Thromboxane A2 receptor-mediated G12/13-dependent glial morphological change

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Abstract

Glial cells express thromboxane A2 receptor, but its physiological role remains unknown. The present study was performed to examine thromboxane A2 receptor-mediated morphological change in 1321N1 human astrocytoma cells. Thromboxane A2 receptor agonists U46619 and STA2 caused a rapid morphological change to spindle shape from stellate form of the cells pretreated with dibutyryl cyclic AMP, but neither carbachol nor histamine caused the change, suggesting that Gq pathway may not mainly contribute to the change. Rho kinase inhibitor Y-27632 inhibited U46619-induced morphological change, and U46619 increased the GTP-bound form of RhoA accompanied with actin stress fiber formation. These responses were reduced by expression of p115-RGS that inhibits G12/13 signaling pathway. U46619 also caused the phosphorylation of extracellular signal-regulated kinase (ERK) and [3H]thymidine incorporation mainly through G12/13-Rho pathway. These results suggest that stimulation of thromboxane A2 receptor causes the morphological change with proliferation mainly through G12/13 activation in glial cells.

Introduction

Glial cells express a number of neurotransmitter and autacoid receptors (Keinanen et al., 1990, Vernadakis, 1996), which might have a role in neuron-glia and/or glia–glia interaction. We first demonstrated that 1321N1 human astrocytoma cells express thromboxane A2 receptor, stimulation of which results in an activation of Gq, which in turn activates phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PI-PLC)(Nakahata et al., 1989). PI-PLC produces inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) followed by an increase in intracellular Ca2+ concentration ([Ca2+]i) and activation of PKC, respectively (Nakahata et al., 1995, Sakai et al., 1996). In addition, it has been shown that rabbit astrocytes (Nakahata et al., 1992), rat oligodendrocytes (Blackman et al., 1998, Lin et al., 2005) and mouse astrocytes (Obara et al., 2005) also express thromboxane A2 receptor. Therefore, it is assumed that thromboxane A2 receptor expressed in glial cells plays an important role under the physiological or pathophysiological condition. Although Gq/PI-PLC is believed to be a main pathway of thromboxane A2 receptor-mediated signaling, it has been shown that thromboxane A2 receptor also couples to G12 family G proteins G12/13 (Offermanns et al., 1994, Allan et al., 1996, Djellas et al., 1999), Gi (Ushikubi et al., 1994), G16 (Van Der Vuurst et al., 1997) and Gh (Vezza et al., 1999). Furthermore, it has been shown that thromboxane A2 receptor stimulation increased cyclic AMP through adenylyl cyclase activation in some cases (Murray et al., 1990), but it remains unclear whether Gs is directly communicated with thromboxane A2 receptor. We previously demonstrated that the partially purified fraction of thromboxane A2 receptor from 1321N1 cells contains Gαq/11 and Gα12 (Honma et al., 1998). The coupling of thromboxane A2 receptor with G12 family G proteins has been reported to be physiologically important in platelet shape change and vascular smooth muscle contraction (Klages et al., 1999, Gohla et al., 2000).

The G12 family G proteins, Gα12 and Gα13, are ubiquitously expressed and share 67% amino acid sequence identity (Dhanasekaran and Dermott, 1996). It is well known that Gα12 and Gα13 regulate the activity of small GTPase Rho and modulate the actin cytoskeleton, cytokinesis, cell motility and contraction (Hall, 1998). As one of guanine nucleotide exchange factors (GEF) for Rho, p115RhoGEF is reported to be downstream of G12/13 (Kozasa et al., 1998). Although thromboxane A2 receptor has been shown to couple with G12 family G proteins, it remains unclear what kinds of cell responses are downstream of G12 family G proteins in glial cells. More recently, we have shown that stimulation of thromboxane A2 receptor facilitates interleukin-6 production through Gq and G13 in human astrocytoma cells (Obara et al., 2005).

It has been shown that dibutyryl cyclic AMP (dbcAMP) causes the morphological change of glial cells to be a process-bearing stellate form, showing cell differentiation (Su et al., 2000). We previously reported that 9,11-epithio-11,12-methano thromboxane A2 (STA2), a thromboxane A2 receptor agonist, caused an activation of p44/42 mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) to a greater extent in 1321N1 human astrocytoma cells differentiated with dbcAMP than in non-differentiated cells (Kobayashi et al., 2000). The ERK activation was not mediated via PI-PLC, but was sensitive to D-609, a phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor. It has been shown that stimulation of thromboxane A2 receptor activates ERK cascade in vascular smooth muscles resulting in cell growth (Morinelli et al., 1994). In platelets, which are not proliferative, the stimulation of thromboxane A2 receptor activates ERK cascade resulting in cPLA2 activation and release of arachidonic acid (Ohkubo et al., 1996). However, the physiological role of ERK activation in glial cells has not been fully understood, so far.

In the course of the study for thromboxane A2 receptor-mediated signaling, we found that stimulation of thromboxane A2 receptor resulted in the rapid morphological change of the differentiated 1321N1 human astrocytoma cells pretreated with dbcAMP. The present study was, therefore, undertaken to investigate the signaling pathway of thromboxane A2 receptor-mediated morphological change in 1321N1 human astrocytoma cells differentiated with dbcAMP. The results suggest that thromboxane A2 receptor-mediated morphological change is due to the activation of RhoA mainly through G12/13, accompanied with cell proliferation through ERK activation.

Section snippets

Materials

Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Fetal calf serum was purchased from General Scientific Laboratory (Los Angels, CA, U.S.A.). [1S-[1α,2β(5Z),3β,4α]]-7-[3-[[2-(Phenylamino)-carbonyl]hydrazino]methyl]-7-[oxabicyclo[2.2.1]hept-2-yl]-5-heptenic acid (SQ29548) and 9,11-dideoxy-9α, 11α,-epoxymethanoprostagrandin F2α (U46619) were purchased from Cayman Chemicals (Ann Arbor, MI, U.S.A.). 9,11-Epithio- 11,12-methano thromboxane A2

Thromboxane A2 receptor-mediated morphological change in dbcAMP-treated 1321N1 cells

Human astrocytoma cells (1321N1) showed the morphology with spindle shape when they were cultivated in the medium containing 5% FCS (Fig. 1A). These cells changed their morphology to be stellate after the culture in the medium containing 0.5 mM dbcAMP for 1 day (Fig. 1B). When the dbcAMP-treated cells were stimulated with U46619, a thromboxane A2 receptor agonist, they rapidly changed their morphology to spindle shape within 20 min (Fig. 1C). This morphological change lasted at least 24 h after

Discussion

We showed here that thromboxane A2 receptor stimulation caused a rapid morphological change mainly through G12/13-Rho-dependent pathway in 1321N1 human astrocytoma cells differentiated with dbcAMP. This thromboxane A2 receptor-mediated morphological change was accompanied with ERK activation and DNA synthesis.

U46619 and LPA, but not carbachol or histamine, cause a morphological change of dbcAMP-treated 1321N1 cells. Carbachol and histamine have been shown to activate Gq/PI-PLC pathway through M3

Acknowledgments

This work was supported, in part, by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (No. 14370737 to N.N.), and from Uehara Memorial Foundation (to S.O.), and from Kuribayashi Educational Work, Scientific Foundation (to S.H.).

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