Stromal Gli signaling regulates the activity anddifferentiation of prostate stem and progenitor cellsReceived for publication, April 1, 2018, and in revised form, May 5, 2018 Published, Papers in Press, May 17, 2018, DOI 10.1074/jbc.RA118.003255

Qianjin Li‡1, Omar A. Alsaidan‡1, Sumit Rai§1, Meng Wu‡, Huifeng Shen‡, Zanna Beharry¶, Luciana L. Almada�,Martin E. Fernandez-Zapico�, X Lianchun Wang§, and Houjian Cai‡2

From the ‡Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia30602, the §Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens,Georgia 30602, the ¶Department of Chemistry and Physics, Florida Gulf Coast University, Fort Myers, Florida 33965, and the�Schulze Center for Novel Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905

Edited by Eric R. Fearon

Interactions between cells in the stroma and epithelium facil-itate prostate stem cell activity and tissue regeneration capacity.Numerous molecular signal transduction pathways, includingthe induction of sonic hedgehog (Shh) to activate the Gli tran-scription factors, are known to mediate the cross-talk of thesetwo cellular compartments. However, the details of how thesesignaling pathways regulate prostate stem and progenitor cellactivity remain elusive. Here we demonstrate that, althoughcell-autonomous epithelial Shh-Gli signaling is essential todetermine the expression levels of basal cell markers and therenewal potential of epithelial stem and progenitor cells, stro-mal Gli signaling regulates prostate stem and progenitor cellactivity by increasing the number and size of prostate spheroidsin vitro. Blockade of stromal Gli signaling also inhibited prostatetissue regeneration in vivo. The inhibition of stromal Gli signal-ing suppressed the differentiation of basal and progenitor cellsto luminal cells and limited prostate tubule secretory capability.Additionally, stromal cells were able to compensate for the defi-ciency of epithelial Shh signaling in prostate tissue regenera-tion. Mechanistically, suppression of Gli signaling increased thesignaling factor transforming growth factor � (TGF�) in stro-mal cells. Elevation of exogenous TGF�1 levels inhibited pros-tate spheroid formation, suggesting that a stromal Gli–TGF�signaling axis regulates the activity of epithelial progenitor cells.Our study illustrates that Gli signaling regulates epithelial stemcell activity and renewal potential in both epithelial and stromalcompartments.

Stromal– epithelial cell interactions are essential for prostatedevelopment and adult prostate tissue regeneration (1). Theprostate gland is composed of numerous connected tubules andthe surrounding stromal microenvironment. Each tubule con-

sists of three types of prostate epithelial cells (PrECs),3 includ-ing secretory luminal cells, basal cells, and neuroendocrine cells(2, 3). Luminal cells typically expressing cytokeratin (CK) 8 or18 locate at the apical region of the epithelium, are sensitive toandrogen stimulation, and produce secretory proteins. Basalcells expressing CK5 or p63 reside underneath the luminal cellsand attach to the basement lamina (4). Both luminal and basalcells in the epithelial compartment contain stem/progenitorcells, which are capable of self-sustentation in tissue regenera-tion (5).

PrECs are surrounded by the stromal microenvironment,providing an important niche to nurse epithelial progenitorcells (6). The stromal compartment comprises a variety of celltypes, including smooth muscle cells, subepithelial cells, wrap-ping cells, interstitial fibroblasts, and others (3, 7). Stromal cellssecrete important signaling factors to stimulate prostate devel-opment and adult prostate tissue regeneration (1, 8). Theseparacrine signaling factors include stromal androgen, fibroblastgrowth factor (FGF), TGF�, bone morphogenetic protein(BMP), Sonic hedgehog (Shh), and others; they induce prostaticsecretion in luminal cells and maintain the self-renewal capac-ity of prostate stem/progenitor cells (9 –12). Shh–Gli signalingexists in both prostate basal cells and stromal cells (7). How-ever, how this signaling pathway controls the renewal capacityof stem/progenitor cells through the stromal– epithelial inter-action remains elusive.

Shh–Gli signaling is an important signal transduction path-way in regulating the normal development of multiple organs,including growth of prostatic tissues and differentiation ofprostate epithelia (13, 14). It has been reported that Shh–Glisignaling facilitates prostate branching morphogenesis throughregulation of hepatocyte growth factor (15). In mammaliancells, the Gli family consists of three members (Gli1, Gli2, andGli3). Although both Gli2 and Gli3 have C-terminal transcrip-

This work was supported by National Institutes of Health GrantsR01CA172495 (to H. C.) and HL-09339 and GM103390 (to L. W.) andDepartment of Defense Grant W81XWH-15-1-0507 (to H. C.). The authorsdeclare that they have no conflicts of interest with the contents of thisarticle. The content is solely the responsibility of the authors and does notnecessarily represent the official views of the National Institutes of Health.

This article contains Figs. S1–S7 and Table S1.1 These authors contributed equally to this work.2 To whom correspondence should be addressed. Fax: 706-542-5358; E-mail:

caihj@uga.edu.

3 The abbreviations used are: PrEC, prostate epithelial cell; CK, cytokeratin;Shh, sonic hedgehog; AR, androgen receptor; shRNA, short hairpin RNA;PEB, prostate epithelial basal; UGSM, urogenital sinus mesenchyme; RFP,red fluorescent protein; H&E, hematoxylin and eosin; DMEM, Dulbecco’smodified Eagle’s medium; PE, phycoerythrin; FBS, fetal bovine serum;SCID, severe combined immunodeficiency; IHC, immunohistochemistry;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; E18.5, embryonicday 18.5; PrEGM, prostate epithelial cell growth medium; APC, allophyco-cyanin; TAMRA, tetramethylrhodamine; BTTP, bis(tert-butyl)-tris(triazolyl-methyl)amine-propanol.

croARTICLE

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tional activation and N-terminal transcriptional repressiondomains, Gli1 contains only a C-terminal transcriptional acti-vation domain. Shh signaling is primarily mediated throughGli2 and Gli3 (16). Gli3T is a mutant missing the C-terminaltranscriptional activation domain and has been used as a con-stitutive transcriptional repressor of Gli signaling (17). In theShh-dependent canonical pathway, the binding of Shh to a12-transmembrane receptor, Patched (Ptc/Ptch/Ptch1), inducestrans-localization of Gli2/3 to the nucleus and regulates expres-sion of downstream target genes such as Gli1, Bcl2, Ptch1, andothers (16, 18, 19).

