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FR-01

ROS-driven COX-2/PGE2 signaling induces p21/CDKN1A-mediated cellular

senescence and inflammation in skin keratinocytes

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2
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Pei-Xuan Wu, Hui-Ching Tseng, Ssu-Yu Chen, Hsi-Lung Hsieh* ,1,2,3

1 Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung
University of Science and Technology, Taoyuan, Taiwan
2 Center for Drug Research and Development, College of Human Ecology, Chang Gung
University of Science and Technology, Taoyuan 33303, Taiwan
3 Department of Neurology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
* E-mail: hlhsieh@mail.cgust.edu.tw

Abstract
Skin inflammation has been shown to involve inflammation-related mediators expression,
including cytokines, cyclooxygenase-2 (COX-2), and matrix metalloproteinase-9 (MMP-9),
which drive extracellular matrix remodeling in response to ultraviolet (UV) irradiation or
reactive oxygen species (ROS)-induced injury. Cellular senescence is closely linked to chronic
inflammation, where activation of the p53-p21/CDKN1A pathway inhibits cyclin-dependent
kinases (CDKs), leading to cell cycle arrest and the development of a senescence-associated
secretory phenotype (SASP). This phenotype is characterized by the release of pro-
inflammatory cytokines such as IL-6, TNF-α, and IL-1β. However, the molecular mechanisms
by which the skin inflammatory mediator COX-2 regulates cell cycle-associated senescence,
and the interplay between inflammation and oxidative stress, remain to be fully elucidated. Here,
we established an in vitro model of skin inflammation and aging using D-galactose (D-gal)-
induced senescence in human keratinocytes (HaCaT cells). Our results demonstrated that D-gal
induced senescence in HaCaT cells, as evidenced by increased senescence-associated-β-
galactosidase activity, elevated expression of the senescence marker p21/CDKN1A, and
enhanced secretion of pro-inflammatory cytokines IL-6 and IL-1β. In addition, D-gal induced
COX-2 expression in a dose- and time-dependent manner, which in turn promoted
prostaglandin E2 (PGE2) biosynthesis. Notably, PGE2 was found to upregulate p53 and
p21/CDKN1A expression, and these effects were inhibited by NS398, a selective COX-2
inhibitor. Furthermore, we demonstrated that D-gal-induced COX-2 expression was mediated
through multiple signaling pathways, including the activation of MAPKs (ERK, JNK, and p38
MAPK), Akt, NF-κB, and AP-1. Importantly, these signaling cascades were activated by
mitochondrial ROS generation in response to D-gal exposure. Collectively, these findings
suggest that COX-2-derived PGE2 contributes to skin inflammation and is linked to the
upregulation of the senescence-associated protein p21/CDKN1A, ultimately leading to cell
cycle arrest and cellular senescence.

Keywords: Cellular senescence; Skin inflammation; Cyclooxygenase-2; Prostaglandin E2;
p21/CDKN1A