F. Others [F. Others] F-1 Nephronophthisis 3 regulates cancer cell growth by primary cilium formation through reactive oxygen species-induced HIF-1alpha and Erk Jae-Wook Lee¹, Eun-Yi Moon¹* ¹Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul 05006, Korea Primary cilium is antenna-like organelle projecting from the surface of cell membrane. We reported previously that primary cilium formation could be regulated by nephronophthisis 3 (NPHP3) expression followed by its interaction with by thymosin β4 using HeLa cervical cancer cells. Here we investigated whether
cancer cell growth could be regulated by NPHP3 expression-associated primary cilium formation. Cancer cell growth was retarded by the incubation without fetal bovine serum (FBS). The number of cells with primary cilia was increased by serum starvation which enhanced NPHP3 expression. While NPHP3 expression was inhibited by siHIF-1α, it was increased by the incubation of cells under hypoxic condition. Under serum starvation, as reactive oxygen species were elevated, the number of cells with primary cilia and an increase in NPHP3 expression were inhibited by N-acetylcysteine treatment. The
number of cells with primary cilia was increased by H2O2 treatment, which was inhibited by siHIF-1α. The number of cells with primary cilia and NPHP3 expression were decreased by the inhibition of Erk with P98059. Cancer cell growth was reduced by the treatment with cilliobrevin A. Taken together, the results imply that the number of cancer cells with primary cilia might be controlled by NPHP3 expression under serum starvation. F-1 Nephronophthisis 3 regulates cancer cell growth by primary cilium formation through reactive oxygen species-induced HIF-1alpha and Erk Jae-Wook Lee and Eun-Yi Moon
Department of Bioscience and Biotechnology, Sejong University, Seoul, 05006, Republic of Korea Abstract Nephrocystin-3 (NPHP3) Primary cilium is antenna-like organelle projecting from the surface of cell membrane. We reported previously that primary cilium formation could be regulated by • Mutations in the mouse ortholog nephronophthisis 3 (NPHP3) expression followed by its interaction with by thymosin β4 of human NPHP3 cause poly using HeLa cervical cancer cells. Here we investigated whether cancer cell growth could cystic kidney disease (pcy) in be regulated by NPHP3 expression-associated
primary cilium formation. Cancer cell mouse model growth was retarded by the incubation without fetal bovine serum (FBS). The number of • Disruption of NPHP3 alters the cells with primary cilia was increased by serum starvation which enhanced NPHP3 frequency and/or the length of expression. While NPHP3 expression was inhibited by siHIF-1α, it was increased by the primary cilia incubation of cells under hypoxic condition. Under serum starvation, as reactive oxygen species were elevated, the number of cells with primary cilia and an increase in NPHP3 • Loss of Nephrocystin-3 function expression
were inhibited by N-acetylcysteine treatment. The number of cells with can cause Embryonic Lethality, primary cilia was increased by H 2 O 2 treatment, which was inhibited by siHIF-1α. The Meckel-Gruber-like and Syndrome, Inversus, Situs Renal- number of cells with primary cilia and NPHP3 expression were decreased by the Hepatic-Pancreatic Dysplasia inhibition of Erk with P98059. Cancer cell growth was reduced by the treatment with Figure 1. Allergic responses in mast cells.. cilliobrevin A. Taken together, the results imply that the number of cancer cells with primary cilia might be
controlled by NPHP3 expression under serum starvation. Data also Methods & Materials demonstrate that NPHP3 expression could be regulated by ROS-induced HIF-1 and Erk in HeLa human cervical cancer cells. It suggests that cancer cell growth under serum Detection of primary cilia was measured as follow. HeLa cells were grown on coverslip and starvation could be associated with the regulation of NPHP3 expression to maintain then incubated with serum-starved DMEM with 0.1% FBS for 36 h. Cells were fixed with 4% paraformaldehyde for 10 min, washed three times with cold PBS, and permeabilized with
