Hh Signaling In Prostate Development

Shh is the most abundantly expressed Hh ligand in the developing mouse prostate (18). Ihh is also expressed but at comparatively low levels. Dhh expression has not been observed. Shh gene expression in the urogenital sinus (UGS) increases before the initiation of ductal budding at embryonic day 17.5 (E17.5). Expression is most abundant during the period of ductal budding in late gestation. Shh expression gradually diminishes through the first l0 days after birth, a period characterized by continued bud formation and by outgrowth and branching of newly formed ducts. Shh gene expression declines progressively, and, by 30 days postnatal, has approached the low level of expression seen in the adult. This decrease parallels the completion of the ductal branching process. A schematic illustration of how Shh expression fits in the timeline of key morphogenetic events in prostate development is presented in Fig. 2.

Shh expression in the human fetal prostate was demonstrated by semiquantitative RT-PCR on RNA prepared from snap-frozen fetal prostate tissues (age 15.5 and 18 weeks). Expression was present at both fetal time points, with relatively greater expression at the earlier time point. Immunostaining for Shh peptide was performed on prostatic tissue sections from seven human male fetuses aged 9.5 to 20 weeks of gestation.

Staining was weak at 9.5 weeks but intense at 11.5, 13, and 16 weeks. The increase in Shh expression was found to coincide with the onset of ductal budding and outgrowth, and particularly robust expression was noted in newly formed prostatic buds (19). Staining diminished at 18 and 20 weeks, in agreement with results obtained by RT-PCR, and staining was absent at 34 weeks. Thus, Shh protein expression in the human prostate is most abundant during the early phase of prostate development when ductal budding occurs (10-17 weeks) and is downregulated before birth. In contrast to the mouse, in which Shh expression remains low in the adult, robust Shh expression is present in both normal adult prostate and benign prostatic hyperplasia (BPH) (20).

Localization of Shh expression during prostate development is correlated with the process of ductal morphogenesis. In the late gestation embryo (E15), Shh is diffusely expressed in the epithelium of the prostatic portion of the UGS. Just before the onset of ductal budding, Shh expression is upregulated and, at the onset of bud formation (E17), expression condenses at the epithelial evaginations that form the nascent ducts. Shh expression in the UGS is not dependent on testosterone, but the redistribution that occurs coincident with ductal budding does occur in response to androgen stimulation (21). As ductal budding proceeds, Shh expression becomes restricted to the developing buds and, by P1, Shh expression is strictly localized to the primary ducts of the developing prostate. Expression is strongest in the advancing distal regions of the ducts and diminishes in the already formed proximal duct segments.

Shh expression during prostate development seems to act primarily in a paracrine fashion. During budding, the genes for the Shh receptor Ptc1 and the transcriptional activator Gli1, both induced targets of Hh signaling, are highly localized in the mesenchyme of the UGS immediately surrounding the nascent buds. Gli2 and Gli3 are also expressed primarily in the UGS mesenchyme (Fig. 3). As Shh expression becomes localized to the apical regions of elongating ducts, the expression for Ptc1





Fig. 2. (A) The mouse lower urogenital tract at embryonic day 15 (E15) and at birth (P1) showing the outgrowth of ductal buds in the region of the coagulating gland (cg), dorsal prostate (dp), and ventral prostate (vp). (B) The expression profile of Sonic Hedgehog (Shh) during mouse prostate morphogenesis. Expression increases before budding at E17.5, remains abundant during late gestation and through birth, and then gradually diminishes after birth to very low levels in the adult. (C) Key events of prostate development, including epithelial budding, ductal branching, and ductal differentiation. T, testis; ur, ureter; b, bladder; u, urethra; sv, seminal vesicle; ugs, urogenital sinus.

seems to be strongest in the mesenchyme surrounding the distal duct segments (21,22). Expression of the three Gli genes in the ductal mesenchyme also exhibits a proximodistal gradient (22). This asymmetric distribution of elements of the Shh-Gli pathway during ductal morphogenesis may signal the early establishment of a proximodistal heterogeneity in the morphology and function of the adult prostatic ducts (23). Although most studies support a primarily paracrine mechanism of Shh action, the presence of low Ptcl, Glil, and Gli3 in the UGS epithelium leaves open the possibility of some autocrine signaling activity (21,22).

