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. 2015 Feb 15;75(3):255-65.
doi: 10.1002/pros.22911. Epub 2014 Oct 18.

Reduced Expression of GDF-15 Is Associated With Atrophic Inflammatory Lesions of the Prostate

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Free PMC article

Reduced Expression of GDF-15 Is Associated With Atrophic Inflammatory Lesions of the Prostate

James R Lambert et al. Prostate. .
Free PMC article

Abstract

Background: Accumulating evidence suggests that chronic prostatic inflammation may lead to prostate cancer development. Growth differentiation factor-15 (GDF-15) is highly expressed in the prostate and has been associated with inflammation and tumorigenesis.

Methods: To examine the relationship between GDF-15 and prostatic inflammation, GDF-15 expression was measured by immunohistochemical (IHC) staining in human prostatectomy specimens containing inflammation. The relationship between GDF-15 and specific inflammatory cells was determined using non-biased computer image analysis. To provide insight into a potential suppressive role for GDF-15 in inflammation, activation of inflammatory mediator nuclear factor of kappa B (NFκB) was measured in PC3 cells.

Results: GDF-15 expression in luminal epithelial cells was decreased with increasing inflammation severity, suggesting an inverse association between GDF-15 and inflammation. Quantification of IHC staining by image analysis for GDF-15 and inflammatory cell markers revealed an inverse correlation between GDF-15 and CD3+, CD4+, CD8+, CD68+, and inos+ leukocytes. GDF-15 suppressed NFκB activity in luciferase reporter assays. Expression of the NFκB target, interleukin 8 (IL-8), was downregulated by GDF-15.

Conclusions: The inverse relationship between GDF-15 and inflammation demonstrates a novel expression pattern for GDF-15 in the human prostate and suppression of NFκB activity may shed light on a potential mechanism for this inverse correlation.

Keywords: GDF-15; NFκB; inflammation; macrophage.

Figures

Fig. 1
Fig. 1
Expression of GDF-15 is inversely associated with prostatic inflammation. Top panel shows prostatic acini containing normal architecture, atrophic, and inflamed regions. IHC staining using an antibody against GDF-15 was performed. Bottom panel, GDF-15 expression and degree of inflammation were scored. GDF-15 expression was scored using the sum of two values—percent of cells staining positive (0= 0%, 1=1–20%, 2= 21–50%, 3=>50%) and staining intensity (0=none, 1=low, 2=moderate, 3=high) by IHC with in a low power field. Degree of inflammation was independently scored as: 0=no inflammation, 1=low inflammation, 2 =moderate inflammation, and 3=severe inflammation on H&E-stained sections of the same regions as stained for GDF-15. Statistical analysis was carried out using Student t-test. *P<0.05.
Fig. 2
Fig. 2
Inverse association between GDF-15 and leukocyte markers. Prostate specimens were immunostained with GDF-15 and leukocyte markers CD3 (T-cells) and CD68 (activated macrophages). Immunostaining is shown for regions containing normal architecture (Normal) and inflammatory atrophy (Atrophic). Photographs were taken at 40×magnification.
Fig. 3
Fig. 3
Computer image quantification of GDF-15 and inflammation marker immunostaining. Consecutive sections from whole prostatectomy specimens were immunostained using antibodies against GDF-15, CD3 (T-cells), CD4 (T-helper cells), CD8 (cytotoxic T-cells), and CD68 (activated macrophages) labeled as A, B, and C, respectively. Upper panel: representative IHC images. Arrows indicate inflammatory cells. Lower panel: example of grid-matching consecutive sections.
Fig. 4
Fig. 4
Scatterplots demonstrate inverse relationship between GDF-15 and inflammation markers in epithelial/peri-epithelial (A) and stromal (B) compartments. Prostate specimens were immunostained for GDF-15, CD3+ (T-cells), CD4+ (T-helper cells), CD8+ (cytotoxic T-cells), and CD68+ (macrophages). Staining intensity was scaled to normalize intensity measurements for markers to be compared. Graph shows staining intensity plotted as a scatterplot. Each panel shows normalized staining intensities for two (indicated) markers that intersect at each graph panel.
Fig. 4
Fig. 4
Scatterplots demonstrate inverse relationship between GDF-15 and inflammation markers in epithelial/peri-epithelial (A) and stromal (B) compartments. Prostate specimens were immunostained for GDF-15, CD3+ (T-cells), CD4+ (T-helper cells), CD8+ (cytotoxic T-cells), and CD68+ (macrophages). Staining intensity was scaled to normalize intensity measurements for markers to be compared. Graph shows staining intensity plotted as a scatterplot. Each panel shows normalized staining intensities for two (indicated) markers that intersect at each graph panel.
Fig. 5
Fig. 5
GDF-15 suppresses NFκB activity in PC3 cells. PC3 prostate cancer cells were co-transfected with the indicated amounts of GDF-15 expression vector (pCMV-GDF-15) and NFκB activated luciferase constructs (NFκB-Luc and COX-2-Luc) and luciferase activity was determined. (A) Immunoblot of GDF-15 expression in co-transfected cells. Statistical analysis was carried out using Student t-test. *P<0.05, **P<0.005.
Fig. 6
Fig. 6
GDF-15 suppresses expression of IL8. PC3 cells were transfected with pCMV-GDF-15 (1 μg/ml) or empty vector control and IL-8 mRNA levels determined by qRT-PCR. β-Actin mRNA levels were used for normalization.

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