Role of HuR and p38MAPK in Ultraviolet B-induced Post-transcriptional Regulation of COX-2 Expression in the Human Keratinocyte Cell Line HaCaT

Abstract

COX-2 (cyclooxygenase-2) is a pivotal player in inflammatory processes, and ultraviolet radiation is a known stimulus for COX-2 expression in skin cells. Here, an induction of COX-2 expression in HaCaT human keratinocytes was observed only upon exposure of cells to UVB (280 -320 nm) but not to UVA radiation (320 -400 nm), as demonstrated by reverse transcription-PCR and Western blotting. Prostaglandin E 2 levels were elevated in cell culture supernatants of HaCaT cells exposed to UVB. COX-2 mRNA stability was dramatically increased by UVB irradiation. Both the stabilization of COX-2 mRNA and the enhancement of COX-2 steady-state mRNA and protein levels caused by UVB were prevented both by inhibition and small interfering RNA-induced depletion of p38 MAPK , a kinase strongly activated upon exposure to UVB, suggesting p38 MAPK -dependent mRNA stabilization as a mechanism of UVB-induced COX-2 expression. A dramatic decrease in COX-2 expression induced by UVB was elicited by small interfering RNA-based depletion of a stress-responsive mRNA stabilizing protein regulated by p38 MAPK , i.e. HuR; UVB-induced elevation of COX-2 mRNA and protein levels coincided with an accumulation of HuR in the cytoplasm and was attenuated in cells depleted of HuR. Moreover, UVB-induced generation of prostaglandin E 2 by HaCaT cells was blunted by HuR depletion, suggesting that stress kinases (such as p38 MAPK ) as well as HuR are excellent targets for approaches aiming at interfering with induction of COX-2 expression by UVB.Cyclooxygenases catalyze the rate-limiting step in prostaglandin biosynthesis, i.e. the conversion of arachidonic acid to prostaglandin (PG) 3 H 2 , which in turn is converted by various synthases to different prostaglandins or thromboxane A 2 , important mediators in inflammatory processes. Two genes coding for isoforms of cyclooxygenase (COX-1 and COX-2) are known (1). Although COX-1 and a COX-1 variant, termed COX-3 (2), are constitutively expressed, expression of COX-2 is strongly inducible by growth factors, cytokines, and other stimuli, resulting in the production of prostaglandins during inflammatory processes. One such potent stimulus for COX-2 induction is UV radiation. Both UVB (280 -320 nm) (3) and UVA (320 -400 nm) (4) were reported previously to enhance the expression of COX-2 in human keratinocytes, followed by an increased production of the inflammatory mediator PGE 2 , a major prostaglandin in skin. Analysis of the relative contributions of UV ranges to the effects of solar light on COX-2 levels demonstrated that UVB is a far more efficient inducer of COX-2 expression; for example, UVB and UVA-2 (320 -350 nm) but not UVA-1 (350 -400 nm) contributed to COX-2 induction by simulated solar light in artificial human epidermis (5). Several lines of evidence link COX-2 and PGE 2 to the development of UV-induced skin cancer, such as the findings that COX-2 and PGE 2 levels are elevated in skin cancer versus normal tissue, that PGE 2 is a promoting factor in skin carcinogenesis, and that depletion or inhib...

Oxidative Stress Mediates Chemical Hypoxia-Induced Injury and Inflammation by Activating NF-κb-COX-2 Pathway in HaCaT Cells

“…Some reports showed that pressure or I/R treatment increased expression of COX-2 (Gomez-Pinilla et al, 2010; Tsutakawa et al, 2009). Additionally, in ultraviolet light B (UVB)-irradiated HaCaT cells COX-2 expression was obviously enhanced (Fernau et al, 2010). In accordance with previous data (Chi and Kim, 2005;Fernau et al, 2010;Tsutakawa et al, 2009), we observed that exposure of HaCaT cells to CoCl 2 upregulated COX-2 expression.…”

“…Additionally, in ultraviolet light B (UVB)-irradiated HaCaT cells COX-2 expression was obviously enhanced (Fernau et al, 2010). In accordance with previous data (Chi and Kim, 2005;Fernau et al, 2010;Tsutakawa et al, 2009), we observed that exposure of HaCaT cells to CoCl 2 upregulated COX-2 expression. Furthermore, inhibition of COX-2 by NS-398, a selective inhibitor of COX-2, significantly attenuated CoCl 2 -induced cytotoxicity and secretion of both IL-6 and IL-8, indicating involvement of COX-2 in CoCl 2 -induced cytotoxicity and inflammation in HaCaT cells.…”

Caspase-mediated Cleavage of RNA-binding Protein HuR Regulates c-Myc Protein Expression after Hypoxic Stress

“…Next, to test whether HuR-depleted cells underwent apoptosis during CoCl 2 treatment, we quantified the percentage of annexin V/propidium iodide-positive cells using flow cytometry. As shown in It has previously been shown that HuR is exported to the cytoplasm under conditions of stress such as UV treatment (36), serum starvation (37), heat shock (34), and staurosporine administration (15). We, therefore, speculated that CoCl 2 treatment, mimicking hypoxic stress, would influence HuR translocation.…”

Inhibition of Caspases Protects Mice from Radiation-induced Oral Mucositis and Abolishes the Cleavage of RNA-binding Protein HuR

“…This observation clearly indicates that HuR is exported from the nucleus in IR-treated cells and could be involved in post-transcriptional regulation. Previously, it has been shown that ultraviolet light (35) and other stresses (34) induce HuR translocation from nucleus to cytoplasm. Herein, we show that in vivo, HuR is exported to the cytoplasm in IR-induced oral mucositis.…”