Activation of Notch signaling by short-term treatment with Jagged-1 enhances store-operated Ca(2+) entry in human pulmonary arterial smooth muscle cells

Abstract

Notch signaling plays a critical role in controlling proliferation and differentiation of pulmonary arterial smooth muscle cells (PASMC). Upregulated Notch ligands and Notch3 receptors in PASMC have been reported to promote the development of pulmonary vascular remodeling in patients with pulmonary arterial hypertension (PAH) and in animals with experimental pulmonary hypertension. Activation of Notch receptors by their ligands leads to the cleavage of the Notch intracellular domain (NICD) to the cytosol by γ-secretase; NICD then translocates into the nucleus to regulate gene transcription. In this study, we examined whether short-term activation of Notch functionally regulates store-operated Ca(2+) entry (SOCE) in human PASMC. Treatment of PASMC with the active fragment of human Jagged-1 protein (Jag-1) for 15-60 min significantly increased the amplitude of SOCE induced by passive deletion of Ca(2+) from the intracellular stores, the sarcoplasmic reticulum (SR). The Jag-1-induced enhancement of SOCE was time dependent: the amplitude was maximized at 30 min of treatment with Jag-1, which was closely correlated with the time course of Jag-1-mediated increase in NICD protein level. The scrambled peptide of Jag-1 active fragment had no effect on SOCE. Inhibition of γ-secretase by N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) significantly attenuated the Jag-1-induced augmentation of SOCE. In addition to the short-term effect, prolonged treatment of PASMC with Jag-1 for 48 h also markedly enhanced the amplitude of SOCE. These data demonstrate that short-term activation of Notch signaling enhances SOCE in PASMC; the NICD-mediated functional interaction with store-operated Ca(2+) channels (SOC) may be involved in the Jag-1-mediated enhancement of SOCE in human PASMC.

Keywords: Jagged; Notch intracellular domain; Notch receptor; pulmonary artery; smooth muscle; store-operated calcium entry.

Fig. 1.

Short-term treatment with Jagged-1 (Jag-1) enhances store-operated Ca²⁺ entry (SOCE) in human pulmonary arterial smooth muscle cells (PASMC). SOCE was induced by passive depletion of Ca²⁺ from the sarcoplasmic reticulum (SR) with 10 μM cyclopiazonic acid (CPA) in human PASMC. The active fragment (aa 188–204) of human Jag-1 protein was used for stimulation of Notch receptors in human PASMC. A: representative records of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) showing CPA-induced increases in [Ca²⁺]_cyt due to Ca²⁺ release and SOCE in control PASMC (left) and PASMC treated with Jag-1 (50 μM). 0Ca, absence of extracellular Ca²⁺. B: representative records of changes in [Ca²⁺]_cyt due to SOCE in PASMC treated with 50 μM Jag-1 for 0 (Control), 5, 10, 30, and 60 min, respectively. C: summarized data (means ± SE) showing the effects of Jag-1 on CPA-induced increases in [Ca²⁺]_cyt due to Ca²⁺ release (left) and SOCE (middle and right) in human PASMC treated with 50 μM Jag-1 for 0 (Control), 5, 10, 30, and 60 min, respectively. Note that the pretreatment with Jag-1 for 30 min dramatically enhanced the amplitude of SOCE. Summarized data were obtained from 17 to 27 cells. *P < 0.05 or **P < 0.01 vs. 0 min. D: Western blot analysis of Notch intracellular domain (NICD) in PASMC treated with Jag-1 (50 μM) for 0, 5, 10, 30, or 60 min (left) using a specific antibody (1:1,000) against the NICD of Notch1. β-Actin (1:500) was used as a control to ensure equal loading. Summarized data (means ± SE, n = 3 experiments) show NICD levels normalized to the level in control PASMC (Jag-1, 0 min).

Fig. 2.

