Activation of cGMP/PKG/p65 signaling associated with PDE5‐Is downregulates CCL5 secretion by CD8 + T cells in benign prostatic hyperplasia

Abstract Background Benign prostatic hyperplasia (BPH) is the most common urological disease in elderly men, but the underlying pathophysiological mechanisms are complex and not fully understood. Phosphodiesterase type 5 inhibitors (PDE5‐Is) used to treat BPH could upregulate the cyclic guanosine monophosphate (cGMP)‐dependent protein kinase G (PKG) signaling, which was shown to blunt inflammation in the prostate. Our previous findings indicate that CD8+ T cells promote the proliferation of BPH epithelial cells (BECs) in low androgen conditions through secretion of CCL5; however, the role of the cGMP/PKG pathway in the process is unclear. Methods Paraffin‐embedded tissues were used for expression quantity of CD8+ T cells, CCL5, cyclin D1, and PDE5 protein by immunohistology in prostate specimens which were/were not treated with finasteride 5 mg daily for at least 6 months before surgery. BPH‐1 cells were cocultured with or without CD8 + T cells or PDE5‐Is in low androgen conditions for 4 days. The conditioned media, BPH‐1 cells, and CD8 + T cells were harvested for the subsequent experiments. The quantitative polymerase chain reaction was used for assaying the level of messenger RNA expression of CCL5. CCL5 in the conditioned media was detected by the enzyme‐linked immunosorbent assay. The effect of PDE5‐Is on cocultured BPH‐1/CD8 + T‐cell proliferation was detected by the cell counting kit‐8. A high‐fat diet (HFD)‐induced prostatic hyperplasia rat model was used to investigate the effect of cGMP/PKG activation in CD8 + T cells in vivo. Results CD8+ T‐cell infiltration into human BPH tissues was positively correlated with the expression of CCL5, cyclin D1, and PDE5, whereas in an HFD‐induced prostatic hyperplasia rat model, the activation of the cGMP/PKG signaling by a PDE5‐I could suppress the CD8 + T‐cell infiltration and the CCL5 and cyclin D1 expression. Furthermore, the activation of the cGMP/PKG pathway inhibited CCL5 secretion by CD8 + T cells by downregulating nuclear factor‐κB p65 phosphorylation, which reduced the growth of BPH‐1 through CCL5/STAT5/CCND1 signaling. Conclusions Our results indicate that the upregulation of the cGMP/PKG/p65 signaling reduces CCL5 secretion in CD8 + T cells, which in turn decreases the proliferation of BECs in low androgen conditions, suggesting that the combination of 5α reductase inhibitors lowering androgen levels and PDE5‐Is may be a novel, more effective treatment for BPH patients.


Conclusions:
Our results indicate that the upregulation of the cGMP/PKG/p65 signaling reduces CCL5 secretion in CD8 + T cells, which in turn decreases the proliferation of BECs in low androgen conditions, suggesting that the combination of 5α reductase inhibitors lowering androgen levels and PDE5-Is may be a novel, more effective treatment for BPH patients. Score, storage and voiding LUTS, and improve quality of life. [2][3][4][5] Most studies suggested that the clinical effect of PDE5-Is is associated with cyclic guanosine monophosphate (cGMP) signaling through cGMPdependent protein kinase G (PKG), which mainly reduces smooth muscle tone of the detrusor, prostate, and urethra, thus relieving LUTS. [6][7][8] Dihydrotestosterone synthesized from testosterone by 5α-reductase (5AR) II plays a critical role in prostate growth, and a 5AR inhibitor (5AR-I) finasteride is known to alleviate BPH symptoms. In our previous studies, we have shown that BPH tissues from finasteride-treated patients had increased CD8 + T-cell infiltration, 9 which could promote the proliferation of BPH epithelial cells (BECs) in low androgen conditions by secreting a chemokine CCL5 (also known as RANTES). 10 Moreover, we also found that PDE5 protein expression positively correlated with CD8 + T-cell infiltration and CCL5 and cyclin D1 levels in human BPH tissues, whereas activation of cGMP/PKG signaling could suppress CD8 + T-cell infiltration and the expression of CCL5 and cyclin D1 in preliminary experiments using a high-fat diet (HFD) BPH rat model. Other studies have also reported that the cGMP/PKG signaling pathway may regulate the inflammatory response implicated in several pathological conditions, including BPH, [11][12][13] but the mechanism of cGMP/PKG signaling and its specific role in inflammation in the prostate is not well understood. In particular, activation of the cGMP/PKG pathway in CD8 + T cells and its effect on BEC proliferation is unclear, and there are few reports on the relationship between the cGMP/PKG pathway and CCL5 secretion. Therefore, the aim of the present study was to clarify the involvement of the cGMP/PKG pathway in the regulation of CCL5 expression in CD8 + T cells and its effect on BEC proliferation in low androgen conditions.

