Dysregulated ASM function underlies the pathogenesis of asthma and AHR (6
). The contraction and relaxation of ASM is primarily mediated through various GPCRs, the aberrant regulation of which can lead to AHR (37
). Cytokines, growth factors, integrins, and other mediators further modulate ASM function. The control of ASM contractility by tyrosine phosphatases has not been characterized. Our work provides evidence that the RPTP CD148 is a critical positive regulator of SFKs in ASM. We observed marked attenuation of AHR in mice lacking CD148 that is not primarily attributable to a dampened immune response. In addition, we report that CD148-deficient mice manifest diminished baseline pulmonary resistance in the absence of allergen sensitization and challenge, implicating decreased intrinsic ASM contractility. We provide compelling genetic, functional, and biochemical evidence that CD148 positively regulates ASM contractility via SFKs. Attenuation of AHR when CD148 is genetically deleted from smooth muscle corroborates its positive role in smooth muscle contractility, but does not exclude the possibility that other cell types, such as airway epithelium, may also be contributing to the attenuation. We show that CD148 deficiency impairs SFK activation, leading to decreased murine tracheal contractility that is recapitulated by chemical inhibition of SFKs in both murine trachea and human bronchi. Our data suggest that inhibition of CD148 phosphatase activity or specific inhibition of SFKs may be attractive therapeutic strategies for the treatment of AHR.
Surprisingly little is understood about the physiologic functions of the RPTP CD148. Most studies have been based on overexpression in cell lines or knockdown approaches, which may not accurately reflect in vivo biology. Overexpression may lead to loss of phosphatase substrate specificity, and incomplete knockdown may result in variable effects, given the complex positive and negative regulatory properties of phosphatases (45
). Here, we have used a mouse carrying a targeted deletion of the CD148 transmembrane domain such that phosphatase activity was abrogated. We have previously shown that CD148, in conjunction with another RPTP, CD45, positively regulates SFKs in B cell and macrophage immunoreceptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of SFKs (19
). In ASM, which does not express CD45, we have discovered that the CD148 phosphatase plays a prominent role in the positive regulation of SFKs. We speculate that the C-terminal tyrosine of SFKs may serve as the direct substrate for CD148, given that it was found to be hyperphosphorylated in CD148 phosphatase–deficient ASM tissue, though this finding does not exclude the possible existence of other CD148 substrates. Of the predominant SFKs expressed in human ASM (SRC, YES, FYN, and LYN) (41
), it is unclear which of these individual SFK members most prominently contributes to the generation of AHR. Future investigation will be needed to define which SFKs are specifically targeted by CD148 in ASM. For instance, our group has recently shown that in neutrophils, CD45 and CD148 play contrasting roles in chemoattractant G protein–coupled receptor signaling, which is in part mediated by the ability of CD45 and CD148 to preferentially regulate different SFK members. Therefore, CD148 may dephosphorylate the C-terminal inhibitory tyrosine residue of specific SFKs that are critical positive regulators of ASM contractility.
Networks of cytokines, growth factors, chemokines, and neurotransmitters work in concert, exerting effects on several different cell types implicated in asthma pathogenesis (46
). Given this complexity, targeting one specific mediator may have limited benefit. Various receptor and nonreceptor tyrosine kinase pathways, in both hematopoietic and structural cells, contribute to asthma pathogenesis. SRC family kinases are enzymes that proximally regulate several pathways relevant to asthma, including antigen receptors, receptor tyrosine kinases, cytokine receptors, GPCRs, and integrins (16
). Classical GPCRs, which are directly implicated in ASM contractile responses, activate serine/threonine kinases or ion channels regulated by second messengers leading to rapid short-term responses. Mounting evidence also implicates GPCR signaling in the control of cell growth, proliferation, and differentiation by activating tyrosine phosphorylation cascades, including SFKs (48
). SFKs can integrate the crosstalk between GPCR and RTK signaling pathways, and many mechanisms have been implicated, including the direct association of GPCRs with SFKs or other receptor-associated proteins, as well as the transactivation of RTKs and focal adhesion complexes by GPCR stimulation (16
The mechanisms by which SFKs are regulated in ASM, and in particular the phosphatases that control their activation status, remain obscure (48
). Much of our knowledge is based on the use of chemical SFK inhibitors, which not only lack selectivity for specific SFK members, but also target other kinases. Our understanding of SFKs is further complicated by the existence of multiple SFK members that may have contrasting functions as well as differing patterns of expression in various tissues. One example is the SFK LYN, which is known to play both positive and negative regulatory roles (51
). Mice genetically deficient for LYN demonstrated an enhanced Th2 response and more severe asthma, likely related to the negative regulatory effects of LYN in the hematopoietic lineage (52
). Therefore, in considering SFK inhibition as a potential asthma therapy, an optimal SFK inhibitor should not target LYN or the negative regulatory kinase CSK. Selective inhibitors of SFKs have been developed. As an example, the SFK inhibitor SU6656 shows selectivity for SRC, YES, FYN, and LYN, but minimally inhibits LCK, an SFK primarily expressed in T cells, or for the kinase CSK (40
). Therefore, a chemical inhibitor that targets specific SFK members may be possible and preferred for therapeutic intervention.
