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Influence of various fatty acids on the activity of protein phosphatase type 2C and apoptosis of endothelial cells and macrophages
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 5 ( 2 0 0 8 ) 397–403
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Influence of various fatty acids on the activity of protein phosphatase type 2C and apoptosis of endothelial cells and macrophages Josef Krieglstein a,b,∗ , Birgit Hufnagel a , Melanie Dworak b , Stefanie Schwarz a , Tobias Kewitz b , Michael Reinbold a , Susanne Klumpp b a b
Institut für Pharmakologie und Toxikologie, Philipps-Universitaet, Marburg, Germany Institut für Pharmazeutische und Medizinische Chemie, Westfaelische Wilhelms-Universitaet, Muenster, Germany
a r t i c l e
i n f o
a b s t r a c t
Article history:
In previous work we have demonstrated that protein phosphatase type 2C (PP2C) ␣ and 
Received 19 November 2007
can be activated by mono-unsaturated fatty acids (MUFAs) leading to apoptosis of cultured
Received in revised form
endothelial cells. In the present paper we could show that saturated fatty acids (SFAs) did
20 August 2008
not activate PP2C and did not cause apoptosis both in endothelial cells and macrophages.
Accepted 20 August 2008
However, long-chain SFAs (>16 C-atoms) were capable of inhibiting both, activation of PP2C
Published on line 4 September 2008
as well as apoptosis of human umbilical vein endothelial cells (HUVECs) and macrophages caused by oleic acid. Interestingly, docosahexaenoic acid (DHA) known to protect arterial
Keywords:
vessels against the progression of atherosclerosis caused apoptosis of HUVECs at high
Atherosclerosis
concentrations (200–400 M) but inhibited the apoptotic damage of HUVECs at a low, phys-
Fatty acids
iologically relevant concentration range (1–10 M). In contrast, oleic acid did not protect
Human umbilical vein endothelial
HUVECs against damage even at low concentrations (1–25 M). It is supposed that an unbal-
cells
anced and chronically increased level of MUFAs in blood has an atherosclerotic potential.
Macrophages
Furthermore, PP2C activated by MUFAs appears as a new target for drugs to prevent or treat
Protein phosphatase type 2C
atherosclerosis. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
We had discovered that MUFAs activate PP2C ␣ and  and that this induces apoptosis of neuronal and endothelial cells (Hufnagel et al., 2005; Schwarz et al., 2006). Apoptosis caused by PP2C could be mediated by dephosphorylation of the proapoptotic BH3-only protein Bad (Klumpp et al., 2003), by activation of the p53 signaling pathway (Ofek et al., 2003) and of the stress-activated protein kinase cascade (Hanada et al., 1998, 2001). PP2C belongs to the PPM phosphatase family whose members depend on divalent cations such as Mg2+
or Mn2+ (McGowan and Cohen, 1998). However, high concentrations of divalent cations (>20 mM) were necessary for measurable activity of PP2C. When MUFAs were added, the PP2C isozymes ␣ and  were maximally active at a physiological range of Mg2+ (0.5–1.5 mM) (Klumpp et al., 1998b). Thus, the combination of Mg2+ and MUFAs seems to play the essential role in regulating PP2C activity under in vivo conditions. The MUFAs stimulating PP2C had to be oxidizable, cis-configurated, at least 15 C-atoms in length and negatively charged. MUFAs that fulfilled all these requirements, e.g. oleic acid, activated PP2C in vitro and caused apoptosis of neu-
∗ Corresponding author at: Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Hittorfstraße 58-62, D-48149 Münster, Germany. Tel.: +49 251 83 32 210; fax: +49 251 83 32 211. E-mail address: [email protected] (J. Krieglstein). 0928-0987/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2008.08.007
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rons and endothelial cells (Klumpp et al., 2002; Zhu et al., 2005). The vascular endothelium is incessantly exposed to MUFAs since the lipoprotein lipase located on the luminal surface of the endothelial cells hydrolyzes triglycerides of the circulating lipoproteins (Stins et al., 1992). We could demonstrate that the physiologic fatty acids obtained from human lipoproteins activated PP2C in vitro and induced apoptosis in HUVECs (Hufnagel et al., 2005; Schwarz et al., 2006). These data suggest PP2C to be a significant player in the complex system of atherogenesis. In this study we attempted to shed some light on the role of saturated fatty acids (SFAs) and docosahexaenoic acid (DHA) in apoptotic damage of endothelial cells and macrophages caused by PP2C activation.
used at passages 3–6 and morphology was controlled prior to treatment. The cells were seeded at a density of 5 × 105 cells/9.6 cm2 dishes. Human THP-1 monocytes were cultured in suspension at a density of 4 × 106 cells/5 ml for 4 days. For differentiation to macrophages, monocytes received 100 ng/ml PMA and 50 M 2-mercaptoethanol (Dory, 1993). After 72 h in culture, the monocytes differentiated to macrophages and became adherent. Experiments were accomplished with adherent and confluent macrophages (>106 cells/9.6 cm2 dishes). Both cell types were cultured at 37 ◦ C in an incubator supplemented with 5% CO2 and 95% air.
2.
Materials and methods
2.1.
Materials
After the replacement of culture medium, HUVECs and macrophages were incubated for an additional 48 h. Cell cultures were then treated for 24 h with oleic acid or DHA, with different concentrations of SFAs or with a combination of both, oleic acid and SFAs. Fatty acids were dissolved in dimethylsulfoxide (DMSO). The final concentration of DMSO in the culture medium did not exceed 0.5%. To induce apoptosis in HUVECs we omitted fetal calf serum from or added staurosporine to the culture medium.
