1 Department of Anatomy, University of California, San Francisco, CA 94143-0452,
USA
2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
* Author for correspondence (e-mail: kurita{at}itsa.ucsf.edu)
Accepted 2 August 2004
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SUMMARY |
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Key words: Androgen, Mucin, Epithelial differentiation, Apoptosis, Urogenital sinus, Cell linage, MAPK, Src
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Introduction |
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Development and growth of the prostate depend upon androgen. The androgen
signals regulating prostatic development (and maintenance) are mediated by
androgen receptor (AR) in stromal cells
(Cunha and Lung, 1978;
Donjacour and Cunha, 1993
;
Kurita et al., 2001
;
Sugimura et al., 1986
).
Androgen receptors in luminal cells are required for secretory protein
production (Donjacour and Cunha,
1993
). The prostate regresses in response to androgen withdrawal,
and restoration of systemic androgen elicits regeneration of the prostate.
Androgen receptors in the stroma regulate these processes
(Kurita et al., 2001
). In male
seasonal breeders such as the ram, the prostate undergoes this
regression/regeneration cycle naturally in response to the seasonal changes in
the systemic androgen levels. This ability of the prostate to go through
multiple cycles of regression/regeneration implies the existence of stem cells
in prostatic epithelium. Although it has not been definitively demonstrated,
basal cells are widely believed to contain prostatic stem cells, which can
differentiate into basal, luminal and neuroendocrine cells
(Collins et al., 2001
;
Foster et al., 2002
;
Hudson et al., 2000
;
Uzgare et al., 2004
;
van Leenders and Schalken,
2001
; Wang et al.,
2001
). In contrast, a recent study has suggested that stem cells
may also reside in luminal cell layer as a slow proliferating/self-reserve
population in the proximal part of prostatic ducts
(Tsujimura et al., 2002
). The
presence and nature of prostatic stem cells continue to be debated because
stem cells may be the target of carcinogenesis.
p63 (also named KET, p51A, p51B, p40 or p73L) is a homologue of the p53
tumor suppressor gene (Yang et al.,
1998). p63 is essential for development of squamous epithelia.
p63/ mice have severe defects in stratified
epithelia, which causes newborn lethality, and lack organs arising from
epidermis such as mammary and salivary glands
(Mills et al., 1999
;
Yang et al., 1999
). In the
female reproductive tract, p63 is an identity switch for cell-fate
determination, and loss of p63 causes transformation of cervical/vaginal
epithelial cells into uterine epithelial cells
(Kurita et al., 2004
). In the
prostate, p63 is expressed in the basal cells
(Yang et al., 1998
).
Signoretti et al. proposed that p63 is the stem cell factor for prostate
because the prostate is not formed in embryonic/newborn
p63/ mice
(Signoretti et al., 2000
).
However, we have generated p63/ prostate
through rescue of p63/ urogenital sinus
(UGS) by transplantation. p63/ prostate
contains luminal and neuroendocrine but not basal cells. In this study, we
elucidate the role of p63 and basal cells in development and maintenance of
the prostate.
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Materials and methods |
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p63/ embryos were produced by
heterozygous intercrosses of p63Brdm2 mice
(Mills et al., 1999). Pregnant
females were sacrificed at 16-18 days postcoitum, and embryos were harvested.
UGSs from p63/ and
p63+/+ embryos were used.
p63/ mice were identified visually by the
limbless phenotype. The genotypes of embryos were confirmed by PCR. The
phenotypes of p63+/+ and p63+/
prostates were essentially identical in the intact host. Therefore, analysis
was concentrated on p63+/+ and
p63/ UGE. Urogenital sinuses were grafted
under the kidney capsule of athymic male nude mice. The results presented here
are based upon analysis of 80 p63/ and 61
p63+/+ UGS grafts.
Some hosts were castrated at the time of grafting or one month after grafting. Compressed pellets of testosterone propionate (Sigma) (25 mg T-pellet) were implanted into castrated host mice one month after the castration.
