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Hypoxia induces up-regulation of progranulin in neuroblastoma cell lines
Neurochemistry International 57 (2010) 893–898
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Hypoxia induces up-regulation of progranulin in neuroblastoma cell lines Paola Piscopo, Roberto Rivabene, Alice Adduci, Cinzia Mallozzi, Lorenzo Malvezzi-Campeggi, Alessio Crestini, Annamaria Confaloni * Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 April 2010 Received in revised form 31 August 2010 Accepted 26 September 2010 Available online 7 October 2010
Progranulin (PGRN) is a widely expressed multifunctional protein, involved in regulation of cell growth and cell cycle progression with a possible involvement in neurodegeneration. We looked for PGRN regulation in three different human neuroblastoma cell lines, following exposure to two different stimuli commonly associated to neurodegeneration: hypoxia and oxidative stress. For gene and protein expression analysis we carried out a quantitative RT-PCR and western blotting analysis. We show that PGRN is strongly up-regulated by hypoxia, through the mitogen-actived protein kinase (MAPK)/ extracellular signal-regulated kinase (MEK) signaling cascade. PGRN is not up-regulated by H2O2induced oxidative stress. These results suggest that PGRN in the brain could exert a protective role against hypoxic stress, one of principal risk factors involved in frontotemporal dementia pathogenesis. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Progranulin Gene expression Hypoxia Oxidative stress Neuroblastoma cell lines
1. Introduction Progranulin (PGRN) is a widely expressed multifunctional protein involved in the regulation of cell growth and cell cycle progression (Bateman et al., 1990; He and Bateman, 2003). In normal peripheral tissues, the PGRN has a complex function, with the full-length form of the protein, which exerts trophic and antiinflammatory activity. Proteolytic cleavage of PGRN generates granulin peptides that promote inflammatory activity. Little is known about the role of PGRN in the central nervous system (CNS). The protein is widely expressed during early neural development (Daniel et al., 2003), but with maturation its expression becomes restricted to defined neuronal populations, such as cortical and hippocampal pyramidal neurons and Purkinje cells (Daniel et al., 2000). PGRN has also been implicated in the sexual differentiation of the brain (Suzuki and Nishiahara, 2002). The protein is up-regulated in activated microglial cells (Baker and Manuelidis, 2003) but not in astrocytes or oligodendrocytes. Recently, Van Damme and colleagues observed that PGRN and GRN E (one of the proteolytic fragments of PGRN) promote neuronal survival and enhance neurite outgrowth in cultured neurons (Van Damme et al., 2008). Several nonsense and frameshift mutations in Abbreviations: FTD, frontotemporal dementia; GLUT-1, glucose transporter 1; GSH, reduced glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide; MAPKs, mitogen-activated protein kinases; MEK, (MAPK)/extracellular signalregulated kinase kinase; PGRN, progranulin. * Corresponding author. Tel.: +39 06 49902930; fax: +39 06 49387143. E-mail address: [email protected] (A. Confaloni). 0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.09.008
the PGRN gene are associated with frontotemporal lobar degeneration (FTLD) (Baker et al., 2006), the third most common cause of neurodegenerative dementia after AD and dementia with Lewy bodies (Bird et al., 2003). FTLD is caused by both genetic and environmental factors, but the exact cause are still unknown. It has been hypothesized that both ischemia/hypoxia and oxidative stress are involved in the pathogenesis of several neurodegenerative diseases, including FTD (Martin et al., 2001; Gerst et al., 1999). In fact, the CNS is particularly susceptible to changes in local O2 ˜ a and Ramirez, 2005) levels, which can affect neuronal activity (Pen and promote the development of disorders, including dementia (Bazan et al., 2002). Interestingly, hypoxia increases progranulin expression in fibroblast cultures (Guerra et al., 2007), but its effect on gene expression in neuronal cells is still unknown. Several signaling pathways have been identified to regulate gene expression during hypoxia (Seta et al., 2002). Among these, the hypoxia-induced activation of the MEK-ERK MAPK cascade could be an important signal transduction pathway of PGRN due to the presence of repeated consensus sequence for the transcription factor activator protein-1 (AP-1) in the promoter region of the gene (Bhandari et al., 1996). On the other side, a possible role for oxygen free radicals in neurodegeneration has been also extensively examined (Mattson, 2002; Fatokun et al., 2008). Here, we investigate the in vitro effect on PGRN regulation of two different stimuli commonly associated with the progression of neurodegenerative processes: hypoxia and oxidative stress. Our
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results demonstrate that chronic hypoxic exposure up-regulates PGRN expression in neuroblastoma cell lines via the mitogenactived protein kinase (MAPK)/extracellular signal-regulated kinase (MEK) signaling cascade. In contrast, PGRN is not modulated by H2O2 treatment. These data, together with those of others (Larade, 2008; Guerra et al., 2007) suggest a possible protective role of PGRN against hypoxic/anoxic insults in the brain. 2. Methods 2.1. Cell lines Human neuroblastoma cell lines SK-N-BE and SK-N-SH (kind gift of Prof. G. Poiana, University ‘‘La Sapienza’’, Rome, Italy) were cultivated in RPMI-1640 medium (Euroclone) and Dulbecco’s modified Eagle’s medium (DMEM, Euroclone) respectively. The human neuroblastoma cell line SH-SY5Y (kindly supplied by Dr. G. Pani, Catholic University, Rome, Italy) was cultivated in Dulbecco’s modified Eagle’s medium with 4.5 g glucose added per liter (DMEM high glucose, Euroclone). All growth media were supplemented with 10% heat-inactivated (v/v) Foetal Bovine Serum (FBS), 5 mM L-glutamine, penicillin (100 IU/ml) and streptomycin (100 mg/ ml). Cell cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2. For experiments, cells were seeded on 60 mm plastic culture dishes at a density of 1 104 cm2 and were grown to 80% confluence, at which point, the medium was changed. The general morphology of cell monolayers, before and after treatments, was monitored by light microscopy. Cell proliferation was estimated by counting the total number of cells in each dish with use of a hematocytometer. Cell viability was determined by Trypan blue dye exclusion test (Sigma–Aldrich). We also evaluated the number of cells that detached from the substrate and were found to be freely floating in cell medium. 