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Intracellular signaling pathways involved in mediating the effects of lead on the transcription factor Sp1
Int. J. Devl Neuroscience 21 (2003) 235–244
Intracellular signaling pathways involved in mediating the effects of lead on the transcription factor Sp1 D.S. Atkins a , Md. Riyaz Basha b , N.H. Zawia b,∗ b
a Department of Pharmacology, Meharry Medical College, Nashville, TN 37028, USA Department of Biomedical Sciences, University of Rhode Island, Kingston, RI 02881, USA
Received 9 January 2003; received in revised form 9 April 2003; accepted 24 April 2003
Abstract It has been well established that exposure to Pb during critical periods of brain development results in both cognitive and behavioral deficits. Although the mechanism by which Pb induces developmental neurotoxicity is unknown, it may involve alterations in transcription of genes that are essential for growth and differentiation. Recent studies reveal that Pb interferes with growth and differentiation by acting on the transcription factor Sp1. Pb-induced changes in the activity of Sp1 may be consequent to alterations in intermediates in signal transduction pathways. This study examines both in vivo and in vitro the role of signaling factors in mediating the effects of Pb on Sp1 DNA-binding. Hippocampal developmental profiles of Sp1 DNA-binding, PKC, and MAPK protein levels were monitored in Pb-exposed rats. Pb exposure resulted in an induction of Sp1 DNA-binding during PND 5–10 followed by a subsequent decline on PND 15–20. The protein expression profiles for PKC␣ and MAPK followed a relatively similar pattern. To examine the interdependence between Sp1 DNA-binding, PKC␣, and MAPK, PC12 cells were exposed to Pb and/or NGF. Pb or NGF exposure increased Sp1 DNA-binding. Addition of the PKC inhibitor (staurosporine) diminished NGF and Pb-induced Sp1 DNA-binding, while the MAPK inhibitor (PD 98059), completely abolished both basal and induced Sp1 DNA-binding. These findings demonstrate that Sp1 DNA-binding is regulated by PKC and MAPK, which may serve as mediators through which Pb may indirectly modulate Sp1 DNA-binding. © 2003 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Pb; Sp1; PKC; MAPK; CNS; PC12
1. Introduction Lead (Pb) is universally accepted as a potent environmental toxic metal which imparts profound effects on brain development. Pb poisoning in children persists as a major public health problem (Rodier, 1990; Silbergeld, 1990, 1992). A variety of studies have revealed that Pb exposure causes deficits in CNS functioning and behavioral disorders including learning disabilities and impaired cognitive development in children (Lyngbye et al., 1990; Needleman et al., 1990; Silbergeld, 1990, 1992). Although the mechanisms involved in Pb-induced developmental neurotoxicity are not fully defined, it has been clearly shown that Pb interferes with the transcription of genes that are essential for brain growth and differentiation (Zawia and Harry, 1996). Transcription factors play a vital role in the orchestration of gene expression during development. Extensive studies from our laboratory have shown that Pb exposure alters the DNA-binding of zinc finger protein (ZFP) ∗
Corresponding author. Tel.: +1-401-874-5909; fax: +1-401-874-5048. E-mail address: [email protected] (N.H. Zawia).
transcription factors such as Sp1 (Zawia et al., 1998, 2000). The DNA-binding of other ZFPs such as Egr1 and TFIID have also been found to be modulated by Pb (Hanas et al., 1999; Zawia et al., 2000). However, it is not clear whether Pb within the cell acts directly on Sp1, thereby modulating its activity or whether the effects of Pb on Sp1 DNA-binding are secondary to the action of Pb on other cellular intermediates. Transcription/transduction coupling is a necessary intermediate in the regulation of gene expression. Therefore, we propose that Pb-induced changes in the activity of Sp1 may be consequent to alterations in intermediates in signal transduction pathways associated with growth and differentiation. Potential candidates for such a mediation are protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). PKC plays a major role in cell growth and differentiation and is a known target for Pb (Murakami et al., 1987; Markovac and Goldstein, 1988a, b). It has also been shown that PKC activity is potently increased by Pb (Murakami et al., 1987; Markovac and Goldstein, 1988a, b). Pb in vitro mimics calcium in the central nervous system by entering the cell via calcium channels (Bressler and Goldstein, 1991); however, in vivo studies that have
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examined the interactions between PKC and Pb in exposed animals are lacking. Studies from our laboratory have revealed that Pb-induced manifestations such as induction in Sp1 DNA-binding and differentiation of pheochromocytoma cells (PC12) were similar to those of nerve growth factor (NGF), suggesting the involvement of the MAPK pathway (Crumpton et al., 2001). The MAPK cascade is a signaling system involved in various physiological processes such as cellular proliferation, differentiation, apoptosis, and development (Chang and Karin, 2001). This pathway is initiated by binding of extracellular mitogens, such as NGF, to receptor tyrosine kinases and then MAPK is phosphorylated and translocated to the nucleus, in turn activating a plethora of transcription factors including Sp1 (Merchant et al., 1999; Liu et al., 2001). The present investigation is aimed at delineating the link between Sp1 DNA-binding, PKC, and MAPK, providing an approach to understand the involvement of intracellular signaling pathways in mediating the effects of Pb on Sp1 DNA-binding and gene expression. 2. Experimental procedures 2.1. Animal exposure Timed-pregnant Long-Evans hooded rats were obtained from Charles River Laboratories (Raleigh, NC). Twenty-four hours following birth was designated as postnatal day 1 (PND 1). All pups were pooled and new litters consisting of 10 males were randomly selected and placed with each dam. Exposure to Pb was initiated on PND 1 with the addition of the acetate form (0.2% lead acetate, Sigma, St. Louis, MO) to the drinking water of the dam and continued until PND 20 (Zawia et al., 1998, 2000; Reddy and Zawia, 2000). Control dams received drinking water. Food and water were freely available throughout the study. The dams with pups were individually housed at constant temperature (21 ± 2 ◦ C) and relative humidity (50 ± 10%) with a 12 h light/dark cycle (07:00–19:00 h). On PND 3, 5, 10, 15, 20, and 30 randomly selected pups were removed from each litter and replaced with filler pups to maintain a constant litter size until weaning. Animals were decapitated following exposure to CO2 and hypothermia and brain regions were dissected, frozen on dry-ice and stored at −80 ◦ C.
