- Research Paper
- Open Access
The zinc matrix metalloproteinase ZMP-2 increases survival of Caenorhabditis elegans through interference with lipoprotein absorption
© Springer-Verlag Berlin Heidelberg 2014
- Received: 11 April 2014
- Accepted: 17 June 2014
- Published: 24 June 2014
Matrix metalloproteinases are zinc-dependent endopeptidases conserved throughout the animal kingdom which primarily degrade components of the extracellular matrix. In the nematode Caenorhabditis elegans, the zinc matrix metalloproteinase (ZMP-2) was demonstrated to increase resistance versus heat and bacterial pathogens. Here, we show that the survival reducing activities caused by the knockdown of zmp-2 in C. elegans essentially requires the presence of vitellogenin-6, a protein homologous to mammalian apolipoprotein B, and RME-2, a receptor mediating endocytosis of cholesterol particles. Measurements of reactive oxygen species inside and outside C. elegans revealed that knockdown of zmp-2 causes a prooxidative extracellular mileu which is a prerequisite for the reduction of survival. Interestingly, RNAi for the foxo transcription factor daf-16 completely prevented those survival reducing effects of zmp-2 RNAi, and RNAi in mutants of the steroid signalling pathway revealed that DAF-16 acts by inhibition of DAF-9 and DAF-12. In conclusion, our study demonstrates survival reducing activities caused by the functional loss of ZMP-2 in C. elegans. Those effects are mediated by the transport of oxidized cholesterol adducts which then trigger the inhibition of DAF-9 and DAF-12 through the activation of DAF-16.
- Zinc matrix metalloproteinase
- Stress resistance
- Steroid signalling
- Foxo transcription factor
- Cholesterol absorption
- Apolipoprotein B
In vertebrates matrix metalloproteinases represent a class of over 26 zinc-dependent enzymes which share a similar structure to each other and are capable of degrading almost every component of the extracellular matrix (Page-McCaw et al. 2007; Hernández-Pérez and Mahalingam 2012; Vu and Werb 2000). Those zinc matrix metalloproteinases are essentially involved in remodelling of the extracellular matrix in both physiological and pathological processes (Siefert and Sarkar 2012). As an example, matrix metalloproteinases (MMPs) are crucial for the generation or regeneration of arteries but their dysregulated activities may also be implicated in the pathogenesis of vascular diseases such as atherosclerosis (Siefert and Sarkar 2012). Interestingly, studies have shown that oxidized low-density lipoproteins and their receptor mediate the up-regulation of vascular factors associated with progression of atherosclerosis, such as MMP-9 (Lund et al. 2011). However, by the progressive accumulation and aggregation of LDL particles in the extracellular matrix, they become targets for oxidative but also enzymatic modifications (Badimon et al. 2011). It is interesting to note in this context that in mammalian keratinocytes, reactive oxygen species (ROS) mediate the effects of heat on tissue destruction and that the expression of several MMPs was induced contributing to tissue regeneration (Shin et al. 2008). Overall this suggests that complex interactions between LDL particles, redox metabolism and MMPs exist.
The nematode Caenorhabditis elegans has been described as an appropriate model to investigate the pathways of lipid storage, transport and signalling (Branicky et al. 2010). In the nematode homologues of apolipoprotein B are expressed in terms of vitellogenins (VITs), a class of egg yolk proteins in invertebrates but also in egg-laying vertebrates (Baker 1988). Since C. elegans is auxotroph for sterols (Chitwood and Lusby 1991), one important function of VITs is the provision of sterols to the organism (Matyash et al. 2001). Accordingly, cholesterol must be absorbed from ingested food in the intestine and later into the oocytes. Besides the VITs, transport at both loci requires the accomplishment of receptor-mediated endocytosis through RME-2 (Matyash et al. 2001; Grant and Hirsh 1999). RME-2 is a member of the LDL receptor superfamily (Grant and Hirsh 1999), and accordingly, VITs and their receptor may be regarded as evolutionary ancestors of LDL and its receptor in higher organisms (Matyash et al. 2001).