2.2. Cell treatments For stimulating hypoxia, we used a hypoxic/anaerobic chamber (BBLTM GasPakTM, USA). The system was set up at 37 8C in 5% CO2, 95% N2. Cells were transferred into the humidified chamber and incubated with the appropriate media for up to 24 h then, lysed for RNA isolation (below). Control cells were maintained in the incubator under normoxic conditions. For stimulating intracellular oxidative stress, sub-confluent cells were exposed to a final concentration of 100 mM H2O2 for 24 h under normoxic conditions, then, washed with PBS, harvested by trypsinization, and processed as described below. For stimulating inhibition of MEK cascades, sub-confluent cells were exposed to a 40 mM 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059; Sigma–Aldrich) inhibitor for 24 h under hypoxic conditions; PD98059-treated cells grown under normoxic conditions were used as controls. Cells were successively washed with PBS, harvested by trypsinization and mRNA was recovered. 2.3. RNA isolation and quantitative real-time PCR Total RNA was extracted from SK-N-BE, SK-N-SH and SH-SY5Y cells using the Invisorb SpinCell RNA kit (Invitek). cDNA was made by retrotranscription using SuperScript III first-strand cDNA synthesis kit (Invitrogen Inc., Carlsbad, CA) with random primers, according to the manufacturer’s protocol. Reverse transcription quantitative real-time PCR (RT-qPCR) was performed using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, USA) with TaqMan Universal PCR Master Mix and TaqMan Gene expression assays (Applied Biosystems). The parameters for PCR amplification were: 50 8C for 2 min, 95 8C for 10 min followed by 40 cycles of 95 8C for 15 s and 60 8C for 1 min. PCR was performed in triplicate for each sample; 18S rRNA was chosen as reference gene. The relative expression of mRNA was calculated using the comparative Ct method. 2.4. RNA interference RNA interference was performed using Thermo Scientific Dharmacon1 Accell1 siRNA Dharmacon (Euroclone S.p.A, Italy), according to manufacturer’s protocol. This protocol is especially modified for delivering small-interfering RNA (siRNA) into cells without use of a transfection reagent; the methodology works at a higher probe concentration than conventional siRNA technology, with a minimal disruption of cell expression profiles. For genetic knockdown experiments, cells were plated at the appropriate cell density and grown overnight, then incubated with 2 ml of fresh complete medium containing 1 mM Accell Smartpool siRNA human PGRN (cat. no. E-009285-00-0050), in which a mixture of four siRNAs targeting human PGRN gene was added. A negative control was used (cat. no. D001910-10-20), with a mixture of four siRNAs that had no significant homology to any known human gene sequence; whereas a positive control (cat. no. D-00193010-20) had a mixture of four siRNAs that targeted the human GAPDH human gene. After 24 h of incubation, cells were transferred into the hypoxic chamber for additional 24 h incubation under hypoxic conditions, as described above. Cells were then harvested, and processed for RNA extraction. The RNA silencing was not toxic to cells: no statistically significant reduction in cellular viability was observed relative to untreated hypoxic cell cultures (data not shown).
2.5. Evaluation of oxidative stress The total intracellular glutathione content was determined, in three different neuroblastoma cell lines, with use of the enzymatic recycling assay with glutathione reductase (type IV, Sigma–Aldrich) and 5,50 -dithiobis-2-nitrobenzoic acid (DTNB, Sigma–Aldrich) (see Anderson, 1985). For the measurement of oxidized glutathione (GSSG), the acidified homogenates were submitted to derivatization with undiluted 2-vinylpyridine (Aldrich, Milwaukee, WI, USA) in the presence of triethanolamine (Sigma–Aldrich), for 1 h at room temperature. Samples were then assayed for total glutathione measurement. The amount of reduced glutathione (GSH) present in the samples was calculated as the difference between total glutathione and GSSG levels. Data were expressed as nmoles of GSH or GSSG per mg of cell protein.
2.6. Protein content Protein concentration was measured by the commercially available Coomassie brilliant blue dye-binding assay (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. Bovine g-globulin was used as a standard. 2.7. Western blot analysis Cells were harvested in RIPA buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1.0% NP-40, 0.1% SDS, 1% Na-deoxycholate), containing a protease inhibitor cocktail (Sigma–Aldrich), 1 mM Na3VO4, 5 mM NaF, 1 for 1 h in ice. The cellular extracts were solubilized in 4X Laemmli sample buffer and boiled for 5 min. Proteins (20 mg) were separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Westran, Schleicher and Schuell). Blots were first blocked with 5% non-fat powdered milk in TBS/Tween 0.1%, then probed overnight at 4 8C with mouse monoclonal anti-Granulin (Abcam no. ab 52557, 1:4000), mouse monoclonal anti-b-actin (Sigma–Aldrich, 1:80,000), polyclonal anti-pERK1/2 (Cell Signalling, 1:1000), polyclonal anti-ERK1/2 (Cell Signalling, 1:1000). Membranes were washed, incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Chemicon, 1:20,000) and developed by ECL (BIORAD) according to manufacturer’s instructions. Densitometric analysis was performed with FX-Imager densitometer and the Quantity One software (Bio-Rad Laboratories). 2.8. Statistical analysis Data are expressed as mean SEM. Comparisons among groups were made using Student’s t-test or using one way ANOVA followed by Bonferroni’s multiple comparison test, with significance set at P < 0.05.