Histochemistry
Alcian blue staining (pH 2.8) was used for detection of acid
mucopolysaccharides (Putt,
1971). Methods for immunohistochemical detection in
paraffin-embedded tissues have been described
(Kurita et al., 1998
). Mouse
monoclonal antibodies were used at the following concentrations: anti-p63 4A4
(1:100) (1:20, Santa Cruz Biotechnology), anti-Ki67 (1:100, Novacastra
Laboratories, Burlingame, CA, USA), anti-K8 LE41 (1:2) and anti-K14 LE001
(1:2, gift from E.B. Lane, University of Dundee, Dundee, UK). Mouse monoclonal
antibody against the active form of Src (clone 28, 1:500) was kindly provided
by Hisaaki Kawakatsu, Lung Biology Center, UCSF (San Francisco, CA, USA)
(Kawakatsu et al., 1996
).
Rabbit polyclonal antibodies were used at the following concentrations:
anti-AR (1:100, Affinity BioReagents, Golden, CO, USA), anti-phospho-histone
H3 rabbit (1:100), anti-phospho-ERK1/2 (1:100), anti-phospho-MEK1/2 (1:100)
and anti-phospho-p65 NF-kB (1:100, Cell Signaling Technology, Beverly MA,
USA), anti-Cdk4 (1:100), anti-maspin (1:200, Santa Cruz Biotechnology),
anti-pan-Ras (1:500, LabVision, Fremont, CA, USA), anti-ERß rabbit (1:30,
BioGenx, San Ramon, CA, USA) and anti-involucrin (1:2000, Covance, Princeton,
NJ, USA). Rabbit antiserum for Nkx3.1 (1:100) was a gift from C. Abate-Shen
(University of Medicine and Dentistry of New Jersey-Robert Wood Johnson
Medical School) and uroplakin (1:2000) was a gift from T. T. Sun (New York
University School of Medicine, New York, USA)
(Wu et al., 1994
). Anti-serum
for secretory proteins from mouse dorsolateral prostate (mDLP), mouse ventral
prostate (mVP), mouse bulburethral gland (mBUG) and mouse seminal vesicle
(mSV) were used at 1:5000 dilution
(Donjacour et al., 1990
).
Anti-K19 rabbit monoclonal antibody (1:1) was obtained from Robert Pytela
(UCSF). Positive signals were visualized as brown precipitates utilizing
3,3'-diaminobenzidine tetra-hydrochloride (Sigma). Some
immunohistochemistry (IHC)-stained slides were also stained with Alcian Blue.
Hematoxylin was used for counter staining. Apoptotic cells were detected by
3'-hydroxyl termini of DNA in situ (TUNEL assay) using ApoTag kit
(Oncor, Gaithersburg, MD, USA).
Preparation of ultra-thin JB4 sections
The tissues were fixed with 1.8% paraformaldehyde, 2.5% glutaraldehyde,
0.1% picric acid in cacodylate buffer (pH 7.2) by perfusion of host mice.
The tissue samples were embedded using a JB4 plus embedding kit (Polysciences,
Warrington, PA, USA) following the manufacturer's instructions. Thin sections
(0.8 µm) were cut with a glass-knife, and stained with Hematoxylin and
Eosin (H&E).
Image analysis
For image analysis, images were captured with CCD camera interfaced with
Macintosh G3 computer (Apple, Cupertino, CA, USA) and at least four specimens
for each group were analyzed. For labeling index of phospho-histone H3 (pH3),
the epithelial cells positive and negative for pH3 were counted on the screen
in at least two frames for each specimen (more than 600 total epithelial
cells). To assess the complexity of ducts, area of the grafts and length of
basement membrane lined with epithelial cells were measured on the screen. The
complexity of the ductal structure was expressed as the total length of
epithelium (basement membrane) (µm) divided by area (µm2) of
prostatic tissue in the graft.