According to homology screening, C. elegans possesses six genes encoding zinc matrix metalloproteinases (ZMPs) (Wada et al. 1998). Recombinant C. elegans ZMPs have been shown to cleave a MMP peptide substrate and this activity was inhibited by human tissue inhibitor of MMP, suggesting that the catalytic sites of C. elegans ZMPs and human MMPs are structurally closely related (Wada et al. 1998). Investigating the effects of all six C. elegans ZMPs in vivo, we found in a previous study that only RNA-interference (RNAi) for zmp-2 (H19M22.3) resulted in nematodes with moulting defects indicating its role in extracellular matrix degradation during ecdysis (Altincicek et al. 2010). Moreover, knockdown of zmp-2 was associated with reduced survival under heat stress or during infection with the nematode pathogenic bacterium Photorhabdus luminescens (Altincicek et al. 2010). In hermaphroditic adults, the strongest expression of zmp-2 was detectable in the two spermathecae and spermathecal-uterine valves but weaker fluorescence was observed in a zmp-2 promoter::gfp transgenic strain along the intestinal tract (Altincicek et al. 2010).
In the present study, we investigated in C. elegans the impact of RNAi for vit-6 on the reduction of survival caused by zmp-2 RNAi under heat stress.
Strains and maintenance
Caenorhabditis elegans strains were maintained on nematode growth medium (NGM) agar plates seeded with E. coli OP50 at 20 °C according to standard protocols (Brenner 1974). Caenorhabditis elegans strains wild-type N2, variation Bristol, the mutants DR2281 daf-9(m540), AA1 daf-12(rh257), CF1038 daf-16(mu86), DH1390 rme-2(b1008), the transgenic strain TJ356 zIs356 Is[daf-16::daf-16-gfp; rol-6] and E. coli OP50 were obtained from the C. elegans Genetics Center (University of Minnesota, MN, US). Methods such as freezing nematodes and obtaining synchronous populations using a bleaching method with hypochlorite treatment of egg-laying adults were performed according to standard protocols. RNAi clones were obtained from MRC GeneService Ltd (Cambridge, UK) and included a negative control (L4440), rme-2 (T11F8.3), daf-9 (T13C5.1), daf-12 (F11A1.3), daf-16 (R13H8.1), sir-2.1 (R11A8.4), ftt-2 (F52D10.3) and par-5 (M117.2). The RNAi clone of vit-6 (K07H8.6) was constructed using standard cloning techniques as described elsewhere (Fischer et al. 2012).
RNAi experiments were performed in liquid cultures as previously described (Lehner et al. 2006; Timmons et al. 2001; Kamath et al. 2001). In brief, expression of gene-specific dsRNA inside the bacterial RNAi feeding strain E. coli HT115 was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 1 h at 37 °C to trigger interference. Subsequently, bacterial cells were washed and resuspended in NGM. A volume of 40 μl of this suspension was dispensed in each well of a 96-well plate (Greiner Bio-One, Frickenhausen, Germany), to which 10–15 synchronized L1 larvae were added. In general, L1 larvae reached the adult stage within 3 days of incubation with agitation at 20 °C.
Survival analysis at 37 °C
Nematode survival was determined using a microplate thermotolerance assay as described (Gill et al. 2003). In brief, nematodes were washed off the wells with M9-buffer into 15 ml tubes followed by additional three washing steps. In each well of a black 384-well low-volume microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 6.5 μl M9-buffer/Tween 20 (1 % v/v) solution was added. Subsequently, one nematode was dispensed in 1 μl M9 buffer under a stereomicroscope (Breukhoven Microscope Systems) into each well and mixed with 7.5 μl SYTOX green (final concentration 1 μM; Life Technologies, Karlsruhe, Germany). To prevent water evaporation, the plates were sealed with Rotilabo sealing film and covered with a lid (Greiner Bio-One, Frickenhausen, Germany). Heat shock (37 °C) was induced and fluorescence was measured with the Fluoroskan Ascent fluorometer (Thermo Labsystems, Bonn, Germany) every 30 min. To detect SYTOX green fluorescence, excitation wavelength was set at 485 nm and emission was measured at 538 nm. All experiments determining the lifespan of nematodes at 37 °C were done at least thrice and representative survival curves are shown in the figures.
