QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 September 2004
doi: 10.1242/dev.01384

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kurita, T. Articles by Cunha, G. R. Search for Related Content PubMed PubMed Citation Articles by Kurita, T. Articles by Cunha, G. R.

Role of p63 and basal cells in the prostate

¹ Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA
² Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA

View larger version (139K): [in a new window]   Fig. 1. Ontogeny of p63 in urogenital sinus (UGS) and phenotype of p63–/– UGS. (A-D) Ontogeny of p63 in embryonic UGS. CA, cloaca; HG, hind gut; MND, mesonephric duct; NT, neural tube. Cross section (A) and saggital section (B-D) of mouse embryos. Scale bar: 50 µm. (E-O) Marker expression was examined in male p63+/+ UGS (E-H,M) and male (I-L,N) and female (O) p63–/– UGSs at E17.5. MD, Müllerian duct; WD, Wolffian duct. Gross morphology of the p63–/– UGS was normal and showed normal sexual dimorphism at E17.5 (compare O [female] with I-L [male]). p63–/– urogenital sinus epithelium (UGE) expressed differentiation markers identical to that of p63+/+ UGE [uroplakin (G,K), involucrin (H,L) and androgen receptor (AR) (M,N)] with the exception that p63 (E,I) and K14 (F,J) were absent in p63–/– UGE. In both male p63+/+ and p63–/– UGSs, notice epithelial outgrowths into stroma in the area where prostates normally form (black and white arrows).

View larger version (131K): [in a new window]   Fig. 2. p63–/– prostate lacks basal cells. Both p63+/+ (A) and p63–/– (B) urogenital sinus (UGS) formed prostate when grafted into intact male hosts. When p63–/– UGS was grafted into castrated male hosts, the UGS developed into a large cyst without ducts (C). The prostatic ducts were surrounded by smooth muscle cells expressing smooth muscle actin (SMA) (D,E). Morphologically definable basal cells (black arrow in F,H) were not observed in p63–/– prostate (G,I). p63–/– prostate contained goblet cells (G,I, white arrows) (see also Fig. 3). Elongated/flat nuclei of stromal cells should not be confused with basal cells. Expression of basal cell markers was examined in p63+/+ (J,L,N,P) and p63–/– (K,M,O,Q) prostates. p63 (J,K), K14 (L,M), transglutaminase II (TGII,N,O) and maspin (P,Q) were detected only in p63+/+ prostate. All basal cell markers were absent in p63–/– prostate (K,M,O,Q). RT-PCR for K14 (R) confirmed results of IHC. K14 mRNA was detected in host prostate (coagulating gland + ventral prostate + dorsolateral prostate, lane 1) and three p63+/+ prostatic grafts (lanes 2-4) but not in p63–/– prostates (lanes 5 and 6). ß-actin was used for control.

View larger version (140K): [in a new window]   Fig. 3. Epithelial differentiation in p63–/– prostate. Scale bars: 50 µm. Goblet mucinous cells were detected in only p63–/– prostate (A,C). (C) The mucinous secretion stained for Alcian blue (pH 2.8). (D) Double staining for p63 and Alcian blue; one week after grafting the mucinous (Alcian blue-positive) cells were undetectable in p63–/– urogenital sinus (UGS). Double staining for androgen receptor (AR) and Alcian blue (E) in p63–/– UGS grafts; a small number of Alcian blue-positive cells was detected in epithelium of proximal ducts at 10 days after grafting (E, arrows). Expression of luminal cell markers was examined in p63+/+ (F,H,J,L) and p63–/– (G,I,K,M) prostates. AR (F,G), Nkx3.1 (Nkx, H,I), and secretory proteins, mouse dorsolateral prostate (mDLP) (J,K) and mouse ventral prostate (mVP) (L,M) were expressed in the luminal cells in p63+/+ and p63–/– prostates. mDLP-positive and mVP-positive cells were found among Alcian blue-positive cells (K,M). mDLP was co-expressed with mucin (K, red and black arrows). Secretory granules contained both mucin and mDLP in some mucinous cells (K, red arrows). Activity of Src was examined in p63+/+ and p63–/– prostates (N-Q). In the p63+/+ prostate, activation of Src was detected in neurons (P, red arrows) and a subset of basal cells (P, black arrows) but not in luminal cells (N,P). In p63–/– prostate, Src activity was detected mainly in Alcian blue-positive cells, but some non-mucinous luminal cells were also strongly positive for active Src (Q, white arrows). Downstream effecters in the Src-Ras-MAP kinase signal transduction pathway (MEK1/2 and ERK1/2) were also activated (phosphorylated) in neurons (not shown) and basal cells (R,S, arrows) in p63+/+ prostate and in (mucinous and non-mucinous) luminal cells in p63–/– prostate (T). Even though MAP kinase signaling was active, p63–/– prostate epithelium was proliferation-quiescent one month after grafting. Phosphorylation of histone H3 (pH3, U,V) was not detected in areas where phosphorylation of MEK was detected in p63+/+ (U) or p63–/– (V). Both p63+/+ and p63–/– contained rare neuroendocrine cells as assessed by expression of synaptophysin (W,X).