RT-PCR
Total RNA was extracted from three p63+/+ and two
p63/ UGS grafts four weeks after grafting
with TRI reagent (Sigma). The cDNA was synthesized from 2 µg total RNA by
using Super Script II (Invitrogen, Carlsbad, CA, USA) with random primer
(Invitrogen). All PCR primers were synthesized at the UCSF Biomolecular
Resource Center. For detection of the keratin 14, forward primer
5'-GCTCTTGTGGTATCGGTGGT and reverse primer 5'-TTGCTCTTCAGGTCCTCGAT
were used (496 bp product). For the detection of the ß-actin, forward
primer 5'-AGCCATGTACGTAGCCATCC and reverse primer
5'-CTCTCAGCTGTGGTGGTGAA were used (392 bp product). RT reaction was
performed in 20 µl reaction mixture and 1 µl of RT-product was used as
template for PCR-reaction. For all primer sets, PCR was performed with the
following protocol: denaturing, 94°C, 15 seconds; annealing, 58°C, 15
seconds; and extension, 72°C, 1 minute; for 40 cycles, with Advan Taq
Platinum Taq polymerase (Clontech Laboratory, Palo Alto, CA, USA).
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Results |
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Androgen induces development of prostate, and androgen action is mediated
by AR in urogenital sinus mesenchyme (Cunha
and Lung, 1979). AR was highly expressed in mesenchyme of
p63/ UGS
(Fig. 1N). The columnar
p63/ UGE appeared to be responding to
androgen-dependent mesenchymal signal to induce prostatic development as
indicated by formation of epithelial evaginations in the regions where
prostate normally forms (Fig.
1I-L,N, arrows). As expected, similar epithelial evaginations were
absent in females (Fig. 1O).
Whereas normal prostatic buds are solid cords of stratified epithelium, the
epithelial cells in the in-folds were simple columnar and had definitive
lumen.
Phenotype of p63/ prostate
p63/ and p63+/+ UGSs
were dissected from E16-18 embryos, transplanted and grown under renal capsule
of adult male nude mice. p63/ UGS developed
ductal structures resembling normal prostate (compare
Fig. 2A and 2B). Development of
p63/ prostate was androgen-dependent,
because p63/ UGS grafted into castrated male
(Fig. 2C) or female (not shown)
hosts formed large cysts and never developed ducts. In general, ducts of
p63/ prostate were less dilated than
p63+/+ prostate, but otherwise, the ductal morphology of
p63/ prostate appeared normal. Ducts of
p63/ prostate were surrounded by smooth
muscle cells as in normal prostate (Fig.
2D,E). Although its gross morphology was similar to
p63+/+ prostate, p63/
prostate lacked morphologically definable basal epithelial cells
(Fig. 2F-I). Instead,
p63/ prostate contained goblet cell-like
cells (Fig. 1G,I, white
arrows). Basal cell markers [p63 (Fig.
2J,K), K14 (Fig.
2L,M), K19 (not shown), transglutaminase II
(Friedrichs et al., 1995)
(Fig. 2N,O)] were undetectable
in p63/ prostate epithelium. Maspin, which
is expressed in the basal cells of normal prostate
(Pierson et al., 2002
)
(Fig. 2P), was also absent in
p63/ prostatic epithelium
(Fig. 1Q). Therefore, p63 is
essential for development of prostatic basal cells. The result of IHC was
confirmed by RT-PCR for K14 (Fig.
2R). K14 mRNA was detected in host prostate and
p63+/+ prostate, but not in
p63/ prostate
(Fig. 2R).
|
|
Activation of Ras-MAPK pathway by Src has been demonstrated to play key
roles in overproduction of mucin in respiratory tract epithelium
(Li et al., 1998). Activation
of Ras and ERK1/2 has been implicated in the mucin production in intestinal
epithelium (Lee et al., 2002
).
Recently, a transgenic mouse model (Pb-RAS mouse) demonstrated that
expression of constitutively active mutant H-Ras (H-RasV12) in
prostatic epithelium transforms luminal cells into mucinous cells
(Scherl et al., 2004
).
