Journal of Neurochemistry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry
Rapid Communication
Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells *-f-Jin-YiChiou and *Edward W. Westhead *Program in Molecular and Cellular Biology, University of Massachusetts at Amherst, Amherst, Massachusetts, U.S.A.; and ?National Laboratories of Foods and Drugs, Department of Health, Taipei, Taiwan, R.O.C.
Abstract: The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PCI 2 cells. PC 12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an of -7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGF. Key Words: Okadaic acid-Neurite outgrowth-PC12 cells. Chiou J.-Y. and Westhead E. W. Okadaic acid, a protein phosphatase inhibitor, inhibits nerve growth factor-directed neurite outgrowth in PC12 cells. J. Neurochem. 59, 1963-1966 (1992).
tion of several intracellular proteins, including histone H 1 and H3, ribosomal protein S6, tyrosine hydroxylase (Halegoua and Patrick, 1980; Mitchell et al., 1990), neurofilament proteins (Lindenbaum et al., 1987), and phospholipase C-7 (Kim et al., 1991). Furthermore, protein kinase C (PKC) has been reported to act at a distal segment of the neurite growth pathway (Hall et al., 1988), and a role for PKC in neurite outgrowth has recently been supported by the inhibitory effect of PKC antibodies (Altin et al., 1992). Sano et al. (1990) have indicated that an NGF-dependent protein kinase is possibly involved in neurite outgrowth in PC12 cells. The NGF receptor has itself been identified as a protein kinase (Ross, 1991;Meakin et al., 1992). Therefore, the activity of protein kinases is thought to be a crucial step in NGF-directed neunte outgrowth. The extent of phosphorylation of serine, threonine, and tyrosine residues in regulated proteins reflects the relative activities of the protein kinases and protein phosphatases acting on them. If the phosphorylation of certain cellular proteins is of physiological significance to NGF-treated PC 12 cells. then it is reasonable to assume that the dephosphorylation of these proteins by protein phosphatase has to be under strict regulation. Okadaic acid (OKA), a polyether fatty acid first isolated from the marine sponges of the genus Halichondria (Suganuma et al., 1988), is a tumor promoter and has been demonstrated in vitro to be a potent inhibitor of protein phosphatase types 2A (ICs0= 0.2-0.5 nM)and 1 (Ks0= 20-70 nM)and a much less potent inhibitor of type 2B. OKA has no effect on protein phosphatase type 2C, alkaline phosphatases, acid phosphatases, phosphotyrosyl phosphatases, and inositol 1,4,5-trisphosphate phosphatase (Bialojan and Takai, 1988; Cohen, 1989; Honkanen et al., 1991). OKA has been widely used with intact cells at micromolar or submicromolar concentrations to inhibit selectively either type 2A or type 2A plus type 1 phosphatases, to study the roles of these enzymes in cellular functions (Haa-
PC12 clonal cell lines are derived from the chromaffin cells of the rat adrenal medulla. PC 12 cells undergo differentiation when exposed to nerve growth factor (NGF), turning from the quiescent chromaffin cell-like state, in which they are round, to the neuronal state, in which they extend processes (Greene and Tischler, 1976) and even form synapses (Schubert et al., 1977). Although some properties of the NGF receptor, such as its structure, function, and regulation, have been elucidated (Hempstead and Chao, 1989), the intracellular transduction signals involved in NGF-directed neurite outgrowth in PC12 cells are still unclear. Previous studies have shown that in NGF-treated PC12 cells there is a selective increase in the phosphorylaResubmitted manuscript received July 24, 1992; accepted July 28, 1992. Address correspondence and reprint requests to Dr. E. W. Westhead at Program in Molecular and Cellular Biology, Lederle Graduate Research Towers, 435 Mom11 Science Center, University of Massachusetts, Amherst, MA 01003, U.S.A.
Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; MAP, microtubule-associated protein; NGF, nerve growth factor; OKA, okadaic acid; PKC, protein kinase C.
1963
J.-Y. CHIOU AND E. W. WESTHEAD
I964
vik et al., 1989; Cohen et al., 1990; Miyasaka et al., 1990; Shenolikar and Nairn, 1991). In this study, OKA was used at nanomolar concentrations to uncover the role of protein phosphatases in mediating NGF-directed neurite outgrowth. We demonstrate that the neurite outgrowth of PC12 cells is reversibly inhibited by 5-10 nMOKA, implying a physiological role for protein phosphatases in neurite extension.