View larger version (92K): [in a new window]   Fig. 4. Effect of castration and androgen treatment. Double staining for androgen receptor (AR) and Alcian blue on p63+/+ (A,C,E) and p63–/– (B,D,F) prostates. After one month of growth in intact male hosts, p63+/+ and p63–/– urogenital sinus (UGS) developed prostate having a complex ductal structure (intact, A,B). The ducts of p63+/+ prostates (A) were more dilated and had larger lumens than that of p63–/– prostate (B). In grafts grown in intact hosts for one month followed by one month of castration, the lumen of ducts in p63+/+ prostate became smaller and the entire graft was reduced in size (C). In p63–/– prostate, castration reduced the number of ducts, and the entire graft became cystic (D, arrows; large cystic ducts). Pools of secretion devoid of epithelium were always observed (D,N,*). One month after the T-pellet implantation to castrated hosts, ducts in p63+/+ prostate became dilated and filled with secretion (E). Ducts of p63–/– prostate also regenerated and exhibited increased complexity of ductal structure (F). (G) Wet weight of prostatic ducts. Data was analyzed with Fisher's PSLD and ANOVA factorial tests. Data are expressed as mean+s.d. Lowercase letters indicate groups statistically distinguished. The data was significantly higher in the following order, c>a>b (P<0.05). (H) Ductal complexity. Total length of epithelial basement membrane (epithelial length, µm) in prostatic tissue was measured in sections of UGS grafts. The epithelial length was divided by the area of prostatic tissue (µm2). Data was analyzed with Fisher's PSLD and ANOVA factorial tests. Data are expressed as mean+s.d. Lowercase letters indicate groups statistically distinguished. There is no significant difference between a versus b, or a versus c. c is significantly higher than b. a, b and c are significantly higher than d (P<0.05). Detection of apoptosis in p63+/+ (I,K) and p63–/– (J,L) prostate. In the intact hosts, apoptotic cells were rarely detected (I,J). Three days after the castration of the host, apoptotic epithelial cells were detected in both p63+/+ and p63–/– prostates (K,L, white arrows). Double staining for p63 and Alcian blue (M,N). The concentration of p63-positive basal cells increased in the shrunken duct of p63+/+ prostate (M). Pools of Alcian blue-positive material devoid of intact epithelium but containing dying/dead cells (N, black arrow) were consistently observed in p63–/– prostate one month following castration (N, *).

View larger version (87K): [in a new window]   Fig. 5. Effect of testosterone. One month after castration (castrated A and B), proliferation markers, phospho-histone H3 (pH3; a,d,g,j), phospho-MEK (pMEK; b,e,h,k) and Cdk4 (c,f,i,l) were very low to undetectable in both p63+/+ (A) and p63–/– (B) prostates. Five days after implantation of a 25 mg T-pellet (castrated+T; C,D), all three proliferation markers were upregulated in both p63+/+ (C) and p63–/– (D) prostates. In the p63–/– prostatic grafts, small bud-like outgrowths were visible (D, red arrows). (E) Labeling index for pH3. Data was analyzed with Fisher's PSLD and ANOVA factorial test. Data was indicated as mean+s.d. The bars with asterisk are significantly higher than others (P<0.05). There's no significant difference among bars without asterisk. Both p63+/+ and p63–/– prostates were proliferation-quiescent in the intact and castrated hosts. T-treatment of castrated hosts increased pH3-positive cells equally in luminal cells of p63+/+ and p63–/– prostates (+T 5 days). However, luminal cells in both p63+/+ and p63–/– prostates became proliferation-quiescent one month after T-pellet implantation (+T one month). One month after T-pellet implantation, regenerated p63–/– prostate expressed markers specific for prostatic luminal epithelium [androgen receptor (AR) (F), and secretory proteins, mouse dorsolateral prostate (mDLP) (G) and mouse ventral prostate (mVP) (H)].