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CXCL1 regulation of oligodendrocyte progenitor cell migration is independent of calcium signaling
Experimental Neurology 236 (2012) 259–267
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
CXCL1 regulation of oligodendrocyte progenitor cell migration is independent of calcium signaling Parvez Vora a, Prakash Pillai a, Joumana Mustapha b, Cory Kowal b, c, Seth Shaffer b, c, Ratna Bose d, Mike Namaka a, Emma E. Frost a,⁎ a
Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada University of Winnipeg, Winnipeg, Manitoba, Canada d Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada b c
a r t i c l e
i n f o
Article history: Received 17 September 2011 Revised 4 April 2012 Accepted 15 April 2012 Available online 24 April 2012 Keywords: CXCL Chemokine PDGF Oligodendrocyte Calcium
a b s t r a c t Cell migration is an indispensable aspect of tissue patterning during embryonic development. Oligodendrocytes, the myelinating cells of the central nervous system, migrate significantly during development of the brain. Several growth factors have been identified as being critical regulators of oligodendrocyte progenitor migration, including platelet derived growth factor-A (PDGFA), and fibroblast growth factor-2 (FGF2). Further, the chemokine CXCL1 has been shown to play a critical role in regulating the dispersal of oligodendrocyte progenitors during development, although the mechanisms underlying this regulation are unknown. Previous studies have also shown that calcium flux is required for oligodendrocyte progenitor migration. CXCL1 induces calcium flux in cells; therefore, we hypothesized that CXCL1 inhibition of oligodendrocyte progenitor migration is regulated via changes in intracellular calcium flux. The current study shows that CXCL1 inhibition of oligodendrocyte progenitor migration is independent of calcium signaling. Further, we show that CXCL1 inhibition of oligodendrocyte progenitor migration is specific to PDGFA induced migration. Finally, we show that CXCL1 inhibition of oligodendrocyte progenitor migration is independent of activation of the cell cycle. Our results provide intriguing results relevant to specific aspects of patterning of white matter tracts in the central nervous system, and may further the understanding of tissue remodeling seen during disease-related processes. © 2012 Published by Elsevier Inc.
Introduction Tissue patterning is a fundamental process required for the development and maintenance of multicellular organisms. Cell migration is a critical part of tissue patterning necessary for normal embryogenesis. Precise regulation by a complex series of biochemical signals drives the directed movement of cells. The entire process of migration is dependent on a series of intracellular signaling cascades that play a crucial role in regulating the timing, duration and localization of the independent events required for controlled cell movement. Calcium (Ca 2+) is a ubiquitous second messenger involved in numerous cellular functions including gene expression, proliferation, differentiation, and apoptosis (Berridge, 2004). Calcium mobilization is critical for directional sensing, cytoskeleton redistribution, traction force generation, and relocation of focal adhesions in migrating cells
Abbreviations: PDGFA, platelet-derived growth factor A; FGF2, fibroblast growth factor-2; CNS, central nervous system; [Ca2+]i, intracellular calcium. ⁎ Corresponding author at: Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0T5. Fax: + 1 204 474 6717. E-mail address: [email protected] (E.E. Frost). 0014-4886/$ – see front matter © 2012 Published by Elsevier Inc. doi:10.1016/j.expneurol.2012.04.012
(Wei, et al., 2009). Precisely controlled cell migration is essential for normal myelin formation in the central nervous system (CNS). Oligodendrocytes are the myelin producing cells of the CNS myelin. There are five basic phases of the oligodendrocyte lineage: generation, migration, proliferation, differentiation and myelination. Four of these five phases are dependent on the successful migration of the precursor cells away from their site of generation, to their site of proliferation and differentiation prior to successful myelination. Understanding the regulatory mechanisms of oligodendrocyte progenitor migration is crucial to being able to dissect out the subsequent processes that culminate in myelination. Oligodendrocyte progenitors originate as pre-progenitor cells in the telencephalon of the developing brain (Rakic and Zecevic, 2003). From the embryonic telencephalon region, the pre-progenitor cells migrate across the sub-pallial layer to the germinal matrix of the ganglionic eminences and the sub-ventricular zone (SVZ), where they proliferate and differentiate into progenitors (LeVine and Goldman, 1988). The oligodendrocyte progenitors then undergo further migration to populate the developing white matter tracts of the brain (Kakita and Goldman, 1999). Several growth factors regulate oligodendrocyte progenitor behavior, including platelet derived growth factor A (PDGFA) (Vora, et al., 2011), and fibroblast growth factor 2 (FGF2) (Baron, et al.,
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2000). PDGFA has been shown to be critical for the formation of normal myelin during development (Soriano, 1997), whereas FGF2 is functionally redundant during CNS development (Murtie et al., 2005a). However, both regulate oligodendrocyte progenitor migration, proliferation and differentiation (Frost, et al., 2003, 2009; McKinnon, et al., 1993; Milner, et al., 1997; Murtie et al., 2005a, 2005b). PDGFA and FGF2 interact with receptor tyrosine kinases, and regulate migration via activation of the extracellular regulated kinase (ERK) pathway (Vora, et al., 2011). Precise positioning of cells in the developing white matter tracts appears to be regulated by the localized expression of signaling cues. Specifically, oligodendrocyte progenitors stop migrating and proliferate in response to a localized concentration of the cytokine CXCL1 (a homologue of interleukin-8, formerly known as Groα) (Robinson, et al., 1998; Tsai, et al., 2002; Tsai and Miller, 2002). CXCL1, at a precise concentration of 0.5 ng/ml, inhibits oligodendrocyte progenitor migration (Tsai, et al., 2002) and induces oligodendrocyte progenitor proliferation, regardless of PDGFA concentration (Robinson, et al., 1998). The exact mechanism of this has yet to be identified. Activation of the CXCL1 receptor, CXCR2, a G-protein coupled receptor, induces intracellular calcium ([Ca2+]i) flux. Calcium mobilization plays a crucial role in the regulation of oligodendrocyte progenitor migration (Simpson and Armstrong, 1999). Thus, we hypothesized that CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration is regulated by changes in intracellular calcium. To examine the mechanisms by which CXCL1 modulates the migratory behaviour of oligodendrocyte progenitors, we assessed [Ca 2+]i flux in response to growth factors with and without CXCL1 treatment. In addition, we assessed the effect of CXCL1 on FGF2 induced oligodendrocyte progenitor migration. Further, we sequestered [Ca 2+]i in the presence of growth factors and CXCL1, to assess the role of [Ca 2+]i in the inhibition process. Interestingly, we show that blocking [Ca 2+]i did not affect the CXCL1 inhibition of growth factor induced oligodendrocyte progenitor migration. Additionally, CXCL1 did not affect [Ca2+]i levels in oligodendrocyte progenitors, in presence or absence of growth factor treatment. We also showed that inhibition of oligodendrocyte progenitor migration by the chemokine CXCL1 was specific to PDGFA induced migration. Finally, we showed that CXCL1 inhibition of oligodendrocyte progenitor migration was independent of cell cycle activation. Materials and methods All materials were from Sigma (St. Louis, MO, USA) unless otherwise stated. Platelet derived growth factor-AA (PDGFA) and fibroblast growth factor 2 (FGF2) were from R&D Systems (Minneapolis, MN, USA). The pharmacological inhibitor 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester (MAPTAM) was dissolved in DMSO at 50 mM stock concentration. CXCL1 was from R&D Systems. Fura-2 AM was from Invitrogen (Life Technologies. Grand Island, NY, USA). The α-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonist, (S)α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA) was from Enzo Life Sciences (Brockville, ON, Canada). Sato defined medium was prepared as previously described (Frost, et al., 2009). Bromo-deoxy-uridine antibody was from Boehringer Mannheim (Indianapolis, IN, USA). Isolation of oligodendrocyte progenitor cells Neonatal oligodendrocyte progenitor cells were prepared from postnatal day 2 rat brains, as previously described (Armstrong, 1998). Approximately, 7–10 days after plating, the oligodendrocyte progenitor cells and microglia were dislodged by shaking the flasks, followed by plating on non-coated tissue culture plastic for 25–40 minutes to allow the differential adhesion of microglial cells. The cell
suspension was collected, concentrated and resuspended in Sato defined medium with high insulin (Bottenstein and Sato, 1979).
Migration assay Cell migration was assayed using the agarose drop assay as previously described (Frost et al., 2000, 2009). Cells were exposed to inhibitory reagents for 30–45 min prior to addition of growth factor. Migration away from the edge of the drop was measured on four sides of the drop every 24 h for three days, using a calibrated eyepiece graticule in which the width of one square was equivalent to 100 μm.
Proliferation assay Bromo-deoxy-uridine (BrdU) incorporation was used to assess cell proliferation as previously described (Frost, et al., 2003). Cell proliferation was assessed as the percentage of 4′,6-Diamidino-2phenylindole Dihydrochloride:Hydrate (DAPI) positive oligodendrocyte progenitors that were also BrdU positive. At least 100 cells per coverslip were counted, with 5 coverslips per experiment with 3 experiments per test group.
Calcium signaling Fura-2 is a fluorescent dye, which binds to free [Ca 2+]i, and is commonly used to assess changes in [Ca 2+]i concentrations (Tucker, et al., 1989). Fura-2-acetoxymethyl ester (Fura-2 AM) is a strongly hydrophobic dye that very easily diffuses through the lipid bilayer of the plasma membrane of cell (Gunter, et al., 1988). Once inside the cell, the AM derivatives are rapidly hydrolyzed by nonspecific cytoplasmic esterases to become hydrophilic free acids, which are nonpermeable and thus are trapped in the cell (Gunter, et al., 1988). Fura2 is excited at 340 nm and 380 nm wavelength. When bound to Ca 2+, Fura-2 is excited at 340 nm and unbound Fura-2 is excited at 380 nm, it emits at 510 nm whether bound or unbound. The ratio of emission at those wavelengths is directly correlated to the concentration of intracellular calcium, using the formula shown in Table 1. The use of a ratio automatically negates uncontrollable variables, such as dye concentration and cell thickness. Purified oligodendrocyte progenitors were incubated with 2 μM Fura-2 AM in Ca 2+ free Locke's buffer (154 mM NaCl; 3.6 mM NaHCO3; 5.6 mM KCl; 1.0 mM MgCl2; 5.0 mM glucose; 5 mM Hepes; pH 7.0) with 0.1% BSA, for 1 hour. After washing, the cells were suspended in Locke's + 0.1% bovine serum albumin, and then placed into a Jasco CAF-110 intracellular ion analyzer for Ca 2+ flux analysis. Adenosine-tri-phosphate (ATP) was used as a positive control, to show that the oligodendrocyte progenitors were able to elicit a calcium response (Agresti et al., 2005). Previous studies have shown that ATP causes the release of intracellular stores of Ca 2+ in oligodendrocyte progenitors (Agresti et al., 2005).