We have shown previously that epithelial Gli signaling regu-lates the expression of p63 and plays an essential role in themaintenance of the homeostasis and renewal potential of pros-tate stem/progenitor cells (20). Dysregulation of Gli signalingby oncogenic events, such as the synergy of Kras and androgenreceptor (AR), promote the pathological expansion of basal/progenitor cells (20, 21). In this study, we demonstrate that theShh–Gli signaling axis mediates the interaction of stromal–epithelial cells in prostate tissue regeneration under physiolog-ical conditions. Particularly, Gli signaling in stromal cells regu-lates the activity of prostate epithelial stem/progenitor cells andpromotes prostate spheroid formation in vitro and tissue regen-eration in vivo. Mechanistically, stromal Gli signaling regulatesTGF�1/2 expression, and exogenous TGF�1 inhibits prostatespheroid formation. Our study emphasizes the importance ofstromal Gli signaling in the regulation of prostate stem cellactivity and tissue regeneration capacity.

Results

Cell autonomous Shh–Gli signaling regulates the renewalpotential of prostate progenitor cells

We have demonstrated previously that epithelial Gli signal-ing is essential in regulating the renewal potential of prostatestem/progenitor cells (20). To further examine the role of thecell-autonomous Shh–Gli signaling axis in controlling prostatestem cell activity, lentiviral vectors overexpressing pre-Shh(WT) or the pre-Shh(C25S) mutant were constructed (Fig.S1, A and B). Shh(WT) undergoes both cholesterol modifica-tion at the C terminus and palmitoylation at the N terminus(Fig. 1A). Mutation of the cysteine to serine blocked palmitoy-lation of Shh without affecting its maturation (Fig. 1, A and B,and Fig. S1C), indicating that both pre-Shh(WT) and pre-Shh(C25S) were processed to form mature Shh protein (19kDa).

The self-renewal potential of prostate stem/progenitor cellscan be examined by prostate sphere formation (Fig. 1C) (22).We examined whether the cell-autonomous Shh–Gli signalingaxis regulates prostate stem cell activity by sphere formationassay. Overexpression of Gli3T and knockdown of Gli1 andGli2 were confirmed by down-regulation of Ptc1 and Bcl2 orGli1/2 expression, respectively (Fig. S2). As reported previously(20), overexpression of Gli3T, a dominant-negative repressor ofGli signaling, in PrECs significantly inhibited primary prostatesphere formation (Fig. 1D). Although overexpression ofShh(C25S) inhibited the number of primary prostate spheres,overexpression of Shh(WT), shRNA-Gli1, or shRNA-Gli2

resulted in no change in sphere number (Fig. 1D). However, thenumber of secondary spheres was significantly inhibited byoverexpression of Gli3T, Shh(C25S), or shRNA-Gli2 but notshRNA-Gli1 (Fig. 1E). The data indicate that suppression ofShh–Gli2/3 signaling by loss of Shh palmitoylation, knockdownof Gli2, or overexpression of the repressor Gli3T inhibits therenewal activity of prostate stem cells.

P63, CK5, and CK14 are well-established markers of prostatebasal/progenitor cells, and CK8 and CK18 are known luminalmarkers. We have shown previously that overexpression ofGli3T and shRNA-Gli2 inhibited p63 expression (20). There-fore, we analyzed whether Shh(WT) or Shh(C25S) regulates thelevels of these proteins in PEB cells, a cell line isolated frommouse prostate basal cells (23). Although overexpression ofShh(WT) in PEB cells did not further increase the expressionlevels of basal or luminal markers, overexpression of Gli3Tdown-regulated expression levels of p63, CK5, CK14, and ARbut not CK8 or CK18. Similarly, Shh(C25S) decreased thelevels of p63, CK5, and CK14 but not CK8, CK18, or AR (Fig.1F). Collectively, the data suggest that the cell-autonomousShh–Gli signaling axis regulates the renewal potential ofprostate spheroids by potential regulation of basal or stem/progenitor cells.

The presence of mesenchymal stromal cells compensates forthe deficiency of epithelial Shh signaling in prostate tissueregeneration

The interactions between stromal and epithelial cells isessential in prostate tissue regeneration (1). Epithelial stem/progenitor cell activity was impaired by overexpression ofShh(C25S) or Gli3T. We examined whether WT mesenchymalcells could compensate for the deficiency of cell-autonomousShh signaling in epithelial cells. In contrast to the inhibition ofprimary prostate sphere formation by Shh(C25S) or Gli3T (Fig.1D), epithelial cells overexpressing Shh(WT) or Shh(C25S)showed no difference in the size of regenerated prostate tissuein the presence of WT urogenital sinus mesenchyme (UGSM)cells (Fig. 2A). RFP-expressing tubules indicated that thetubules were infected with a control vector, Shh(WT), orShh(C25S). Histologically, regenerated prostate tubulesshowed no difference in expression levels of CK5/CK8 or p63(Fig. 2B). Similarly, regenerated prostate tissues derived fromoverexpression of Gli3T in epithelial cells showed a similartubule structure as the control vector (Fig. S3). The data suggestthat impaired epithelial Shh signaling could be rescued by thepresence of normal mesenchymal cells.

Mesenchymal stromal cells enhance prostate spheroidformation and elevate epithelial Gli and p63 expression

To evaluate the contribution of stromal cells to the regula-tion of prostate epithelial stem/progenitor cell activity, an invitro stromal cell sphere co-culture assay was developed inwhich the formation of prostate spheres is under the stimula-tion of mesenchymal stromal cells (Fig. 3A). The number andsize of prostate spheroids were significantly increased whenPrECs were co-cultured with UGSM cells (Fig. 3, B and C).Additionally, stromal cells significantly increased the number