Therefore, we examined activation of Src
(Fig. 3N-Q) and MEK1/2
(Fig. 3R,T). In the
p63+/+ prostate, activated Src was detected in neurons
(Fig. 3P, red arrows) and in a
subset of basal cells (Fig. 3P,
back arrows) but not in luminal cells (Fig.
3N,P) after one month of growth in intact male hosts. In contrast,
in p63/ prostate, Src activity was detected
in luminal epithelium (Fig.
3O,Q). Active Src was detected mainly in Alcian blue-positive
cells, but some non-mucinous luminal cells were also strongly positive for
active Src (Fig. 3Q, white
arrows). In p63/ prostate, focal
upregulation of Ras was also detected in the luminal cells which appeared to
be in the process of trans-differentiation into mucinous cells (not shown). In
the p63+/+ prostate, downstream effecters in the
Src-Ras-MAPK signal transduction (MEK1/2 and ERK1/2) were activated
(phosphorylated) in the same cell types as Src activation; in neurons (not
shown) and a subset of basal cells (Fig.
3R,S). In p63/ prostate, MEK1/2
was phosphorylated and translocated into nucleus in both mucinous and
non-mucinous luminal cells (Fig.
3T). Even though MAP kinase signaling was active,
p63/ prostate was quiescent in regard to
proliferation one month after grafting. Phosphorylation of histone H3 was
equally low in p63+/+ and
p63/ prostate in intact male hosts
(Fig. 3U,V).
Neuroendocrine cells developed independent of basal cells
Both p63+/+ and p63/
prostates contained rare neuroendocrine cells as assessed by expression of
synaptophysin (Fig. 3W,X).
There was no distinctive difference between p63+/+ and
p63/ prostates in the distribution and
concentration of synaptophysin-positive cells.
Effect of castration and testosterone-treatment
One month after the grafting, p63+/+ and
p63/ UGSs developed prostate with complex
ductal structure (Fig. 4A,B). The p63+/+ prostate was negative for Alcian blue
(Fig. 4A), whereas ducts of
p63/ prostate were filled with Alcian
blue-positive mucin (Fig. 4B).
The wet weight of prostate is determined mostly by the water content.
Therefore, p63+/+ prostate, which had more dilated ducts,
was significantly heavier than p63/
prostatic gland (Fig. 4G). In
the intact hosts, apoptotic cells were almost undetectable in both
p63+/+ and p63/ prostate
(Fig. 4I,J). In response to
androgen deprivation epithelial apoptosis was detected in both the
p63+/+ and p63/
prostates three days after the castration of the host
(Fig. 4J,K). One month after
the castration, p63+/+ prostatic regression was complete
with marked reduction in luminal cells, reduction in the content of the ducts,
and reduced ductal size (Fig.
4C). As a result, the entire p63+/+ prostatic
graft was reduced in size with approximately 60% reduction in the original
weight (Fig. 4G). Even though
castration reduced its size, ductal structure remained and retained its
complexity in p63+/+ prostate
(Fig. 4C,H). In the shrunken
ducts, p63-positive basal cells were enriched
(Fig. 4M). In
p63/ prostatic grafts, castration elicited a
much more severe reduction in the number of ducts, and in all cases the entire
graft became cystic (compare Fig. 4B with
4D, arrow indicates large cystic ducts). Although the wet weight
was maintained following castration, the complexity of duct reduced
dramatically one month after castration
(Fig. 4H). In some areas of
p63/ prostate, there was a complete loss of
luminal cells leaving pools of mucinous material in the stroma
(Fig. 4D,N,*). The
mucinous pools and cysts filled with Alcian blue-positive material were
observed in all p63/ prostate grafts
subjected to androgen depletion by castration (18/18). The mucinous pools in
the stroma were connected to residual ducts or cysts lined at least in part by
epithelial cells (not shown). Dead cells were also observed in the mucinous
pools (Fig. 4N, black arrows),
suggesting that the mucin was originally circumscribed by epithelium. In the
surviving ducts of p63/ prostate, both
luminal and atypical mucinous cells were present. Mucinous cells were stained
for Alcian blue in the castrated host (Fig.