washing nuclei in TKM buffer with 0.3% Triton X-100 followed by TKM buffer plus 0.15% Triton X-100 and finally TKM buffer alone to remove excess traces of detergents. After each wash, nuclei were sedimented by centrifugation at 3300 × g for 12 min at 4 ◦ C, and all supernatants were pooled. Nuclear pellets were resuspended in 0.5 ml of TKM buffer and nuclei disrupted by repeated passage through a 22-gauge needle. The combined supernatants were centrifuged at 100,000 × g for 1 h at 4 ◦ C, and the supernatant obtained was the cytosolic fraction. The pellet obtained (i.e. the particulate or membrane fraction) was resuspended in TKM buffer plus 0.2% Igepal, rocked on ice for 30 min, then centrifuged at 100,000 × g for 30 min at 4 ◦ C. The supernatant was saved as the membrane extract. All reagents mentioned in this procedure were obtained from Sigma (St. Louis, MO). 2.3. Western blot analysis Initially five isozymes of PKC, namely, ␣, I, II, ␥, and ε were screened for their ability to show a developmental profile in all three cellular fractions. Only nuclear PKC␣ levels exhibited a developmental profile that was prominently modulated by Pb. Therefore, subsequent studies focused on this isozyme. The levels of PKC␣ and MAPK were estimated in nuclear extracts of hippocampal tissue as well as PC12 cells by Western blot analysis. Twenty micrograms of protein extracts were fractionated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) using 1× Tris–glycine–SDS buffer (BioRad, Hercules, CA), then transferred electrophoretically into 0.2 m nitrocellulose membranes (Sigma, St. Louis, MO) using a semi-dry electroblotter and Tris buffer (25 mM Tris–HCl, 192 mM glycine and 20% (v/v) methanol). Membranes were blocked for 1 h with 5% evaporated milk, then incubated with a polyclonal rabbit anti-PKC or anti-MAPK (1:1000; Santa Cruz Immunochemicals, Santa Cruz, CA) for 1 h, followed by subsequent incubation with secondary antibody (1:20,000; goat anti-rabbit IgG (H + L) horseradish peroxidase; Pierce, Rockford, IL) for 1 h. Blots were incubated for 1 min in a system of chemiluminescent reagents (Pierce) then exposed to X-ray film for 5 s. Development of the film produced immunoreactive bands whose densities were later quantified with the Alpha Innotech imaging system. 2.4. Preparation of in vitro samples
2.2. Subcellular fractionation Subcellular fractions were prepared according to the method described by Adunyah et al. (1991) with slight modifications. Tissue or cells were homogenized in 10 volumes of TKM buffer (10 mM Tris–HCl, 1.5 mM MgCl2 , 10 mM KCl, 2 mM EDTA, 2.5 mM EGTA, 5% (w/v) glycerol, 0.1% (v/v) ME, 10 g/ml aprotinin, 10 g/ml leupeptin, 0.5 mM PMSF, and 1 mM DTT), and then centrifuged at 750 × g for 10 min at 4 ◦ C. Perinuclear proteins were removed by
Pheochromocytoma (PC12) cells (ATCC, Manassas, VA) maintained under conditions of 5% CO2 and 37 ◦ C were grown to semi-confluency in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Logan, UT) supplemented to a final concentration with 10% fetal bovine serum (Hyclone), 5% horse (equine) serum (Hyclone) and 1% penicillin– streptomycin solution (Sigma, St. Louis, MO). Subsequently, cells were exposed to various doses of Pb (final concentrations 0.025, 0.1, and 1 M), NGF (final
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concentrations 0.3, 3, and 50 ng/ml; Calbiochem, San Diego, CA) or a combination of the two (0.1 M Pb and 50 ng/ml NGF). Either the PKC inhibitor staurosporine (STAURO, 0.1 M) or the MAPK inhibitor, PD 98059 (25 M) were added to separate groups of control and Pb or NGF-exposed cells. Forty-eight hours later, nuclear extracts were prepared following trypsinization to remove adherent cells. 2.5. Nuclear protein extraction Nuclear proteins were extracted from both hippocampal tissue and PC12 cells according to the method given by Dignam et al. (1983), with slight modifications. Cells/tissues were homogenized in phosphate buffered saline (PBS) and the homogenates were homogenized with 1 ml PBS, pH 7.5, and centrifuged at 2500 × g for 10 min. The pellet obtained was resuspended in five volumes of buffer A (10 mM HEPES at pH 7.9, 1.5 mM MgCl2 , 0.5 mM DTT, 0.5 mM EDTA, and 0.2 mM PMSF) and centrifuged at 6000 rpm for 2 min at 4 ◦ C. Again, the pellet was resuspended in three volumes of buffer A and centrifuged at 6000 rpm for 2 min at 4 ◦ C. The pellet was then resuspended in five volumes of buffer C (20 mM HEPES at pH 7.9, 1.5 mM MgCl2 , 0.5 mM DTT, 0.5 mM EDTA, 420 mM NaCl, 20% glycerol, 0.2 mM PMSF, 0.002 mg/ml aprotinin, and 0.0005 mg/ml leupeptin) and homogenized. The final suspensions were centrifuged at 12,000 × g for 10 min. The supernatants were transferred to 1.5 ml tube, snap frozen in an ethanol dry-ice bath, and then stored at −80 ◦ C. 2.6. Electrophoretic mobility shift assay (EMSA) The Sp1 oligonucleotide (1.75 pmol/l) (Promega) was incubated with 1 l of [␥-32 P] ATP (3000 Ci/mmol) and 5–10 units of T4 polynucleotide kinase in a final volume of 10 l, for 10 min at 37 ◦ C. A reaction mixture containing 20 l of gel shift binding buffer (2 mM HEPES, 2.5 mM MgCl2 , 2.5 mM KCl, 0.5 mM DTT, 2.5% glycerol, 0.001% NP40, 50 g/ml BSA, 0.2% protease inhibitor cocktail (Sigma), 0.2% ficoll, and 20 ng/ml polydIdC) and 1 l of the labeled oligonucleotide (Sp1) probe was prepared. Then, 5–10 g of protein from the nuclear extracts of hippocampal tissue or PC12 cells was added to the reaction mixture. All reactions were done on ice unless otherwise stated. After 10 min incubation at room temperature, 1 l gel loading buffer was added and the final reaction mixture was then loaded onto a 4% polyacrylamide gel and electrophoretically resolved. The gel was exposed to X-ray film overnight, and the resulting autoradiograms were analyzed for shifted bands and quantified using an image acquisition and analysis software (UVP, CA).
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and hippocampus. The metal analysis was carried out following the method as described by Smith et al. (1998). Animals were deeply anesthetized and perfused with sterile saline via cardiac puncture; the brain was excised and the hippocampus was isolated. Samples were pooled from three animals and three to four sample determinations were made for each exposure condition. Pb levels in the hippocampal tissue and blood were analyzed by RI Analytical Laboratory, Warwick, RI, using atomic absorption spectroscopy following EPA approved methodology (Method: 200.9). 2.8. Statistical analysis Data were analyzed by analysis of variance (ANOVA) and that was followed by Dunnet’s post hoc test to compare the effects among various groups and P < 0.05 was considered significant.
3. Results Experiments were conducted to examine the relationship between Sp1 DNA-binding and signaling intermediates such as PKC and MAPK both in vivo and in vitro. As we had previously published (Zawia and Harry, 1996; Zawia et al., 1998), we did not observe any changes in body or brain weights of Pb-exposed animals, which might be indicative of gross nutritional abnormalities. Consistent with our previously published values (Harry et al., 1996; Basha et al., 2003), the Pb levels in the hippocampus of these animals were measured on PND 20 and found to be <0.2 g/g in control animals and 0.34 ± 0.012 g/g in Pb-exposed animals. Similarly, the concentrations of Pb used in vitro did not result in any cytotoxicity to the cells (Crumpton et al., 2001). 3.1. Sp1 DNA-binding in the hippocampus Previous studies from our laboratory had revealed that Pb exposure results in perturbations in Sp1 DNA-binding in the cerebellum (Zawia et al., 1998, 2000). In the present study, Sp1 DNA-binding was monitored in the hippocampus of control and Pb-exposed rat pups from PND 3 to PND 30. Consistent with our earlier studies in the cerebellum (Zawia et al., 1998), Sp1 DNA-binding was low in control animals at PND 3 and gradually increased on PND 15–20 decreasing thereafter. In Pb-exposed animals, premature peaks of Sp1 DNA-binding appeared on PND 5–10 and then declined significantly to below those of control animals on subsequent days (Fig. 1).
2.7. Lead determination
3.2. PKC
On postnatal day 20, pups from both the control and Pb groups were used to determine the levels of Pb in the blood
Since it has been shown that Pb alters PKC activity, changes in hippocampal PKC levels were monitored
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Fig. 1. Developmental profiles of Sp1 DNA-binding in the hippocampus of control and Pb-exposed rats. Sp1 DNA-binding was monitored using the gel mobility shift assay. Shifted bands were scanned and quantified using image acquisition and analysis software (UVP Laboratory Products, CA). Values shown are for the mean ± S.E. from four independent experiments. Values indicated with an “∗” are significant (P < 0.05) over controls at the same postnatal age, as determined by ANOVA. A representative autoradiogram is shown in the box.