In vitro studies have confirmed the paracrine mechanism of action. Exogenous Shh peptide exerts an inductive effect on both Ptc and Glil gene expression (known downstream targets of the pathway) in isolated mouse male E14 UGS, and this effect is direct and inhibited by cyclopamine, a specific and potent chemical inhibitor of Hh action. The target genes of Hh signaling in the prostate are not yet described, but work in other systems has identified targets that may be conserved. Hepatocyte nuclear factor-3p is a well-established target of Gli1 regulation (10). Using a cell-based assay, Yoon

Fig. 3. Whole-mount in situ hybridization for Sonic Hedgehog (Shh), patched (Ptc)-1 and Glil expression in the developing mouse prostate. (A) In the whole-mount E15 mouse specimen, little expression is seen. (B) Sectioning reveals uniform Shh expression in epithelium (e) lining the lumen of the urethra (u). (C) At P1, Shh expression is focused to the nascent buds of the dorsal prostate (dp), coagulating gland (cg), and ventral prostate (vp). (D) Apparent concentration of Shh expression is exhibited in the epithelium (e) of the distal duct (long arrow) relative to the proximal duct (short arrow). Note the diminished expression in the epithelium (e*) of the urethra (u). No Shh expression is detected in the mesenchyme (m) at any stage of prostate development. Expression of Ptcl (E) and Glil (G) surround the prostatic buds, and expression of both genes is more concentrated in the mesenchyme immediately surrounding the epithelium source of the Shh ligand (F,H). B, bladder; sv, seminal vesicle. (Photographs reprinted with permission from ref. 21).

Fig. 3. Whole-mount in situ hybridization for Sonic Hedgehog (Shh), patched (Ptc)-1 and Glil expression in the developing mouse prostate. (A) In the whole-mount E15 mouse specimen, little expression is seen. (B) Sectioning reveals uniform Shh expression in epithelium (e) lining the lumen of the urethra (u). (C) At P1, Shh expression is focused to the nascent buds of the dorsal prostate (dp), coagulating gland (cg), and ventral prostate (vp). (D) Apparent concentration of Shh expression is exhibited in the epithelium (e) of the distal duct (long arrow) relative to the proximal duct (short arrow). Note the diminished expression in the epithelium (e*) of the urethra (u). No Shh expression is detected in the mesenchyme (m) at any stage of prostate development. Expression of Ptcl (E) and Glil (G) surround the prostatic buds, and expression of both genes is more concentrated in the mesenchyme immediately surrounding the epithelium source of the Shh ligand (F,H). B, bladder; sv, seminal vesicle. (Photographs reprinted with permission from ref. 21).

et al. (24) identified approx 30 targets of Gli-1, including cyclin D2, osteopontin, insulin-like growth factor binding protein 6, mitogen-activated protein kinase 6c, and plakoglobin. One of these, insulinlike growth factor binding protein 6, has recently been shown to be a Shh-regulated gene in the fetal mouse prostate (16).

Functional studies of Hh signaling in prostate development have used antibody blockade, chemical inhibition, and genetic loss of function models. These studies have yielded somewhat conflicting data on the requirement of Shh for normal prostate development. A polyclonal antibody to Shh seemed to block prostate development in a subcapsular renal graft model (18). However, the UGS of the Shh-null transgenic mouse exhibited budding morphogenesis in organ culture and prostatic glandular morphogenesis when grafted under the renal capsule of an adult male host mouse (25). This apparent discrepancy could be explained if Ihh, which is also expressed in the UGS, provides functional redundancy for Shh. A polyclonal antibody would likely block the action of both Shh and Ihh, whereas the Shh-null would manifest a selective loss of Shh function.

The compound cyclopamine is a steroidal alkaloid derived from the subalpine flower Veratrum californicum. Cyclopamine is a specific a chemical inhibitor of Hh signaling that acts by interfering with Shh signal transduction by the transmembrane protein, Smo, and blocks signaling by all Hh ligands. Cyclopamine treatment of cultured UGS tissues has produced a variety of observations that may be a function of the stage in prostate development when signaling is disrupted. Cyclopamine blockade initiated in the prebud E14 mouse UGS inhibits epithelial cell proliferation and seems to decrease the total number of discrete prostatic buds (21). Together with the effect of Shh antibody blockade on prostate development, these data may suggest an early requirement for Hh signaling in prostate development and budding morphogenesis. Cyclopamine blockade later in development, in E16.5 mouse UGS or the neonate rat ventral prostate, produces apparently opposite effects. Epithelial cell proliferation is increased, prostate growth is enhanced, and the number of ducts is either increased or not significantly affected (25,26). Exogenous Shh produces the opposite effects, inhibiting cell proliferation and decreasing the number of prostatic ducts (25,26). Collectively, these observations suggest a shift in the role for Shh signaling in prostate morphogenesis: from promoting bud formation and outgrowth via increased epithelial cell proliferation to inhibiting cell proliferation and branching morphogenesis in the postnatal prostate.