Quantitative real-time PCR
Total RNA was extracted from 10,000 worms using Trizol (Life Technologies). One-step real-time PCRs were performed in triplicate using 1 µl of RNA template, Brilliant II SYBR Green QRT-PCR Mastermix-Kit (Stratagene Products, Waldbronn, Germany) and appropriate primers in a CFX™ Real-Time PCR Detection System (BioRad, München, Germany). Cycling conditions were 1 × [15 min 50 °C], 1 × [10 min 95 °C], 40 × [30 s 95 °C, 15 s 53 °C, 30 s 60 °C], 1 × [1 min 95 °C], 1 × [30 s 53 °C] and 1 × [1 min 95 °C]. Changes in the target gene expression were calculated according to Pfaffl (2001) using equation 2−ΔΔCT. For each sample, the fold change in the target gene was normalized to 18S rRNA and relative to the control expression. For determination of RNAi efficiency, unique primer pairs (Eurofins MWG Operon, Ebersberg, Germany) recognizing only cDNA derived from endogenous mRNA were designed to avoid cross-reaction with genomic DNA and bacterially generated dsRNA and were as follows: 18S rRNA-fw 5′-ATG GTT GCA AAG CTG AAA CT-3′, 18S rRNA-rev 5′-TCC CGT GTT GAG TCA AAT TA-3′; zmp-2-fw 5′-GCT CAG ATT AGA AGA CGA AG-3′, zmp-2-rev 5′-GAA CTT CAC TTG GAA GAA CA-3′; vit-6-fw 5′-ACC CCA TGC TAC TCC GTT CTC-3′, vit-6-rev 5′-CAA CCT TGA CGT TCT TCC ACT CC-3′; daf-9-fw 5′-TTC TGT CTA GCG TTC TCG-3′, daf-9-rev 5′-CCA AGA ACT TTG GCT TAT GT-3′; daf-12-fw 5′-CAA CGT GGA TGA TAT GTT TG-3′, daf-12-rev 5′-GAA GAA ACC GAA GAA CTC TA-3′; daf-16-fw 5′-CGG AAG GCT TAA ACT CAA TG-3′; daf-16-rev 5′-GAC GGA TCG AGT TCT TCC AT-3′.
Relative mRNA levels in nematodes cultured on RNAi bacteria were significantly lower (p < 0.01) than mRNA levels of the control that was fed L4440 and were below 0.25 for all genes tested.
For detection of VIT-6 at the protein level, a YP88 rat-anti-vit-6 polyclonal antibody (kindly provided by Prof. Thomas Blumenthal, University of Colorado) at a 1:5,000 dilution was used and β-actin, as a loading control, was detected with a rabbit-anti-actin polyclonal antibody (Sigma-Aldrich, Hamburg, Germany) at 1:500. As secondary antibodies goat-anti-rat IgG polyclonal (Dianova, Hamburg, Germany) at 1:5,000, and goat-anti-rabbit IgG polyclonal (Santa Cruz, Heidelberg, Germany) at 1:500, respectively, were used as horseradish peroxidase (HRP) conjugate. Groups of 10,000 nematodes were treated with lysis buffer (saccharose 0.8 M, EDTA 1 mM, Tris–HCL 10 mM, PMSF 0.5 mM) and twice freeze-thawed at −80 °C and room temperature, respectively. Lysates were separated on 8.5 % SDS-PAGE gels and proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Schwalbach, Germany) for 1 h at 1.0 mA/cm2 membrane area. The blots were preblocked in 15 ml of 1 % milk in TBS/0.05 % Tween 20 and probed with antibodies diluted in blocking solution for 1 h. Following 2 × 5 min washes with TBS-T and 2 × 5 min TBS, and incubation in enhanced chemiluminescence (ECL) solution, the radiographic film was exposed to detect the immunoreactive bands. Band intensities were quantified using ImageJ (NIH).