Dose dependence of Jag-1-mediated enhancement of SOCE in human PASMC. SOCE was induced by passive depletion of Ca²⁺ from the SR with 10 μM CPA in human PASMC. The active fragment (aa 188–204) of human Jag-1 protein was used for activation of Notch receptors. A: representative records showing the increases in [Ca²⁺]_cyt due to SOCE in control PASMC (Vehicle control) and PASMC treated with 5, 50, or 500 μM Jag-1 (for 30 min). B: summarized data (means ± SE, n = 17–20 cells) showing the amplitude of the increase in [Ca²⁺]_cyt due to Ca²⁺ release or leakage (left) and the amplitude of the peak (middle) and plateau (right) increases in [Ca²⁺]_cyt in PASMC treated with 5, 50, or 500 μM Jag-1 (for 30 min). *P < 0.05, **P < 0.01 vs. control PASMC (0 μM) treated with vehicle (1% distilled water).

Fig. 3.

Inhibition of Notch signaling by the γ-secretase inhibitor DAPT attenuates Jag-1-mediated SOCE enhancement in human PASMC. SOCE was induced by passive depletion of Ca²⁺ from the SR with 10 μM CPA in human PASMC. The active fragment (aa 188–204) of human Jag-1 protein was used for activation of Notch receptors. A: representative records showing the increases in [Ca²⁺]_cyt due to SOCE in control PASMC and PASMC treated with scrambled Jag-1 (scJag-1, 50 μM for 30 min), Jag-1 (50 μM for 30 min), and Jag-1 (50 μM) plus DAPT (10 μM, an inhibitor of γ-secretase, for 30 min). B: summarized data (means ± SE, n = 17–20 cells) showing the amplitude of the increase in [Ca²⁺]_cyt due to Ca²⁺ release or leakage (Release, left) and the amplitude of the peak (middle) and plateau (right) increases in [Ca²⁺]_cyt in control PASMC and PASMC treated with scJag-1, Jag-1, or Jag-1 + DAPT (for 30 min). **P < 0.01 vs. vehicle control (Control, PASMC treated with 1% distilled water); ##P < 0.01 vs. Jag-1.

Fig. 4.

Prolonged treatment with Jag-1 enhances SOCE in human PASMC. SOCE was induced by passive depletion of Ca²⁺ from the SR with 10 μM CPA in human PASMC. The active fragment (aa 188–204) of human Jag-1 protein was used for stimulation of Notch receptors. A: representative records showing the increase in [Ca²⁺]_cyt due to SOCE in control PASMC and PASMC treated with Jag-1 (50 μM for 24 h) and scrambled Jag-1 (scJag-1, 50 μM for 24 h). B: summarized data (means ± SE, n = 17–30 cells) showing the amplitude of the increase in [Ca²⁺]_cyt due to Ca²⁺ release or leakage (left) and the amplitude of the peak (middle) and plateau (right) increases in [Ca²⁺]_cyt in control PASMC and PASMC treated with Jag-1 or scJag-1 (for 24 h). C: representative records showing the increase in [Ca²⁺]_cyt due to SOCE in control PASMC and PASMC treated with Jag-1 (50 μM for 48 h), Jag-1 + DAPT (50 μM and 10 μM, respectively, for 48 h), and scrambled Jag-1 (scJag-1, 50 μM for 48 h). D: summarized data (means ± SE, n = 17–30 cells) showing the amplitude of the increase in [Ca²⁺]_cyt due to Ca²⁺ release or leakage (left) and the amplitude of the peak (middle) and plateau (right) increases in [Ca²⁺]_cyt in control PASMC and PASMC with Jag-1, Jag-1 + DAPT, and scJag-1 (for 48 h). E: representative records showing the increase in [Ca²⁺]_cyt due to SOCE in control PASMC and PASMC treated with Jag-1 (50 μM for 72 h) and scrambled Jag-1 (scJag-1, 50 μM for 72 h). F: summarized data (means ± SE, n = 17–30 cells) showing the amplitude of the increase in [Ca²⁺]_cyt due to Ca²⁺ release or leakage (left) and the amplitude of the peak (middle) and plateau (right) increases in [Ca²⁺]_cyt in control PASMC and PASMC treated with Jag-1 or scJag-1 (for 72 h). **P < 0.01 vs. vehicle control (Control, PASMC treated with 1% distilled water); ##P < 0.01 vs. Jag-1.