| Patients
Patients were selected using the electronic medical record system containing the data on 921 patients with BPH who underwent transurethral resection of the prostate between January 2007 and December 2011 in the Department of Urology, Peking University First Hospital, Beijing, China, and it was approved by the ethical committee of our institution. Patients who had urinary tract infection, prostatitis, earlier prostate-related surgery, or the history of urethral catheterization were excluded from this study. Prostate tissues were obtained from 34 BPH patients treated or not with finasteride (5 mg daily) for at least 6 months before surgery and examined microscopically by two pathologists to confirm the diagnosis of BPH not associated with prostate cancer or prostatic intraepithelial neoplasia.

| Animals
An HFD-induced prostatic hyperplasia model was established as previously described. [14][15][16] Male Sprague-Dawley rats (7-to 9-week old; 200-220 g) (Beijing Keao Xieli Feed Co, Ltd, Beijing, China) were housed in individual cages under standard conditions in a temperature-and humidity-controlled room with a 12-hour light/dark cycle and had free access to food and water. After a week on a standard rat diet, animals were randomly assigned to control (n = 6), HFD (n = 6) or HFD + PDE5-Is (n = 6) groups according to their weight. The control group continued on the regular diet (3.85 kcal/g; carbohydrate, 70%; protein, 20%; fat, 10%, kcal), HFD and HFD + PDE5-Is groups were fed HFD (5. 24

| Immunohistochemistry
Serial paraffin sections of the prostate samples were stained with specific antibodies using IHC as previously described. 10

BPH-1 and Molt-3 cells were incubated for 3 days and collected for
Western blot analysis performed as described previously. 10

| cGMP/PKG activation downregulated NF-κB phosphorylation in CD8 + T cells and CCL5/STAT5/ CCND1 signaling in BECs
Previous studies indicate that PDE5 inhibition by PDE5-Is activates the cGMP/PKG signaling pathway in BECs. [18][19][20] Therefore, we  Prostate inflammation has been suggested as an etiological factor for BPH, and emerging evidence indicates that inflammation may contribute to prostate growth. 28,29 It was shown that downregulation of androgen receptor signaling in mouse prostate luminal cells could upregulate secretion of cytokines and chemokines in a cell-autonomous F I G U R E 4 Activation of cGMP/PKG signaling suppressed CCL5 secretion by CD8 + T cells and reversed the induction of BEC proliferation in vivo. A, The degree of prostatic hyperplasia in rats receiving regular diet (RD), HFD, or HFD + PDE5-Is was evaluated by hematoxylin and eosin staining of rat prostate samples (n = 6 rats per group). B, The serum testosterone levels of RD and HFD groups were measured using an automated chemiluminescence system at week 12; *P < 0.05, **P < 0.01, and ***P < 0.001 (by t test). C, All rat prostate weight of three groups were tested at last; *P < 0.05, **P < 0.01, and ***P < 0.001 (by one-way ANOVA in BEC monocultures ( Figure 2). Furthermore, cGMP/PKG activation blocked CCL5 secretion by CD8 + T cells but not by BECs. In addition, a PKG inhibitor restored BEC proliferation and the secretion of CCL5 induced by CD8 + T cells in low androgen conditions.
To further investigate the molecular mechanism underlying CCL5 secretion by CD8 + T cells in low androgen conditions, we focused on the signaling cascade downstream of cGMP/PKG. Several studies indicated that NF-κB plays an important regulatory role in the immune response, [21][22][23] and that cGMP/PKG activation inhibited NF-κB in hyperthermia-exposed MCF-7 cells, resulting in apoptosis. 21 In this study, we showed, for the first time, that the cGMP/PKG pathway regulates the function of CD8 + T cells by controlling NF-κB p65 phosphorylation, which affects the secretion of CCL5. A PKG inhibitor could re-establish NF-κB phosphorylation in CD8 + T cells and STAT5 phosphorylation and CCND1 expression in BECs, and restore BEC proliferation enhanced by CD8 + T cells, thereby implicating cGMP/PKG signaling in BPH pathogenesis.
Our previous studies deemed that there were more CD8 However, some limitations of this study should be recognized.
The major one is the deficiency of clinical BPH samples which were treated with both 5AR-Is and PDE5-Is. In addition, CD8 + T cells in rat prostate tissues were not separated by fluorescence-activated cell sorting to further certify the function of the cGMP/PKG pathway.

| CONCLUSIONS
The important finding of the present study is that the activation of the cGMP/PKG/p65 pathway associated with PDE5-Is in CD8 + T cells under low androgen conditions reduces the secretion of CCL5, which results in the inhibition of BEC proliferation via CCL5/STAT5/ CCND1 signaling ( Figure 5). As PDE-Is block CCL5 secretion by CD8 + T cells and 5AR-Is the reduce androgen levels, the combination of these drugs may effectively suppress the growth of BECs in the prostate, and therefore, can be considered as a candidate therapeutic approach for BPH patients in the future.

ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (grant no. 81470984 to JP and grant nos. 81770755 and 81570683 to JJ).