HUVECs and endothelial cell growth medium containing 2% heat-inactivated fetal bovine serum, 0.1 ng/ml epidermal growth factor, 1 g/ml hydrocortisone, 1 ng/ml basic fibroblast growth factor and antibiotics were purchased from Promocell (Heidelberg, Germany). For serum deprivation studies HUVECs were cultured in OptiMEM (Gibco, Germany). THP-1 monocytes were a gift from the Institute for Arteriosclerosis Research (Muenster, Germany). THP-1 cells were cultured in a medium containing RPMI-1640, 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM l-glutamine, 100 mM sodium pyruvate. Phorbol-12-myristate-13-acetate (PMA), 2mercaptoethanol, Hoechst 33258, Nile blue, fatty acids (oleic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, arachidic acid, behenic acid, and DHA) and bovine serum albumin (BSA) were obtained from Sigma–Aldrich (Taufkirchen, Germany). Nile blue staining was determined with the confocal laser-scanning microscope Zeiss LM510 (Jena, Germany) and analysis of apoptotic cells was accomplished with the fluorescent microscope Axiovert 100 Zeiss (Jena, Germany). Recombinant PP2C was prepared as described previously (Klumpp et al., 1998a).
2.2.
Measurement of PP2C activity
Activity of PP2C was measured using [32 P]-labelled casein as a substrate (McGowan and Cohen, 1998; Klumpp et al., 1998b). PP2C assays contained 30 mM Tris–HCl (pH 7.0), 0.1% 2-mercaptoethanol, 0.6 mg/ml BSA, 1 M [32 P]casein (5 × 104 cpm), and 10–200 ng PP2C in a final volume of 30 l. PP2C is characterized by its dependence on divalent cations for activity. If not indicated otherwise, 1 mM magnesium acetate was present. Reactions were terminated after 10 min at 30 ◦ C by adding trichloroacetic acid. The samples were centrifuged, and the supernatant was analyzed for [32 P]phosphate content.
2.3.
Cell culture
HUVECs were cultured in the endothelial cell growth medium to 90% confluence. In all experiments cells were
2.4.
2.5.
Fatty acid treatment of HUVECs and macrophages
Staining with Hoechst 33258 and Nile blue
Fatty acid-treated cells were washed with PBS (phosphate buffered saline, pH 7.4), fixed for 30 min with paraformaldehyde (4%) and then incubated for 30 min with the DNA fluorochrome Hoechst 33258 (10 g/ml) in methanol at room temperature in the dark. After washing with PBS, the nuclear morphology of HUVECs and macrophages was analyzed under a fluorescent microscope at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Cells showing shrunken or fragmented nuclei or chromatin condensation were counted as apoptotic cells. Nile blue was used to reveal uptake of fatty acids. After staining with Hoechst 33258, HUVECs and macrophages were washed with PBS, stained with Nile blue solution (10 g/ml) for 2 h and analyzed under a confocal laser-scanning microscope at wavelengths of 488/525 nm.
2.6.
Protein determination
The concentration of proteins was determined by BCAor Lowry-assay (Lowry et al., 1951) using BSA as a standard.
2.7.
Statistical methods
All values, except the PP2C activity values in Fig. 2, are given as means ± S.D. One-way analysis of variance (ANOVA) followed by Scheffé’s test was applied. PP2C-assays were performed in duplicate and independently repeated at least twice.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 5 ( 2 0 0 8 ) 397–403
399
Fig. 1 – Uptake of oleic acid and stearic acid into HUVECs (A–C) and macrophages (D–F). Cells were incubated for 24 h with the fatty acids (200 M) and stained with Nile blue. The two fatty acids (yellow) were taken up by the cells but, notably, oleic acid-treated cells were partially damaged and stearic acid-treated were not 10 m.
3.
Results
3.1. Uptake of fatty acids into HUVECs and macrophages The uptake of SFAs into HUVECs and macrophages was similar to that of MUFAs (Fig. 1). Here we were using Nile blue to study the uptake of fatty acids into HUVECs and macrophages. Control cells received 0.5% DMSO and subsequent staining resulted in a weak fluorescence reflecting endogenous lipids. Addition of oleic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid and stearic acid resulted in a bright fluorescence of the cells within 3 h of otherwise intact cells indicating a similar uptake of all fatty acids. After an incubation period of 24 h, the oleic acid-treated cells revealed apoptosis whereas stearic acid-treated cells remained intact (Fig. 1).
3.2.
Influence of fatty acids on PP2Cˇ activity
Searching for regulatory components of PP2C we discovered that the activity of PP2C increased 10–15-fold after the addition of certain MUFAs, e.g. oleic acid but also DHA, if measured at a physiological concentration of 0.5–1 mM free Mg2+ (Klumpp et al., 1998b). In contrast, SFAs were not capable of stimulating the activity of PP2C (Fig. 2A).
To further study the regulation of PP2C, we wanted to elucidate whether SFAs of different chain length might be able to abolish the oleic acid-induced activation of PP2C. Treatment of PP2C with SFAs with chain lengths from 14 to 22 C-atoms showed no activation of the enzyme (Fig. 2A). PP2C in the presence of both, oleic acid and SFAs, revealed interesting new results: SFAs with a chain length of 16 C-atoms or less, e.g. myristic acid (14 C-atoms), pentadecanoic acid (15 C-atoms) and palmitic acid (16 C-atoms), were not able to inhibit the activation-capacity of oleic acid. In contrast, SFAs with a chain length of more than 16 C-atoms, e.g. heptadecanoic acid (17 Catoms), stearic acid (18 C-atoms), arachidic acid (20 C-atoms) and behenic acid (22 C-atoms) reduced the oleic acid-induced activation of PP2C (Fig. 2B) in vitro.
3.3. Apoptosis caused by oleic acid and DHA in HUVECs and macrophages Oleic acid but also DHA induced apoptosis in HUVECs at a concentration of 200–400 M (Fig. 3). The same was true for macrophages exposed to oleic acid (250 M) for 24 h (Fig. 4C). Similar to HUVECs (Hufnagel et al., 2005) the corresponding trans-derivative and the oleic acid methylester both were not harmful to macrophages when administered in the same concentration range as oleic acid (data not shown).
