Original Article
Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 overexpression induces beta-2 adrenergic receptor desensitization in airway smooth muscle cells
Abstract
Background: Beta-2 adrenergic receptor (β2AR) downregulation is critical to asthma rescue therapy; however, tolerance, also known as β2AR or bronchodilator desensitization, mechanisms potentially resulting in life-threatening rescue treatment failure remain poorly understood.
Methods: Airway smooth muscle cells (ASMCs) from BALB/c mice were primarily cultured. The full-length Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI2) gene from ASMCs was amplified by RT-PCR, and RhoGDI2 gene was subcloned into the digested PWPXL plasmid. The recombinant lentivirus PWPXL-RhoGDI2 expression plasmid was packaged into mature lentivirus by 293T cells and used to infect ASMCs. Fluorescent quantitation RT-PCR and Western Blot were used to detect the level of mRNA and protein expression of RhoGDI2, β2AR, guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP) and G protein-coupled receptor kinases (GRKs) in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. Membrane receptor numbers of β2AR was observed by radioligand receptor binding assay in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group.
Results: RhoGDI2vector successfully transfected ASMCs, with infection efficiency (the percentage of GFP-positive cells) >80%. RhoGDI2, GEF and G-protein-coupled receptor kinase 2 (GRK2) expressions significantly increased in the RhoGDI2 overexpression group compared to control and negative control groups (all P<0.05). Conversely, β2AR and GAP expressions were significantly lower in the RhoGDI2 overexpression group (both P<0.05), exhibiting an inverse correlation with RhoGDI2 expression. Control and negative control groups exhibiting β2AR density more than 2-fold higher than that observed in the RhoGDI2 overexpression group.
Conclusions: RhoGDI2 reduces β2AR density, potentially by reducing β2AR and GAP expressions and increase GEF and GRK2 expressions. Thus, RhoGDI2 is central in cellular β2AR desensitization, though this full mechanism and intermediates merit further investigation.
Methods: Airway smooth muscle cells (ASMCs) from BALB/c mice were primarily cultured. The full-length Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI2) gene from ASMCs was amplified by RT-PCR, and RhoGDI2 gene was subcloned into the digested PWPXL plasmid. The recombinant lentivirus PWPXL-RhoGDI2 expression plasmid was packaged into mature lentivirus by 293T cells and used to infect ASMCs. Fluorescent quantitation RT-PCR and Western Blot were used to detect the level of mRNA and protein expression of RhoGDI2, β2AR, guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP) and G protein-coupled receptor kinases (GRKs) in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. Membrane receptor numbers of β2AR was observed by radioligand receptor binding assay in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group.
Results: RhoGDI2vector successfully transfected ASMCs, with infection efficiency (the percentage of GFP-positive cells) >80%. RhoGDI2, GEF and G-protein-coupled receptor kinase 2 (GRK2) expressions significantly increased in the RhoGDI2 overexpression group compared to control and negative control groups (all P<0.05). Conversely, β2AR and GAP expressions were significantly lower in the RhoGDI2 overexpression group (both P<0.05), exhibiting an inverse correlation with RhoGDI2 expression. Control and negative control groups exhibiting β2AR density more than 2-fold higher than that observed in the RhoGDI2 overexpression group.
Conclusions: RhoGDI2 reduces β2AR density, potentially by reducing β2AR and GAP expressions and increase GEF and GRK2 expressions. Thus, RhoGDI2 is central in cellular β2AR desensitization, though this full mechanism and intermediates merit further investigation.
