Korean J Ophthalmol.  2016 Dec;30(6):468-478. 10.3341/kjo.2016.30.6.468.

Tunicamycin-induced Endoplasmic Reticulum Stress Upregulates the Expression of Pentraxin 3 in Human Retinal Pigment Epithelial Cells

• Hwang N
• Kwon MY
• Cha JB
• Chung SW
• Woo JM

• KMID: 2374005
• DOI: http://doi.org/10.3341/kjo.2016.30.6.468

Abstract

PURPOSE
To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19.
METHODS
PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells.
RESULTS
The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells.
CONCLUSIONS
These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.

Keyword

Endoplasmic reticulum stress; Human retinal pigment epithelial cells; Inositol-requiring enzyme 1α; Pentraxin 3

MeSH Terms

Anti-Bacterial Agents/pharmacology
Apoptosis
Blotting, Western
C-Reactive Protein/biosynthesis/*genetics
Cells, Cultured
Endoplasmic Reticulum Stress/*drug effects/genetics
Enzyme-Linked Immunosorbent Assay
*Gene Expression Regulation
Humans
Polymerase Chain Reaction
RNA, Messenger/*genetics
Retinal Pigment Epithelium/*metabolism/pathology
Serum Amyloid P-Component/biosynthesis/*genetics
Tunicamycin/*pharmacology
Anti-Bacterial Agents
C-Reactive Protein
RNA, Messenger
Serum Amyloid P-Component
Tunicamycin

Figure

• 

  Fig. 1 (A) ARPE-19 cells, a human retinal pigment epithelial cell line, that show morphologic retinal pigment epithelium (RPE). The image was obtained using an optical microscope at 100× magnification without staining. (B) Real-time polymerase chain reaction analysis at sequential time points for the expression of key genes in RPE differentiation and development. Undifferentiated HT29 cells (human colon carcinoma cell line) were used as a negative control. Expression of paired box protein 6, microphthalmia-associated transcription factor, and orthodenticle homeobox 2 were higher in human retinal pigment epithelium (hRPE) compared to HT29 cells. hPAX6 = human paired box protein 6; hMITF = human microphthalmia-associated transcription factor; hOTX2 = human orthodenticle homeobox 2.

• 

  Fig. 2 Tunicamycin (TM)-induced endoplasmic reticulum stress accelerates cell death in the human retinal pigment epithelial cell line, ARPE-19. (A) ARPE-19 cells were treated with vehicle or TM (1 µg/mL). After 48 hours, cell nuclei were stained to detect live cells using Hoechst 33342. (B) Quantification of the amount of live cells. Values are mean ± standard deviation, n = 6. *p < 0.05 vs. vehicle. (C) The viability of ARPE-19 cells was measured 48 hours after vehicle or TM (0.01, 0.1, and 1 µg/mL) treatment using the Ez-Cytox Cell Viability Assay Kit. Values are mean ± standard deviation, n = 12. *p < 0.05 vs. vehicle.

• 

  Fig. 3 Pentraxin 3 (PTX3) production is enhanced by tunicamycin (TM)-induced endoplasmic reticulum stress in ARPE-19 cells. (A) ARPE-19 cells were stimulated with increasing concentrations of TM (0.01, 0.1, and 1 µg/mL). After 48 hours, supernatants were harvested and assessed for PTX3 production. (B) ARPE-19 cells were incubated for various time-periods in the absence or presence of TM (0.1 µg/mL). Supernatants were harvested and measured for PTX3 production at the indicated time points. Values are mean ± standard deviation, n = 10. *p < 0.05 vs. vehicle.

