Induction of Antiproliferative Connective Tissue Growth Factor Expression in Wilms' Tumor Cells by Sphingosine-1-Phosphate Receptor 2

Abstract

Connective tissue growth factor (CTGF), a member of the CCN family of secreted matricellular proteins, regulates fibrosis, angiogenesis, cell proliferation, apoptosis, tumor growth, and metastasis. However, the role of CTGF and its regulation mechanism in Wilms' tumor remains largely unknown. We found that the bioactive lipid sphingosine-1-phosphate (S1P) induced CTGF expression in a concentration-and time-dependent manner in a Wilms' tumor cell line (WiT49), whereas FTY720-phosphate, an S1P analogue that binds all S1P receptors except S1P 2 , did not. Further, the specific S1P 2 antagonist JTE-013 completely inhibited S1P-induced CTGF expression, whereas the S1P 1 antagonist VPC44116 did not, indicating that this effect was mediated by S1P 2 . This was confirmed by adenoviral transduction of S1P 2 in WiT49 cells, which showed that overexpression of S1P 2 increased the expression of CTGF. Induction of CTGF by S1P was sensitive to ROCK inhibitor Y-27632 and c-Jun NH 2 -terminal kinase inhibitor SP600125, suggesting the requirement of RhoA/ROCK and c-Jun NH 2 -terminal kinase pathways for S1P-induced CTGF expression. Interestingly, the expression levels of CTGF were decreased in 8 of 10 Wilms' tumor tissues compared with matched normal tissues by quantitative real-time PCR and Western blot analysis. In vitro, human recombinant CTGF significantly inhibited the proliferation of WiT49 cells. In addition, overexpression of CTGF resulted in significant inhibition of WiT49 cell growth. Taken together, these data suggest that CTGF protein induced by S1P 2 might act as a growth inhibitor in

Sphingosine 1-Phosphate in Renal Diseases

“…In this context, Katsuma et al [36] suggested that S1P 2 and/or S1P 3 , are involved in the S1P-induced CTGF expression in renal MCs. More recent data in tumor cells rather suggested the S1P 2 to be involved in the pro-fibrotic effects of eS1P [37]. This conclusion is derived from the finding that phospho-FTY720, which cannot bind to the S1P 2 [38], had no effect on CTGF expression in the Wilms' tumor cell line WiT49 ([37], see below).…”

Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma

“…By coupling primarily to the G 12/13 heterotrimeric G protein pathway, S1P 2 mediates different cellular functions and pathologies critical to immune, nervous, metabolic, cardiovascular, musculoskeletal, and renal systems . Although it is well known that S1P 2 regulates the Rho/Rho kinase pathway to inhibit tumor cell migration and lymphoma development (Cattoretti et al, 2009;Muppidi et al, 2014), studies from our group (Li et al, 2008a(Li et al, , 2009a(Li et al, , 2011(Li et al, , 2014 as well as others (Young et al, 2009;Ponnusamy et al, 2012;Orr Gandy et al, 2013) have found that S1P 2 plays important roles in tumor growth and progression in a variety of cancers, indicating that S1P 2 also acts as a protumorigenic receptor. The latter findings suggest potential therapeutic avenues that exploit S1P 2 to inhibit tumor growth in some situations.…”

“…In our previous studies, we demonstrated that S1P 2 controlled CTGF expression in Wilms' tumor, another pediatric solid malignancy, resulting in antiproliferative effects on the cancer cell itself (Li et al, 2008a). However, CTGF is known to play a significant role in tumor cell epithelial to mesenchymal transition, a well understood process that underlies fibrosis and can facilitate metastasis in the context of the tumor microenvironment (Singh and Settleman, 2010).…”

“…A SYBR Green-based quantitative real-time polymerase chain reaction was carried out as described (Li et al, 2009a). Primers were designed using Primer Express 2.0 (Applied Biosystems, Foster City, CA) and the detected genes were CTGF (Li et al, 2008a) and chemokine (C-C motif) ligand 2 (CCL2) (Li et al, 2014).…”

“…S1P 2 exerts diverse cellular functions by regulating different downstream effector molecules. Our prior studies as well as studies performed by others have shown that such molecules include intracellular signaling mediators (p-Akt, p-ERK), as well as growth and differentiation modulators such as CTGF (Sanchez et al, 2007;Li et al, 2008a). To further compare the efficiency of JTE-013 and AB1 in vitro, Western blot analysis was performed.…”