In the developing nervous system, TIMP-2 is expressed coincident with the onset of neuronal differentiation and its expression is maintained at high levels into adulthood (Blavier and DeClerck 1997; Vaillant et al. 1999; Fager and Jaworski 2000), suggesting TIMP-2 plays a role in the acquisition and maintenance of a differentiated neuronal phenotype. Previously, we demonstrated that TIMP-2 induces cell cycle arrest and neurite outgrowth in PC12 cells independent of its role in MMP inhibition (Pérez-Martínez & Jaworski, 2005). Furthermore, immunoprecipitation studies revealed that TIMP-2 interacts with α3β1 integrin, suggesting TIMP-2 might exert its MMP-independent activities via integrins. To determine whether these observations are unique to PC12 cells and/or NGF-mediated differentiation, the regulation of TIMP-2 expression was examined in cell lines responsive to various neurogenic signals. Here, we demonstrate that TIMP-2 expression is up-regulated coincident with neuronal differentiation in all lines examined, but that cell surface TIMP-2 expression is primarily detected on PC12 cells, cells that express α3β1 integrin.
TIMP-2 expression is up-regulated during neuronal differentiation in a variety of cell lines
The most widely used model system for investigating the molecular mechanism underlying neuronal differentiation is the PC12 pheochromocytoma cell line. Exposure to NGF induces differentiation into sympathetic-like neurons (Greene and Tischler 1976). In contrast, proliferation is stimulated in the presence of EGF (Huff et al. 1981). bFGF induces proliferation (Kawamata et al. 2001) or differentiation (Rydel and Greene 1987) in a concentration dependent manner. Log phase growth PC12 cells were serum-starved for 12 h prior to growth factor addition. To induce differentiation, cells were treated with either NGF or bFGF at 100 ng/mL. Since glycosaminoglycans can potentiate the ability of bFGF to induce neurite outgrowth (Damon et al. 1988), heparin (1 µg/mL) was included in bFGF treated cultures. To induce proliferation, medium containing either EGF (100 ng/mL) or bFGF (10 ng/mL) was added. To determine basal levels of TIMP-2, proliferating PC12 cells maintained in 15% serum were harvested 12 h after plating. TIMP-2 expression was up-regulated in response to all growth factor treatments (Figs 1a, e). Overnight serum starvation induced a 2.7-fold increase in TIMP-2 expression from proliferating conditions. Thus, growth factor deprivation itself regulates TIMP-2 expression. For NGF treated cells, TIMP-2 expression gradually increased to peak at 5 DIV. bFGF/heparin (bFGFb) treatment initially (1 and 2 DIV) resulted in increased TIMP-2 levels comparable to that observed with NGF. However, after 3 DIV TIMP-2 expression dramatically declined. In contrast to the transient TIMP-2 increase by bFGF/heparin, bFGF alone (bFGFa), at concentrations that promote proliferation, resulted in sustained TIMP-2 expression to 5 DIV. Moreover, both the time course and extent of TIMP-2 up-regulation in response to bFGF was similar to that with EGF. These data demonstrate that TIMP-2 expression in PC12 cells is regulated by multiple growth factors.
Figure 1. TIMP-2 expression is up-regulated by multiple neurogenic signals. Western blot analysis of cell lysates for TIMP-2 expression in PC12 (a), Neuro-2a (b), N1E-115 (c) and P19 (d) cells. TIMP-2 expression in PC12 cells was up-regulated by overnight serum starvation (T0) relative to proliferating cells maintained in 15% serum (P). While EGF promotes proliferation and NGF induces differentiation, bFGF regulates both proliferation (+ bFGFa, 10 ng/mL) and differentiation (+ bFGFb, 100 ng/mL) of PC12 cells in a dose-dependent manner. Neuro-2a and N1E-115 cells were induced to differentiate by serum depletion (to 0.5%). P19 cells were maintained in suspension for 4 days in the presence of RA (+ RA). Neuronal differentiation proceeds upon re-plating in the absence of RA. (e) Densitometric analysis of TIMP-2 expression. Expression was first normalized to actin at each time point, then the extent of TIMP-2 up-regulation was determined relative to basal TIMP-2 levels prior to growth factor addition (PC12, P19) or depletion (Neuro-2a, N1E-115) (T0). Since proliferating P19 cells possessed a high basal level of TIMP-2 and expression was down-regulated by RA, the extent of TIMP-2 up-regulation in P19 cells was determined relative to RA-treated cells. Results are representative of at least two independent experiments.
