The authors have declared that no competing interests exist.
Conceived and designed the experiments: QH. Performed the experiments: QH JL. Analyzed the data: QH JL WH. Contributed reagents/materials/analysis tools: QH JF XM. Wrote the paper: QH JL ML.
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) (
C1q tumor necrosis factor-related proteins (CTRPs) are members of the highly conserved family of adiponectins. Each of the 15 known members (CTRP1–CTRP15) exhibits homologous structures composed of four distinct domains: a signal peptide at the N terminus, a short variable region, a collagenous domain and a C-terminal globular domain [
CTRP3 (also known as cartducin, cartonectin) was first reported as a growth plate cartilage-derived secretory protein and identified as a novel adipokine [
Though CTRP3 is acknowledged as a novel cytokine, its other functions in metabolism and endocrine are still unknown. Based on the findings that CTRP3 stimulates proliferation and anti-apoptosis of several types of cells, in this study, we investigated the potential functions of CTRP3 in regulating cell growth, differentiation and apoptosis of prostate cells.
The human prostate epithelial cell line RWPE-1 was purchased from the American Type Culture Collection (ATCC Number CRL-11609). RWPE-1 cells were maintained in keratinocyte serum-free medium (KSFM; GIBCO Laboratories, Grand Island, NY) supplemented with 50 mg/L bovine pituitary extract, 5% l-glutamine and 5 μg/L EGF. RWPE-1 cells were maintained in a humidified incubator (5% CO2) at 37°C.
Cells were treated with various concentrations of CTRP3 as indicated and analyzed as described below. In the control experiments, 0.1 M phosphate buffer (pH 7.2) containing 0.1% gelatin and 40% glycerol was added to the culture.
To detect the effects of a PKC inhibitor, staurosporine (Santa Cruz, CA, USA), on CTRP3-induced proliferation, anti-apoptosis and change of cell cycle, RWPE-1 cells were pretreated with 0.25μmol/L of staurosporine before and during the stimulation with 10 μg/mL CTRP3.
Monoclonal anti-FLAG M2 antibody was purchased from Sigma-Aldrich (St. Louis, MO, MA USA). Goat anti-human CTRP3 antibody was purchased from Abcam (ab36870, Cambridge, MA USA). GLRX3, DDAH1 and 14-3-3 sigma antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-(ser) PKC substrate antibody 2261 was purchased from Cell Signaling Technology (CST, MA, USA).
A pcDNA3.1 expression construct encoding a C-terminal FLAG-tagged CTRP3 was used to generate CTRP3 protein. HEK 293T cells were cultured in FreeStyle293 expression medium, then transfected using 293fectin (Invitrogen) according to the manufacturer’s instructions. Four days later, an anti-FLAG affinity gel (Sigma-Aldrich) was used to purify the supernatants. Purified protein was dialyzed against 20 mmol/L HEPES buffer (pH 8.0).
Prostate cancer tissue was used as a positive control in western blot analysis of purified CTRP3 protein. The specimen was collected from a prostate cancer patient confirmed by pathology. This study was ratified by the ethics committees of Central Hospital of Longgang District. The piece of tissue was crushed in liquid nitrogen, and then used to extract protein. Total cellular proteins were prepared from treated or untreated cells by lysing cells in lysis buffer (CelLytic, Sigma-Aldrich). Lysates were resolved on 10% or 12.5% polyacrylamide gels. Proteins were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA), which was blocked with 5% milk containing TBS-T buffer (0.05% Tween-20) and incubated with primary antibodies at 4°C overnight. After washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, donkey anti-goat IgG, or goat anti-mouse IgG antibodies (Jackson, USA). Immunoreactive bands were visualized with the Western Breeze chromogenic detection system (Invitrogen).
RWPE-1 cells were seeded in a 96-well plate at a density of 1 x 103 cells/well and treated with various concentrations of CTRP3 (3 μg/mL, 10 μg/mL and 30 μg/mL) in triplicate at 37°C, with 5% CO2. After incubation, cell proliferation was determined by the MTT assay. MTT (20 μl; 5 mg/mL PBS, pH 7.4; Sigma-Aldrich) was added to the cells for 4 h. Then, MTT-containing medium was removed and 100 μL dimethyl sulfoxide (Sigma-Aldrich) was added to each well for 10 min. The optical density (OD) of the samples was measured at an absorbance of 490 nm (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA).
The number of apoptotic cells was measured by flow cytometry using the Annexin V-FITC apoptosis detection kit (Roche, Germany). Approximately 1–5 x 105 cells were acquired in each sample. Samples were analyzed by bivariate flow cytometry using a BD FACSCanto cytometer equipped with FlowJo software.