4D,N).
|
The expression of proliferation makers clearly demonstrated the ability of p63+/+ and p63/ prostate to regenerate. In the castrated hosts, pH3, phospho-MEK1/2 (pMEK) and Cdk4 were very low to undetectable in both p63+/+ (Fig. 5A) and p63/ (Fig. 5B) prostates. Therefore, phosphorylation of MEK in the epithelium of p63/ prostate in the intact hosts (Fig. 3T) is androgen-dependent. Five days after 25 mg T-pellet implantation, all three proliferation markers were upregulated in both p63+/+ (Fig. 5C) and p63/ (Fig. 5D) prostates. In p63+/+ prostate, the lumen of existing ducts became enlarged, and the luminal epithelial cells increased in height as expected (compare Fig. 5A with Fig. 5C). In the p63/ prostate, most ducts disappeared after the castration. Therefore, regeneration of p63/ prostate started as outgrowths of tightly packed ducts from the large cysts, similar to the early prostatic development (Fig. 5D). Epithelial labeling indices for pH3 clearly showed regeneration of prostatic tissue in both p63+/+ and p63/ prostates. Epithelial pH3 labeling index was low (<1%) in the intact and castrated host in both p63+/+ and p63/ prostates. T-pellet treatment significantly increased pH3 labeling index in both p63+/+ and p63/ prostates (Fig. 5E,*). One month after T-pellet treatment to the castrated hosts, p63+/+ and p63/ prostates were fully regenerated and the pH3 labeling index returned to the basal level (Fig. 5E, +T one month).
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Discussion |
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Tsujimura et al. have suggested that luminal epithelium contains a
self-renewing population of putative stem cells that are presumably required
for prostatic regeneration (Tsujimura et
al., 2002). Our results also demonstrated regenerative capacity of
prostate lacking basal cells. Therefore, stem cell function (at least
regenerative capacity) is not necessarily associated with `basal cells'.
However, our results do not exclude possible trans-differentiation of basal to
luminal cells in the normal adult prostate as suggested previously
(Collins et al., 2001
;
Foster et al., 2002
;
Hudson et al., 2000
;
Uzgare et al., 2004
;
van Leenders and Schalken,
2001
; Wang et al.,
2001
). Moreover, our results do not exclude the presence of
prostatic stem cells in the basal cells of normal wild-type prostate.
Although basal cells are not required for the differentiation of luminal
cells, basal cells appear to be essential to maintain normal luminal cell
differentiation. Luminal cells of p63/
prostate transformed into mucinous cells. Activation of Src, Ras and MAPK
signaling can induce mucinous differentiation or overproduction of mucin in
epithelial tissues (Lee et al.,
2002; Li et al.,
1998
; Meerzaman et al.,
2001
; Perrais et al.,
2002
), and in the p63/ prostate
Src and MEK were activated in the luminal cells. The activation of Src and
MAPK signaling almost certainly plays a key role in mucinous transformation of
luminal cells in p63/ prostate. Src can be
activated by signals via adhesion molecules, cytokines or growth factors
(Abram and Courtneidge, 2000
;
Thomas and Brugge, 1997
). It
is notable that in human prostate laminin 5 (
3ß3
2) is
produced exclusively by basal cells
(Calaluce et al., 2001
) and
downregulation of laminin 5 affects membrane stability of integrin
6ß4 and gene expression in the prostatic luminal cells
(Hao et al., 2001
). These
observations clearly demonstrate that loss of basal cells and/or change in
cell adhesion molecule indeed affect gene expression and phenotype of luminal
cells in the prostate. Moreover, in the adult prostate, epithelial growth and
functional differentiation are regulated by androgen via stromal paracrine
factors. In the normal prostate, p63-positive basal cells reside on the
basement membrane, and may mediate or modulate interactions between stromal
cells and luminal cells. Therefore, it is likely that loss of basal cells
disturbs the interaction between stromal cells and epithelial cells, and
causes activation of Src in luminal cells. In the normal prostate, Src was
active in a subset of basal cells in the proliferation-quiescent adult state,
indicating the existence of Src-activating signals in the normal prostatic
epithelium. The factors activating Src in luminal cells of
p63/ prostate are yet to be identified, even
though a wide spectrum of autocrine and paracrine growth factors have been
described in the prostate (Cunha et al.,
1998
).
