Astrocyte Progression from G1 to S Phase of the Cell Cycle Depends upon Multiple Protein Interaction*

Ali PedramDagger , Mahnaz RazandiDagger , Ren-Ming HuDagger , and Ellis R. LevinDagger §

From the Division of Endocrinology, Veteran Affairs Medical Center, Long Beach, California 90822 and the Departments of Dagger  Medicine and § Pharmacology, University of California, Irvine, California 92717

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The proliferation of cultured astrocytes is positively and negatively regulated, respectively, by the endogenous neuropeptides, endothelin-3 (ET-3) and atrial natriuretic peptide (ANP). Here, we determined the important steps for the modulation by ET and ANP of G1 to S phase cell cycle progression. ET-3 stimulated an increased number of fetal rat diencephalic astrocytes to progress through G1/S, and this was blocked significantly by ANP. ET augmented the gene expression and/or protein production of D-type, A and E cyclins, whereas ANP inhibited these events significantly. ET also stimulated the activation of the cyclin-dependent kinases Cdk2, Cdk4, and Cdk6, directed against the retinoblastoma protein pRb, and this was inhibited by as much as 80% by ANP. As an additional mechanism of cell cycle restraint, ANP stimulated the production of multiple cyclin-dependent kinase inhibitory (CKI) proteins, including p16, p27, and p57. This was critical because antisense oligonucleotides to each CKI reversed ANP-induced inhibition of ET-stimulated DNA synthesis by as much as 85%. CKI antisense oligonucleotides also reversed the ANP inhibition of Cdk phosphorylation of pRb. In turn, ET inhibited ANP-stimulated production of the CKIs, thereby promoting cell cycle progression. Specific and changing associations of the CKI with Cdk2 and Cdk4 were stimulated by ANP and inhibited by ET. Our findings identify several mechanisms by which endogenous modulators of astrocyte proliferation can control the G1-S progression and indicate that multiple CKIs are necessary to restrain cell cycle progression in these cells.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Astrocytes are important cells in the central (and peripheral) nervous system where they guide the development and migration of neurons during brain development, produce growth factors, maintain the integrity of the blood-brain barrier, and participate in the immune and repair responses to disease and brain injury. Astrocytes within the central nervous system mainly proliferate only during embryogenesis, but these cells divide in the adult human after brain insult. For instance, intense migration, hypertrophy, and division of astroglia characterize the typical gliotic response to inflammatory diseases such as multiple sclerosis. Transformation of astrocytes into the most prevalent primary tumor of the brain, the glioma, results from undefined steps that lead to relatively unregulated cell proliferation. Similarly, the key events that lead to astrocyte proliferation are not well understood.

In general, the regulation of cellular proliferation involves the production and action of a variety of proteins that modulate progression through the cell cycle (1). The transition through G1 to S phase and the commitment of the cell to divide are modulated importantly by the activity of phase-specific threonine/serine kinases, the cyclin-dependent protein kinases (Cdks)1 (2). The activity of these kinases is regulated in part by the actions of the cyclin proteins (3) and is restrained by the actions of a separate family of cyclin-dependent kinase-inhibitory proteins (CKI). These latter proteins include p15 and p16 (4, 5), p27 (6), p21 (7), and p57 (8). The CKI can be induced by growth-inhibitory proteins such as transforming growth factor (TGF)-beta (for review, see Ref. 9) and p53 (10) and functionally prevent cell passage from G1 into S phase by reducing the activity of cyclin-Cdk complexes and preventing the inactivating phosphorylation of the retinoblastoma (pRb) and related proteins (11).

The coordinated production and activation of the cyclin-Cdk protein complexes induced in response to a growth stimulus occur through several mechanisms. These include the stimulation of cyclin or kinase protein production and association (11), the activating phosphorylation of the Cdks by cyclin-activating kinases (12), or activating dephosphorylation of the Cdks by the cdc25A phosphatase (13). Growth factors have also been reported in a few studies to inhibit the production and association of the CKI proteins with the Cdks (14). Modulation of each of these events is a theoretical target by which anti-growth proteins could inhibit cell proliferation.

