Osteopontin depletion in macrophages perturbs proteostasis via regulating UCHL1-UPS axis and mitochondria-mediated apoptosis

Abstract

We identified 631 DEPs in OPN<i>^KO</i> or GA-stimulated macrophages as compared to WT macrophages. The two topmost downregulated DEPs in OPN<i>^KO</i> macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of the ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We found that UCHL1, previously described as a neuron-specific protein, is expressed by BMMΦ and its regulation in macrophages was OPN-dependent. Moreover, UCHL1 interacted with OPN in a protein complex. The effects of GA activation on inducing UCHL1 and anti-inflammatory macrophage profiles were mediated by OPN. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (<i>e.g</i>., ROS, Lamp1-2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (<i>e.g</i>., 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages-inhibiting translation and protein turnover and inducing apoptosis-whereas OPN induction by GA restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via the regulation of protein synthesis, UCHL1-UPS axis, and mitochondria-mediated apoptotic processes, indicating its potential application in immune-based therapies.