p63/ prostate expressed proper secretory proteins specific for mouse prostate. Therefore, expression of these androgen-regulated genes in luminal cells does not require basal cells. However, ducts in 63/ prostate were less dilated than ducts in the p63+/+ prostate, and the ducts in p63/ prostate were unable to reduce luminal secretory products in response to castration. This phenotype may be secondary to the mucinous differentiation of luminal cells, otherwise, basal cells may play a role in regulating secretory activity of luminal cells.
Basal cells also play key roles in maintaining ductal structure of the
prostate in the absence of androgen. In response to castration, normal
prostatic ducts lose luminal secretion content, and a substantial portion of
luminal cells die via apoptosis. The entire process is highly coordinated, and
therefore, normal prostatic ducts are able to shrink while maintaining their
morphologic integrity. In contrast, in p63/
prostate complete loss of ducts occurred leaving only residual cystic
structures or pools of mucinous secretion in the stroma following castration.
In normal prostate, a substantial portion of luminal cells dies in response to
castration, but always a subset of luminal cells survive to maintain ductal
structure and to permit regeneration when androgens are restored. The
mechanism controlling death or survival of luminal cells following castration
is not known. Because castration selectively eliminates luminal (versus basal)
cells, the ratio of basal cells to luminal cells increases
(Rouleau et al., 1990), and
after castration most surviving luminal cells are in direct contact with basal
cells. Taken together, the phenotypes of the
p63/ prostate strongly suggest that basal
cells play an important role in the control of luminal cell differentiation
and survival/apoptosis.
Adult prostate is a proliferation-quiescent organ in the mouse. Although androgen is a potent mitogen for developing and regenerating prostatic epithelia, androgen maintains prostatic tissue but does not stimulate epithelial proliferation in adult prostate. Luminal epithelial cells in developing or regenerating p63+/+ and p63/ prostatic grafts became proliferation-quiescent once the grafts reached a certain size. Therefore, the mode switch for androgen function, growth to maintenance, appears to be intact in p63/ prostate. Moreover, the labeling index for pH3 was identical between p63+/+ and p63/ prostates in response to hormonal manipulation, suggesting that p63 and basal cells are not essential for regulation of luminal epithelial proliferation.
Mucinous metaplasia of prostate has been suggested to be a pre-neoplastic
condition (Scherl et al.,
2004). Loss of basal cell is also a common characteristic in
prostatic carcinogenesis. These observations suggest that changes caused by
loss of basal cells may have an impact on carcinogenesis of the prostate. The
effect of the loss of basal cells in carcinogenesis of the prostate is
currently under investigation utilizing p63/
UGS and the hormonal carcinogenesis model with T+E2-treatment
(Wang et al., 2000
).
In conclusion, p63 is essential for differentiation of prostatic basal
cells, and basal cells are essential in maintaining normal differentiation of
luminal cells and integrity of prostatic ducts. However, basal cells
(therefore p63) are not required for development and regeneration of prostate.
Further experimentation is required to define the role of p63 in basal cell
differentiation. p63 isoforms are functionally distinct in regard to cell fate
commitment, particularly in epidermal differentiation
(Koster et al., 2004;
McKeon, 2004
). The
differentiation of epidermis appears to be regulated by the balance between
isoforms containing and lacking the transactivation domain. To understand the
function of p63 in basal cell differentiation in prostate may require detailed
analysis of isoform expression in the developing UGS and the adult
prostate.
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ACKNOWLEDGMENTS |
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