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Fig. 2 – Effect of oleic acid and SFAs on PP2C activity. (A) Dephosphorylation of [32 P]casein by PP2C was measured after addition of various fatty acids in the presence of 1 mM Mg2+ . Oleic acid stimulated the activity of PP2C ninefold, whereas SFAs with different chain lengths failed to activate the enzyme. SFAs added were myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid (17:0), stearic acid (18:0), arachidic acid (20:0) and behenic acid (22:0). (B) Effect of various SFAs on PP2C activated by oleic acid (18:1). Phosphatase activity was measured after addition of oleic acid and oleic acid in combination with different SFAs. SFAs less than 17 C-atoms could not clearly affect the oleic acid-induced activation of PP2C, whereas SFAs with a chain length longer than 16 C-atoms did. Dephosphorylation of [32 P]casein was determined with 15–160 ng PP2C per incubation. Activity in the absence of fatty acids (control) was 5.1 nmol Pi /(min mg) (100%). Values are given as means of two independent determinations in duplicate, respectively.
3.4.
Inhibition of oleic acid-induced apoptosis by SFAs
In further experiments we tested whether long-chain SFAs capable of reducing oleic acid-activated PP2C were able to abolish the oleic acid-induced apoptosis in HUVECs and macrophages. First, cells were treated with various SFAs (14–22 C-atoms). Those SFAs tested did not influence the viability of HUVECs and macrophages (Fig. 4A and C). Next, and in parallel to the results described on PP2C in vitro, we attempted to elucidate whether treatment of HUVECs and macrophages with SFAs in the presence of oleic acid might affect oleic acid-induced apoptosis. Myristic acid and pentadecanoic acid were not able to reduce the apoptotic effect of oleic acid in HUVECs and macrophages. In contrast, heptadecanoic acid and stearic acid significantly reduced the oleic acid-induced apoptosis in both cell types (Fig. 4B and D). Palmitic acid with a chain length right in between protecting (≥C-17) and nonprotecting (≤C-15) SFAs had a different effect on the oleic
Fig. 3 – Oleic acid and DHA induce apoptosis in HUVECs. The cells were incubated with different concentrations of oleic acid (A) or DHA (B). Morphology was analyzed under a fluorescence microscope after nuclear staining with Hoechst 33258. Cells showing fragmented nuclei or condensed chromatin were counted as apoptotic cells. Values are given as means ± S.D. of 5 experiments. ***p < 0.001, compared with vehicle-treated control.
acid-induced apoptosis in HUVECs and macrophages (Fig. 4B and D). Arachidic acid and behenic acid were almost insoluble in the endothelial cell growth medium and thus could not be tested.
3.5.
Influence of DHA on the viability of HUVECs
Omega-3 fatty acids are particularly suggested to inhibit atherogenesis and to lower cardiovascular mortality (for reviews see von Eckardstein, 2005). Therefore, we tested the influence of DHA on the viability of HUVECs. It turned out that 200 M DHA damaged HUVECs similar to oleic acid (Fig. 3). To elucidate a putative protective effect of DHA we reduced the viability of HUVECs by removing fetal calf serum from the culture medium for about 60 h and the percentage of apoptotic cells increased in the controls up to 20%. However, when DHA was added to the culture medium to give a low concentration (1–10 M) the percentage of apoptotic HUVECs was significantly reduced to around 10% (Fig. 4A). This protective effect could not be achieved by oleic acid (Fig. 5B).
4.
Discussion
Reversible phosphorylation of proteins, catalyzed by kinases and phosphatases, is a major mechanism to regulate apoptotic pathways. Our previous studies have demonstrated that serine/threonine phosphatases PP2C ␣ and  are activated by certain MUFAs (Klumpp et al., 1998b). We could demonstrate
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 5 ( 2 0 0 8 ) 397–403
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Fig. 4 – Effect of oleic acid and SFAs with different chain lengths on the viability of HUVECs (A) and macrophages (C). Cells were incubated with oleic acid (18:1), myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid (17:0) and stearic acid (18:0) at a concentration of 200 M, respectively. Controls were treated with vehicle only (0.2% DMSO). Heptadecanoic acid and stearic acid protected HUVECs from oleic acid-induced apoptosis. (B) Treatment of HUVECs with myristic acid (14:0), pentadecanoic acid (15:0) and palmitic acid (16:0) could not abolish the toxic effect of oleic acid whereas heptadecanoic acid (17:0) and stearic acid (18:0) could. (D) Treatment of macrophages with myristic acid (14:0) and pentadecanoic acid (15:0) could not abolish the toxic effect of oleic acid whereas palmitic acid (16:0), heptadecanoic acid (17:0) and stearic acid (18:0) could. Morphology was analyzed under a fluorescence microscope after nuclear staining with Hoechst 33258. Cells showing fragmented nuclei or condensed chromatin were counted as apoptotic cells. Values are given as means ± S.D. of 5 experiments. ***p < 0.001, compared with vehicle-treated control. ### p < 0.001, compared with oleic acid-treated cells.
