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: RhoGDI2 vector 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.

Keywords: Beta-2 adrenergic receptor (β2AR); G-protein-coupled receptor kinase 2 (GRK2); GTPase-activating protein (GAP); Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI2); desensitization; guanine nucleotide exchange factor (GEF).

Figure 1

Typical airway smooth muscle cells (ASMCs). (A) Third generation ASMCs showing typical peak and valley formations under inverted phase contrast microscope (×100); (B) ASMCs positive for smooth muscle α-actin filaments (green: FITC) under fluorescence microscopy (×200).

Figure 2

ASMCs displaying green fluorescence (GFP). ASMCs in the negative control group in visible light (A) (×100) and fluorescence (B) (×100) microscope, respectively. ASMCs in RhoGDI₂ overexpression group in visible light (C) (×100) and fluorescence (D) (×200) microscope, respectively. Negative control group: ASMCs transfected with GFP lentivirus; RhoGDI₂ overexpression group: ASMCs transfected with lentivirus-mediated RhoGDI₂ overexpression.

Figure 3

RhoGDI₂ mRNA and protein expression levels in ASMCs. (A) 4 days after transfection, total RNA of ASMCs was extracted and assayed for RhoGDI₂ mRNA by real-time RT-PCR. α-tubulin was used as an internal control; (B1,2) 4 days after transfection, total protein of ASMCs was extracted and assayed for RhoGDI₂ protein by western blot. α-tubulin was used as an internal control. The data represents the means ± standard deviation (SD) of three independent experiments. *P<0.05 vs. control group; ^#P<0.05 vs. negative control group. Control group, the untreated ASMCs; Negative control group, ASMCs transfected with GFP lentivirus; RhoGDI₂ overexpression group, ASMCs transfected with lentivirus-mediated RhoGDI₂ overexpression.

Figure 4

The effects of RhoGDI₂ overexpression on mRNA and protein expressions of β₂AR, GEF, GAP and GRK₂ in ASMCs. (A) 4 days after transfection, total RNA of ASMCs was extracted and assayed for β₂AR, GEF, GAP and GRK₂ mRNA by real-time RT-PCR. α-tubulin was used as an internal control; (B1,2) 4 days after transfection, total protein of ASMCs was extracted and assayed for β₂AR, GEF, GAP and GRK₂ protein by western blot. α-tubulin was used as an internal control. The data represents the means ± SD of three independent experiments. *P<0.05 vs. control group; ^#P<0.05 vs. negative control group.

Figure 5

The effects of RhoGDI₂ overexpression on β₂AR density in ASMCs. 4 days after transfection, β₂AR density was determined by radioligand receptor binding assay. The data represents the means ± SD of three independent experiments. *P<0.05 vs. control group; ^#P<0.05 vs. negative control group.

Figure 6

Schematic diagram for regulation of β₂AR by RhoGDI₂. [1]. A signal localizes the complex proximal to a membrane compartment; [2]. RhoGDI_2, GEF and GAP regulate Rho; [3]. Exchange of RhoGDP for RhoGTP; [4]. And allows the GTPase to associate with GRK₂; [5]. GRK₂ mediates homologous desensitization.