Cigarette smoke-related hydroquinone induces filamentous actin reorganization and heat shock protein 27 phosphorylation through p38 and extracellular signal-regulated kinase 1/2 in retinal pigment epithelium: implications for age-related macular degeneration

Abstract

Retinal pigment epithelium (RPE)-derived membranous debris named blebs, may accumulate and contribute to sub-RPE deposit formation, which is the earliest sign of age-related macular degeneration (AMD). Oxidative injury to the RPE might play a significant role in AMD. However, the underlying mechanisms are unknown. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, foodstuff, and atmospheric pollutants, induces actin rearrangement and membrane blebbing in RPE cells as well as sub-RPE deposits in mice. Here, we show for the first time that phosphorylated Heat shock protein 27 (Hsp27), a key regulator of actin filaments dynamics, is up-regulated in RPE from patients with AMD. Also, HQ-induced nonlethal oxidative injury led to Hsp27mRNA up-regulation, dimer formation, and Hsp27 phosphorylation in ARPE-19 cells. Furthermore, we found that a cross talk between p38 and extracellular signal-regulated kinase (ERK) mediates HQ-induced Hsp27 phosphorylation and actin aggregate formation, revealing ERK as a novel upstream mediator of Hsp27 phosphorylation. Finally, we demonstrated that Hsp25, p38, and ERK phosphorylation are increased in aging C57BL/6 mice chronically exposed to HQ, whereas Hsp25 expression is decreased. Our data suggest that phosphorylated Hsp27 might be a key mediator in AMD and HQ-induced oxidative injury to the RPE, which may provide helpful insights into the early cellular events associated with actin reorganization and bleb formation involved in sub-RPE deposits formation relevant to the pathogenesis of AMD.

Figure 1

Increased phosphorylated Hsp27 (p-Hsp27) expression in human RPE from patient donors with dry AMD. A: Representative Western blot for p-Hsp27, total Hsp27, and GAPDH on RPE lysates from three donors with dry AMD and three controls with no known history of eye disease. B–G: Representative immunofluorescent double staining of p-Hsp27 (green) and nuclei (bleu) in retina sections from human donor eyes with no known eye disease (D), and human donor eyes with dry AMD (E). Negative controls were generated by omission of the primary antibody (B and C). Higher magnification showing RPE and BrM in control (F) and patients with AMD (G). Sections were analyzed by using confocal microscopy (original magnification, ×40 and ×400). INL, inner nuclear layer; ONL, outer nuclear layer; PIS, photoreceptor inner segments; POS, photoreceptor outer segments; Ch, choroid.

Figure 2

Effect of HQ on Hsp27 in ARPE-19 cells. Confluent serum-starved ARPE-19 cells were treated with or without 100 μmol/L HQ for six hours. A: Hsp27 monomer and dimer expression in ARPE-19 cells before and after treatment with HQ for six hours. Hsp27 expression was assessed by Western blot analysis. Top: Western blot from a representative experiment showing change in Hsp27 dimer protein expression. Number on the left represents protein molecular weight in kilodaltons. Bottom: Average results of three independent experiments run in duplicate. Total Hsp27 protein expression was normalized to β-actin. B: Up-regulation of Hsp27 mRNA expression in ARPE-19 cells exposed to HQ for six hours. Total RNA was extracted and Hsp27 mRNA expression was analyzed by real-time PCR. GAPDH was used as the housekeeping gene. Data represent average results of five independent experiments run in duplicate. Results are expressed as mean ± SE. ***P < 0.001 versus control (−HQ).

