Hedgehog pathway responsiveness correlates with the presence of primary cilia on prostate stromal cells
© Zhang et al; licensee BioMed Central Ltd. 2009
Received: 20 November 2008
Accepted: 7 October 2009
Published: 7 October 2009
Hedgehog (Hh) signaling from the urogenital sinus (UGS) epithelium to the surrounding mesenchyme plays a critical role in regulating ductal formation and growth during prostate development. The primary cilium, a feature of most interphase vertebrate cell types, serves as a required localization domain for Hh signaling transducing proteins.
Immunostaining revealed the presence of primary cilia in mesenchymal cells of the developing prostate. Cell-based assays of a urongenital sinus mesenchymal cell line (UGSM-2) revealed that proliferation-limiting (serum starvation and/or confluence) growth conditions promoted cilia formation and correlated with pathway activation associated with accumulation of Smoothened in primary cilia. The prostate cancer cell lines PC-3, LNCaP, and 22RV1, previously shown to lack demonstrable autocrine Hh signaling capacity, did not exhibit primary cilia even under proliferation-limiting growth conditions.
We conclude that paracrine Hedgehog signaling activity in the prostate is associated with the presence of primary cilia on stromal cells but that a role in autocrine Hh signaling remains speculative.
The prostate is a multi-lobed male accessory sex gland composed of complex secretory ductal structures that drain into the prostatic urethra. The prostate develops from the prostatic anlagen of the urogenital sinus (UGS) where the hallmark event is budding of UGS epithelium into the surrounding mesenchyme and initiation of ductal growth and morphogenesis. Hedgehog signaling plays a key role in this process, being required for normal budding and ductal outgrowth [1–4]. Of the three vertebrate Hh ligands [Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh)], Shh mRNA is the most abundantly expressed in the developing mouse prostate . Expression in the epithelium of the urogenital sinus (UGS), increases prior to the initiation of ductal budding at embryonic day 17.5 (E17.5) and then localizes to the tips of the nascent ducts . During prostate development Shh appears to act primarily in a paracrine fashion, inducing expression of Gli1, Ptc1, and other recently identified Hh target genes in the adjacent mesenchyme [, unpublished observations]. However, the presence of low Ptc1, Gli1 and Gli3 expression in the urogenital sinus epithelium leaves open the possibility of limited autocrine signaling activity . Hh ligand expression and pathway activity is common in localized prostate cancer (PCa) and may promote tumor cell proliferation by a combination of autocrine and paracrine signaling [7–9] via canonical ligand-mediated signal transduction and/or genetic mutations affecting the regulation of Hh pathway activity in the tumor cells as suggested by Sheng et al., . Hh pathway activity is dramatically increased in advanced, metastatic PCa  but whether this represents mutational activation or an increased responsiveness of the tumor cell or ectopic stroma to Hh ligand is not known (For review, see ).
Primary non-motile cilia are microtubule-based organelles formed by active interflagellar transport (IFT) present on most vertebrate cells . Recent evidence indicates that the primary cilium is a required cellular feature for canonical vertebrate Hh signal transduction . Hedgehog ligand binding to the 12-pass transmembrane protein Patched relieves its repression of the 7-pass transmembrane protein Smoothened (Smo), followed by accumulation of Smo along with the transcription factors Gli2 and Gli3 in the primary cilium [14, 15]. Functional ciliary IFT is required for regulating the activity of Gli2 and Gli3 , which coordinately modulate the expression of Hh target genes.
We recently described the generation and characterization of an immortalized cell line (UGSM-2) isolated from the E16.5 UGS mesenchyme . The Hh responsiveness of this cell line was noted to increase with conditions of growth arrest - confluence and serum starvation - conditions associated with the formation of primary cilia. This observation prompted us to examine the expression of primary cilia in the mesenchyme of the developing prostate and to examine the functional requirement for primary cilia in the response of prostate mesenchymal and stromal cells to Hh ligand.
Human prostate myofibroblast WPMY-1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in recommended media. Mouse prostate mesenchymal UGSM-2 cells were maintained as described previously . For gene expression assays, cells were plated in media containing 10% FBS at 2.0×104 (subconfluent) or 1×105 cell/well (confluent) in 400 μl media in a 24-well plate and allowed to attach overnight. Following, cells were washed in media containing 0.1% FBS, which was then replaced with fresh medium containing 10% FBS (high serum) or 0.1% FBS (low serum) ± 1 nM octylated (Curis, Inc., Cambridge, MA) and ± 10 μM cyclopamine (Toronto Research chemicals, Ontario, Candada). Human prostate cancer cell lines were maintained as described previously . For immunocytochemistry assays, cells were plated at 1.0 × 104/well in 4 well chamber slides.