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Because TIMP-2 expression was also up-regulated in response to growth factor depletion, TIMP-2 expression was examined in two cells lines that differentiate upon serum withdrawal. Murine Neuro-2a neuroblastoma cells (Klebe and Ruddle 1969) differentiate into cholinergic neuron-like cells following retinoic acid treatment (Shea et al. 1985) or serum removal that is associated with a rapid and persistent increase in the cellular levels of ceramide (Tsuji et al. 1988; Riboni et al. 1995). Similar to its regulation in PC12 cells, TIMP-2 expression increased in Neuro-2a cells in a time dependent manner (Figs 1b, e). This temporal increase was observed even in cells maintained in 10% serum. At both 1 and 7 DIV, TIMP-2 expression in serum reduced cells was not different from cells in 10% serum. However, serum depletion resulted in a 2.4-fold increase of TIMP-2 at 2 DIV, a 2.5-fold increase at 3 DIV, and a 1.6-fold increase at 5 DIV relative to cells in 10% serum. The murine N1E-115 neuronal cell line (Amano et al. 1972) exhibits neurite outgrowth in response to serum deprivation, presumably due to removal of lipophosphatidic acid in serum (Jalink et al. 1994; Kozma et al. 1997). Serum depletion of N1E-115 cells resulted in a 1.5-fold increase of TIMP-2 at 1 DIV and a 2-fold increase at 2 DIV (Figs 1c, e). Afterwards, TIMP-2 expression was greater in cells maintained in 10% serum than in serum reduced cells. Thus, TIMP-2 expression in N1E-115 cells is regulated transiently, similar to TIMP-2 regulation observed in PC12 cells.
To determine the ontogenic expression of TIMP-2 during neurogenesis, pluripotent murine P19 embryonal carcinoma cells were examined. In contrast to the other cell lines examined, a significant basal level of TIMP-2 expression was detected in undifferentiated cells (Fig. 1d). P19 cells can be induced to differentiate in neurons/glia, myocytes, and fibroblast-like cells by retinoic acid (RA) (McBurney et al. 1982), with the cell type generated depending on the concentration of RA used (Jones-Villeneuve et al. 1982; Edwards and McBurney 1983). RA-induced P19 differentiation requires two phases. In the induction phase, cells are allowed to aggregate with RA for 4 days, during which time the cells are determined into neuronal progenitors. Interestingly, TIMP-2 expression was down-regulated during this phase (Fig. 1d, + RA lane). Neuronal differentiation is then initiated upon plating cells in the absence of RA. TIMP-2 expression was up-regulated in differentiated P19 cells in a time course similar to that of NGF-differentiated PC12 cells (Figs 1d, e). Expression peaked at 5 DIV and then declined. Taken together, these data demonstrate that TIMP-2 expression is modulated by changes in growth factor conditions and signaling pathways that regulate neuronal differentiation.
Cell surface expression of TIMP-2 is differentially regulated in neuronal cell lines
To more closely examine the regulation of TIMP-2 expression during neuronal differentiation, its spatial distribution was determined immunocytochemically. TIMP-2 is secreted into the extracellular milieu were it functions as a soluble molecule. In addition, TIMP-2 acts at the cell surface via specific, saturable, high-affinity receptors (Hayakawa et al. 1994; Hoegy et al. 2001). To determine whether TIMP-2 serves as a soluble regulator or acts at the cell surface, both live labeling and Triton-permeabilized cells were subjected to immunolabeling. In contrast to the intracellular TIMP-2 expression detected in all cell lines examined, cell surface TIMP-2 expression was only observed in PC12 cells.
