Coordinated induction of cell survival signaling in the inflamed microenvironment of the prostate

Abstract

Purpose: Both prostate cancer and benign prostatic hyperplasia are associated with inflammatory microenvironments. Inflammation is damaging to tissues, but it is unclear how the inflammatory microenvironment protects specialized epithelial cells that function to proliferate and repair the tissue. The objective of this study is to characterize the cell death and cell survival response of the prostatic epithelium in response to inflammation.

Methods: We assessed induction of cell death (TNF, TRAIL, TWEAK, FasL) and cell survival factors (IGFs, hedgehogs, IL-6, FGFs, and TGFs) in inflamed and control mouse prostates by ELISA. Cell death mechanisms were determined by immunoblotting and immunofluorescence for cleavage of caspases and TUNEL. Survival pathway activation was assessed by immunoblotting and immunofluorescence for Mcl-1, Bcl-2, Bcl-XL, and survivin. Autophagy was determined by immunoblotting and immunofluorescence for free and membrane associated light chain 3 (LC-3).

Results: Cleavage of all four caspases was significantly increased during the first 2 days of inflammation, and survival protein expression was substantially increased subsequently, maximizing at 3 days. By 5 days of inflammation, 50% of prostatic epithelial cells expressed survivin. Autophagy was also evident during the recovery phase (3 days). Finally, immunofluorescent staining of human specimens indicates strong activation of survival proteins juxtaposed to inflammation in inflamed prostate specimens.

Conclusions: The prostate responds to deleterious inflammation with induction of cell survival mechanisms, most notably survivin and autophagy, demonstrating a coordinated induction of survival factors that protects and expands a specialized set of prostatic epithelial cells as part of the repair and recovery process during inflammation.

Keywords: cell death; cell survival; inflammation; prostate; repair.

Fig. 1

Hematoxylin and Eosin staining of 3 day inflamed dorsolateral prostates in the mouse prostatic inflammation model. A: (200×) Non-inflamed control prostate shows pseudostratified epithelium and very few apoptotic or autophagic cells. B: 200× Inflamed prostatic duct shows layering of epithelium characteristic of reactive hyperplasia during inflammation, but also shows numerous damaged and apoptotic cells as evidenced by pyknotic nuclei and retracted cytoplasm (arrows). C: 400× Image of damaged epithelial cells in hyperplastic epithelium in inflamed prostate.

Fig. 2

Inflammation of the mouse prostate results in induced apoptotic signaling. A: Fluorescent image (200×) of cleaved caspase 3 (green) in the epithelium (PanCK-red) of mouse prostates inflamed 3 days. B: Calculated data of epithelial cells for cleaved caspases 3, 7, and 6 from 6 mouse dorsal-lateral prostate lobes inflamed to the time points shown, expressed as percentage of cleaved caspase-positive cells within the epithelial compartment. C: Fluorescent image (200×) of TUNEL-positive cells. D: Calculated data of epithelial cells for TUNEL from 6 mouse dorsal-lateral prostate lobes inflamed to the time points shown, expressed as percentage of TUNEL-positive cells within the epithelial compartment. All data are expressed as mean ±s.e.m. ^*P <0.05 versus PBS-instilled prostate; comparisons using analysis of variance (ANOVA), n =6.

Fig. 3

Inflammation induces the expression and release of known cell death factors, followed by an induction of a panel of known cell survival-inducing factors. A: Total tissue content of TNFα, TWEAK, TRAIL, and FAS ligand are all induced rapidly upon induction of inflammation in the dorsolateral prostate, maximizing at 1–2 days after induction. B: Correspondingly, the released fraction of these death ligands increases, indicating that release of the factor occurs allowing their function. C: Subsequently, the induction of known cell survival factors Shh, IGF-1, IL-1α, IL-6, TGFβ1, and TGFβ2 begins in the second day of inflammation, and maximizes at day 3. IL-1α, IL-6, and TGFβ1 remained induced for 5 days after inflammation induction. D: The released fraction of cell survival factors increases correspondingly to their production. All peptides were assessed by ELISA of whole dorsolateral prostate lobes and calculated as picogram of peptide per gram of tissue. Data were then normalized as a ratio to control non-inflamed prostates at the given time point of induction, for presentation. All data are expressed as mean ±s.e.m. ^*P <0.05 versus PBS-instilled prostate; comparisons using analysis of variance (ANOVA), n =6. Asterisk-labeled time points are those that show significant inducibility of all death or survival factors.

