Urocortin affects migration of hepatic cancer cell lines via differential regulation of cPLA2 and iPLA2

doi:10.1016/j.cellsig.2014.02.002

Cellular Signalling

Volume 26, Issue 5, May 2014, Pages 1125-1134

https://doi.org/10.1016/j.cellsig.2014.02.002 Get rights and content

Highlights

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UCN decreased iPLA2 expression via CRFR2 receptor and inhibited cell migration.
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UCN increased cPLA2 expression via CRFR1 receptor and promoted cell migration.
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Stable transfection of CRFR1 or CRFR2 into SMMC-7721 verified the effects of CRFRs.
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NF-κB played a role in the UCN-induced cPLA2 expression.

Abstract

Urocortin (UCN) is a member of corticotrophin-releasing factor (CRF) family, which has been reported to play a role in many biological processes, including inflammation and cancer development. Growing evidence shows that PLA2 (phospholipase A2) enzymes also participate in inflammation and tumor development. The primary aim of the present study was to identify a novel signaling pathway of CRF receptor activation leading to migration of two kinds of hepatoma carcinoma cell lines, HepG2 and SMMC-7721, linking the stimulation of PLA2 expression by UCN to UCN-induced tumor cell migration. Pharmacological inhibitors and genetic approaches (such as stable transfection and siRNAs) were used in this study. Unlike HepG2 cells which express both CRF receptors themselves, SMMC-7721 cells which hardly express these two CRF receptors needed stable transfection with CRFR1 or CRFR2 to observe the effect of UCN. Two types of PLA2 enzymes, cPLA2 and iPLA2, were found to be regulated by UCN. Our data showed that UCN raised cPLA2 expression but lowered iPLA2 expression. Moreover, UCN was found to act on the certain region of iPLA2 promoter to reduce its transcription. UCN promoted tumor cell migration by up-regulating cPLA2 expression via CRFR1 whereas it suppressed tumor cell migration by down-regulating iPLA2 expression via CRFR2. These results indicate the dual roles for UCN in the hepatoma carcinoma cell migration, which involve the regulation of both cPLA2and iPLA2.

Introduction

Corticotrophin-releasing factor (CRF) was first isolated in1981 [1], and its natural receptors include CRFR1 and CRFR2 [2]. CRF acts through these two G-protein-coupled transmembrane receptors to affect various physiological and pathological processes [3]. Afterwards, three mammalian CRF-like paralogs, including UCN, UCN2 and UCN3 [4], [5], [6], [7], have been identified. Among CRF-related family peptides, UCN can bind to two types of CRF receptor with similar affinities. By contrast, UCN2 binds to CRFR2 with higher affinities than CRFR1 [3].

Our previous work demonstrated that UCN suppresses tumor growth via CRFR2 [8]. Others obtained similar outcomes in different tumor cells [9]. Many researches showed that activation of CRFR1 can also inhibit the proliferation of multiple tumor cells such as Ishikawa (IK) human endometrial carcinoma cell line [10], mouse melanoma cells [11] and human breast cancer cells [12]. Although most reports support that activation of CRFR1 or CRFR2 inhibit tumor growth, there were many studies indicating that CRFR activation promotes angiogenesis, tumor cell proliferation and migration [13], [14]. These opposite results made the effect of CRF system in the study of tumor more complex. Up to date, it is still not clear whether and how CRFR1 and CRFR2 are differently involved in tumor development and progression.

Phospholipase A2(PLA2) is a wide class of enzyme that catalyzes deacylation of the sn-2 position of glycerophospholipids producing two subclasses of lipid mediators, fatty acid derivatives and lysophospholipids. So far, based on their cellular localization, substrate specificity, and calcium dependence, more than 20 identified PLA2 have been classified into four categories: secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca^2 +-independent PLA2 (iPLA2) and PAF acetylhydrolases (PAFAH) [15]. Many researches showed that PLA2 took part in a lot of physiological and pathological processes, such as inflammation, cell growth and death, cellular signaling pathway, cancer metastasis, tumor angiogenesis and so on [16], [17], [18].