One such family of anti-growth proteins, the natriuretic peptides, inhibits the proliferation of a wide variety of cells, including kidney mesangial, vascular endothelial and smooth muscle, and astrocytic cells (15). The anti-growth effects of atrial natriuretic peptide (ANP) have been studied most completely in astrocytes, where this peptide inhibits endothelin (ET) and other growth factor signaling through mitogen-activated protein kinase to nuclear events that are critical for glial proliferation (16). The nuclear events mediating anti-growth include the inhibition of ET-stimulated production of the immediate-early gene/protein egr-1 and the autologous growth factor, basic fibroblast growth factor (17). It is conceivable that ANP affects actions to restrain G1 to S phase progression, in some situations by modulating the actions of ET which lead to cell cycle progression. One important potential target is the stimulation of the production of the CKI proteins and their association with cyclin-Cdk complexes to inhibit kinase activity. We report here that ANP can stimulate the production of several CKI proteins and that this is crucial for the anti-growth actions of these peptides in the astrocyte. We further found that ET-ANP interactions modulate cyclin production, Cdk activity, and cyclin-Cdk protein association, as additional mechanisms regulating G1-S progression in these important neural cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture and Northern Analysis-- Fetal rat hypothalamic astrocytes were prepared as described previously (17, 18) and utilized in passages 2 and 3 for the experiments. The cells were serum deprived for 48 h before experimentation to synchronize the cells into G1 phase. For Northern analysis, total RNA was extracted after the cells were then incubated with ET-3 or ANP, 100 nM, or in combination, for up to 16 h, and 20 µg from each experimental condition was separated on a denaturing 1.2% agarose gel, transferred to nitrocellulose, and prehybridized as we described previously (16). The blots were hybridized for 12-18 h at 42 °C with 32P-labeled, antisense cRNAs, or end-labeled cDNAs for the CKI, by methods described previously (16, 19). The probes for p27 (6), p16 (5), and p57 (8) were kindly provided by Drs. J. Massague, D. Beach, and W. Harper and have been described as referenced. Cyclin D1 and E gene expression was determined experimentally in a similar manner (19). The cDNA probes used were constructed as described previously (20, 21) and were the kind gifts of Dr. D. Beach and S. Reed, respectively. Hybridization bands were quantified by laser densitometry after autoradiography. Sense probes produced no hybridization.

Cell Cycle Distribution-- Astrocytes were synchronized for 48 h in the absence of serum, then incubated in the absence of serum but in the presence or absence of ET, ANP, or both peptides for 4, 8, 12, 16, and 24 h. Ethanol (70%) was added in cold phosphate-buffered saline for 30 min, removed, and then 1 ml of propidium iodide/RNase solution was added for 20 min. The percentage of cells in each phase of the cell cycle was determined by a flow cytometer.

Cyclin-dependent Kinase Inhibitors-- To assess the effects of ET and ANP on CKI protein, metabolic labeling studies for protein synthesis were performed. Astrocytes were synchronized by serum deprivation for 48 h, then incubated in methionine-free Dulbecco's modified Eagle's medium with dialyzed 10% fetal bovine serum for 1 h before experimentation, as described previously (19). Each dish of cells was then incubated in the absence of serum or unlabeled methionine but with 250 µCi of [35S]methionine in the presence or absence of 100 nM ET-3, ANP, or both for up to 24 h. In additional studies, antisense oligonucleotides (ASO) or missense constructs were added for 1 h before the addition of growth or anti-growth factor. The cells were then lysed, precleared, and specific labeled CKI protein was immunoprecipitated using polyclonal antibody for p27, p57, and p16 (Santa Cruz Biotechnology, Santa Cruz, CA). The immunoprecipitated protein was solubilized and denatured in SDS-reducing buffer and electrophoretically resolved by PAGE using a glycine buffer system. For comparison, molecular weight markers were resolved under the same circumstances. The gel was then stained and destained and subjected to flurography and then autoradiography for 1 day. Each translation experiment was performed at least three times. The effects of ANP or ET on cyclin D1, D3, and E protein expression were determined similarly. To understand whether ANP or ET influenced the amount of Cdk protein that complexed with the various cyclins, SDS-PAGE-resolved Cdk proteins were then immunoblotted with antibody specific to each of the cyclins, as described previously (18).