MATERIALS AND METHODS Materials NGF (7s NGF from mouse submaxillary glands) and rabbit anti-NGF (2.5s) were purchased from Sigma. OKA was from Kamiya Biomedical Co.
Cell culture After removal from liquid nitrogen, PC12 cells were adapted to grow in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum (GIBCO), 5% fetal calf serum (Intergen), 100 units/ ml of penicillin, 100 pg/ml of streptomycin, and 0.25 &ml of amphotericin B (Fungizone) in a 37"C, water-saturated atmosphere of 95% air and 5% COz. Cells were plated on rat tail collagen-coated culture dishes (35 X 10 mm; Falcon) at a density of 1 X lo3cells/cm2 3 days before NGF and OKA were added. Medium was changed every day, and cells were treated with NGF (50 ng/ml) and/or OKA as indicated in the figure legends.
OKA treatment OKA was kept in dimethyl forniamide at 4°C as a stock solution of 100 Fg/ml. It was diluted to 1 &ml with sterile phosphate-buffered saline (pH 7.4) before it was added to medium to make its various final concentrations. To wash out NGF and/or OKA, cell cultures were rinsed thoroughly with warm DMEM without supplement but with serum for three times after medium was removed. Cells were examined by phase-contrast microscopy (Nikon) under 200x magnification.
response to NGF was suppressed by one-third with 5 nM and by two-thirds with 10 nMOKA (Fig. 1). On washout of OKA, this suppression was reversed, and neurites grew at a rate similar to the initial rate of growth in cells exposed to NGF alone. When cells were treated with NGF and various concentrations of OKA for 3 days, the half-maximal inhibition (IC5,,) was 7 nM (Fig. 2). NGF-treated cells appeared larger and flatter than untreated cells (Fig. 3A and B), suggesting that the treated cells have greater adhesion to the culture surface. Apparently, OKA inhibited the neurite outgrowth but not the other morphological changes during 3 days of exposure to NGF (Fig. 3C). When OKA treatment was terminated after 2 days but NGF remained in the culture medium, cells extended neurites promptly during the next 24-h interval (Fig. 3D). OKA concentrations of 1 5 &had no effect on cell viability within 3 days, as determined by trypan blue exclusion and phase-contrast microscopy. Approximately 10- 15% of the cells lost their viability after 3 days of concurrent treatment with NGF and 10 nMOKA. OKA appears to have a cytotoxic effect at a higher concentration than needed for suppression of neurite outgrowth. OKA not only inhibited neurite outgrowth but also enhanced neurite degeneration (Fig. 4). After priming with NGF for 4 days, -33% of the cells were neurite bearing. After the washout of NGF, control cells showed a continuing increase in neurite outgrowth during the first day. In contrast, if cells were treated with 10 nMOKA at 6 h after NGF washout, the number of neurite-bearing cells dropped by almost one-third in the next 18 h. Neurite degeneration of individual cells was also observed and photographed by culturing cells on CELLocate coverslips (Eppendorf), which have labeled grids for cell location. An experiment exactly replicating that of Fig. 4 was also carried out, but adding a third group of cells to the experiment. In the third group, after NGF was washed out, antiNGF antibody was added to the medium to ensure inactivation of any residual NGF. The results with and without antibody were identical. Neurite outgrowth without antibody was as follows: at 24 h, 39 & 5.6% (n = 3); at 48 h, 35
Anti-NGF treatment Lyophilized anti-NGF powder was dissolved in 0.1 ml of deionized water to make a stock solution. An aliquot of reconstituted rabbit anti-NGF was added to medium containing 50 ng/ml of NGF to bring the final concentration of antibody to 1:50 dilution from the stock and incubated for 30 min at room temperature before it was added to PC12 cells. The activity of anti-NGF in blocking the ability of NGF to stimulate neurite outgrowth from PC12 cells was monitored by phase-contrast microscopy over a 4-day penod.
RESULTS AND DISCUSSION PC 12 cells extend neurites gradually and reach a maximal response after a 1-week exposure to NGF (Greene and Tischler, 1976). In our experiments, PC12 cells exhibited characteristic changes in cellular morphology after 1 day in the presence of 50 ng/ml ofNGF. After 3 days, -20-25% of the NGF-treated cells extended neurites that were longer than their cell bodies, whereas
Rapid Communication
Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells *-f-Jin-YiChiou and *Edward W. Westhead *Program in Molecular and Cellular Biology, University of Massachusetts at Amherst, Amherst, Massachusetts, U.S.A.; and ?National Laboratories of Foods and Drugs, Department of Health, Taipei, Taiwan, R.O.C.