primary cilium formation. PBST (0.1% (v/v) Triton X-100 in PBS) for 10 min. Then, cells were washed three times, and incubated with monoclonal anti-acetylated tubulin antibodies diluted (1:1000) in PBST for 1 Acknowledgement: This work was supported by grants from the Basic Research Laboratory h at room temperature. After washing three times with PBS, cells were incubated with FITC- Program (#2021R1A4A5033289) through the National Research Foundation of Korea (NRF) conjugated goat anti-mouse IgG-secondary antibody or goat anti-mouse IgG-Alexa 568 funded by the Ministry of Education, Science
and Technology (MEST), Korea. diluted (1:1000) in PBST for 1h at room temperature. Nucleus was visualized by staining cells with DAPI. After washing with PBS, cells were mounted on glass slide. Primary cilia were Keywords: Nephronophthisis 3, Primary cilium, HIF-1, Erk, Reactive oxygen species observed and photographed at 1000 x magnification under a fluorescence microscope (Nikon, Tokyo, Japan). Cytotoxicity was measured by luminescence assay.using Celltiter-glo substrate. Results A B C D Figure 2. Total cell number was reduced by serum starvation (A) but no changes in cell viability (B).
Ciliogenesis was increased by serum starvation (C). NPHP3 expression was enhanced by serum starvation (D). A C D E Figure 3. HIF- A B C B 1 increased Figure 4. HIF-1 by serum under hypoxic starvation (B) condition (A-B) or DMOG (D) contribute to contribute to ciliogenesis (C). ciliogenesis (A, C, E). A B C D E Figure 5. Reactive oxygen species (ROS) enhanced ciliogenesis. ROS was increased under serm starvation (A). ROS-mediated cilliogenesis was reduced by N-acetylcysteine (NAC) (B). NAC also inhibited ciliogenesis-associated NPHP3 (C). ROS-mediated cilliogenesis was confirmed by the
treatment with hydrogen peroxide (H 2 O 2 ) (D). Cilliogenesis by H 2 O 2 treatment was mediated by HIF-1, which was confirmed by the treatment with HIF-1 -siRNA (E). A C D B Figure 6. Serum starvation increased Erk phosphorylation (A), which was inhibited by NAC treatment (B). PD98059, Erk inhibitor, reduced NPHP3 transcriptional activity (C) and ciliogenesis (D).. A B C D D Figure 7. Cilliobrevin A, inhibitor for primary cilium formation, inhibited ciliogenesis (A), NPHP3 expression and transcriptional activity (B), and cell viability (C). This scheme demonstrates that NPHP3 expression
could be regulated by ROS-induced HIF-1 and Erk which lead to the control of cancer cell viability (D) Summaries 1. The number of cancer cells with primary cilia might be controlled by NPHP3 expression under serum starvation. 2. Data demonstrate that NPHP3 expression could be regulated by ROS-induced HIF-1 and Erk in HeLa human cervical cancer cells. 3. It suggests that cancer cell growth under serum starvation could be associated with the regulation of NPHP3 expression to maintain primary cilium formation. 4. Therefore, it is required the comprehensive studies on the regulation of cancer
cell viability. [F. Others] F-2 Expression of c-Jun and KROX-20 on facial nerve degeneration and regeneration in a rat model Seung Geun Yeo¹, In Hyeok Kim¹, Jae Min Lee¹, Dong Choon Park²* ¹Otorhinolaryngology, Head and Neck Surgery, College of Medicine, Kyung Hee University, Seoul 02447, Korea, ²Obstetrics and Gynecology, St. Vincent’s Hospital, The Catholic University of Korea, Suwon 93, Korea Purpose: This study evaluated the expression of Krox-20 and c-Jun, which have been associated with facial nerve regeneration after facial nerve compression and cutting, on recovery from facial
