CB1 Cannabinoid Receptors Stimulate Gßγ-GRK2-Mediated FAK Phosphorylation at Tyrosine 925 to Regulate ERK Activation Involving Neuronal Focal Adhesions.

CB1 cannabinoid receptors (CB1) are abundantly expressed in the nervous system where they regulate focal adhesion kinase (FAK) and the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1 and 2 (ERK1/2). However, the role of CB1-stimulated FAK 925 tyrosine phosphorylation (Tyr-P) in regulating ERK1/2 activation remains undefined. Here, immunoblotting analyses using antibodies against FAK phospho-Tyr 925 and ERK2 phospho-Tyr 204 demonstrated CB1-stimulated FAK 925 Tyr-P and ERK2 204 Tyr-P (0-5 min) which was followed by a decline in Tyr-P (5-20 min). CB1 stimulated FAK-Grb2 association and Ras-mediated ERK2 activation. The FAK inhibitors Y11 and PF 573228 abolished FAK 925 Tyr-P and partially inhibited ERK2 204 Tyr-P. FAK 925 Tyr-P and ERK2 204 Tyr-P were adhesion-dependent, required an intact actin cytoskeleton, and were mediated by integrins, Flk-1 vascular endothelial growth factor receptors, and epidermal growth factor receptors. FAK 925 Tyr-P and ERK2 204 Tyr-P were blocked by the Gßγ inhibitor gallein, a GRK2 inhibitor, and GRK2 siRNA silencing, suggesting Gßγ and GRK2 participate in FAK-mediated ERK2 activation. Together, these studies indicate FAK 925 Tyr-P occurs concurrently with CB1-stimulated ERK2 activation and requires the actin cytoskeleton and Gi/oßγ-GRK2-mediated cross-talk between CB1, integrins, and receptor tyrosine kinases (RTKs).

Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts.

BACKGROUND: In vivo studies have demonstrated the ability of multi-walled carbon nanotubes (MWCNT) to induce airway remodeling, a key feature of chronic respiratory diseases like asthma and chronic obstructive pulmonary disease. However, the mechanism leading to remodeling is poorly understood. Particularly, there is limited insight about the role of airway epithelial injury in these changes. OBJECTIVES: We investigated the mechanism of MWCNT-induced primary human bronchial epithelial (HBE) cell injury and its contribution in inducing a profibrotic response. METHODS: Primary HBE cells were exposed to thoroughly characterized MWCNTs (1.5-24 µg/mL equivalent to 0.37-6.0 µg/cm2) and MRC-5 human lung fibroblasts were exposed to 1:4 diluted conditioned medium from these cells. Flow cytometry, ELISA, immunostainings/immunoblots and PCR analyses were employed to study cellular mechanisms. RESULTS: MWCNT induced NLRP3 inflammasome dependent pyroptosis in HBE cells in a time- and dose-dependent manner. Cell death and cytokine production were significantly reduced by antioxidants, siRNA to NLRP3, a caspase-1 inhibitor (z-WEHD-FMK) or a cathepsin B inhibitor (CA-074Me). Conditioned medium from MWCNT-treated HBE cells induced significant increase in mRNA expression of pro-fibrotic markers (TIMP-1, Tenascin-C, Procollagen 1, and Osteopontin) in human lung fibroblasts, without a concomitant change in expression of TGF-beta. Induction of pro-fibrotic markers was significantly reduced when IL-1ß, IL-18 and IL-8 neutralizing antibodies were added to the conditioned medium or when conditioned medium from NLRP3 siRNA transfected HBE cells was used. CONCLUSIONS: Taken together these results demonstrate induction of a NLRP3 inflammasome dependent but TGF-beta independent pro-fibrotic response after MWCNT exposure.