Fig. 2. Pb-induced changes in the developmental profiles of nuclear PKC␣ levels in the rat hippocampus. Control and Pb-exposed rat pups were sacrificed on various days and their brains were removed and dissected. Nuclear protein extracts were examined by Western blot analysis using a polyclonal PKC␣ antibody (1:1000). The density of immunoreactive bands was quantified using the Alpha Innotech Imaging System. Values shown are for the mean ± S.E. from three independent experiments. Values indicated with an “∗” are significant (P < 0.05) over controls of the same postnatal age, as evaluated by ANOVA.
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Fig. 3. Changes in the developmental profiles of nuclear MAPK levels in the rat hippocampus following Pb exposure. Control and Pb-exposed rat pups were sacrificed on various days and their brains removed and dissected. Nuclear protein extracts were examined by Western blot analysis using a polyclonal MAPK (ERK2) antibody (1:1000). The density of immunoreactive bands was quantified using the Alpha Innotech Imaging System. Values shown are for the mean ± S.E. from three independent experiments. Values indicated with “∗” are significant at P < 0.05 over controls of the same postnatal age, as evaluated by ANOVA.
following exposure to Pb. In a pilot study, five isozymes of PKC, namely, ␣, I, II, ␥, and ε were initially screened for their ability to show a developmental profile, and their responses to Pb exposure. We found that only PKC␣ exhibited a definite profile which was modulated by Pb exposure. Further studies were performed to determine the levels of PKC␣ in hippocampal nuclear extracts. We observed that the levels of PKC␣ in control animals exhibited a developmental profile (Fig. 2) that was similar to Sp1 DNA-binding (Fig. 1). In Pb-exposed animals minor changes were observed in the early days after birth followed by a significant decrease at later time points, that is, PND 20–30. 3.3. MAPK MAPK immunoreactivity in hippocampal nuclear extracts was examined in the same period as Sp1 DNA-binding and PKC␣. We found that the developmental profiles of MAPK levels (Fig. 3) were similar (in both control and Pb-exposed animals) to those of PKC␣ (Fig. 2), as well as Sp1 DNA-binding (Fig. 1). However, while all patterns were generally similar, there was a greater resemblance among the profiles of the levels of the two enzymes (PKC␣ and MAPK).
3.4. Sp1 DNA-binding, PKCα, and MAPK in PC12 cells The similarities in Sp1 DNA-binding, PKC␣, and MAPK developmental patterns in the hippocampus implied that they may be linked to each other. In order to examine the relationship between these three different proteins, Sp1 DNA-binding, PKC␣, and MAPK levels were measured in NGF-stimulated and Pb-exposed PC12 cells in the presence or absence of enzyme inhibitors. As illustrated in Fig. 4, both Pb and NGF increased Sp1 DNA-binding in a concentration-dependent manner, 48 h following exposure. Administration of staurosporine significantly reduced the induction of Sp1 DNA-binding caused by both Pb and NGF, but failed to influence basal Sp1 DNA-binding. Studies were further conducted to examine whether the levels of the isoform PKC␣ were altered following exposure to either Pb or NGF, in the presence or absence of staurosporine. We found that NGF significantly increased the levels of PKC␣ in a dose-dependent manner, however, Pb alone had minor effects on PKC␣ levels (Fig. 5). Interestingly co-administration of 0.1 M Pb and 50 ng/ml NGF produced an effect that was intermediate to the effects shown individually, but significantly greater than that of Pb alone (Fig. 6). Consistent with the lack of effect of staurosporine on basal Sp1 DNA-binding in PC12 cells, the presence of
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Fig. 4. Modulation of the effects of Pb and NGF on Sp1 DNA-binding in PC12 cells by staurosporine (STAURO). Cells grown to semi-confluency were exposed to various concentrations of either NGF (0.3, 3, and 50 ng/ml), Pb (0.025, 0.1, and 1 M), or both, in the presence or absence of the potent PKC inhibitor STAURO (0.1 M). Forty-eight hours later, nuclear protein extracts were prepared and Sp1 binding to a specific DNA consensus sequence was observed by EMSA. Values shown are for the mean ± S.E. from three independent experiments. Values marked with letters are significant at P < 0.05, as evaluated by ANOVA: “a” over control, “b” over Pb (0.1 M), and “c” over NGF (50 ng/ml).
staurosporine had no effect on basal PKC␣ levels. On the other hand exposure to staurosporine tended to decrease PKC␣ levels when co-administered with either Pb or NGF (Fig. 6). To evaluate the involvement of MAPK in mediating the effects of Pb on Sp1 activity, we monitored the changes in Sp1 DNA-binding following exposure to either Pb or NGF in the presence of the MAPK inhibitor PD 98059 (MEKI). MEKI dramatically decreased basal Sp1 DNA-binding, but
Fig. 5. PKC␣ immunoreactive bands in control and exposed PC12 cells. PC12 cells grown to semi-confluency were exposed to various concentrations of NGF, Pb, and STAURO. Forty-eight hours later, cell extracts were prepared and the levels of PKC␣ were determined by Western blot analysis.
this effect could not be reversed by the addition of either Pb or NGF. However, unlike staurosporine, MEKI dramatically reduced basal Sp1 activity (Fig. 7).
4. Discussion Sp1 is important for the transcription of genes involved in growth and differentiation of the nervous system as evidenced by its high expression in the developing brain (Saffer et al., 1991). Exposure to Pb has been shown to result in perturbations in the regulation of gene expression and these disturbances in transcriptional events appear to be partially mediated by Sp1 (Zawia and Harry, 1996; Zawia et al., 1998, 2000). Both in vivo and in vitro studies from this laboratory have indicated that exposure to Pb induces premature inductions of Sp1 activity with a corresponding effect on its target genes (Zawia et al., 1998). These premature peaks in Sp1 activity are likely to force cells to differentiate leading to an early termination of the developmental program. While there is sufficient evidence to show that Pb interferes with Sp1 DNA-binding, the mechanisms associated
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Fig. 6. Modulation of the effects of Pb and NGF on PKC␣ levels in PC12 cells by STAURO. PC12 cells grown to semi-confluency were exposed to various concentrations of either NGF (0.3, 3, and 50 ng/ml), Pb (0.025, 0.1, and 1 M), or both, in the presence or absence of the potent PKC inhibitor STAURO (0.1 M). Forty-eight hours later, whole cell extracts were prepared, and effects on the levels of PKC␣ were determined by Western blot analysis. Values shown are for the mean ± S.E. from three independent experiments. Values marked with letters are significant at P < 0.05, as evaluated by ANOVA: “a” over control, “b” over Pb (0.1 M), and “c” over STAURO alone (0.1 M).
with this phenomenon are not understood. However, the involvement of signal transduction pathways are highly suspected. Several lines of evidence implicating PKC as a mediator in the effects of Pb (Markovac and Goldstein, 1988a, b; Laterra et al., 1992; Long et al., 1994) and PKC’s role in the control of growth and differentiation (Burgess et al., 1986) nominated it as a candidate for this study (Murakami et al., 1987; Markovac and Goldstein, 1988a, b; Jadhav et al., 2000; Nihei et al., 2001; Hwang et al., 2002; Deng and Poretz, 2002). The ability of Pb to stimulate neurite outgrowth and Sp1 DNA-binding in a manner similar to NGF also suggested that Pb may interfere with growth factor signaling pathways that are normally coupled to MAPK (Crumpton et al., 2001). The hippocampus is a brain region which undergoes major development after birth. Moreover this region is a center for the coordination of certain functions such as learning and memory and cognitive functions, which are impaired by Pb. Consistent with our earlier studies in the cerebellum (Zawia et al., 1998), exposure to Pb resulted in premature peaks of Sp1 DNA-binding during PND 5–10. These findings confirm that the effects of Pb are widespread in the brain and that gene expression in other areas may be disturbed by Pb.
