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High-throughput screening discovers antifibrotic properties of haloperidol by hindering myofibroblast activation
Michael Rehman, … , Paolo Carloni, Serena Zacchigna
Michael Rehman, … , Paolo Carloni, Serena Zacchigna
Published April 18, 2019
Citation Information: JCI Insight. 2019;4(8):e123987. https://doi.org/10.1172/jci.insight.123987.
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Research Article Cell biology Pulmonology

High-throughput screening discovers antifibrotic properties of haloperidol by hindering myofibroblast activation

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Abstract

Fibrosis is a hallmark in the pathogenesis of various diseases, with very limited therapeutic solutions. A key event in the fibrotic process is the expression of contractile proteins, including α-smooth muscle actin (αSMA) by fibroblasts, which become myofibroblasts. Here, we report the results of a high-throughput screening of a library of approved drugs that led to the discovery of haloperidol, a common antipsychotic drug, as a potent inhibitor of myofibroblast activation. We show that haloperidol exerts its antifibrotic effect on primary murine and human fibroblasts by binding to sigma receptor 1, independent from the canonical transforming growth factor-β signaling pathway. Its mechanism of action involves the modulation of intracellular calcium, with moderate induction of endoplasmic reticulum stress response, which in turn abrogates Notch1 signaling and the consequent expression of its targets, including αSMA. Importantly, haloperidol also reduced the fibrotic burden in 3 different animal models of lung, cardiac, and tumor-associated fibrosis, thus supporting the repurposing of this drug for the treatment of fibrotic conditions.

Authors

Michael Rehman, Simone Vodret, Luca Braga, Corrado Guarnaccia, Fulvio Celsi, Giulia Rossetti, Valentina Martinelli, Tiziana Battini, Carlin Long, Kristina Vukusic, Tea Kocijan, Chiara Collesi, Nadja Ring, Natasa Skoko, Mauro Giacca, Giannino Del Sal, Marco Confalonieri, Marcello Raspa, Alessandro Marcello, Michael P. Myers, Sergio Crovella, Paolo Carloni, Serena Zacchigna

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Figure 5

Modulation of ER stress and Notch pathway by haloperidol.

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Modulation of ER stress and Notch pathway by haloperidol.
(A) Representa...
(A) Representative images of cardiac fibroblasts stained for calreticulin (green) and treated with haloperidol (Halo), thapsigargin (Thapsi) and tunicamycin (Tunica). Nuclei are stained blue with Hoechst. (B) Quantification of cytosolic calreticulin (n = 5/gp). (C) Western blot for PERK in cardiac fibroblasts treated with haloperidol, thapsigargin, or tunicamycin for 48 hours. Loading control: actin. (D) Western blot for PERK, eIF2α, and their phosphorylated forms (p-PERK and p-eIF2α) in cardiac fibroblasts at the indicated time points after treatment with haloperidol. Hsc70: additional loading control. The 3 blots were run in parallel. (E) Quantification of p-PERK/PERK and p-eIF2α/eIF2α ratios in cardiac fibroblasts at the indicated time points after treatment with haloperidol (n = 3/gp). (F) Western blot for αSMA and PERK in cardiac fibroblasts treated with haloperidol, siPERK, or their combination. Loading control: tubulin. The 3 blots were run in parallel. Lower panels show representative immunofluorescence for αSMA (red). Nuclei are stained blue with Hoechst. (G) Quantification of αSMA levels in cardiac fibroblasts treated with siPERK, haloperidol, or their combination (n = 3/gp). (H) Levels of mRNA expression of Postn, Acta2, and Col1a1 after treatment with haloperidol, thapsigargin, or tunicamycin (n = 3/gp). (I) Western blot for αSMA upon treatment of cardiac fibroblasts with haloperidol, thapsigargin, or tunicamycin. Loading control: actin. (J) Western blot for the transmembrane form of Notch1 (TM-Notch1), SMAD2 and its phosphorylated form (p-SMAD2) in cardiac fibroblasts treated with either haloperidol or thapsigargin. Loading control: tubulin. The 3 blots were run in parallel. (K) Western blot for TM-Notch1 upon treatment of cardiac fibroblasts with TGF-β, haloperidol, or their combination. Loading control: tubulin. (L) Western blot for the intracellular form of Notch1 (Notch1-ICD), αSMA, and Sigmar1 upon transfection of a constitutively active form of Notch1 (N1ICD) either alone or in combination with haloperidol. Loading control: tubulin. The 3 blots were run in parallel. Scale bars in A and H: 50 μm. Values in B, E, G, and H are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t test.

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