that Bad, a pro-apoptotic member of the Bcl-2 family, is a substrate of PP2C ␣ and , indicating that PP2C can trigger the pro-apoptotic function of Bad (Schwarz et al., 2006). In its dephosphorylated state, Bad promotes apoptosis by heterodimerization with the antiapoptotic oncogene Bcl-XL (Zha et al., 1996). Immunocytochemistry revealed that PP2C and its substrate Bad are co-localized within the cytosol of HUVECs (Hufnagel et al., 2005) and macrophages (data not shown). Extensive studies with a variety of fatty acids demonstrated a striking correlation between activation of PP2C and induction of apoptosis in HUVECs (Hufnagel et al., 2005). This correlation could be extended to macrophages: SFAs (14–22 C-atoms), trans-fatty acids like eladaic acid (18:1 trans-9 ) or methylester derivatives like oleic acid methylester (18:1 cis-9 ester) do not fulfil the structural requirements for PP2C activation and could not damage cultured macrophages. When PP2C was downregulated by the RNAi technique the apoptotic effect of oleic acid on HUVECs was significantly reduced indicating a causal role of PP2C in this process (Schwarz et al., 2006). Since SFAs are not able to activate PP2C in vitro (Fig. 2A), we wanted to elucidate in this study if they could inhibit the oleic acid-induced activation of PP2C. They have a free carboxyl
group and are lipophilic; theoretically they could displace oleic acid from a putative binding site on PP2C without activating the enzyme thus preventing the oleic acid-induced activation. Another explanation could be that SFAs form mixed micelles with oleic acid and thereby decrease the concentration of free oleic acid under the level of efficacy. We tested the effect of SFAs with different chain lengths (14–22 C-atoms) in combination with oleic acid on PP2C activity in vitro. Interestingly, SFAs with a chain length of 16 C-atoms or less, like myristic acid (14 C-atoms), pentadecanoic acid (15 C-atoms), and palmitic acid (16 C-atoms), could not clearly influence the oleic acid-induced activation of PP2C, whereas SFAs with a chain length longer than 16 C-atoms, like heptadecanoic acid (17 Catoms), stearic acid (18 C-atoms), arachidic acid (20 C-atoms), and behenic acid (22 C-atoms), could (Fig. 2B). Obviously, SFAs with a chain length of ≥16 C-atoms and a free carboxyl group were able to prevent the activation of PP2C caused by oleic acid. Double staining with the DNA fluorochrome Hoechst 33258 and Nile blue revealed that the SFAs and oleic acid were taken up by HUVECs and macrophages to a similar extent (Fig. 1), however, only heptadecanoic acid and stearic acid prevented
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Fig. 5 – Physiological concentrations of DHA protect HUVECs against apoptosis. HUVECs were incubated with serum-free medium (OptiMEM) and DHA (A) or oleic acid (B) for about 60 h, respectively. The percentage of apoptotic cells was determined by nuclear staining with Hoechst 33258. Values are given as means ± S.D. of 5 experiments. *p < 0.05 and ***p < 0.001, compared to serum-free vehicle-treated control, respectively.
some of them inhibit oleic acid-induced activation of PP2C and apoptosis of HUVECs and macrophages. Since most of the fatty acids are transported in vivo by lipoproteins and have to pass the endothelium to be distributed into the tissues, the PP2C-induced apoptosis could be crucial for atherogenesis in organs such as the heart which covers about 70% of its energy demand by fatty acids. Since this huge amount of fatty acids has to pass through the endothelium of that organ, there is a better chance for PP2C to be activated, for endothelial cells to be damaged, and consequently for the induction of atherosclerosis. Thus, our hypothesis could explain why atherosclerosis is more pronounced in coronary arteries than, for instance, in cerebral arteries because the brain uses only glucose for its energy metabolism. Furthermore, it also becomes understandable now why lipid loaded macrophages in atherosclerotic plaques undergo apoptosis thus finally induce plaque instability and rupture, and why patients with diabetes mellitus exhibit micro- and macroangiopathies. A balanced mixture of MUFAs and SFAs should be present in the diet to avoid a predominant increase of MUFAs level in the blood which could intensify the development of atherosclerosis. On the other hand, the inhibition of MUFAs-activated PP2C may provide a new strategy for prevention and therapy of atherosclerosis.
Acknowledgement Studies were supported by grants from the Deutsche Forschungsgesellschaft (DFG; Kr 354/17-1 and Kl 601/9-3).
references the damaging effect of oleic acid (Fig. 4). The capability of SFAs to inhibit oleic acid-activated PP2C could pave the way to develop appropriate inhibitors suitable for a new therapeutic strategy against atherosclerosis. Furthermore, the results suggest that a balanced mixture of various fatty acids in the blood could prevent the activation of PP2C and subsequently atherogenesis. There is some evidence in the literature that MUFAs lower the atherosclerotic risk (for reviews see von Eckardstein, 2005). Our results do not support this view. However, data on the anti-atherosclerotic activity of omega-3 fatty acids are more convincing (von Eckardstein, 2005). Since activation of PP2C by DHA already has been shown (Klumpp et al., 1998b) we now tested the effect of DHA on endothelial cells and found intriguing results. Only high concentrations of DHA (200–400 M) in the culture medium – which are most likely not achieved in the blood – could activate PP2C and induce apoptosis of HUVECs comparable to that obtained with oleic acid used at a similar concentration range (Fig. 3). In contrast, at low concentrations (1–10 M) DHA protected HUVECs against damage caused by serum deprivation (Fig. 5A) or staurosporine (data not shown) whereas oleic acid did not (Fig. 5B). The protective effect of DHA obviously is not mediated by PP2C but by another mechanism of action. Taken together we could show that (i) MUFAs activate PP2C and cause apoptosis in HUVECs and macrophages, (ii) DHA at low concentrations protects HUVECs against damage, and (iii) SFAs do not affect PP2C activity and do not cause apoptosis but
Dory, L., 1993. Post-transcriptional regulation of apolipoprotein E expression in mouse macrophages by phorbol ester. Biochem. J. 292, 105–111. Hanada, M., Kobayashi, T., Ohnishi, M., Ikeda, S., Wang, H., Katsura, K., Yanagawa, Y., Hiraga, A., Kanamaru, R., Tamura, S., 1998. Selective suppression of stress-activated protein kinase pathway by protein phosphatase 2C in mammalian cells. FEBS Lett. 437, 172–176. Hanada, M., Ninomyia-Tsuji, J., Komaki, K., Ohnishi, M., Katsura, K., Kanamaru, R., Matsumoto, K., Tamura, S., 2001. Regulation of the TAK1 signaling pathway by protein phosphatase 2C. J. Biol. Chem. 276, 5753–5759. Hufnagel, B., Dworak, M., Soufi, M., Mester, Z., Zhu, Y., Schaefer, J.R., Klumpp, S., Krieglstein, J., 2005. Unsaturated fatty acids isolated from human lipoproteins activate protein phosphatase type 2C and induce apoptosis in endothelial cells. Atherosclerosis 180, 245–254. Klumpp, S., Selke, D., Fischer, D., Baumann, A., Müller, F., Thanos, S., 1998a. Protein phosphatase type-2 isozymes present in vertebrate retinae: purification, characterization, and localization in photoreceptors. J. Neurosci. Res. 51, 328–338. Klumpp, S., Selke, D., Hermesmeier, J., 1998b. Protein phosphatase type 2C active at physiological Mg2+ : stimulation by unsaturated fatty acids. FEBS Lett. 437, 229–232. Klumpp, S., Selke, D., Ahlemeyer, B., Schaper, C., Krieglstein, J., 2002. Relationship between protein phosphatase type-2C activity and induction of apoptosis in cultured neuronal cells. Neurochem. Int. 41, 251–259. Klumpp, S., Selke, D., Krieglstein, J., 2003. Protein phosphatase type 2C dephosphorylates Bad. Neurochem. Int. 42, 555–560.