Further studies were performed to examine the developmental patterns and changes in the levels of PKC and MAPK in this brain region to see whether they are temporally and characteristically related to Sp1. An initial pilot study demonstrated that brain PKC␣ isoform was developmentally regulated and its developmental profile was altered by Pb (data not shown). This was consistent with published reports (Hashimoto et al., 1988; Yoshida et al., 1988; Huang et al., 1990) showing that PKC␣ is present in the rat brain at birth and progressively increases during the first 3 weeks of age (Figs. 2 and 3). It is also consistent with the in vitro effects of Pb on human astrocytoma cells (Lu et al., 2001). However, other studies have suggested that PKC␥ is selectively altered by Pb in older animals (Nihei et al., 2001). Although PKC in the brain is found mainly associated with synaptic membranes and the cytosol (Kikkawa et al., 1982), there is evidence that it is also present in other cellular compartments (Kraft and Anderson, 1983). This is further corroborated by the demonstration that PKC␣ translocates to the nucleus (Leach et al., 1992). Although we have screened all three compartments (membrane, cytosolic, nuclear) for PKC levels, we found that only nuclear PKC␣ exhibited a developmental profile and was subject to modulation by Pb. Since the majority of studies in the literature
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Fig. 7. Effects of the MAP kinase inhibitor PD 98059 on the modulation of Sp1 DNA-binding by Pb and NGF. PC12 cells grown to semi-confluency were exposed to Pb (0.1 M), NGF (50 ng/ml), PD 98059 (MEKI, 25 M), or combinations of these as shown. Forty-eight hours later nuclear protein extracts were prepared, and Sp1 DNA-binding was observed by EMSA. Values shown are for the mean ± S.E. of three independent experiments. Values indicated with “a”, “b”, “c”, and “d” are significantly different (P < 0.05) from their corresponding controls, as evaluated by ANOVA.
measure PKC activity or isoforms in crude cellular extracts or the cytosol, the role of PKC on transcriptional events that are resident to the nucleus is usually missed. While PKC has a biological function in the cytosol, MAPK’s substrates are exclusively present in the nucleus. Therefore, MAPK must translocate to the nucleus upon activation, thereby phosphorylating a variety of proteins that are important for transcription (Merchant et al., 1999; Liu et al., 2001). In Pb-exposed animals, MAPK levels were decreased in the nucleus 2 weeks following exposure, and this decrement was sustained thereafter (Fig. 3). The overall developmental patterns for Sp1, PKC␣, and MAPK were generally similar. However, on PND 30, while both PKC␣ and MAPK levels remained low, Sp1 DNA-binding recovered to control levels (Fig. 1). These departures among the profiles of the enzymes and Sp1 DNA-binding indicates that Sp1 may also be subject to regulation by other factors and alternate signaling pathways. However, the striking similarities between the developmental profiles of these three proteins during specific developmental stages suggested that a link between them may exist. While there is no point to point agreement, there are resemblances among the profiles particularly after PND 15. PC12 cells are a commonly used in vitro model to explore the mechanisms of action of toxicants. Therefore, we examined the potential interactions of these three proteins in these cells. When added to PC12 cells, the potent PKC inhibitor
staurosporine had no effect on basal Sp1 DNA-binding or PKC␣ levels (Fig. 4). However, staurosporine effectively reduced both Sp1 DNA-binding as well as PKC␣ levels only when cells were either stimulated to differentiate by NGF or were exposed to Pb (Figs. 4 and 6). This suggested that PKC␣ does not regulate basal levels of Sp1 DNA-binding, but rather plays a role only under conditions of active cell differentiation. However, it is important to note that this may be cell-specific. Unlike PC12 cells, Pb exposure attenuates PKC␣ levels in human astrocytoma cells (Lu et al., 2001). This down-regulation in PKC␣ levels seen in human astrocytoma cells is similar to that observed in the brain of Pb-exposed rats (Fig. 2). On the other hand, the MAPK inhibitor (MEKI PD 98059) produced a dramatic decline in basal as well as stimulated Sp1 DNA-binding activity (Fig. 7). This would be consistent with MAPK’s later position in the signaling pathway, which allows it to receive converging signals from multiple sources. These data clearly indicate that MAPK plays a pivotal role in the nucleus to regulate Sp1 DNA-binding. However, both PKC␣ and MAPK appear to be involved in regulating the DNA-binding activity of Sp1 in a process subject to modulation by Pb. Since interruption in either PKC␣ or MAPK results in a lowering of Sp1 DNA-binding, it is quite possible that both these signaling intermediates are somehow linked. In support of this hypothesis, PKC␣ has been shown to
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phosphorylate, and therefore, activate Raf (MAP kinase kinase kinase). This is the first kinase activated following NGF binding to the high affinity TrkA receptors and is thus ultimately responsible for precipitating MAPK activation. Furthermore, inhibition of MAPK activity blocks differentiation in PC12 cells by NGF (Pang et al., 1995). More recent studies have demonstrated the direct phosphorylation of Sp1 by MAPK (ERK2) (Merchant et al., 1999; Liu et al., 2001). Although the in vivo and in vitro effects of Pb exposure on PKC␣ levels are divergent, the involvement of PKC␣ in the regulation of Sp1 DNA-binding is highly suspected. Tissue PKC␣ levels are products of the contributions of multiple cells at different stages of development, while PKC␣ levels obtained from PC12 cells are a manifestation of a single population of cells at identical stages of growth. Together, both in vivo and in vitro lines of evidence provide a link between NGF, PKC, MAPK, and Sp1. Pb may interact with this pathway by modulating PKC under conditions of active growth and thus, precipitating a chain of events leading to alterations in Sp1 DNA-binding activity. However, it is clear that PKC plays a partial role in regulating Sp1 DNA-binding in vitro, while MAPK is fully involved in maintaining Sp1 DNA-binding. Although Pb can directly alter the DNA-binding of a recombinant Sp1 protein (Razmiafshari and Zawia, 2000), these findings provide an important clue in understanding the mechanism by which Pb produces cellular neurotoxicity. Future studies should be aimed at controlling the activity of these pathways as a means of arresting the detrimental effects of Pb on gene expression and subsequent cellular growth and differentiation.
Acknowledgements The authors thank S. Bakheet for his technical help for the preparation of this manuscript. The authors are grateful to Dr. X. Sun, Research Asst. Professor (Statistics), Cancer Prevention Research Center, U.R.I. for providing assistance in the Statistical analysis. This work was partially funded by a Grant from NIEHS (ES08104). D. Atkins was supported by a fellowship from the EPA.