3. Results 3.1. Preliminary assessment of hypoxia We analysed the expression of glucose transporter GLUT1, which is regulated under hypoxic condition, as a marker of hypoxia (Fisk et al., 2006). As shown in Fig. 1A, GLUT1 levels were up-regulated in response to hypoxia in all cell lines tested, with a dramatical increase at 24 h of incubation. Moreover, 24 h of hypoxic treatment was not cytotoxic for the cells, at least under the assay conditions we have used here. In fact, the number of cells treated with hypoxia did not differ by more than 5% from the number of control cells, without any decrease in cell viability (96 2%). Twenty-four hours of H2O2 treatment did not alter GLUT1 gene expression in any of the cell lines. 3.2. Preliminary assessment of oxidative stress As a marker of oxidative stress we used the GSH/GSSG ratio, considered to be an indicator of redox potential of the cells (Esteve et al., 1999). Twenty-four hours of H2O2 treatment induced a consistent oxidative stress in SK-N-BE cells, as indicated by a sustained decrease in GSH and a parallel decrease in GSSG, with a reduction of the GSH/GSSG ratio, (Table 1). However, under our experimental conditions, 24 h of chronic hypoxia did not alter glutathione levels. A significant reduction in cell number (42 4.5%) accompanied by a moderate decrease in cell viability (88 4%), with respect to controls, was observed in the H2O2 treated
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Fig. 1. Twenty-four hours of hypoxia treatment, affect GLUT-1 and PGRN gene expression in neuroblastoma cell lines. (A and B) The histograms show mRNA levels of GLUT-1 (A) and PGRN (B) after hypoxia administration. Data, compared to 18S rRNA, are expressed as fold changes relative to the control at each treatment. Mean SEM of six experiments is shown. (C) Representative Western blot analysis of PGRN protein. Densitometric analysis data are presented as ratios normalized by b-actin. The histogram shows the mean SEM of at least four preparations. **P < 0.01 and *P < 0.05 vs. untreated normoxic cells.
cells. In parallel the number of cells detached from the substrate and freely floating in the medium, increased. Similar results were obtained in both SK-N-SH and SH-5YSY cells (data not shown). 3.3. Hypoxia, but not oxidative stress, induces PGRN up-regulation in neuroblastoma cell lines To test the effect of hypoxia and hydrogen peroxide treatment s on the regulation of the PGRN gene, we analysed the PGRN transcripts of multiple neuroblastoma cell lines by quantitative real-time PCR, under normoxic and hypoxic conditions. Fig. 1B shows a modulation in PGRN transcription after 24 h of hypoxic compared to normoxic conditions. In particular, we found that PGRN mRNA was increased 2.44 0.15-fold in SK-N-BE, 1.39 0.1-fold in SK-N-SH and 1.96 0.19-fold in SH-SY5Y cells. Since the best hypoxic/normoxic PGRN expression ratio was displayed by SK-N-BE cells, we decided to perform all subsequent analyses in this cell line. To test another degenerative stimulus, we treated SK-N-BE cells for 24 h with 100 mM H2O2. No effects on PGRN expression were observed after exposure to hydrogen peroxide (Fig. 2). Moreover, to investigate whether the differences noted in mRNA levels were also reflected at the protein level, a specific immunoblot analysis was performed in SK-N-BE cells, revealing a consistent and significant increase of PGRN proteins after 24 h of hypoxic treatment, relative to normoxic conditions (Fig. 1C).
3.4. siRNA-mediated down regulation of PGRN expression is associated with altered cell morphology In order to verify if and how much, PGRN protected neuroblastoma cells from hypoxic damage we knocked down PGRN with use of small-interfering RNA (siRNA) technology. The efficacy of silencing treatment was monitored by both RT-PCR and western blot analysis. Fig. 3A shows that, PGRN siRNA exposure depressed PGRN expression in hypoxic SK-N-BE cells by 50%, with respect to untreated hypoxic cells. Gene silencing was also confirmed by the reduced expression of down-stream PGRN protein, as revealed by western blot analysis (Fig. 3B). A parallel analysis of cell monolayers performed by phase contrast microscopy showed that cells under normoxic conditions (CTRL), displayed a flat and polygonal aspect with very short fillopodia; no significantly modifications were visible after 24 h of hypoxic conditions (hyp); when siRNA for PGRN was added for 24 h (siRNA PGRN), the cells displayed greater tendency to form clumps; a number of cells displayed elongated cell bodies (Fig. 3C, arrows). In addition, a moderate decrease in the number of adherent cells was appreciablely lower (25 3%) compared to untreated cells, with a parallel increase (+12 2%) in the number of detached cells observed floating freely in the medium. Finally, a significant reduction in cell
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Table 1 Twenty-four hours of 100 mM H2O2 treatment, but not hypoxia, induce oxidative stress in SK-N-BE cells. Parameter
Control
H2O2
Hypoxia
GSH (nmol/mg protein) GSSG(nmol/mg protein) GSH/GSSG
11.7 0.99 0.37 0.09 32.66 2.0
6.88 0.72** 1.23 0.18** 5.59 0.49***
13.5 1.47 0.45 0.07 30 2.19
The means SEM of six separate experiments, each performed in duplicate are shown. ** P < 0.01. *** P < 0.001 vs. untreated normoxic cells.
Fig. 2. Twenty-four hours of H2O2 did not affect PGRN and GLUT-1 gene expression in SK-N-BE cells. Data, compared to 18S rRNA, are expressed as fold changes relative to the control at each treatment. Mean SEM of six experiments is shown.
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Fig. 3. PGRN silencing determines cell number and morphology alterations. (A) siRNA PGRN transcripts were evaluated by quantitative Real time PCR. Data, compared to 18S rRNA, are expressed as fold changes relative to the normoxic control at each treatment. Mean SEM of three experiments is shown. *P < 0.05 vs. untreated normoxic cells. (B) Western blot analysis in SK-N-BE cells silenced or not for PGRN protein for 48 h in hypoxic and normoxic conditions. (C) Phase contrast images of SK-N-BE showed cells in normoxic, hypoxic and silencing conditions. Pictures are representative of three independent experiments and at least 10 fields of view.