Cyclin-dependent Kinase Activity-- For determination of the effects of ANP or ET on Cdk activity, the cells were cultured with 100 nM ET-3, with or without equimolar natriuretic peptides for up to 16 h, or they were treated with no added substance (control kinase expression). Cells were lysed, and the supernatant was frozen. Cell lysate (200 µl) was then added to Cdk antiserum (10 µl) conjugated to prewashed protein A-Sepharose; the mixture was then incubated for 2 h at 4 °C in microcentrifuge tubes. After washing, each bead-antibody-antigen complex was then incubated for 30 min at 30 °C with 40 µl of kinase buffer (25 mM HEPES, 10 mM magnesium acetate, 40 µM ATP, 2 mM dithiothreitol), 10 µCi of [32P]ATP, and either glutathione S-transferase (GST)-pRb (Santa Cruz) or histone, as substrate for kinase activity. Reactions were terminated by adding SDS reducing buffer, samples were boiled, and the proteins were separated by SDS-PAGE. After autoradiography, the bands were compared by laser densitometry.

Cyclin Proteins-- The regulation of the production of cyclin D1, D3, A, and E proteins was determined in response to ET, ANP, or both peptides over a 24-h time course, as described above.

[3H]Thymidine Incorporation-- Subconfluent astrocytes were synchronized for 24 h in serum-free medium and then incubated for 20 h in the absence or presence of ET-3, with and without ANP, each at 100 nM, all conditions without serum. ASO or missense oligonucleotides (scrambled but the same base composition) to p16, p27, or p57 were also added for 1 h before the addition of ET or ANP. The ASO were 18-20-mer in length and were directed at the initiation codon and the following sequences for the proteins: p16, 5'-GCTCCATGCTGCCTCGTGCC-3'; p27, 5'-CACTCGCACGTTTGACAT-3'; and p57, 5'-ATGGAACGCTTGGCCTCCAG-3'. The ability of the various ASO to inhibit CKI protein synthesis specifically was validated by protein translation studies (see above). After the addition of 0.5 µCi of [3H]thymidine for 4 h, cells were washed, incubated with 10% trichloroacetic acid to precipitate nuclear thymidine, and lysed with 0.2 N NaOH and neutralized with 0.2 N HCl; the lysates were counted in a beta -counter. Data were interpreted by analysis of variance plus Schefe's test.

Endogenous pRb Phosphorylation-- To determine whether ET increased and ANP inhibited the phosphorylation of endogenous pRb, cells were incubated with the peptides in the presence or absence of the CKI ASO for 16 h. After cell lysis, endogenous pRb was separated by SDS-PAGE, transferred to nitrocellulose, then blotted with an antibody that recognizes phosphorylated and nonphosphorylated forms of pRb (Santa Cruz). The study was repeated three times.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Cycle Analysis-- We determined the effects of ET and ANP on cell cycle progression in the astrocytes. The percentages of cells in all phases of the cell cycle were comparably unchanged over 24 h in both the control (serum-deprived) and ANP alone states, indicating that at least 90% of cells did not progress under these conditions. Under control or ANP alone conditions, the percentage of cells in G1 (90%) or S (5%) at 4 h was very similar to the ET alone (Fig. 1) or ET + ANP-treated cells (data not shown). ET then stimulated the progression of the astrocytes from G1 to S phase, commencing at 12-16 h, with 5-fold more cells reaching S phase between 16 and 24 h (Fig. 1) compared with control cells. ANP inhibited this progression, causing a 40% reduction in the number of cells reaching S phase at 24 h. This was very comparable to the overall extent of anti-proliferation induced by ANP (see below) and corresponds to the 45% reduction in cell number caused by ANP + ET-3, compared with ET-3 alone, which we described previously (22).


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Fig. 1.   Cell cycle staging by flow cytometry of cultured fetal rat diencephalic astrocytes after synchronization by serum deprivation followed by incubation with ET-3 (square ) or ET-3 + ANP (black-square) for 24 h. G1 and S phase cell percentages are shown at the different time points. ANP in the presence of ET-3 decreased the number of cells progressing to S phase by 40% compared with ET-3 alone. Cell percentages and standard deviations from three determinations are shown in G1 and S phase. Cultures incubated with ANP alone or maintained in the absence of serum were comparable to ET or ET + ANP-treated cells at 4 h and did not change over 24 h.

