Inhibition of apoptosis by p26: implications for small heat shock protein function during Artemia development

Abstract

p26, an abundantly expressed small heat shock protein, is thought to establish stress resistance in oviparously developing embryos of the crustacean Artemia franciscana by preventing irreversible protein denaturation, but it might also promote survival by inhibiting apoptosis. To test this possibility, stably transfected mammalian cells producing p26 were generated and their ability to resist apoptosis induction determined. Examination of immunofluorescently stained transfected 293H cells by confocal microscopy demonstrated p26 is diffusely distributed in the cytoplasm with a minor amount of the protein in nuclei. As shown by immunoprobing of Western blots, p26 constituted approximately 0.6% of soluble cell protein. p26 localization and quantity were unchanged during prolonged culture, and the protein had no apparent ill effects on transfected cells. Molecular sieve chromatography in Sepharose 6B revealed p26 oligomers of about 20 monomers, with a second fraction occurring as larger aggregates. A similar pattern was observed in sucrose gradients, but overall oligomer size was smaller. Mammalian cells containing p26 were more thermotolerant than cells transfected with the expression vector only, and as measured by annexin V labeling, Hoescht 33342 nuclear staining and procaspase-3 activation, transfected cells effectively resisted apoptosis induction by heat and staurosporine. The ability to confer thermotolerance and limit heat-induced apoptosis is important because Artemia embryos are frequently exposed to high temperature in their natural habitat. p26 also blocked apoptosis in transfected cells during drying and rehydration, findings with direct relevance to Artemia life history characteristics because desiccation terminates cyst diapause. Thus, in addition to functioning as a molecular chaperone, p26 inhibits apoptosis, an activity shared by other small heat shock proteins and with the potential to play an important role during Artemia embryo development.

Fig 1.

Immunostaining of E11′L cells. E11′L cells immunostained with antibody to p26 and Alexa Fluor®488-labeled anti-rabbit IgG secondary antibody (green) were incubated with propidium iodide to reveal nuclei (red) and examined by confocal microscopy. c, chromosomes; d, dividing cell. Bar = 25 μm (D). All figures are the same magnification

Fig 2.

p26 from E11′L cells. (A) Protein extracts from E11′L cells were electrophoresed in 12.5% SDS polyacrylamide gels and either stained with Coomassie blue (upper panel) or blotted to nitrocellulose and immunostained with antibody to p26 (lower panel). Lane 1: 10.0 and 5.0 μg of Artemia embryo protein for gel and blot, respectively; lane 2: 30 μg of protein extract from 293H cells transfected with vector only; lane 3: 30 μg of protein extract from E11′L cells early in culture; lane 4: 30 μg of protein extract from E11′L cells after 50 days in culture. (B) E11′L protein extract and purified p26 produced in bacteria were electrophoresed concurrently in 12.5% gels, blotted to nitrocellulose, probed with anti-p26 antibody, and scanned with the UMAX Astra 1200S scanner. The amount of p26 was determined by comparing the p26 band pixel density in the E11′L lane to the standard curve derived with bacterially produced p26. Insert lane 1: 0.2 μg; lane 2: 0.3 μg; lane 3: 0.4 μg; lane 4: 0.5 μg; lane 5: 0.6 μg; lane 6: 0.7 μg of protein. Lane 7 contained 40 μg of E11′L cell protein. (C) Protein extracts from E11′L cells were either chromatographed in Sepharose 6B columns (upper panel) or centrifuged in 10–50% continuous sucrose gradients (lower panel). Collected fractions were electrophoresed in 12.5% SDS polyacrylamide gels, blotted to nitrocellulose, and immunostained with antibody to p26 (inserts). Labeled arrowheads, molecular mass markers × 10⁻³

Fig 3.

p26-dependent thermotolerance in transfected mammalian cells. (A) E11′L cells and cells transfected with vector only were heat shocked for 0, 15, 30, 45, and 60 min at 46°C; incubated in T75 vented tissue culture flasks for 2 weeks; and stained with crystal violet. Nonshaded bar, E11′L; shaded bar, cells lacking p26. The data are the mean ± standard error of 3 independent experiments. (B) Crystal violet–stained flasks containing E11′L (left side) and control (right side) cells heat shocked for 30 min before incubation for 2 weeks. (C) Nomarski images of cells either containing (E11′L) or lacking (control) p26 after heat shock for 30 minutes. The bar in the lower right panel represents 50 μm, and all pictures are the same magnification

Fig 4.

p26 reduces heat-induced apoptosis. E11′L and cells transfected with vector only were heat shocked at 46°C for the times indicated, incubated at 37°C for 24 hours, and stained with either annexin V-Alexa Fluor®488 conjugate (A) or Hoescht 33342 (B). Nonshaded bar, E11′L cells; shaded bar, cells lacking p26. The data are the mean ± standard error of 3 independent experiments. Cells heat shocked for 30 minutes were either double-stained with propidium iodide (red) and annexin V-Alexa Fluor®488 conjugate (green) (C, D) or stained with Hoescht 33342 (E). Arrows in panel E indicate apoptotic cell nuclei. Bar = 40 μm (D), which has the same magnification as (C). Bar = 15 μm (E)

Fig 5.

p26 blocks apoptosis on cell drying and rehydration. E11′L cells and cells transfected with vector only were vacuum dried to 0.35 g H₂O per gram dry weight, rehydrated, incubated in either the presence or absence of the pan-caspase inhibitor OPH-109, then stained with FITC-DEVD-FMK, a fluorescently labeled inhibitor of activated caspase-3, and propidium iodide before analysis by flow cytometry. (A) 293H cells transfected with vector only; (B) 293H cells transfected with vector only and incubated with OPH-109; (C) E11′L cells; (D) E11′L cells incubated with OPH-109. (E) Experiments in panels A–D were done in triplicate; the results were averaged and plotted with standard error. N1, dead cells; N2, apoptotic and dead cells; N3, live cells; N4, apoptotic cells

Fig 6.

p26 reduces staurosporine-induced apoptosis. E11′L cells and cells transfected with vector only were treated with 0.2 M staurosporine for the times indicated and then stained with either annexin V-Alexa Fluor®488 conjugate (A) or Hoescht 33342 (B). Nonshaded bar, E11′L cells; shaded bar, cells lacking p26

Fig 7.

Inhibition of procaspase-3 activation by p26. E11′L cells (A) and cells transfected with vector only (B) were either heat shocked at 46°C or exposed to staurosporine for 6 hours, as described in Materials and Methods. Protein extracts were prepared from these cells, electrophoresed in 12.5% SDS polyacrylamide gels, blotted to nitrocellulose, and probed with anti–caspase-3 antibody. Lanes 1– 5: protein extracts from cells heat shocked for 0, 15, 30, 45, and 60 minutes, respectively. Lanes 6, 7: extracts from cells exposed to 0.0 and 0.2 μm, respectively, of staurosporine for 6 hours. Approximately equal amounts of protein were applied to each lane. Pro, procaspase-3; 17 kDa, 17-kDa fragment from procaspase-3; 12 kDa, 12-kDa fragment from procaspase-3