Expression and functional roles of caspase-5 in inflammatory responses of human retinal pigment epithelial cells

Abstract

Purpose: To investigate the expression, activation, and functional involvement of caspase-5 in human retinal pigment epithelial (hRPE) cells.

Methods: Expression and activation of caspase-5 in primary cultured hRPE cells, telomerase-immortalized hTERT-RPE1 cells (hTERT-RPE1), or both, were measured after stimulation with proinflammatory agents IL-1β, TNF-α, lipopolysaccharide (LPS), interferon-γ, monocyte coculture, adenosine triphosphate (ATP), or endoplasmic reticulum (ER) stress inducers. Immunomodulating agents dexamethasone (Dex), IL-10, and triamcinolone acetonide (TA) were used to antagonize proinflammatory stimulation. Cell death ELISA and TUNEL staining assays were used to assess apoptosis.

Results: Caspase-5 mRNA expression and protein activation were induced by LPS and monocyte-hRPE coculture. Caspase-5 activation appeared as early as 2 hours after challenge by LPS and consistently increased to 24 hours. Meanwhile, caspase-1 expression and protein activation were induced by LPS. Activation of caspase-5 was blocked or reduced by Dex, IL-10, and TA. Activation of caspase-5 and -1 was also enhanced by ATP and ER stress inducers. Expression and activation of caspase-5 were inhibited by a caspase-1-specific inhibitor. Caspase-5 knockdown reduced caspase-1 protein expression and activation and inhibited TNF-α-induced IL-8 and MCP-1. In contrast to caspase-4, the contribution of caspase-5 to stress-induced apoptosis was moderate.

Conclusions: Caspase-5 mRNA synthesis, protein expression, and catalytic activation were highly regulated in response to various proinflammatory stimuli, ATP, and ER stress inducers. Mutual activation between caspase-5 and -1 suggests caspase-5 may work predominantly in concert with caspase-1 in modulating hRPE inflammatory responses.

Figure 1.

Stimulation of caspase-5 mRNA synthesis (A, B) and caspase-5 and -1 protein cleavage (C, D) in hRPE cells. The hRPE cells were cultured either without (untreated; Ctl) or with IL-1β (IL-1, 2 ng/mL), TNF-α (TNF, 20 ng/mL), LPS (1000 ng/mL), IFN-γ (500 U/mL), or overlaid monocytes (RM) and were incubated for 6 hours (A), 0, 2, 4, 6, and 8 hours (B), or 24 hours (C, D). The data shown represent results from a typical experiment. (A, B) The steady state caspase-5 mRNA levels determined by RT-PCR. (C, D) Western blot analysis of caspase-5 protein. The fold changes were calculated by normalization against β-actin and comparison with untreated control (A) or with treated control at 0 hours (B) of mRNA levels in RT-PCR, or by normalization against actin and making a comparison with untreated control levels of caspase-5 in Western blot analysis (C, D). The bands at approximately 30 kDa (C) are presumably either nonspecific bands or intermediately cleaved caspase-5 protein.

Figure 2.

Induction of caspase-5 catalytic activity by LPS. The hRPE cells were stimulated by LPS (1000 ng/mL, A, B) for 0, 1, 2, 7 (A), and 24 (A, B) hours, with or without caspase -1 inhibitor Z-YVAD-fm (2 μM) or Dex (10⁻⁶ M). Two hundred micrograms of protein from the hRPE cell lysate was analyzed in each assay using a caspase-5 assay kit. The data are representative of three independent experiments with similar results, as shown by fold increases in caspase-5 activity. Values represent mean ± SEM. *P < 0.05; ***P < 0.001.

Figure 3.

Stimulation of human RPE caspase-5 mRNA synthesis (C) and protein maturation (A, B, D) by ATP and ER stress. hRPE cells were pretreated with LPS (1000 ng/mL, A, B) for 4 hours and cultured either with ATP (3 mM) or BzATP (300 μM) for 0, 5, 10, 30, 60, and 100 minutes, tunicamycin (10 μM), or thapsigargin (25 ng/mL) for 6 (C), 0, 24, and 48 hours (D). The data shown represent results from a typical experiment. (C) Steady state caspase-5 mRNA, as determined by RT-PCR. The fold changes were calculated by normalization against β-actin and comparison with control cells without the treatment. (A, B, D) Western blot analysis of caspase-5, caspase-1, and actin proteins. (D) The bands at approximately 30 kDa are presumably either nonspecific bands or intermediately cleaved caspase-5 protein.