Abstract: The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PCI 2 cells. PC 12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an of -7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGF. Key Words: Okadaic acid-Neurite outgrowth-PC12 cells. Chiou J.-Y. and Westhead E. W. Okadaic acid, a protein phosphatase inhibitor, inhibits nerve growth factor-directed neurite outgrowth in PC12 cells. J. Neurochem. 59, 1963-1966 (1992).
tion of several intracellular proteins, including histone H 1 and H3, ribosomal protein S6, tyrosine hydroxylase (Halegoua and Patrick, 1980; Mitchell et al., 1990), neurofilament proteins (Lindenbaum et al., 1987), and phospholipase C-7 (Kim et al., 1991). Furthermore, protein kinase C (PKC) has been reported to act at a distal segment of the neurite growth pathway (Hall et al., 1988), and a role for PKC in neurite outgrowth has recently been supported by the inhibitory effect of PKC antibodies (Altin et al., 1992). Sano et al. (1990) have indicated that an NGF-dependent protein kinase is possibly involved in neurite outgrowth in PC12 cells. The NGF receptor has itself been identified as a protein kinase (Ross, 1991;Meakin et al., 1992). Therefore, the activity of protein kinases is thought to be a crucial step in NGF-directed neunte outgrowth. The extent of phosphorylation of serine, threonine, and tyrosine residues in regulated proteins reflects the relative activities of the protein kinases and protein phosphatases acting on them. If the phosphorylation of certain cellular proteins is of physiological significance to NGF-treated PC 12 cells. then it is reasonable to assume that the dephosphorylation of these proteins by protein phosphatase has to be under strict regulation. Okadaic acid (OKA), a polyether fatty acid first isolated from the marine sponges of the genus Halichondria (Suganuma et al., 1988), is a tumor promoter and has been demonstrated in vitro to be a potent inhibitor of protein phosphatase types 2A (ICs0= 0.2-0.5 nM)and 1 (Ks0= 20-70 nM)and a much less potent inhibitor of type 2B. OKA has no effect on protein phosphatase type 2C, alkaline phosphatases, acid phosphatases, phosphotyrosyl phosphatases, and inositol 1,4,5-trisphosphate phosphatase (Bialojan and Takai, 1988; Cohen, 1989; Honkanen et al., 1991). OKA has been widely used with intact cells at micromolar or submicromolar concentrations to inhibit selectively either type 2A or type 2A plus type 1 phosphatases, to study the roles of these enzymes in cellular functions (Haa-
PC12 clonal cell lines are derived from the chromaffin cells of the rat adrenal medulla. PC 12 cells undergo differentiation when exposed to nerve growth factor (NGF), turning from the quiescent chromaffin cell-like state, in which they are round, to the neuronal state, in which they extend processes (Greene and Tischler, 1976) and even form synapses (Schubert et al., 1977). Although some properties of the NGF receptor, such as its structure, function, and regulation, have been elucidated (Hempstead and Chao, 1989), the intracellular transduction signals involved in NGF-directed neurite outgrowth in PC12 cells are still unclear. Previous studies have shown that in NGF-treated PC12 cells there is a selective increase in the phosphorylaResubmitted manuscript received July 24, 1992; accepted July 28, 1992. Address correspondence and reprint requests to Dr. E. W. Westhead at Program in Molecular and Cellular Biology, Lederle Graduate Research Towers, 435 Mom11 Science Center, University of Massachusetts, Amherst, MA 01003, U.S.A.
Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; MAP, microtubule-associated protein; NGF, nerve growth factor; OKA, okadaic acid; PKC, protein kinase C.
1963
J.-Y. CHIOU AND E. W. WESTHEAD
I964
vik et al., 1989; Cohen et al., 1990; Miyasaka et al., 1990; Shenolikar and Nairn, 1991). In this study, OKA was used at nanomolar concentrations to uncover the role of protein phosphatases in mediating NGF-directed neurite outgrowth. We demonstrate that the neurite outgrowth of PC12 cells is reversibly inhibited by 5-10 nMOKA, implying a physiological role for protein phosphatases in neurite extension.
