RvD1n-3 DPA Downregulates the Transcription of Pro-Inflammatory Genes in Oral Epithelial Cells and Reverses Nuclear Translocation of Transcription Factor p65 after TNF-α Stimulation.

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1n-3 DPA and that cultured OECs respond to RvD1n-3 DPA addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1n-3 DPA-signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1n-3 DPA and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1n-3 DPA inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1n-3 DPA to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1n-3 DPA increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

Expression and function of resolvin RvD1n-3 DPA receptors in oral epithelial cells.

Chronic inflammatory responses can inflict permanent damage to host tissues. Specialized pro-resolving mediators downregulate inflammation but also can have other functions. The aim of this study was to examine whether oral epithelial cells express the receptors FPR2/ALX and DRV1/GPR32, which bind RvD1n-3 DPA , a recently described pro-resolving mediator derived from omega-3 docosapentaenoic acid (DPA), and whether RvD1n-3 DPA exposure induced significant responses in these cells. Gingival biopsies were stained using antibodies to FPR2/ALX and DRV1/GPR32. Expression of FPR2/ALX and DRV1/GPR32 was examined in primary oral epithelial cells by qRT-PCR, flow cytometry, and immunofluorescence. The effect of RvD1n-3 DPA on intracellular calcium mobilization and transcription of beta-defensins 1 and 2, and cathelicidin was evaluated by qRT-PCR. FPR2/ALX and DRV1/GPR32 were expressed by gingival keratinocytes in situ. In cultured oral epithelial cells, FPR2/ALX was detected on the cell surface, whereas FPR2/ALX and DRV1/GPR32 were detected intracellularly. Exposure to RvD1n-3 DPA induced intracellular calcium mobilization, FPR2/ALX internalization, DRV1/GPR32 translocation to the nucleus, and significantly increased expression of genes coding for beta-defensin 1, beta-defensin 2, and cathelicidin. This shows that the signal constituted by RvD1n-3 DPA is recognized by oral keratinocytes and that this can strengthen the antimicrobial and regulatory potential of the oral epithelium.

Cyclic Di-adenosine Monophosphate Regulates Metabolism and Growth in the Oral Commensal Streptococcus mitis.

Cyclic di-adenosine monophosphate (c-di-AMP) has emerged as an important bacterial signaling molecule that functions both as an intracellular second messenger in bacterial cells and an extracellular ligand involved in bacteria-host cross-talk. In this study, we identify and characterize proteins involved in controlling the c-di-AMP concentration in the oral commensal and opportunistic pathogen Streptococcusmitis (S. mitis). We identified three known types of c-di-AMP turnover proteins in the genome of S. mitis CCUG31611: a CdaA-type diadenylate cyclase as well as GdpP-, and DhhP-type phosphodiesterases. Biochemical analyses of purified proteins demonstrated that CdaA synthesizes c-di-AMP from ATP whereas both phosphodiesterases can utilize c-di-AMP as well as the intermediary metabolite of c-di-AMP hydrolysis 5'-phosphadenylyl-adenosine (pApA) as substrate to generate AMP, albeit at different catalytic efficiency. Using deletion mutants of each of the genes encoding c-di-AMP turnover proteins, we show by high resolution MS/MS that the intracellular concentration of c-di-AMP is increased in deletion mutants of the phosphodiesterases and non-detectable in the cdaA-mutant. We also detected pApA in mutants of the DhhP-type phosphodiesterase. Low and high levels of c-di-AMP were associated with longer and shorter chains of S. mitis, respectively indicating a role in regulation of cell division. The deletion mutant of the DhhP-type phosphodiesterase displayed slow growth and reduced rate of glucose metabolism.

OPA1-anchored PKA phosphorylates perilipin 1 on S522 and S497 in adipocytes differentiated from human adipose stem cells.

Optic atrophy 1 (OPA1) is the A-kinase anchoring protein targeting the pool of protein kinase A (PKA) responsible for perilipin 1 phosphorylation, a gatekeeper for lipolysis. However, the involvement of OPA1-bound PKA in the downstream regulation of lipolysis is unknown. Here we show up-regulation and relocation of OPA1 from mitochondria to lipid droplets during adipocytic differentiation of human adipose stem cells. We employed various biochemical and immunological approaches to demonstrate that OPA1-bound PKA phosphorylates perilipin 1 at S522 and S497 on lipolytic stimulation. We show that the first 30 amino acids of OPA1 are essential for its lipid droplet localization as is OMA1-dependent processing. Finally, our results indicate that presence of OPA1 is necessary for lipolytic phosphorylation of downstream targets. Our results show for the first time, to our knowledge, how OPA1 mediates adrenergic control of lipolysis in human adipocytes by regulating phosphorylation of perilipin 1.