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Figure 1. Cell-autonomous Shh signaling inhibits the renewal potential of prostate epithelial stem/progenitor cells. A, schematic of the maturation ofShh(WT) and the Shh(C25S) mutant, a mutation of cysteine to serine. Pre-Shh(WT) and pre-Shh(C25S) were cloned into a lentiviral vector. Pre-Shh(C25S) isprocessed and cholesterolized at the C terminus as the pre-Shh(WT) but not palmitoylated at the N terminus. B, loss of palmitoylation in the Shh(C25S) mutant.PEB cells, a mouse prostate basal cell line, were transduced with pre-Shh(WT) and pre-Shh(C25S) by lentiviral infection. The cells were grown with 17-octade-cynoic acid (17-ODYA, 60 �M). The expression of pre-Shh protein (�50 kDa) or mature protein (19 kDa) was detected by immunoblotting (right panel), lysateswere subjected to click chemistry, and palmitoylated-proteins were detected by fluorescence (left panel). C, schematic of primary and secondary spheroidformation. Prostate tissues were isolated from BL6 mice and dissociated into single cells. PrECs were transduced with pre-Shh(WT), pre-Shh(C25S), Gli3T, orshRNA-Gli1/2. Only PrECs with stem cell activity (indicated in orange) could form primary spheroids after embedding within Matrigel. Primary spheres weredigested into spheroid cells and embedded in Matrigel to form secondary spheres. D, primary PrECs transduced with control vector, pre-Shh(WT), pre-Shh(C25S), Gli3T, shRNA-Gli1, or shRNA-Gli2 were mixed with Matrigel and plated in separate wells in a 12-well plate. Sphere number was counted after 10 daysof incubation. E, the primary spheres were dissociated and replated in the same way as described in D to generate secondary spheres. F, PEB cells weretransduced with control vector, pre-Shh(WT), pre-Shh(C25S), or Gli3T (positive control). The protein lysates were analyzed for p63, CK5, CK14, AR, CK8, CK18,Gli3T, Shh (mature form), and tubulin expression by immunoblotting. *, p � 0.05; ***, p � 0.001; N.S., not significant.

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of prostate spheroid aggregates. The aggregates contained twoto six spheroids, with some aggregates having more than sixspheroids (Fig. 3B).

Further analysis showed that the expression levels of progen-itor and basal markers (p63, CK5, or CK14), AR, and luminalmarkers (CK8 or CK18) were increased in the PrECs�UGSMgroup compared with the PrECs alone group (Fig. 3D).The expression levels of Gli1, Gli2, and Gli3 in prostatespheres grown with or without the stimulation of UGSM

cells were also compared. Although Gli1 was barely detectedin spheres (data not shown), the levels of Gli2 and Gli3 weresignificantly elevated in the PrECs�UGSM group comparedwith the PrECs alone group (Fig. 3D). Additionally, the num-bers of single spheres and spheroid aggregates derived fromthe PrECs�UGSM group were significantly higher thanthose from PrECs alone in the secondary spheroid passage,suggesting that UGSM cells enhance sphere renewal poten-tial (Fig. 3E). Collectively, the data suggest that mesenchy-

Figure 2. Mesenchymal stromal cells compensate for deficiency of epithelial Shh signaling in prostate tissue regeneration. A, primary PrECs weretransduced with control vector, Shh(WT), or Shh(C25S), mixed with UGSM cells, and implanted under the renal capsule. After regenerated prostate tissues wereharvested, phase and RFP fluorescence images were taken (scale bars � 2 mm). Grafts derived from Shh(WT) or Shh(C25S) had less RFP, likely because of a lowertransfection efficiency compared with the control vector. B, H&E, RFP fluorescence (a–f, scale bars � 100 �m), and IHC staining of CK5 (red)/CK8 (green)/DAPI(blue), p63, and AR of the generated tissue (g– o, scale bars � 50 �m). Arrows indicate p63� cells. Of note, the RFP signal is usually bleached after antigen retrievalso that the staining for CK5 (red) will not interfere with the RFP in tissues (see also Fig. S7).

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mal stromal cells enhance the activity of prostate stem/progenitor cells, which is associated with epithelial Glisignaling.

Stromal Gli signaling promotes prostate stem/progenitoractivity

We studied the contribution of stromal Gli signaling in sup-porting epithelial stem/progenitor cell activity. To exclude theendogenous stromal cells from the PrEC preparation, prostatebasal cells were isolated based on Lin�CD49f�Sca1� markers(4) (Fig. 4, A–C). Additionally, UGSM cells were transducedwith Gli3T, shRNA-Gli1, or shRNA-Gli2 by lentiviral infection

(Fig. 4A). The number of spheres increased more than 2-fold inthe basal cells�UGSM or UGSM vector groups compared withbasal cells alone or basal cells�3T3 cells (Fig. 4D). The size ofspheres increased in the presence of UGSM, UGSM�Gli3T/shRNA-Gli2/shRNA-Gli1, or 3T3 cells compared with basalcells alone (Fig. 4D). The UGSM-induced sphere number wassignificantly inhibited by overexpression of Gli3T or shRNA-Gli2 but not by shRNA-Gli1 (Fig. 4D). Additionally, the numberof spheres and spheroid aggregates significantly decreased inthe PrECs�UGSM-Gli3T group compared with the PrECs�UGSM control group (Fig. 4E), suggesting that overexpressionof Gli3T inhibited spheroid renewal potential. The data suggest

Figure 3. Mesenchymal stromal cells enhance prostate spheroid formation and increase the expression levels of Gli transcription activators and basaland luminal markers of prostate spheres. A, schematic of the UGSM–sphere co-culture assay. Primary PrECs were isolated from prostate tissue. UGSM cellswere isolated from E16.5 mouse embryos. PrECs were mixed with 5000 UGSM cells and Matrigel and plated in the rim of a 12-well plate, and another 5000 UGSMcells were inoculated in the center of a well. Of note, only PrECs with stem cell activity (indicated in orange) are able to form spheres. UGSM cells remained ascells and were not able to form spheres in the co-culture assay. B and C, the numbers of single spheres and spheroid aggregates (two, three, four, five, six, andmore than six spheroid aggregates, B) and size of single spheres (C) were recorded on day 10. Representative images are shown. The presence of UGSM cellsincreased the size and number of prostate spheres and spheroid aggregates. Scale bar is 100 �m. D, prostate spheres grown with or without UGSM cells werecollected. After UGSM cells were removed from the mixture of spheres and UGSM cells by filtering through 40-�m mesh, spheres from both groups werecollected and compared. Proteins were extracted from the isolated prostate spheroids (UGSM cells were excluded) for analysis of Gli2/3 and GAPDH expression.Cell lysate derived from embryonic stem cells was used as a positive control for the expression of Gli2 and Gli3. Expression levels of AR, p63, CK8, CK18, CK5,CK14, and GAPDH in two sets of experiments are shown. E, prostate spheres from the PrECs and PrECs�UGSM groups were dissociated into single cells. 6000dissociated cells were mixed with Matrigel and evaluated for secondary sphere formation. The number of single spheres (labeled 1 on the x axis) and spheroidaggregates (with two, three, or four spheres) were recorded. *, p � 0.05; ***, p � 0.001; ND, not detected.