viability was detected in siRNA-treated cells under hypoxic conditions, with respect to untreated hypoxic cells (90 3% vs. 96 2%; P < 0.05). Interestingly, the progranulin decrease induced by siRNA under normoxic conditions did not significantly reduce cell viability (94 3%), nor cause any cellular shape modification. 3.5. PD98059 inhibitor reverts hypoxia-dependent PGRN up-regulation To evaluate if the MEK pathway is involved in hypoxia-induced PGRN up-regulation, cells were treated with the specific MEK inhibitor PD98059. As showed in Fig. 4B the treatment with the inhibitor induces a 60% decrease of ERK 1/2 phosphorylation. PD98059 treatment reverted the PGRN mRNA transcripts, which
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increased after 24 h of hypoxia, to levels similar to those of controls (Fig. 4A). Moreover, the use of the inhibitor did not modify PGRN transcription in SK-N-BE cells that were maintained under normoxic conditions (Fig. 4A). To evaluate if the MEK pathway is involved in hypoxia-induced PGRN up-regulation, cells were treated with the specific MEK inhibitor PD98059. This treatment reverted the PGRN mRNA transcripts, which were increased after 24 h of hypoxia, to levels similar to those of controls (Fig. 4A). Moreover, the use of the inhibitor did not modify PGRN transcription in SK-N-BE cells that were maintained under normoxic conditions (Fig. 4A). To establish the strength of the inhibition we also carried out an analysis on activation of MEK/ERK pathway in SK-N-BE cells after 24 h of hypoxic treatment. As showed in Fig. 4B the inhibitor induces a 60% decrease of ERK 1/2 phosphorylation. 4. Discussion
Fig. 4. (A) MEK inhibitor PD98059 reverts the PGRN hypoxia-induced up-regulation in SK-N-BE neuron-like cells. Data, compared to 18S rRNA, are expressed as fold changes relative to the normoxic control at each treatment. Mean SEM of almost three experiments is shown; **P < 0.01. (B) Activation of MEK/ERK pathway in SK-N-BE cells after 24 h of hypoxic treatment. The specific MEK1 inhibitor PD98059 abolishes ERK activation (representative Western blot). The histogram shows the data obtained from densitometric analysis of the p-ERK/ERK ratio (n = 4). Bars represent the mean SEM; *P < 0.05.
Ischemia/hypoxia and oxidative stress play a role in the pathogenesis of several neurodegenerative diseases, including FTD (Martin et al., 2001; Gerst et al., 1999). To evaluate a possible involvement for PGRN in neurodegeneration, we evaluated the modulation of the PGRN gene following exposure to these two neurodegenerative stimuli. Here, we report that levels of both PGRN gene and protein expression were significantly elevated following chronic hypoxia. In contrast, neither gene expression nor the protein, were altered following hydroperoxide-induced oxidative stress. Thus, each of the two stress conditions, associated with neurodegeneration, has distinct effects on gene expression. Interestingly, Yin et al. described that PGRN-deficient hippocampal slices were hypersusceptible to deprivation of oxygen (Yin et al., 2010). Moreover, a study on a marine gastropod reports accumulation of l-grn transcripts as part of the response to anoxia (Larade, 2008). Guerra and colleagues also describe an upregulation of PGRN transcripts in fibroblasts under condition of hypoxia (Guerra et al., 2007). These findings are in agreement with the present data, wherein we demonstrate a modulation not only of PGRN mRNA level, but also of its protein expression. Moreover, PGRN silencing was associated with changes in cell morphology and a fewer adherent cells, suggesting a possible protective role for this gene under hypoxic conditions. This neuroprotective action is also confirmed by a study on serum-deprivation in motor neurons describing that progranulin is cytoprotective over prolonged
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periods when over-expressed in a neuronal cell line (Ryan et al., 2009). Oxygen-deprived stress induces a number of spatially and temporally regulated intracellular responses, ranging from reduced channel activity to altered gene expression. In fact, depending on the duration and severity of oxygen deprivation, cellular oxygen-sensor responses activate a variety of short- and long-term energy-saving and cellular protection mechanisms. Hypoxic adaptation encompasses an immediate depolarization block by changing potassium, sodium and chloride ion fluxes across the cellular membrane. Moreover, the physiologic response to hypoxia modulates gene expression and protein synthesis, with a HIF-mediated up-regulation of enzymes or growth factors inducing angiogenesis, anaerobic glycolysis, cell survival or neural stem cell growth (Van Elzen et al., 2008; Acker and Acker, 2004). Many potential regulatory elements associated with the functional activity of promoters of other eukaryotic genes are scattered throughout the 50 flanking region of the human PRGN. However, among these, the consensus sequences for the family of hypoxia-inducible factors are not included. Notably, 25 sequences show a 7 out of 8 bp match with the AP-1 consensus sequence TGA(C/G)TCAG (Bhandari et al., 1996). Members of the AP-1 transcription factor family are well-known targets of the MEK signaling cascade. Moreover, the MAPKs respond to a variety of environmental stresses (Gaitanaki et al., 2004) including hypoxia (Haddad, 2004) and have a primary role in mediating stressinduced gene expression. Our data, for the first time, provide strong evidence that the highly selective inhibition of the MEK signal transduction pathway abolishes the up-regulation of PGRN in SK-N-BE human neuron-like cell culture, as stimulated by hypoxia treatment. These findings demonstrate a direct involvement of MAPKs in the induction of mRNA transcription. Moreover, the inhibition of MEK activity does not alter PGRN mRNA expression under normoxic conditions, suggesting that the PGRN gene transcription is specifically triggered by the hypoxic stimulus. As PGRN expression also promotes neuronal survival, and enhances neurite outgrowth in vitro, (Van Damme et al., 2008) it is reasonable that the observed cellular response to hypoxia could be adaptative; in this case our data agree with the reported PGRN blocking effect on tamoxifen-induced apoptosis in breast tumors (Tangkeangsirisin et al., 2004). Interestingly, PGRN activates the extracellular regulated kinases, and the phosphatidyl inositol-3 kinase signal cascades and also increases expression of cyclins D and B (Lu and Serrero, 2001). So, regulation of PGRN expression, exerted by the MAPKs under hypoxia, could be indicative of a survival loop which is switched on by cells to blunt pro-apoptotic environmental conditions, preserving neuronal viability. Interestingly, a similar hypothesis regarding the reciprocal functional interaction of PGRN and MEK pathway was formulated by Kamrava et al. in their study on lysophosphatidic acid and endothelin-induced proliferation of ovarian cancer cell lines suggesting a diffused proliferation and survival loop shared by different cell types and unrelated environmental stimuli (Kamrava et al., 2005). In conclusion, our results suggest that PGRN is highly susceptible to changes in local O2 tension and, as a specific hypoxic sensor, could have an adaptative/protective role against one of principal risk factors involved in AD and FTD pathogenesis as hypoxic stress.
Acknowledgments The authors would address particular thanks to Dr. Sonal Jhaveri for reviewing the manuscript. This study was supported by a grant from the Italian Ministry of Health: ‘‘Genetic determinants
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and modulator factors in neurodegenerative diseases: clinical and experimental model’’ to A.C.
