Role of Dynamin 2 in mitochondrial fission and cell cycle regulation: Dysregulation of a miR-124-3p-STAT3-DNM2-Drp1-RGCC pathway connects fission and cell proliferation in pulmonary arterial hypertension
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Introduction
Dynamin-related protein 1 (Drp1) activation increases mitochondrial fission and cell cycle progression in hyperproliferative cells, termed mitotic fission . However, the diameter of a fission apparatus comprised solely of Drp1 and its binding partners appears insufficient to complete fission. Moreover, the mechanism linking fission to cell cycle progression is unknown, suggesting an additional mediator in the terminal steps of mitotic fission.
Hypothesis
The large GTPase, dynamin 2 (DNM2), interacts with Drp1 to complete mitochondrial fission and regulate cell cycle progression.
Corollary
DNM2 is epigenetically upregulated in pulmonary arterial smooth muscle cells in pulmonary arterial hypertension (PAH PASMC).
Methods
Mitochondrial morphology, protein colocalization and mitochondrial fission sites were assessed using super-resolution microscopy. Protein-protein interaction was confirmed using immunoprecipitation. The role of DNM2’s GTPase domain in mitochondrial targeting was studied by heterologous expression of truncated constructs. Transcriptomic changes from silencing DNM2 in PAH PASMC were measured by RNA-seq. DNM2 expression was quantified in normal and PAH PASMC and lungs from PAH patients and rats with monocrotaline (MCT) and SU5416/hypoxia (Su/Hx)-induced PAH. The effects of manipulating DNM2 on cell proliferation, cell cycle progression and apoptosis were assessed by flow cytometry. siRNA targeting DNM2 was nebulized to rats with MCT-PAH.
Results
DNM2 is increased in PAH PASMC in humans and rodents and interacts with Drp1 via its GTPase domain, permitting mitochondrial translocation. Silencing DNM2 in PAH PASMC inhibits fission and slows cell proliferation by causing G1/G0 phase blockade. Augmenting DNM2 in normal PASMC induces fission and accelerates proliferation. siDNM2 downregulates the positive cell cycle regulator, Regulator of Cell Cycle ( RGCC ), in PAH PASMC. Moreover, siRGCC causes G1/G0 cell cycle arrest. miR-124-3p negatively regulates DNM2 and is decreased in PAH PASMC. Augmenting miR-124-3p in PAH PASMC decreases DNM2, inhibits proliferation and induces apoptosis. STAT3 is also negatively regulated by miR-124-3p and siSTAT3 reduces DNM2 and mitochondrial fission in PAH PASMC. Nebulized siDNM2 regresses PAH in MCT-PAH.
Conclusion
DNM2’s GTPase domain binds Drp1, mediating mitochondrial translocation and tighter mitochondrial fission. DNM2 is upregulated in PAH by miR-124a-3p deficiency and STAT3 activation. The miR-124a-3p-STAT3-DNM2-Drp1-RGCC pathway underlies accelerated mitotic fission in PAH and offers novel therapeutic targets.
