Immunohistochemistry of activated macrophage marker F4/80 in mouse jejunum.

<p>Jejunal tissues were collected 7 days after irradiation and embedded in paraffin. After fixation and processing, cross sections of jejunum were immunostained with F4/80 antibodies and number of F4/80 positive cells per mm² area of tissue was counted using HistoQuest image analysis software. Bars represent the means ± SEMs for tissue sections of mice in each group. *Statistically significant differences between R and RP groups (P<0.05).</p

The effect of p<i>2TA</i> peptide on COX-2 expression in jejunal crypts.

<p>Expression of COX-2 in mouse intestine: a–h) overview, i–p) details. a–d, i–l) A/J mice; e–h, m–p) BALB/c mice; a,e,i,m) sham irradiated controls; b,f,j,n) mice treated with p<i>2TA</i> peptide six days before tissue harvest; c,g,k,o) mice exposed to 8 Gy gamma rays total body irradiation seven days before the sacrifice; d,h,l,p) mice exposed to 8 Gy gamma rays that received p<i>2TA</i> peptide 24 hours after radiation exposure. In irradiated mice (c,g,k,o) villus integrity is disrupted, their crypts show disorganization, and COX-2 staining is observable in cells in the surface layer of villi. Intestines of irradiated mice that were also treated with 5 mg/kg p<i>2TA</i> peptide 24 hours after irradiation show a more normal morphology with respect to villus height and crypt appearance, and they also show fewer COX-2 positive cells in villi surface cell layer, while COX-2 staining pattern inside villi replicates the pattern seen in sham irradiated mice.</p

The effect of p<i>2TA</i> peptide on jejunal crypts.

<p>Jejunal tissues were collected 7 days after irradiation and embedded in paraffin. After fixation and processing, cross sections of jejunum were stained with hematoxylin and eosin and analyzed for the number of surviving crypts (panel <b>A</b>) and villus height (panel <b>B</b>). Bars represent the means ± SEM of 4 cross-sections for each mouse, and 4–5 mice in each group. *Statistically significant difference between R and RP groups (P<0.05).</p

The effect of p<i>2TA</i> peptide on Cyclin D1 expression in mouse intestine.

<p>Expression of Cyclin D1 in mouse intestine: a–h) overview, i–p) details. a–d, i–l) A/J mice; e–h, m–p) BALB/c mice; a,e,i,m) sham irradiated controls; b,f,j,n) mice treated with p<i>2TA</i> peptide six days before tissue harvest; c,g,k,o) mice exposed to 8 Gy gamma rays total body irradiation seven days before the sacrifice; d,h,l,p) mice exposed to 8 Gy gamma rays that received 5 mg/kg p<i>2TA</i> peptide 24 hours after radiation exposure. In irradiated mice (c,g,k,o) villus integrity is disrupted, their crypts show disorganization, and very little Cyclin D1 staining can be observed outside the crypts. Intestines of irradiated mice that were treated with p<i>2TA</i> peptide 24 h later show a more normal morphology with respect to villus height and crypt appearance; they also show more numerous CD1 positive cells inside villi.</p

Src regulates motility and PG down-regulates Src activity in prostate cancer.

<p>A–B. PC3-M, C4-2B (A), and LNCaP cells (B) were plated in 6 well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA (a pool of 4 sequences). PC3-M, C4-2B, and LNCaP cells were treated with medium containing PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the dispase assay. Inhibition of Src strengthens cell-cell adhesion in PCa cells in general and is able to rescue cell-cell adhesion in PG-deficient cells. C. PC3-M and ARCaP_M cells were plated in 24-well plates and allowed to attach and spread. The cells were treated with medium containing the selective Src- family kinase inhibitor PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the scratch wound assay. D. ARCaP_E cells were plated in 24-well plates and allowed to attach and spread. The cells were transduced with GFP-containing adenovirus or caSrc-containing adenovirus, and after 24 hours a scratch wound was made. Activity of Src is directly correlated with motility of PCa cells. E. ARCaP_M, PC3-M, and LNCaP cells were plated in 6-well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA pool and after 96 hours, the cells were lysed. ARCAP_M and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours the cells were lysed. The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against PG, pSrc, Src, and GAPDH. Levels of pSrc were determined by normalizing to Src levels for each cell line using Image J. Phosphorylation (activation) of Src is inversely correlated with PG levels. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized pSrc/Src ratio for the blot shown. The average ratio and standard deviation of pSrc/Src normalized for loading by GAPDH is as follows: ARCaP_M 0.6+/−0.2, LNCaP 1.7+/−0.2, PC3M 0.7+/−0.1. Graphs represent averages +/− SEM. *P<0.04; **P<003; ****P<0.0001, by paired Student t test.</p

PG-dependent Src activity is regulated by VN.