Detection of H2O2
Detection of H2O2 was done by the use of the N-acetyl-3,7-dihydroxyphenoxazine (Amplex® Ultra Red, Sigma-Aldrich, Hamburg, Germany) assay. In the presence of H2O2, Amplex® Ultra Red is oxidized by horseradish peroxidase (HRP) to a red-fluorescent oxidation product (Mohanty et al. 1997). To assess the H2O2 production from C. elegans, 700 young adult worms per condition were washed four times with 0.25 M Na2HPO4. Approximately 100 nematodes per 50 µl were transferred into flat-bottomed 96-well plates (Greiner Bio-One, Frickenhausen, Germany) and subsequently 50 µl Amplex® Ultra Red reaction buffer solution (50 µl 10 mM Amplex® Ultra Red, 100 µl HRP; 10 U/ml, 4.85 ml 0.25 M Na2HPO4) was added to each well. After 3-h incubation, fluorescence was measured by the use of the microplate reader with excitation at 550 nm and fluorescence emission detection at 592 nm, respectively.
The Millipore OxyBlot™ Protein Oxidation Detection Kit was used for immunoblot detection of carbonylated proteins. Therefore, groups of 10,000 nematodes were treated with lysis buffer (0.8 M sucrose, 1 mM EDTA, 10 mM Tris‐HCL, 0.5 mM PMSF), twice freeze-thawed at −80 °C and room temperature, respectively, and subsequently sonicated on ice. The concentration of isolated proteins was adjusted to 1.5 mg/ml before derivatization was performed according to the instruction manual, using 5 µl protein sample. Derivatized proteins were separated by 8.5 % SDS-PAGE and blotted onto a PVDF membrane (1 h, 1.0 mA/cm2). The membrane was blocked with 1 % milk in TBS/0.05 % Tween®20 and probed with antibodies as described in the instruction manual of the OxyBlot kit. β-Actin from non-derivatized samples was detected as a loading control for the normalization of intensities of the bands between the different treatments by the use of a horseradish peroxidase-conjugated goat-anti-rabbit IgG (Santa Cruz, Heidelberg, Germany). For chemiluminescence detection, the membrane was incubated for 1 min with freshly prepared enhanced chemiluminescent solution (1 M Tris pH 8.5, 90 mM p-coumaric acid, 250 mM luminol, 0.01 % H2O2). An autoradiography film was exposed for 5 min to detect immunoreactive bands. Band intensities were quantified using ImageJ (NIH).
To visualize ROS stress located in mitochondria, worms were grown in liquid culture and treated for 24 h at 20 °C in the young adult stage with 0.5 μg/ml MitoTracker CM-H2XRos (Life Technologies). CM-H2XRos produces a ROS-specific staining of mitochondria within cells that is amenable to both qualitative and quantitative measurements of mitochondrial production of H2O2 (Degli Esposti 2002). It does not get fluorescent until it enters an actively respiring cell, where it is oxidized predominantly by reactions involving hydrogen peroxide production (Degli Esposti 2002). According to its positive charge acquired on oxidation by intracellular ROS, the probe covalently binds to mitochondrial proteins and thus produces a permanent organelle-specific stain (Degli Esposti 2002). The MitoTracker fluorescence was measured at 580 nm after excitation at 530–585 nm by the use of an Axioskop 2 epifluorescence microscope (Zeiss, Jena). The microscopic images were generated as 24-bit-jpeg format, which were quantified by using ImageJ (NIH).
Dehydroergosterol (Sigma, Deisehofen, Germany) uptake was measured using the Axioskop2 as described for MitoTracker staining. Therefore, L1 larvae were grown as described for RNAi experiments in the presence of 100 µM dehydroergosterol. Fluorescence was measured after excitations at 340–380 nm and emissions at 425 nm and quantified by using ImageJ (NIH).
Calculations and statistics
Statistical analyses were performed with GraphPad Prism 5.0 software (GraphPad, La Jolla, CA, USA). Log-rank test was performed to compare survival rates under heat stress with p values <0.05 being significant. Kaplan–Meier survival curves are shown for lifespan experiments, which all have been repeated more than once with similar effects. For group comparisons, t test or analysis of variance (ANOVA) was performed. Following ANOVA, differences between groups were determined by Bonferroni-Holm multiple comparison test. Results are presented as mean ± SD.