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Saffer, J.D., Jackson, S.P., Annarella, M.B., 1991. Developmental expression of Sp1 in the mouse. Mol. Cell. Biol. 11, 2189–2199. Silbergeld, E.K., 1990. Toward the twenty-first century: lessons from lead and lessons yet to learn. Environ. Health Perspect. 86, 191–196. Silbergeld, E.K., 1992. Mechanisms of lead neurotoxicity, or looking beyond the lamppost. FASEB J. 6, 3201–3206. Smith, D., Bayer, L., Strupp, B.J., 1998. Efficacy of succimer chelation for reducing brain Pb levels in rodent model. Environ. Res. 78, 168–176. Yoshida, Y., Huang, F.L., Nakabayashi, H., Huang, K.P., 1988. Tissue distribution and developmental expression of protein kinase C isozymes. J. Biol. Chem. 263, 9868–9873. Zawia, N.H., Harry, G.J., 1996. Developmental exposure to lead interferes with glial and neuronal differential gene expression in the rat cerebellum. Toxicol. Appl. Pharmacol. 138, 43–47. Zawia, N.H., Sharan, R., Brydie, M., Oyama, T., Crumpton, T., 1998. Sp1 as a target site for metal-induced perturbations of transcriptional regulation of developmental brain gene expression. Dev. Brain Res. 107, 291–298. Zawia, N.H., Crumpton, T., Brydie, M., Reddy, G.R., RazmiAfshari, M., 2000. Disruption of the zinc finger domain: a common target that underlies many of the effects of lead. Neurotoxicology 21, 1–11.
Intracellular signaling pathways involved in mediating the effects of lead on the transcription factor Sp1 D.S. Atkins a , Md. Riyaz Basha b , N.H. Zawia b,∗ b
a Department of Pharmacology, Meharry Medical College, Nashville, TN 37028, USA Department of Biomedical Sciences, University of Rhode Island, Kingston, RI 02881, USA
Received 9 January 2003; received in revised form 9 April 2003; accepted 24 April 2003
Abstract It has been well established that exposure to Pb during critical periods of brain development results in both cognitive and behavioral deficits. Although the mechanism by which Pb induces developmental neurotoxicity is unknown, it may involve alterations in transcription of genes that are essential for growth and differentiation. Recent studies reveal that Pb interferes with growth and differentiation by acting on the transcription factor Sp1. Pb-induced changes in the activity of Sp1 may be consequent to alterations in intermediates in signal transduction pathways. This study examines both in vivo and in vitro the role of signaling factors in mediating the effects of Pb on Sp1 DNA-binding. Hippocampal developmental profiles of Sp1 DNA-binding, PKC, and MAPK protein levels were monitored in Pb-exposed rats. Pb exposure resulted in an induction of Sp1 DNA-binding during PND 5–10 followed by a subsequent decline on PND 15–20. The protein expression profiles for PKC␣ and MAPK followed a relatively similar pattern. To examine the interdependence between Sp1 DNA-binding, PKC␣, and MAPK, PC12 cells were exposed to Pb and/or NGF. Pb or NGF exposure increased Sp1 DNA-binding. Addition of the PKC inhibitor (staurosporine) diminished NGF and Pb-induced Sp1 DNA-binding, while the MAPK inhibitor (PD 98059), completely abolished both basal and induced Sp1 DNA-binding. These findings demonstrate that Sp1 DNA-binding is regulated by PKC and MAPK, which may serve as mediators through which Pb may indirectly modulate Sp1 DNA-binding. © 2003 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Pb; Sp1; PKC; MAPK; CNS; PC12
1. Introduction Lead (Pb) is universally accepted as a potent environmental toxic metal which imparts profound effects on brain development. Pb poisoning in children persists as a major public health problem (Rodier, 1990; Silbergeld, 1990, 1992). A variety of studies have revealed that Pb exposure causes deficits in CNS functioning and behavioral disorders including learning disabilities and impaired cognitive development in children (Lyngbye et al., 1990; Needleman et al., 1990; Silbergeld, 1990, 1992). Although the mechanisms involved in Pb-induced developmental neurotoxicity are not fully defined, it has been clearly shown that Pb interferes with the transcription of genes that are essential for brain growth and differentiation (Zawia and Harry, 1996). Transcription factors play a vital role in the orchestration of gene expression during development. Extensive studies from our laboratory have shown that Pb exposure alters the DNA-binding of zinc finger protein (ZFP) ∗
Corresponding author. Tel.: +1-401-874-5909; fax: +1-401-874-5048. E-mail address: [email protected] (N.H. Zawia).
transcription factors such as Sp1 (Zawia et al., 1998, 2000). The DNA-binding of other ZFPs such as Egr1 and TFIID have also been found to be modulated by Pb (Hanas et al., 1999; Zawia et al., 2000). However, it is not clear whether Pb within the cell acts directly on Sp1, thereby modulating its activity or whether the effects of Pb on Sp1 DNA-binding are secondary to the action of Pb on other cellular intermediates. Transcription/transduction coupling is a necessary intermediate in the regulation of gene expression. Therefore, we propose that Pb-induced changes in the activity of Sp1 may be consequent to alterations in intermediates in signal transduction pathways associated with growth and differentiation. Potential candidates for such a mediation are protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). PKC plays a major role in cell growth and differentiation and is a known target for Pb (Murakami et al., 1987; Markovac and Goldstein, 1988a, b). It has also been shown that PKC activity is potently increased by Pb (Murakami et al., 1987; Markovac and Goldstein, 1988a, b). Pb in vitro mimics calcium in the central nervous system by entering the cell via calcium channels (Bressler and Goldstein, 1991); however, in vivo studies that have
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examined the interactions between PKC and Pb in exposed animals are lacking. Studies from our laboratory have revealed that Pb-induced manifestations such as induction in Sp1 DNA-binding and differentiation of pheochromocytoma cells (PC12) were similar to those of nerve growth factor (NGF), suggesting the involvement of the MAPK pathway (Crumpton et al., 2001). The MAPK cascade is a signaling system involved in various physiological processes such as cellular proliferation, differentiation, apoptosis, and development (Chang and Karin, 2001). This pathway is initiated by binding of extracellular mitogens, such as NGF, to receptor tyrosine kinases and then MAPK is phosphorylated and translocated to the nucleus, in turn activating a plethora of transcription factors including Sp1 (Merchant et al., 1999; Liu et al., 2001). The present investigation is aimed at delineating the link between Sp1 DNA-binding, PKC, and MAPK, providing an approach to understand the involvement of intracellular signaling pathways in mediating the effects of Pb on Sp1 DNA-binding and gene expression. 2. Experimental procedures 2.1. Animal exposure Timed-pregnant Long-Evans hooded rats were obtained from Charles River Laboratories (Raleigh, NC). Twenty-four hours following birth was designated as postnatal day 1 (PND 1). All pups were pooled and new litters consisting of 10 males were randomly selected and placed with each dam. Exposure to Pb was initiated on PND 1 with the addition of the acetate form (0.2% lead acetate, Sigma, St. Louis, MO) to the drinking water of the dam and continued until PND 20 (Zawia et al., 1998, 2000; Reddy and Zawia, 2000). Control dams received drinking water. Food and water were freely available throughout the study. The dams with pups were individually housed at constant temperature (21 ± 2 ◦ C) and relative humidity (50 ± 10%) with a 12 h light/dark cycle (07:00–19:00 h). On PND 3, 5, 10, 15, 20, and 30 randomly selected pups were removed from each litter and replaced with filler pups to maintain a constant litter size until weaning. Animals were decapitated following exposure to CO2 and hypothermia and brain regions were dissected, frozen on dry-ice and stored at −80 ◦ C.
