I. 英文摘要
This study was designed to investigate the effect of YC-1 upon the proliferation of rat mesangial cells and its underlying mechanism. YC-1 inhibited cell proliferation and DNA synthesis in a dose and time-dependent manner. Flow-cytometry cell-cycle studies revealed that YC-1 prevented the entry of cells from G1 into S phase. The expression of cyclin D1 and the kinase activity of cyclin D1/CDK 4 were lower within YC-1-treated cells, revealed by Western blotting, Northern blotting and kinase assays. YC-1 did not increase the intracellular cGMP concentration in mesangial cells. Inhibitors of sGC, PKG, or PKA also did not reverse the inhibitory effect elicited by YC-1, while co-treatment with p38 MAPK inhibitor could partially reversed the suppressive effect. Conclusion: YC-1 inhibited proliferation and induced cell-cycle arrest by the reduction of cyclin D1 synthesis and cyclin D1/CDK4 kinase activity. This effect acts partially through p38 MAPK signal transduction activation and is independent of cGMP-signaling pathways.
Key words: YC-1, mesangial cells, cyclin D1, proliferation, p38 mitogen-activated protein kinase
Abbreviation: BrdU, 5-bromo-2-deoxyuridine; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; cAMP, 3’,5’-cyclic adenosine
monophosphate; cGMP, 3’,5’-cyclic guanosine monophosphate; ERK, extracellular signal-regulated kinase; GSK3β, glycogen synthase kinase-3 β; H89,
N-[2-(4-bromocinnamylamino)-ethyl]-5-isoquinoline ; KT5823, (9S,10R,12R)-2,3,9,10,11,12,
hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylicacid methyl ester; MAPK, mitogen activated protein kinase; ODQ,
1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one; PDGF, platelet derived growth factor; PI3K, phosphatidylinositol 3 kinase; PKA, cyclic AMP-dependent protein kinase; PKG, cyclic GMP-dependent protein kinase; Rb, retinoblastoma; SB203580, [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole]; sGC, soluble guanylate cyclase; YC-1, 1-Benzyl-3-(5’-hydroxymethyl-2’-furyl) indazole. 中文摘要
II. 前言、研究目的、文獻探討
For many examples of glomerular disease, mesangial-cell proliferation is the important pathological change following the initiation, by the primary insult, of an inflammatory reaction and intraglomerular thrombosis (Peace et al., 1991 and Floege et al., 1992). The increased mesangial-cell numbers that result could be responsible for the secretion of a greater quantity of various growth factors, inflammatory cytokines and an extracellular matrix substance which will further damage the glomeruli. Such an outcome may result in a vicious circle incorporating the ultimate outcome of significant renal damage. The administration of certain reagents which elicit a growth-inhibition effect upon proliferating mesangial cells, such as a
platelet-derived growth factor (PDGF) antagonist, could attenuate the proliferation of mesangial cells and the extent of glomerular damage for many cases of proliferative glomerulonephritis (Lehrke et al., 2002, Floege et al., 1999, and Johnson et al., 1992).
YC-1, a soluble guanylate cyclase activator, facilitates and increases the rate of nitric oxide-mediated cyclic GMP formation (Ko et al., 1994 and Mulsch et al., 1997). Several earlier studies have demonstrated that YC-1 was able to inhibit the
proliferation of certain cells including vascular smooth-muscle cells in-vivo (Wu et al., 2004 and Tulis et al., 2002) and human umbilical-vein endothelial cells in-vitro (Hsu et al., 2003). In addition to its effect upon cell proliferation, YC-1 also demonstrates an inhibitory effect upon platelet function, and therefore also, thrombus formation (Wu et al., 1995 and Teng et al., 1997), a stimulatory effect upon the synthesis of endothelial nitric oxide within the endothelium (Wohlfart et al., 1999),and an inhibitory effect upon white blood-cell function (Hwang et al., 2003) and the
generation of superoxide anions by neutrophils (Wang et al., 2002). All these effects might be construed to be beneficial for cases of renal disease. To evaluate the possible application of YC-1 to cases of renal disease, we first examined the effect of YC-1 upon the proliferation of mesangial cells in vitro. The transition between different phases of the normal cell cycle depends upon changes to and behavior of many cell-cycle proteins. In this study, we also evaluated whether these changes occurred for YC-1 treated cells. The possible mediating signaling pathways involved,
especially a cyclic GMP-mediated pathway, were also evaluated. III. 研究方法
Materials and methods:
Cell Cultures
Mesangial-cell cultures were established as has been described previously (Tsai et al., 1995). Following the first cell passage, mesangial cells were maintained in
incubator. Cells deriving from passages eight to fifteen were used for the following experiments. In these experiments, cells were subcultured into tissue-culture grade 10-cm dishes, and/or six- or 96-well plates. Sub-confluent cells were cultured in serum-free M199 for 24 hours in order to achieve cell quiescence. Cells were
stimulated with 10% fetal calf serum in the cell-proliferation experiments or 20ng/ml of rat platelet-derived growth factor BB for all other experiments. Stimulated cells were treated simultaneously with stimulation with test agents or vehicle. For experiments that lasted longer than 48 hours, the media, PDGF, and/or test agents were changed completely in order to ensure an adequate culture environment for cells to proliferate. Pentobarbital was used as the anesthetic for all procedures conducted with test rats. The management of rats fulfilled the laws of the Republic of China and all animal-management and experimental protocols were approved by the Committee of Experimental Animal Management, the College of Medicine, National Taiwan University. For all experiments, the final concentration at which dimethyl sulfoxide (DMSO) was used for both the control and treatment groups was purposely adjusted to be the same value (0.1%).