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Hypoxia induces up-regulation of progranulin in neuroblastoma cell lines Paola Piscopo, Roberto Rivabene, Alice Adduci, Cinzia Mallozzi, Lorenzo Malvezzi-Campeggi, Alessio Crestini, Annamaria Confaloni * Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 April 2010 Received in revised form 31 August 2010 Accepted 26 September 2010 Available online 7 October 2010
Progranulin (PGRN) is a widely expressed multifunctional protein, involved in regulation of cell growth and cell cycle progression with a possible involvement in neurodegeneration. We looked for PGRN regulation in three different human neuroblastoma cell lines, following exposure to two different stimuli commonly associated to neurodegeneration: hypoxia and oxidative stress. For gene and protein expression analysis we carried out a quantitative RT-PCR and western blotting analysis. We show that PGRN is strongly up-regulated by hypoxia, through the mitogen-actived protein kinase (MAPK)/ extracellular signal-regulated kinase (MEK) signaling cascade. PGRN is not up-regulated by H2O2induced oxidative stress. These results suggest that PGRN in the brain could exert a protective role against hypoxic stress, one of principal risk factors involved in frontotemporal dementia pathogenesis. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Progranulin Gene expression Hypoxia Oxidative stress Neuroblastoma cell lines
1. Introduction Progranulin (PGRN) is a widely expressed multifunctional protein involved in the regulation of cell growth and cell cycle progression (Bateman et al., 1990; He and Bateman, 2003). In normal peripheral tissues, the PGRN has a complex function, with the full-length form of the protein, which exerts trophic and antiinflammatory activity. Proteolytic cleavage of PGRN generates granulin peptides that promote inflammatory activity. Little is known about the role of PGRN in the central nervous system (CNS). The protein is widely expressed during early neural development (Daniel et al., 2003), but with maturation its expression becomes restricted to defined neuronal populations, such as cortical and hippocampal pyramidal neurons and Purkinje cells (Daniel et al., 2000). PGRN has also been implicated in the sexual differentiation of the brain (Suzuki and Nishiahara, 2002). The protein is up-regulated in activated microglial cells (Baker and Manuelidis, 2003) but not in astrocytes or oligodendrocytes. Recently, Van Damme and colleagues observed that PGRN and GRN E (one of the proteolytic fragments of PGRN) promote neuronal survival and enhance neurite outgrowth in cultured neurons (Van Damme et al., 2008). Several nonsense and frameshift mutations in Abbreviations: FTD, frontotemporal dementia; GLUT-1, glucose transporter 1; GSH, reduced glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide; MAPKs, mitogen-activated protein kinases; MEK, (MAPK)/extracellular signalregulated kinase kinase; PGRN, progranulin. * Corresponding author. Tel.: +39 06 49902930; fax: +39 06 49387143. E-mail address: [email protected] (A. Confaloni). 0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.09.008
the PGRN gene are associated with frontotemporal lobar degeneration (FTLD) (Baker et al., 2006), the third most common cause of neurodegenerative dementia after AD and dementia with Lewy bodies (Bird et al., 2003). FTLD is caused by both genetic and environmental factors, but the exact cause are still unknown. It has been hypothesized that both ischemia/hypoxia and oxidative stress are involved in the pathogenesis of several neurodegenerative diseases, including FTD (Martin et al., 2001; Gerst et al., 1999). In fact, the CNS is particularly susceptible to changes in local O2 ˜ a and Ramirez, 2005) levels, which can affect neuronal activity (Pen and promote the development of disorders, including dementia (Bazan et al., 2002). Interestingly, hypoxia increases progranulin expression in fibroblast cultures (Guerra et al., 2007), but its effect on gene expression in neuronal cells is still unknown. Several signaling pathways have been identified to regulate gene expression during hypoxia (Seta et al., 2002). Among these, the hypoxia-induced activation of the MEK-ERK MAPK cascade could be an important signal transduction pathway of PGRN due to the presence of repeated consensus sequence for the transcription factor activator protein-1 (AP-1) in the promoter region of the gene (Bhandari et al., 1996). On the other side, a possible role for oxygen free radicals in neurodegeneration has been also extensively examined (Mattson, 2002; Fatokun et al., 2008). Here, we investigate the in vitro effect on PGRN regulation of two different stimuli commonly associated with the progression of neurodegenerative processes: hypoxia and oxidative stress. Our
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results demonstrate that chronic hypoxic exposure up-regulates PGRN expression in neuroblastoma cell lines via the mitogenactived protein kinase (MAPK)/extracellular signal-regulated kinase (MEK) signaling cascade. In contrast, PGRN is not modulated by H2O2 treatment. These data, together with those of others (Larade, 2008; Guerra et al., 2007) suggest a possible protective role of PGRN against hypoxic/anoxic insults in the brain. 2. Methods 2.1. Cell lines Human neuroblastoma cell lines SK-N-BE and SK-N-SH (kind gift of Prof. G. Poiana, University ‘‘La Sapienza’’, Rome, Italy) were cultivated in RPMI-1640 medium (Euroclone) and Dulbecco’s modified Eagle’s medium (DMEM, Euroclone) respectively. The human neuroblastoma cell line SH-SY5Y (kindly supplied by Dr. G. Pani, Catholic University, Rome, Italy) was cultivated in Dulbecco’s modified Eagle’s medium with 4.5 g glucose added per liter (DMEM high glucose, Euroclone). All growth media were supplemented with 10% heat-inactivated (v/v) Foetal Bovine Serum (FBS), 5 mM L-glutamine, penicillin (100 IU/ml) and streptomycin (100 mg/ ml). Cell cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2. For experiments, cells were seeded on 60 mm plastic culture dishes at a density of 1 104 cm2 and were grown to 80% confluence, at which point, the medium was changed. The general morphology of cell monolayers, before and after treatments, was monitored by light microscopy. Cell proliferation was estimated by counting the total number of cells in each dish with use of a hematocytometer. Cell viability was determined by Trypan blue dye exclusion test (Sigma–Aldrich). We also evaluated the number of cells that detached from the substrate and were found to be freely floating in cell medium. 