Figure 1.

Sialidase activity is associated with CB1 GPCR agonist treatments of live RAW-blue macrophage cells. (A) We propose that the CB1 GPCRs heterodimerize with neuromedin B (NMBR) tethered to TLR in a multimeric receptor complex with matrix metalloproteinase-9 (MMP-9) and neuraminidase-1 (Neu1) in naïve (unstimulated) TLR-expressing cells. Here, a novel molecular signaling platform regulating the interaction and signaling mechanism between these molecules on the cell surface uncovers a functional selectivity of CB1 GPCR biased heteromers with NMBR to induce TLR activation signaling axis mediated by Neu1 sialidase activation and the modification of TLR glycosylation. This signaling platform potentiates MMP9 and Neu1 crosstalk on the cell surface, which is essential for activating TLR. Citation: Taken in part from Bunsick et al. [52]. (B) Cells were allowed to adhere on 12 mm circular glass slides for 24 h at 37 °C in a humidified incubator. After removing the media, 0.318 mM 4-MUNANA substrate in Tris-buffered saline pH 7.4 was added to cells alone (control background) or with either AM-404, Arvanil, and Olvanil at the indicated dosage. Fluorescent images were taken 2 min after adding substrate using epi-fluorescent microscopy (20× objective). The sialidase hydrolyzed product of 4-MUNANA (4-MU) has an emission at 450 nm (blue color) when excited at 365 nm. The mean fluorescence of 50 multi-point replicates surrounding the live cells’ periphery was calculated using Image J software, 1.5g, Java 1.8.0_345 (64-bit). The mean fluorescence ± S.E.M is represented by error bars. (C) Different components of the signaling paradigm were inhibited using BIM-23127, an antagonist of NMBR, MMP9i, an inhibitor of MMP-9, and oseltamivir phosphate (OP), an inhibitor of Neu-1 at the indicated predetermined concentrations. The quantified data represent two to three independent experiments displaying similar results. Statistical significance, as indicated by asterisks, was calculated with ANOVA and Fisher’s LSD uncorrected multiple comparisons post hoc test at a confidence level of 95%. ns = non-significant, **** p < 0.0001, * p < 0.05.