Cyclin mRNA/Protein-- We then determined the effects of the neuropeptides on cyclin production. The expression of cyclin D1 and cyclin E mRNA, as representative of two important G1 phase cyclins, was seen at low levels in both the basal state and in ANP-treated cells (Fig. 2A). ET-3 stimulated cyclin D1 mRNA by 8 h of incubation and maximally at 12 h, returning toward basal levels by 16 h. Cyclin E message was induced at 12-16 h (lanes 4 versus 12), coincident with this protein playing an important role in the later stages of G1. ANP significantly reversed the ET stimulation of either cyclin mRNA, indicating one mechanism by which ANP could exert anti-growth effects.


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Fig. 2.   Panel A, time course of cyclins D1 and E mRNA levels in cultured astrocytes, induced by ET-3, and inhibited by ANP, determined by Northern blot. Hybridization of the same RNA with a probe for beta -actin is shown below. Panel B, cyclin protein production determined by [35S]methionine labeling of astrocytes, stimulated by ET, and inhibited by ANP. After incubation with ET or ANP, cell lysate was precleared, and labeled proteins were immunoprecipitated with monoclonal antibody against each cyclin and separated by SDS-PAGE. The figures shown are representative of three experiments.

At the protein level, the production of cyclins D1, D3, E, and A was stimulated by ET-3 compared with basal cell expression of these proteins (Fig. 2B). For cyclin D1 and E, this occurred as early as 8 h, peaked at 12-16 h, and returned toward control levels by 24 h. The stimulation of cyclin E protein, earlier than mRNA, may indicate that the regulation of this protein occurs partly at a post-transcriptional level. Interestingly, cyclin D3 production was somewhat delayed, relative to D1, and cyclin E protein was expressed earlier than cyclin A (the latter is felt to be more important for late G1-S or early S phase events). ANP significantly inhibited the ability of ET to stimulate production of each of these proteins under coincubation conditions. Therefore, at both the mRNA and protein synthesis levels, ET stimulated and ANP inhibited G1 cyclins, indicating mechanisms of growth promotion or restraint, respectively.

CKI Protein Synthesis-- Knowing the kinetics of cell cycle progression in this setting, we determined the time-dependent effects of ANP and ET on CKI mRNA and protein synthesis. Examining the mRNA for p16, p27, and p57, we found that ANP stimulated but ET inhibited this stimulation for each CKI, seen during the period of G1 and especially at 4-12 h (Fig. 3A). ANP also induced an increase in the production of p16, p27, and p57 proteins (Fig. 3B). This was first seen at 12 h of incubation, corresponding to the mid-late G1 phase in the astrocytes, and was maximal at this time but persisted at 16 h. Consistent with the passage of cells through G1 to S at 16-24 h, the production of the three CKI proteins then waned, returning to basal levels by 24 h. Interestingly, the growth factor, ET-3, inhibited basal CKI production at 12 and 16 h (compared with control) and also inhibited ANP-stimulated CKI substantially over this same time course. Thus, the anti-proliferation factor ANP stimulates CKI production as a potential mechanism to restrain passage through G1 to S phase. The mitogen ET could overcome these growth-inhibitory actions of ANP by inhibiting CKI synthesis.


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Fig. 3.   Stimulation of CKI (panel A) mRNA and (panel B) protein by ANP and inhibition by ET over time. Northern blot and metabolic labeling (translation) over 16-24 h for the CKI proteins p16, p27, and p57 are shown, as described under "Materials and Methods." The study was carried out as described for Fig. 2B and was repeated twice. Hybridization of RNA with a beta -actin cRNA probe is shown below panel A as loading control.

