CTRP3 Stimulates Proliferation and Anti-Apoptosis of Prostate Cells through PKC Signaling Pathways.

Qi Hou,Jinyan Lin,Wentao Huang,Maoyin Li,Jianhua Feng,Xiangming Mao,Lucia R Languino

doi:10.1371/journal.pone.0134006

Abstract

C1q/TNF-related protein-3 (CTRP3) is a novel adipokine with roles in multiple cellular processes. However, little is known about its function in prostate cells. This study investigated the effects and mechanisms of CTRP3 in prostate cells. We first generated and purified CTRP3 protein in HEK 293T cells. Proliferation of RWPE-1 prostate cells was evaluated by MTT analyses under treatment with different concentrations of CTRP3 for various exposure times. The results revealed maximum enhancement of proliferation with 10 μg/mL CTRP3 for 72 h. Cell apoptosis and cell cycle were determined by TUNEL staining and flow cytometry analysis. TUNEL assay showed decreased TUNEL-positive cells in RWPE-1 prostate cells treated with CTRP3, and flow cytometry showed significantly decreased apoptotic cells upon CTRP3 treatment (treated cells, 8.34±1.175 vs. controls, 20.163±0.35) (P < 0.01). Moreover, flow cytometry analysis also showed a significant decrease of cells in the G1 phase and an increase of cells in the S and G2 phase upon CTRP3 treatment (treated cells, 42.85±1.40 vs. control, 52.77±0.90; 28.41±0.57 vs. 23.49±1.13; 27.08±1.97 vs. 22.20±1.32, respectively) (all P < 0.05). Two-dimensional gel electrophoresis and mass spectrometry identified differentially expressed proteins, including cytokeratin-19, GLRX3 and DDAH1, which were upregulated in CTRP3 treated cells, and cytokeratin-17 and 14-3-3 sigma, which were downregulated. GLRX3, DDAH1 and 14-3-3 sigma were confirmed using western blot analysis. A PKC inhibitor, staurosporine, was used to inhibit PKC activity in CTRP3 treated RWPE-1 cells. Staurosporine completely abolished the CTRP3-induced increased phosphorylation of intracellular PKC substrates and CTRP3-stimulated effect by RWPE-1 cells. Our results provide the first evidence for a physiological role of the novel adipokine, CTRP3, in prostate cells. Our findings suggest that CTRP3 could improve proliferation and anti-apoptosis of prostate cells through protein kinase C signaling pathways.

Highlights

C1q tumor necrosis factor-related proteins (CTRPs) are members of the highly conserved family of adiponectins
Compared with the control group, the numbers of RWPE-1 cells treated with 3 μg/mL, 10 μg/mL and 30 μg/mL C1q/TNF-related protein-3 (CTRP3) were increased by 62.75%, 89.07%, 106.98% at 24 h, respectively; 68.32%, 104.05%, 80.24% at 48 h; and 28.64%, 80.25%, 28.64% at 72 h
The differences between 10 μg/mL and 3 μg/mL, and 10 μg/mL and 30 μg/mL treatment groups at both 48 h and 72 h were significant. Together these results indicate that 10 μg/mL was the optimal concentration for CTRP3 in promoting proliferation of RWPE-1 prostate cells (Fig 2)

Summary

Introduction

C1q tumor necrosis factor-related proteins (CTRPs) are members of the highly conserved family of adiponectins. CTRP3 significantly inhibited LPS-induced IL-8 release without affecting IL-6 and TNF, lowered TGFβ levels in the supernatants of these cells, and reduced connective tissue growth factor expression [7]. CTRP3 was shown to be an important cytokine which plays crucial roles in regulating the growth of diverse types of cells, for instance, chondrogenic precursor cells [10], endothelial cells [11], osteosarcoma cells [12], vascular smooth cells [13] and cardiomyocytes [14]. By participating in adipokine secretion, fatty acid oxidation, inflammation, cell proliferation, differentiation and apoptosis, CTRP3 has broad functions in regulating various biological processes

Methods

Results

Conclusion