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Results: 4

1.
Fig. 1

Fig. 1. From: Altered Prostate Epithelial Development and IGF-1 Signal in Mice Lacking the Androgen Receptor in Smooth Muscle Cells.

Using a Cre-loxP conditional knockout strategy, we mated female heterozygous flox-AR mice with male Tgln-Cre mice to generate SM-ARKO male mice. The DNA fragments of Cre and floxed AR exon 2 were detected in tail genomic DNA (Fig. 1A).

Shengqiang Yu, et al. Prostate. ;71(5):517-524.
2.
Fig. 2

Fig. 2. From: Altered Prostate Epithelial Development and IGF-1 Signal in Mice Lacking the Androgen Receptor in Smooth Muscle Cells.

We compared the prostates of SM-ARKO mice and WT littermates, but found no significant difference in gross appearance (Fig. 2A-B) and branching morphogenesis (Fig. S1). However, H&E staining showed defective structures in the SM-ARKO AP with fewer epithelial infoldings into the lumens (Fig. 2C-H). In the VP, we also found fewer epithelial infolding structures and thinner epithelium layer especially in 18-week-old SM-ARKO mice (Fig. 2I-L). In the DLP, we did not find histological changes in SM-ARKO mice (data not shown). The histological changes in different prostate lobes are consistent with the Cre activity: AP > VP >> DLP. Previously, we characterized SM-ARKO mice and their testicular function, and found there is no alteration of the serum testosterone, luteinizing hormone, and follicle stimulating hormone levels [15]. Thus, the morphology changes of SM-ARKO mouse prostates are not due to the hormone level changes.

Shengqiang Yu, et al. Prostate. ;71(5):517-524.
3.
Fig. 4

Fig. 4. From: Altered Prostate Epithelial Development and IGF-1 Signal in Mice Lacking the Androgen Receptor in Smooth Muscle Cells.

IGF-1 is an important growth factor in prostate for the proliferation and morphogenesis, which was secreted principally in the stromal SMCs [18–20], and is regulated by the AR signal [20–21]. To probe the effect of AR loss in SMCs, we examined the IGF-1 expression levels by IHC staining, and showed a dramatically reduced IGF-1 expression in AP and VP of SM-ARKO mice (Fig. 4A). Then we used an AR positive rat prostate smooth muscle cells line, PS-1 [13], to confirm the in vivo findings. First, we introduced the pSuperior-AR-shRNA/scramble to PS-1 cells, and established the PS-1 AR-shRNA (PS-1-ARsh) and scramble (PS-1-sc) stable cells. Western blotting and Q-PCR were used to confirm the AR knockdown efficiency in PS-1-ARsh cells (Fig. 4B, C). We then compared cellular growth rate between PS-1-sc and PS-1-ARsh cells, and found loss of AR could inhibit the growth of SMCs (Fig. 4D). Furthermore, we used Q-PCR to examine relative gene expression levels of IGF-1, HGF, and FGF10, and found the IGF-1 gene expression level was obviously reduced in PS-1-ARsh cells (Fig. 4C). To confirm the Q-PCR data, we performed the cytohistochemical staining, and also found the lower expression level of IGF-1 in PS-1-ARsh cells (Fig. 4E). These data suggested that SMCs’ AR could regulate the IGF-1 expression level in the prostate, then further regulate the epithelium proliferation.

Shengqiang Yu, et al. Prostate. ;71(5):517-524.
4.
Fig. 3

Fig. 3. From: Altered Prostate Epithelial Development and IGF-1 Signal in Mice Lacking the Androgen Receptor in Smooth Muscle Cells.

To dissect the mechanism why the SM-ARKO mouse prostates have altered structure with fewer epithelial folding structures, we detected the proliferation, apoptosis, and differentiation of epithelium. Ki67 IHC staining was performed to determine the epithelial proliferation of AP and VP of 6 wks old mice (Fig. 3A-D). Then, we counted 500 epithelial cells to determine the Ki67 positive numbers. In AP lobes, the average Ki67 positive cells were 7.6 ± 1.5 in WT mice, but only 2.6 ± 1.2 in SM-ARKO mice (*P<0.05) (Fig. 3G); in VP lobes, the average of Ki67 positive cells was 5.8 ± 2.03 in WT mice, but 1.8 ± 0.97 in SM-ARKO mice (*P<0.05). The proliferative signals in mouse prostates are high from birth to young adult age (8 weeks). After 12 wks, only few cells are positive for proliferation signals. Therefore, the Ki67 staining at 6 wks old prostates was presented here. In contrast to the proliferation, the apoptotic signal is barely detectable in prostates at a young age. Cellular apoptosis was determine by TUNEL assay, the data did not show significant differences in the AP (Fig. 3E, F, H), VP, or DLP (data not shown) of WT and SM-ARKO mice at 26 wks old. Moreover, we detected the basal cell marker P63 (Fig. 3I-L) and the luminal cell marker keratin 8 (data not shown) using IHC staining. By counting the positive cells per 100 epithelial cells, we did not find any difference of basal cell and luminal epithelial cell ratio between the SM-ARKO and WT mice. Together, the above data indicated that the morphological changes of SM-ARKO mouse prostates are mainly due to the defected epithelium proliferation, not via apoptosis or differentiation.

Shengqiang Yu, et al. Prostate. ;71(5):517-524.

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