Cdk Activity-- We then determined the effects of ET, ANP, or coincubation with both peptides on Cdk activity, directed against pRb (Fig. 4, upper panel). Over the 16-h period of mainly G1, basal Cdk activity was fairly stable (control). ANP caused a reduction in the basal activity of Cdk4 and Cdk6 by 8 h, persisting throughout the experiment. Cdk2 activity was inhibited, but at a later time (between 12 and 16 h). In contrast, ET stimulated all three kinase activities, beginning as early as 4 h and persisting throughout the G1 phase. ANP potently inhibited by as much as 80% the ability of ET to stimulate all three kinase activities against pRb, indicating a potential anti-growth effect of this peptide. Similar results were found in determining the regulation of kinase activity as directed against histone (data not shown). The protein levels of the kinases at 16 h, determined by [35S]methionine labeling, were not affected by any peptide treatment of the astrocytes (Fig. 4, lower panel).


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Fig. 4.   Cdk activities directed against recombinant pRb, stimulated by ET, and inhibited by ANP. Cells were incubated with ET or ANP over 16 h and lysed, and specific kinase activity (Cdk2, Cdk4, Cdk6) was immunoprecipitated and activity determined against pRb (upper panel) as described under "Materials and Methods." Lower panel, effects of ET or ANP on cyclin new protein synthesis (determined by [S35]methionine labeling) is shown, unchanged by peptide treatment. The figure is representative of three studies.

CKI Association with Cdks-- We next examined the alterations by ANP or ET of the interactions of the CKI proteins with the various Cdks (Fig. 5). This was done to support the idea that modulation of CKI·Cdk complexing is a potential mechanism to restrain astrocyte cell cycle progression. Individual CKI proteins were immunoprecipitated, followed by blotting of Cdk proteins. For Cdk4, we found that ANP increased the amount of p15 and p16 proteins specifically associated with this kinase, whereas ET inhibited these associations. We also found that ANP stimulated, but ET inhibited, total p15 protein synthesis, with time kinetics similar to the other CKI proteins (data not shown).


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Fig. 5.   Association of CKI proteins with Cdk4 or Cdk2 proteins, as modulated by ET or ANP. Immunoprecipitation of CKI proteins was followed by blotting with antibody against Cdk proteins. The study was repeated twice, and a representative experiment is shown.

Similarly, the association of p27 with Cdk2 was promoted by ANP and inhibited by ET. These effects of ANP could reflect the general ability of this peptide to stimulate CKI production, but this is also seen specifically in the relevant Cdk-associated faction. In fact, the amount of the CKI protein associated with the Cdks in the setting of ANP was only approximately 10% the total CKI protein produced during incubation of astrocytes with the anti-mitogen, determined by protein quantification. We speculate that the augmented CKI not associated with the Cdk proteins has additional functions in restraining growth. We also found that ANP does not increase the amount of p57 that associates with Cdk2 (Fig. 5).

Proliferation Studies-- We then sought to understand whether ANP stimulation of any or all of the CKI is critical to growth restraint induced by this peptide. We incubated the glia with ET or ANP peptides plus antisense, or missense oligonucleotides for the p16, p27, and p57 proteins. We first validated the ability of each of the ASO constructs to inhibit only its specific corresponding CKI (Fig. 6). We found that a specific CKI ASO caused a 60-80% reduction in the ability of ANP to stimulate the protein synthesis of only that CKI. A particular CKI ASO, for instance to p16, had no effect on the stimulation of other CKI proteins by ANP, indicating the specificity of action. Scrambled, missense ASO (MSO) had no effects on ANP-stimulated CKI protein synthesis (data not shown).


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Fig. 6.   Specificity of ASO to inhibit ANP-induced synthesis of CKI proteins p16, p27, and p57. This representative study was repeated three times. Missense constructs for each CKI produced no effects on CKI synthesis (data not shown).

We then carried out DNA synthesis studies. Nuclear thymidine incorporation in the glia was stimulated significantly by ET, whereas ANP inhibited this proliferation/S phase response by 47% (Table I). Incubation of the astrocytes with the two peptides plus an antisense oligonucleotide to p16 reversed the ANP inhibition of ET-stimulated DNA synthesis by 63 and 38%, at two concentrations. Independently, ASO to p27 or p57 also prevented the anti-growth effects of ANP, by 53 and 85%, respectively, again in dose-related fashion. MSO for the three CKI had no effect on the anti-growth properties of ANP. We also incubated the astrocytes with ET and ANP plus all combinations of two CKI ASO and found little additional effect compared with the maximal effect of the dominant CKI ASO. These data indicate that the progression of astrocytes into S phase, as stimulated by ET, is inhibited by ANP and that several CKI are crucial to this inhibitory effect.