Lawrence et al. reported that UCN could attenuate Ca^2 +-independent PLA2 gene expression to protect cardiac myocytes from ischemia/reperfusion injury in 2003 [19]. Lately, direct evidence showed that UCN2 down-regulated iPLA2 expression in the dystrophic muscle through both cAMP and Epac pathways [20]. In contrast, Gao et al. found that CRF or UCN could increase the expression of cPLA2 in cultured human placental trophoblasts [21].

In this work we investigated whether UCN took part in tumor cell migration through regulating the expression of PLA2. At first, we observed the effect of UCN in HepG2 which expresses both the two CRF receptors. In particular, since SMMC-7721 hardly expresses CRFR1 or CRFR2 [8], we performed stable transfection of CRFR1 or CRFR2 plasmid into SMMC-7721 to better observe the separate effects of the two receptors. Hence with these two kinds of cell lines, we could observe either the effect of two CRF receptors at the same time or the effect of single CRF receptor separately. Interestingly, our results showed that UCN increased cPLA2 expression by CRFR1, whereas it decreased iPLA2 expression by CRFR2. As a result, UCN promoted tumor cell migration via CRFR1 while suppressed tumor cell migration via CRFR2. These data provide a new insight into the importance of the dual characters of UCN and may contribute to the establishment of a potential therapeutic target for hepatoma carcinoma cell migration.

Section snippets

Cell culture and reagents

Human hepatocellular carcinoma cell lines HepG2 and SMMC-7721 were maintained in DMEM high glucose medium containing 10% fetal bovine serum at 37 °C with 5% CO₂ (Gibco). After starvation for more than 6 h in DMEM high glucose medium containing 0% serum, cells were treated with indicated drugs in DMEM high glucose medium containing 2% serum. In Wound-healing assay, cells were maintained in DMEM containing 10% serum after starvation.

UCN (10^− 8 M), UCN2 (10^− 8 M), CRFR1 antagonist Antalarmin (Anta, 10^− 7

UCN decreased iPLA2 expression via CRFR2 receptor

HepG2 cells express both CRFR1 and CRFR2 (Fig. 1G). iPLA2 mRNA and proteins were analyzed by real-time PCR and western blot. Treatment with UCN made HepG2 cells express lower mRNA after 8 h and lower proteins after 24 h (Fig. 1A and D). The decrease of iPLA2 at protein level treated with UCN was attenuated by the Anti-30 (selective CRFR2 inhibitor) pretreatment. However, Anta (selective CRFR1 inhibitor) addition made no changes (Fig. 1E). By means of reporter assay, we found that UCN decreased

Discussion

The data presented in this study indicated that UCN could regulate tumor cell migration via two types of its natural receptor. Two independent PLA2 enzymes, iPLA2 and cPLA2, were involved in the regulation of cell migration by UCN. In HepG2 cells, UCN decreased iPLA2 expression via CRFR2 to suppress cell migration while it increased cPLA2 expression via CRFR1 to promote cell migration. The suppression of cell migration was augmented by the addition of BEL and the promotion of cell migration was

Conflict of interest

There is no conflict of interests in this work.

Acknowledgments

This work was supported by the Natural Science Foundation of China [81072668, 91129711]; the Jiangsu Provincial Funds [No. 10KJA350031], and also funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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¹: These authors contributed equally to this work.

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Urocortin affects migration of hepatic cancer cell lines via differential regulation of cPLA2 and iPLA2

Highlights

Abstract

Introduction

Section snippets

Cell culture and reagents

UCN decreased iPLA2 expression via CRFR2 receptor

Discussion

Conflict of interest

Acknowledgments

Front. Neuroendocrinol.

Mol. Cell. Endocrinol.

Am. J. Pathol.

Eur. J. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Mol. Immunol.

J. Biol. Chem.

J. Biol. Chem.

Science

Proc. Natl. Acad. Sci. U. S. A.

Nature

Nat. Med.

Proc. Natl. Acad. Sci. U. S. A.

Proc. Natl. Acad. Sci. U. S. A.

Cancer Invest.

Proc. Natl. Acad. Sci. U. S. A.

Oncol. Rep.

Anticancer Res.

Exp. Dermatol.

Mol. Cancer

IUBMB Life

Cardiovasc. Drugs Ther.

Am. J. Physiol. Cell Physiol.

J. Natl. Cancer Inst.

FASEB J.

Endocrinology