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• W2022893645 abstract "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. 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. 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 (1Murray A.W. Kirschner M.W. Science. 1989; 246: 614-621Crossref PubMed Scopus (508) Google Scholar). 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) 1The 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).1The 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). (2Pines J. Hunter T. Trends Cell Biol. 1991; 1: 117-121Abstract Full Text PDF PubMed Scopus (96) Google Scholar). The activity of these kinases is regulated in part by the actions of the cyclin proteins (3Hunt T. Semin. Cell Biol. 1991; 2: 213-222PubMed Google Scholar) and is restrained by the actions of a separate family of cyclin-dependent kinase-inhibitory proteins (CKI). These latter proteins include p15 and p16 (4Hannon G.J. Beach D. Nature. 1994; 371: 257-261Crossref PubMed Scopus (1875) Google Scholar, 5Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3340) Google Scholar), p27 (6Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2041) Google Scholar), p21 (7Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5201) Google Scholar), and p57 (8Matsuoka S. Edwards M.C. Bai C. Parker S. Zhang P. Baldini A. Harper J.W. Elledge S.J. Genes Dev. 1995; 9: 650-662Crossref PubMed Scopus (900) Google Scholar). The CKI can be induced by growth-inhibitory proteins such as transforming growth factor (TGF)-β (for review, see Ref. 9Peter M. Herskowitz I. Cell. 1994; 79: 181-184Abstract Full Text PDF PubMed Scopus (448) Google Scholar) and p53 (10El-Deiry W.S. Tokino T. Velculescu V.E. Levby D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7869) Google Scholar) and functionally prevent cell passage from G1into S phase by reducing the activity of cyclin-Cdk complexes and preventing the inactivating phosphorylation of the retinoblastoma (pRb) and related proteins (11Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2916) Google Scholar). 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 (11Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2916) Google Scholar), the activating phosphorylation of the Cdks by cyclin-activating kinases (12Wu L. Yee A. Liu L. Carbonaro-Hall D. Venkatesan N. Tolo V.T. Hall F.L. Oncogene. 1994; 9: 2089-2096PubMed Google Scholar), or activating dephosphorylation of the Cdks by the cdc25A phosphatase (13Jinno S. Suto K. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (398) Google Scholar). Growth factors have also been reported in a few studies to inhibit the production and association of the CKI proteins with the Cdks (14Coats S. Flannagan W.M. Nourse J. Roberts J. Science. 1996; 272: 877-880Crossref PubMed Scopus (646) Google Scholar). 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 (15Appel R.G. Am. J. Physiol. 1992; 262: F911-F918PubMed Google Scholar). 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 (16Hu R.-M. Levin E.R. J. Clin. Invest. 1994; 93: 1820-1827Crossref PubMed Scopus (50) Google Scholar). 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 (17Biesiada E. Razandi M. Levin E.R. J. Biol. Chem. 1996; 271: 18576-18581Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). 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. Fetal rat hypothalamic astrocytes were prepared as described previously (17Biesiada E. Razandi M. Levin E.R. J. Biol. Chem. 1996; 271: 18576-18581Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 18Prins B. Weber M.J. Hu R.-M. Pedram A. Daniels M. Levin E.R. J. Biol. Chem. 1996; 271: 14156-14162Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) 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 (16Hu R.-M. Levin E.R. J. Clin. Invest. 1994; 93: 1820-1827Crossref PubMed Scopus (50) Google Scholar). 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 (16Hu R.-M. Levin E.R. J. Clin. Invest. 1994; 93: 1820-1827Crossref PubMed Scopus (50) Google Scholar, 19Nazario B. Hu R.-M. Pedram A. Prins B. Levin E.R. J. Clin. Invest. 1995; 95: 1151-1157Crossref PubMed Scopus (81) Google Scholar). The probes for p27 (6Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2041) Google Scholar), p16 (5Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3340) Google Scholar), and p57 (8Matsuoka S. Edwards M.C. Bai C. Parker S. Zhang P. Baldini A. Harper J.W. Elledge S.J. Genes Dev. 1995; 9: 650-662Crossref PubMed Scopus (900) Google Scholar) 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 (19Nazario B. Hu R.-M. Pedram A. Prins B. Levin E.R. J. Clin. Invest. 1995; 95: 1151-1157Crossref PubMed Scopus (81) Google Scholar). The cDNA probes used were constructed as described previously (20Xiong Y. Menninger J. Beach D. Ward D.C. Genomics. 