                              
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Table I
DNA synthesis in astrocytes is stimulated by ET and the inhibition by ANP is dependent upon the synthesis of CKI proteins
Data are the mean ± S.E. from four experiments combined, triplicate determinations in each experiment.

CKI Restraint of Cdk Activity-- We proposed that an important effect of ANP-stimulated CKI production would be to inhibit Cdk activity against pRb. We found that the ability of ANP to inhibit Cdk 4 activity at 16 h of incubation was reversed significantly by the ASO to p16, but not to p27 or p57 (Fig. 7, upper panel, Cdk4, lanes 3-7). ET stimulated Cdk4 activity 3.5-fold, and this was inhibited 52% by ANP. This inhibition was reversed 80% by the ASO to p16 but not by the ASO for p27 and p57. This indicates the importance and selectivity of p16 to this restraining action on cell cycle progression induced by ANP. Missense constructs for these proteins had no reversal effects (lanes 11-13), and the Cdk proteins were unaffected by the experimental treatments (Fig. 7, lower panel). Similarly, the restraint of Cdk2 activity by ANP (55%) was reversed 85% by the ASO to p27 and not by the ASO to p57 or to p16 (Fig. 7, upper panel, Cdk2, lanes 3-7). These latter data identify p27 as being crucial to the inhibition of Cdk2 activity against pRb and support this as a mechanism by which ANP restrains G1-S progression.


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Fig. 7.   Effects of ASO to CKIs on modulation by ANP or ET of Cdk2 and Cdk4 activity against pRb. Cells were incubated with ET, ANP, or both ± ASO or MSO to CKIs for 16 h. Cdk activity (upper panel) and protein levels (lower panel) are shown. Results are representative of an additional study.

Phosphorylation of Endogenous pRb-- To support this mechanism further, we examined the effects of ET and ANP on the phosphorylation of the endogenous pRb via CKI. Compared with control, ET strongly stimulated the increased amount and phosphorylation of pRb (hence inactivated), whereas ANP inhibited this stimulation almost completely (Fig. 8). The ASO to p16 and p27, but not p57, reversed this inhibition of pRb phosphorylation by 78 and 63%, respectively. These results affirm the targeting of endogenous pRb phosphorylation by ET and ANP as a mechanism for the promotion or restraint of G1-S progression in these cells, mediated significantly through CKI. A diagram of these interactions is shown in Fig. 9.


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Fig. 8.   ET stimulates the phosphorylation of endogenous pRb, inhibited by ANP. Cells were incubated with ET, ANP, or neither for 16 h, and then the cell lysate was immunoblotted for pRb, as described under "Materials and Methods." The ASO to p16 or p27, but not p57, reversed the inhibitory effect of ANP. A representative experiment, repeated twice, is shown.


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Fig. 9.   Model of ANP-induced inhibition of G1-S progression in astrocytes. ANP stimulates the production and association of p15 and p16 with Cdk4, leading to the inhibition of Cdk4 phosphorylation of pRb. Increased binding of p15 and p16 might displace p27, contributing to the demonstrated increased binding of this CKI to Cdk2 and further inhibition of pRb phosphorylation, as stimulated by ET. ET inhibits ANP stimulation of the CKI.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The mitogen ET-3 and the anti-proliferative ANP modulate several key steps in the progression of astrocytes through G1 to S stages of the proliferation cycle. First, ET-3 stimulates the production of cyclins D1, D3, E, and A, and ANP inhibits each of these. Second, ANP stimulates the production of key CKI proteins, including p15, p16, p27, and p57, maximally just before the G1-S transition and is generally preceded by increased gene expression. Based on the antisense studies, the stimulation of CKI production was critical to ANP-induced restraint of ET-stimulated DNA synthesis, a marker of S phase progression. ET, conversely, inhibited ANP-stimulated production of the CKI proteins. Therefore, astroglial growth factors can promote cell cycle progression by opposing the production of cell cycle-restraining proteins induced by anti-growth factors.

