Examining Mechanisms for Voltage-Sensitive Calcium Channel-Mediated Secretion Events in Bone Cells

Abstract

In addition to their well-described functions in cell excitability, voltage-sensitive calcium channels (VSCCs) serve a critical role in calcium (Ca²⁺)-mediated secretion of pleiotropic paracrine and endocrine factors, including those produced in bone. Influx of Ca²⁺ through VSCCs activates intracellular signaling pathways to modulate a variety of cellular processes that include cell proliferation, differentiation, and bone adaptation in response to mechanical stimuli. Less well understood is the role of VSCCs in the control of bone and calcium homeostasis mediated through secreted factors. In this review, we discuss the various functions of VSCCs in skeletal cells as regulators of Ca²⁺ dynamics and detail how these channels might control the release of bioactive factors from bone cells. Because VSCCs are druggable, a better understanding of the multiple functions of these channels in the skeleton offers the opportunity for developing new therapies to enhance and maintain bone and to improve systemic health.

Keywords: Bone secretion; Calcium homeostasis; Extracellular vesicles; Mechanosensation; Voltage-sensitive calcium channels.

Fig. 1. VSCCs activate signaling pathways that directly or indirectly regulate bone cell secretion and function.

Upon mechanical stimulation, voltage-sensitive calcium channels (VSCCs) initiate a myriad of intracellular signaling events that result in the release of prostaglandin E₂ (PGE₂), ATP, and nitric oxide (NO). Secretion of these molecules activates various signaling pathways that alter gene expression and induce the synthesis of osteogenic and resorptive factors to modulate skeletal adaptation. Ca²⁺-influx via VSCCs also trigger release of intracellular Ca²⁺ from ER stores and activate downstream Ca²⁺-dependent pathways that regulate cell migration, proliferation, and differentiation. See text for reference. Straight line: Direct activation; Dotted line: Indirect activation. AA arachidonic acid; Akt protein kinase B; BMP2 Bone morphogenetic protein 2; CAM calmodulin: CAM; CAMK Ca²⁺/calmodulin-dependent protein kinase; CollA1 Collagen 1α1; COX-2 Cyclooxygenase-2; CRE cAMP-response elements; CREB cAMP-response element-binding protein; DAG Diacyl glycerol; Dlx5 Distal-Less Homeobox 5; eNOS endothelial nitric oxide synthase; NFATc nuclear factor of activated T cells c; NO nitric oxide; Ocn osteocalcin; OPG osteoprotegerin; OPN osteopontin; Osx osterix; PI3K phosphoinositide 3 kinase; PKA protein kinase A; PKC protein kinase C; PLA2 phospholipase A2; PLC phospholipase C. Figure was created using icons from BioRender.com

Fig. 2. Proposed mechanism by which VSCCs might mediate extracellular vesicle release in bone cells.

Studies in other cell types suggest that VSCCs regulate EV release by initiating a localized increase in Ca²⁺. This increase controls docking and fusion of the machinery necessary for EV release. The Rab family of molecules and the SNARE motor assembly are also involved in coordinating various stages of vesicular transport. Therefore, by regulating Rab-mediated vesicular release, VSCCs could indirectly or directly influence bone cell function. Interaction of vesicles with VSCCs is enabled by Rab3 proteins through a series of molecular connections. Rab3 binds to Rab3-interacting molecules (RIMs) that then associate with RIMbinding proteins (RBPs). RBPs attach to the β subunit of VSCCs, enabling physical tethering of vesicles to VSCCs. Thus, control of Rab-mediated vesicular release is a means by which VSCCs could regulate bone cell function. Similarly, binding of Ca²⁺ to Synaptotagmin leads to vesicular fusion, while Ca²⁺-influx via VSCCs also triggers release of intracellular Ca²⁺ from ER stores to stimulate vesicular release. At the cell membrane, the α₂δ₁ subunit interacts with extracellular matrix molecules, including perlecan, representing a means by which this auxiliary subunit may scaffold various extracellular ligands to regulate EV release. These interactions could be important to initiate vesicular docking, secretion, and subsequent intercellular signaling. Finally, secreted microvesicles and exosomes containing molecular cargo act on bone cells to control their function and activity. Figure was created using icons from BioRender.com