Lysophosphatidic Acid Upregulates Recepteur D'origine Nantais Expression and Cell Invasion via Egr-1, AP-1, and NF-κB Signaling in Bladder Carcinoma Cells

Abstract

Muscle invasive bladder carcinoma is a highly malignant cancer with a high mortality rate, due to its tendency to metastasize. The tyrosine kinase recepteur d'origine nantais (RON) promotes bladder carcinoma metastasis. Lysophosphatidic acid (LPA) is a phospholipid derivative, which acts as a signaling molecule to activate three high affinity G-protein coupled receptors, LPA1, LPA2, and LPA3. This in turn leads to cell proliferation and contributes to oncogenesis. However, little is known about the effects of LPA on invasive bladder cancer (IBC). In this study, we discovered that LPA upregulated RON expression, which in turn promoted cell invasion in bladder cancer T24 cells. As expected, we found that the LPA receptor was essential for the LPA induced increase in RON expression. More interestingly, we discovered that LPA induced RON expression via the MAPK (ERK1/2, JNK1/2), Egr-1, AP-1, and NF-κB signaling axes. These results provide experimental evidence and novel insights regarding bladder malignancy metastasis, which could be helpful for developing new therapeutic strategies for IBC treatment.

Keywords: AP-1; Egr-1; NF-κB; bladder cancer; lysophosphatidic acid; recepteur d’origine nantais.

Figure 1

RON expression is higher in invasive bladder cancer than in normal bladder tissue. (A) The expression level of RON from a clinical sample database (https://www.oncomine.org). (B) H&E staining of normal bladder tissue (upper) and invasive bladder carcinoma (lower) sections. The photographs were taken under a microscope at 100× magnification. (C) Immunofluorescence staining was performed to detect RON protein in normal bladder and invasive bladder carcinoma tissue. RON positive cells were stained green, and the nuclei were stained blue. The photographs were taken with a fluorescence microscope at 400× magnification.

Figure 2

Induction of RON by lysophosphatidic acid (LPA) in T24 bladder cancer cells. (A,B) RT-PCR and Western blot analyses of the effect of LPA on RON mRNA and protein expression in T24 cells, respectively. Cells were incubated with 5 μM LPA for the indicated durations. (C) T24 cells were transiently transfected with the pGL3–RON reporter construct overnight. The transfected cells were incubated with 5 μM LPA for 0–8 h, and luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements.

Figure 3

Involvement of LPA receptors in LPA induced RON expression in T24 human bladder cancer cells. (A) Total RNA was extracted from T24 cells, and the mRNA levels of LPA1, LPA2, LPA3, and GAPDH were analyzed by RT-PCR. (B–D) T24 cells were transiently transfected with specific LPA1, LPA2, and LPA3 siRNA oligonucleotides (0–30 nM) for 5 h. After stabilization for 48 h, the amount of LPA1, LPA2, LPA3, and GAPDH mRNAs was determined by RT-PCR. (E) T24 cells transfected with 20 nM LPA1, LPA2, and LPA3 siRNA oligonucleotides were incubated with 5 μM LPA for 8 h, and then, RT-PCR was performed. (F) T24 cells transfected with specific LPA1, LPA2, and LPA3 siRNAs for 48 h were transiently transfected with a RON promoter reporter construct overnight. The transfected cells were incubated with 5 μM LPA for 8 h, and luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements. * p < 0.05 vs. LPA only.

Figure 4

Involvement of Erk-1/2 and JNK in LPA induced RON expression in human bladder cancer T24 cells. (A) T24 cells were incubated with 5 μM LPA for various periods, and the levels of phosphorylated Erk-1/2, P38 and JNK MAPK in the cell lysates were determined by Western blot analysis. (B) T24 cells, after being pretreated with PD98059 (PD, 50 μM), SB203580 (SB, 10 μM), and SP600125 (SP, 10 μM) for 1 h, were incubated with 5 μM LPA for 8 h. After incubation, the RON mRNA in the cell lysates was determined by RT-PCR analysis. (C) T24 cells transfected with Erk-1/2 (K97M) and JNK (TAM67) dominant negative mutants for 48 h were then co-transfected with a RON promoter reporter overnight. After incubation with 5 μM LPA PMA for 8 h, luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements. * p < 0.05 vs. LPA only.