Given this inhibitory action of Shh signaling on epithelial proliferation and ductal branching during postnatal prostate development, the decline in Shh expression after birth can be viewed as permissive for branching morphogenesis. Shh upregulates the expression of transforming growth factor-31 and activin A. These factors are both expressed in prostatic mesenchyme and inhibit prostate branching morphogenesis (26-28). Bone morphogenetic factor (Bmp) 4 is another member of the transforming growth factor family that inhibits branching and is expressed in the prostatic mesenchyme (29). Whether Bmp4 is a direct target of Shh signaling in the prostate remains to be resolved (22,26). Shh was shown to downregulate the expression of mesenchymal fibroblast growth factor (Fgf)-10 in rat ventral prostate, and exogenous Fgf-10 was shown to reverse Shh-mediated inhibition of prostate growth and branching. A model for ductal branching that involves the interaction of Shh, FGF-10, and Bmp4 was recently proposed (22).

Two studies suggest that Shh may influence differentiation of ductal epithelium into basal and luminal cells, but these studies have yielded conflicting observations. One study showed that exogenous Shh increased the proportion of epithelial cells that did not express CK14 and p63. This was interpreted as showing that Shh increased luminal cell differentiation. Cyclopamine was shown to have the opposite effect (26). The other study found that cyclopamine accelerated both ductal canalization and epithelial cell differentiation (30). The apparent discrepancy between these two studies could result from unique features of the experimental design, which differentially highlight particular aspects of the complex actions of Shh during ductal development. Clearly, a better understanding of the role of Shh signaling in the process of differentiation is of considerable interest.


Shh expression and pathway activity is very low in the normal adult mouse prostate. To examine the role of Hh signaling in prostate regeneration, Karhadkar et al. (31) tested the effect of pathway blockade on androgen-induced regrowth of the castrate ventral prostate. Both Hh-neutralizing antibody and chemical inhibition of Hh signaling abolished prostate regeneration in this model. This finding suggested that Shh signaling might be involved in the recruitment and/or renewal of progenitor cell populations.

An emerging paradigm for cancer development holds that malignant transformation occurs in stem cells or progenitor cells that have an unlimited proliferation potential. It is postulated that injury and inflammation activate proliferation of stem cells as part of the repair process. In the presence of inflammation, these proliferating progenitor cells are exposed to oncogenic forces, such as oxidative stress, that may induce genetic or epigenetic changes that lead to persistent stem cell activation. Depending on the tissue type, either Hh or Wnt signaling seem to figure prominently in the process of stem cell activation, and tumor development is postulated to involve persistent and autonomous activation of Hh or Wnt signaling that drives tumor growth.

Shh has recently been postulated to regulate stem cell recruitment in tissues where it plays a key role in regulating growth during development, including the central nervous system, retina, bile duct, bone, lung, and prostate (32). Evidence for such a role has recently been obtained in studies of a model of lung injury and repair (33,34). Given the clear-cut role of Shh in prostate development and the recently identified role of chronic inflammation in prostate carcinogenesis, it is reasonable to speculate that Hh signaling could play a central role in the development and progression of prostate cancer.


Analysis of specimens from young adult men, from men with BPH, and from men undergoing radical prostatectomy for prostate cancer revealed Shh expression in all three groups (20). The level of Shh expression and Gli1 activation in these tissues, as determined by quantitative RT-PCR, was surprisingly robust-comparable to the level of expression in the fetal brain. This was an unexpected observation that contrasts with the low level of expression in the adult mouse prostate. Whether this reflects a persistent generalized growth activity in the postpubertal human prostate or is associated with multifocal processes of inflammation and hyperplasia in the human prostate remains to be determined. Several studies have examined the expression of Shh in localized prostate cancer and in metastatic prostate cancer (20,31,35,36). They have yielded a picture of variably increased Shh expression in localized tumors exerting a combination of autocrine and paracrine signaling activity and showing dramatically increased pathway activation in metastatic disease.