paralysis. Methods: The left facial nerves of 24 male Sprague-Dawley (SD) rats aged six weeks were subjected to crushing or cutting injury. The facial nerves on both sides were removed, and the expression of c-Jun and Krox-20 proteins was evaluated by Western blotting. Results: Both whisker movements of the vibrissae muscle and blink reflexes of the eyelids 4 and 14 days after facial nerve injury showed greater improvements in the crushing than in the cutting group, with the difference in facial nerve recovery on day 14 being significantly higher in the crushing group, as shown by whisker
movements and blink reflexes (p 관관 ID관 rId5관 관관관 관관관 관관관관 관관 관 관관관관. Expression of c-Jun and KROX-20 on facial nerve degeneration and regeneration in a rat model 1 1 1 2 Seung Geun Yeo , In Hyeok Kim , Jae Min Lee , Dong Choon Park 1 Department of Otorhinolaryngology - Head & Neck Surgery, School of Medicine, Kyung Hee University, Seoul, Korea 2 Department of Obstetrics and Gynecology, St. Vincent’s Hospital, The Catholic University of Korea, Suwon, Korea Introduction • Facial paralysis, although not a life-threatening condition, is one of the most important conditions requiring a complete
cure because it has devastating effects on patients’ emotional and social lives. Various treatments have been tested to cure facial paralysis, and considerable research has attempted to identify the mechanisms underlying damage to and regeneration of facial nerves. • The present study sought to identify Figure 2. Induction of facial nerve injury in Sprague-Dawley rats. (a) Ihhalation anesthesia (b) A some of the biological factors involved retroauricular incision was made in the skin and subcutaneous tissue, the tendon of the clavotrapezius muscle was identified and its position moved,
exposing the facial nerve trunk (c) Facial nerve branch in nerve regeneration after damage to (d) The proximal portion of facial nerve trunk was subjected to crushing injury for 30 seconds, or (e) The the facial nerve. Specifically, the expression proximal portion of facial nerve trunk was cut with scissors, and the facial nerve trunk was cut off after of two regulatory proteins was assessed: the cutting injury. Krox-20, a positive regulator, and c-Jun, a negative regulator, of nerve regeneration. • The expression patterns of these proteins in damaged areas distal to facial nerve injury, and
the relationship of these proteins to facial nerve regeneration, were determined in rats. Methods 2.1. Subjects and study design Figure 3. Levels of expression of Krox-20 and c-Jun in rat facial nerves (FN). (Left panel) Western Twenty-four male Sprague-Dawley (SD) rats, aged six weeks and weighing 200-250 g, were subjected blotting results showing levels of expression of Krox-20, c-Jun, and -actin proteins in intact (control) to a 1-week quarantine and adaptation period. FNs and in FNs subjected to crushing and cutting injuries 4 and 14 days later. Results were quantified by Of these 40 SD
rats, 12 were subjected to crushing injury and 12 to cutting injury of the left facial Image J software, and levels of (middle panel) Krox-20 and (right panel) c-Jun normalized to those of - nerve. Six rats in each group were sacrificed 4 days after injury and six in each group were sacrificed actin were compared. 14 days after injury. The control group consisted of the uninjured normal right facial nerves of these 24 • The level of expression of Krox-20 proteins in facial nerves collected on 4 days after injury was lower SD rats. in the crushing (0.70) than in the control group (0.82), and
significantly lower in the cutting (0.57) than in the control group (p=0.009). In contrast, there were no statistically significant differences on 2.2. Crushing injury / Cutting injury day 14 (crushing, cutting and control respectively, 0.58, 0.67, 0.71, p=0.436). The proximal part of the facial nerve trunk was subjected to crushing for 30 seconds or was completely • The levels of expression of c-Jun were significantly higher in facial nerves collected from the cut with scissors. The wound was subsequently closed and the rats allowed to recover from anesthesia. crushing and cutting groups than
in the normal group on days 4 (crushing, cutting and control