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Figure 4. Mesenchymal stromal Gli signaling promotes the activity of prostate stem/progenitor cells. A, schematic of the co-culture of prostate basalcells with UGSM cells. Prostate tissues were dissociated into PrECs. PrECs were co-stained with Lin-FITC, CD49f-PE, and Sca1-APC antibodies. Lin�CD49f�Sca1�

basal cells were further isolated by flow cytometry as shown in B and C. Additionally, UGSM cells were transduced with control vector (FUCRW), Gli3T,shRNA-Gli1, or shRNA-Gli2 by lentiviral infection. Basal cells and transduced UGSM cells were mixed in the co-culture assay. B and C, isolation of primary prostatebasal cells. Primary PrECs were first gated for the negative staining of Lin-FITC (B), and then the Lin� cell population was further gated for positive staining ofSca1-APC and CD49f-PE (C). The Lin�CD49f�Sca1� cells represent the basal cells. SSC, side scatter. D, Lin�CD49f�Sca1� basal cells were mixed with UGSM-vector/Gli3T/shRNA-Gli1/shRNA-Gli2 or 3T3 (control) for sphere formation. The sphere number (left panel) and size (right panel) were recorded on day 10, andsphere images were taken (bottom panel). Scale bar is 100 �m. E, the prostate spheroids derived from the UGSM-control or UGSM-Gli3T groups were dissociatedinto single cells. Cells were mixed with Matrigel and subjected to the secondary sphere assay. The x axis shows single spheres (labeled 1) or spheroid aggregates(spheroid aggregates with two, three, or four spheres). The y axis represents the ratio of sphere formation. 1000:1 means 1000 epithelial cells forming oneprostate sphere. F, schematic of the co-culture of Lin�CD49f�Sca1� basal cells with adult stromal cells. Adult stromal cells were isolated based on theLin�CD49f�Sca1� population as shown in C. The stromal cells were grown in the same medium as used for growing UGSM cells. The adult stromal cells weretransduced with control vector (FUCRW) or Gli3T by lentiviral infection. Lin�CD49f�Sca1� basal cells were mixed with adult stromal cells in the co-cultureassay. G, sphere number and size were recorded on day 10, and sphere images were taken. **, p � 0.01; ***, p � 0.001; N.S., not significant; ND, not detected.

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that the expression of stromal Gli2/3 promotes epithelial stemcell activity.

Next, we further examined whether sphere formation couldalso be regulated by adult prostate stromal cells. Primary adultstromal cells were isolated based on Lin�CD49f�Sca1� mark-ers (Fig. 4, B and C). The isolated adult stromal cells grew sim-ilarly as UGSM cells. Stromal cells were transduced with con-trol or Gli3T by lentiviral infection (Fig. 4F). Similar to theUGSM sphere assay (Fig. 4D), adult stromal cells promoted thenumber and size of prostate spheres. The number, but notthe size, of prostate spheres was inhibited by the suppression ofGli signaling through overexpression of Gli3T (Fig. 4G). Collec-tively, the data indicate that adult stromal Gli signaling regu-lates epithelial stem cells as well.

Blockade of stromal Gli signaling inhibits prostate tissueregeneration in vivo

We examined the contribution of stromal Gli signaling inprostate tissue regeneration in vivo. To exclude the potential ofendogenous stromal contribution in tissue regeneration, basalcells were isolated based on Lin�CD49f�Sca1� markers (Fig. 5,A–C). The isolated cells were mixed with or without UGSM-expressing control vector or Gli3T (Fig. 5A) and subjected to aprostate tissue regeneration assay. The number of regeneratedtubules was significantly elevated in regenerated tissue derivedfrom the basal cells�UGSM group compared with the basalcells alone group (Fig. 5, D and E, and Fig. S4). However, it wassignificantly inhibited in the basal cells�UGSM-Gli3T groupcompared with the basal cells�UGSM group (Fig. 5, D and E).Additionally, the secretion (stained as a pinkish color by H&Estaining) was largely not visible in the lumen of tubules in thebasal cells�UGSM-Gli3T group compared with those in thebasal cells�UGSM group (Fig. 5D, b and c).

We characterized cell types in the regenerated tubules fromthe different groups. The number of p63� and/or CK5� cells inthe tubules derived from the basal cells alone or basal cells�UGSM-Gli3T group were significantly elevated compared withthe basal cells�UGSM group (Fig. 5, D, F, and H, and Fig. S4). Incontrast, the number of CK8� cells in the tubules derived fromthe basal cells alone or basal cells�UGSM-Gli3T groups weresignificantly lower (Fig. 5, D and G, and Fig. S4). Collectively,the data indicate that blockade of Gli signaling in UGSM cellsinhibited the tissue regeneration potential and the differentia-tion of prostate basal/progenitor cells into luminal cells.

Similarly, by using PrECs (unsorted cells, endogenous stro-mal cells not excluded) for prostate tissue regeneration, thenumber of tubules and branching morphogenesis of the regen-erated tubules were also inhibited in the PrECs�UGSM-Gli3Tgroup compared with the PrECs�UGSM control group(Fig. S5).

Mesenchymal stromal Gli signaling regulates TGF� expressionlevels to affect stem cell activity

We investigated how blockade of stromal Shh–Gli signalingregulates other signaling pathways that are related to stem cellactivity in stromal cells. Overexpression of Gli3T or knock-down of Gli3 was confirmed by decreased mRNA levels of Glidownstream genes such as Gli1 and Bcl2 (Fig. 6, A and C).

Although overexpression of Gli3T resulted in a significantincrease in TGF�2 levels in UGSM cells (Fig. 6B), shRNA-Gli3increased the expression levels of TGF�1 and TGF�R1/2/3(Fig. 6D). The difference in regulation of TGF�1 or TGF�2expression levels might be due to a broader effect of Gli3T thatcould block both Gli1/2 or Gli3 signaling, considering they havedifferent downstream targets (24, 25). Interference of Gli sig-naling in UGSM cells also affected AR, Notch, and BMP expres-sion (Fig. S6).