Figure 3

HQ activates Hsp27 and p38 MAPK phosphorylation. A: Time-dependent Hsp27 phosphorylation assessed by Western blot on cell lysates from ARPE-19 cells treated with 100 μmol/L HQ for various periods of time. Top: Western blot from a representative experiment. Number on the left represents protein molecular weight in kilodaltons. Bottom: Average results of three independent experiments run in duplicate. Phospho-Hsp27 (p-Hsp27) protein expression was normalized to total Hsp27. GAPDH was used as loading control for total Hsp27. B: Time-dependent p38 MAPK phosphorylation assessed by Western blot on cell lysates from ARPE-19 exposed to 100 μmol/L HQ for various periods of time. Top: Western blot from a representative experiment. Number on the left represents protein molecular weight in kilodaltons. Bottom: Average results of three independent experiments run in duplicate on ARPE-19 cells. Phospho-p38 (p-p38) MAPK protein expression was normalized to GAPDH. Data are expressed as percentage of control and are means ± SE of three independent experiments run in duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control.

Figure 4

Inhibition of p38 MAPK blocks HQ-induced Hsp27 phosphorylation and focal aggregates formation in ARPE-19 cells. A: Inhibition of HQ-induced Hsp27 phosphorylation by the selective inhibitor of the kinase activity of p38α and p38β MAPK isoforms, SB203580 (SB). ARPE-19 cells were pretreated for one hour with 20 μmol/L of SB, and then exposed to 100 μmol/L HQ for five minutes. Total Hsp27, p-Hsp27, and GAPDH protein expression was assessed by Western blot analysis. Top: Representative Western blot for p-Hsp27. Numbers on the left represent protein molecular mass in kilodaltons. p-Hsp27 protein expression was normalized to total Hsp27 protein. GAPDH was used as loading control for total Hsp27. Bottom: Average densitometry results of three independent experiments run in duplicate. Data are expressed as percentage of control and are means ± SE. **P < 0.01 versus control; ***P < 0.001 versus HQ alone. B: Decreased formation of F-actin aggregates showed by staining for F-actin in ARPE-19 cells pretreated for one hour with or without 20 μmol/L of SB, before they were exposed to 100 μmol/L HQ for six hours. Cells were stained with rhodamine-phalloidin and examined by confocal microscopy by using magnification ×40. White arrowheads show formation of focal aggregates. C: Quantification of F-actin aggregates from three independent experiments run in duplicate. Data are expressed as a percentage of HQ-treated cells and are means ± SE. ***P < 0.001 versus HQ-treated cells.

Figure 5

Inhibition of Hsp27 dephosphorylation increases HQ-induced Hsp27 phosphorylation and reorganization of F-actin filaments in ARPE-19 cells. A: Increased HQ-induced Hsp27 phosphorylation by okadaic acid. Confluent serum-starved ARPE-19 cells were pretreated for one hour with 60 nmol/L of okadaic acid, and then exposed to 100 μmol/L HQ for five minutes. Total Hsp27, p-Hsp27, and GAPDH protein expression was evaluated by Western blot analysis. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. p-Hsp27 protein expression was normalized to total Hsp27 protein. Bottom: Average densitometry results of five independent experiments run in duplicate. Data are expressed as percentage of control and are means ± SE. **P < 0.01 and ***P < 0.001 versus control; *P < 0.05 versus okadaic acid alone; ****P < 0.01 versus HQ alone. B: Increased formation of F-actin aggregates showed by staining for F-actin in ARPE-19 cells pretreated for on hour with or without 60 nmol/L of okadaic acid and then exposed to 100 μmol/L HQ for six hours. Cells were stained with rhodamine-phalloidin and examined by confocal microscopy by using magnification ×40. White arrowheads show formation of focal aggregates. C: Quantification of F-acting aggregates from three independent experiments run in duplicate. Data are expressed as a percentage of HQ-treated cells and are means ± SE. ***P < 0.001 versus HQ-treated cells.