Table 1 Calculation of intracellular calcium concentration. h
Ca2þ
i i
¼ Kd
RRmin Rmax R
λ380nm atminimum λ380nm atmaximum
Where R is the ratio of 510 nm emission intensity, exciting at 340 nm, to 510 nm emission intensity, exciting at 380 nm. Rmin is the ratio at zero free Ca2+ (i.e. prior to the addition of CaCl2 to the Locke's buffer); Rmax is the ratio at saturating Ca2+ (i.e. after the addition of digitonin to perforate the cell membranes allowing extracellular Ca2+ to flood into the cell). λ380 nm at minimum is the fluorescence intensity, exciting at saturating free Ca2+; λ380 nm at maximum is the fluorescence intensity, exciting at zero free Ca2+. Kd is an empirical value determined from Fura-2 containing Ca2+ standards run on the analyzer prior to the use of the Fura-2 for each experiment (Grynkiewicz, et al., 1985).
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Statistics Statistical analysis was performed using GraphPad Prism version 4.03 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. One-way ANOVA with Dunnett's posttest was used to test differences between migration curves. Student t-test was used to test the difference between means of cell count experiments. Results Calcium is required for PDGFA and FGF2 induced oligodendrocyte progenitor migration Previous studies have shown that chelation of extracellular Ca 2+ does not affect PDGFA induced oligodendrocyte progenitor migration, whereas chelation of intracellular Ca 2+ does inhibit PDGFA induced oligodendrocyte progenitor migration (Simpson and Armstrong, 1999). We used the calcium chelator MAPTAM to confirm the requirement for Ca 2+ signaling in PDGFA and FGF2 induced oligodendrocyte progenitor migration in the agarose drop assay (Pende, et al., 1997). MAPTAM, is a cell permeant analog of EGTA which chelates [Ca 2+]i. Chelation is the formation of soluble, complex molecules that inactivates the ions so that they cannot react with other molecules. Oligodendrocyte progenitors were pre-incubated with 45 μM MAPTAM for 30 min prior to the addition of growth factor. Cell migration requires process extension and adhesion to the substratum (Horwitz and Webb, 2003). Oligodendrocyte progenitors are unable to migrate in the absence of processes (Frost et al., 1996, 1999). In order to ensure that [Ca 2+]i chelation did not inhibit migration by preventing process formation we assessed cell morphology. Morphological analysis of the oligodendrocyte progenitors treated with MAPTAM ± growth factor showed that chelation of [Ca 2+]i did not affect cell processes (Fig. 1A–D). Migration was measured 72 hours
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after addition of growth factor. In the presence of MAPTAM, both PDGFA and FGF2 induced migration was inhibited significantly (Fig. 1E). PDGFA induced oligodendrocyte progenitors to migrate 973.25 ± 42.61 μm (compared to 262.15 ± 31.79 μm in control wells), and in the presence of MAPTAM migration was significantly reduced by 45% to 536.62 ± 38.32 μm (p b 0.001). FGF2 induced oligodendrocyte progenitors to migrate 893.25 ± 32.14 μm, and in the presence of MAPTAM, oligodendrocyte progenitor migration was inhibited by 44% to 502.44± 36.324 μm (p b 0.001). However, the levels of migration seen in response to growth factor, in the presence of MAPTAM, were still significantly higher than in untreated cells. Oligodendrocyte progenitors migrated 536.6 ±38.32 μm in the presence of PDGFA + MAPTAM compared to control cells (262.1 ± 31.79 μm: p b 0.0001), and 502.4 ± 36.32 μm in the presence of FGF2 + MAPTAM (p b 0.0001). MAPTAM treatment alone had no effect on oligodendrocyte migration (277.7± 10.1 μm). Active inhibition of PDGFA induced oligodendrocyte progenitor migration by the chemokine CXCL1 is specific to CXCL1 concentration We have previously shown that the chemokine CXCL1 acts as a stop signal for oligodendrocyte progenitors migrating in response to PDGFA, using a microchemotaxis chamber migration assay (Tsai, et al., 2002). For this study, we switched to an agarose drop assay, which allowed us to manipulate the media during the assay, as well as to study migration over a longer period of time (Frost, et al., 2000). To further understand the inhibition of PDGFA induced oligodendrocyte progenitor migration by CXCL1 we assessed the effect of two concentrations of CXCL1 (0.5 and 5 ng/ml) on a dose response curve to PDGFA (0.5–10 ng/ml). Concurring with the previous findings, we showed that in presence of 0.5 ng/ml CXCL1, PDGFA induced oligodendrocyte progenitor migration was inhibited regardless of growth factor concentration. However, in presence of 5 ng/ml
Fig. 1. Effect of MAPTAM on oligodendrocyte progenitors.The morphology of oligodendrocyte progenitors exposed to PDGFA (panel A) or FGF2 (panel C) is unaffected by treatment with MAPTAM (Panels B and D respectively). Panel E — oligodendrocyte progenitors pretreated for 20 minutes with 45μm MAPTAM show significantly reduced migration in response to PDGFA and FGF2 compared to growth factor treatment alone. (n = 6, with 4–6 replicates) ***p b 0.001.
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CXCL1, there was no inhibition of PDGFA induced oligodendrocyte progenitor migration, regardless of growth factor concentration (Fig. 2A). To analyze the effect of CXCL1 on growth factor treated oligodendrocyte progenitors further, we used 10 ng/ml PDGFA, and added CXCL1 48 hours later. Cells were left for migrate for a further 24 hours. Oligodendrocyte progenitors exposed to CXCL1 were prevented from migrating by the addition of CXCL1, indicating that CXCL1 is an active inhibitor of migration, even after the activation of the migration machinery (Fig. 2B). After 24 h of PDGFA exposure, oligodendrocyte progenitors migrated 401.21 ± 55.30 μm compared to 263.43 ± 38.35 μm in control (untreated) wells. After 48 hours of PDGFA exposure, oligodendrocyte progenitors migrated 688.98 ± 62.25 μm compared to 344.07 ± 64.23 μm in control (untreated) wells. CXCL1 (0.5 ng/ml) was added to the wells at T48, and 24 h later (T72), oligodendrocyte progenitor migration in the presence of PDGFA stopped at 671.75 ± 89.16 μm, compared to 989.00 ± 61.65 μm in the presence of PDGFA alone. Inhibition of oligodendrocyte progenitor migration by the chemokine CXCL1 is specific to PDGFA induced migration PDGFA acts through the PDGF receptor-alpha (PDGFRα), which is a receptor tyrosine kinase (RTK). Another growth factor known to induce oligodendrocyte progenitor migration is FGF2. The members of the FGF receptor family (FGFR) are also RTKs, with similar intracellular signaling cascades activated by ligand binding. In order to assess the potential for CXCL1 as the primary stop signal for oligodendrocyte progenitor migration, we analysed the effects of CXCL1 on FGF induced oligodendrocyte progenitor migration. In contrast to PDGFA, the addition of CXCL1 did not affect FGF2 induced oligodendrocyte
progenitor migration. In the presence of 1 ng/ml FGF2, oligodendrocyte progenitors were induced to migrate 578.10 ± 95.85 μm compared to 512.89 ± 70.13 μm in the presence of CXCL1 (0.5 ng/ml) p = 0.395. In the presence of 10 ng/ml FGF2, oligodendrocyte progenitors were induced to migrate 849.21 ± 40.80 μm compared to 725.38 ± 144.23 μm in the presence of 0.5 ng/ml CXCL1 p = 0.150 (Fig. 3). PDGFA induces significant, dose-dependent calcium flux, while FGF2 does not induce calcium flux in oligodendrocytes In order to analyze the calcium flux in oligodendrocyte progenitors, we preincubated freshly isolated oligodendrocyte progenitors with Fura-2 for 2 h, and then assessed changes in intracellular calcium concentration in the presence and absence of PDGFA. We found that in the presence of both 1 ng/ml and 10 ng/ml, PDGFA evoked a significant increase in [Ca 2+]i of 230.35 nM compared to baseline of 183.2 nM; and 338.668 nM compared to a baseline of 208.6 nM (Fig. 4). This represented 82.83% of the peak ATP value and 98.82% of the peak ATP value respectively. Previously published studies on FGF receptor signaling, have indicated that the [Ca2+]i is altered in response to FGF ligand binding (Eswarakumar, et al., 2005; Tsuda, et al., 1985). However, several other studies have shown that the effect of receptor activation differs between cell types, with different intracellular signaling pathways being activated by ligand binding (Murakami, et al., 2008; Satoh, et al., 1993; Stork and Schmitt, 2002). Therefore, for completeness, we assessed the Ca2+ flux in response to FGF2 in oligodendrocyte progenitors. Interestingly, even at 10 ng/ml concentration FGF2 did not induce Ca2+ flux in the oligodendrocyte progenitors (Fig. 5). We assessed changes in [Ca2+]i in response to 0.1 ng/ml FGF2 in case a lower concentration threshold is required for Ca2+ flux. However, there was no apparent change in [Ca 2+]i in response to any concentration tested (Fig. 5). CXCL1 does not affect intracellular calcium levels in oligodendrocyte progenitor in the presence or absence of PDGFA CXCL1 acts through the type 2 CXC receptor. CXCR2 is a G-protein coupled receptor, which induces Ca 2+ flux in other cell types (Puma,
Fig. 2. Inhibition of PDGFA induced oligodendrocyte progenitor migration by CXCR2 activation. Panel A — CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration is concentration dependent. At 0.5 ng/ml, CXCL1 inhibits oligodendrocyte progenitor migration induced by all concentrations of PDGFA (open diamonds, dashed line), whereas at 5 ng/ml, CXCL1 has no effect on PDGFA induced oligodendrocyte progenitor migration (open circles, dotted line), at any concentration. (n = 3 with 4–6 replicates). Panel B — Oligodendrocyte progenitor migration induced by PDGFA (solid circle, solid line) is inhibited by CXCL1 added 24 hours after the initiation of migration (open square, dashed line). Untreated oligodendrocyte progenitors do not migrate in the absence of PDGFA (closed circles, dotted line) (n = 5, with 4–5 replicates per treatment).