We examined how exogenous TGF�1 affects the activity ofprostate progenitor cells using the sphere formation assay. Anincrease in TGF�1 in the medium inhibited the number (Fig.6E) and size (Fig. 6F) of prostate spheres and spheroid aggre-gates (Fig. 6G) stimulated by UGSM cells. A high concentrationof TGF�1 (10 ng/ml) completely inhibited UGSM-stimulatedprostate spheroid formation (Fig. 6E). The results were consis-tent with the inhibitory effect of sphere formation in vitro (Fig.4, D and G) and tubule formation in vivo (Fig. 5D). The dataindicate that stromal Shh–Gli signaling modulates the TGF�signaling pathway in stromal cells, thereby regulating epithelialstem cell activity.

Discussion

Our study demonstrates that cell-autonomous Shh–Gli sig-naling in the epithelial compartment dictates the renewalpotential of prostate stem/progenitor cells. Prostate basal cellsare a major pool of stem/progenitor cells in prostate tissue (4).The cells maintain high expression levels of Shh and Gli3 (7).Blockade of Shh signaling by overexpression of the Shh(C25S)mutant inhibits expression of p63 or CK5, a marker of progen-itor/basal cells in prostate tissue (26, 27), and prostate spheroidformation. This result is consistent with our previous experi-mental evidence showing that overexpression of Gli3T, a dom-inant-negative suppressor of Gli signaling, inhibits the renewalcapacity of prostate progenitor cells (20). Our studies illustratethat the Shh–Gli2/3–p63 signal transduction pathway is essen-tial for maintaining homeostasis of prostate stem cells in theepithelial compartment (Fig. 7).

Our study also illustrates that stromal Gli signaling regulatesepithelial stem cell activity (Fig. 7). Stromal– epithelial cellinteractions are important for prostate development and adultprostate regeneration (1). We show that mesenchymal stromalcells elevate expression of Gli2/3 and p63/CK5 in prostatespheroids from the co-culture assay. The epithelial CK8/CK18levels are also elevated through stimulation of stromal cells,suggesting that stromal cells promote the differentiation pro-cess as well. Blockade of stromal Gli signaling inhibits the num-ber of spheres in vitro and regenerated prostate tubules in vivo.Our data support Shh–Gli signaling as an important pathwaythat facilitates cross-talk of stromal cells with epithelial basal/progenitor cells in prostate development and tumor initiation(7). In particular, stromal Gli2/3–TGF�1/2 signaling promotesthe activity of prostate stem/progenitor cells. In contrast to pre-vious reports (28), the experimental design of this study allowedus to clearly dissect the contribution of Shh signaling in eitherthe epithelial or stromal compartment toward the regulation ofprostate tissue regeneration. Similar to other epithelial stem

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cells, the microenvironment is essential for promoting therenewal activity of prostate progenitor cells (6, 29).

Prostate epithelium and reactive stromal cells co-evolve toregulate prostate development (3). TGF� and hedgehog signal-

ing are two well-characterized pathways in embryonic develop-ment (30). Numerous studies have reported that TGF� regu-lates hedgehog signaling and Gli expression. Reciprocally, Glisignaling positively regulates TGF� levels (24, 31). Our data

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Figure 5. Blockade of Gli signaling in mesenchymal stromal cells inhibits prostate regeneration potential. A, schematic of the in vivo prostate regener-ation assay. Lin�CD49f�Sca1� basal cells isolated by flow cytometry shown in B and C were mixed with or without UGSM-vector or UGSM-Gli3T. The cellmixture was implanted under the renal capsule of SCID mice. B and C, isolation of primary prostate basal cells. Primary PrECs were first gated for the negativestaining of Lin-FITC (B), and then the Lin� cell population was further gated for positive staining of Sca1-APC and CD49f-PE (C). Lin�CD49f�Sca1� basal cellswere used for prostate tissue regeneration experiments. SSC, side scatter. D, the regenerated tissues were subjected to histological analysis. H&E (a– c, scalebars � 100 �m) and IHC staining (d–i, scale bars � 50 �m) of CK5 (red)/CK8 (green)/DAPI (blue) and p63 were analyzed in the regenerated tissues. Stars indicatesecretion in the lumen of regenerated prostate tubules by H&E staining, and arrows indicate p63� cells. E, the number of tubules in the regenerated grafts wascounted. The tubule number in the basal cells�UGSM-vector was set as 100%. F–H, the number of CK5� (F), CK8� (G), or p63� (H) cells per tubule was counted,and the percentage was calculated. *, p � 0.05; ***, p � 0.001.

Figure 6. Mesenchymal–stromal Gli signaling regulates the expression level of TGF�. A–D, UGSM cells were transduced with vector control, Gli3T (A andB), or shRNA-Gli3 (C and D) by lentiviral infection. Total RNA in UGSM-control, UGSM-Gli3T, or shRNA-Gli3 cells was extracted. Overexpression of Gli3T (A) ordown-regulation of Gli3 (C) was confirmed by decreased expression levels of Gli1, Gli3, Ptc1, and/or Bcl2 by RT-PCR. Expression levels of TGF�1/2/3 andTGF�R1/2/3 were measured by RT-PCR (B and D). E and F, prostate tissues were dissociated into PrECs. PrECs alone or PrECs mixed with UGSM cells in theco-culture sphere assay were grown in prostate sphere growth medium with control or 1, 5, or 10 ng/ml of TGF�1. The number (E) and size (F) of spheres werecounted on day 10. G, the number of single spheres (labeled 1 on the x axis) and spheroid aggregates (aggregates with two, three, four, five, or six spheres) inPrECs cultured with UGSM with/without 1 ng/ml of TGF�1 were recorded. *, p � 0.05; **, p � 0.01; ***, p � 0.001; ND, not detected.

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reveal transcriptional regulation of TGF� by Gli signaling instromal cells. Blockade of Gli signaling by overexpression ofGli3T inhibits stroma-stimulated basal cell differentiation inprostate tissue regeneration. Particularly stromal Gli signalingregulates numerous signaling routes, including TGF� expres-sion levels, which inhibits the differentiation of basal/progeni-tor cells (p63� or CK5�) to CK8� luminal cells. As a result, itfurther controls the fate of epithelial stem cells.

