Abstract
In addition to maintaining cellular ER Ca2+ stores, store-operated Ca2+ entry (SOCE) regulates several Ca2+-sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca2+, acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca2+-sensitive enzymes. Here, we report that depletion of ER Ca2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca2+/calmodulin–mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca2+-insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca2+ release activated Ca2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca2+/cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.
Highlights
Store-operated Ca2+ entry (SOCE) allows extracellular Ca2+ to replenish depleted endoplasmic reticulum (ER) (Ca2+) stores in nonexcitable and certain excitable cells [1,2,3]
Decreases in fluorescence resonance energy transfer (FRET) were recorded in H134-expressing human arterial smooth muscle cells (HASMCs) incubated with the adenylyl cyclases (ADCYs) activator forskolin (Fsk), and these responses were blocked by the transmembrane ADCY inhibitor, SQ22536 (Fig. S1, A and B)
Because the data presented here indicated that PDE1C activation was necessary for optimal SOCE-dependent ADCY8 activation (Fig. 4), we investigated whether PDE1C regulated HASMC leading-edge protrusions (LEPs) formation by virtue of its effects on ADCY8 activity
Summary
Store-operated Ca2+ entry (SOCE) allows extracellular Ca2+ to replenish depleted endoplasmic reticulum (ER) (Ca2+) stores in nonexcitable and certain excitable cells [1,2,3]. While our analysis confirmed that ER(Ca2+) store depletion did promote formation of Orai-1/Stim-1-containing puncta in HASMCs, silencing PDE1C did not alter their numbers or the expression of the ER luminal Ca2+-sensing protein, Stim-1 (Fig. S5, A–D).
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