Calcineurin Regulates Homologous Desensitization of Natriuretic Peptide Receptor-A and Inhibits ANP-Induced Testosterone Production in MA-10 Cells

<div><p>Receptor desensitization is a ubiquitous regulatory mechanism that defines the activatable pool of receptors, and thus, the ability of cells to respond to environmental stimuli. In recent years, the molecular mechanisms controlling the desensitization of a variety of receptors have been established. However, little is known about the molecular mechanisms that underlie desensitization of natriuretic peptide receptors, including natriuretic peptide receptor-A (NPR-A). Here we report that calcineurin (protein phosphatase 2B, PP2B, PPP3C) regulates homologous desensitization of NPR-A in murine Leydig tumor (MA-10) cells. We demonstrate that both pharmacological inhibition of calcineurin activity and siRNA-mediated suppression of calcineurin expression potentiate atrial natriuretic peptide (ANP)-induced cGMP synthesis. Treatment of MA-10 cells with inhibitors of other phosphoprotein phosphatases had little or no effect on ANP-induced cGMP accumulation. In addition, overexpression of calcineurin blunts ANP-induced cGMP synthesis. We also present data indicating that the inhibition of calcineurin potentiates ANP-induced testosterone production. To better understand the contribution of calcineurin in the regulation of NPR-A activity, we examined the kinetics of ANP-induced cGMP signals. We observed transient ANP-induced cGMP signals, even in the presence of phosphodiesterase inhibitors. Inhibition of both calcineurin and phosphodiesterase dramatically slowed the decay in the response. These observations are consistent with a model in which calcineurin mediated dephosphorylation and desensitization of NPR-A is associated with significant inhibition of cGMP synthesis. PDE activity hydrolyzes cGMP, thus lowering intracellular cGMP toward the basal level. Taken together, these data suggest that calcineurin plays a previously unrecognized role in the desensitization of NPR-A and, thereby, inhibits ANP-mediated increases in testosterone production.</p> </div

Simulations depict effects of altering the <i>K</i>_m or <i>V</i>_max of PDE5 activity on ANP-induced cGMP signals in the presence and absence of receptor desensitization.

<p>Solid lines indicate the simulated response with a <i>K</i>_m of 4 µM and an unstimulated <i>V</i>_max of 1.17 µM/s. (A,B) Effects of altering the <i>K</i>_m of PDE5 activity from 0.1 to 5 * <i>K</i>_m on ANP-induced cGMP signals with (A) and without (B) receptor desensitization. (C,D) Effects of altering the <i>V</i>_max-PDEs (ANP-stimulated <i>V</i>_max) from 1 to 10 * <i>V</i>_max on ANP-induced cGMP signals with (C) and without (D) receptor desensitization.</p

ANP-triggered increased NFAT reporter activity in MA-10 cells.

<p>Cells were transiently transfected with plasmids containing NFAT-luciferase reporter (NFAT/LUC) or control plasmid. Forty-eight hours post transfection, cells were treated with vehicle or 50 µM CIP (60 min) followed by 10 nM ANP (20 min). Cells were then washed and allowed to incubate for 1 hour, at which point they were collected for assay of luciferase activity. Results were normalized to protein levels and expressed as fold induction over vehicle control. Experiments were conducted in the presence of 500 µM IBMX. * P<0.05. Data are from at least three experiments.</p

Pretreatment with CIP inhibits dephosphorylation of NPR-A.

<p>(A) A representative experiment demonstrating that treatment of MA-10 cells with 10 nM ANP caused a reduction in p-Ser content. Pretreatment with CIP substantially augmented NPR-A phosphorylation. (B) Quantitation of NPR-A phosphorylation demonstrates that pretreatment with CIP caused a two-fold increase in the relative intensity of p-Ser labeled bands from ANP-treated MA-10 cells. Experiments were conducted in the presence of 500 µM IBMX. * P<0.05. Data are representative of three experiments.</p

Calcineurin and PDE5 activities underlie the decay in transient ANP-induced cGMP signals in MA-10 cells.

