Lysophosphatidic acid stimulates gastric cancer cell proliferation via ERK1-dependent upregulation of sphingosine kinase 1 transcription

Abstract

In MKN1 gastric cancer cells, lysophosphatidic acid (LPA) upregulates expression of sphingosine kinase 1 (SphK1) and its downregulation or inhibition suppresses LPA mediated proliferation. Although LPA activates numerous signaling pathways downstream of its receptors, including extracellular-signal-regulated kinase 1/2, p38, JNK, and Akt, and the transactivation of the epidermal growth factor receptor, pharmacological and molecular approaches demonstrated that only activation of ERK1, in addition to the CCAAT/enhancer-binding protein β transcription factor, is involved in transcriptional upregulation of SphK1 by LPA. Our data implicate ERK1 as an important mediator of LPA signaling leading to upregulation of SphK1 and point to SphK1 and sphingosine-1-phosphate production as potential therapeutic targets in gastric cancer.

Fig. 1. LPA induced proliferation of MKN1 cells requires SphK1

(A) MKN1 cells transfected with siControl or siSphK1 were stimulated without or with 10 μM LPA and cell growth determined with WST-1 on the indicated days. (B) MKN1 cells were cultured in the absence or presence of LPA (10 μM) and 5 μM SK1-I and cell growth determined with WST-1 on the indicated days. (C) Cells were cultured for 5 days in the absence or presence of LPA (10 μM) without or with 1, 3, 5, and 10 μM SK1-I and proliferation determined by crystal violet staining. Data are expressed as fold ± SD of triplicate determinations. *, P < 0.05, compared to LPA treated cells.

Fig. 2. Effects of inhibitors of LPA signaling pathways on SphK1 upregulation

(A) MKN1 cells were stimulated with 1 μM LPA for the indicated times, equal amounts of cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Blots were stripped and immunoblotted with anti-ERK2, anti-Akt, and anti-tubulin to ensure equal loading and transfer. (B) MKN1 cells were pretreated with vehicle or with Ki16425 (10 μM), AG1478 (1 μM), Go6983 (10 μM), SB202190 (1 μM), SP600125 (10 μM), BAY11-7082 (1 μM), CAY10470 (1 μM), LY294002 (10 μM), PX-866 (100 nM), Akt VIII (10 μM), U0126 (10 μM), or PD184352 (10 μM), for 1 hour, and subsequently stimulated with LPA (10 μM) for 6 hours. RNA was isolated, reverse-transcribed, and SphK1 mRNA measured by QPCR. Data are expressed as fold induction after normalization to GAPDH. *, P < 0.001, ^#, P< 0.05 compared to LPA treated cells. (C) Cultures were pretreated with the indicated inhibitors for 1 hour prior to stimulation with LPA (10 μM) for 20 min. Equal amounts of lysate proteins were analyzed by immunoblotting with anti-phospho-Akt and anti-phospho-ERK1/2 antibodies. Blots were stripped and reprobed with anti-calnexin antibody to ensure equal loading and transfer.

Fig. 3. ERK1 is important for upregulation of SphK1 by LPA

(A) MKN1 cells were transfected with ON TARGET plus SMART pool siRNA targeted to Akt-1, Akt-2, Akt-3 or with control siRNA. RNA was isolated and mRNA levels were measured by QPCR. (B) Duplicate cultures were stimulated for 6 hours without or with LPA and SphK1 mRNA measured by QPCR. (C,D) MKN1 cells were transfected with ON TARGET plus SMART pool siRNA targeted to ERK1, ERK2 or control siRNA. (C) RNA was isolated and mRNA levels were measured by QPCR. (D) Western blotting analysis of ERK1/2 expression. (E) Duplicate cultures were stimulated for 6 hours without or with LPA and SphK1 mRNA measured by QPCR. (A,B,C,E) Data are expressed as fold change after normalization to GAPDH. *, P < 0.05, compared to LPA stimulated siControl.

Fig. 4. Involvement of C/EPBβ in LPA-induced transcriptional upregulation of SphK1

(A) MKN1 cells were pretreated for 1 hour with actinomycin D (5 μg/ml) and then stimulated for 6 hours without or with LPA and SphK1 mRNA measured by QPCR. (B) Cells were stimulated with LPA for the indicated times and equal amounts of lysates analyzed by immunoblotting with anti-phospho-C/EPBβ (Thr235) antibody or with antiC/EPBβ antibody. The LAP and LIP subunits are indicated. (C) MKN1 cells transfected with vector or LIP were stimulated without or with LPA for 6 hours and SphK1 mRNA measured by QPCR (Insert: western blotting demonstrating LIP expression). (D,E) MKN1 cells were pretreated for 30 min with vehicle, AG1478 (1 μM) (D,E), or U0126 (10 μM) (E), as indicated. Cells were then stimulated for 5 min with vehicle, LPA (10 μM), or EGF (10 ng/ml) (D), or for the designated times (E), as indicated. Proteins were analyzed by immunoblotting with antibodies against phospho-ERK1/2, phospho-Akt, tubulin, phospho-C/EPBβ (Thr235), or ERK1/2.