Fig. 3. FGF2 induced oligodendrocyte progenitor migration is not inhibited by CXCL1.Oligodendrocyte progenitors migrate significant distances compared to control in response to 1 ng/ml and 10 ng/ml FGF2 (p = 0.0019 and p b 0.0001 respectively). However, the chemokine CXCL1 does not inhibit FGF2 induced migration (ns), in contrast to PDGFA induced migration. Oligodendrocyte progenitor migration was measured 72 hours after the addition of growth factor. (n = 3 with 4–6 replicates; ** = p = 0.0019 *** = p b 0.0001).
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Fig. 4. Calcium flux in response to PDGFA.Calcium flux in primary oligodendrocyte progenitors is induced by PDGFA in a dose dependent manner. Panel A — PDGFA is added to the cells at 1 ng/ml. A significant increase in intracellular calcium is seen. Panel B — PDGFA is added to the cells at 10 ng/ml concentrations. A significant increase in intracellular calcium is seen. ATP is added to the cells to show that they are responding as expected. Arrows indicate the addition of PDGFA and ATP. The values given for calcium concentration are nM. (n = 3).
et al., 2001; Shibata, et al., 2002). Moreover, other chemokines invoke rapid mobilization of Ca 2+ from intracellular store in neurons (Giovannelli, et al., 1998; Meucci, et al., 1998). However, in oligodendrocyte progenitors, contrary to expectations, we found that at 0.5 ng/ml, CXCL1 had no effect on Ca 2+ flux (Fig. 6A). Even at concentrations as high as 50 ng/ml there was no changes in [Ca 2+]i (Fig. 6B). In order to test the hypothesis that CXCL1 inhibits oligodendrocyte progenitor migration via a Ca 2+ dependent pathway, CXCL1 was added prior to the addition of PDGFA, and [Ca 2+]i changes were assessed. We found that CXCL1 had no effect on PDGFA induced oligodendrocyte progenitor Ca 2+ flux (Fig. 7). CXCL1 was added to the oligodendrocyte progenitor at 0.5 ng/ml and 30 seconds later 1 ng/ml PDGFA was added to the cells. PDGFA elicited a significant increase in [Ca 2+]i to 340.50 nM after pre-treatment with CXCL1, which represents 87.6% of the ATP response (388.9 nM), compared to 338.7 nM in the presence of 10 ng/ml PDGFA alone (Fig. 4).
Inhibition of cell cycle activation does not affect CXCL1 induced inhibition of oligodendrocyte progenitor migration To check whether CXCL1 induced inhibition of oligodendrocyte progenitor migration is via activation of the cell cycle, we used SAMPA. The cyclin E-cyclin-dependant kinase 2 (cdk2) complex is a major regulator of G1–S transition in OP cells (Ghiani and Gallo, 2001). Activation of the AMPA receptor by S-AMPA, inhibits cyclin
Fig. 5. Effect of FGF2 on oligodendrocyte progenitor calcium flux in vitro.Calcium flux in primary oligodendrocyte progenitors is unaffected by FGF2. ATP is added to the cells to show that they are responding as expected. Panel A — FGF2 at 0.1 ng/ml, Panel B — FGF2 added at 1 ng/ml and Panel C — FGF2 added at 10 ng/ml. Arrows indicate the addition of PDGFA and ATP. The values are given for calcium concentration are nM. (n = 3).
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E–cdk2 formation, leading to cell cycle arrest (Ghiani and Gallo, 2001), without affecting oligodendrocyte progenitor migration (Frost, et al., 2009; Gallo, et al., 1996). The current study shows that S-AMPA had no effect on the CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration (Fig. 8). Results were normalized and shown as a percentage of the migration elicited in response to 10 ng/ml PDGFA. Thus, PDGFA alone produced 100 ± 6.23% migration. PDGFA in combination with S-AMPA resulted in a slight reduction in migration to 94.57 ± 6.66%. In the presence of CXCL1, PDGFA treatment resulted in 44.7 ± 6.99% migration. In the presence of S-AMPA and CXCL1, PDGFA induced 40.91 ± 7.08% migration (Fig. 8). Oligodendrocyte progenitor proliferation and survival is not affected by AMPA treatment Bromo-deoxyuridine (BrdU) incorporation was used to assess the effects of AMPA on oligodendrocyte progenitor proliferation (Frost et al., 2003, 2009). The percentage of control (untreated) oligodendrocyte progenitor cells that incorporated BrdU was 26.58 ± 3.22%. Whereas in the presence of PDGFA, BrdU incorporation increased to 68.54 ± 4.69%. Pretreatment of the cells with AMPA reduced BrdU incorporation to 34.15 ± 3.21%. BrdU incorporation was unaffected by treatment with AMPA alone (29.9 ± 4.11%). The effects of AMPA treatment on cell survival were assessed by propidium iodide incorporation over a 24-hour period (Frost, et al., 2003). In the presence of PDGFA 78.5 ± 2.0% of cells survived, compared to 52.3 ± 2.3% of untreated cells. In the presence of AMPA, PDGFA induced oligodendrocyte progenitor survival was 79.9 ± 2.8%. In the presence of AMPA treatment alone, 54.6 ± 3.6% of the cells survived. Thus, AMPA inhibited the cell cycle, without affecting survival in oligodendrocyte progenitors. Discussion Normal myelination is critical for the successful functioning of the nervous system. Aberrant myelination is characteristic of several disorders of the brain, including cerebral palsy (Dammann and Leviton,
Fig. 6. Calcium signaling in response to CXCL1.Calcium flux in primary oligodendrocyte progenitors is unaffected by CXCL1. Panel A — CXCL1 is added to the cells at 0.5 ng/ml concentration. Panel B — CXCL1 is added to the cells at 50 ng/ml concentration. ATP is added to the cells to show that they are responding as expected. Arrows indicate the addition of CXCL1 and ATP. The values given for calcium concentration are nM. (n = 3).
Fig. 7. Calcium flux in response to PDGFA after CXCL1 treatment.PDGFA induced calcium flux in primary oligodendrocyte progenitors is unaffected by the addition of CXCL1 at 0.5 ng/ml prior to the addition of the PDGFA. ATP is added to the cells to show that they are responding as expected. Arrows indicated the addition of CXCL1, PDGFA, and ATP. The values given for calcium concentration are nM. (n = 3).
Fig. 8. Cell cycle inhibition does not block CXCL1 inhibition of oligodendrocyte progenitor migration.Oligodendrocyte progenitors are treated with AMPA and or CXCL1 and PDGFA to assess the effect of inhibiting the cell cycle on CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration. PDGFA induced migration was significantly increased compared to control (p b 0.0001), in the presence of AMPA PDGFA induced migration was significantly increased over control (p b 0.0001). CXCL1 inhibited PDGFA induced migration back to control levels. In the presence of AMPA, CXCL1 inhibition of PDGFA induced migration was unaffected. (n = 3 with 4–6 replicates; *** = p b 0.0001).