TUNEL staining was performed using the In Situ Cell Death Detection Kit-TMR red (Roche, Germany) following the manufacturer’s instructions. Both TMR (red) and DAPI (blue) fluorescence were visualized and imaged using an IX53 inverted phase contrast microscope (OLYMPUS Inc., Japan).
RWPE-1 cells were harvested by trypsin-EDTA and fixed in ethanol (70% in PBS). At least 1 to 2 h before flow cytometry, cells were resuspended and then stained with 10 μg/mL RNase A and 5 μg/mL propidium iodide in PBS. Cell distribution in the different phases of the cell cycle was analyzed by flow cytometry (FACSCalibur-S System, BD Bioscience) equipped with FlowJo software.
Cells (2.5 × 107 cells) were lysed in 100 μL cell lysis buffer (C2360, CelLytic, Sigma-Aldrich). The samples were placed at room temperature for 60 min and then centrifuged for 20 min at 4°C, 14,000 rpm. The supernatant was harvested and proteins were collected and stored at -80°C. For first D electrophoresis, the protein samples mixed with 350 μL rehydration buffer were loaded onto each IPG strip in a 2D gel system. Then, strips were transferred with 12% SDS-polyacrylamide gel on the Amersham Dalt II system.
Protein identification via mass spectrometric peptide sequencing was conducted at an 4700 Proteomics Analyzer using MALDI TOF/TOF (Applied Biosystems, Foster City, CA). The resulting MS and MS/MS peptides were matched to the Swiss-Prot and NCBI protein databases.
Data were expressed as mean ± SE and evaluated statistically using t-test and repeated measures ANOVA with SPSS (version 19.0) software. A value of P < 0.05 was considered statistically significant.
To generate purified CTRP3 protein, we transiently transfected HEK 293T cells with a pcDNA3.1 expression construct encoding a C-terminal FLAG-tagged CTRP3 (
(A) RT-PCR analysis of the pcDNA3.1 expression construct encoding a C-terminal FLAG-tagged CTRP3. (B) Western blot analysis of HEK 293T cells expressing ectopic CTRP3 protein. (C) Western blot analysis of purified CTRP3 protein. CTRP3 protein expressed in prostate cancer tissue was used as a positive control. Lane 1 and Lane 2 were the results from the same sample.
Previous studies showed that CTRP3 could promote proliferation of several types of cells. Thus, we first determined whether CTRP3 also could promote the proliferation of RWPE-1 prostate cells. We evaluated the number of cells treated with different concentrations of CTRP3 (0, 3, 10, 30 μg/mL) for different incubation times (0, 24, 48, 72 h). Compared with the control group, the numbers of RWPE-1 cells treated with 3 μg/mL, 10 μg/mL and 30 μg/mL 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 (all
Cells were treated with various concentrations of CTRP3: (A) 0 μg/mL, (B) 3 μg/mL, (C) 10 μg/mL, (D) 30 μg/mL. The optimum concentration of CTRP3 for the growth of RWPE-1 was 10 μg/mL.
The above results showed that the maximal effect of stimulating cell proliferation occurred with 10 μg/mL CTRP3 in RWPE-1 prostate cells for 72 h. We next investigated whether CTRP3 protected RWPE-1 cells from apoptosis to promote cell cycle progression under these conditions by TUNEL staining and flow cytometry analyses. We observed a decreased number of TUNEL-positive cells among the DAPI-positive cells in cells treated with CTRP3 (
(A) TUNEL-positive cells among DAPI-positive cells were decreased in cells treated with CTRP3. TUNEL-positive apoptotic nuclei and DAPI-stained nuclei were visualized at ×200 magnification. (B) Flow cytometry analysis demonstrated a significantly decreased ratio of apoptotic cells in cells treated with CTRP3. Data are representative of three independently performed experiments. Mean±SD. *
We next sought to identify differentially expressed proteins in CTRP3-treated prostate cells. We performed 2D-PAGE analyses with lysates from RWPE-1 cells treated with 10 μg/mL CTRP3 for 72 h and control cells, using three pairs of samples. Overall, the protein spot patterns of individual samples were mostly consistent, indicating no heterogeneity of each sample (
Data are representative of three independent performed experiments.
Peptide sequence | Protein | |
---|---|---|
Down-regulated | keratin 17 | |
14-3-3 Sigma | ||
Up-regulated | keratin 19 | |
GLRX3 | ||
DDAH1 |
(A) 14-3-3 sigma, (B) DDAH1, (C) GLRX3.
