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Osteopontin depletion in macrophages perturbs proteostasis via regulation of UCHL1-UPS pathway and activation of mitochondria-mediated apoptosis

ABSTRACT: Osteopontin depletion in macrophages perturbs proteostasis via regulation of UCHL1-UPS pathway and activation of mitochondria-mediated apoptosis Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow derived macrophages (BMM), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMM upregulates OPN expression, promoting an anti inflammatory, phagocytic, pro-healing phenotype, whereas OPN inhibition triggered a pro-inflammatory phenotype. Here, we applied a global proteome profiling via mass spectrometry analysis to gain a mechanistic understanding of OPN suppression versus induction in macrophages. We identified over 630 differentially expressed proteins (DEPs) in OPN knockout (OPNKO) or GA stimulated versus wild type (WT) macrophages. Two topmost downregulated DEPs in OPN deficient macrophages were ubiquitin C terminal hydrolase L1 (UCHL1), a crucial component of ubiquitin proteasome system (UPS), and the anti inflammatory Heme oxygenase 1 (HMOX 1), whereas GA stimulation upregulated their expression. We further confirmed UCHL1 expression in BMM, and its direct interaction with OPN in protein complex. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome mitochondria-mediated apoptosis (e.g. ROS, Lamp1/2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g. 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, inducing apoptosis and inhibiting translation, whereas GA stimulated OPN induction restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via regulation of cell viability, UCHL1 UPS pathway, and protein synthesis, indicating its potential application in immune-based therapy.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Jennifer Van Eyk

PROVIDER: MSV000091221 | MassIVE | Wed Feb 08 03:32:00 GMT 2023

SECONDARY ACCESSION(S): PXD039950

REPOSITORIES: MassIVE

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Osteopontin depletion in macrophages perturbs proteostasis via regulation of UCHL1-UPS pathway and activation of mitochondria-mediated apoptosis

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