OPN‐a induces muscle inflammation by increasing recruitment and activation of pro‐inflammatory macrophages

Abstract

New Findings What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full‐length human OPN‐a isoform is the most pro‐inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD‐integrin‐dependent manner. OPN‐a upregulates expression of tenascin‐C (TNC), a known Toll‐like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro‐inflammatory effects of OPN‐a, suggesting that a potential mechanism of OPN action is by promoting TNC–TLR4 signalling. Although osteopontin (OPN) is an important mediator of muscle remodelling in health and disease, functional differences in human spliced OPN variants in the muscle microenvironment have not been characterized. We thus sought to define the pro‐inflammatory activities of human OPN isoforms (OPN‐a, OPN‐b and OPN‐c) on cells present in regenerating muscle. OPN transcripts were quantified in normal and dystrophic human and dog muscle. Human macrophages and myoblasts were stimulated with recombinant human OPN protein isoforms, and cytokine mRNA and protein induction was assayed. OPN isoforms were greatly increased in dystrophic human (OPN‐a > OPN‐b > OPN‐c) and dog muscle (OPN‐a = OPN‐c). In healthy human muscle, mechanical loading also upregulated OPN‐a expression (eightfold; P < 0.01), but did not significantly upregulate OPN‐c expression (twofold; P > 0.05). In vitro, OPN‐a displayed the most pronounced pro‐inflammatory activity among isoforms, acting on both macrophages and myoblasts. In vitro and in vivo data revealed that OPN‐a upregulated tenascin‐C (TNC), a known Toll‐like receptor 4 (TLR4) agonist. Inhibition of TLR4 signalling attenuated OPN‐mediated macrophage cytokine production. In summary, OPN‐a is the most abundant and functionally active human spliced isoform in the skeletal muscle microenvironment. Here, OPN‐a promotes pro‐inflammatory signalling in both macrophages and myoblasts, possibly through induction of TNC–TLR4 signalling. Together, our findings suggest that specific targeting of OPN‐a and/or TNC signalling in the damaged muscle microenvironment may be of therapeutic relevance.

Osteopontin is linked with AKT, FoxO1, and myostatin in skeletal muscle cells

“…The rs28357094 genotype was associated with an increase in biceps muscle size in women but not men, in keeping with an effect of estrogens on OPN expression . In healthy human muscle, OPN expression increased with acute mechanical loading, further suggesting a role of OPN in muscle injury and hypertrophic remodeling . The same rs28357094 polymorphism tracked with loss of muscle strength, motor function, and independent ambulation in 3 separate cohorts of dystrophin‐deficient Duchenne muscular dystrophy (DMD) patients .…”

“…Quantitative RT‐PCR data from the GRMD CS muscle at 4–9 weeks (with cellular hypertrophy evident but before gross hypertrophy) showed a strong negative correlation between OPN (increased) and MSTN (decreased) mRNAs (Fig. A).…”

“…Human (Hu) WT OPN and Hu‐RGD→KAE OPN proteins were a generous gift from Dr. Larry Fisher and were prepared as previously described . In Hu‐RGD→KAE OPN, the RGD amino acids were mutated to lysine (K), alanine (A), and glutamic acid (E), respectively .…”

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury

“…Myoblasts were harvested for qPCR of ChIP-qPCR analyses 48 hours after treatment onset. Chemical compounds were used at a final concentration of 10μM, while rOPN was diluted to a final concentration of 1μg/ml [ 40 , 58 ]. Effectiveness of dosing in cells and muscles was tested in pilot experiments by means of qPCR analysis of Slug , Snail , and Mzf1 mRNA at 24 hours after treatment onset.…”

Osteopontin contributes to effective neutrophil recruitment, IL‐1β production and apoptosis in Aspergillus fumigatus keratitis

“…[8][9][10][11] First, OPN has chemotactic properties that promote cell recruitment to inflammatory sites. 12,13 It also functions as an adhesion protein involved in cell attachment and wound healing. 14,15 In addition, OPN mediates cell activation and cytokine production, as well as promoting cell survival by regulating apoptosis.…”