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Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265–275. McGowan, C.H., Cohen, P., 1998. Protein phosphatase-2C from rabbit skeletal muscle and liver: an Mg2+ -dependent enzyme. Methods Enzymol. 159, 416–426. Ofek, P., Ben-Meir, D., Kariv-Inbal, Z., Oren, M., Lavi, S., 2003. Cell cycle regulation and p53 activation by protein phosphatase 2C alpha. J. Biol. Chem. 278, 14299–14305. Schwarz, S., Hufnagel, B., Dworak, M., Klumpp, S., Krieglstein, J., 2006. Protein phosphatase type 2C␣ and 2C are involved in fatty-acid-induced apoptosis of neuronal and endothelial cells. Apoptosis 11, 1111–1119.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Influence of various fatty acids on the activity of protein phosphatase type 2C and apoptosis of endothelial cells and macrophages Josef Krieglstein a,b,∗ , Birgit Hufnagel a , Melanie Dworak b , Stefanie Schwarz a , Tobias Kewitz b , Michael Reinbold a , Susanne Klumpp b a b
Institut für Pharmakologie und Toxikologie, Philipps-Universitaet, Marburg, Germany Institut für Pharmazeutische und Medizinische Chemie, Westfaelische Wilhelms-Universitaet, Muenster, Germany
a r t i c l e
i n f o
a b s t r a c t
Article history:
In previous work we have demonstrated that protein phosphatase type 2C (PP2C) ␣ and 
Received 19 November 2007
can be activated by mono-unsaturated fatty acids (MUFAs) leading to apoptosis of cultured
Received in revised form
endothelial cells. In the present paper we could show that saturated fatty acids (SFAs) did
20 August 2008
not activate PP2C and did not cause apoptosis both in endothelial cells and macrophages.
Accepted 20 August 2008
However, long-chain SFAs (>16 C-atoms) were capable of inhibiting both, activation of PP2C
Published on line 4 September 2008
as well as apoptosis of human umbilical vein endothelial cells (HUVECs) and macrophages caused by oleic acid. Interestingly, docosahexaenoic acid (DHA) known to protect arterial
Keywords:
vessels against the progression of atherosclerosis caused apoptosis of HUVECs at high
Atherosclerosis
concentrations (200–400 M) but inhibited the apoptotic damage of HUVECs at a low, phys-
Fatty acids
iologically relevant concentration range (1–10 M). In contrast, oleic acid did not protect
Human umbilical vein endothelial
HUVECs against damage even at low concentrations (1–25 M). It is supposed that an unbal-
cells
anced and chronically increased level of MUFAs in blood has an atherosclerotic potential.
Macrophages
Furthermore, PP2C activated by MUFAs appears as a new target for drugs to prevent or treat
Protein phosphatase type 2C
atherosclerosis. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
We had discovered that MUFAs activate PP2C ␣ and  and that this induces apoptosis of neuronal and endothelial cells (Hufnagel et al., 2005; Schwarz et al., 2006). Apoptosis caused by PP2C could be mediated by dephosphorylation of the proapoptotic BH3-only protein Bad (Klumpp et al., 2003), by activation of the p53 signaling pathway (Ofek et al., 2003) and of the stress-activated protein kinase cascade (Hanada et al., 1998, 2001). PP2C belongs to the PPM phosphatase family whose members depend on divalent cations such as Mg2+
or Mn2+ (McGowan and Cohen, 1998). However, high concentrations of divalent cations (>20 mM) were necessary for measurable activity of PP2C. When MUFAs were added, the PP2C isozymes ␣ and  were maximally active at a physiological range of Mg2+ (0.5–1.5 mM) (Klumpp et al., 1998b). Thus, the combination of Mg2+ and MUFAs seems to play the essential role in regulating PP2C activity under in vivo conditions. The MUFAs stimulating PP2C had to be oxidizable, cis-configurated, at least 15 C-atoms in length and negatively charged. MUFAs that fulfilled all these requirements, e.g. oleic acid, activated PP2C in vitro and caused apoptosis of neu-
∗ Corresponding author at: Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Hittorfstraße 58-62, D-48149 Münster, Germany. Tel.: +49 251 83 32 210; fax: +49 251 83 32 211. E-mail address: [email protected] (J. Krieglstein). 0928-0987/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2008.08.007
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rons and endothelial cells (Klumpp et al., 2002; Zhu et al., 2005). The vascular endothelium is incessantly exposed to MUFAs since the lipoprotein lipase located on the luminal surface of the endothelial cells hydrolyzes triglycerides of the circulating lipoproteins (Stins et al., 1992). We could demonstrate that the physiologic fatty acids obtained from human lipoproteins activated PP2C in vitro and induced apoptosis in HUVECs (Hufnagel et al., 2005; Schwarz et al., 2006). These data suggest PP2C to be a significant player in the complex system of atherogenesis. In this study we attempted to shed some light on the role of saturated fatty acids (SFAs) and docosahexaenoic acid (DHA) in apoptotic damage of endothelial cells and macrophages caused by PP2C activation.
used at passages 3–6 and morphology was controlled prior to treatment. The cells were seeded at a density of 5 × 105 cells/9.6 cm2 dishes. Human THP-1 monocytes were cultured in suspension at a density of 4 × 106 cells/5 ml for 4 days. For differentiation to macrophages, monocytes received 100 ng/ml PMA and 50 M 2-mercaptoethanol (Dory, 1993). After 72 h in culture, the monocytes differentiated to macrophages and became adherent. Experiments were accomplished with adherent and confluent macrophages (>106 cells/9.6 cm2 dishes). Both cell types were cultured at 37 ◦ C in an incubator supplemented with 5% CO2 and 95% air.