2.2. Cell treatments For stimulating hypoxia, we used a hypoxic/anaerobic chamber (BBLTM GasPakTM, USA). The system was set up at 37 8C in 5% CO2, 95% N2. Cells were transferred into the humidified chamber and incubated with the appropriate media for up to 24 h then, lysed for RNA isolation (below). Control cells were maintained in the incubator under normoxic conditions. For stimulating intracellular oxidative stress, sub-confluent cells were exposed to a final concentration of 100 mM H2O2 for 24 h under normoxic conditions, then, washed with PBS, harvested by trypsinization, and processed as described below. For stimulating inhibition of MEK cascades, sub-confluent cells were exposed to a 40 mM 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059; Sigma–Aldrich) inhibitor for 24 h under hypoxic conditions; PD98059-treated cells grown under normoxic conditions were used as controls. Cells were successively washed with PBS, harvested by trypsinization and mRNA was recovered. 2.3. RNA isolation and quantitative real-time PCR Total RNA was extracted from SK-N-BE, SK-N-SH and SH-SY5Y cells using the Invisorb SpinCell RNA kit (Invitek). cDNA was made by retrotranscription using SuperScript III first-strand cDNA synthesis kit (Invitrogen Inc., Carlsbad, CA) with random primers, according to the manufacturer’s protocol. Reverse transcription quantitative real-time PCR (RT-qPCR) was performed using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, USA) with TaqMan Universal PCR Master Mix and TaqMan Gene expression assays (Applied Biosystems). The parameters for PCR amplification were: 50 8C for 2 min, 95 8C for 10 min followed by 40 cycles of 95 8C for 15 s and 60 8C for 1 min. PCR was performed in triplicate for each sample; 18S rRNA was chosen as reference gene. The relative expression of mRNA was calculated using the comparative Ct method. 2.4. RNA interference RNA interference was performed using Thermo Scientific Dharmacon1 Accell1 siRNA Dharmacon (Euroclone S.p.A, Italy), according to manufacturer’s protocol. This protocol is especially modified for delivering small-interfering RNA (siRNA) into cells without use of a transfection reagent; the methodology works at a higher probe concentration than conventional siRNA technology, with a minimal disruption of cell expression profiles. For genetic knockdown experiments, cells were plated at the appropriate cell density and grown overnight, then incubated with 2 ml of fresh complete medium containing 1 mM Accell Smartpool siRNA human PGRN (cat. no. E-009285-00-0050), in which a mixture of four siRNAs targeting human PGRN gene was added. A negative control was used (cat. no. D001910-10-20), with a mixture of four siRNAs that had no significant homology to any known human gene sequence; whereas a positive control (cat. no. D-00193010-20) had a mixture of four siRNAs that targeted the human GAPDH human gene. After 24 h of incubation, cells were transferred into the hypoxic chamber for additional 24 h incubation under hypoxic conditions, as described above. Cells were then harvested, and processed for RNA extraction. The RNA silencing was not toxic to cells: no statistically significant reduction in cellular viability was observed relative to untreated hypoxic cell cultures (data not shown).
2.5. Evaluation of oxidative stress The total intracellular glutathione content was determined, in three different neuroblastoma cell lines, with use of the enzymatic recycling assay with glutathione reductase (type IV, Sigma–Aldrich) and 5,50 -dithiobis-2-nitrobenzoic acid (DTNB, Sigma–Aldrich) (see Anderson, 1985). For the measurement of oxidized glutathione (GSSG), the acidified homogenates were submitted to derivatization with undiluted 2-vinylpyridine (Aldrich, Milwaukee, WI, USA) in the presence of triethanolamine (Sigma–Aldrich), for 1 h at room temperature. Samples were then assayed for total glutathione measurement. The amount of reduced glutathione (GSH) present in the samples was calculated as the difference between total glutathione and GSSG levels. Data were expressed as nmoles of GSH or GSSG per mg of cell protein.
2.6. Protein content Protein concentration was measured by the commercially available Coomassie brilliant blue dye-binding assay (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. Bovine g-globulin was used as a standard. 2.7. Western blot analysis Cells were harvested in RIPA buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1.0% NP-40, 0.1% SDS, 1% Na-deoxycholate), containing a protease inhibitor cocktail (Sigma–Aldrich), 1 mM Na3VO4, 5 mM NaF, 1 for 1 h in ice. The cellular extracts were solubilized in 4X Laemmli sample buffer and boiled for 5 min. Proteins (20 mg) were separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Westran, Schleicher and Schuell). Blots were first blocked with 5% non-fat powdered milk in TBS/Tween 0.1%, then probed overnight at 4 8C with mouse monoclonal anti-Granulin (Abcam no. ab 52557, 1:4000), mouse monoclonal anti-b-actin (Sigma–Aldrich, 1:80,000), polyclonal anti-pERK1/2 (Cell Signalling, 1:1000), polyclonal anti-ERK1/2 (Cell Signalling, 1:1000). Membranes were washed, incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Chemicon, 1:20,000) and developed by ECL (BIORAD) according to manufacturer’s instructions. Densitometric analysis was performed with FX-Imager densitometer and the Quantity One software (Bio-Rad Laboratories). 2.8. Statistical analysis Data are expressed as mean SEM. Comparisons among groups were made using Student’s t-test or using one way ANOVA followed by Bonferroni’s multiple comparison test, with significance set at P < 0.05.