washing nuclei in TKM buffer with 0.3% Triton X-100 followed by TKM buffer plus 0.15% Triton X-100 and finally TKM buffer alone to remove excess traces of detergents. After each wash, nuclei were sedimented by centrifugation at 3300 × g for 12 min at 4 ◦ C, and all supernatants were pooled. Nuclear pellets were resuspended in 0.5 ml of TKM buffer and nuclei disrupted by repeated passage through a 22-gauge needle. The combined supernatants were centrifuged at 100,000 × g for 1 h at 4 ◦ C, and the supernatant obtained was the cytosolic fraction. The pellet obtained (i.e. the particulate or membrane fraction) was resuspended in TKM buffer plus 0.2% Igepal, rocked on ice for 30 min, then centrifuged at 100,000 × g for 30 min at 4 ◦ C. The supernatant was saved as the membrane extract. All reagents mentioned in this procedure were obtained from Sigma (St. Louis, MO). 2.3. Western blot analysis Initially five isozymes of PKC, namely, ␣, I, II, ␥, and ε were screened for their ability to show a developmental profile in all three cellular fractions. Only nuclear PKC␣ levels exhibited a developmental profile that was prominently modulated by Pb. Therefore, subsequent studies focused on this isozyme. The levels of PKC␣ and MAPK were estimated in nuclear extracts of hippocampal tissue as well as PC12 cells by Western blot analysis. Twenty micrograms of protein extracts were fractionated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) using 1× Tris–glycine–SDS buffer (BioRad, Hercules, CA), then transferred electrophoretically into 0.2 m nitrocellulose membranes (Sigma, St. Louis, MO) using a semi-dry electroblotter and Tris buffer (25 mM Tris–HCl, 192 mM glycine and 20% (v/v) methanol). Membranes were blocked for 1 h with 5% evaporated milk, then incubated with a polyclonal rabbit anti-PKC or anti-MAPK (1:1000; Santa Cruz Immunochemicals, Santa Cruz, CA) for 1 h, followed by subsequent incubation with secondary antibody (1:20,000; goat anti-rabbit IgG (H + L) horseradish peroxidase; Pierce, Rockford, IL) for 1 h. Blots were incubated for 1 min in a system of chemiluminescent reagents (Pierce) then exposed to X-ray film for 5 s. Development of the film produced immunoreactive bands whose densities were later quantified with the Alpha Innotech imaging system. 2.4. Preparation of in vitro samples
2.2. Subcellular fractionation Subcellular fractions were prepared according to the method described by Adunyah et al. (1991) with slight modifications. Tissue or cells were homogenized in 10 volumes of TKM buffer (10 mM Tris–HCl, 1.5 mM MgCl2 , 10 mM KCl, 2 mM EDTA, 2.5 mM EGTA, 5% (w/v) glycerol, 0.1% (v/v) ME, 10 g/ml aprotinin, 10 g/ml leupeptin, 0.5 mM PMSF, and 1 mM DTT), and then centrifuged at 750 × g for 10 min at 4 ◦ C. Perinuclear proteins were removed by
Pheochromocytoma (PC12) cells (ATCC, Manassas, VA) maintained under conditions of 5% CO2 and 37 ◦ C were grown to semi-confluency in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Logan, UT) supplemented to a final concentration with 10% fetal bovine serum (Hyclone), 5% horse (equine) serum (Hyclone) and 1% penicillin– streptomycin solution (Sigma, St. Louis, MO). Subsequently, cells were exposed to various doses of Pb (final concentrations 0.025, 0.1, and 1 M), NGF (final
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concentrations 0.3, 3, and 50 ng/ml; Calbiochem, San Diego, CA) or a combination of the two (0.1 M Pb and 50 ng/ml NGF). Either the PKC inhibitor staurosporine (STAURO, 0.1 M) or the MAPK inhibitor, PD 98059 (25 M) were added to separate groups of control and Pb or NGF-exposed cells. Forty-eight hours later, nuclear extracts were prepared following trypsinization to remove adherent cells. 2.5. Nuclear protein extraction Nuclear proteins were extracted from both hippocampal tissue and PC12 cells according to the method given by Dignam et al. (1983), with slight modifications. Cells/tissues were homogenized in phosphate buffered saline (PBS) and the homogenates were homogenized with 1 ml PBS, pH 7.5, and centrifuged at 2500 × g for 10 min. The pellet obtained was resuspended in five volumes of buffer A (10 mM HEPES at pH 7.9, 1.5 mM MgCl2 , 0.5 mM DTT, 0.5 mM EDTA, and 0.2 mM PMSF) and centrifuged at 6000 rpm for 2 min at 4 ◦ C. Again, the pellet was resuspended in three volumes of buffer A and centrifuged at 6000 rpm for 2 min at 4 ◦ C. The pellet was then resuspended in five volumes of buffer C (20 mM HEPES at pH 7.9, 1.5 mM MgCl2 , 0.5 mM DTT, 0.5 mM EDTA, 420 mM NaCl, 20% glycerol, 0.2 mM PMSF, 0.002 mg/ml aprotinin, and 0.0005 mg/ml leupeptin) and homogenized. The final suspensions were centrifuged at 12,000 × g for 10 min. The supernatants were transferred to 1.5 ml tube, snap frozen in an ethanol dry-ice bath, and then stored at −80 ◦ C. 2.6. Electrophoretic mobility shift assay (EMSA) The Sp1 oligonucleotide (1.75 pmol/l) (Promega) was incubated with 1 l of [␥-32 P] ATP (3000 Ci/mmol) and 5–10 units of T4 polynucleotide kinase in a final volume of 10 l, for 10 min at 37 ◦ C. A reaction mixture containing 20 l of gel shift binding buffer (2 mM HEPES, 2.5 mM MgCl2 , 2.5 mM KCl, 0.5 mM DTT, 2.5% glycerol, 0.001% NP40, 50 g/ml BSA, 0.2% protease inhibitor cocktail (Sigma), 0.2% ficoll, and 20 ng/ml polydIdC) and 1 l of the labeled oligonucleotide (Sp1) probe was prepared. Then, 5–10 g of protein from the nuclear extracts of hippocampal tissue or PC12 cells was added to the reaction mixture. All reactions were done on ice unless otherwise stated. After 10 min incubation at room temperature, 1 l gel loading buffer was added and the final reaction mixture was then loaded onto a 4% polyacrylamide gel and electrophoretically resolved. The gel was exposed to X-ray film overnight, and the resulting autoradiograms were analyzed for shifted bands and quantified using an image acquisition and analysis software (UVP, CA).
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and hippocampus. The metal analysis was carried out following the method as described by Smith et al. (1998). Animals were deeply anesthetized and perfused with sterile saline via cardiac puncture; the brain was excised and the hippocampus was isolated. Samples were pooled from three animals and three to four sample determinations were made for each exposure condition. Pb levels in the hippocampal tissue and blood were analyzed by RI Analytical Laboratory, Warwick, RI, using atomic absorption spectroscopy following EPA approved methodology (Method: 200.9). 2.8. Statistical analysis Data were analyzed by analysis of variance (ANOVA) and that was followed by Dunnet’s post hoc test to compare the effects among various groups and P < 0.05 was considered significant.
3. Results Experiments were conducted to examine the relationship between Sp1 DNA-binding and signaling intermediates such as PKC and MAPK both in vivo and in vitro. As we had previously published (Zawia and Harry, 1996; Zawia et al., 1998), we did not observe any changes in body or brain weights of Pb-exposed animals, which might be indicative of gross nutritional abnormalities. Consistent with our previously published values (Harry et al., 1996; Basha et al., 2003), the Pb levels in the hippocampus of these animals were measured on PND 20 and found to be <0.2 g/g in control animals and 0.34 ± 0.012 g/g in Pb-exposed animals. Similarly, the concentrations of Pb used in vitro did not result in any cytotoxicity to the cells (Crumpton et al., 2001). 3.1. Sp1 DNA-binding in the hippocampus Previous studies from our laboratory had revealed that Pb exposure results in perturbations in Sp1 DNA-binding in the cerebellum (Zawia et al., 1998, 2000). In the present study, Sp1 DNA-binding was monitored in the hippocampus of control and Pb-exposed rat pups from PND 3 to PND 30. Consistent with our earlier studies in the cerebellum (Zawia et al., 1998), Sp1 DNA-binding was low in control animals at PND 3 and gradually increased on PND 15–20 decreasing thereafter. In Pb-exposed animals, premature peaks of Sp1 DNA-binding appeared on PND 5–10 and then declined significantly to below those of control animals on subsequent days (Fig. 1).
