Metabolic Adaptation of Airway Smooth Muscle Cells to an SPHK2 Substrate Precedes Cytostasis

Thickening of the airway smooth muscle is central to bronchial hyperreactivity. We have shown that the sphingosine analog (R)-2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol (AAL-R) can reverse preestablished airway hyperreactivity in a chronic asthma model. Because sphingosine analogs can be metabolized by SPHK2 (sphingosine kinase 2), we investigated whether this enzyme was required for AAL-R to perturb mechanisms sustaining airway smooth muscle cell proliferation. We found that AAL-R pretreatment reduced the capacity of live airway smooth muscle cells to use oxygen for oxidative phosphorylation and increased lactate dehydrogenase activity. We also determined that SPHK2 was upregulated in airway smooth muscle cells bearing the proliferation marker Ki67 relative to their Ki67-negative counterpart. Comparing different stromal cell subsets of the lung, we found that high SPHK2 concentrations were associated with the ability of AAL-R to inhibit metabolic activity assessed by conversion of the tetrazolium dye MTT. Knockdown or pharmacological inhibition of SPHK2 reversed the effect of AAL-R on MTT conversion, indicating the essential role for this kinase in the metabolic perturbations induced by sphingosine analogs. Our results support the hypothesis that increased SPHK2 levels in proliferating airway smooth muscle cells could be exploited to counteract airway smooth muscle thickening with synthetic substrates.