<p>A. Confocal images showing PG and pSrc expression in normal and malignant prostate tissue (using 68 cores from the prostate TMA). PG is in green and pSrc is red. Bar, 50 µm. B. Scatterplot demonstrating the inverse correlation between pSrc expression and PG expression in prostate tissue (using 68 cores). C. Confocal images showing PG and VN expression in normal and malignant tissue (T2 samples were shown for both pSrc and VN as representative of average expression levels in all malignant tissue samples). PG is in green and VN is red. Bar, 50 µm. D. Scatterplot showing the inverse correlation between VN expression and PG expression in prostate tissue (using 68 cores). E. Scatterplot showing the direct correlation between VN expression and pSrc in prostate tissue (using 68 cores). F. Western blot showing PG, pSrc, and Src expression in LNCaP cells transfected with control or PG siRNA plated on uncoated or VN-coated plastic. VN rescues Src activation in the absence of PG.</p

PG alters the profile of expression of ECM molecules in PCa cell lines.

<p>A–E. ARCaP_E, ARCaP_M, LNCaP, C4-2B, and PC3-M cells were plated in 6-well plates and allowed to attach and spread. The cells were either transduced with GFP- or PG-containing adenovirus and were lysed after 24 hours (PC3-M, C4-2B, ARCaP_M); or the cells were transfected with control siRNA or PG siRNA pool and after 96 hours lysed (ARCaP_E, LNCaP, C4-2B). The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against VN (A), FN (B), LN (C), Col. I (D), Col IV (E) and GAPDH. Levels of ECM proteins were determined by normalizing to GAPDH levels for each cell line using Image J. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG siRNA/Con siRNA or PG OE/GFP ratio for the blot shown. The average ratio and standard deviation of GAPDH normalized PG siRNA/Con siRNA and PG OE/GFP is as follows: (A) ARCaP_E 3.1+/−1.3, LNCaP 2.0+/−0.9, PC3-M 0.3+/−0.1; (B) LNCaP 0.7+/−0.05, C4-2B KD 0.8+/−0.1, C4-2B OE 1.2+/−0.2, PC3-M 1.5+/−0.05; (C) ARCaP_M 0.6+/−0.1, PC3-M 0.4+/−0.3; (D) 0.3+/−0.1; (E) 0.4+/−0.2.</p

The effect of p<i>2TA</i> peptide on systemic inflammatory mediators.

<p>Plasma levels of IL-6 (panel <b>A</b>), KC (panel <b>B</b>) or fibrinogen (panel <b>C</b>) were measured on day 7 after irradiation. C: sham-irradiated mice, P: sham-irradiated mice that received 5 mg/kg of the p<i>2TA</i> peptide, R: 8 Gy-irradiated mice, RP: 8 Gy-irradiated mice that received 5 mg/kg of the p<i>2TA</i> peptide 24 h after irradiation. Bars represent the means ± SEM of 4–5 mice. *Statistically significant difference between R and RP groups (P<0.05).</p

The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

<div><p>Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG’s effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.</p> </div

Model of the role of PG in prostate cancer adhesion and motility.

<p>Loss of PG results in a double negative hit on cell-cell junctions, while orchestrating alterations in extracellular matrix that promote motility. PG levels correspond directly to the strength of cell-cell adhesion in PCa cells. Down-regulation of PG leads to loss of cell-cell adhesion strength. Concomitantly, loss of PG causes up-regulation of VN and activation of Src. Together, these cell-cell and cell-ECM dependent effects of PG down-regulation lead to an increase in PCa cell motility.</p