Fig 1.
Detection of PAK isoforms and markers in human prostate tissue, and in WPMY-1 cells.
Detection was performed by RT-PCR (A), or Western blot analyses (B, C). Shown are ratios of Ct values (target/GAPDH) for each sample (from n = 7 patients, or from WPMY-1 cells from 4 experiments) (A), or bands for all investigated samples (from n = 12 patients, or WPMY-1 cells from 4 experiments (B). In (B) and (C), indicated molecular weights are the expected sizes of proteins. Detection of each PAK isoform in (B) was performed with two different antibodies for each isoform (mouse, and rabbit or goat) (ab, antibody). Western blots included calponin as a marker for smooth muscle cells, pan-cytokeratin as a marker of endothelial cells (glands), prostate-specific antigen (PSA) as a marker for hyperplasia, and α1A-adrenoceptors as an important feature of prostate smooth muscle cells.
Table 1.
Summarized results from detection of mRNA and protein of different PAK isoforms.
Detections were performed by RT-PCR, Western blot analysis, and immunofluorescence staining. Detection by Western blot was performed with to different.
Fig 2.
Immunofluorescence stainings of human prostate tissues.
Sections were double labeled for different PAK isoforms, together with calponin (marker for smooth muscle cells), pan-cytokeratin (marker for epithelial cells of glands), or tyrosine hydroxylase (TH, marker for catecholaminergic nerves). Yellow color in merged pictures may indicate colocalization of targets. Shown are representative stainings from series with tissues from n = 5 patients for each combination.
Fig 3.
Effects of FRAX486 and IPA3 in low concentrations on contraction of human prostate strips.
Contractions of isolated human prostate strips were induced as indicated, after addition of 10 μM FRAX48, 100 μM IPA3, or DMSO (control). Tensions have been expressed as % of contraction by highmolar KCl, being assessed before application of inhibitors or solvent. This normalization allows comparisons despite different conditions of tissues or patients, e. g. due to varying degree of BPH, different content of smooth muscle, or any other heterogeneity (compare Fig 1C). Shown are means ± SEM from experiments with tissues from n = 5 (endothelin-1, EFS, each with FRAX486), n = 4 (endothelin-2), or n = 3 (EFS with IPA3) patients for each group.
Fig 4.
Effects of FRAX486 and IPA3 in high concentrations on contraction of human prostate strips.
Contractions of isolated human prostate strips were induced as indicated, after addition of FRAX48 (30 μM), IPA3 (300 μM), or DMSO (control). Tensions have been expressed as % of contraction by highmolar KCl, being assessed before application of inhibitors or solvent. This normalization allows comparisons despite different conditions of tissues or patients, e. g. due to varying degree of BPH, different content of smooth muscle, or any other heterogeneity (compare Fig 1C). Shown are means ± SEM from experiments with tissues from n = 5–8 patients for each group (# p<0.05).
Fig 5.
Effects of FRAX486 and IPA3 on actin organization in WPMY-1 cells.
Actin filaments were visualized by staining with FITC-coupled phalloidin, after incubation of WPMY-1 cells with DMSO, FRAX486 (1–10 μM), or IPA3 (1–10 μM) for 24 h. Shown are representative images from series with 5 independent experiments, with similar results.
Fig 6.
Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells.
Survival of WPMY-1 cells was assessed using CCK-8 assay, after incubation with DMSO, FRAX486 (1–10 μM), or IPA3 (1–10 μM) for 24–72 h. Shown are means ±SEM from series with 5 independent experiments for each setting (# p<0.05 vs. control).
Fig 7.
Effects of FRAX486 and IPA3 on proliferation of WPMY-1 cells.
Proliferation rate was assessed by EdU assay after incubation with DMSO (control), FRAX486 (1–10 μM), or IPA3 (1–10 μM) for 24 h. The number of cells showing proliferation (= red nuclei) was referred to the total number of cells (= red + blue nuclei), to correct for reduced number of cells after longer incubation periods (compare with Fig 5). Shown are representative images and means ±SEM, from series with 8 independent samples for each setting (# p<0.05 vs. control).
Fig 8.
Effects of agonist stimulation on PAK phosphorylation in WPMY-1 cells.
WPMY-1 cells were stimulated with agonists as indicated, or remained without stimulation (controls). Subsequently, Western blot analyses were performed for phospho-PAK (using a phospho-specific antibody recognizing PAK1-3 phosphorylated at threonine 402 or corresponding sites), PAK1, and β-actin. Shown are representative Western blots and densitometric quantification of all experiments (series with n = 5 independent experiments for each agonist) (means ±SEM).
Fig 9.
Assumed PAK signaling in the hyperplastic human prostate, and intervention by PAK-specific small molecule inhibitors.
The presented model is based on findings of this study. At least two different PAK functions may critically determine adrenergic and endothelin-mediated contraction of prostate smooth muscle. First, PAK promotes the release of noradrenaline during sympathic neurotransmission to smooth muscle cells, followed by contraction by activation of postsynaptic α1-adrenoceptors. Consequently, PAK inhibitors inhibited EFS-induced contractions of prostate strips. Secondly, PAK in smooth muscle cells mediates contraction, where PAK is selectively activated by endothelin receptors (ET-A/B), but not by α1-adrenceptors. Possibly, PAK mediates the contractile signal by ET-A/B, in parallel to established intracellular pathways, which include Ca2+, protein kinase C (PKC), and Rho kinase. This might explain, why PAK inhibitors inhibited endothelin-induced contractions of prostate strips, but not contractions by α1-agonists. Besides its role for smooth muscle tone, PAK inhibition reduced proliferation of stromal cells, suggesting a role for prostate growth.