Blockade of Gli signaling by Gli3T (Fig. 6, A and B) orshRNA-Gli3 (Fig. 6, C and D) results in an increase in TGF�2and TGF�1 levels in UGSM cells, respectively. The differentialeffect of Gli3T and shRNA-Gli3 on the regulation of TGF�isoforms might be due to the different genetic approaches forsuppression of Gli signaling. Gli3T is a constitutive repressorform of Gli3 that antagonizes, in a dominant fashion, the tran-scription of Gli factors, including Gli1 and Gli2. Overexpres-sion of Gli3T leading to elevation of TGF�2 expression has alsobeen reported for pancreatic cancer cells (32) or with activationof Smoothened (24). On the other hand, microarray analysissuggests that Gli3 alone regulates TGF�1 expression levels inthymocytes at the embryonic day 18.5 (E18.5) (33). Gli3 isbifunctional, meaning that it acts as a transcriptional activatoras Gli3A or as a repressor as Gli3R. Gli3 is mostly expressed inthe fetal stage (34) and is predominantly the Gli3R isoform instromal cells (35). In contrast to Gli3T, shRNA-Gli3 potentially

suppresses Gli3R levels, leading to up-regulation of TGF�1 inUGSM cells (Fig. 6, C and D).

Targeting palmitoylation of Shh is a therapeutic approach forinhibition of Shh–Gli signaling. Our studies demonstrate thatloss of Shh palmitoylation inhibits the renewal potential ofprostate epithelial stem/progenitor cells. Although palmitoyla-tion is not required for the autocleavage of pre-Shh protein tomature Shh (36), it is essential for the formation of the multi-meric form of Shh to perform its physiological activity, such asthe regulation of embryonic development (37, 38). Shh–Gli sig-naling is associated with numerous types of cancer, includingprostate cancer (39). Up-regulation of Shh in prostate cancercells modulates the tumor microenvironment and facilitatesosteoblastic cells in metastatic prostate cancer (40). The major-ity of inhibitors targeting Shh–Gli signaling are based on block-ade of Shh downstream signaling, including Smo, Ptch, or Glitranscriptional activators (41, 42). Therefore, targeting palmi-toylation of Shh provides a direct therapeutic approach toinhibit the ligand activity. Palmitoylation of Shh is catalyzed byHhat, which transfers palmitoyl-CoA to the N-terminal cys-teine of Shh (36). It has been shown that mutation of Hhat,which disrupts the palmitoylation of Shh, interferes with testic-ular organogenesis (43). Recently identified inhibitors, such asRU-SKI 39/41/43/50, show promising inhibitory effects on Shhsignaling (44, 45). Our results provide biological evidence that,

Figure 7. Shh–Gli signaling mediates cross-talk of the stromal– epithelial interaction to regulate the activity of prostate stem/progenitor cells. Incell-autonomous Shh–Gli signaling, prostate basal cells secrete epithelial Shh and induce the dissociation of Ptch1 from Smo, which then activates thetranscriptional activator Gli2/3 to regulate the expression of p63, Gli1, Bcl2, and Ptc1. Overexpression of Gli3T blocks Gli signaling and inhibits p63 expressionand the renewal potential of prostate stem/progenitor cells (20). Additionally, epithelial stem cell activity is influenced by stromal Gli signaling. Blockingstromal Gli signaling (by overexpression of Gli3T or shRNA-Gli3) increases the expression levels of TGF�1 or TGF�2 and other signaling. Elevation of TGF�inhibits proliferation and/or differentiation of progenitor cells, subsequently inhibiting prostate tissue regeneration or spheroid formation. As a result, stromalGli signaling, through regulation of TGF� levels, contributes to the differentiation and activity of epithelial stem cells.

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although inhibition of Shh palmitoylation suppresses cell-au-tonomous Shh–Gli signaling, the normal stromal microenvi-ronment is sufficient to overcome the deficit of epithelial Shh–Gli signaling. Therefore, the efficacy of the inhibitors intargeting stromal Gli signaling should also be investigated.

Experimental procedures

Plasmid and lentiviral production

The ORF of mouse Shh was PCR-amplified from the parentalvector, pBS-Shh(WT) (Addgene, plasmid 13999) (primers arelisted in Table S1) and inserted into the XbaI site of the FUCRWvector. The C25S mutation was introduced by sequential PCRsite-directed mutagenesis technique. In the first round of thePCR reaction, pre-mShh-5�(XbaI)/pre-mShh-C25S-R and pre-mShh-C25S-F/pre-mShh-3�(XbaI) fragments were amplifiedindividually with pBS-mShh as the template. In the secondPCR, full-length Shh-C25S was amplified by the two flankingprimers, pre-mShh-5�(XbaI) and pre-mShh-3�(XbaI), with thetwo fragments generated in the first PCR as the template. PCRwas performed in a 20-�l reaction mixture containing 100 ng ofDNA template and 20 ng of each primer. The thermocycler stepswere 2 min at 95 °C, 30 cycles of 30 s (s) at 95 °C, 30 s at 58 °C, and1 min extension at 72 °C, followed by a final extension at 72 °C for5 min. The PCR products were gel-purified, digested by XbaI, andinserted into the XbaI site of the FUCRW vector. Because all len-tiviral vectors were derived from the FUCRW parental vector, theycarry an RFP marker under the cytomegalovirus promoter. Lenti-virus production and infection were performed as described pre-viously (21). All procedures followed the safety guidelines and reg-ulations of the University of Georgia.

Isolation of primary prostate epithelial cells and prostatebasal cells

Whole prostate tissues were isolated from five C57BL/6J mice (2months old) and minced. The minced tissues were furtherdigested by collagenase in DMEM for at least 1 h at 37 °C. Thecollagenase-digested cell suspension was further digested with0.05% trypsin for 5 min to dissociate cell clusters into single cells(22). After washing with DMEM to remove trypsin, the dissociatedprimary PrECs were resuspended in 1 ml of PrEGM medium.