Plasmids and Retroviral infection
Vectors pLTR-hGli1, pCMV-hGli2β were kindly provided by Dr. Philip Iannaccone (Northwestern University, Chicago, IL) and gDHuSMO-M2 containing cDNA of activated human Smoothened was a generous gift from Genentech, Inc. (South San Francisco, CA). Plasmids containing Myc-tagged mouse wild type Smoothened (WT-Smo) and ciliary localization defective smoothened (CLD-Smo) were kindly provided by Dr. Jeremy Reiter (University of California, San Francisco, CA). Each was subcloned into a retrovirus vector pCMV-IRES-GFP (Gift from Dr. Michael Hoffman, University of Wisconsin, Madison, WI) using standard molecular cloning techniques. All the constructions were confirmed by gel electrophoretic analyses and sequencing. Retroviruses were generated as described . Cells were infected with virus and following one week of passage, GFP+ cells were collected by flow cytometry.
Cells grown in four-well chamber slides were fixed in 4% paraformaldehyde for 30 minutes at room temperature. Prostate tissues were isolated from P1 CD-1 mice. Formalin-fixed, paraffin embedded sections were dewaxed, rehydrated, and processed for antigen retrieval. Immunohistochemistry was performed using the following primary antibodies; Rabbit anti-Myc (Abcam, ab9106, 1:200); Mouse anti-acetylated tubulin (Sigma, T6793, 1:1000). The following secondary antibodies were purchased from Molecular Probes and used at 1:200 dilution; Alexa Fluor 546 goat anti- rabbit IgG; Alexa fluor 488 goat anti mouse IgG; Alexa fluor 546 goat anti-mouse IgG. Mouse anti-p63 (Santa Cruz, sc-8431, 1:100) antibody was directly labeled with Alexa Fluor 488 using a monoclonal antibody labeling kit (Invitrogen, A-20181). Slides were mounted with Vectashield Hardset + DAPI mounting media (Vector, Burlingame, CA) and imaged using an Olympus BX51 or BD pathway fluorescent microscope. The tissue sections were imaged using Bio-Rad Radiance 2100 MP Rainbow confocal/multiphoton microscope with LaserSharp software.
Scanning electron microscopy
Scanning electron microscopy was performed as previously described .
RNA isolation and Real time-PCR
Sequences of real time RT-PCR Primers
Data presented is the mean and standard error of three replicate experiments and assessed for significant differences by unpaired t-test. Reported differences have a P-value of ≤ 0.05.
Results and Discussion
We have shown previously that overexpression of an activated form of Gli2 activates Hh target genes in LNCaP and PC-3 cells, while overexpression of an activated form of Smo does not . These data suggest that a defect in the pathway between Smo and the Gli transcription factors exists in both cell lines, Our finding here that these cells do not exhibit cilia when grown in vitro suggests a plausible mechanism for the defect in signal transduction but further investigation will be required to substantiate that paradigm. Future work assessing the role of autocrine Hh signaling in these widely-used cancer cell lines should consider the apparent essential role of cilia in Hh signal transduction and in vitro and in vivo growth conditions that may affect cilia formation.
These studies demonstrate the presence of cilia in both epithelial and mesenchymal cells of the developing prostate. As we found that in vitro culture conditions of UGSM-2 cells which promote ciliary formation enable functional Hh signal transduction, our observation that cilia are a feature of peri-ductal mesenchyme cells is consistent with the previously described paracrine signaling paradigm where Hh ligand secreted by prostate epithelium acts on adjacent mesenchymal cells and a previous report identifying cilia as a feature of human prostate stromal cells . To our knowledge, work investigating how serum and density conditions of cells grown in vitro correlate with the in vivo microenvironment of the developing prostate has not been presented and should be an area of future study given these findings. The absence of cilia on several prostate cancer cell lines and a correlative lack of Hh-responsiveness further argues against a role for cell-autonomous Hh signaling and plausibly explains the observation that pathway activation can be achieved by expressing activated Gli2 but not activated Smo. A role for autocrine signaling in cilia-expressing epithelial cells remains uncertain.
The authors would like to Kathleen Sulik, Deborah Dehart, Crist Cook and Kathy Schell and the UW-CCC Flow Cytometry facility for technical assistance. This work was supported from the National Institute of Health (DK065303-01 and DK056238) and the Robert and Delores Schnoes Chair in Urologic Research.
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