Sonic hedgehog-responsive genes in the fetal prostate

Abstract

The Hedgehog (Hh) signaling pathway plays an important role in prostate development and appears to play an equally important role in promoting growth of advanced prostate cancer. During prostate development, epithelial cells in the urogenital sinus (UGS) express Sonic Hedgehog (Shh) and secrete Shh peptide. The secreted Hh peptide acts on adjacent mesenchymal cells to activate the Hh signal transduction pathway and elicit paracrine effects on epithelial proliferation and differentiation. To identify mesenchymal targets of Shh signaling, we performed microarray analysis on a Shh-responsive, immortalized urogential sinus mesenchymal cell line. We found 68 genes that were up-regulated by Shh and 21 genes that were down-regulated. Eighteen of those were selected for further study with Ptc1 and Gli1 serving as reference controls. We found 10 of 18 were also Hh-regulated in primary UGS mesenchymal cells and 13 of 18 in the cultured UGS. Seven of 18 exhibited Shh-regulated expression in both assays (Igfbp-6, Igfbp-3, Fbn2, Ntrk3, Agpt4, Dmp1, and Mmp13). Three of the 18 genes contained putative Gli binding motifs that bound Gli1 peptide in electrophoretic mobility shift assays. With the exception of Tiam1, target gene expression generally showed no differences in the concentration dependence of ligand-induced expression, but we observed strikingly different responses to direct pathway activation by transfection with activated Smo, Gli1, and Gli2.

FIGURE 1.

Localization of target gene expressions in the newborn prostate. Whole-mount in situ hybridization reveals focused domains of Timp3 (A), Cxcl14 (C), Plxna2 (E), Dner (G), and Igfbp3 (I) expression in the mesenchyme surrounding the nascent prostate buds. Sectioning confirms the expression of Timp3 (B), Cxcl14 (D), Plxna2 (F), Dner (H), and Igfbp3 (J) localized to the mesenchyme (m) surrounding the epithelial buds (e). Scale bar = 500 μm for A, C, E, G, and I. Scale bar = 40 μm for B, D, F, H, and J. Data are representative of 3 specimens per gene analyzed.

FIGURE 2.

UGSM-2 cells, MEF cells, and 3T3 cells were treated with 50 nm Shh. Gene expression was analyzed by real time PCR analysis on total RNA extracted after 24 h treatment. Gli1 and Ptc1 were induced in cell lines tested. Three genes (Dner, Tnmd, and Timp3) showed induced expression after Shh treatment in UGSM-2 cells, but not in MEF cells or 3T3 cells (n = 3; **, p < 0.01; *, p < 0.05).

FIGURE 3.

Tiam1 expression in the developing prostate. Immunofluorescence staining of Tiam1 and smooth muscle actin (SMA) were performed on sections of P10 and adult prostate. Expression of Tiam1 (green) co-localized with smooth muscle actin (red) expression. Scale bar = 100 μm.

FIGURE 4.

Target gene expression in UGSM-2 cells transfected with Hh pathway components. UGSM-2 cells were transfected with Gli1, activated Gli2, activated Smo, or green fluorescent protein alone. Expression of the selected 18 genes was analyzed by real time PCR on the extracted RNA from transfected cells (*, p < 0.05, n = 3). GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogeanse.

FIGURE 5.

Identification of Gli1-binding motifs in the 5′ region of the Shh target genes. Electrophoretic mobility shift assays demonstrated two shifted bands (arrows) in Igfbp-3, Fbn2, and Rgs-4 genes. Specific shifted bands were abrogated by non-radiolabeled oligonucleotide specific to each gene. Control protein or a mutant oligonucleotide DNA did not affect the mobility shift, indicating the specificity of the Gli binding interaction.

FIGURE 6.

Regulation of target gene expression by forskolin treatment. UGSM-2 cells were treated with Shh in the presence or absence of forskolin. RNA were extracted after 24 h, followed by performing real time PCR to analyze selected gene expression (*, p < 0.05, n = 3). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.