MATERIALS AND METHODS Materials NGF (7s NGF from mouse submaxillary glands) and rabbit anti-NGF (2.5s) were purchased from Sigma. OKA was from Kamiya Biomedical Co.
Cell culture After removal from liquid nitrogen, PC12 cells were adapted to grow in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum (GIBCO), 5% fetal calf serum (Intergen), 100 units/ ml of penicillin, 100 pg/ml of streptomycin, and 0.25 &ml of amphotericin B (Fungizone) in a 37"C, water-saturated atmosphere of 95% air and 5% COz. Cells were plated on rat tail collagen-coated culture dishes (35 X 10 mm; Falcon) at a density of 1 X lo3cells/cm2 3 days before NGF and OKA were added. Medium was changed every day, and cells were treated with NGF (50 ng/ml) and/or OKA as indicated in the figure legends.
OKA treatment OKA was kept in dimethyl forniamide at 4°C as a stock solution of 100 Fg/ml. It was diluted to 1 &ml with sterile phosphate-buffered saline (pH 7.4) before it was added to medium to make its various final concentrations. To wash out NGF and/or OKA, cell cultures were rinsed thoroughly with warm DMEM without supplement but with serum for three times after medium was removed. Cells were examined by phase-contrast microscopy (Nikon) under 200x magnification.
response to NGF was suppressed by one-third with 5 nM and by two-thirds with 10 nMOKA (Fig. 1). On washout of OKA, this suppression was reversed, and neurites grew at a rate similar to the initial rate of growth in cells exposed to NGF alone. When cells were treated with NGF and various concentrations of OKA for 3 days, the half-maximal inhibition (IC5,,) was 7 nM (Fig. 2). NGF-treated cells appeared larger and flatter than untreated cells (Fig. 3A and B), suggesting that the treated cells have greater adhesion to the culture surface. Apparently, OKA inhibited the neurite outgrowth but not the other morphological changes during 3 days of exposure to NGF (Fig. 3C). When OKA treatment was terminated after 2 days but NGF remained in the culture medium, cells extended neurites promptly during the next 24-h interval (Fig. 3D). OKA concentrations of 1 5 &had no effect on cell viability within 3 days, as determined by trypan blue exclusion and phase-contrast microscopy. Approximately 10- 15% of the cells lost their viability after 3 days of concurrent treatment with NGF and 10 nMOKA. OKA appears to have a cytotoxic effect at a higher concentration than needed for suppression of neurite outgrowth. OKA not only inhibited neurite outgrowth but also enhanced neurite degeneration (Fig. 4). After priming with NGF for 4 days, -33% of the cells were neurite bearing. After the washout of NGF, control cells showed a continuing increase in neurite outgrowth during the first day. In contrast, if cells were treated with 10 nMOKA at 6 h after NGF washout, the number of neurite-bearing cells dropped by almost one-third in the next 18 h. Neurite degeneration of individual cells was also observed and photographed by culturing cells on CELLocate coverslips (Eppendorf), which have labeled grids for cell location. An experiment exactly replicating that of Fig. 4 was also carried out, but adding a third group of cells to the experiment. In the third group, after NGF was washed out, antiNGF antibody was added to the medium to ensure inactivation of any residual NGF. The results with and without antibody were identical. Neurite outgrowth without antibody was as follows: at 24 h, 39 & 5.6% (n = 3); at 48 h, 35
Anti-NGF treatment Lyophilized anti-NGF powder was dissolved in 0.1 ml of deionized water to make a stock solution. An aliquot of reconstituted rabbit anti-NGF was added to medium containing 50 ng/ml of NGF to bring the final concentration of antibody to 1:50 dilution from the stock and incubated for 30 min at room temperature before it was added to PC12 cells. The activity of anti-NGF in blocking the ability of NGF to stimulate neurite outgrowth from PC12 cells was monitored by phase-contrast microscopy over a 4-day penod.
RESULTS AND DISCUSSION PC 12 cells extend neurites gradually and reach a maximal response after a 1-week exposure to NGF (Greene and Tischler, 1976). In our experiments, PC12 cells exhibited characteristic changes in cellular morphology after 1 day in the presence of 50 ng/ml ofNGF. After 3 days, -20-25% of the NGF-treated cells extended neurites that were longer than their cell bodies, whereas
