(150) Altered Regulation of Prostate Smooth Muscle Relaxation and Bladder Function Associated with Aging and Long-term Increased Androgen Signaling

William H. Walker¹, Erin Wilderoter¹, Kim K. Steitz², Terry I. Medrano³, Chad M.Vezina², Paul S. Cooke³

1. Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA

2. Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA.

3. Department of Physiological Sciences, University of Florida, Gainesville, FL, USA

 

Abstract Text:

Benign prostate hyperplasia (BPH) incidence increases with age and is common in older men. Quality of life is severely impacted by BPH, which leads to bladder outlet obstruction (BOO) and lower urinary tract symptoms (LUTS). BPH/LUTS occur in response to increased cell proliferation, immune responses and/or sustained contraction of smooth muscle in the prostate that impinge on the urethra and bladder. We focused on the regulation of smooth muscle relaxation, which occurs after dephosphorylation of myosin light chain kinase (MLCK) by the myosin light chain phosphatase (MLCP). MLCP is composed of the PP1C phosphatase, a regulatory PPP1R protein and a 20kDa protein of unknown function. Immunofluorescence assays determined that PPP1R proteins 12A, 12C, 14C, 16A and16B are expressed in mouse prostate smooth muscle cells and are the most likely proteins regulating smooth MLCP activity and muscle contraction. Although PPP1R12A is thought to be the major positive regulator of PP1C in most tissues, we found Ppp1r12c mRNA was the most highly expressed mRNA encoding a promoter of PPP1c activation and muscle relaxation. mRNA levels of Ppp1r16b, encoding an inhibitor of PP1C, were 50% lower than Ppp1r12c in control (7-month-old) mouse prostate but, levels increased by two-fold in aged (20-month-old) mice. Thus, PPP1R16B is a potential target for treating LUTS in aging men. Studies of primary cultures of prostate cells from BPH tissue and adjacent normal prostate found that expression of Ppp1r14a mRNA encoding an inhibitor of PP1C and muscle relaxation was induced 33-fold in BPH cells (RNA analyses by RT-qPCR, n=3 to 5). These results suggest that PPP1R14A is as a potential marker of, and contributor to, BPH/LUTS. In mouse control prostate tissue sections and untreated prostate cell cultures from normal human prostate, PPP1R14A protein expression was restricted to prostate epithelial cells. However, in normal prostate cell cultures treated with DHT for 48 h, immunostaining for phosphorylated PPP1R14A was strongly induced in the perinuclear regions of smooth muscle cells. Because phosphorylation of PPP1R14A increases inhibition of PP1C by 1,000-fold, these results suggest that elevated androgen levels promote sustained muscle contraction via PPP14A-mediated blockage of PP1C-mediated muscle relaxation. Older transgenic mice (7- to 11-months-old) with increased androgen signaling function have qualities consistent with polyuria as they produced 30% more urine spots of the smallest size and two-fold greater total urine output than wild-type mice during a 4 h period (Void spot assays, n=5). These transgenic mice express endogenous androgen receptors (ARs) plus additional mutated ARs having only nonclassical activity that increase cellular kinase activity. Interestingly, transgenic mice expressing endogenous AR plus an AR mutant having only classical activity limited to regulating gene expression did not have altered voiding characteristics. These results suggest nonclassical activity is important in mediating the observed phenotypes. Similarly aged transgenic mice lacking endogenous estrogen receptor 1 (ESR1) but expressing ESR1 in only the nucleus or at the plasma membrane did not have altered voiding or bladder characteristics. No differences were observed for cytometry of anesthetized transgenic mice with varying ER or AR signaling activities. Together, our results provide information regarding factors causing alterations of smooth muscle contraction in aged and BPH prostate, bladder function, voiding characteristics and BPH/LUTS disease. Supported by NIH grant 1R21AG0777740.