Figure 6

Induction of cellular changes in GFP-ARPE-19 cells exposed to HQ. Membrane blebbing (A–D) and formation of F-actin aggregates (E–H). Confluent GFP-ARPE-19 cells were pretreated with or without 20 μmol/L of SB203580 (SB) or 60 nmol/L of okadaic acid, and then exposed to 100 μmol/L HQ for six hours. Cells were directly observed under a fluorescence microscope (top) or stained with rhodamin-phalloidin and then examined under a fluorescent microscope (bottom). A: Control GFP-ARPE-19 cells in which GFP was localized to the membrane. Membrane-derived blebs (thin arrows) after exposure to 100 μmol/L HQ for six hours (B), 20 μmol/L of SB for one hour followed by 100 μmol/L HQ for six hours (C), and 60 nmol/L of okadaic acid for one hour followed by 100 μmol/L HQ for six hours (D). E: Control GFP-ARPE-19 cells showing normal F-actin filaments. F-actin aggregates (thick arrows) after exposure to 100 μmol/L HQ for six hours (F), 20 μmol/L of SB for one hour followed by 100 μmol/L HQ for six hours (G), and 60 nmol/L of okadaic acid for one hour followed by 100 μmol/L HQ for six hours (H). Images are representative of three independent experiments. Original magnification, ×400.

Figure 7

Effect of Hsp27 gene silencing on HQ-induced Hsp27 phosphorylation and F-actin aggregates formation. ARPE-19 cells were transfected with siRNA against Hsp27 or scrambled siRNA. Forty-eight hours later, cells were exposed to 100 μmol/L HQ for either five minutes or six hours. A: Reduction in endogenous Hsp27 protein expression by siRNA against Hsp27. The efficiency of siRNA against Hsp27 in reducing endogenous Hsp27 protein was confirmed by Western blot analysis. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Hsp27 protein expression was normalized to GAPDH. Bottom: Average densitometry results of four independent experiments run in duplicate. Data are expressed as percentage of scrambled siRNA and are means ± SE. *P < 0.05 versus scrambled siRNA. B: Effect of Hsp27 siRNA on HQ-induced Hsp27 phosphorylation. Hsp27 phosphorylation was assessed by Western blot analysis and p-Hsp27 protein expression normalized to GAPDH. Top: Western blot from a representative experiment. Number on the left represents protein molecular weight in kilodaltons. Bottom: Average densitometry results of four independent experiments run in duplicate. Data are expressed as percentage of control (−HQ) and are means ± SE. *P < 0.05 versus control (−HQ). C: Inhibition of focal aggregates formation by Hsp27 gene silencing in ARPE-19 cells treated with HQ. Cells were stained with rhodamine-phalloidin and examined by confocal microscopy by using magnification ×40. Arrows show formation of focal aggregates. D: Quantification of F-acting aggregates from three independent experiments run in duplicate. Data are expressed as a percentage of HQ-treated cells transfected with scrambled siRNA and are means ± SE. ***P < 0.001 versus scrambled siRNA.

Figure 8

HQ activates ERK1/2 phosphorylation. Time-dependent ERK1/2 phosphorylation assessed by Western blot on cell lysates from ARPE-19 cells treated with 100 μmol/L HQ for various periods of time. Top: Western blot from a representative experiment. Number on the left represents protein molecular weight in kilodaltons. Bottom: Average densitometry results of three independent experiments run in duplicate. Phospho-ERK (p-ERK) protein expression was normalized to total ERK. GAPDH was used as loading control for total ERK. Data are expressed as percentage of control and are means ± SE. *P < 0.05, **P < 0.01, and ***P > 0.001 versus control.

Figure 9

Cross talk between p38 and ERK1/2 MAPK signaling pathways in ARPE-19 cells treated with HQ. A: Inhibition of p38 MAPK pathway potentiates HQ-induced ERK1/2 phosphorylation in ARPE-19 cells. Confluent serum-starved ARPE-19 cells were pretreated for one hour with 20 μmol/L of p38 MAPK inhibitor SB203580 (SB), and then exposed to 100 μmol/L HQ for five minutes. Phospho-ERK (p-ERK) was examined by Western blot and normalized to total ERK. GAPDH was used as loading control for total ERK. B: Inhibition of ERK MAPK pathway with PD98059 (PD) leads to a decrease in HQ-induced p38 phosphorylation in ARPE-19 cells. ARPE-19 cells were pretreated for one hour with 40 μmol/L of ERK inhibitor PD, and then exposed to 100 μmol/L HQ for five minutes. Phospho-p38 (p-p38) was examined by Western blot and normalized to total p38. GAPDH was used as loading control for total p38. Top: Western blot from a representative experiment. Number on the left represents protein molecular weight in kilodaltons. Bottom: Average densitometry results of three independent experiments run in duplicate. Data are expressed as percentage of control and are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control; ****P < 0.01 and *P < 0.05 versus HQ alone.