Few studies have directly interrogated the role of SFKs in ASM, but to date none have used for in vivo genetic approaches. The SFK inhibitor PP1, which is not highly selective, reduced the migration of human ASM cells and diminished the contractility of rat tracheal smooth muscle (50
). SRC has been implicated in ASM proliferation and migration in response to various GPCR and receptor tyrosine kinase agonists (55
). Interestingly, angiotensin II–augmented contractility of the rat left main bronchus following carbachol stimulation was attenuated by pretreatment with the SFK inhibitor SU6656 (56
). However, in contrast to our studies, no inhibition of baseline rat bronchial contractility was seen with SU6656 pretreatment, which we observed in both mouse tracheal and human bronchial contractility. Perhaps species differences or contrasting technical approaches may underlie this discrepancy. In rat pulmonary artery vascular smooth muscle, the selective SRC family kinase inhibitors SU6656 and PP2 inhibited PGF2a
-induced contraction and MLC phosphorylation, implicating a positive regulatory role for SFKs in GPCR-mediated vascular smooth muscle contraction (57
). We found that CD148-deficient ASM demonstrated more inhibition of SFKs (hyperphosphorylation of the C-terminal inhibitory tyrosine of SRC Y527) than WT ASM, and diminished the frequency of calcium oscillations and contraction following stimulation with muscarinic agonists, underscoring a critical and perhaps proximal role for SFKs in the activation of this contractile pathway.
ASM calcium oscillations induced by muscarinic agonists are, in part, mediated by the binding of IP3 to the IP3 receptor (IP3R), calcium release from the sarcoplasmic reticulum, and complex feedback circuitry (58
). We provide strong evidence that the tyrosine phosphatase CD148 modulates this pathway, although the exact mechanisms at play remain unclear. CD148 may regulate GPCR sensitization and/or desensitization through its effects on SFKs or other substrates. Another possibility could be modification of the IP3 receptor itself via tyrosine phosphorylation, which was shown to occur in T cells by the SFK FYN (59
). Oscillation frequency is also regulated by sarcoplasmic reticulum (SR) calcium content and is dependent on the refilling of SR stores via the sarcoplasmic/endoplasmic reticulum Ca2+
ATPase (SERCA) pumps and via calcium influx from extracellular sources. How CD148 influences these pathways remains an important area for future investigation, and, notably, the intersection of tyrosine phosphorylation pathways with pathways regulating calcium oscillations has not been well characterized. One exciting possibility is that the positive regulatory role of CD148 on SFKs may regulate transient receptor potential (TRP) channels, which are phosphorylated by SRC (60
), and by this mechanism, CD148 may regulate calcium oscillation frequency. This is certainly a high priority for future investigation.
SFKs are proximally involved in modulating several receptor tyrosine kinase (RTK) growth factors that have been implicated in asthma pathogenesis such as EGFR, VEGFR, PDGFR, and c-KIT (2
). There exists evidence for both negative and positive regulation of RTK signaling by CD148 (63
), suggesting that the role of CD148 may be context dependent. Consistent with our studies, other groups have reported that CD148 is a positive regulator of SFKs via dephosphorylation of the C-terminal inhibitory tyrosine of SFKs in thyroid carcinoma (66
) as well as in endothelial cells (65
). Although we did not directly evaluate the effect of the CD148 phosphatase on RTK signaling pathways in ASM, it is conceivable that CD148 could be playing an important regulatory role through its influence on SFKs.
SFKs are critical regulators of integrins, which transmit signals via focal adhesion kinase (FAK) and focal adhesion signaling complexes that affect the actin cytoskeleton, an important determinant of ASM contractility (67
). CD148 has been shown to play a positive regulatory role in platelet integrin function via SFK regulation (25
). Future studies will more closely examine the role of CD148 in ASM integrin function and activation of focal adhesion complexes, other mechanisms by which CD148 could impact ASM contractility. CD148 function in ASM contractility could potentially be regulated by binding its recently identified ligands, thrombospondin-1 (TSP1) and syndecan-2, proteins that modulate interactions with extracellular matrix components (14
). For example, TSP1 stimulates vascular smooth muscle cell migration through FAK, so it is plausible that TSP1 interactions with CD148 could regulate ASM contractility (68
Although the exact mechanisms by which the CD148 phosphatase and SFKs are involved in AHR remain incompletely defined, our data strongly suggest that the CD148 phosphatase is a critical positive regulator of SFK activity in ASM. These data present exciting new strategies for asthma treatment, focusing on the inhibition of ASM contractility. We propose two potential therapeutic approaches for asthma, directed at ASM: (a) inhibition of CD148 phosphatase activity through targeting the CD148 extracellular domain and/or modulating interactions with its ligands; or (b) inhibition of SFKs. Strategically designed inhaled inhibitors of the CD148 phosphatase or of specific SFKs could directly impact ASM function and AHR with minimal systemic side effects.