Assessment of Cell Proliferation and DNA synthesis
Twenty thousands of mesangial cells were seeded in each well of the six-well culture plates. Subsequent to 24 hours of quiescence, cells were stimulated with 10% FCS in the presence of various concentrations of YC-1 or vehicle (DMSO). At the following time points (24, 48, 72, or 96 hours subsequent to stimulation) culture medium was removed and cells trypsinized. The trypsinized cells were centrifuged, supernatant aspirated and the cells suspended with 3ml of culture medium. Twenty microliters of this cell suspension was removed and mixed with the same volume of trypan blue. An aliquot of the mixture was applied to a hemocytometer, and the density of surviving cells (cells the cytoplasm of which was devoid of the
characteristic trypan-blue stain) and dead cells (cells featuring trypan-blue staining of the cytoplasm) were enumerated under a microscope. The total number of surviving cells per well was estimated by the estimated cell density multiplied by the quantity of diluting medium. The proportion (percentage) of dead cells was calculated relative to the total cell count. To evaluate the toxicity of YC-1, lactate dehydrogenase (LDH) levels present in the collected supernatant were determined on day four subsequent to YC-1 stimulation and compared with those cells not treated with YC-1. In order to evaluate the cell loss by detachment from the culture surface, the number of cells present in the supernatants was determined daily.
BrdU-incorporation analysis was performed to attempt to assess the level of DNA synthesis. Three thousands of mesangial cells were plated in each well of the 96-well
culture plates. Cells were stimulated with PDGF (20ng/ml) containing various concentrations of YC-1 or vehicle. BrdU was added 18 hours subsequent to PDGF stimulation. The quantity of incorporated BrdU was analyzed using an ELISA kit according to the protocol provided by the manufacturer (Amersham, Piscataway, NJ, USA).
Flow Cytometry
One hundred and fifteen thousand of mesangial cells were seeded into
10cm-diameter tissue-culture dishes. Cells were allowed to proliferate to a level of subconfluency, following which cells were treated with PDGF and YC-1 or DMSO subsequent to 24 hours of cell quiescence. Flow-cytometry analysis of the cell-cycle distribution of mesangial cells was performed before and 18 hours subsequent to treatment of cells with the various test agents. Briefly, cells were washed twice with phosphate buffered saline (PBS), harvested by trypsinization, centrifuged (2000 rpm for 5 minutes), and suspended with 1ml of cold PBS, and then fixed in methanol for 30 minutes on ice. Following two washes with PBS, fixed cells were incubated in RNase (1mg/ml) at 37°C for 30 minutes, followed by staining of the DNA with propidium iodide (1µg/µL) at 4°C for 30 minutes in the dark; each sample was analyzed with a Coulter EPICS 753 flow cytometer, and the proportion (percentage) of cells within the G1, S, and G2/M phases of the cell cycle were determined
(Matsushime et al., 1991). Western-Blot Analysis
Western-blot analysis was performed using a protocol that has been previously described (Cheng et al., 2004). Mesangial cells were washed and lysed in RIPA buffer. Forty micrograms of cell lysate were heated at 100oC for ten minutes, applied to sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), and electrophoresed, following which, the electrophoresed proteins in gels were transferred onto polyvinylidine difluoride membranes (Millipore, Bedford, MA, USA) using a transblot chamber with Tris buffer. Subsequent to blocking, Western blots were incubated at 4oC overnight with the appropriate primary antibody, and in the morning of the following day, membranes were washed with PBS/Tween-20, and incubated for 1 hour with peroxidase-conjugated secondary antibodies at room temperature. The dilution for both the primary and secondary antibody was 1: 1000 (or 1: 500 for the primary anti-phospho-p38 MAPK and anti-total p38 MAPK antibody). Subsequent to washing with PBS/Tween-20, membranes were developed with enhanced
chemilumination(Amarshem, Piscataway, NJ, USA). Northern-blot analysis
Total RNA was isolated using the acid quanidinium thiocyanate-phenol-chloroform method and sample concentration as has been described previously (Tsai et al., 1995). Total RNA was electrophoresed on formaldehyde-denatured 1% agarose gels in MOPS buffer (morpholinopropanesulfonic acid [0.2M], sodium acetate [0.05M], EDTA [0.01M]), and transferred to nylon membranes (Genescreen, Boston, MA, USA). Hybridization was performed with digoxigenin-labeled RNA probes. The blots were developed using CSPD® (Roche Molecular Biochemicals, Mannheim, Germany) as the substrate for alkaline phosphatase and results normalized against the signal of glyceraldehyde-3-phosphate dehydrogenase (GADPH) messages.