We also found that ANP stimulated the association of specific CKI proteins with specific Cdks, and this would be predicted to inhibit kinase activity. To prove this, we demonstrated a requirement of p16 and p27, respectively, for the ability of ANP to inhibit ET-stimulated Cdk4 or Cdk2 activity against both exogenous or endogenous pRb. This indicates a mechanism of how G1 kinase activity is regulated in this setting. There was no effect of ET or ANP on the levels of the Cdk proteins, as shown in several experiments, indicating that the changes in CKI proteins associated with Cdk2 or 4 reflect modulation of only CKI concentrations. ET-induced phosphorylation inactivates pRb (and related proteins p107 and p130), leading to the release of the E2F family of transcription factor proteins (23), which then transactivate genes that promote passage from G1 to S phase (24). We propose that the ability of ANP to prevent pRb phosphorylation via the production of CKI is one mechanism to restrain cell cycle progression. Because the association of the CKI proteins with the cyclin-Cdk complexes was perturbed by ANP (and prevented by ET), an overall titration of CKI binding to the Cdks probably regulates pRb phosphorylation (see below) or other effects of these kinases. Conversely, ET stimulates Cdk activity, in part through inhibiting ANP-stimulated CKI production and association with the Cdks. pRb is probably not the only target because in some cells pRb phosphorylation or E2-F-mediated transactivation is unnecessary for cyclin E-induced G1-S progression (25).

What upstream events precede G1 protein activity/synthesis and potentially modulate cell cycle passage in astrocytes? We showed previously that ANP inhibits ET-stimulated mitogen-activated protein kinase (17), leading to the inhibition of ET-stimulated egr-1 transcription and transactivation of the basic fibroblast growth factor gene by Egr-1 (18). These proteins are required for ET- stimulated astrocyte proliferation (16). Growth factor-induced Ras participation in the G1-S transition is critically dependent on pRb, associated with the induction of cyclin D protein (26), or cyclin E-dependent kinase activity (in conjunction with Myc) (27). Mitogen-activated protein kinase (Erk) is a downstream effector of Ras-induced cell proliferation, Myc protein is a substrate for Erk (28), and cyclin D induction can be stimulated by this kinase (29). Ras and Erk are activated by ET (17, 30), and Erk is inhibited by ANP (17). Hence, signaling interactions through Erk probably contribute to cell cycle progression in the astrocyte, perhaps in conjunction with Egr-1 or basic fibroblast growth factor actions. The precise nuclear targets and interactive effects require definition. Very recently, it has been shown that TGF-alpha stimulation of cyclin D1 production is mediated through Egr-1 binding sites on the cyclin promoter (31). We postulate that ET-3 may be stimulating a similar induction pathway.

In addition to ANP, other anti-growth factors potentially limit the G1-S transition in various cells through activating CKI production. TGF-beta inhibits cell proliferation and cyclin E-Cdk2 activity (32). Similarly to ANP, TGF-beta stimulates p15 production and binding to cyclin D-Cdk4, releasing p27 that exists basally complexed with cyclin D-Cdk4. p27 then binds and decreases cyclin E-Cdk2 activity (33). TGF-beta does not alter p27 protein levels, and thus ANP acts differently in this respect. Modulation of cyclin D-Cdk4 complexes and activity by ANP is important because this complex, as well as cyclin D-Cdk6, phosphorylates pRb in vivo (4, 34) and precedes cyclin E-dependent phosphorylation of this protein (27) as one target. p27 may interrupt cell cycle progression independently of pRb by inhibiting cyclin D, E2F, or cyclin A transcription/activity (35). p27 may also restrain cyclin E-related kinase activity directed against another target because cyclin E stimulates G1-S progression in pRb- cells (36). Induction of the p21 CKI by TGF-beta or ionizing irradiation allows some cells to pause at the G1-S transition (10, 37). However, we found no effect of ANP to induce p21 in the astrocyte.2

There is uncertainty about the role of individual CKIs to inhibit G1 progression. Homozygous inactivation of p27 yields abnormalities of growth and development in defined tissues where p27 is highly expressed (38-40). However, cell cycle arrest, as induced by multiple agents, is not affected by the deletion of this CKI (40), and so in vivo, p27 is not uniquely/generally required for cell cycle restraint. Similarly, p21 knockout mice have some but far from complete G1 arrest, in contrast to p53-deficient mice that have severe abnormalities of G1-S progression (41, 42). Our results indicate that, at least in the astrocyte, there are multiple CKIs that play a necessary role in the modulation of growth/anti-growth.