In contrast to proliferating cells (15% serum) (Figs 2a1 and 2b1), cell surface (Fig. 2a) and intracellular (Fig. 2b) TIMP-2 expression was present in PC12 cells under all treatment conditions. Serum deprived PC12 cells prior to growth factor addition (T0) did not show cell surface labeling (Fig. 2a8). With an additional day of growth factor deprivation, cell surface TIMP-2 was detectable (Fig. 2a2), but the labeling declined with time (Fig. 2a3−7). Cells maintained under proliferating conditions, either with EGF (Fig. 2a9−14) or bFGF (Fig. 2a15−20) showed cell surface TIMP-2 expression. In NGF differentiated cells, cell surface TIMP-2 was present on the cell soma and neuritic processes (Fig. 2a21−26). Cells differentiated with bFGF/heparin (bFGFb) had TIMP-2 on the soma, but none detectable on processes (Fig. 2a27−32). Intracellular TIMP-2 expression, in Triton-permeabilized cells, was much greater than cell surface expression (Fig. 2b). A low level of intracellular TIMP-2 was present in overnight serum deprived PC12 cells (Fig. 2b8). As observed for cell surface TIMP-2, an additional day of growth factor deprivation increased intracellular TIMP-2 expression (Fig. 2b2). However, unlike cell surface labeling which declined, intracellular labeling was maintained (Fig. 23−7). Even in the absence of exogenous growth factor, PC12 cells spontaneously differentiated and expressed TIMP-2 in the soma and processes (Fig. 2b6,7). Intracellular TIMP-2 expression in EGF (Fig. 2b9−14) and bFGF (Fig. 2b15−20) treated cells was much more intense than cell surface expression. Expression of TIMP-2 in neuritic processes of NGF differentiated cells was detectable as early as 1 DIV (Fig. 2b21) as opposed to 3 DIV for cell surface TIMP-2 (Fig. 2a23). TIMP-2 expression in bFGF/heparin differentiated cells (Fig. 2b27−32) was not as intense as NGF differentiated cells. These data demonstrate that TIMP-2 expression is up-regulated by both proliferative and neurogenic signals. Most importantly, cell surface TIMP-2 expression coincides with PC12 cell differentiation indicative of the interaction of TIMP-2 with specific PC12 cell surface receptor(s).
Figure 2. Regulation of TIMP-2 expression in PC12 cells. (a) Live-labeling immunocytochemically was performed to detect cell surface TIMP-2 expression. TIMP-2 was not detected on the cell surface of proliferating cells maintained in 15% serum (1) or after 12 h of serum deprivation (8). Otherwise, cell surface TIMP-2 expression was present in response to all growth factor treatments. TIMP-2 expression was even up-regulated in the absence of exogenous growth factors (2–7). (b) Cells were permeabilized with Triton X-100 to detect intracellular TIMP-2. Although no TIMP-2 was detected in proliferating cells (1), TIMP-2 expression was up-regulated by serum depletion (8). Expression was further up-regulated by growth factor treatment. In all cases, TIMP-2 expression was greater intracellularly than on the cell surface. TIMP-2 expression is particularly enriched in the soma and neuritic processes of NGF differentiated cells. Scale bar = 25 µm.
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In contrast to PC12 cells, cell surface TIMP-2 expression was not detected in either Neuro-2a (Fig. 3) or N1E-115 (Fig. 4) cells. TIMP-2 was not present on cells maintained in 10% serum (Figs 3 and 4a–f) or differentiated cells in 0.5% serum (Figs 3 and 4m–q). However, intracellular TIMP-2 expression was abundant in both cell types. TIMP-2 expression was greater in serum depleted cells (Figs 3 and 4r–v) than cells maintained in 10% serum (Figs 3 and 4g–l) and resembled that of NGF differentiated PC12 cells, with intense expression in both the soma and neuritic processes. To determine whether the lack of cell surface TIMP-2 labeling in these cells was due to an absence of TIMP-2 secretion, Western blot analysis was performed with conditioned media from cells grown in differentiation media (Fig. 5a). Neuro-2a and N1E-115 cells secreted considerably less TIMP-2 than PC12 cells. Only a weak TIMP-2 signal was observed in Neuro-2a cells after 7 DIV. TIMP-2 expression was first detected in N1E-115 conditioned media at 2 DIV, increased at 3 DIV, and then declined at 5 DIV. To determine whether cell surface labeling could be achieved by increasing the level of TIMP-2 released into the media, TIMP-2 was over-expressed via transient transfection using a TIMP-2 expression vector previously described (Pérez-Martínez & Jaworski, 2005). A significant amount of Flag-tagged TIMP-2 was present in the conditioned media of Neuro-2a and N1E-115 cells 3 days after transfection (Fig. 5b). In contrast to the abundant cell surface Flag labeling detected on GFP-positive transfected PC12 cells (Fig. 5c1,4,7,10), neither Neuro-2a (Fig. 5c2, 5, 8, 11) nor N1E-115 (Fig. 5c3,6,9,12) cells showed cell surface Flag labeling, suggesting that secreted TIMP-2 did not bind cells in an autocrine manner. Furthermore, the extent of Flag immunolabeling on untransfected cells was no greater than in vector transfected controls (data not shown), suggesting that secreted TIMP-2 did not bind cells in a paracrine manner. Thus, TIMP-2 is secreted, but unlike PC12 cells where it acts at the cell surface, TIMP-2 likely serves as a soluble regulator of matrix proteolysis in Neuro-2a and N1E-115 cells.