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1998; Juliet, et al., 2009), Schizophrenia (Carter, 2006; Peirce, et al., 2006), and leukodystrophies (Costello, et al., 2009; Schiffmann and van der Knaap, 2004). Further, the demyelinating disease, multiple sclerosis (MS) is also characterized by damage to the CNS myelin. Understanding the mechanisms that regulate the formation of myelin in the developing CNS may provide insights into potential therapeutic strategies for diseases associated with myelin dysfunction. Oligodendrocyte progenitors migrate extensively during development of the CNS (Jakovcevski and Zecevic, 2005; Marshall and Goldman, 2002; Rakic and Zecevic, 2003). Developmental migration is thought to be regulated by chemotactic gradients of mitogens, such as FGF2 (Hardy, et al., 2011; Sato, et al., 2011), and PDGFA (Yang, et al., 2008). However, there is no evidence of such a phenomenon in the developing CNS. Cell migration is therefore likely to be precisely regulated by the presence of localized morphogens, which alter the cells’ response to its local environment based on ligand concentration, and/or receptor occupancy (Freeman and Gurdon, 2002). However, the exact molecular mechanisms underlying the regulation of oligodendrocyte progenitor migration have yet to be fully elucidated. The chemokine CXCL1 was shown previously to regulate the spatial distribution of oligodendrocyte progenitors in the developing CNS (Tsai, et al., 2002). CXCL1 cooperates with PDGFRα to regulate the dispersal and proliferation of oligodendrocyte progenitors (Robinson, et al., 1998; Tsai, et al., 2002). Despite numerous studies on the expression patterns of CXCR2 in development and disease (Omari et al., 2005, 2006; Padovani-Claudio, et al., 2006), the mechanism of action of the chemokine remains unknown. In order to test our hypothesis, that CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration is regulated by changes in intracellular calcium, we studied the effect of CXCL1 on oligodendrocyte progenitor Ca 2+ mobilization in the presence and absence of PDGFA. In addition, to assess further the role of CXCL1 on oligodendrocyte progenitor behaviour, we assessed its effect on FGF2 induced oligodendrocyte progenitor migration. Numerous studies have shown that PDGFA and FGF2 both regulate oligodendrocyte progenitor migration (Armstrong, et al., 1990; Milner, et al., 1997; Vora, et al., 2011). Previously, we have shown that they regulate migration via the ERK signaling pathway (Frost, et al., 2009; Vora, et al., 2011). However, there are significant differences between the response of oligodendrocyte progenitors to the two receptor tyrosine-kinases (Vora, et al., 2011). Past research has shown that CXCL1 binding to its receptor influences PDGFRα signaling to regulate the dispersal and proliferation of oligodendrocyte progenitors (Robinson, et al., 1998; Tsai, et al., 2002). To better understand the role of CXCL1 in regulating oligodendrocyte progenitor dispersal, we assessed the effect of CXCL1 on PDGFA and FGF2 induced oligodendrocyte progenitor migration. The CXCL1 receptor, CXCR2, is a pertussis toxin sensitive G-protein coupled receptor (Richardson, et al., 1998). Activation of CXCR2 activates several intracellular signaling pathways, including the mobilization of [Ca 2+]i (Shibata, et al., 2002) and the Rho kinase, and phosphatidyl inositol-3-kinase (PI3K) signaling cascades (L'Heureux, et al., 1995; Richardson, et al., 1998; Schraufstatter, et al., 2001). Numerous studies have shown that Ca 2+ mobilization is required for cell migration (Wei, et al., 2009). Calcium plays a critical role as secondary messenger in the process the myosin-actin complex activation, which is required for the cytoskeletal reorganization that accompanies migration (Adelstein and Hathaway, 1979). Further, Ca 2+ mobilization is associated with the G1 phase of the cell cycle, although the complex interactions between [Ca 2+]i dependent signaling pathways and cell proliferation are still not fully elucidated (Capiod, 2011). Previous studies have shown a role for [Ca 2+]i in the regulation of oligodendrocyte progenitor migration (Agresti et al., 2005; Simpson and Armstrong, 1999). Thus, we hypothesised that CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration is regulated
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by changes in intracellular calcium, which induces oligodendrocyte progenitor proliferation. The present study clearly shows that PDGFA induces an immediate increase in [Ca 2+]i in a dose-dependent manner. Further, in our study, there is no increase in [Ca 2+]i in response to FGF2 treatment. This concurs with a previous study that showed FGF2 treatment of oligodendrocyte progenitors resulted in a low to non-existent Ca 2+ flux (Pende, et al., 1997). Our findings fit with previous studies, which show that FGF2 regulates oligodendrocyte progenitor migration via different signaling pathways to PDGFRα (Vora, et al., 2011), and that FGF2 is not critical for the development of myelin (Armstrong, et al., 2002). The factors that evoke changes in the [Ca 2+]i levels at different stages of oligodendrocyte development are also known to regulate their development and pathology. The key hypothesis of this study is that one such factor CXCL1, which is previously shown to inhibit oligodendrocyte progenitor migration (Tsai, et al., 2002), uses [Ca 2+]i stores to bring about its effect. Interestingly, we did not see any changes in oligodendrocyte progenitor [Ca 2+]i levels even at high concentrations of CXCL1 treatment. Furthermore, we hypothesized that Ca 2+ stores are released upon inhibition of growth factor induced oligodendrocyte progenitor migration by CXCL1. However, the current study shows that oligodendrocyte progenitor migration is not completely dependent on Ca 2+ signaling. Moreover, blocking the [Ca 2+]i with the calcium chelating agent, MAPTAM failed to inhibit CXCL1 action on oligodendrocyte progenitor migration. The role of Ca 2+ in oligodendrocyte progenitor migration has been controversial (Clyman, et al., 1994; Simpson and Armstrong, 1999). Chelation of [Ca 2+]i significantly reduced the extent of migration of oligodendrocyte progenitors in response to both PDGFA and FGF2. Interestingly, the oligodendrocyte progenitors treated with MAPTAM still migrated significantly further in the presence of growth factor, than those cells left untreated. This indicates that, although Ca 2+ plays a role in oligodendrocyte progenitor migration, it is not the only signaling pathway involved. Our previous studies have shown that ERK is critical for oligodendrocyte progenitor migration (Vora and Frost, 2009; Vora, et al., 2011), and that cross-talk between signaling cascades also plays an important role in maintaining oligodendrocyte progenitor migration in response to growth factors (Frost, et al., 2009). Thus, further studies are required to assess the nature of the Ca 2+ independent pathways that are regulating oligodendrocyte progenitor migration. A possible explanation for the CXCL1 inhibition of migration is induction of the cell cycle. In order to proliferate, cells must stop migrating, to allow the restructuring of the cytoskeleton necessary for spindle formation and cytokinesis. Calcium plays a critical role in the reorganisation of the cytoskeleton during cell division (Capiod, 2011). Thus, we hypothesized that CXCL1 induction of the cell cycle results in inhibition of migration. To test the hypothesis, a known inhibitor of oligodendrocyte progenitor proliferation, the glutamate receptor agonist AMPA was used (Gallo, et al., 1996; Ghiani and Gallo, 2001). We show that AMPA blocks proliferation of oligodendrocyte progenitors without affecting migration or cell survival. In the presence of CXCL1, the expected outcome of inhibition of cell cycle activation would be no change in oligodendrocyte progenitor migration. However, our results show that in the presence of AMPA, CXCL1 still blocks oligodendrocyte progenitor migration. These results indicate that inhibition of the cell cycle does not appear to prevent the inhibitory effects of CXCL1. Thus, we show for the first time that CXCL1 actively blocks oligodendrocyte progenitor migration via a cell cycle independent pathway. The exact mechanisms by which CXCL1 inhibit migration require further investigation. Other pathways potentially involved include the activation of RhoK, which inhibits myosin phosphatase (Hall, 2005). Myosin phosphatase inactivates myosin light chain kinase, which is critical for the reorganisation of actin filaments in
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oligodendrocyte (Thomas, et al., 2002). Further, activation of CXCR2 induces the JAK/STAT and PLC/PI3K pathways, which are both involved in the induction of PDGFA induced oligodendrocyte progenitor proliferation (Dell'Albani, et al., 1998; Ebner, et al., 2000). Conclusions The mechanism by which CXCL1 inhibits oligodendrocyte progenitor migration remains an enigma. The inhibitory effect of CXCL1 on oligodendrocyte progenitors is directly regulated via CXCR2 (Tsai, et al., 2002). The current study shows that this essential inhibition of oligodendrocyte progenitor migration is not arbitrary, but may be specific to PDGFA activity in the developing brain. Further, this study shows that oligodendrocyte progenitor migration requires Ca2+ flux, but is not dependent on Ca2+ flux. Understanding the downstream signaling pathways involved in CXCL1 inhibition of oligodendrocyte progenitor migration will further our understanding of molecular and cellular mechanisms involved in developmental myelination, and will provide information relevant to remyelination during adult tissue repair and other disease related processes. Acknowledgments We would like to acknowledge our funding agencies: The Manitoba Institute for Child Health (EEF), the Manitoba Health Research Council (EEF), The Manitoba Medical Services Foundation (EEF & MPN), Pfizer Global UK (MPN & EEF), Biogen Idec (MPN & EEF). PV was recipient of an MHRC studentship. CK and SS were recipients of Manitoba Institute for Child Health Summer Studentships. References Adelstein, R.S., Hathaway, D.R., 1979. Role of calcium and cyclic adenosine 3′:5′ monophosphate in regulating smooth muscle contraction. Mechanisms of excitation– contraction coupling in smooth muscle. Am. J. Cardiol. 44, 783–787. Agresti, C., Meomartini, M.E., Amadio, S., Ambrosini, E., Volonte, C., Aloisi, F., Visentin, S., 2005. ATP regulates oligodendrocyte progenitor migration, proliferation, and differentiation: involvement of metabotropic P2 receptors. Brain Res. Brain Res. Rev. 48, 157–165. Armstrong, R.C., 1998. Isolation and characterization of immature oligodendrocyte lineage cells. Methods 16, 282–292. Armstrong, R.C., Harvath, L., Dubois-Dalcq, M.E., 1990. Type 1 astrocytes and oligodendrocyte-type 2 astrocyte glial progenitors migrate toward distinct molecules. J. Neurosci. Res. 27, 400–407. Armstrong, R.C., Le, T.Q., Frost, E.E., Borke, R.C., Vana, A.C., 2002. Absence of fibroblast growth factor 2 promotes oligodendroglial repopulation of demyelinated white matter. J. Neurosci. 22, 8574–8585. Baron, W., Metz, B., Bansal, R., Hoekstra, D., de Vries, H., 2000. PDGF and FGF-2 signaling in oligodendrocyte progenitor cells: regulation of proliferation and differentiation by multiple intracellular signaling pathways. Mol. Cell. Neurosci. 15, 314–329. Berridge, M.J., 2004. Calcium signal transduction and cellular control mechanisms. Biochim. Biophys. Acta 1742, 3–7. Bottenstein, J.E., Sato, G.H., 1979. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. Natl. Acad. Sci. U. S. A. 76, 514–517. Capiod, T., 2011. Cell proliferation, calcium influx and calcium channels. Biochimie 93, 2075–2079. Carter, C.J., 2006. Schizophrenia susceptibility genes converge on interlinked pathways related to glutamatergic transmission and long-term potentiation, oxidative stress and oligodendrocyte viability. Schizophr. Res. 86, 1–14. Clyman, R.I., Peters, K.G., Chen, Y.Q., Escobedo, J., Williams, L.T., Ives, H.E., Wilson, E., 1994. Phospholipase C gamma activation, phosphotidylinositol hydrolysis, and calcium mobilization are not required for FGF receptor-mediated chemotaxis. Cell Adhes. Commun. 1, 333–342. Costello, D.J., Eichler, A.F., Eichler, F.S., 2009. Leukodystrophies: classification, diagnosis, and treatment. Neurologist 15, 319–328. Dammann, O., Leviton, A., 1998. Infection remote from the brain, neonatal white matter damage, and cerebral palsy in the preterm infant. Semin. Pediatr. Neurol. 5, 190–201. Dell'Albani, P., Kahn, M.A., Cole, R., Condorelli, D.F., Giuffrida-Stella, A.M., de Vellis, J., 1998. Oligodendroglial survival factors, PDGF-AA and CNTF, activate similar JAK/ STAT signaling pathways. J. Neurosci. Res. 54, 191–205. Ebner, S., Dunbar, M., McKinnon, R.D., 2000. Distinct roles for PI3K in proliferation and survival of oligodendrocyte progenitor cells. J. Neurosci. Res. 62, 336–345. Eswarakumar, V.P., Lax, I., Schlessinger, J., 2005. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 16, 139–149.
