The initial observation of prostatic epithelial development via mesenchymal-epithelial interaction was reported two decades ago [3
]. Based on IHC results, AR was detected in urogenital sinus mesenchyme during early development, but not in epithelium [23
], indicating the importance of mesenchymal AR in the prostate early development. The tissue recombinants of AR-deficient UGM and WT-epithelium could not form prostate in the nude mice sub-renal capsules, but the recombinants of WT-UGM and AR-deficient epithelium could develop to a prostate-like organ [24
], which indicated the critical roles of mesenchymal AR in the prostate development. However, the previous tissue recombination assays were conducted in immune-deficient mice without proper prostate microenvironment, and could not determine the AR roles in different cell types of the prostate stroma. In this work, we focused on the SMCs AR role in the prostate development by using the Cre-loxP gene knockout system.
We generated the SM-ARKO mice by mating floxed AR mice [11
] with Tgln-Cre mice [14
]. The expression pattern in the organs of Tgln-Cre transgenic mice has been reported previously [14
]. In the prostate, the AP has the highest Cre expression level, thus has the highest AR knockout efficiency. Based on the H&E staining, we found that there are reduced infolding structures mostly in AP. Further studies showed the phenotype change is mainly due to the defected epithelium proliferation, not apoptosis. Surprisingly, there is no significant difference in the basal cells ratio between SM-ARKO and WT mice. This suggests that SMCs AR might play more important roles in the epithelium proliferation, rather than in apoptosis or differentiation. However, the AR knockout efficiency, even in AP, is only around 50% (data not shown). The remaining AR positive SMCs might partially maintain the basic AR function, thus the prostates could still develop and grow with normal gross appearance.
The molecular pathways of prostate stromal AR regulating epithelial development are mediated, at least in part, by stromal growth factors under the influence of androgen signals [28
]. The IGF-1, primarily mediated through the IGF-1 receptor 1 (IGF-R1), is important for the prostate normal development and cancer [29
], and is synthesized principally by the SMCs [19
]. Androgen/AR signal could regulate IGF-1 expression directly at the transcriptional level via potential androgen response elements located on the IGF-1 5′ promoter [21
]. In this study, we found the IGF-1 expression level (based on IHC staining data) was obviously reduced in the SM-ARKO prostates, which might contribute to the altered prostate infolding structure and reduced epithelial proliferation. To further confirm the in vivo
findings, we cultured the prostate smooth muscle PS-1 cells, and found knockdown of AR could also decrease the IFG-1 expression level, but not the FGF10 or HGF.
We determined the IGF-1, FGF7, FGF10, HGF, and smooth muscle alpha-actin (SMA) expression levels in 26-week-old mouse APs using Q-PCR, but only found a significant difference of SMA expression (Fig. S2
, n=5). The decreased SMA expression in SM-ARKO AP could be due to the growth inhibition of SMCs when knocking out the AR. The in vitro PS-1-ARsh/sc growth data () further support this result. There are two possible explanations for why we did not observe the changes of those growth factors: (1) SMCs are not the only cell source to produce those growth factors in prostate, and (2) the SMC population in the whole prostate is no more than 10%, and the presence of many other cell types could mask the result. Therefore, the Q-PCR data collected from the whole AP did not show the difference.
Taken together, by using this SM-ARKO mouse model, we found the AR in SMCs plays important roles in the epithelial cells normal development. The morphological changes are mainly due to the reduced epithelium proliferation mediated by IGF-1 signal.