Figure 3. Regulation of TIMP-2 expression in Neuro-2a cells. Cell surface TIMP-2 was not detected on proliferating Neuro-2a cells (10% serum, a–f) or cells differentiated by serum depletion (0.5% serum, M-Q). However, intracellular TIMP-2 was present in both proliferating (g–l) and Triton-permeabilized differentiated cells (r–v). TIMP-2 was even detected in neurites of spontaneously differentiated cells maintained in 10% serum (j, k arrows). In differentiated cells, TIMP-2 was present in both fibrous processes and lamellapodial processes (u). Scale bar = 50 µm.
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Figure 4. Regulation of TIMP-2 expression in N1E-115 cells. Cell surface TIMP-2 was not detected on proliferating N1E-115 cells (10% serum, a–f) or cells differentiated by serum depletion (0.5% serum, m–q). Intracellular TIMP-2 was present in both proliferating (g–l) and Triton-permeabilized differentiated cells (r–v). TIMP-2 was present in neuritic processes of spontaneously differentiated cells in 10% serum (k arrows) and serum depleted cells (r–v). Scale bar = 50 µm.
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Figure 5. TIMP-2 is secreted from Neuro-2a and N1E-115 cells, but does not bind to the cell surface. (a) TIMP-2 expression in conditioned media (100 µL) from differentiated PC12, Neuro-2a and N1E-115 cells. The presence of TIMP-2 in the media of Neuro-2a and N1E-115 cells demonstrates that the lack of cell surface labeling is not due to the lack of TIMP-2 secretion, but may be due to reduced secretion relative to PC12 cells or the lack of TIMP-2 receptor(s) expression. (b) Western blot analysis of conditioned media (100 µL) from Neuro-2a and N1E-115 cells 3 days after transient transfection to over-express TIMP-2. Exogenously produced TIMP-2 (TIMP-2Flag) was immunoprecipitated with Flag antibody. TIMP-2 was abundantly released from both cell lines. (c) Live label immunocytochemistry with Flag (red) 3 days after TIMP-2 transfection (GFP-positive green cells) demonstrates that cell surface TIMP-2 ‘receptors’ are expressed on PC12 cells, but not on Neuro-2a or N1E-115 cells. Scale bar = 50 µm.
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Like PC12 cells, P19 cells express cell surface receptors for TIMP-2 (Fig. 6). However, unlike PC12 cells where cell surface labeling was not detected in undifferentiated cells (Fig. 2a1,8), cell surface labeling was only detected in pluripotent P19 cells (Fig. 6a). Cell surface labeling was lost after 4 days of RA treatment (Fig. 6b–g). Significant intracellular TIMP-2 expression was present in P19 cells. Similar to cell surface TIMP-2, intracellular levels declined as pluripotent cells (Fig. 6h) were exposed to RA (Fig. 6i). To determine whether cell surface or intracellular TIMP-2 was expressed at a greater level in embryoid bodies than adherent cells (Figs 6b and i), immunocytochemistry was performed in suspension. The embryoid bodies showed no cell surface labeling and no greater intracellular TIMP-2 than that observed for adherent cells (data not shown). Intracellular TIMP-2 expression increased as cells differentiated into neurons (Fig. 6j–n).
Figure 6. Regulation of TIMP-2 expression in P19 cells. In contrast to other cells lines examined which showed little or no TIMP-2 expression in proliferating cells, TIMP-2 expression was enriched in pluripotent progenitors (T0). Expression was detected both on the cell surface (a) and intracellularly (h). Exposure to RA for 4 days abrogated cell surface TIMP-2 expression (b) and significantly reduced intracellular expression (i). After re-plating cells in the absence of RA, cell surface TIMP-2 was not detectable at any time point. Intracellular TIMP-2 expression was detected as early as 1 DIV in process bearing neurons (j, arrows). Scale bar = 20 µ m.