Among the tissues studied by Fan et al. (20), there was a trend toward higher Shh expression in the cancer specimens as a group and a mean level of expression nearly 10-fold higher than that observed in the nontumor specimens. Karhadkar et al. (31) performed PCR analysis of 12 localized tumors and found substantial Shh expression in all of them. They also detected the presence of lower level Ihh expression. Sheng et al. (36) performed immunostaining for Shh peptide and found protein expression in more than half the tumors they examined. Sanchez et al. (35) found Shh expression in both normal prostate tissue and prostate cancer, but found more intense staining in 33% of the tumors. These studies all agree that Shh expression is a common feature of localized prostate cancer and suggest that very high expression is present in a subset of tumors. However, they also indicate that the range of expression in these tumors is quite broad and occurs against a background of robust and variable expression in the normal and hyperplastic prostate. Consequently, the specificity of Shh overexpression for cancerous tissue is an important but as yet unresolved issue. Fan et al. (20) compared expression by RT-PCR in 11 histologically confirmed tumors and benign tissues from the same gland and found no significant difference in Shh expression between the cancer specimens and zone homologous benign tissue. This suggests that the robust level of Shh expression in localized prostate cancer is generally mirrored by equally strong expression in histologically benign tissue from the same gland. On the other hand, Sanchez et al. (35) showed a variable (0- to 9.8-fold) increase in Shh expression measured by RT-PCR in six matched specimens. This observation suggests that tumor-specific increased Shh expression may occur in a subset of prostate cancers. On the basis of immunostaining, Sheng et al. (36) suggested that Shh overexpression may occur in a subset of high Gleason grade tumors. Interestingly, our unpublished studies revealed very high levels of Shh expression in locally advanced tumors that required transurethral resection for outlet obstruction. This is consistent with the notion that Shh overexpression may be associated with increased tumor progression and/or growth.

The activity of Hh signaling pathway in normal and tumor tissue depends both on the expression of the Hh ligands and on the responsiveness of the target cell. Therefore, the true activity of the pathway is best measured by the expression of target genes Glil and Ptc that are reliably induced by Hh ligand. Studies of Shh expression in benign prostate tissue and localized cancer generally suggest a correlation between Shh expression and Gli1 expression, but two studies suggest that changes in pathway regulation in tumor cells may result in disproportionate pathway activation for the level of ligand. Fan et al. (20) examined Gli1 expression by real time RT-PCR in adult normal, BPH, and cancer specimens and found expression in all tissues examined. In addition, they found an extremely tight correlation of Shh and Gli1 expression in all of these tissues, indicating a generally tight linkage between expression of Shh ligand and pathway activation. They also observed a tight correlation between Shh expression and the levels of Gli2 and Gli3 expression. Sanchez et al. (35) observed both Gli1 and Ptc expression in all six matched normal and tumor tissues they examined. Increased Shh expression in specimens of prostate cancer was generally accompanied by increased expression of Ptc1, Gli1, Gli2, and Gli3. In contrast to these studies, Karhadkar et al. (31) did not observe either Ptc or Gli1 expression in 12 specimens of normal prostate tissue. These authors observed substantial Ptc and Gli1 expression in only 3 of 12 localized tumors, but found abundant Ptc and Gli1 expression in their survey of 15 metastatic prostate cancers. This was attributed to enhanced pathway responsiveness caused by increased SMO expression in the metastatic lesions. Sheng et al. (36) presented evidence that Hh pathway activation may also occur through mutation in the regulatory protein, Suppressor of Fused. Collectively, the studies to date suggest that Shh expression and pathway activation in benign tissues is tightly correlated, but that tumor tissue is characterized by variable degrees of increased Shh expression and increased pathway responsiveness that may correlate with tumor grade, growth, and metastasis.

Was this article helpful?

0 0
Getting Back Into Shape After The Pregnancy

Getting Back Into Shape After The Pregnancy

Once your pregnancy is over and done with, your baby is happily in your arms, and youre headed back home from the hospital, youll begin to realize that things have only just begun. Over the next few days, weeks, and months, youre going to increasingly notice that your entire life has changed in more ways than you could ever imagine.

Get My Free Ebook

Post a comment