Vit-6 RNAi blocks the reduction of thermotolerance caused by zmp-2 RNAi
Vit-6 RNAi increases the ROS burden inside and outside of C. elegans
Zmp-2 RNAi reduces survival under heat stress depending on DAF-16 which inhibits DAF-9 and DAF-12
RNAi for sir-2.1, ftt-2 or par-5 prevent the reduction of survival under heat stress caused by zmp-2 RNAi
Average lifespan ± SD [h]
Number of nematodes
9.8 ± 1.3
7.2 ± 2.3
9.3 ± 2.0
9.4 ± 1.9
9.3 ± 1.4
9.1 ± 1.1
9.0 ± 1.0
8.9 ± 1.1
A basic supply of cholesterol is mandatory for activation of steroid signalling and the increase in thermotolerance under zmp-2 RNAi
It was surprising to note that the reductions of thermotolerance caused by the knockdown of zmp-2 were completely dependent on the presence of the foxo transcription factor DAF-16, which has been described to transcriptionally trigger several stress response mechanisms in association with longevity (McColl et al. 2010). On the other hand, we have previously demonstrated that the prevention of nuclear DAF-16 localization by VIT-6 is associated with defence mechanisms against a nematode pathogen (Fischer et al. 2013) demonstrating that enhanced resistance, as achieved, e.g. by the prevention of faulty water homoeostasis (Evans et al. 2008), can also derive from the absence of transcriptional activity of DAF-16. Here, however, it is not that the absence of DAF-16 transcriptional activity provides stress resistance but that its nuclear localization is found to be associated with the survival reductions caused by zmp-2 RNAi. That indeed the zmp-2 RNAi effects on thermotolerance are mediated through transcriptional activity of DAF-16 is further stressed by the fact that the knockdown of its transcriptional co-activators sir-2.1, ftt-2 and par-5 (Berdichevsky et al. 2006; Berdichevsky and Guarente 2006) prevented the reduced thermotolerance as caused by zmp-2 RNAi in an identical manner as daf-16 RNAi.
Besides the insulin signalling pathway with its key factor DAF-16, the steroid signalling pathway including its key components DAF-9, a cytochrome P450-dependent enzyme forming dafachronic acids, and the nuclear hormone receptor DAF-12, recognizing dafachronic acids as ligands, has been shown to exert significant effects on lifespan and survival (Rottiers et al. 2006; Gerisch et al. 2001). Also in this regard, the picture is not completely clear, given that dafachronic acid supplementation shortened the lifespan of long-lived daf-9 mutants and abolished their stress resistance, whereas they extended the lifespan of germ-line ablated daf-9 mutants, demonstrating that the steroid signalling pathway may regulate C. elegans lifespan according to signalling state (Gerisch et al. 2007). In the present study, we demonstrate that both DAF-9 and DAF-12 are involved in the protection against the toxicity mediated by zmp-2 RNAi (Fig. 7). Strains mutant for either daf-9 or daf-12 were hypersensitive to zmp-2 RNAi. Moreover, in these strains, daf-16 RNAi no longer provided any protection from zmp-2 RNAi, demonstrating that both factors act downstream from DAF-16 and are inhibited by the foxo transcription factor (Fig. 7).
Since cholesterol, however, provides the substrate for steroid signalling, it has to be assumed that a minimal supply of sterols is mandatory in order to enable DAF-9 to generate ligands for DAF-12 and to compensate for the detrimental effects of zmp-2 RNAi (Fig. 7). This hypothesis is substantiated by the results obtained in wild-type nematodes which were fed low cholesterol concentrations because they did not respond with an extended survival when vit-6 was knocked down in zmp-2 RNAi exposed nematodes. Also the daf-16 RNAi-mediated resistance to the reductions of survival by zmp-2 RNAi proved to be dependent on an adequate minimal cholesterol supply since double RNAi for zmp-2 and vit-6 reduced the survival of daf-16 mutants dramatically.
In conclusion, we provide evidence that ZMP-2 in C. elegans prevents the accumulation of oxidized lipoproteins containing VIT-6 which are absorbed into the nematodes by receptor-mediated endocytosis. A lack of ZMP-2 activity leads to the absorption of toxic adducts which then trigger DAF-16 to inhibit DAF-9 and DAF-12 and lead to diminished thermotolerance.
Conflict of interest
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