Figure 4.

The effect of Dex, IL-10, and TA on caspase-5 protein production and maturation by IL-1β (A) and LPS (B, C) in hRPE cells. hRPE cells were pretreated with Dex (10⁻⁸ or 10⁻⁷ to 10⁻⁶ M), IL-10 (100 U/mL), or TA (0.1 or 0.01 mg/mL) for 30 minutes and then coincubated with IL-1β (2 ng/mL) or LPS (1000 ng/mL) for an additional 24 hours. Proteins from the whole hRPE cell lysates were detected by anti–caspase-5 antibody specific for pro-caspase-5 and cleaved caspase-5. Fold changes of the cleaved caspase-5 were calculated by relative density between treated and untreated samples, as determined by densitometry after normalization with actin protein.

Figure 5.

Blockade of caspase-5 activation by caspase-1 inhibitors (A) and caspase-5 and -1 activation by caspase-5 knockdown (B, C). (A) hRPE cells were pretreated with caspase-1 inhibitor Z-YVAD-fmk or caspase-1 and -5 inhibitor Z-WEHD-fmk for 30 minutes and then coincubated with or without LPS (1000 ng/mL) for an additional 24 hours. Proteins from the whole hRPE cell lysates were subjected to Western blot analysis using anti–caspase-5 antibody. (B, C) Whole cell lysates from stably knockdown hTERT-RPE 1 cells with caspase-5 shRNA and control shRNA were detected by antibodies specific for pro-caspase-5 or cleaved caspase-5 or -1. Fold changes of cleaved caspase-5 and -1 were calculated by relative density between scrambled shRNA002 and caspase-5–specific shRNA3554 using densitometry after normalization to actin protein.

Figure 6.

Inhibition of human RPE IL-8 and MCP-1 production by caspase inhibitors (A, B, E, F) and caspase-5 shRNA knockdown (C, D). hRPE cells (A, B, E, F) were pretreated with caspase-5 and -1 inhibitor Z-WEHD-fmk (2 μM), caspase-1 inhibitor Z-YVAD-fmk (2 μM), or pan-caspase inhibitor Z-VAD-fmk (50 μM) and were costimulated with IL-1β (2 ng/mL), TNF-α (20 ng/mL), or LPS (1000 ng/mL) for 24 hours. (C, D) hTERT-RPE 1 cells were transfected with scrambled shRNA002 or caspase-5–specific shRNA3554. Secretion of IL-8 and MCP-1 was determined by ELISA. Values represent mean ± SEM (n = 3). Statistical analysis was carried out for all values with inhibitor versus the corresponding control without inhibitor (A, B, E, F) or for all values with scrambled shRNA versus caspase-5–specific shRNA. Data are representative of three independent experiments with similar results. Ctrl, untreated; IL-1, IL-1β; TNF, TNF-α.

Figure 7.

ER stress-induced hRPE apoptotic cell death. hRPE cells were cultured with or without tunicamycin (10 μM) in the presence or absence of caspase-4 inhibitor Z-LEVD-fmk (Z-LEVD, 2 μM) or caspase-5 and -1 inhibitor Z-WEHD-fmk (Z-WEHD, 2 μM) for 48 or 72 hours. Apoptosis was determined by the absorbance difference between A_405nm and A_490nM using a cell death detection ELISA kit (A). For comparison, tunicamycin-treated cells were assigned as 100% apoptotic cell death (positive control). (B, C) Quantification of the effects of ER stress-induced hRPE cell death by TUNEL assays. (B) TUNEL staining (dark brown), shown at 400× magnification. The hRPE cells were stained by vimentin (red). Left, middle: unstimulated hRPE cells (normal cultures and culture in TUNEL assay); right: hRPE cells treated with tunicamycin showing nuclear condensation and cell shrinkage. (C) Data are expressed as percentage of TUNEL-positive hRPE cells. Values represent mean ± SEM. ***P <0.001, **P < 0.01, and *P < 0.05, compared with tunicamycin treatment without inhibitors. The findings are representative of three independent experiments.