2.
Materials and methods
2.1.
Materials
After the replacement of culture medium, HUVECs and macrophages were incubated for an additional 48 h. Cell cultures were then treated for 24 h with oleic acid or DHA, with different concentrations of SFAs or with a combination of both, oleic acid and SFAs. Fatty acids were dissolved in dimethylsulfoxide (DMSO). The final concentration of DMSO in the culture medium did not exceed 0.5%. To induce apoptosis in HUVECs we omitted fetal calf serum from or added staurosporine to the culture medium.
HUVECs and endothelial cell growth medium containing 2% heat-inactivated fetal bovine serum, 0.1 ng/ml epidermal growth factor, 1 g/ml hydrocortisone, 1 ng/ml basic fibroblast growth factor and antibiotics were purchased from Promocell (Heidelberg, Germany). For serum deprivation studies HUVECs were cultured in OptiMEM (Gibco, Germany). THP-1 monocytes were a gift from the Institute for Arteriosclerosis Research (Muenster, Germany). THP-1 cells were cultured in a medium containing RPMI-1640, 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM l-glutamine, 100 mM sodium pyruvate. Phorbol-12-myristate-13-acetate (PMA), 2mercaptoethanol, Hoechst 33258, Nile blue, fatty acids (oleic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, arachidic acid, behenic acid, and DHA) and bovine serum albumin (BSA) were obtained from Sigma–Aldrich (Taufkirchen, Germany). Nile blue staining was determined with the confocal laser-scanning microscope Zeiss LM510 (Jena, Germany) and analysis of apoptotic cells was accomplished with the fluorescent microscope Axiovert 100 Zeiss (Jena, Germany). Recombinant PP2C was prepared as described previously (Klumpp et al., 1998a).
2.2.
Measurement of PP2C activity
Activity of PP2C was measured using [32 P]-labelled casein as a substrate (McGowan and Cohen, 1998; Klumpp et al., 1998b). PP2C assays contained 30 mM Tris–HCl (pH 7.0), 0.1% 2-mercaptoethanol, 0.6 mg/ml BSA, 1 M [32 P]casein (5 × 104 cpm), and 10–200 ng PP2C in a final volume of 30 l. PP2C is characterized by its dependence on divalent cations for activity. If not indicated otherwise, 1 mM magnesium acetate was present. Reactions were terminated after 10 min at 30 ◦ C by adding trichloroacetic acid. The samples were centrifuged, and the supernatant was analyzed for [32 P]phosphate content.
2.3.
Cell culture
HUVECs were cultured in the endothelial cell growth medium to 90% confluence. In all experiments cells were
2.4.
2.5.
Fatty acid treatment of HUVECs and macrophages
Staining with Hoechst 33258 and Nile blue
Fatty acid-treated cells were washed with PBS (phosphate buffered saline, pH 7.4), fixed for 30 min with paraformaldehyde (4%) and then incubated for 30 min with the DNA fluorochrome Hoechst 33258 (10 g/ml) in methanol at room temperature in the dark. After washing with PBS, the nuclear morphology of HUVECs and macrophages was analyzed under a fluorescent microscope at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Cells showing shrunken or fragmented nuclei or chromatin condensation were counted as apoptotic cells. Nile blue was used to reveal uptake of fatty acids. After staining with Hoechst 33258, HUVECs and macrophages were washed with PBS, stained with Nile blue solution (10 g/ml) for 2 h and analyzed under a confocal laser-scanning microscope at wavelengths of 488/525 nm.
2.6.
Protein determination
The concentration of proteins was determined by BCAor Lowry-assay (Lowry et al., 1951) using BSA as a standard.
2.7.
Statistical methods
All values, except the PP2C activity values in Fig. 2, are given as means ± S.D. One-way analysis of variance (ANOVA) followed by Scheffé’s test was applied. PP2C-assays were performed in duplicate and independently repeated at least twice.
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Fig. 1 – Uptake of oleic acid and stearic acid into HUVECs (A–C) and macrophages (D–F). Cells were incubated for 24 h with the fatty acids (200 M) and stained with Nile blue. The two fatty acids (yellow) were taken up by the cells but, notably, oleic acid-treated cells were partially damaged and stearic acid-treated were not 10 m.
3.
Results
3.1. Uptake of fatty acids into HUVECs and macrophages The uptake of SFAs into HUVECs and macrophages was similar to that of MUFAs (Fig. 1). Here we were using Nile blue to study the uptake of fatty acids into HUVECs and macrophages. Control cells received 0.5% DMSO and subsequent staining resulted in a weak fluorescence reflecting endogenous lipids. Addition of oleic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid and stearic acid resulted in a bright fluorescence of the cells within 3 h of otherwise intact cells indicating a similar uptake of all fatty acids. After an incubation period of 24 h, the oleic acid-treated cells revealed apoptosis whereas stearic acid-treated cells remained intact (Fig. 1).
3.2.
Influence of fatty acids on PP2Cˇ activity
Searching for regulatory components of PP2C we discovered that the activity of PP2C increased 10–15-fold after the addition of certain MUFAs, e.g. oleic acid but also DHA, if measured at a physiological concentration of 0.5–1 mM free Mg2+ (Klumpp et al., 1998b). In contrast, SFAs were not capable of stimulating the activity of PP2C (Fig. 2A).