<p>(A,B) Exposure to ANP triggered transient cGMP responses in the absence and presence of 500 µM IBMX. (C) Pretreatment with both 50 µM CIP and 500 µM IBMX substantially increased the peak response and reduced the extent of the decay in the response. (D) 14 and 40 nM sildenafil as well as 200 µM IBMX significantly inhibited cGMP PDE activity (assayed at 0.1 µM ³HcGMP) in total cell lysates from vehicle or ANP (10 nM, 20 min) treated MA-10 cells, indicating that PDE5 is primarily responsible for cGMP hydrolysis in these cells. (E) 10 nM ANP triggered an increase in peak cGMP PDE activity under these experimental conditions. (F) Treatment with both IBMX and ANP did not induce significant increases in extracellular cGMP, even after 100 min. * P<0.05.</p

Schematic depicts the regulation of ANP-induced cGMP signals in MA-10 cells.

<p>ANP binds to phosphorylated receptors, triggering increased cGMP synthesis. Calcineurin dephosphorylates NPR-A, blunting cGMP synthesis. Cyclic GMP is hydrolyzed by PDE5. The resulting transient increase in cGMP levels is believed to lead to an increase in steroidogenic acute regulatory protein (StAR) activity – the rate limiting step in steroidogenesis. The molecular mechanisms underlying ANP-induced increases in StAR activity remain unclear. In the absence of ligand, phosphorylated NPR-A has a low level of cGMP synthesis. Thus, a balance of phosphorylation by an unknown kinase and dephosphorylation by calcineurin determines the levels of basal and ANP-induced cGMP production.</p

Pretreatment with CIP potentiated ANP-induced cGMP production.

<p>(A) MA-10 cells were exposed to the phosphatase inhibitors calyculin (20 nM, 140 min), okadaic acid (100 nM, 140 min) or CIP (50 µM, 80 min) prior to exposure to IBMX (500 µM) and ANP (10 nM, 20 min), and intracellular cGMP was assessed. Pretreatment with CIP caused significant increases in ANP-induced intracellular cGMP accumulation. (B) Pretreatment with CIP triggered a 50% increase in IBMX-induced cGMP levels. (C) Cells were exposed to vehicle or CIP prior to exposure to IBMX and ANP. In cells exposed to CIP, three-fold increases in ANP-induced intracellular cGMP (open bars) but no decreases in extracellular cGMP (solid bars) were observed. (D) Exposure to CIP did not significantly alter cGMP PDE activity (assayed at 0.1 µM ³HcGMP). The basal cGMP level (i.e., in the absence of ANP and IBMX) was 1.3±0.2 pmol/mg protein. Data represent at least four experiments. * P<0.05.</p

siRNA-mediated knockdown of calcineurin potentiates ANP-induced cGMP accumulation.

<p>(A) MA-10 cells transfected with siRNA targeted against calcineurin α, β, and γ catalytic domains displayed two-fold greater levels of ANP-induced cGMP accumulation compared to cells transfected with scrambled siRNA. Following pretreatment with CIP, cells transfected with either scrambled siRNA or siRNA targeted against the α, β, and γ catalytic subunits of calcineurin displayed similar levels of cGMP accumulation. Basal cGMP levels for cells transfected with scrambled and targeted siRNA were 1.5±0.1 and 1.9±0.1 pmol/mg protein, respectively. (B) Cells treated with siRNA targeted against the α, β, and γ catalytic subunits of calcineurin had substantially lower calcineurin protein levels than cells transfected with scrambled siRNA. (C) Densitometry reveals ∼ 50% lower calcineurin levels in cells treated with siRNA targeted against α, β, and γ catalytic subunits of calcineurin. (D,E) Targeted knockdown of calcineurin had little or no effect on PP1, PP2A, PP4, or PP5 protein levels. Experiments were conducted in the presence of 500 µM IBMX. * P<0.05. Data are representative of at least three experiments.</p