2.7. Lead determination
3.2. PKC
On postnatal day 20, pups from both the control and Pb groups were used to determine the levels of Pb in the blood
Since it has been shown that Pb alters PKC activity, changes in hippocampal PKC levels were monitored
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Fig. 1. Developmental profiles of Sp1 DNA-binding in the hippocampus of control and Pb-exposed rats. Sp1 DNA-binding was monitored using the gel mobility shift assay. Shifted bands were scanned and quantified using image acquisition and analysis software (UVP Laboratory Products, CA). Values shown are for the mean ± S.E. from four independent experiments. Values indicated with an “∗” are significant (P < 0.05) over controls at the same postnatal age, as determined by ANOVA. A representative autoradiogram is shown in the box.
Fig. 2. Pb-induced changes in the developmental profiles of nuclear PKC␣ levels in the rat hippocampus. Control and Pb-exposed rat pups were sacrificed on various days and their brains were removed and dissected. Nuclear protein extracts were examined by Western blot analysis using a polyclonal PKC␣ antibody (1:1000). The density of immunoreactive bands was quantified using the Alpha Innotech Imaging System. Values shown are for the mean ± S.E. from three independent experiments. Values indicated with an “∗” are significant (P < 0.05) over controls of the same postnatal age, as evaluated by ANOVA.
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Fig. 3. Changes in the developmental profiles of nuclear MAPK levels in the rat hippocampus following Pb exposure. Control and Pb-exposed rat pups were sacrificed on various days and their brains removed and dissected. Nuclear protein extracts were examined by Western blot analysis using a polyclonal MAPK (ERK2) antibody (1:1000). The density of immunoreactive bands was quantified using the Alpha Innotech Imaging System. Values shown are for the mean ± S.E. from three independent experiments. Values indicated with “∗” are significant at P < 0.05 over controls of the same postnatal age, as evaluated by ANOVA.
following exposure to Pb. In a pilot study, five isozymes of PKC, namely, ␣, I, II, ␥, and ε were initially screened for their ability to show a developmental profile, and their responses to Pb exposure. We found that only PKC␣ exhibited a definite profile which was modulated by Pb exposure. Further studies were performed to determine the levels of PKC␣ in hippocampal nuclear extracts. We observed that the levels of PKC␣ in control animals exhibited a developmental profile (Fig. 2) that was similar to Sp1 DNA-binding (Fig. 1). In Pb-exposed animals minor changes were observed in the early days after birth followed by a significant decrease at later time points, that is, PND 20–30. 3.3. MAPK MAPK immunoreactivity in hippocampal nuclear extracts was examined in the same period as Sp1 DNA-binding and PKC␣. We found that the developmental profiles of MAPK levels (Fig. 3) were similar (in both control and Pb-exposed animals) to those of PKC␣ (Fig. 2), as well as Sp1 DNA-binding (Fig. 1). However, while all patterns were generally similar, there was a greater resemblance among the profiles of the levels of the two enzymes (PKC␣ and MAPK).
3.4. Sp1 DNA-binding, PKCα, and MAPK in PC12 cells The similarities in Sp1 DNA-binding, PKC␣, and MAPK developmental patterns in the hippocampus implied that they may be linked to each other. In order to examine the relationship between these three different proteins, Sp1 DNA-binding, PKC␣, and MAPK levels were measured in NGF-stimulated and Pb-exposed PC12 cells in the presence or absence of enzyme inhibitors. As illustrated in Fig. 4, both Pb and NGF increased Sp1 DNA-binding in a concentration-dependent manner, 48 h following exposure. Administration of staurosporine significantly reduced the induction of Sp1 DNA-binding caused by both Pb and NGF, but failed to influence basal Sp1 DNA-binding. Studies were further conducted to examine whether the levels of the isoform PKC␣ were altered following exposure to either Pb or NGF, in the presence or absence of staurosporine. We found that NGF significantly increased the levels of PKC␣ in a dose-dependent manner, however, Pb alone had minor effects on PKC␣ levels (Fig. 5). Interestingly co-administration of 0.1 M Pb and 50 ng/ml NGF produced an effect that was intermediate to the effects shown individually, but significantly greater than that of Pb alone (Fig. 6). Consistent with the lack of effect of staurosporine on basal Sp1 DNA-binding in PC12 cells, the presence of
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Fig. 4. Modulation of the effects of Pb and NGF on Sp1 DNA-binding in PC12 cells by staurosporine (STAURO). Cells grown to semi-confluency were exposed to various concentrations of either NGF (0.3, 3, and 50 ng/ml), Pb (0.025, 0.1, and 1 M), or both, in the presence or absence of the potent PKC inhibitor STAURO (0.1 M). Forty-eight hours later, nuclear protein extracts were prepared and Sp1 binding to a specific DNA consensus sequence was observed by EMSA. Values shown are for the mean ± S.E. from three independent experiments. Values marked with letters are significant at P < 0.05, as evaluated by ANOVA: “a” over control, “b” over Pb (0.1 M), and “c” over NGF (50 ng/ml).
staurosporine had no effect on basal PKC␣ levels. On the other hand exposure to staurosporine tended to decrease PKC␣ levels when co-administered with either Pb or NGF (Fig. 6). To evaluate the involvement of MAPK in mediating the effects of Pb on Sp1 activity, we monitored the changes in Sp1 DNA-binding following exposure to either Pb or NGF in the presence of the MAPK inhibitor PD 98059 (MEKI). MEKI dramatically decreased basal Sp1 DNA-binding, but
Fig. 5. PKC␣ immunoreactive bands in control and exposed PC12 cells. PC12 cells grown to semi-confluency were exposed to various concentrations of NGF, Pb, and STAURO. Forty-eight hours later, cell extracts were prepared and the levels of PKC␣ were determined by Western blot analysis.
this effect could not be reversed by the addition of either Pb or NGF. However, unlike staurosporine, MEKI dramatically reduced basal Sp1 activity (Fig. 7).
4. Discussion Sp1 is important for the transcription of genes involved in growth and differentiation of the nervous system as evidenced by its high expression in the developing brain (Saffer et al., 1991). Exposure to Pb has been shown to result in perturbations in the regulation of gene expression and these disturbances in transcriptional events appear to be partially mediated by Sp1 (Zawia and Harry, 1996; Zawia et al., 1998, 2000). Both in vivo and in vitro studies from this laboratory have indicated that exposure to Pb induces premature inductions of Sp1 activity with a corresponding effect on its target genes (Zawia et al., 1998). These premature peaks in Sp1 activity are likely to force cells to differentiate leading to an early termination of the developmental program. While there is sufficient evidence to show that Pb interferes with Sp1 DNA-binding, the mechanisms associated
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Fig. 6. Modulation of the effects of Pb and NGF on PKC␣ levels in PC12 cells by STAURO. PC12 cells grown to semi-confluency were exposed to various concentrations of either NGF (0.3, 3, and 50 ng/ml), Pb (0.025, 0.1, and 1 M), or both, in the presence or absence of the potent PKC inhibitor STAURO (0.1 M). Forty-eight hours later, whole cell extracts were prepared, and effects on the levels of PKC␣ were determined by Western blot analysis. Values shown are for the mean ± S.E. from three independent experiments. Values marked with letters are significant at P < 0.05, as evaluated by ANOVA: “a” over control, “b” over Pb (0.1 M), and “c” over STAURO alone (0.1 M).
with this phenomenon are not understood. However, the involvement of signal transduction pathways are highly suspected. Several lines of evidence implicating PKC as a mediator in the effects of Pb (Markovac and Goldstein, 1988a, b; Laterra et al., 1992; Long et al., 1994) and PKC’s role in the control of growth and differentiation (Burgess et al., 1986) nominated it as a candidate for this study (Murakami et al., 1987; Markovac and Goldstein, 1988a, b; Jadhav et al., 2000; Nihei et al., 2001; Hwang et al., 2002; Deng and Poretz, 2002). The ability of Pb to stimulate neurite outgrowth and Sp1 DNA-binding in a manner similar to NGF also suggested that Pb may interfere with growth factor signaling pathways that are normally coupled to MAPK (Crumpton et al., 2001). The hippocampus is a brain region which undergoes major development after birth. Moreover this region is a center for the coordination of certain functions such as learning and memory and cognitive functions, which are impaired by Pb. Consistent with our earlier studies in the cerebellum (Zawia et al., 1998), exposure to Pb resulted in premature peaks of Sp1 DNA-binding during PND 5–10. These findings confirm that the effects of Pb are widespread in the brain and that gene expression in other areas may be disturbed by Pb.
