Association of the α2δ1 subunit with Cav3.2 enhances membrane expression and regulates mechanically induced ATP release in MLO-Y4 osteocytes

Abstract

Voltage sensitive calcium channels (VSCCs) mediate signaling events in bone cells in response to mechanical loading. Osteoblasts predominantly express L-type VSCCs composed of the α1 pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs, but a relatively small amount of L-type α1 subunits. Auxiliary VSCC subunits have several functions including modulating gating kinetics, trafficking of the channel and phosphorylation events. The influence of the α2δ auxiliary subunit on T-type VSCCs and the physiological consequences of that association are incompletely understood and have yet to be investigated in bone. In this study, we postulated that the auxiliary α2δ subunit of the VSCC complex modulates mechanically-regulated ATP release in osteocytes via its association with the T-type, Cav3.2 (α1H) subunit. We demonstrated by RT-PCR, Western blotting, and immunostaining that MLO-Y4 osteocyte-like cells express the T-type, Cav3.2 (α1H) subunit more abundantly than the L-type, Cav1.2 (α1C). We also demonstrated that the α2δ1 subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Cav3.2 (α1H) subunit in MLO-Y4 cells. Interestingly, siRNA mediated knockdown of α2δ1 completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the α2δ1 subunit and resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following α2δ1 knockdown showed decreased membrane localization of Cav3.2 (α1H) at the plasma membrane, suggesting that the diminished ATP release and ERK1/2 activation in response to membrane stretch resulted from a lack of Cav3.2 (α1H) at the cell membrane.

Ca v 3.2 T-type calcium channel is required for the NFAT-dependent Sox9 expression in tracheal cartilage

“…T-type Ca 2+ channels are highly expressed in many developing tissues and are implicated in regulating cellular events leading to cell proliferation and differentiation (50). Calcium influx through T-type Ca 2+ channels activates several cellular pathways, including calcineurin/NFAT (29,39), extracellular signal-regulated kinase (31,(51)(52)(53), nitric oxide synthase 3 (54), and Ca 2+ /calmodulindependent protein kinase II (55). However, the detailed mechanisms linking these T-type Ca 2+ channel-activated signaling pathways to cell proliferation and differentiation are not completely clear.…”

Matrix-dependent adhesion mediates network responses to physiological stimulation of the osteocyte cell process

“…Reports of force-induced mobilization and action of such molecules as PGE 2 (41,42), NO (25,43), VEGF (20), RANKL, OPG (44), ATP (30,45), and Ca 2+ (18,22) affirm autocrine/paracrine signaling in MLO-Y4 cells. Our evaluation of P2R signaling dynamics in MLO-Y4 cells after incubation with the broad-spectrum P2 receptor antagonist suramin demonstrated suppressed effector responses of MLO-Y4 cells, consistent with previously reported suramin-induced interference with P2Y and P2X receptor binding sites that blocked the action of ATP on purinergic receptors and subsequent signaling cascades (46,47).…”

Fluid Flow-Induced Calcium Response in Osteoclasts: Signaling Pathways

“…7,15,19 Lots of studies indicated that ATP could release from the mechanically stimulated osteoblasts or osteocytes and further binds the purinergic P2 receptor on the neighboring cells to cause the calcium release from ER. 10,40 It has been found that the P2X7 receptor is expressed in osteoclasts, 21 which may contribute to cell-cell calcium transfer. The influence of P2 receptors on osteoclast physiology and bone physiology in general is beginning to be understood.…”