Inosine is an endogenous purine nucleoside that is produced by catabolism
of adenosine. Adenosine has a short half-life (approximately 10 s) and is
rapidly deaminated to inosine, a stable metabolite with a half-life of
approximately 15 h. Resembling adenosine, inosine acting through adenosine
receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory
effects in vivo. The immunomodulatory effects of inosine in vivo, at least in
part, are mediated via the adenosine A2A receptor (A2AR),
an observation that cannot be explained fully by in vitro pharmacological
characterization of inosine at the A2AR. It is unclear whether the in
vivo effects of inosine are due to inosine or a metabolite of inosine engaging
the A2AR. Here, utilizing a combination of label-free, cell-based,
and membrane-based functional assays in conjunction with an equilibrium
agonist-binding assay we provide evidence for inosine engagement at the
A2AR and subsequent activation of downstream signaling events.
Inosine-mediated A2AR activation leads to cAMP production with an
EC50 of 300.7 μM and to extracellular signal-regulated
kinase-1 and -2 (ERK1/2) phosphorylation with an EC50 of 89.38
μM. Our data demonstrate that inosine produces ERKl/2-biased signaling
whereas adenosine produces cAMP-biased signaling at the A2AR,
highlighting pharmacological differences between these two agonists. Given the
in vivo stability of inosine, our data suggest an additional, previously
unrecognized, mechanism that utilizes inosine to functionally amplify and
prolong A2AR activation in vivo.
Summary
Darier’s Disease (DD), caused by mutations in the endoplasmic reticulum (ER) Ca2+ ATPase ATP2A2 (SERCA2b), is a skin disease that exhibits impaired epidermal cell-to-cell adhesion and altered differentiation. Although previous studies have shown that keratinocyte Ca2+ sequestration and fluxes are controlled by sphingolipid signaling, the role of this signaling pathway in DD previously has not been investigated. We show here that sphingosine levels increase and sphingosine kinase (SPHK1) expression decreases after inactivating SERCA2b with the specific SERCA2 inhibitors thapsigargin (TG) or siRNA to SERCA2b. Conversely, inhibiting sphingosine lyase rescues the defects in keratinocyte differentiation, E-cadherin localization, Desmoplakin (DP) translocation, and ER Ca2+ sequestration seen in TG-treated keratinocytes. To our knowledge, it was previously unreported that the keratinocyte sphingolipid and Ca2+ signaling pathways intersect in ATP2A2- controlled ER Ca2+ sequestration, E-cadherin and desmoplakin localization and Ca2+ - controlled differentiation, and thus may be important mediators in DD.
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