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CXCL1 regulation of oligodendrocyte progenitor cell migration is independent of calcium signaling Parvez Vora a, Prakash Pillai a, Joumana Mustapha b, Cory Kowal b, c, Seth Shaffer b, c, Ratna Bose d, Mike Namaka a, Emma E. Frost a,⁎ a
Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada University of Winnipeg, Winnipeg, Manitoba, Canada d Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada b c
a r t i c l e
i n f o
Article history: Received 17 September 2011 Revised 4 April 2012 Accepted 15 April 2012 Available online 24 April 2012 Keywords: CXCL Chemokine PDGF Oligodendrocyte Calcium
a b s t r a c t Cell migration is an indispensable aspect of tissue patterning during embryonic development. Oligodendrocytes, the myelinating cells of the central nervous system, migrate significantly during development of the brain. Several growth factors have been identified as being critical regulators of oligodendrocyte progenitor migration, including platelet derived growth factor-A (PDGFA), and fibroblast growth factor-2 (FGF2). Further, the chemokine CXCL1 has been shown to play a critical role in regulating the dispersal of oligodendrocyte progenitors during development, although the mechanisms underlying this regulation are unknown. Previous studies have also shown that calcium flux is required for oligodendrocyte progenitor migration. CXCL1 induces calcium flux in cells; therefore, we hypothesized that CXCL1 inhibition of oligodendrocyte progenitor migration is regulated via changes in intracellular calcium flux. The current study shows that CXCL1 inhibition of oligodendrocyte progenitor migration is independent of calcium signaling. Further, we show that CXCL1 inhibition of oligodendrocyte progenitor migration is specific to PDGFA induced migration. Finally, we show that CXCL1 inhibition of oligodendrocyte progenitor migration is independent of activation of the cell cycle. Our results provide intriguing results relevant to specific aspects of patterning of white matter tracts in the central nervous system, and may further the understanding of tissue remodeling seen during disease-related processes. © 2012 Published by Elsevier Inc.
Introduction Tissue patterning is a fundamental process required for the development and maintenance of multicellular organisms. Cell migration is a critical part of tissue patterning necessary for normal embryogenesis. Precise regulation by a complex series of biochemical signals drives the directed movement of cells. The entire process of migration is dependent on a series of intracellular signaling cascades that play a crucial role in regulating the timing, duration and localization of the independent events required for controlled cell movement. Calcium (Ca 2+) is a ubiquitous second messenger involved in numerous cellular functions including gene expression, proliferation, differentiation, and apoptosis (Berridge, 2004). Calcium mobilization is critical for directional sensing, cytoskeleton redistribution, traction force generation, and relocation of focal adhesions in migrating cells
Abbreviations: PDGFA, platelet-derived growth factor A; FGF2, fibroblast growth factor-2; CNS, central nervous system; [Ca2+]i, intracellular calcium. ⁎ Corresponding author at: Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0T5. Fax: + 1 204 474 6717. E-mail address: [email protected] (E.E. Frost). 0014-4886/$ – see front matter © 2012 Published by Elsevier Inc. doi:10.1016/j.expneurol.2012.04.012
(Wei, et al., 2009). Precisely controlled cell migration is essential for normal myelin formation in the central nervous system (CNS). Oligodendrocytes are the myelin producing cells of the CNS myelin. There are five basic phases of the oligodendrocyte lineage: generation, migration, proliferation, differentiation and myelination. Four of these five phases are dependent on the successful migration of the precursor cells away from their site of generation, to their site of proliferation and differentiation prior to successful myelination. Understanding the regulatory mechanisms of oligodendrocyte progenitor migration is crucial to being able to dissect out the subsequent processes that culminate in myelination. Oligodendrocyte progenitors originate as pre-progenitor cells in the telencephalon of the developing brain (Rakic and Zecevic, 2003). From the embryonic telencephalon region, the pre-progenitor cells migrate across the sub-pallial layer to the germinal matrix of the ganglionic eminences and the sub-ventricular zone (SVZ), where they proliferate and differentiate into progenitors (LeVine and Goldman, 1988). The oligodendrocyte progenitors then undergo further migration to populate the developing white matter tracts of the brain (Kakita and Goldman, 1999). Several growth factors regulate oligodendrocyte progenitor behavior, including platelet derived growth factor A (PDGFA) (Vora, et al., 2011), and fibroblast growth factor 2 (FGF2) (Baron, et al.,
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2000). PDGFA has been shown to be critical for the formation of normal myelin during development (Soriano, 1997), whereas FGF2 is functionally redundant during CNS development (Murtie et al., 2005a). However, both regulate oligodendrocyte progenitor migration, proliferation and differentiation (Frost, et al., 2003, 2009; McKinnon, et al., 1993; Milner, et al., 1997; Murtie et al., 2005a, 2005b). PDGFA and FGF2 interact with receptor tyrosine kinases, and regulate migration via activation of the extracellular regulated kinase (ERK) pathway (Vora, et al., 2011). Precise positioning of cells in the developing white matter tracts appears to be regulated by the localized expression of signaling cues. Specifically, oligodendrocyte progenitors stop migrating and proliferate in response to a localized concentration of the cytokine CXCL1 (a homologue of interleukin-8, formerly known as Groα) (Robinson, et al., 1998; Tsai, et al., 2002; Tsai and Miller, 2002). CXCL1, at a precise concentration of 0.5 ng/ml, inhibits oligodendrocyte progenitor migration (Tsai, et al., 2002) and induces oligodendrocyte progenitor proliferation, regardless of PDGFA concentration (Robinson, et al., 1998). The exact mechanism of this has yet to be identified. Activation of the CXCL1 receptor, CXCR2, a G-protein coupled receptor, induces intracellular calcium ([Ca2+]i) flux. Calcium mobilization plays a crucial role in the regulation of oligodendrocyte progenitor migration (Simpson and Armstrong, 1999). Thus, we hypothesized that CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration is regulated by changes in intracellular calcium. To examine the mechanisms by which CXCL1 modulates the migratory behaviour of oligodendrocyte progenitors, we assessed [Ca 2+]i flux in response to growth factors with and without CXCL1 treatment. In addition, we assessed the effect of CXCL1 on FGF2 induced oligodendrocyte progenitor migration. Further, we sequestered [Ca 2+]i in the presence of growth factors and CXCL1, to assess the role of [Ca 2+]i in the inhibition process. Interestingly, we show that blocking [Ca 2+]i did not affect the CXCL1 inhibition of growth factor induced oligodendrocyte progenitor migration. Additionally, CXCL1 did not affect [Ca2+]i levels in oligodendrocyte progenitors, in presence or absence of growth factor treatment. We also showed that inhibition of oligodendrocyte progenitor migration by the chemokine CXCL1 was specific to PDGFA induced migration. Finally, we showed that CXCL1 inhibition of oligodendrocyte progenitor migration was independent of cell cycle activation. Materials and methods All materials were from Sigma (St. Louis, MO, USA) unless otherwise stated. Platelet derived growth factor-AA (PDGFA) and fibroblast growth factor 2 (FGF2) were from R&D Systems (Minneapolis, MN, USA). The pharmacological inhibitor 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester (MAPTAM) was dissolved in DMSO at 50 mM stock concentration. CXCL1 was from R&D Systems. Fura-2 AM was from Invitrogen (Life Technologies. Grand Island, NY, USA). The α-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonist, (S)α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA) was from Enzo Life Sciences (Brockville, ON, Canada). Sato defined medium was prepared as previously described (Frost, et al., 2009). Bromo-deoxy-uridine antibody was from Boehringer Mannheim (Indianapolis, IN, USA). Isolation of oligodendrocyte progenitor cells Neonatal oligodendrocyte progenitor cells were prepared from postnatal day 2 rat brains, as previously described (Armstrong, 1998). Approximately, 7–10 days after plating, the oligodendrocyte progenitor cells and microglia were dislodged by shaking the flasks, followed by plating on non-coated tissue culture plastic for 25–40 minutes to allow the differential adhesion of microglial cells. The cell
suspension was collected, concentrated and resuspended in Sato defined medium with high insulin (Bottenstein and Sato, 1979).
Migration assay Cell migration was assayed using the agarose drop assay as previously described (Frost et al., 2000, 2009). Cells were exposed to inhibitory reagents for 30–45 min prior to addition of growth factor. Migration away from the edge of the drop was measured on four sides of the drop every 24 h for three days, using a calibrated eyepiece graticule in which the width of one square was equivalent to 100 μm.
Proliferation assay Bromo-deoxy-uridine (BrdU) incorporation was used to assess cell proliferation as previously described (Frost, et al., 2003). Cell proliferation was assessed as the percentage of 4′,6-Diamidino-2phenylindole Dihydrochloride:Hydrate (DAPI) positive oligodendrocyte progenitors that were also BrdU positive. At least 100 cells per coverslip were counted, with 5 coverslips per experiment with 3 experiments per test group.
Calcium signaling Fura-2 is a fluorescent dye, which binds to free [Ca 2+]i, and is commonly used to assess changes in [Ca 2+]i concentrations (Tucker, et al., 1989). Fura-2-acetoxymethyl ester (Fura-2 AM) is a strongly hydrophobic dye that very easily diffuses through the lipid bilayer of the plasma membrane of cell (Gunter, et al., 1988). Once inside the cell, the AM derivatives are rapidly hydrolyzed by nonspecific cytoplasmic esterases to become hydrophilic free acids, which are nonpermeable and thus are trapped in the cell (Gunter, et al., 1988). Fura2 is excited at 340 nm and 380 nm wavelength. When bound to Ca 2+, Fura-2 is excited at 340 nm and unbound Fura-2 is excited at 380 nm, it emits at 510 nm whether bound or unbound. The ratio of emission at those wavelengths is directly correlated to the concentration of intracellular calcium, using the formula shown in Table 1. The use of a ratio automatically negates uncontrollable variables, such as dye concentration and cell thickness. Purified oligodendrocyte progenitors were incubated with 2 μM Fura-2 AM in Ca 2+ free Locke's buffer (154 mM NaCl; 3.6 mM NaHCO3; 5.6 mM KCl; 1.0 mM MgCl2; 5.0 mM glucose; 5 mM Hepes; pH 7.0) with 0.1% BSA, for 1 hour. After washing, the cells were suspended in Locke's + 0.1% bovine serum albumin, and then placed into a Jasco CAF-110 intracellular ion analyzer for Ca 2+ flux analysis. Adenosine-tri-phosphate (ATP) was used as a positive control, to show that the oligodendrocyte progenitors were able to elicit a calcium response (Agresti et al., 2005). Previous studies have shown that ATP causes the release of intracellular stores of Ca 2+ in oligodendrocyte progenitors (Agresti et al., 2005).
