Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.
We reported the expression of phosphorylated HSP27 during epithelial wound healing in murine corneas (Jain et al., 2012) in July of 2012. This in vivo investigation demonstrated that the expression levels of phosphorylated HSP27 were greater in wounded corneal epithelial cells than in unwounded controls and that the localization of phosphorylated HSP27 was in the basal and superficial epithelia three days following corneal epithelial wounding. We suggested that phosphorylated HSP27 had a role in the early phase of corneal epithelial wound healing. The purpose of this study was to investigate the exact role of heat shock protein 27 (HSP27) phosphorylation for the wound healing of cultured human corneal epithelial cells (HCECs). HSP27-specific siRNAs and control-siRNAs, with no known homologous targets in HCECs, were created. The cultured HCECs were divided into two groups: Scrambled control-siRNA-transfected group vs. HSP27-specific siRNA-transfected group. The scratch-induced directional wounding assay, Western blotting, using antibodies against non-phosphorylated and phosphorylated HSP27, non-phosphorylated and phosphorylated Akt, and Bcl-2-associated X protein (Bax), immunofluorescence staining to determine the filament actin, flow cytometry to measure apoptosis, and proliferation assay were performed to determine the role of HSP27. Western blot assay showed that the expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding, compared with those in unwounded HCECs (all p < 0.05). Western blot assay also showed HSP27-specific siRNAs effectively blocked the expression of non-phosphorylated HSP27. The HSP27-specific siRNA-transfected group had more Bax expression, less phosphorylated Akt expression, and less non-phosphorylated and phosphorylated HSP27 expression (all p < 0.05). The scratch-induced directional wounding assay showed the HSP27-specific siRNA-transfected group with a less migrating cell number than the control-siRNA-transfected group (p < 0.05). Immunofluorescence staining showed that reorganization of actin cytoskeleton prominently decreased in the HSP27-specific siRNA-transfected group, compared with the control siRNA-tranfected group. Flow cytometry revealed that the HSP27-specific siRNA-transfected group had more HCEC apoptosis. Proliferation assay showed no difference between the two groups. In conclusion, the role of HSP27 in corneal epithelial wound healing can be epithelial cell apoptosis, as well as epithelial migration. HSP27 is involved in HCEC migration by the reorganization of actin cytoskeleton.
Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
In Seok
IS
Song
Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
Soon-Suk
SS
Kang
Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
Eun-Soon
ES
Kim
Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
Hyun-Min
HM
Park
Department of Ophthalmology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.
Chul Young
CY
Choi
Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea; Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
Hungwon
H
Tchah
Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea; Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea. Electronic address: jykim2311@gmail.com.
Jae Yong
JY
Kim
D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement; D049109:Cell Proliferation; D019573:Epithelium, Corneal / Q000293:injuries / Q000378:metabolism* / Q000473:pathology; D005131:Eye Injuries / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D055551:HSP27 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology*
D055551:HSP27 Heat-Shock Proteins; D034741:RNA, Small Interfering
apoptosis; corneal epithelial migration; corneal epithelial wound healing; heat shock protein 27