Abstract
Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C δ (PKCδ) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKCδ or ERK1/2 inhibitors due to intrinsic inhibition of PKCδ and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-κB binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-κB for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E2. As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKCδ/ERK1/2 signaling pathway.
Highlights
Quiescent cells are traditionally considered to be small, dense cells passively exiting from the cell cycle
The action of protein kinase C d (PKCd) is signaled via extracellular signal regulated protein kinase 1/2 (ERK1/2) Since phorbol 12myristate 13-acetate (PMA)- and tumor necrosis factor a (TNFa)-induced COX-2 expression was suppressed by the MEK-1/ERK1/2 inhibitor PD in SF- and not in SR-Human foreskin fibroblasts (HsFb), we investigated the involvement of ERK1/2 in PKCd-mediated COX-2 expression in SF-and SR-cells
PMA-induced mRNA increases were much lower in SRHsFb, consistent with the microarray data. These results indicate that the expression of a selective group of proinflammatory genes is controlled in a fashion similar to that of COX-2: the expression level is robust in quiescent cells and becomes suppressed in proliferative fibroblasts
Summary
Quiescent cells are traditionally considered to be small, dense cells passively exiting from the cell cycle. Quiescent fibroblasts exhibit high metabolic rates tilting towards pentose phosphate pathway and NADPH generation [4]. In response to stimulation by cytokines, lipopolysaccharides, mitogenic and growth factors, they express a much higher level of a prototypic inflammatory gene, cyclooxygenase-2 (COX-2) [5,6], than proliferative fibroblasts [7]. It is unclear whether other proinflammatory genes are controlled in quiescent vs proliferative cells in a manner similar to COX-2 nor is it known how the differential response is signaled. A low COX-2 expression in proliferative fibroblasts is attributed to shutdown of the PKCdRERK1/2 signaling pathway
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