For the isolation of prostate basal cells, the above PrECs weresorted based on Lin�Sca1�CD49f� markers as described pre-viously (4). In brief, 10 �l of cell suspension (around 5 � 104

cells) was aliquoted into four tubes, each containing 0.5 ml ofPrEGM medium. One microliter of CD49f-PE (0.2 mg/ml), 1 �lof Sca-1–APC (0.2 mg/ml), 1 �l of Lin-FITC containing a mix-ture of CD45 (0.17 mg/ml), CD31 (0.17 mg/ml), and Ter119(0.17 mg/ml) or none were added to each tube, respectively.The samples were used for a gating control in FACS. Additionally,a mixture of 5 �l of CD49f-PE (0.2 mg/ml), 4 �l Sca-1–APC (0.2mg/ml), and 5 �l of Lin-FITC containing a mixture of CD45 (0.17mg/ml), CD31 (0.17 mg/ml), and Ter119 (0.17 mg/ml) was addedto the parental tubes. All tubes were incubated on ice for 30 min.Stained cells were subjected to cell sorting by the MoFlo XDP(Beckman Coulter). Basal cells (Lin�CD49f�Scal�) were collectedand counted for the sphere formation assay.

Primary sphere formation from PrECs and secondary sphereformation from dissociated primary spheroid cells (withoutstromal cells)

Isolated primary PrECs from BL6 mice as described abovewere counted and seeded at 5 � 105 cells/well in a 12-wellplate. A lentivirus carrying the control vector or overexpres-sion of Shh(WT), Shh(C25S), Gli3T, shRNA-Gli1, orshRNA-Gli2 was added to dissociated primary prostate cellswith a multiplicity of infection of 10 –20, respectively. After2 h of spin infection (at 1500 rpm), the lentivirus wasremoved from each well. The transduced cells were resus-pended from the well, washed twice, and finally resuspendedin 50 �l of PrEGM medium (Lonza, catalog no. CC-3166).Fifty microliters of cell suspension was mixed with 50 �l ofMatrigel and plated around the rims of the wells in a 12-wellplate. The rims of the wells allowed the Matrigel to create a3D space for PrECs to grow as prostate spheres. After thecell–Matrigel mixture solidified at 37 °C for 20 min, 1 mlof PrEGM was added. The sphere number and size wererecorded after 10 days of incubation.

For the experiments of sphere formation in the presenceof TGF�1 ligand, isolated primary PrECs from BL6 mice asdescribed above were counted. 7.5 � 103 cells were resus-pended in 50 �l of PrEGM medium, mixed with 50 �l of Matri-gel, and plated around the rims of the wells in a 12-well plate.When solidified at 37 °C for 20 min, 1 ml of PrEGM was added.After overnight incubation, the medium was replaced withfresh PrEGM medium containing 0, 1, 5, or 10 ng/ml of TGF�1(a gift from Dr. Peter Sun’s laboratory). The number and size ofspheres were recorded at day 10.

For the secondary spheres, dispase was added to digest theMatrigel matrix. The primary spheres were collected andtreated sequentially with collagenase and trypsin as describedabove. Digested cells were passed through a 22-gauge syringe 3times and filtered with a 40-�m cell strainer. Cells were thenresuspended in 400 �l of PrEGM medium and sorted for RFP-positive cells by flow cytometry. Fifty microliters of 8 � 103

RFP� cells in PrEGM were mixed with 50 �l of Matrigel andreseeded in a well of a 12-well plate. The number of spheres wascounted after 10 days of incubation. Growth medium andMatrigel conditions were the same as for the primary sphereassay (22).

Sphere–stromal cell co-culture assay

In this assay, spheres were formed under the induction ofUGSM cells or adult stromal cells. Of note, UGSM or stromalcells alone did not form spheres in the Matrigel in the co-cul-ture assay.

For sphere–UGSM cell co-culture, UGSM cells were isolatedfrom E16.5 of BL6 mouse embryos (46). 5 � 103 unsorted pri-mary PrECs or Lin�CD49f�Scal� basal cells as isolated abovewere aliquoted into a tube and mixed with PrEGM medium orPrEGM medium containing 5 � 103 UGSM, UGSM-vector,UGSM-Gli3T, UGSM-shRNA-Gli1, UGSM-shRNA-Gli2, orNIH3T3 cells (as a control) to a final volume of 50 �l. The basalcells�UGSM cell mixture were then mixed with 50 �l of Matri-gel and plated around the rims of the wells in a 12-well plate.

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After the cell–Matrigel mixture solidified at 37 °C for 20 min, 1ml of PrEGM medium or PrEGM medium containing 5 � 103

of UGSM, UGSM-vector, UGSM-Gli3T, UGSM-shRNA-Gli1,UGSM-shRNA-Gli2, or NIH3T3 cells was added to the corre-sponding wells, respectively.

For sphere–adult stromal cells co-culture, UGSM cellsisolated as described above were replaced with adult stromalcells to perform similar experiments. Adult stromal cellswere isolated based on Lin�CD49f�Scal�. The isolated cellswere grown in the same medium as UGSM cells (DMEM, 5%Nu-serum, 5% FBS, 0.05% insulin, 0.01 �M dihydrotestoster-one, and penicillin–streptomycin) and could be passaged incell culture for three to five passages. Adult stromal cellswere transduced with control vector, Gli3T, shRNA-Gli1, orshRNA-Gli2 by lentiviral infection depending on the exper-imental settings. The number of spheres was counted 10 dayslater.

Cell culture and antibodies for Western blot analysis

293T and 3T3 cells were grown in DMEM with 10% FBS.Normal mouse prostate epithelial basal (PEB) cells were iso-lated and immortalized as a cell line (a gift from Dr. Wilson’slaboratory) (23). The cells were grown in PrEGM medium withsupplemental growth factors (Lonza) and 5% FBS. PEB cellstransduced with control vector, Shh(WT), Shh(C25S), or Gli3T(control) by lentiviral infection were grown in PrEGM with 5%FBS. To exclude uninfected cells, RFP-positive cells were iso-lated by cell sorting (BD Biosciences). Three days after cellswere transduced, the culture medium was removed, and cellswere washed with PBS. Cells were lysed by radioimmune pre-cipitation assay buffer containing protease inhibitors. Celllysate was subjected to Western blot analysis for expressionlevels of Gli3/Gli3T (Abcam, Ab69838, 1:1000), ERK2 (SantaCruz Biotechnology, sc-154, 1:5000), p63 (Santa Cruz Biotech-nology, 8431, 1:250), CK5 (Biolegend, 905501, 1:500), Shh (CellSignaling Technology, 2207, 1:1000), AR (Santa Cruz Biotech-nology, sc-816, 1:1000), Gli1 (Cell Signaling Technology, 3538,1:1000), Gli2 (Abcam, ab26056, 1:1000), GAPDH (Cell Signal-ing Technology, 2118, 1:5000), and �-tubulin (Sigma, T6557,1:5000).