3. Results 3.1. Preliminary assessment of hypoxia We analysed the expression of glucose transporter GLUT1, which is regulated under hypoxic condition, as a marker of hypoxia (Fisk et al., 2006). As shown in Fig. 1A, GLUT1 levels were up-regulated in response to hypoxia in all cell lines tested, with a dramatical increase at 24 h of incubation. Moreover, 24 h of hypoxic treatment was not cytotoxic for the cells, at least under the assay conditions we have used here. In fact, the number of cells treated with hypoxia did not differ by more than 5% from the number of control cells, without any decrease in cell viability (96 2%). Twenty-four hours of H2O2 treatment did not alter GLUT1 gene expression in any of the cell lines. 3.2. Preliminary assessment of oxidative stress As a marker of oxidative stress we used the GSH/GSSG ratio, considered to be an indicator of redox potential of the cells (Esteve et al., 1999). Twenty-four hours of H2O2 treatment induced a consistent oxidative stress in SK-N-BE cells, as indicated by a sustained decrease in GSH and a parallel decrease in GSSG, with a reduction of the GSH/GSSG ratio, (Table 1). However, under our experimental conditions, 24 h of chronic hypoxia did not alter glutathione levels. A significant reduction in cell number (42 4.5%) accompanied by a moderate decrease in cell viability (88 4%), with respect to controls, was observed in the H2O2 treated
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Fig. 1. Twenty-four hours of hypoxia treatment, affect GLUT-1 and PGRN gene expression in neuroblastoma cell lines. (A and B) The histograms show mRNA levels of GLUT-1 (A) and PGRN (B) after hypoxia administration. Data, compared to 18S rRNA, are expressed as fold changes relative to the control at each treatment. Mean SEM of six experiments is shown. (C) Representative Western blot analysis of PGRN protein. Densitometric analysis data are presented as ratios normalized by b-actin. The histogram shows the mean SEM of at least four preparations. **P < 0.01 and *P < 0.05 vs. untreated normoxic cells.
cells. In parallel the number of cells detached from the substrate and freely floating in the medium, increased. Similar results were obtained in both SK-N-SH and SH-5YSY cells (data not shown). 3.3. Hypoxia, but not oxidative stress, induces PGRN up-regulation in neuroblastoma cell lines To test the effect of hypoxia and hydrogen peroxide treatment s on the regulation of the PGRN gene, we analysed the PGRN transcripts of multiple neuroblastoma cell lines by quantitative real-time PCR, under normoxic and hypoxic conditions. Fig. 1B shows a modulation in PGRN transcription after 24 h of hypoxic compared to normoxic conditions. In particular, we found that PGRN mRNA was increased 2.44 0.15-fold in SK-N-BE, 1.39 0.1-fold in SK-N-SH and 1.96 0.19-fold in SH-SY5Y cells. Since the best hypoxic/normoxic PGRN expression ratio was displayed by SK-N-BE cells, we decided to perform all subsequent analyses in this cell line. To test another degenerative stimulus, we treated SK-N-BE cells for 24 h with 100 mM H2O2. No effects on PGRN expression were observed after exposure to hydrogen peroxide (Fig. 2). Moreover, to investigate whether the differences noted in mRNA levels were also reflected at the protein level, a specific immunoblot analysis was performed in SK-N-BE cells, revealing a consistent and significant increase of PGRN proteins after 24 h of hypoxic treatment, relative to normoxic conditions (Fig. 1C).
3.4. siRNA-mediated down regulation of PGRN expression is associated with altered cell morphology In order to verify if and how much, PGRN protected neuroblastoma cells from hypoxic damage we knocked down PGRN with use of small-interfering RNA (siRNA) technology. The efficacy of silencing treatment was monitored by both RT-PCR and western blot analysis. Fig. 3A shows that, PGRN siRNA exposure depressed PGRN expression in hypoxic SK-N-BE cells by 50%, with respect to untreated hypoxic cells. Gene silencing was also confirmed by the reduced expression of down-stream PGRN protein, as revealed by western blot analysis (Fig. 3B). A parallel analysis of cell monolayers performed by phase contrast microscopy showed that cells under normoxic conditions (CTRL), displayed a flat and polygonal aspect with very short fillopodia; no significantly modifications were visible after 24 h of hypoxic conditions (hyp); when siRNA for PGRN was added for 24 h (siRNA PGRN), the cells displayed greater tendency to form clumps; a number of cells displayed elongated cell bodies (Fig. 3C, arrows). In addition, a moderate decrease in the number of adherent cells was appreciablely lower (25 3%) compared to untreated cells, with a parallel increase (+12 2%) in the number of detached cells observed floating freely in the medium. Finally, a significant reduction in cell
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Table 1 Twenty-four hours of 100 mM H2O2 treatment, but not hypoxia, induce oxidative stress in SK-N-BE cells. Parameter
Control
H2O2
Hypoxia
GSH (nmol/mg protein) GSSG(nmol/mg protein) GSH/GSSG
11.7 0.99 0.37 0.09 32.66 2.0
6.88 0.72** 1.23 0.18** 5.59 0.49***
13.5 1.47 0.45 0.07 30 2.19
The means SEM of six separate experiments, each performed in duplicate are shown. ** P < 0.01. *** P < 0.001 vs. untreated normoxic cells.
Fig. 2. Twenty-four hours of H2O2 did not affect PGRN and GLUT-1 gene expression in SK-N-BE cells. Data, compared to 18S rRNA, are expressed as fold changes relative to the control at each treatment. Mean SEM of six experiments is shown.
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Fig. 3. PGRN silencing determines cell number and morphology alterations. (A) siRNA PGRN transcripts were evaluated by quantitative Real time PCR. Data, compared to 18S rRNA, are expressed as fold changes relative to the normoxic control at each treatment. Mean SEM of three experiments is shown. *P < 0.05 vs. untreated normoxic cells. (B) Western blot analysis in SK-N-BE cells silenced or not for PGRN protein for 48 h in hypoxic and normoxic conditions. (C) Phase contrast images of SK-N-BE showed cells in normoxic, hypoxic and silencing conditions. Pictures are representative of three independent experiments and at least 10 fields of view.
