Acceleration Of Dermal Wound Healing By Regulation Of A Circadian Clock Gene, Neuronal Pas Domain 2 (Npas2)
Akishige Hokugo, DDS PhD1, Hodaka Sasaki, DDS, PhD2,3, Lixin Wang, DMD1, Kenzo Morinaga, DDS, PhD2,4, Hiroko Okawa, DDS, PhD2, Daniel Khalil, MD1, Reza Jarrahy, MD1, Ichiro Nishimura, DDS, DMSc, DMD2.
1David Geffen School of Medicine at UCLA, Los Angeles, CA, USA, 2UCLA School of Dentistry, Los Angeles, CA, USA, 3Tokyo Dental College, Tokyo, Japan, 4Fukuoka Dental College, Fukuoka, Japan.
PURPOSE: Circadian rhythms that maintain cellular homeostasis during a 24-hour cycle have been shown to regulate a wide range of peripheral tissues. We have hypothesized that dermal wound healing is also under the regulation of circadian gene. In this study, we investigated the role of one of the circadian genes, Neuronal PAS domain 2 (Npas2) in the homeostasis of dermal structure using in vivoand in vitrowound healing models. MATERIALS AND METHODS: Primary fibroblasts were isolated from homozygous knock out (KO) (Npas2-/-), heterozygous KO (Npas2+/-) and normal C57Bl6J (WT) mice. The expression of core clock genes was determined by quantitative RT-PCR (qPCR). Fibroblast behaviors were characterized in terms of cell proliferation (WST-1 test), cell migration (Scratch test), and cell contraction (floating collagen gel culture). In addition, gene expression of alpha-smooth muscle actin (alpha-SMA) and extracellular matrix collagens (type I, III, XII, and XIV) was determined by qPCR and in vitro collagen accumulation was evaluated by Picrosirius red staining. The time-course healing of full-thickness punched-out wounds was monitored in WT and Npas2 KO mice. Moreover, we screened 1,120 FDA-approved compounds for Npas2 expression and fibroblast migration. One candidate compound exhibited the increased fibroblast migration in vitro and the accelerated full-thickness dorsal skin punched-out wound healing in vivo. RESULTS: There was no effect on the core clock gene expression by Npas2 KO mutation. The KO fibroblasts showed higher cell proliferation, migration and contraction capabilities. While alpha-SMA expression was not affected, FACIT collagen XII and XIV gene expression was significantly increased in Npas2 KO fibroblasts. Picrosirus red staining was strongly positive in Npas2 KO fibroblasts. Npas2-/- mice demonstrated faster dermal wound closure than the other groups (p<0.01). Furthermore, candidate compound-treated dermal wounds suggested accelerated wound healing. DISCUSSION AND CONCLUSION: Our study demonstrated that Npas2 suppression in dermal fibroblasts modified cell behaviors demonstrated by accelerated cell proliferation, cell migration and cell contraction force in vitro. Moreover, Npas2 suppression resulted in accelerated dermal wound healing. This study suggests that Npas2 may be a novel therapeutic target for dermal homeostasis and wound healing.
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