To further study the regulation of PP2C, we wanted to elucidate whether SFAs of different chain length might be able to abolish the oleic acid-induced activation of PP2C. Treatment of PP2C with SFAs with chain lengths from 14 to 22 C-atoms showed no activation of the enzyme (Fig. 2A). PP2C in the presence of both, oleic acid and SFAs, revealed interesting new results: SFAs with a chain length of 16 C-atoms or less, e.g. myristic acid (14 C-atoms), pentadecanoic acid (15 C-atoms) and palmitic acid (16 C-atoms), were not able to inhibit the activation-capacity of oleic acid. In contrast, SFAs with a chain length of more than 16 C-atoms, e.g. heptadecanoic acid (17 Catoms), stearic acid (18 C-atoms), arachidic acid (20 C-atoms) and behenic acid (22 C-atoms) reduced the oleic acid-induced activation of PP2C (Fig. 2B) in vitro.
3.3. Apoptosis caused by oleic acid and DHA in HUVECs and macrophages Oleic acid but also DHA induced apoptosis in HUVECs at a concentration of 200–400 M (Fig. 3). The same was true for macrophages exposed to oleic acid (250 M) for 24 h (Fig. 4C). Similar to HUVECs (Hufnagel et al., 2005) the corresponding trans-derivative and the oleic acid methylester both were not harmful to macrophages when administered in the same concentration range as oleic acid (data not shown).
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Fig. 2 – Effect of oleic acid and SFAs on PP2C activity. (A) Dephosphorylation of [32 P]casein by PP2C was measured after addition of various fatty acids in the presence of 1 mM Mg2+ . Oleic acid stimulated the activity of PP2C ninefold, whereas SFAs with different chain lengths failed to activate the enzyme. SFAs added were myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid (17:0), stearic acid (18:0), arachidic acid (20:0) and behenic acid (22:0). (B) Effect of various SFAs on PP2C activated by oleic acid (18:1). Phosphatase activity was measured after addition of oleic acid and oleic acid in combination with different SFAs. SFAs less than 17 C-atoms could not clearly affect the oleic acid-induced activation of PP2C, whereas SFAs with a chain length longer than 16 C-atoms did. Dephosphorylation of [32 P]casein was determined with 15–160 ng PP2C per incubation. Activity in the absence of fatty acids (control) was 5.1 nmol Pi /(min mg) (100%). Values are given as means of two independent determinations in duplicate, respectively.
3.4.
Inhibition of oleic acid-induced apoptosis by SFAs
In further experiments we tested whether long-chain SFAs capable of reducing oleic acid-activated PP2C were able to abolish the oleic acid-induced apoptosis in HUVECs and macrophages. First, cells were treated with various SFAs (14–22 C-atoms). Those SFAs tested did not influence the viability of HUVECs and macrophages (Fig. 4A and C). Next, and in parallel to the results described on PP2C in vitro, we attempted to elucidate whether treatment of HUVECs and macrophages with SFAs in the presence of oleic acid might affect oleic acid-induced apoptosis. Myristic acid and pentadecanoic acid were not able to reduce the apoptotic effect of oleic acid in HUVECs and macrophages. In contrast, heptadecanoic acid and stearic acid significantly reduced the oleic acid-induced apoptosis in both cell types (Fig. 4B and D). Palmitic acid with a chain length right in between protecting (≥C-17) and nonprotecting (≤C-15) SFAs had a different effect on the oleic
Fig. 3 – Oleic acid and DHA induce apoptosis in HUVECs. The cells were incubated with different concentrations of oleic acid (A) or DHA (B). Morphology was analyzed under a fluorescence microscope after nuclear staining with Hoechst 33258. Cells showing fragmented nuclei or condensed chromatin were counted as apoptotic cells. Values are given as means ± S.D. of 5 experiments. ***p < 0.001, compared with vehicle-treated control.
acid-induced apoptosis in HUVECs and macrophages (Fig. 4B and D). Arachidic acid and behenic acid were almost insoluble in the endothelial cell growth medium and thus could not be tested.
3.5.
Influence of DHA on the viability of HUVECs
Omega-3 fatty acids are particularly suggested to inhibit atherogenesis and to lower cardiovascular mortality (for reviews see von Eckardstein, 2005). Therefore, we tested the influence of DHA on the viability of HUVECs. It turned out that 200 M DHA damaged HUVECs similar to oleic acid (Fig. 3). To elucidate a putative protective effect of DHA we reduced the viability of HUVECs by removing fetal calf serum from the culture medium for about 60 h and the percentage of apoptotic cells increased in the controls up to 20%. However, when DHA was added to the culture medium to give a low concentration (1–10 M) the percentage of apoptotic HUVECs was significantly reduced to around 10% (Fig. 4A). This protective effect could not be achieved by oleic acid (Fig. 5B).
4.
Discussion
Reversible phosphorylation of proteins, catalyzed by kinases and phosphatases, is a major mechanism to regulate apoptotic pathways. Our previous studies have demonstrated that serine/threonine phosphatases PP2C ␣ and  are activated by certain MUFAs (Klumpp et al., 1998b). We could demonstrate
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Fig. 4 – Effect of oleic acid and SFAs with different chain lengths on the viability of HUVECs (A) and macrophages (C). Cells were incubated with oleic acid (18:1), myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid (17:0) and stearic acid (18:0) at a concentration of 200 M, respectively. Controls were treated with vehicle only (0.2% DMSO). Heptadecanoic acid and stearic acid protected HUVECs from oleic acid-induced apoptosis. (B) Treatment of HUVECs with myristic acid (14:0), pentadecanoic acid (15:0) and palmitic acid (16:0) could not abolish the toxic effect of oleic acid whereas heptadecanoic acid (17:0) and stearic acid (18:0) could. (D) Treatment of macrophages with myristic acid (14:0) and pentadecanoic acid (15:0) could not abolish the toxic effect of oleic acid whereas palmitic acid (16:0), heptadecanoic acid (17:0) and stearic acid (18:0) could. Morphology was analyzed under a fluorescence microscope after nuclear staining with Hoechst 33258. Cells showing fragmented nuclei or condensed chromatin were counted as apoptotic cells. Values are given as means ± S.D. of 5 experiments. ***p < 0.001, compared with vehicle-treated control. ### p < 0.001, compared with oleic acid-treated cells.