Figure 5

Involvement of the transcription factor Egr-1 in LPA induced RON expression. (A,B) T24 cells were incubated with 5 μM LPA for indicated durations, and Egr-1 mRNA and protein expression were determined by RT-PCR and Western blot analysis, respectively. (C) T24 cells transfected with Egr-1 expression plasmids (0–2 μg) for 48 h were transiently co-transfected with a RON promoter reporter overnight, and luciferase activity was then measured using a luminometer. Bars show the mean standard deviation from three measurements. (D,E) T24 cells transfected with Egr-1 siRNA oligonucleotides for 48 h were treated with 5 μM LPA for the indicated durations, and RON mRNA and protein expression were determined by RT-PCR and Western blot analysis, respectively. (F) T24 cells transfected with Egr-1 siRNA oligonucleotides for 48 h were co-transfected with a RON promoter reporter overnight. After incubation with 5 μM LPA for 8 h, luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements. * p < 0.05 vs. LPA only.

Figure 6

Involvement of the AP-1 transcription factors in LPA induced RON expression. (A) Cells were transiently transfected with the pAP-1 luciferase reporter construct and then incubated with 5 μM LPA for 0–8 h. After incubation, the cells were lysed, and luciferase activity was measured. Bars show the mean standard deviation from three measurements. (B) T24 cells were incubated with 5 μM LPA for 0–8 h, and the mRNA levels of c-fos and c-jun were determined by RT-PCR. (C) T24 cells were incubated with 5 μM LPA for 0–120 min, and the levels of c-fos and c-jun phosphorylation were determined by Western blot analysis. (D) T24 cells pretreated with SR11032 (SR, AP-1 inhibitor, 0–1 μM) were incubated with 5 μM LPA for 8 h, and RON mRNA levels were determined by RT-PCR. (E) T24 cells were transfected with an AP-1 decoy for 48 h and co-transfected with a RON promoter reporter overnight. After incubation with 5 μM LPA for 8 h, luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements. * p < 0.05 vs. LPA only.

Figure 7

Involvement of the NF-κB transcription factor in LPA induced RON expression. (A) Cells were transiently transfected with the pNF-κB luciferase reporter construct and then incubated with 5 μM LPA for 0–8 h. After incubation, the cells were lysed, and luciferase activity was measured. Bars show the mean standard deviation from three measurements. (B) T24 cells were incubated with 5 μM LPA for 0–120 min, and the protein level of IκBa and level of IκBa and NF-κB-p65 phosphorylation were determined by Western blot analysis. (C) T24 cells were pretreated with BAY11-7082 (BAY, an NF-κB inhibitor, 0–20 μM) were incubated with 5 μM LPA for 8 h, and RON mRNA levels were determined by RT-PCR. (D) T24 cells transfected with I-κBα, I-κBβ, and NIK dominant negative mutants were co-transfected with a RON promoter reporter overnight. After incubation with 5 μM LPA for 8 h, luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements. * p < 0.05 vs. LPA only.

Figure 8

LPA effect on T24 cell invasiveness. (A) T24 cells (10⁵) were seeded onto Matrigel coated membranes of a chamber with 5 μM LPA for 24 h, in the presence or absence of anti-RON antibody (200 ng/mL). After incubation, the cells that had invaded the lower chambers were fixed with 100% methanol and stained with a Diff-Quick stain kit. Images of the invading T24 cells were obtained using a phase contrast light microscope. (B) T24 cells (10⁵) were seeded onto Matrigel coated membranes of a chamber with 5 μM LPA for 24 h, in the presence or absence of non-specific IgG (200 ng/mL), anti-uPA antibody (200 ng/mL), anti-uPAR antibody (200 ng/mL), siEgr-1 (20 nM, cells transfected with Egr-1 siRNA oligonucleotides for 48 h before LPA treatment), PD98059 (PD, 50 μM), SB203580 (SB, 10 μM), SP600125 (SP, 10 μM), SR11032 (SR, 1 μM), and BAY11-7082 (BAY, 10 μM). After incubation, the cells that had invaded the lower chambers were counted. The bars show the mean and SD from three measurements. * p < 0.05 vs. LPA only.