Table 1 Calculation of intracellular calcium concentration. h
Ca2þ
i i
¼ Kd
RRmin Rmax R
λ380nm atminimum λ380nm atmaximum
Where R is the ratio of 510 nm emission intensity, exciting at 340 nm, to 510 nm emission intensity, exciting at 380 nm. Rmin is the ratio at zero free Ca2+ (i.e. prior to the addition of CaCl2 to the Locke's buffer); Rmax is the ratio at saturating Ca2+ (i.e. after the addition of digitonin to perforate the cell membranes allowing extracellular Ca2+ to flood into the cell). λ380 nm at minimum is the fluorescence intensity, exciting at saturating free Ca2+; λ380 nm at maximum is the fluorescence intensity, exciting at zero free Ca2+. Kd is an empirical value determined from Fura-2 containing Ca2+ standards run on the analyzer prior to the use of the Fura-2 for each experiment (Grynkiewicz, et al., 1985).
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Statistics Statistical analysis was performed using GraphPad Prism version 4.03 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. One-way ANOVA with Dunnett's posttest was used to test differences between migration curves. Student t-test was used to test the difference between means of cell count experiments. Results Calcium is required for PDGFA and FGF2 induced oligodendrocyte progenitor migration Previous studies have shown that chelation of extracellular Ca 2+ does not affect PDGFA induced oligodendrocyte progenitor migration, whereas chelation of intracellular Ca 2+ does inhibit PDGFA induced oligodendrocyte progenitor migration (Simpson and Armstrong, 1999). We used the calcium chelator MAPTAM to confirm the requirement for Ca 2+ signaling in PDGFA and FGF2 induced oligodendrocyte progenitor migration in the agarose drop assay (Pende, et al., 1997). MAPTAM, is a cell permeant analog of EGTA which chelates [Ca 2+]i. Chelation is the formation of soluble, complex molecules that inactivates the ions so that they cannot react with other molecules. Oligodendrocyte progenitors were pre-incubated with 45 μM MAPTAM for 30 min prior to the addition of growth factor. Cell migration requires process extension and adhesion to the substratum (Horwitz and Webb, 2003). Oligodendrocyte progenitors are unable to migrate in the absence of processes (Frost et al., 1996, 1999). In order to ensure that [Ca 2+]i chelation did not inhibit migration by preventing process formation we assessed cell morphology. Morphological analysis of the oligodendrocyte progenitors treated with MAPTAM ± growth factor showed that chelation of [Ca 2+]i did not affect cell processes (Fig. 1A–D). Migration was measured 72 hours
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after addition of growth factor. In the presence of MAPTAM, both PDGFA and FGF2 induced migration was inhibited significantly (Fig. 1E). PDGFA induced oligodendrocyte progenitors to migrate 973.25 ± 42.61 μm (compared to 262.15 ± 31.79 μm in control wells), and in the presence of MAPTAM migration was significantly reduced by 45% to 536.62 ± 38.32 μm (p b 0.001). FGF2 induced oligodendrocyte progenitors to migrate 893.25 ± 32.14 μm, and in the presence of MAPTAM, oligodendrocyte progenitor migration was inhibited by 44% to 502.44± 36.324 μm (p b 0.001). However, the levels of migration seen in response to growth factor, in the presence of MAPTAM, were still significantly higher than in untreated cells. Oligodendrocyte progenitors migrated 536.6 ±38.32 μm in the presence of PDGFA + MAPTAM compared to control cells (262.1 ± 31.79 μm: p b 0.0001), and 502.4 ± 36.32 μm in the presence of FGF2 + MAPTAM (p b 0.0001). MAPTAM treatment alone had no effect on oligodendrocyte migration (277.7± 10.1 μm). Active inhibition of PDGFA induced oligodendrocyte progenitor migration by the chemokine CXCL1 is specific to CXCL1 concentration We have previously shown that the chemokine CXCL1 acts as a stop signal for oligodendrocyte progenitors migrating in response to PDGFA, using a microchemotaxis chamber migration assay (Tsai, et al., 2002). For this study, we switched to an agarose drop assay, which allowed us to manipulate the media during the assay, as well as to study migration over a longer period of time (Frost, et al., 2000). To further understand the inhibition of PDGFA induced oligodendrocyte progenitor migration by CXCL1 we assessed the effect of two concentrations of CXCL1 (0.5 and 5 ng/ml) on a dose response curve to PDGFA (0.5–10 ng/ml). Concurring with the previous findings, we showed that in presence of 0.5 ng/ml CXCL1, PDGFA induced oligodendrocyte progenitor migration was inhibited regardless of growth factor concentration. However, in presence of 5 ng/ml
Fig. 1. Effect of MAPTAM on oligodendrocyte progenitors.The morphology of oligodendrocyte progenitors exposed to PDGFA (panel A) or FGF2 (panel C) is unaffected by treatment with MAPTAM (Panels B and D respectively). Panel E — oligodendrocyte progenitors pretreated for 20 minutes with 45μm MAPTAM show significantly reduced migration in response to PDGFA and FGF2 compared to growth factor treatment alone. (n = 6, with 4–6 replicates) ***p b 0.001.
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CXCL1, there was no inhibition of PDGFA induced oligodendrocyte progenitor migration, regardless of growth factor concentration (Fig. 2A). To analyze the effect of CXCL1 on growth factor treated oligodendrocyte progenitors further, we used 10 ng/ml PDGFA, and added CXCL1 48 hours later. Cells were left for migrate for a further 24 hours. Oligodendrocyte progenitors exposed to CXCL1 were prevented from migrating by the addition of CXCL1, indicating that CXCL1 is an active inhibitor of migration, even after the activation of the migration machinery (Fig. 2B). After 24 h of PDGFA exposure, oligodendrocyte progenitors migrated 401.21 ± 55.30 μm compared to 263.43 ± 38.35 μm in control (untreated) wells. After 48 hours of PDGFA exposure, oligodendrocyte progenitors migrated 688.98 ± 62.25 μm compared to 344.07 ± 64.23 μm in control (untreated) wells. CXCL1 (0.5 ng/ml) was added to the wells at T48, and 24 h later (T72), oligodendrocyte progenitor migration in the presence of PDGFA stopped at 671.75 ± 89.16 μm, compared to 989.00 ± 61.65 μm in the presence of PDGFA alone. Inhibition of oligodendrocyte progenitor migration by the chemokine CXCL1 is specific to PDGFA induced migration PDGFA acts through the PDGF receptor-alpha (PDGFRα), which is a receptor tyrosine kinase (RTK). Another growth factor known to induce oligodendrocyte progenitor migration is FGF2. The members of the FGF receptor family (FGFR) are also RTKs, with similar intracellular signaling cascades activated by ligand binding. In order to assess the potential for CXCL1 as the primary stop signal for oligodendrocyte progenitor migration, we analysed the effects of CXCL1 on FGF induced oligodendrocyte progenitor migration. In contrast to PDGFA, the addition of CXCL1 did not affect FGF2 induced oligodendrocyte
progenitor migration. In the presence of 1 ng/ml FGF2, oligodendrocyte progenitors were induced to migrate 578.10 ± 95.85 μm compared to 512.89 ± 70.13 μm in the presence of CXCL1 (0.5 ng/ml) p = 0.395. In the presence of 10 ng/ml FGF2, oligodendrocyte progenitors were induced to migrate 849.21 ± 40.80 μm compared to 725.38 ± 144.23 μm in the presence of 0.5 ng/ml CXCL1 p = 0.150 (Fig. 3). PDGFA induces significant, dose-dependent calcium flux, while FGF2 does not induce calcium flux in oligodendrocytes In order to analyze the calcium flux in oligodendrocyte progenitors, we preincubated freshly isolated oligodendrocyte progenitors with Fura-2 for 2 h, and then assessed changes in intracellular calcium concentration in the presence and absence of PDGFA. We found that in the presence of both 1 ng/ml and 10 ng/ml, PDGFA evoked a significant increase in [Ca 2+]i of 230.35 nM compared to baseline of 183.2 nM; and 338.668 nM compared to a baseline of 208.6 nM (Fig. 4). This represented 82.83% of the peak ATP value and 98.82% of the peak ATP value respectively. Previously published studies on FGF receptor signaling, have indicated that the [Ca2+]i is altered in response to FGF ligand binding (Eswarakumar, et al., 2005; Tsuda, et al., 1985). However, several other studies have shown that the effect of receptor activation differs between cell types, with different intracellular signaling pathways being activated by ligand binding (Murakami, et al., 2008; Satoh, et al., 1993; Stork and Schmitt, 2002). Therefore, for completeness, we assessed the Ca2+ flux in response to FGF2 in oligodendrocyte progenitors. Interestingly, even at 10 ng/ml concentration FGF2 did not induce Ca2+ flux in the oligodendrocyte progenitors (Fig. 5). We assessed changes in [Ca2+]i in response to 0.1 ng/ml FGF2 in case a lower concentration threshold is required for Ca2+ flux. However, there was no apparent change in [Ca 2+]i in response to any concentration tested (Fig. 5). CXCL1 does not affect intracellular calcium levels in oligodendrocyte progenitor in the presence or absence of PDGFA CXCL1 acts through the type 2 CXC receptor. CXCR2 is a G-protein coupled receptor, which induces Ca 2+ flux in other cell types (Puma,
Fig. 2. Inhibition of PDGFA induced oligodendrocyte progenitor migration by CXCR2 activation. Panel A — CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration is concentration dependent. At 0.5 ng/ml, CXCL1 inhibits oligodendrocyte progenitor migration induced by all concentrations of PDGFA (open diamonds, dashed line), whereas at 5 ng/ml, CXCL1 has no effect on PDGFA induced oligodendrocyte progenitor migration (open circles, dotted line), at any concentration. (n = 3 with 4–6 replicates). Panel B — Oligodendrocyte progenitor migration induced by PDGFA (solid circle, solid line) is inhibited by CXCL1 added 24 hours after the initiation of migration (open square, dashed line). Untreated oligodendrocyte progenitors do not migrate in the absence of PDGFA (closed circles, dotted line) (n = 5, with 4–5 replicates per treatment).