A requirement for multiple CKI or a critical mass of these protein(s) could theoretically arise from the need to saturate/inactivate the cyclin-Cdk complexes completely. Many molecules of p27 are required to cause significant inactivation of just cyclin E-Cdk2 (32). In other cells, multiple CKI proteins are needed to restrain cell cycle progression (43). These results support a model in which the actions of multiple CKI proteins may in some way be linked to each other. For instance, full phosphorylation and (presumed) inactivation of pRb require the sequential actions of multiple kinases (44), each regulated by unique CKI. We found that the addition of two antisense constructs to the astrocytes did not reverse the anti-proliferative action of ANP further, significantly more than the inhibition of production of one CKI. We therefore propose that the loss of one CKI protein disrupts a chain of events which cannot be compensated for by another CKI. For example, the production of p15 stimulated by ANP could be necessary to allow the release of p27 from cyclin D-Cdk4 and -6 complexes and its subsequent binding to cyclin E-Cdk2, as supported by our association experiments. Near complete inhibition of p27 production would make p15 unimportant, if this were its major function.

Linkage of CKI effects may not center upon restraining Cdk activity. We found that inhibition of ANP-induced p57 synthesis with a specific ASO reversed the growth-inhibitory effects of ANP by 85%. However, p57-induced restraint of Cdk2 activity was not the mechanism involved because this CKI did not associate with Cdk2 increasingly, nor did the ASO to p57 alter Cdk2 activity against pRb. We found recently that ANP inhibition of Cdk4 activity was also not altered by the p57 ASO.2 We also determined here that the inhibition of one CKI by a specific ASO does not affect the ANP-stimulated production of other CKI and therefore is not a mechanism for linkage of multiple CKI interactions. In specific cells, the actions of a single CKI might predominate (38-40, 45). The lack of a uniform mechanism of cell cycle restraint imposed by the CKIs in various organs is not surprising because requirements for proliferation are likely to differ among distinct cell types and between undifferentiated versus lineage-committed cells.

There may be additional steps in S, G2, or M phases which are also regulated by individual CKI. p21 helps uncouple S and M phases (46). Cdk activity is required for the transition between several phases of the cell cycle (11), and the kinases mediating these steps are targets for CKI. In a more general sense, ANP and other natriuretic peptide family members inhibit the growth of a variety cells (15, 22, 47), and ET is a mitogen for vascular, kidney mesangial, fibroblastic, and several neoplastic cells (for review, see Ref. 48), so our findings may be relevant in other organ systems.

In summary, we report several mechanisms by which ANP can restrain astrocyte G1-S cell cycle progression, often through modulating the actions of the mitogen ET. ET can promote growth through both stimulating positive effectors (cyclin production, Cdk activity) and inhibiting negative regulators (CKI production). The restraint of cell cycle progression by ANP necessarily involves the stimulated production of multiple CKI proteins. The precise steps mediated by the CKI indicate potential targets to restrain proliferating and perhaps transforming cells. As a model, understanding the steps necessary for growth factor activation of quiescent astrocytes could lead to strategies to stimulate neural regeneration.

    FOOTNOTES

* This work was supported by a Veterans Affairs merit review grant and National Institutes of Health Grants HL-50161 and NS-30521 (to E. R. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Medical Service (111-I), Long Beach VA Medical Center, 5901 E. 7th St., Long Beach, CA 90822. Tel.: 562-494-5748; Fax: 562-494-5515; E-mail: elevin{at}pop.long-beach.va.gov.

1 The abbreviations used are: Cdk, cyclin-dependent kinase(s); CKI, cyclin-dependent kinase inhibitor; TGF, transforming growth factor; pRb, retinoblastoma protein; ANP, atrial natriuretic peptide; ET, endothelin; ASO, antisense oligonucleotide(s); PAGE, polyacrylamide gel electrophoresis; MSO, scrambled, missense oligonucleotide(s).

2 E. R. Levin, A. Pedram, M. Razandi, and R.-M. Hu, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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