Figure 10

Inhibition of ERK MAPK pathway blocks HQ-induced Hsp27 phosphorylation and focal aggregates formation. A: Abrogation of HQ-induced Hsp27 phosphorylation by PD98059. Confluent serum-starved ARPE-19 cells were pretreated for one hour with 40 μmol/L of ERK inhibitor PD98059 (PD), and then exposed to 100 μmol/L HQ for five minutes. Total Hsp27, p-Hsp27, and GAPDH protein expression was evaluated by Western blot analysis. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. p-Hsp27 protein expression was normalized to total Hsp27 protein. Bottom: Average densitometry results of three independent experiments run in duplicate. Data are expressed as percentage of control and are means ± SE. *P < 0.01 versus HQ alone; **P < 0.01 versus control. B: Decreased formation of F-actin aggregates showed by staining for F-actin in ARPE-19 cells pretreated for one hour with or without 40 μmol/L of PD and then exposed to 100 μmol/L HQ for six hours. Cells were stained with rhodamine-phalloidin and examined by confocal microscopy by using magnification ×40. Arrows show formation of focal aggregates. C: Quantification of F-acting aggregates from three independent experiments run in duplicate. Data are expressed as percentage of HQ-treated cells and are means ± SE. ***P < 0.001 versus HQ-treated cells.

Figure 11

Regulation of Hsp25 and phosphorylated Hsp27 (p-Hsp25) in dissected RPE sheets from mice treated with HQ (0.8%) in drinking water for seven months. Protein and total RNA were extracted from RPE sheets (n = 6 eyes/group). A: Hsp25 mRNA expression analyzed by real-time PCR. GAPDH was used as the housekeeping gene. Data represent average results from six eyes normalized to the housekeeping gene. Results are expressed as mean ± SE. ***P < 0.001 versus control. B: Ratio p-Hsp25/Hsp25 protein expression evaluated by Western blot. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. p-Hsp25 protein expression was normalized to total Hsp25 protein. Bottom: Data are the average results from six eyes. Data are expressed as percentage of control and shown are mean results ± SE. ***P < 0.001 versus control. C: Ratio Hsp25/GAPDH protein expression evaluated by Western blot. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Hsp25 protein expression was normalized to GAPDH. Bottom: Data are the average results from six eyes. Data are expressed as percentage of control and shown are mean results ± SE. ***P < 0.001 versus control.

Figure 12

Regulation of phosphorylated p38 (p-p38), and phosphorylated ERK (p-ERK) in dissected RPE sheets from mice treated with HQ (0.8%) in drinking water for seven months. Protein was extracted from RPE sheets (n = 6 eyes/group). A: p-p38 protein expression determined by Western blot. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. p-p38 protein expression was normalized to total p38 protein. Bottom: Data are the average results from six eyes. Data are expressed as percentage of control and shown are mean results ± SE. ***P < 0.001 versus control. B: Ratio p38/GAPDH protein expression evaluated by Western blot. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Data are the average results from six eyes. Data are expressed as percentage of control and shown are mean results ± SE. C: pERK protein expression determined by Western blot. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. p-ERK protein expression was normalized to total ERK protein. Bottom: Data are the average results from six eyes. Data are expressed as percentage of control and shown are mean results ± SE. ***P < 0.001 versus control. D: Ratio ERK/GAPDH protein expression evaluated by Western blot. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Data are the average results from six eyes. Data are expressed as percentage of control and shown are mean results ± SE.