that Bad, a pro-apoptotic member of the Bcl-2 family, is a substrate of PP2C ␣ and , indicating that PP2C can trigger the pro-apoptotic function of Bad (Schwarz et al., 2006). In its dephosphorylated state, Bad promotes apoptosis by heterodimerization with the antiapoptotic oncogene Bcl-XL (Zha et al., 1996). Immunocytochemistry revealed that PP2C and its substrate Bad are co-localized within the cytosol of HUVECs (Hufnagel et al., 2005) and macrophages (data not shown). Extensive studies with a variety of fatty acids demonstrated a striking correlation between activation of PP2C and induction of apoptosis in HUVECs (Hufnagel et al., 2005). This correlation could be extended to macrophages: SFAs (14–22 C-atoms), trans-fatty acids like eladaic acid (18:1 trans-9 ) or methylester derivatives like oleic acid methylester (18:1 cis-9 ester) do not fulfil the structural requirements for PP2C activation and could not damage cultured macrophages. When PP2C was downregulated by the RNAi technique the apoptotic effect of oleic acid on HUVECs was significantly reduced indicating a causal role of PP2C in this process (Schwarz et al., 2006). Since SFAs are not able to activate PP2C in vitro (Fig. 2A), we wanted to elucidate in this study if they could inhibit the oleic acid-induced activation of PP2C. They have a free carboxyl
group and are lipophilic; theoretically they could displace oleic acid from a putative binding site on PP2C without activating the enzyme thus preventing the oleic acid-induced activation. Another explanation could be that SFAs form mixed micelles with oleic acid and thereby decrease the concentration of free oleic acid under the level of efficacy. We tested the effect of SFAs with different chain lengths (14–22 C-atoms) in combination with oleic acid on PP2C activity in vitro. Interestingly, SFAs with a chain length of 16 C-atoms or less, like myristic acid (14 C-atoms), pentadecanoic acid (15 C-atoms), and palmitic acid (16 C-atoms), could not clearly influence the oleic acid-induced activation of PP2C, whereas SFAs with a chain length longer than 16 C-atoms, like heptadecanoic acid (17 Catoms), stearic acid (18 C-atoms), arachidic acid (20 C-atoms), and behenic acid (22 C-atoms), could (Fig. 2B). Obviously, SFAs with a chain length of ≥16 C-atoms and a free carboxyl group were able to prevent the activation of PP2C caused by oleic acid. Double staining with the DNA fluorochrome Hoechst 33258 and Nile blue revealed that the SFAs and oleic acid were taken up by HUVECs and macrophages to a similar extent (Fig. 1), however, only heptadecanoic acid and stearic acid prevented
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Fig. 5 – Physiological concentrations of DHA protect HUVECs against apoptosis. HUVECs were incubated with serum-free medium (OptiMEM) and DHA (A) or oleic acid (B) for about 60 h, respectively. The percentage of apoptotic cells was determined by nuclear staining with Hoechst 33258. Values are given as means ± S.D. of 5 experiments. *p < 0.05 and ***p < 0.001, compared to serum-free vehicle-treated control, respectively.
some of them inhibit oleic acid-induced activation of PP2C and apoptosis of HUVECs and macrophages. Since most of the fatty acids are transported in vivo by lipoproteins and have to pass the endothelium to be distributed into the tissues, the PP2C-induced apoptosis could be crucial for atherogenesis in organs such as the heart which covers about 70% of its energy demand by fatty acids. Since this huge amount of fatty acids has to pass through the endothelium of that organ, there is a better chance for PP2C to be activated, for endothelial cells to be damaged, and consequently for the induction of atherosclerosis. Thus, our hypothesis could explain why atherosclerosis is more pronounced in coronary arteries than, for instance, in cerebral arteries because the brain uses only glucose for its energy metabolism. Furthermore, it also becomes understandable now why lipid loaded macrophages in atherosclerotic plaques undergo apoptosis thus finally induce plaque instability and rupture, and why patients with diabetes mellitus exhibit micro- and macroangiopathies. A balanced mixture of MUFAs and SFAs should be present in the diet to avoid a predominant increase of MUFAs level in the blood which could intensify the development of atherosclerosis. On the other hand, the inhibition of MUFAs-activated PP2C may provide a new strategy for prevention and therapy of atherosclerosis.
Acknowledgement Studies were supported by grants from the Deutsche Forschungsgesellschaft (DFG; Kr 354/17-1 and Kl 601/9-3).
references the damaging effect of oleic acid (Fig. 4). The capability of SFAs to inhibit oleic acid-activated PP2C could pave the way to develop appropriate inhibitors suitable for a new therapeutic strategy against atherosclerosis. Furthermore, the results suggest that a balanced mixture of various fatty acids in the blood could prevent the activation of PP2C and subsequently atherogenesis. There is some evidence in the literature that MUFAs lower the atherosclerotic risk (for reviews see von Eckardstein, 2005). Our results do not support this view. However, data on the anti-atherosclerotic activity of omega-3 fatty acids are more convincing (von Eckardstein, 2005). Since activation of PP2C by DHA already has been shown (Klumpp et al., 1998b) we now tested the effect of DHA on endothelial cells and found intriguing results. Only high concentrations of DHA (200–400 M) in the culture medium – which are most likely not achieved in the blood – could activate PP2C and induce apoptosis of HUVECs comparable to that obtained with oleic acid used at a similar concentration range (Fig. 3). In contrast, at low concentrations (1–10 M) DHA protected HUVECs against damage caused by serum deprivation (Fig. 5A) or staurosporine (data not shown) whereas oleic acid did not (Fig. 5B). The protective effect of DHA obviously is not mediated by PP2C but by another mechanism of action. Taken together we could show that (i) MUFAs activate PP2C and cause apoptosis in HUVECs and macrophages, (ii) DHA at low concentrations protects HUVECs against damage, and (iii) SFAs do not affect PP2C activity and do not cause apoptosis but
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