scholarly journals Inhibition of geranylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells

2001 ◽  
Vol 281 (4) ◽  
pp. L824-L831 ◽  
Author(s):  
Ryan E. Lesh ◽  
Charles W. Emala ◽  
H. Thomas Lee ◽  
Defen Zhu ◽  
Reynold A. Panettieri ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Fiber Formation ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Actin Reorganization ◽  
Human Airway ◽  
Geranylgeranyltransferase I

To determine whether RhoA isoprenylation (geranylgeranylation) is required for agonist-induced actin cytoskeleton reorganization (measured by an increase in the filamentous F- to monomeric G-actin ratio), human airway smooth muscle cells were treated for 72 h with inhibitors of geranylgeranyltransferase I. Geranylgeranyltransferase inhibitor (GGTI)-2147 or -286 pretreatment completely blocked the increase in the F- to G-actin fluorescence ratio when cells were stimulated with lysophosphatidic acid (LPA), endothelin, or carbachol. In contrast, LPA or endothelin induced actin cytoskeletal reorganization in cells treated with farnesyltransferase inhibitor (FTI)-277 to inactivate Ras. Forskolin-induced adenylyl cyclase activity was inhibited by carbachol in GGTI-2147-pretreated cells, demonstrating that the effect of geranylgeranyltransferase I inhibition on stress fiber formation was not due to uncoupling of signaling between the heterotrimeric Gi protein (the Gγ subunit is isoprenylated) and distal effectors. These results demonstrate that selective GGTIs can inhibit agonist-induced actin reorganization.

Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells

1998 ◽  
Vol 274 (5) ◽  
pp. L803-L809 ◽  
Author(s):  
Hideaki Togashi ◽  
Charles W. Emala ◽  
Ian P. Hall ◽  
Carol A. Hirshman
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Human Airway ◽  
Stress Fiber Formation

To determine whether M2 muscarinic receptors are linked to the monomeric G protein Rho, we studied the effect of carbachol on actin reorganization (stress fiber formation) in cultured human airway smooth muscle cells that expressed mainly M2 muscarinic receptors by dual- fluorescence labeling of filamentous (F) and monomeric (G) actin. F-actin was labeled with FITC-labeled phalloidin, and G-actin was labeled with Texas Red-labeled DNase I. Carbachol stimulation induced stress fiber formation (increased F-actin staining) in the cells and increased the F- to G-actin ratio 3.6 ± 0.4-fold (mean ± SE; n = 5 experiments). Preincubation with pertussis toxin, Clostridium C3 exoenzyme, or tyrosine kinase inhibitors reduced the carbachol-induced increase in stress fiber formation and significantly decreased the F- to G-actin ratio, whereas a mitogen-activated protein kinase inhibitor, a phosphatidylinositol 3-kinase inhibitor, and a protein kinase C inhibitor were without effect. This study demonstrates that in cultured human airway smooth muscle cells, muscarinic-receptor activation induces stress fiber formation via a pathway involving a pertussis-sensitive G protein, Rho proteins, and tyrosine phosphorylation.

Urokinase potentiates PDGF-induced chemotaxis of human airway smooth muscle cells

2003 ◽  
Vol 284 (6) ◽  
pp. L1020-L1026 ◽  
Author(s):  
Stephen M. Carlin ◽  
Michael Roth ◽  
Judith L. Black
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Kinase Inhibitor ◽  
Airway Remodeling ◽  
Muscle Cells ◽  
Mek Inhibitor ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Human Airway

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-μm perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGFBB, PDGFAA, and PDGFABwere all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3′-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE2, formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.

Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho

1999 ◽  
Vol 277 (3) ◽  
pp. L653-L661 ◽  
Author(s):  
Carol A. Hirshman ◽  
Charles W. Emala
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Muscle Cells ◽  
Fiber Formation ◽  
Airway Smooth Muscle Cells ◽  
Actin Reorganization ◽  
Endothelin 1 ◽  
In Cells

Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells and Ca2+ sensitization in intact smooth muscle preparations by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. In airway smooth muscle, the agonists responsible for cytoskeletal reorganization via actin polymerization are poorly understood. Carbachol-, lysophosphatidic acid (LPA)-, and endothelin-1-induced increases in filamentous actin staining are indicative of actin reorganization (filamentous-to-globular actin ratios of 2.4 ± 0.3 in control cells, 6.7 ± 0.8 with carbachol, 7.2 ± 0.8 with LPA, and 7.4 ± 0.9 with endothelin-1; P < 0.001; n = 14 experiments). Although the effect of all agonists was blocked by C3 exoenzyme (inactivator of Rho), only carbachol was blocked by pertussis toxin. Although carbachol-induced actin reorganization was blocked in cells pretreated with antisense oligonucleotides directed against Gαi-2 alone, LPA- and endothelin-1-induced actin reorganization were only blocked when both Gαi-2 and Gqα were depleted. These data indicate that in human airway smooth muscle cells, carbachol induces actin reorganization via a Gαi-2pathway, whereas LPA or endothelin-1 induce actin reorganization via either a Gαi-2 or a Gqα pathway.

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