Cell Surface ATP Synthase: A Novel Mechanism for ATP Signaling in Osteoblasts

Anabolism of bone at the cellular level is controlled by the bone forming osteoblasts where purinergic signaling plays an important role. ATP which is mostly known for its role in energy metabolism has emerged as a signaling molecule whose mitogenic potential has been demonstrated in several cell types including osteoblasts. One of the earliest responses of osteoblasts to fluid shear stress (FSS) is a calcium dependent release of ATP via vesicles. However, static or unstimulated cells have also been shown to release ATP. Recently ATP synthase was discovered on the cell membrane of endothelial cells where it was shown to interact with caveolin 1 and to be involved in FSS mediated ATP release. Based on our previous studies and these observations, I hypothesized that osteoblasts express cell surface ATP synthase and that the activity of this molecular complex is responsible for the basal release of ATP. I further postulated that ATP synthase interacts with caveolin 1 and that this interaction aids in the regulation of ATP synthase localization and activity. I demonstrated that that osteoblasts indeed express cell surface ATP synthase where it is catalytically active and utilizes a proton gradient similar to the mitochondrial ATP synthase to synthesize ATP I also found that FSS increased the presence of membrane bound ATP synthase. Membrane bound ATP synthase was found to stimulate cellular proliferation and that this proliferation could be blocked with Piceatannol, an inhibitor of ATP synthase. I found that ATP synthase resides in the lipid raft region of the cell membrane where it interacts with caveolin 1. Furthermore, in silico analysis revealed that the c subunit of the F₀ portion of the ATP synthase was found to contain the caveolin binding domain. Interestingly, disruption of caveolae with Methyl-βcyclodextrin (MβCD), inhibiting caveolin 1 with Antennapaedia-caveolin 1 scaffolding domain fusion construct or suppression of caveolin 1 with siRNA failed to impair the catalytic activity of ATP synthase. Caveolin 1 siRNA also failed to alter membrane localization of ATP synthase. Thermal induction of endocytosis effectively removed ATP synthase membrane localization and activity could be restored 30 minutes after endocytosis induction. These studies suggest a novel mechanism of basal release of ATP in non-stimulated osteoblasts through ATP production which is mitogenic and may be an important contributor of bone formation. My studies also suggest that plasma membrane ATP synthase can be regulated by caveolin 1 and through this interaction may regulate osteoblast mechanosensitivity

Year	Entry
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Year

Entry

2003

Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. May 15, 2003;423(6937):337-342.

2002

Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. January 11, 2002;108(1):17-29.

1990

Reich KM, Gay CV, Frangos JA. Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. J Cell Physiol. April 1990;143(1):100-103.

2001

Ryder KD, Duncan RL. Parathyroid hormone enhances fluid shear-induced [Ca²⁺]_i signaling in osteoblastic cells through activation of mechanosensitive and voltage-sensitive Ca²⁺ channels. J Bone Miner Res. February 2001;16(2):240-248.

1993

Mori S, Burr DB. Increased intracortical remodeling following fatigue damage. Bone. March–April 1993;14(2):103-109.