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J. Biol. Chem., Vol. 282, Issue 5, 3403-3412, February 2, 2007

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P2X7 Nucleotide Receptors Mediate Blebbing in Osteoblasts through a Pathway Involving Lysophosphatidic Acid^*

Nattapon Panupinthu¹, Lin Zhao, Fred Possmayer, Hua Z. Ke^¶2, Stephen M. Sims, and S. Jeffrey Dixon³

From the Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology and the Department of Obstetrics and Gynaecology and Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada and ^¶Pfizer Global Research and Development, Groton, Connecticut 06340

Extracellular nucleotides, released in response to mechanical or inflammatory stimuli, signal through P2 receptors in many cell types, including osteoblasts. P2X7 receptors are ATP-gated cation channels that can induce formation of large membrane pores. Disruption of the gene encoding the P2X7 receptor leads to decreased periosteal bone formation and insensitivity of the skeleton to mechanical stimulation. Our purpose was to investigate signaling pathways coupled to P2X7 activation in osteoblasts. Live cell imaging showed that ATP or 2 ',3 '-O-(4-benzoylbenzoyl)-ATP (BzATP), but not UTP, UDP, or 2-methylthio-ADP, induced dynamic membrane blebbing in calvarial osteoblasts. Blebbing was observed in calvarial cells from wildtype but not P2X7 knock-out mice. P2X7 receptors coupled to activation of phospholipase D and A₂, inhibition of which suppressed BzATP-induced blebbing. Activation of these phospholipases leads to production of lysophosphatidic acid (LPA). LPA caused dynamic blebbing in osteoblasts from both wild-type and P2X7 knock-out mice, similar to that induced by BzATP in wildtype cells. However, LPA-induced blebbing was more rapid in onset and was not affected by inhibition of phospholipase D or A₂. Blockade or desensitization of LPA receptors suppressed blebbing in response to LPA and BzATP, without affecting P2X7-stimulated pore formation. Thus, LPA functions downstream of P2X7 receptors to induce membrane blebbing. Furthermore, inhibition of Rho-associated kinase abolished blebbing induced by both BzATP and LPA. In summary, we propose a novel signaling axis that links P2X7 receptors through phospholipases to production of LPA and activation of Rho-associated kinase. This pathway may contribute to P2X7-stimulated osteogenesis during skeletal development and mechanotransduction.

Received for publication, June 12, 2006 , and in revised form, November 7, 2006.

^* This work was supported by the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Movies 1 and 2.

¹ Supported by a scholarship from the Faculty of Science, Mahidol University, Bangkok, Thailand.

² Present address: Amgen Inc., Thousand Oaks, CA 91320.

³ To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada. E-mail: jeff.dixon{at}schulich.uwo.ca.

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