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Finally, experiments were conducted to determine the nature of the TIMP-2 receptor. Some TIMPs are tethered to the cell by membrane type MMPs (MT-MMPs). Once secreted, TIMP-2 forms a ternary complex with pro-MMP-2 and MT1-MMP (Butler et al. 1998; Zucker et al. 1998). To determine whether cells lacking surface TIMP-2 labeling possessed MT1-MMP, its expression in undifferentiated (–) and differentiated (+) PC12, Neuro-2a, N1E-115, and P19 cells was examined at 5 DIV. By western blot analysis, all cells expressed MT1-MMP (Fig. 7a). Interestingly, MT1-MMP expression was greater in proliferating PC12, Neuro-2a, and N1E-115 cells and down-regulated upon differentiation, similar to its expression pattern as myoblasts differentiate into mytotubes (Lluri and Jaworski 2005). To determine whether MT1-MMP was present at the cell surface to serve as a TIMP-2 ‘receptor’, live label immunocytochemistry was performed (Fig. 7b). In PC12 cells, both TIMP-2 (Fig. 7b1) and MT1-MMP (Fig. 7b5) were expressed at the cell surface (Fig. 7b9,13). Neither TIMP-2 nor MT1-MMP was detected on the cell surface of Neuro-2a (Fig. 7b2,6,10,14) or P19 (Fig. 7b4,8,12,16). However, cell surface MT1-MMP, but not TIMP-2 was present on N1E-115 cells (Fig. 7b3, 7, 11, 15).
Figure 7. MT1-MMP expression in neuronal cell lines. (a) Western blot analysis of cell lystates for MT1-MMP expression in cells grown in the absence or presence of differentiation signal for 5 days. Although Neuro-2a, N1E-115, and P19 cells lack TIMP-2 cell surface expression, these cell lines express abundant amounts of MT1-MMP. (b) Live label immunocytochemistry for TIMP-2 (red) and MT1-MMP (green) demonstrates that both PC12 cells and N1E-115 cells possess cell surface MT1-MMP, but only PC12 cells have detectable cell surface TIMP-2 expression. Scale bar = 50 µm.
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TIMP-2 also binds the cell surface independent of MT-MMPs, suggesting a distinct receptor mechanism (Corcoran and Stetler-Stevenson 1995; Hoegy et al. 2001). TIMP-2 inhibits endothelial cell proliferation by binding α3β1 integrin (Seo et al. 2003). In addition, we recently demonstrated that TIMP-2 binds to α3β1 integrin in vitro and in vivo (Pérez-Martínez & Jaworski, 2005). Thus, we determined whether cell surface TIMP-2 expression was associated with integrin expression during neuronal differentiation. Live label immunocytochemistry was performed for α3 integrin at 5 DIV, a time point when TIMP-2 expression was greatest in most cell lines. With a few exceptions, cell surface TIMP-2 expression correlated with α3 integrin expression (Fig. 8a). In PC12 cells, α3 integrin was expressed under all treatment conditions (Fig. 8a1−6). Although surface TIMP-2 was not detected prior to growth factor addition (Fig. 2a8), α3 integrin was observed (Fig. 8a1). Similar to TIMP-2, α3 integrin expression was not detected on Neuro-2a (Fig. 8a7-9) or N1E-115 (Fig. 8a10-12) cells. Surface TIMP-2 was present on pluripotent P19 cells (Fig. 6a), yet no α3 integrin expression was detectable (Fig. 8a13). Because PC12 cells express α3β1 and α1β1 integrins (Tomaselli et al. 1990; Arregui et al. 1994), live label immunocytochemistry was performed to determine whether TIMP-2 colocalized with these integrins in PC12 cells. TIMP-2 colocalized with α1 (Fig. 8b9), α3 (Fig. 8b10), and β1 (Fig. 8b12) integrins on the soma and neuritic processes. Labeling specificity is demonstrated by the lack of α5 integrin expression (Fig. 8b11). These data corroborate our previous observation that TIMP-2 binds to α3β1 in NGF treated PC12 cells (Pérez-Martínez & Jaworski, 2005). In sum, TIMP-2 expression is regulated by multiple factors coincident with neuronal differentiation and its cell surface expression principally correlates with the expression of α3β1 integrin.
Figure 8. Cell surface TIMP-2 expression correlates with α3 integrin expression. (a) Live label immunocytochemistry with α3 integrin at 5 DIV revealed that, like TIMP-2, cell surface labeling for α3 integrin was only present on PC12 cells. α3 integrin expression (a) was greater than cell surface TIMP-2 (Fig. 2a8) in serum starved PC12 cells. Like TIMP-2, α3 integrin expression was up-regulated by overnight growth factor deprivation (b) and further increased by growth factor addition (3, 4, 5 and 6). In NGF differentiated PC12 cells, α3 integrin was expressed on the soma and neuritic processes (c). Neuro-2a, N1E-115 and P19 cells lack detectable α3 integrin expression. (b) Live label immunocytochemistry for TIMP-2 (1–4) and integrin (5–8) in PC12 cells treated with NGF for 5 days. TIMP-2 colocalizes with α1 (9), α3 (10), and β1 (12) integrin. Scale bar = 50 µm (a), 25 µm (b).
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