Fig. 4

Inflammation induces three primary cell survival signaling pathways in mouse dorsolateral prostates, most prominently, survivin. A: Immunoblot example of induction of survivin, Bcl-2, and Mcl-1 during the time course of inflammation in mouse prostates. B: Quantified data of relative expression of all three survival proteins during prostatic inflammation; data were calculated as ratio of pixel intensity of the given survival protein, relative to β-actin, and expressed as ratio of expression to control prostates at the corresponding time of induction. Data are expressed as mean ±s.e.m. ^*P <0.05 versus PBS-instilled (control) prostate; comparisons using analysis of variance (ANOVA), n =6. C: Immunofluorescence of survivin expression (green) in 5 day instilled control (top) and inflamed (bottom) dorsolateral prostates demonstrating epithelial cell (PanCK, red) expression in the nucleus during inflammation. D: Quantified cell counting of epithelial cells positive for survivin expression; data expressed are the percentage of epithelial cells expressing survivin in control and inflamed prostates at each time point of inflammation. All data are expressed as mean ±s.e.m. ^*P <0.05 versus PBS-instilled prostate; comparisons using analysis of variance (ANOVA), n =6.

Fig. 5

Inflammation autophagy marker expression in mouse dorsolateral prostates. A: Immunoblot example of induction of the autophagy marker LC3 at inflammation time points indicated; the higher running band (cyt) represents cytosolic LC3 while the lower running band (Aph) indicates LC3 associated in the autophagasome, and is indicative of autophagy induction. B: Quantified data of relative expression of all cytosolic and autophagasome-associated LC3; data were calculated as ratio of pixel intensity of the LC3 form, relative to β-actin, and expressed as ratio of expression to control prostates at the corresponding time of induction. Data are expressed as mean ±s.e.m. ^*P <0.05 versus PBS-instilled (control) prostate; comparisons using analysis of variance (ANOVA), n =6. C: Immunofluorescence of LC3 (green) localization in 3 day instilled control (top) and inflamed (bottom) dorsolateral prostates demonstrating the characteristic punctate LC3 Localization with the autophagasome of autophagic epithelial cells, a further indicator of autophagic cells (PanCK, red). D: Quantified cell counting of epithelial cells positive for punctate LC3 Localization; data expressed are the percentage of epithelial cells expressing punctate LC3 in control and inflamed prostates at each time point of inflammation. All data are expressed as mean ±s.e.m. ^*P <0.05 versus PBS-instilled prostate; comparisons using analysis of variance (ANOVA), n =6.

Fig. 6

Human prostate specimens demonstrate intense survivin staining juxtaposed to regions of inflammation. A: Immunofluorescent images of non-inflamed or inflamed human prostates representing non-diseased controls (peripheral zone) taken from cystoprostatectomy cancer and BPH-free prostate specimens, BPH specimens (transition zone from TURP), or prostate cancer specimens, as indicated. Sections were stained for survivin (green) and CD45, a pan leukocyte marker (red) to identify regions of inflammation. Sections were deemed non-inflamed if they exhibited less than 10 Leukocytes per 20× field, and inflamed if they exhibited greater than 30 Leukocytes per field. B: Quantified cell counting of epithelial cells positive for survivin expression in human prostates; data expressed are the percentage of epithelial cells expressing survivin in non-inflamed and inflamed prostates at each time point of inflammation—three 20× fields per prostate section were averaged for each data point, and all data are expressed as mean ±s.e.m. ^*P <0.05 inflamed versus non-inflamed prostate; #P <0.05 disease condition versus non-diseased control. Analysis of variance (ANOVA), n =12 human prostates. C: percent of sections from each human prostate group (non-diseased, BPH, and cancer) that exhibited 30 Leukocytes per 20× section. Three 20× fields per prostate section were averaged for each data point, and all data are expressed as mean ±s.e.m. ^#P <0.05, disease condition versus non-diseased control. Analysis of variance (ANOVA), n =12 human prostates.