Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation

Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I).

Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.

We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed.

DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition. Conclusions: Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.