The above results strongly indicated that CTRP3 stimulated proliferation and anti-apoptosis of prostate cells through PKC signaling pathways. To further confirm that, PKC activity in CTRP3 treated RWPE-1 cells was inhibited using a PKC inhibitor, staurosporine. Western blot analysis showed that the phosphorylation of intracellular PKC substrates increased in CTRP3 treated RWPE-1 cells and went back to normal when pretreated with PKC inhibitor (
(A) Phosphorylation of intracellular PKC substrates increased in CTRP3 treated RWPE-1 cells and went back to normal when pretreated with PKC inhibitor. (B) RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by a MTT assay. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated proliferation in RWPE-1 cells.
RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by flow cytometry. The percent of RWPE-1 cells in the G1 phase decreased significantly upon CTRP3 treatment whereas the percentage of cells in the S and G2 phase increased. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated effect. Mean±SD. *
CTRP3 and adiponectin have highly homologous structures, which is a characteristic of the CTRP family. While structurally related to the other 15 members of the CTRP family, CTRP3 emerges as a novel adipokine with potential functions in the regulation of glucose metabolism and lipid metabolism. However, some studies also showed that CTRP3 is related with tumor development [
In the present study, we provide the first data on the biological effects of CTRP3 in prostate cells. We showed that CTRP3 promoted RWPE-1 prostate cell proliferation in a concentration-dependent and time-dependent manner. We found that the most effective concentration in inducing proliferation of RWPE-1 cells was 10 μg/mL. In addition, upon treatment of RWPE-1 cells with 10 μg/mL CTRP3, we found that the levels of apoptotic cells significantly decreased. Therefore, our results show that CTRP3 could stimulate proliferation and anti-apoptosis activity in prostate cells.
So far, little is known about CTRP3-specific receptor or signaling pathways. Hou
2D gel electrophoresis has been developed as a standardized and widely-used technique, thus offering new opportunities for pathways detection. Its advantages include high resolving power and the ability to display several thousand proteins at once. Because of these advantages, we used 2D gel electrophoresis to explore the possible mechanisms of CTRP3 impact on the prostate cells. We identified three proteins (cytokeratin-19, GLRX3, DDAH1) that showed upregulated expression in response to CTRP3 and two proteins (cytokeratin-17 and 14-3-3 sigma) that were downregulated. We focused on GLRX3, DDAH1 and 14-3-3 sigma, as these proteins exhibit specific functions in signaling pathways, and confirmed the 2D results using western blot analysis.
14-3-3 sigma is an important regulator involved in signaling transduction, stress response, apoptosis, transcriptional regulation and coordination of cell adhesion and motility [
GLRX3, also known as PICOT (PKC-interacting cousin of thioredoxin), is a member of the glutaredoxin family, which includes oxidoreductase enzymes that reduce a variety of substrates using glutathione as a cofactor. GLRX3 contains an N-terminal thioredoxin homology domain that binds and modulates the function of PKC theta [
Members of the PKC family of intracellular serine/threonine kinases play key roles in the regulation of cellular differentiation and proliferation in diverse cell types and in response to varied cytokines and hormones. Both 14-3-3 sigma and GLRX3 identified in this study play a part in PKC signaling pathways. Furthermore, 14-3-3 sigma, which exhibits inhibitory effects in PKC signaling pathways, was downregulated in response to CTRP3, and GLRX3, which is a direct regulator in PKC signaling pathways, was upregulated. The pattern of differential protein expression strongly indicates that CTRP3 stimulates proliferation and anti-apoptosis of prostate cells through PKC signaling pathways. In the present study, CTRP3 could increase phosphorylation of intracellular PKC substrates in RWPE-1 cells. Moreover, inhibition of PKC activity by a PKC inhibitor staurosporine completely abolished the increased phosphorylation of intracellular PKC substrates and CTRP3-stimulated effect in RWPE-1 cells. Thus, our findings that the stimulatory effect of CTRP3 on proliferation and anti-apoptosis is mediated through PKC signaling pathway is undoubted. In addition, 14-3-3 sigma, GLRX3 and DDAH1 have been shown to function in various kinds of tumors as well as prostate cancer. Together this suggests that CTRP3 may promote the transformation from prostate cells to cancer cells.
In summary, our data support CTRP3 as a novel cytokine for stimulating proliferation and anti-apoptosis of prostate cells through PKC signaling pathways. Furthermore, the potential involvement between CTRP3 and prostate cancer may provide new insights into the molecular mechanism underlying prostate cancer.
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