Real-time PCR

UGSM cells were infected with a lentivirus depending on theexperimental setting and cultured for 5 days. Cells were washedwith PBS for RNA or protein extraction. Total RNA was iso-lated using the RNeasy Kit (Qiagen) following the protocol ofthe manufacturer. Complementary DNA was reverse-tran-scribed from 1.5 �g of total RNA in a 20-�l reaction with ahigh-capacity cDNA reverse transcription kit (Life Technolo-gies). The reverse transcription products were diluted 30 timeswith distilled H2O, and 2 �l was used as template for each real-time PCR reaction. The reactions were performed usingPerfeCTa SYBR Green FastMix (Quanta Biosciences). Thethermal cycling conditions were composed of an initial dena-turation step at 95 °C for 1 min, 40 cycles at 95 °C for 10 s, and60 °C for 50 s. The experiments were carried out in triplicate.Relative quantification of -fold changes of gene expression wasobtained by the 2�Ct method, with GAPDH as the internal

reference gene. The sequences of primers used for RT-PCR arelisted in Table S1.

Click chemistry for the detection of Shh palmitoylation

PEB or 293T cells infected with a virus carrying a controlvector, Shh(WT), or Shh(C25S) were grown in 6-cm dishes to80 –90% confluence. To metabolically label palmitoylated pro-teins, cells were further cultured in medium containing 2% BSA(fatty acid–free) and 50 �M palmitic acid azide (15-azidopenta-decanoic acid) (Thermo Fisher, C10265) for 293T cells or 50 �M

17-octadecynoic acid (Cayman, 90270) for PEB cells for 24 h.Cells were washed twice with PBS, and proteins wereextracted with lysis buffer (100 mM sodium phosphate (pH7.5), 150 mM NaCl, and 1% Nonidet P-40) containing prote-ase and protein thioesterase inhibitors (0.2 mM hexadecyl-sulfonyl fluoride and 10 �M palmostatin B). To perform clickchemistry reactions, 50 �g of cell lysate was incubated withreaction buffer (0.2 mM TAMRA-azide, 5 mM sodiumL-ascorbate, 1 mM BTTP, and 2 mM CuSO4) at a 1:1 ratio(v/v) for 1 h at room temperature in the dark. 4� SDS gelloading buffer containing 150 mM �-mercaptoethanol wasadded, and samples were heated for 10 min at 70 °C andseparated by SDS-PAGE. After gel electrophoresis, the gelwas soaked in a fixation solution (40% methanol, 10% aceticacid, and 50% water) for 1 h, followed by rinsing with deion-ized water (three times, 5 min each time) at room tempera-ture. The fluorescence signal was detected using a TyphoonTRIO� variable mode imager (GE Healthcare). The imagewas analyzed with ImageQuant (GE Healthcare). The ex-pression of Shh was also confirmed by Western blotting, and�-tubulin was used as the loading control.

Prostate regeneration assay and immunohistochemistry

For the prostate regeneration assay, primary prostate cellswere isolated from 8- to 12 week-old BL6 male mice. The iso-lated primary prostate cells were transduced with control vec-tor, Shh(WT), or Shh(C25S) by lentiviral infection. Basal cellswere also isolated based on Lin�CD49f�Sca1� from primaryprostate cells as described above. The transduced cells (2 � 105

cells/graft), including control vector or Gli3T, isolated basalcells, or unsorted cells, were combined with UGSM cells (2–3 �105 cells/graft), followed by 25 �l of collagen type I (adjusted topH 7.0). For Shh-induced transformation, UGSM cells weretransduced with control, Shh(WT), or Shh(C25S) by lentiviralinfection. The transduced UGSM cells were combined withPrECs. After overnight incubation, grafts were implantedunder the kidney capsule in CB.17SCID/SCID (SCID) mice bysurvival surgery. After 8 weeks, regenerated prostate tissueswere obtained for histological and immunohistochemistryanalysis.

All animals were maintained and used according to the sur-gical and experimental procedures of protocol A2013-03-008,approved by the Institutional Animal Care and Use Committeeof the University of Georgia.

Formalin-fixed/paraffin-embedded specimens were sec-tioned at 4-�m thickness and mounted on positively chargedslides. Sections were stained with H&E and immunohisto-

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chemistry (IHC) analysis was performed as described previ-ously (21, 46, 47).

Statistical analysis

Prism software was used to carry out statistical analysis. Thedata are presented as mean S.E. and were analyzed byStudent’s t test. All t tests were performed at the two-sided 0.05level for significance.

Author contributions—Q. L., O. A. A., S. R., and H. C. conceptual-ization; Q. L., O. A. A., S. R., H. S., Z. B., L. L. A., and H. C. data cura-tion; Q. L., O. A. A., S. R., and H. C. formal analysis; Q. L., O. A. A.,S. R., M. W., H. S., and H. C. investigation; Q. L. and H. C. visualiza-tion; Q. L., O. A. A., S. R., M. E. F.-Z., and H. C. methodology; Q. L.,O. A. A., S. R., Z. B., M. E. F.-Z., L. W., and H. C. writing-review andediting; Z. B. and H. C. writing-original draft; L. L. A., M. E. F.-Z.,and H. C. resources; M. E. F.-Z., L. W., and H. C. supervision; L. W.and H. C. project administration; H. C. funding acquisition; H. C.validation.

Acknowledgment—Dr. Peter Sun provided TGF�1 ligand.

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Role of Gli signaling in prostate tissue development

10560 J. Biol. Chem. (2018) 293(27) 10547–10560

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Luciana L. Almada, Martin E. Fernandez-Zapico, Lianchun Wang and Houjian CaiQianjin Li, Omar A. Alsaidan, Sumit Rai, Meng Wu, Huifeng Shen, Zanna Beharry,

progenitor cellsStromal Gli signaling regulates the activity and differentiation of prostate stem and

doi: 10.1074/jbc.RA118.003255 originally published online May 17, 20182018, 293:10547-10560.J. Biol. Chem. 

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