration of the role of selected ORS keratinocytes in the control of catagen development.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported in part by National Institutes of Health grant AR42689/Harvard Skin Disease Research Center (S. M.-R., H. R., T. S. K.) and by grants from the Deutsche Forschungsgemeinschaft (Pa 345/6-1, 8-1) and Wella AG, Darmstadt. R. P. and H. R. were supported for part of this work by the Center de Recherche et l’Investigation Epidermique et Sensorielle (CERIES) Neuilly.
Drs. Müller-Röver and Rossiter contributed equally to the present study.
Accepted for publication December 22, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Pradhan, H. K. Kim, C. J. Thrash, M. A. Cox, S. K. Mantena, J.-H. Wu, M. Athar, S. K. Katiyar, C. A. Elmets, and L. Timares A Critical Role for the Proapoptotic Protein Bid in Ultraviolet-Induced Immune Suppression and Cutaneous Apoptosis J. Immunol., September 1, 2008; 181(5): 3077 - 3088. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. C. Arck, R. Overall, K. Spatz, C. Liezman, B. Handjiski, B. F. Klapp, M. A. Birch-Machin, and E. M. J. Peters Towards a "free radical theory of graying": melanocyte apoptosis in the aging human hair follicle is an indicator of oxidative stress induced tissue damage FASEB J, July 1, 2006; 20(9): 1567 - 1569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Morgan and P. Rose An Investigation of Apoptosis in Androgenetic Alopecia Ann. Clin. Lab. Sci., January 1, 2003; 33(1): 107 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Rossiter, S. Beissert, C. Mayer, M. P. Schön, B. G. Wienrich, E. Tschachler, and T. S. Kupper Targeted Expression of bcl-2 to Murine Basal Epidermal Keratinocytes Results in Paradoxical Retardation of Ultraviolet- and Chemical-induced Tumorigenesis Cancer Res., May 1, 2001; 61(9): 3619 - 3626. [Abstract] [Full Text]
|
|
|
|
 |
|
 K. S. Stenn and R. Paus Controls of Hair Follicle Cycling Physiol Rev, January 1, 2001; 81(1): 449 - 494. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