Fig. 3. FGF2 induced oligodendrocyte progenitor migration is not inhibited by CXCL1.Oligodendrocyte progenitors migrate significant distances compared to control in response to 1 ng/ml and 10 ng/ml FGF2 (p = 0.0019 and p b 0.0001 respectively). However, the chemokine CXCL1 does not inhibit FGF2 induced migration (ns), in contrast to PDGFA induced migration. Oligodendrocyte progenitor migration was measured 72 hours after the addition of growth factor. (n = 3 with 4–6 replicates; ** = p = 0.0019 *** = p b 0.0001).
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Fig. 4. Calcium flux in response to PDGFA.Calcium flux in primary oligodendrocyte progenitors is induced by PDGFA in a dose dependent manner. Panel A — PDGFA is added to the cells at 1 ng/ml. A significant increase in intracellular calcium is seen. Panel B — PDGFA is added to the cells at 10 ng/ml concentrations. A significant increase in intracellular calcium is seen. ATP is added to the cells to show that they are responding as expected. Arrows indicate the addition of PDGFA and ATP. The values given for calcium concentration are nM. (n = 3).
et al., 2001; Shibata, et al., 2002). Moreover, other chemokines invoke rapid mobilization of Ca 2+ from intracellular store in neurons (Giovannelli, et al., 1998; Meucci, et al., 1998). However, in oligodendrocyte progenitors, contrary to expectations, we found that at 0.5 ng/ml, CXCL1 had no effect on Ca 2+ flux (Fig. 6A). Even at concentrations as high as 50 ng/ml there was no changes in [Ca 2+]i (Fig. 6B). In order to test the hypothesis that CXCL1 inhibits oligodendrocyte progenitor migration via a Ca 2+ dependent pathway, CXCL1 was added prior to the addition of PDGFA, and [Ca 2+]i changes were assessed. We found that CXCL1 had no effect on PDGFA induced oligodendrocyte progenitor Ca 2+ flux (Fig. 7). CXCL1 was added to the oligodendrocyte progenitor at 0.5 ng/ml and 30 seconds later 1 ng/ml PDGFA was added to the cells. PDGFA elicited a significant increase in [Ca 2+]i to 340.50 nM after pre-treatment with CXCL1, which represents 87.6% of the ATP response (388.9 nM), compared to 338.7 nM in the presence of 10 ng/ml PDGFA alone (Fig. 4).
Inhibition of cell cycle activation does not affect CXCL1 induced inhibition of oligodendrocyte progenitor migration To check whether CXCL1 induced inhibition of oligodendrocyte progenitor migration is via activation of the cell cycle, we used SAMPA. The cyclin E-cyclin-dependant kinase 2 (cdk2) complex is a major regulator of G1–S transition in OP cells (Ghiani and Gallo, 2001). Activation of the AMPA receptor by S-AMPA, inhibits cyclin
Fig. 5. Effect of FGF2 on oligodendrocyte progenitor calcium flux in vitro.Calcium flux in primary oligodendrocyte progenitors is unaffected by FGF2. ATP is added to the cells to show that they are responding as expected. Panel A — FGF2 at 0.1 ng/ml, Panel B — FGF2 added at 1 ng/ml and Panel C — FGF2 added at 10 ng/ml. Arrows indicate the addition of PDGFA and ATP. The values are given for calcium concentration are nM. (n = 3).
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E–cdk2 formation, leading to cell cycle arrest (Ghiani and Gallo, 2001), without affecting oligodendrocyte progenitor migration (Frost, et al., 2009; Gallo, et al., 1996). The current study shows that S-AMPA had no effect on the CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration (Fig. 8). Results were normalized and shown as a percentage of the migration elicited in response to 10 ng/ml PDGFA. Thus, PDGFA alone produced 100 ± 6.23% migration. PDGFA in combination with S-AMPA resulted in a slight reduction in migration to 94.57 ± 6.66%. In the presence of CXCL1, PDGFA treatment resulted in 44.7 ± 6.99% migration. In the presence of S-AMPA and CXCL1, PDGFA induced 40.91 ± 7.08% migration (Fig. 8). Oligodendrocyte progenitor proliferation and survival is not affected by AMPA treatment Bromo-deoxyuridine (BrdU) incorporation was used to assess the effects of AMPA on oligodendrocyte progenitor proliferation (Frost et al., 2003, 2009). The percentage of control (untreated) oligodendrocyte progenitor cells that incorporated BrdU was 26.58 ± 3.22%. Whereas in the presence of PDGFA, BrdU incorporation increased to 68.54 ± 4.69%. Pretreatment of the cells with AMPA reduced BrdU incorporation to 34.15 ± 3.21%. BrdU incorporation was unaffected by treatment with AMPA alone (29.9 ± 4.11%). The effects of AMPA treatment on cell survival were assessed by propidium iodide incorporation over a 24-hour period (Frost, et al., 2003). In the presence of PDGFA 78.5 ± 2.0% of cells survived, compared to 52.3 ± 2.3% of untreated cells. In the presence of AMPA, PDGFA induced oligodendrocyte progenitor survival was 79.9 ± 2.8%. In the presence of AMPA treatment alone, 54.6 ± 3.6% of the cells survived. Thus, AMPA inhibited the cell cycle, without affecting survival in oligodendrocyte progenitors. Discussion Normal myelination is critical for the successful functioning of the nervous system. Aberrant myelination is characteristic of several disorders of the brain, including cerebral palsy (Dammann and Leviton,
Fig. 6. Calcium signaling in response to CXCL1.Calcium flux in primary oligodendrocyte progenitors is unaffected by CXCL1. Panel A — CXCL1 is added to the cells at 0.5 ng/ml concentration. Panel B — CXCL1 is added to the cells at 50 ng/ml concentration. ATP is added to the cells to show that they are responding as expected. Arrows indicate the addition of CXCL1 and ATP. The values given for calcium concentration are nM. (n = 3).
Fig. 7. Calcium flux in response to PDGFA after CXCL1 treatment.PDGFA induced calcium flux in primary oligodendrocyte progenitors is unaffected by the addition of CXCL1 at 0.5 ng/ml prior to the addition of the PDGFA. ATP is added to the cells to show that they are responding as expected. Arrows indicated the addition of CXCL1, PDGFA, and ATP. The values given for calcium concentration are nM. (n = 3).
Fig. 8. Cell cycle inhibition does not block CXCL1 inhibition of oligodendrocyte progenitor migration.Oligodendrocyte progenitors are treated with AMPA and or CXCL1 and PDGFA to assess the effect of inhibiting the cell cycle on CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration. PDGFA induced migration was significantly increased compared to control (p b 0.0001), in the presence of AMPA PDGFA induced migration was significantly increased over control (p b 0.0001). CXCL1 inhibited PDGFA induced migration back to control levels. In the presence of AMPA, CXCL1 inhibition of PDGFA induced migration was unaffected. (n = 3 with 4–6 replicates; *** = p b 0.0001).