Further studies were performed to examine the developmental patterns and changes in the levels of PKC and MAPK in this brain region to see whether they are temporally and characteristically related to Sp1. An initial pilot study demonstrated that brain PKC␣ isoform was developmentally regulated and its developmental profile was altered by Pb (data not shown). This was consistent with published reports (Hashimoto et al., 1988; Yoshida et al., 1988; Huang et al., 1990) showing that PKC␣ is present in the rat brain at birth and progressively increases during the first 3 weeks of age (Figs. 2 and 3). It is also consistent with the in vitro effects of Pb on human astrocytoma cells (Lu et al., 2001). However, other studies have suggested that PKC␥ is selectively altered by Pb in older animals (Nihei et al., 2001). Although PKC in the brain is found mainly associated with synaptic membranes and the cytosol (Kikkawa et al., 1982), there is evidence that it is also present in other cellular compartments (Kraft and Anderson, 1983). This is further corroborated by the demonstration that PKC␣ translocates to the nucleus (Leach et al., 1992). Although we have screened all three compartments (membrane, cytosolic, nuclear) for PKC levels, we found that only nuclear PKC␣ exhibited a developmental profile and was subject to modulation by Pb. Since the majority of studies in the literature
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Fig. 7. Effects of the MAP kinase inhibitor PD 98059 on the modulation of Sp1 DNA-binding by Pb and NGF. PC12 cells grown to semi-confluency were exposed to Pb (0.1 M), NGF (50 ng/ml), PD 98059 (MEKI, 25 M), or combinations of these as shown. Forty-eight hours later nuclear protein extracts were prepared, and Sp1 DNA-binding was observed by EMSA. Values shown are for the mean ± S.E. of three independent experiments. Values indicated with “a”, “b”, “c”, and “d” are significantly different (P < 0.05) from their corresponding controls, as evaluated by ANOVA.
measure PKC activity or isoforms in crude cellular extracts or the cytosol, the role of PKC on transcriptional events that are resident to the nucleus is usually missed. While PKC has a biological function in the cytosol, MAPK’s substrates are exclusively present in the nucleus. Therefore, MAPK must translocate to the nucleus upon activation, thereby phosphorylating a variety of proteins that are important for transcription (Merchant et al., 1999; Liu et al., 2001). In Pb-exposed animals, MAPK levels were decreased in the nucleus 2 weeks following exposure, and this decrement was sustained thereafter (Fig. 3). The overall developmental patterns for Sp1, PKC␣, and MAPK were generally similar. However, on PND 30, while both PKC␣ and MAPK levels remained low, Sp1 DNA-binding recovered to control levels (Fig. 1). These departures among the profiles of the enzymes and Sp1 DNA-binding indicates that Sp1 may also be subject to regulation by other factors and alternate signaling pathways. However, the striking similarities between the developmental profiles of these three proteins during specific developmental stages suggested that a link between them may exist. While there is no point to point agreement, there are resemblances among the profiles particularly after PND 15. PC12 cells are a commonly used in vitro model to explore the mechanisms of action of toxicants. Therefore, we examined the potential interactions of these three proteins in these cells. When added to PC12 cells, the potent PKC inhibitor
staurosporine had no effect on basal Sp1 DNA-binding or PKC␣ levels (Fig. 4). However, staurosporine effectively reduced both Sp1 DNA-binding as well as PKC␣ levels only when cells were either stimulated to differentiate by NGF or were exposed to Pb (Figs. 4 and 6). This suggested that PKC␣ does not regulate basal levels of Sp1 DNA-binding, but rather plays a role only under conditions of active cell differentiation. However, it is important to note that this may be cell-specific. Unlike PC12 cells, Pb exposure attenuates PKC␣ levels in human astrocytoma cells (Lu et al., 2001). This down-regulation in PKC␣ levels seen in human astrocytoma cells is similar to that observed in the brain of Pb-exposed rats (Fig. 2). On the other hand, the MAPK inhibitor (MEKI PD 98059) produced a dramatic decline in basal as well as stimulated Sp1 DNA-binding activity (Fig. 7). This would be consistent with MAPK’s later position in the signaling pathway, which allows it to receive converging signals from multiple sources. These data clearly indicate that MAPK plays a pivotal role in the nucleus to regulate Sp1 DNA-binding. However, both PKC␣ and MAPK appear to be involved in regulating the DNA-binding activity of Sp1 in a process subject to modulation by Pb. Since interruption in either PKC␣ or MAPK results in a lowering of Sp1 DNA-binding, it is quite possible that both these signaling intermediates are somehow linked. In support of this hypothesis, PKC␣ has been shown to
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phosphorylate, and therefore, activate Raf (MAP kinase kinase kinase). This is the first kinase activated following NGF binding to the high affinity TrkA receptors and is thus ultimately responsible for precipitating MAPK activation. Furthermore, inhibition of MAPK activity blocks differentiation in PC12 cells by NGF (Pang et al., 1995). More recent studies have demonstrated the direct phosphorylation of Sp1 by MAPK (ERK2) (Merchant et al., 1999; Liu et al., 2001). Although the in vivo and in vitro effects of Pb exposure on PKC␣ levels are divergent, the involvement of PKC␣ in the regulation of Sp1 DNA-binding is highly suspected. Tissue PKC␣ levels are products of the contributions of multiple cells at different stages of development, while PKC␣ levels obtained from PC12 cells are a manifestation of a single population of cells at identical stages of growth. Together, both in vivo and in vitro lines of evidence provide a link between NGF, PKC, MAPK, and Sp1. Pb may interact with this pathway by modulating PKC under conditions of active growth and thus, precipitating a chain of events leading to alterations in Sp1 DNA-binding activity. However, it is clear that PKC plays a partial role in regulating Sp1 DNA-binding in vitro, while MAPK is fully involved in maintaining Sp1 DNA-binding. Although Pb can directly alter the DNA-binding of a recombinant Sp1 protein (Razmiafshari and Zawia, 2000), these findings provide an important clue in understanding the mechanism by which Pb produces cellular neurotoxicity. Future studies should be aimed at controlling the activity of these pathways as a means of arresting the detrimental effects of Pb on gene expression and subsequent cellular growth and differentiation.
Acknowledgements The authors thank S. Bakheet for his technical help for the preparation of this manuscript. The authors are grateful to Dr. X. Sun, Research Asst. Professor (Statistics), Cancer Prevention Research Center, U.R.I. for providing assistance in the Statistical analysis. This work was partially funded by a Grant from NIEHS (ES08104). D. Atkins was supported by a fellowship from the EPA.
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