• 

  Fig. 4 Pentraxin 3 (PTX3) mRNA expression is increased by tunicamycin (TM)-induced endoplasmic reticulum stress in ARPE-19 cells. (A) Total RNA was extracted from ARPE-19 cells after 24-hour treatment with various doses of TM. (B) ARPE-19 cells were incubated for various time-periods in the absence or presence of TM (0.1 µg/mL). PTX3 mRNA expression levels were assessed by real-time reverse transcription-polymerase chain reaction analysis. Three independent experiments were performed. PTX3 expression values were divided by the expression of the control gene, β-actin, and represented as fold increase relative to the vehicle control. *p < 0.05 vs. vehicle. For the real-time polymerase chain reaction experiments, values are presented as mean ± standard deviation, n = 3.

• 

  Fig. 5 NF-κB signaling induces pentraxin 3 (PTX3) production. (A) ARPE-19 cells were stimulated with vehicle or tunicamycin (TM, 0.1 or 1 µg/mL) in the absence or presence of NF-B (BAY 11-7085) and c-Jun NH2-terminal kinase (SP600125) signaling inhibitors. After 48 hours, supernatants were harvested and assessed for PTX3 production. Results are shown as means ± standard deviation (SD), n = 12. *p < 0.05 vs. vehicle. (B) ARPE-19 cells were stably transfected with a control (CON) vector or small hairpin RNA (shRNA) for inositol-requiring enzyme 1α (IRE1α). The cells were then exposed to vehicle or TM (0.1 µg/mL) for 48 hours. Quantitative reverse transcription-polymerase chain reaction analyses were performed for IRElα and β-actin (loading CON) to assess the degree of IRElα silencing. Three independent experiments were performed. Results are shown as means ± SD, n = 4. *p < 0.05 vs. CON shRNA. (C) PTX3 production was assessed in CON shRNA or IRE1α shRNA transfected ARPE-19 cells in the absence or presence of TM (0.1 µg/mL). Results are shown as means ± SD, n = 4. *p < 0.05 vs. CON shRNA.

• 

  Fig. 6 Cell viability is decreased in pentraxin 3 (PTX3) knock-down ARPE-19 cells. (A) ARPE-19 cells were transfected with a control (CON) vector or with PTX3 small hairpin RNA (shRNA). Western blot analyses for PTX3 expression were also performed to assess the degree of PTX3 silencing. (B) Cell viability of CON shRNA or PTX3 shRNA stable cells was measured 48 hours after vehicle or tunicamycin (TM, 0.5 and 1 µg/mL) treatment using the Ez-Cytox Cell Viability Assay Kit (Itsbio, Seoul, Korea). *p < 0.05: decreased cell viability of PTX3 shRNA cells vs. CON shRNA cells. Values are shown as mean ± standard deviation, n = 12.

• 

  Fig. 7 Tunicamycin (TM)-induced endoplasmic reticulum stress activates prolonged CCAAT-enhancer-binding protein homologous protein (CHOP) expression in pentraxin 3 (PTX3) knockdown ARPE-19 cells. (A) ARPE-19 cells were transfected with a control (CON) vector or with PTX3 small hairpin RNA (shRNA). Total RNA was extracted after various time periods in the absence or presence of tunicamycin (0.1 µg/mL). CHOP mRNA expression was quantified by real-time reverse transcription-polymerase chain reaction analysis, and three independent experiments were performed. The values for CHOP expression were divided by expression of the CON gene, β-actin, and presented as fold increase relative to the vehicle CON. *p < 0.05 increased mRNA levels of CHOP in PTX3 shRNA cells vs. CON shRNA cells. For the real-time polymerase chain reaction experiments, values are presented as mean ± standard deviation (SD), n = 3. (B) CON shRNA and PTX3 shRNA transfected cells were incubated for various time periods in the absence or presence of tunicamycin (0.1 µg/mL). Total protein was harvested and protein levels of CHOP were analyzed by western blot analysis using a human anti-CHOP polyclonal antibody. Results are representative of three independent experiments. (C) The fold change of signal intensity of protein expression was calculated and corrected for loading in vehicle treated cells. *p < 0.05: enhanced CHOP protein levels vs. CON shRNA; †p < 0.05: decreased CHOP protein levels vs. CON shRNA. Values are shown as mean ± SD, n = 3.

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