Severe asthma encompasses a variety of phenotypes that are characterized by ages of onset 
, duration of disease, degree of airflow impairment, presence of co-morbidity and types of inflammation 
. The majority of subjects with severe asthma manifest a degree of irreversible airway obstruction despite maximal bronchodilation and, in some, a lack of methacholine responsiveness 
. Severe asthma patients also experience more frequent and sustained exacerbations as compared with that of mild/moderate patients 
. Given the irreversible component of the disease, investigators have suggested in part that airway remodeling and ASM hyperplasia may contribute to fixed airway obstruction 
. We now show that human ASM proliferation requires expression of RGS4 protein, which interacts with the p85 subunit of PI3K. RGS4 also inhibits agonist-induced bronchoconstriction and calcium mobilization. Further, RGS4 expression in ASM cells is associated with increasing disease severity and may serve as a unique biomarker and/or therapeutic target to abrogate ASM hyperplasia and irreversible airway obstruction in asthma.
The most well-known function of RGS proteins is to reduce signaling output from GPCR activation. The importance of RGS proteins in the dynamic control of signaling is supported by changes in mRNA for these proteins under a variety of conditions 
. Different RGS proteins interact with varying preference with members of the Gi/o
families to reduce signaling. In addition to the RGS domain, RGS proteins have a variety of domains for non-GPCR protein-protein interactions, and thus selectivity for activation of particular pathways may be obtained by scaffolding mechanisms. Although few investigators have explored whether RGS proteins modulate RTK signaling pathways 
, our data suggest that RGS4 is essential to regulate RTK-mediated ASM growth. RGS expression is highly tissue- and cell-specific and, as such, imparts unique control of cellular function 
However, although the RGS family includes GPCR kinases (GRKs) such as β-adrenergic receptor kinase, about which much is known, the function of the smallest RGS molecules, namely, the B/R4 subfamily (RGS1–5, 8, 13, 16, 18, 21), remains unclear. In HASM, we show that the B/R4 RGS3, 4 and 5 proteins are the dominant RGS molecules expressed. Since RGS proteins can profoundly modulate GPCR signaling downstream from the receptor, conceptually B/R4 RGS proteins may modulate agonist-induced ASM contractility at multiple levels. In aortic smooth muscle, knockdown of RGS3 increased muscarinic 3 receptor (M3R)-dependent ERK activation but had no effect on angiotensin II-evoked signaling, suggesting receptor selectivity 
Given the necessity of RGS4 in regulating ASM proliferation, we posited that patients with severe asthma, who manifested marked increases in bronchial smooth muscle mass and irreversible airway obstruction, would manifest increased numbers of RGS4+
ASM cells. The numbers of RGS4+
ASM cells correlated with increasing disease severity. Interestingly, the RGS4+
myocytes localized in discrete areas of the bronchial smooth muscle bundle suggesting heterogeneity of expression among myocytes. The recent development of bronchial thermoplasty, which delivers a thermal injury to the bronchial wall, appears to decrease ASM mass and improve clinical outcomes in severe asthma 
. The location of the RGS4+
ASM cells and the requirement of RGS expression in mediating ASM proliferation suggest a particular susceptibility of these cells to such an injury. Since the ASM cells fail to regenerate after the thermal injury, RGS4+
cells may represent a unique population of ASM akin to skeletal myoblasts that serve to replenish differentiated muscle.
Although phenotypic plasticity of smooth muscle has been recognized for decades, the precise signaling pathways that inhibit contractile responses and that promote smooth muscle growth remain unknown. In cultured vascular smooth muscle, mostly RGS2 expression, but also 1, 3 and 4, are increased after stimulation with angiotensin II, PDGF, IL-1β or TGFβ 
. RGS5 is expressed in rat and human aortic smooth muscle but not coronary or venous myocytes 
. In cardiac muscle, RGS4 plays a critical role in regulating the chronotropic actions of acetylcholine. Lack of RGS4 enhances sensitivity to carbachol-induced bradycardia and evokes arrhythmias 
. Others reported that increases in cardiac muscle expression of RGS4 decreases cardiac inotropy that promotes heart failure 
. In myometrial smooth muscle, RGS12 expression is markedly increased at term while other RGS proteins are unchanged 
. We now show that RGS4 promotes mitogen-induced ASM growth through regulation of PI3K activity yet inhibits agonist-induced contractile function by decreasing calcium mobilization stimulated by agonists. Importantly, PDGF had little effect on β-agonist-induced bronchodilation. Increases in RGS4 expression by growth factors, which enhance ASM mitogenesis, may impart a susceptibility to ASM hyperplasia in asthma. In our study, cultured ASM cells were derived from the proximal airway while the contractile responses were measured in the distal airway. To address whether the proximal and distal ASM responded differentially to agonists, cytosolic calcium mobilization to agonists was also studied in the cultured ASM and demonstrated that PDGF modulated agonist-induced calcium levels in the cultured ASM. Despite these studies, we recognize that proximal and distal ASM in vivo
may undergo differential growth responses, and further experiments are needed to definitively demonstrate whether growth factors modulate RGS4 expression, contraction and proliferation in vivo
Although asthma is considered a disease of reversible airway obstruction and inflammation, patients with severe disease experience irreversible airflow obstruction refractory to current therapies. Given the strikingly increased morbidity seen in this subset of patients compared to those with mild/moderate asthma, the need for new therapeutic approaches remains dire. We have identified growth factor-mediated upregulation of RGS4 in ASM as a deleterious event in severe asthma. RGS4 was required for ASM hyperplasia and rendered cells poorly contractile, consistent with a maladaptive phenotypic switch as shown in . Although asthma is characterized by airway hyperresponsiveness, the data herein suggest that in severe disease, ASM becomes less responsive, which fixes the airway luminal diameter. Therapeutic approaches that decrease RGS4 expression or antagonize RGS4 function may prevent ASM hyperplasia and irreversible airway obstruction while promoting a more responsive smooth muscle phenotype.
A schematic illustration of the role of RGS4 in modulating human ASM excitation-contraction coupling and mitogen-induced proliferation.