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1998; Juliet, et al., 2009), Schizophrenia (Carter, 2006; Peirce, et al., 2006), and leukodystrophies (Costello, et al., 2009; Schiffmann and van der Knaap, 2004). Further, the demyelinating disease, multiple sclerosis (MS) is also characterized by damage to the CNS myelin. Understanding the mechanisms that regulate the formation of myelin in the developing CNS may provide insights into potential therapeutic strategies for diseases associated with myelin dysfunction. Oligodendrocyte progenitors migrate extensively during development of the CNS (Jakovcevski and Zecevic, 2005; Marshall and Goldman, 2002; Rakic and Zecevic, 2003). Developmental migration is thought to be regulated by chemotactic gradients of mitogens, such as FGF2 (Hardy, et al., 2011; Sato, et al., 2011), and PDGFA (Yang, et al., 2008). However, there is no evidence of such a phenomenon in the developing CNS. Cell migration is therefore likely to be precisely regulated by the presence of localized morphogens, which alter the cells’ response to its local environment based on ligand concentration, and/or receptor occupancy (Freeman and Gurdon, 2002). However, the exact molecular mechanisms underlying the regulation of oligodendrocyte progenitor migration have yet to be fully elucidated. The chemokine CXCL1 was shown previously to regulate the spatial distribution of oligodendrocyte progenitors in the developing CNS (Tsai, et al., 2002). CXCL1 cooperates with PDGFRα to regulate the dispersal and proliferation of oligodendrocyte progenitors (Robinson, et al., 1998; Tsai, et al., 2002). Despite numerous studies on the expression patterns of CXCR2 in development and disease (Omari et al., 2005, 2006; Padovani-Claudio, et al., 2006), the mechanism of action of the chemokine remains unknown. In order to test our hypothesis, that CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration is regulated by changes in intracellular calcium, we studied the effect of CXCL1 on oligodendrocyte progenitor Ca 2+ mobilization in the presence and absence of PDGFA. In addition, to assess further the role of CXCL1 on oligodendrocyte progenitor behaviour, we assessed its effect on FGF2 induced oligodendrocyte progenitor migration. Numerous studies have shown that PDGFA and FGF2 both regulate oligodendrocyte progenitor migration (Armstrong, et al., 1990; Milner, et al., 1997; Vora, et al., 2011). Previously, we have shown that they regulate migration via the ERK signaling pathway (Frost, et al., 2009; Vora, et al., 2011). However, there are significant differences between the response of oligodendrocyte progenitors to the two receptor tyrosine-kinases (Vora, et al., 2011). Past research has shown that CXCL1 binding to its receptor influences PDGFRα signaling to regulate the dispersal and proliferation of oligodendrocyte progenitors (Robinson, et al., 1998; Tsai, et al., 2002). To better understand the role of CXCL1 in regulating oligodendrocyte progenitor dispersal, we assessed the effect of CXCL1 on PDGFA and FGF2 induced oligodendrocyte progenitor migration. The CXCL1 receptor, CXCR2, is a pertussis toxin sensitive G-protein coupled receptor (Richardson, et al., 1998). Activation of CXCR2 activates several intracellular signaling pathways, including the mobilization of [Ca 2+]i (Shibata, et al., 2002) and the Rho kinase, and phosphatidyl inositol-3-kinase (PI3K) signaling cascades (L'Heureux, et al., 1995; Richardson, et al., 1998; Schraufstatter, et al., 2001). Numerous studies have shown that Ca 2+ mobilization is required for cell migration (Wei, et al., 2009). Calcium plays a critical role as secondary messenger in the process the myosin-actin complex activation, which is required for the cytoskeletal reorganization that accompanies migration (Adelstein and Hathaway, 1979). Further, Ca 2+ mobilization is associated with the G1 phase of the cell cycle, although the complex interactions between [Ca 2+]i dependent signaling pathways and cell proliferation are still not fully elucidated (Capiod, 2011). Previous studies have shown a role for [Ca 2+]i in the regulation of oligodendrocyte progenitor migration (Agresti et al., 2005; Simpson and Armstrong, 1999). Thus, we hypothesised that CXCL1 inhibition of PDGFA induced oligodendrocyte progenitor migration is regulated
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by changes in intracellular calcium, which induces oligodendrocyte progenitor proliferation. The present study clearly shows that PDGFA induces an immediate increase in [Ca 2+]i in a dose-dependent manner. Further, in our study, there is no increase in [Ca 2+]i in response to FGF2 treatment. This concurs with a previous study that showed FGF2 treatment of oligodendrocyte progenitors resulted in a low to non-existent Ca 2+ flux (Pende, et al., 1997). Our findings fit with previous studies, which show that FGF2 regulates oligodendrocyte progenitor migration via different signaling pathways to PDGFRα (Vora, et al., 2011), and that FGF2 is not critical for the development of myelin (Armstrong, et al., 2002). The factors that evoke changes in the [Ca 2+]i levels at different stages of oligodendrocyte development are also known to regulate their development and pathology. The key hypothesis of this study is that one such factor CXCL1, which is previously shown to inhibit oligodendrocyte progenitor migration (Tsai, et al., 2002), uses [Ca 2+]i stores to bring about its effect. Interestingly, we did not see any changes in oligodendrocyte progenitor [Ca 2+]i levels even at high concentrations of CXCL1 treatment. Furthermore, we hypothesized that Ca 2+ stores are released upon inhibition of growth factor induced oligodendrocyte progenitor migration by CXCL1. However, the current study shows that oligodendrocyte progenitor migration is not completely dependent on Ca 2+ signaling. Moreover, blocking the [Ca 2+]i with the calcium chelating agent, MAPTAM failed to inhibit CXCL1 action on oligodendrocyte progenitor migration. The role of Ca 2+ in oligodendrocyte progenitor migration has been controversial (Clyman, et al., 1994; Simpson and Armstrong, 1999). Chelation of [Ca 2+]i significantly reduced the extent of migration of oligodendrocyte progenitors in response to both PDGFA and FGF2. Interestingly, the oligodendrocyte progenitors treated with MAPTAM still migrated significantly further in the presence of growth factor, than those cells left untreated. This indicates that, although Ca 2+ plays a role in oligodendrocyte progenitor migration, it is not the only signaling pathway involved. Our previous studies have shown that ERK is critical for oligodendrocyte progenitor migration (Vora and Frost, 2009; Vora, et al., 2011), and that cross-talk between signaling cascades also plays an important role in maintaining oligodendrocyte progenitor migration in response to growth factors (Frost, et al., 2009). Thus, further studies are required to assess the nature of the Ca 2+ independent pathways that are regulating oligodendrocyte progenitor migration. A possible explanation for the CXCL1 inhibition of migration is induction of the cell cycle. In order to proliferate, cells must stop migrating, to allow the restructuring of the cytoskeleton necessary for spindle formation and cytokinesis. Calcium plays a critical role in the reorganisation of the cytoskeleton during cell division (Capiod, 2011). Thus, we hypothesized that CXCL1 induction of the cell cycle results in inhibition of migration. To test the hypothesis, a known inhibitor of oligodendrocyte progenitor proliferation, the glutamate receptor agonist AMPA was used (Gallo, et al., 1996; Ghiani and Gallo, 2001). We show that AMPA blocks proliferation of oligodendrocyte progenitors without affecting migration or cell survival. In the presence of CXCL1, the expected outcome of inhibition of cell cycle activation would be no change in oligodendrocyte progenitor migration. However, our results show that in the presence of AMPA, CXCL1 still blocks oligodendrocyte progenitor migration. These results indicate that inhibition of the cell cycle does not appear to prevent the inhibitory effects of CXCL1. Thus, we show for the first time that CXCL1 actively blocks oligodendrocyte progenitor migration via a cell cycle independent pathway. The exact mechanisms by which CXCL1 inhibit migration require further investigation. Other pathways potentially involved include the activation of RhoK, which inhibits myosin phosphatase (Hall, 2005). Myosin phosphatase inactivates myosin light chain kinase, which is critical for the reorganisation of actin filaments in
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oligodendrocyte (Thomas, et al., 2002). Further, activation of CXCR2 induces the JAK/STAT and PLC/PI3K pathways, which are both involved in the induction of PDGFA induced oligodendrocyte progenitor proliferation (Dell'Albani, et al., 1998; Ebner, et al., 2000). Conclusions The mechanism by which CXCL1 inhibits oligodendrocyte progenitor migration remains an enigma. The inhibitory effect of CXCL1 on oligodendrocyte progenitors is directly regulated via CXCR2 (Tsai, et al., 2002). The current study shows that this essential inhibition of oligodendrocyte progenitor migration is not arbitrary, but may be specific to PDGFA activity in the developing brain. Further, this study shows that oligodendrocyte progenitor migration requires Ca2+ flux, but is not dependent on Ca2+ flux. Understanding the downstream signaling pathways involved in CXCL1 inhibition of oligodendrocyte progenitor migration will further our understanding of molecular and cellular mechanisms involved in developmental myelination, and will provide information relevant to remyelination during adult tissue repair and other disease related processes. Acknowledgments We would like to acknowledge our funding agencies: The Manitoba Institute for Child Health (EEF), the Manitoba Health Research Council (EEF), The Manitoba Medical Services Foundation (EEF & MPN), Pfizer Global UK (MPN & EEF), Biogen Idec (MPN & EEF). PV was recipient of an MHRC studentship. CK and SS were recipients of Manitoba Institute for Child Health Summer Studentships. References Adelstein, R.S., Hathaway, D.R., 1979. Role of calcium and cyclic adenosine 3′:5′ monophosphate in regulating smooth muscle contraction. Mechanisms of excitation– contraction coupling in smooth muscle. Am. J. Cardiol. 44, 783–787. Agresti, C., Meomartini, M.E., Amadio, S., Ambrosini, E., Volonte, C., Aloisi, F., Visentin, S., 2005. ATP regulates oligodendrocyte progenitor migration, proliferation, and differentiation: involvement of metabotropic P2 receptors. Brain Res. Brain Res. Rev. 48, 157–165. Armstrong, R.C., 1998. Isolation and characterization of immature oligodendrocyte lineage cells. Methods 16, 282–292. Armstrong, R.C., Harvath, L., Dubois-Dalcq, M.E., 1990. Type 1 astrocytes and oligodendrocyte-type 2 astrocyte glial progenitors migrate toward distinct molecules. J. Neurosci. Res. 27, 400–407. Armstrong, R.C., Le, T.Q., Frost, E.E., Borke, R.C., Vana, A.C., 2002. Absence of fibroblast growth factor 2 promotes oligodendroglial repopulation of demyelinated white matter. J. Neurosci. 22, 8574–8585. Baron, W., Metz, B., Bansal, R., Hoekstra, D., de Vries, H., 2000. PDGF and FGF-2 signaling in oligodendrocyte progenitor cells: regulation of proliferation and differentiation by multiple intracellular signaling pathways. Mol. Cell. Neurosci. 15, 314–329. Berridge, M.J., 2004. Calcium signal transduction and cellular control mechanisms. Biochim. Biophys. Acta 1742, 3–7. Bottenstein, J.E., Sato, G.H., 1979. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. Natl. Acad. Sci. U. S. A. 76, 514–517. Capiod, T., 2011. Cell proliferation, calcium influx and calcium channels. Biochimie 93, 2075–2079. Carter, C.J., 2006. Schizophrenia susceptibility genes converge on interlinked pathways related to glutamatergic transmission and long-term potentiation, oxidative stress and oligodendrocyte viability. Schizophr. Res. 86, 1–14. Clyman, R.I., Peters, K.G., Chen, Y.Q., Escobedo, J., Williams, L.T., Ives, H.E., Wilson, E., 1994. Phospholipase C gamma activation, phosphotidylinositol hydrolysis, and calcium mobilization are not required for FGF receptor-mediated chemotaxis. Cell Adhes. Commun. 1, 333–342. Costello, D.J., Eichler, A.F., Eichler, F.S., 2009. Leukodystrophies: classification, diagnosis, and treatment. Neurologist 15, 319–328. Dammann, O., Leviton, A., 1998. Infection remote from the brain, neonatal white matter damage, and cerebral palsy in the preterm infant. Semin. Pediatr. Neurol. 5, 190–201. Dell'Albani, P., Kahn, M.A., Cole, R., Condorelli, D.F., Giuffrida-Stella, A.M., de Vellis, J., 1998. Oligodendroglial survival factors, PDGF-AA and CNTF, activate similar JAK/ STAT signaling pathways. J. Neurosci. Res. 54, 191–205. Ebner, S., Dunbar, M., McKinnon, R.D., 2000. Distinct roles for PI3K in proliferation and survival of oligodendrocyte progenitor cells. J. Neurosci. Res. 62, 336–345. Eswarakumar, V.P., Lax, I., Schlessinger, J., 2005. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 16, 139–149.
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