1992; 13: 575-584Crossref PubMed Scopus (180) Google Scholar, 21Lew D.J. Dulic V. Reed S.I. Cell. 1991; 66: 1197-1206Abstract Full Text PDF PubMed Scopus (655) Google Scholar) 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. 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. 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 (19Nazario B. Hu R.-M. Pedram A. Prins B. Levin E.R. J. Clin. Invest. 1995; 95: 1151-1157Crossref PubMed Scopus (81) Google Scholar). 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 (18Prins B. Weber M.J. Hu R.-M. Pedram A. Daniels M. Levin E.R. J. Biol. Chem. 1996; 271: 14156-14162Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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. 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. 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 β-counter. Data were interpreted by analysis of variance plus Schefe's test. 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. 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 (22Levin E.R. Frank H.J.L. Am. J. Physiol. 1991; 261: R453-R457PubMed Google Scholar). 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.2 A). 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. 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. 2 B). 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 D3production 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. 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. 3 A). ANP also induced an increase in the production of p16, p27, and p57 proteins (Fig.3 B). 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, inhibitedbasal 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. 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). 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). 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 notassociated 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). 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). 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% (TableI). 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.Table IDNA synthesis in astrocytes is stimulated by ET and the inhibition by ANP is dependent upon the synthesis of CKI proteinsNuclear thymidinecpm × 10−3Control4.4 ± 0.12ANP 100 nm4.1 ± 0.14ET-3 100 nm17.3 ± 0.861-ap < 0.05 for ET versuscontrol by analysis of variance and Schefe's test.ANP + ET-311.3 ± 0.751-bp < 0.05 for ET versus ET plus ANP (±MSO).ANP + ET-3 + p16ASO 0.5 μm15.1 ± 0.751-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).ANP + ET-3 + p16ASO 0.05 μm13.6 ± 0.731-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).ANP + ET-3 + p16MSO 0.5 μm11.0 ± 0.711-bp < 0.05 for ET versus ET plus ANP (±MSO).ANP + ET-3 + p27ASO 0.5 μm14.5 ± 0.871-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).ANP + ET-3 + p27ASO 0.05 μm13.5 ± 0.881-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).ANP + ET-3 + p27MSO 0.5 μm11.9 ± 0.621-bp < 0.05 for ET versus ET plus ANP (±MSO).ANP + ET-3 + p57ASO 0.5 μm16.4 ± 0.541-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).ANP + ET-3 + p57ASO 0.05 μm13.3 ± 0.171-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).ANP + ET-3 + p57MSO 0.5 μm11.4 ± 0.221-bp < 0.05 for ET versus ET plus ANP (±MSO).ANP + ET-3 + p16ASO 0.5 μm + p27ASO 0.5 μm15.0 ± 0.621-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).ANP + ET-3 + p57ASO 0.5 μm + p27ASO 0.5 μm16.5 ± 0.461-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).ANP + ET-3 + p16ASO 0.5 μm + p57ASO 0.5 μm16.4 ± 0.381-cp < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown).Data are the mean ± S.E. from four experiments combined, triplicate determinations in each experiment.1-a p < 0.05 for ET versuscontrol by analysis of variance and Schefe's test.1-b p < 0.05 for ET versus ET plus ANP (±MSO).1-c p < 0.05 for ET + ANPversus ET + ANP + ASO. ASO oligonucleotides had no effects by themselves (data not shown). Open table in a new tab Data are the mean ± S.E. from four experiments combined, triplicate determinations in each experiment. 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. 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.Figure 9Model 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 de" @default.
• W2022893645 created "2016-06-24" @default.
• W2022893645 creator A5032390983 @default.
• W2022893645 creator A5052170610 @default.
• W2022893645 creator A5071114812 @default.
• W2022893645 creator A5086799584 @default.
• W2022893645 date "1998-05-01" @default.
• W2022893645 modified "2023-10-12" @default.
• W2022893645 title "Astrocyte Progression from G1 to S Phase of the Cell Cycle Depends upon Multiple Protein Interaction" @default.
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