Exaggerated responses to agonists of G protein–coupled receptors expressed on airway smooth muscle represent a central feature of human asthma. Although the core pathway linking receptor ligation to airway smooth muscle contraction has been well established, much remains unknown about how this core pathway is modulated and why this pathway is hyperresponsive in asthma. Interactions between integrins and the surrounding ECM have been shown to regulate airway smooth muscle proliferation (34
) and to connect smooth muscle cells to the matrix, allowing contraction to be translated into the force generation required for airway narrowing (14
). Here, we describe a role for ligation of integrin α9
in preventing exaggerated contraction of airway smooth muscle, which we believe to be novel. Integrin-mediated prevention of smooth muscle contraction depends on the previously described association of the integrin α9
cytoplasmic domain with SSAT and is caused by catabolism of higher-order polyamines (spermine and spermidine). A reduction in local concentrations of polyamines appears to decrease the contractile response to G protein–coupled receptor agonists by reducing the activity of the integrin-associated lipid kinase PIP5K1γ, which reduces the concentration of available PIP2 and thus inhibits IP3
-mediated calcium release from the smooth muscle SR (Figure ). These conclusions are supported by our findings that exogenous PIP2 enhanced contraction only in control airway smooth muscle and that overexpression of WT PIP5K1γ, but not a kinase-dead version or a version that cannot localize to integrins, also enhanced contraction only in smooth muscle containing functional, ligated integrin α9
Integrin α9β1 inhibits airway smooth muscle contraction by interacting with SSAT and regulating PIP5K1γ kinase activity.
is highly expressed in airway smooth muscle. This integrin has been shown to interact with a wide variety of ligands, including the endothelial counter receptor VCAM-1 (36
), tenascin C (37
), osteopontin (38
), cellular fibronectin (40
), several members of the a
etalloprotease (ADAM) family (41
), coagulation factor XIII (44
), tissue transglumatinase (44
), VEGFA (45
), VEGFC and VEGFD (47
), and von Willebrand factor (44
). Our current findings suggest that integrin α9
ligation is required for inhibition of airway smooth muscle contraction, since blocking antibody that inhibits interactions with ligands was as effective as KO or knockdown of the integrin in enhancing airway smooth muscle contraction. However, given the large number of potential ligands, we have not attempted here to identify the relevant ligand on or near airway smooth muscle.
We have previously shown that another effect of integrin α9
, accelerated cell migration, depends on the interaction between the integrin α9
cytoplasmic domain and SSAT (24
). SSAT is the rate-limiting step in catabolism of higher-order polyamines (48
), which converts spermine to spermidine and then spermidine to putrescine. Because SSAT is rapidly ubiquitinated and degraded in cells, for in vitro experiments it was necessary to use the polyamine analog BE3-3-3, which binds to SSAT and prevents its degradation. By preventing SSAT degradation, this drug effectively enhances SSAT activity, and it has thus proven a useful tool to probe SSAT-mediated effects in cells. In this study, we found that airway narrowing in lung slices and force generation by tracheal rings were decreased by BE3-3-3 treatment only in tissues in which integrin α9
was expressed and functional; BE3-3-3 had no effect on tissues of α9
smKO mice or animals treated with integrin α9
–blocking antibody. Furthermore, exogenous spermine also enhanced airway narrowing and force generation only in tissues in which integrin α9
was expressed and functional on airway smooth muscle. Together, these results strongly suggest that integrin α9
normally inhibits airway smooth muscle contraction through interaction with SSAT and local breakdown of higher-order polyamines.
The 2 best-characterized cytosolic effects of spermidine and spermine that are not shared by putrescine are inward rectification of inwardly rectifying potassium (Kir) channels (49
) and dramatic enhancement of PIP5K1γ activity (31
). We have previously shown that integrin α9
–mediated enhancement of cell migration is caused by altered rectification of a specific Kir channel, Kir4.2 (25
), and have found that Kir4.2 is also the major Kir channel expressed in airway smooth muscle (our unpublished observations). However, we found that inhibiting Kir channels by barium chloride had no effect on force generation by tracheal rings or lung slices (our unpublished observations). PIP5K1γ is the major source of PIP2 in airway smooth muscle cells (31
). Conversion of PIP2 to IP3
is a critical step in the pathway by which G protein–coupled receptor agonists induced calcium efflux from the SR, an important step regulating smooth muscle contraction. We found that increased cellular PIP2 concentration or increased PIP5K1γ kinase activity reversed the inhibitory effects of integrin α9
. Furthermore, the absence of this integrin enhanced the frequency of calcium oscillations in airway smooth muscle, and this effect was inhibited by BE3-3-3 and enhanced by spermine only in smooth muscle that expressed integrin α9
. These data support the hypothesis that integrin α9
and SSAT prevent airway narrowing at least in part through modulation of PIP5K1γ activity and PIP2 generation, and that these effects are mediated by enhanced calcium release from intracellular stores. The absence of any effects of integrin α9
loss on the contractile response to KCl, which bypasses all these steps by inducing influx of extracellular calcium through voltage-gated calcium channels, provided further support for this interpretation.
PIP2 also plays an important role in linking actin to integrin-mediated adhesion sites, a key step in agonist-induced force generation by airway smooth muscle (52
). However, as noted above, loss of integrin α9
on airway smooth muscle specifically inhibited responses to G protein–coupled receptor ligation, with no effect on contractile responses to smooth muscle depolarization by KCl. If integrin α9
was principally modulating smooth muscle contraction by enhancing interactions between actin and other anchoring integrins, we would have also expected effects on KCl-induced contraction. However, we cannot exclude some contribution of PIP2’s effects on integrin anchoring as an amplifier of the inhibitory response we describe herein.
It is tempting to speculate that the pathway we describe could have relevance to the exaggerated airway narrowing that characterizes human asthma. For example, our results would predict that genetic or acquired defects in integrin α9β1, its relevant ligands, or SSAT could result in functionally important airway hyperresponsiveness in humans. Furthermore, these results suggest that stabilization of SSAT or inhibition of PIP5K1γ could represent novel therapies for prevention of airway smooth muscle contraction in asthma.