viability was detected in siRNA-treated cells under hypoxic conditions, with respect to untreated hypoxic cells (90 3% vs. 96 2%; P < 0.05). Interestingly, the progranulin decrease induced by siRNA under normoxic conditions did not significantly reduce cell viability (94 3%), nor cause any cellular shape modification. 3.5. PD98059 inhibitor reverts hypoxia-dependent PGRN up-regulation To evaluate if the MEK pathway is involved in hypoxia-induced PGRN up-regulation, cells were treated with the specific MEK inhibitor PD98059. As showed in Fig. 4B the treatment with the inhibitor induces a 60% decrease of ERK 1/2 phosphorylation. PD98059 treatment reverted the PGRN mRNA transcripts, which
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increased after 24 h of hypoxia, to levels similar to those of controls (Fig. 4A). Moreover, the use of the inhibitor did not modify PGRN transcription in SK-N-BE cells that were maintained under normoxic conditions (Fig. 4A). To evaluate if the MEK pathway is involved in hypoxia-induced PGRN up-regulation, cells were treated with the specific MEK inhibitor PD98059. This treatment reverted the PGRN mRNA transcripts, which were increased after 24 h of hypoxia, to levels similar to those of controls (Fig. 4A). Moreover, the use of the inhibitor did not modify PGRN transcription in SK-N-BE cells that were maintained under normoxic conditions (Fig. 4A). To establish the strength of the inhibition we also carried out an analysis on activation of MEK/ERK pathway in SK-N-BE cells after 24 h of hypoxic treatment. As showed in Fig. 4B the inhibitor induces a 60% decrease of ERK 1/2 phosphorylation. 4. Discussion
Fig. 4. (A) MEK inhibitor PD98059 reverts the PGRN hypoxia-induced up-regulation in SK-N-BE neuron-like cells. Data, compared to 18S rRNA, are expressed as fold changes relative to the normoxic control at each treatment. Mean SEM of almost three experiments is shown; **P < 0.01. (B) Activation of MEK/ERK pathway in SK-N-BE cells after 24 h of hypoxic treatment. The specific MEK1 inhibitor PD98059 abolishes ERK activation (representative Western blot). The histogram shows the data obtained from densitometric analysis of the p-ERK/ERK ratio (n = 4). Bars represent the mean SEM; *P < 0.05.
Ischemia/hypoxia and oxidative stress play a role in the pathogenesis of several neurodegenerative diseases, including FTD (Martin et al., 2001; Gerst et al., 1999). To evaluate a possible involvement for PGRN in neurodegeneration, we evaluated the modulation of the PGRN gene following exposure to these two neurodegenerative stimuli. Here, we report that levels of both PGRN gene and protein expression were significantly elevated following chronic hypoxia. In contrast, neither gene expression nor the protein, were altered following hydroperoxide-induced oxidative stress. Thus, each of the two stress conditions, associated with neurodegeneration, has distinct effects on gene expression. Interestingly, Yin et al. described that PGRN-deficient hippocampal slices were hypersusceptible to deprivation of oxygen (Yin et al., 2010). Moreover, a study on a marine gastropod reports accumulation of l-grn transcripts as part of the response to anoxia (Larade, 2008). Guerra and colleagues also describe an upregulation of PGRN transcripts in fibroblasts under condition of hypoxia (Guerra et al., 2007). These findings are in agreement with the present data, wherein we demonstrate a modulation not only of PGRN mRNA level, but also of its protein expression. Moreover, PGRN silencing was associated with changes in cell morphology and a fewer adherent cells, suggesting a possible protective role for this gene under hypoxic conditions. This neuroprotective action is also confirmed by a study on serum-deprivation in motor neurons describing that progranulin is cytoprotective over prolonged
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periods when over-expressed in a neuronal cell line (Ryan et al., 2009). Oxygen-deprived stress induces a number of spatially and temporally regulated intracellular responses, ranging from reduced channel activity to altered gene expression. In fact, depending on the duration and severity of oxygen deprivation, cellular oxygen-sensor responses activate a variety of short- and long-term energy-saving and cellular protection mechanisms. Hypoxic adaptation encompasses an immediate depolarization block by changing potassium, sodium and chloride ion fluxes across the cellular membrane. Moreover, the physiologic response to hypoxia modulates gene expression and protein synthesis, with a HIF-mediated up-regulation of enzymes or growth factors inducing angiogenesis, anaerobic glycolysis, cell survival or neural stem cell growth (Van Elzen et al., 2008; Acker and Acker, 2004). Many potential regulatory elements associated with the functional activity of promoters of other eukaryotic genes are scattered throughout the 50 flanking region of the human PRGN. However, among these, the consensus sequences for the family of hypoxia-inducible factors are not included. Notably, 25 sequences show a 7 out of 8 bp match with the AP-1 consensus sequence TGA(C/G)TCAG (Bhandari et al., 1996). Members of the AP-1 transcription factor family are well-known targets of the MEK signaling cascade. Moreover, the MAPKs respond to a variety of environmental stresses (Gaitanaki et al., 2004) including hypoxia (Haddad, 2004) and have a primary role in mediating stressinduced gene expression. Our data, for the first time, provide strong evidence that the highly selective inhibition of the MEK signal transduction pathway abolishes the up-regulation of PGRN in SK-N-BE human neuron-like cell culture, as stimulated by hypoxia treatment. These findings demonstrate a direct involvement of MAPKs in the induction of mRNA transcription. Moreover, the inhibition of MEK activity does not alter PGRN mRNA expression under normoxic conditions, suggesting that the PGRN gene transcription is specifically triggered by the hypoxic stimulus. As PGRN expression also promotes neuronal survival, and enhances neurite outgrowth in vitro, (Van Damme et al., 2008) it is reasonable that the observed cellular response to hypoxia could be adaptative; in this case our data agree with the reported PGRN blocking effect on tamoxifen-induced apoptosis in breast tumors (Tangkeangsirisin et al., 2004). Interestingly, PGRN activates the extracellular regulated kinases, and the phosphatidyl inositol-3 kinase signal cascades and also increases expression of cyclins D and B (Lu and Serrero, 2001). So, regulation of PGRN expression, exerted by the MAPKs under hypoxia, could be indicative of a survival loop which is switched on by cells to blunt pro-apoptotic environmental conditions, preserving neuronal viability. Interestingly, a similar hypothesis regarding the reciprocal functional interaction of PGRN and MEK pathway was formulated by Kamrava et al. in their study on lysophosphatidic acid and endothelin-induced proliferation of ovarian cancer cell lines suggesting a diffused proliferation and survival loop shared by different cell types and unrelated environmental stimuli (Kamrava et al., 2005). In conclusion, our results suggest that PGRN is highly susceptible to changes in local O2 tension and, as a specific hypoxic sensor, could have an adaptative/protective role against one of principal risk factors involved in AD and FTD pathogenesis as hypoxic stress.
Acknowledgments The authors would address particular thanks to Dr. Sonal Jhaveri for reviewing the manuscript. This study was supported by a grant from the Italian Ministry of Health: ‘‘Genetic determinants
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and modulator factors in neurodegenerative diseases: clinical and experimental model’’ to A.C.
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