Rat cyclin D1 RNA probe was synthesized as is described below. The cDNA fragments were generated by reverse transcription-polymerase chain reaction from mesangial-cell DNA using the specific primer pairs:
5’-CTGACACCAATCTCCTCAAC-3’ (corresponding to bases 178 to 197) and 5’-GTAGATGCACAACTTCTCGG-3’ (complementary to bases 506 to 487)
(Tamura et al., 1993). The products were subsequently subcloned into the pGEM-dT vectors (Promega; Madison, WI, USA). The cloned cDNA was then linearized and used as the templates for the in-vitro transcription of antisense
digoxigenin-conjugated riboprobes, according to the supplier's instructions (Roche, Mannhein, Germany).
Immunoprecipitation and CyclinD1/CDK4 kinase assay
Mesangial cells were stimulated with PDGF and the various appropriate test agents or vehicle, following which cell lysates were collected with specific lysis buffer (Tris [20mM; pH = 7.5], NaCl [150mM], EDTA [1mM], EGTA [1mM], Triton-100 [1%], Na2H2P2O4, [2.5mM],β-glycerophosphate [1mM], Na3VO4 [1mM], leupeptin
[1µg/ml], PMSF [1mM]). Immunoprecipitating antibody (anti-CDK4 [2.5µg]) was added to cell-lysate protein (500µg/500µl) and rocked on a shaking table for two hours at 4oC. The immunocomplex was captured using 25µl of protein A/G plus agarose beads. Following subsequent centrifugation (3000 rpm for 30 seconds), the complex was washed twice with lysis buffer and then twice with kinase buffer (Tris [25mM; pH = 7.5], β-glycerophosphate [2.5mM], DTT [2mM], Na3VO4 [0.1mM],
MgCl2 [10mM]). The immunoprecipitation bead pellet was suspended in 40µL kinase
buffer supplemented with ATP (200µM) and 1µg of substrate Rb fusion protein fragment (amino acid 769-921), and then incubated for 30 minutes at 30oC. The reaction was terminated using 10µl of 5X SDS sample buffer. The mixture was then boiled at 100oC for five minutes then cooled on ice and then pellet down. The
supernatant was then used for SDS-PAGE electrophoresis, and subsequently analyzed by a Western-blot technique using an anti-pRb antibody (Ser 780).
Measurement of intracellular cGMP
Three thousands of mesangial cells were plated onto 96-well plates, after 24 hours of quiescence, cells were stimulated with PDGF and various concentration of YC-1. Intracellular cGMP was measured one hour after PDGF stimulation as protocol provided (Amarshem, Piscataway, NJ, USA). The relative intracellular cGMP level to vehicle treated group was used to compare cGMP level between groups.
Reagents
Medium 199, fetal calf serum (FCS), and other tissue-culture reagents were purchased from Gibco BRL (Rockville, MD, USA). Culture flasks and plates were purchased from Costa Corning (Cambridge, MA, USA). Rat recombinant PDGF-BB was
purchased from R & D Systems (Minneapolis, MN, USA). Antibodies against β-actin were obtained from Sigma (The Sigma Chemical Co., Saint Louis, MO, USA). Antibodies against cyclin D1, cyclin E, total Akt1/2(Akt), CDK2, p21waf1, and p27Kip1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phosphorylated-ERK1/2 [p-ERK], total ERK1/2, phosphorylated-p38 MAPK [p-p38 MAPK], total p38 MAPK, Ser473-phosphorylated Akt [p-Akt],
Ser780-phosphorylated retinoblastoma [p-Rb], and Ser9-phosphorylated GSK3β [p-GSK3β] were acquired from Cell Signaling Technology (Beverly, MA, USA). Antibody against CDK4 was purchased from BD Transduction Laboratories
(Lexington, KY, USA). Protein A/G plus agarose beads, and Rb fusion protein was a product of Santa Cruz Biotechnology. RNase was purchased from Calbiochem (San Diego, CA, USA). YC-1 was generously provided by Yung-Shin Pharma Ind. Co. (Taipei, Taiwan).H89 (N-[2-(4-bromocinnamylamino)-ethyl]-5-isoquinoline), KT5823 ((9S,10R,12R)-2,3,9,10,11,12,
hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylicacid methyl ester),SB203580 ([4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole]) and ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one) were obtained from
Calbiochem (San Diego, CA, USA).All chemicals used for total RNA isolation, reverse transcription-polymerase chain reaction, Northern-blot analysis, whole-cell lysate extraction, and Western-blot analysis were of molecular grade and were obtained from either Sigma or Roche unless otherwise specified. YC-1, ODQ and SB203580 were dissolved in dimethyl sulfoxide (DMSO).
Statistical analysis
All values described in the text and figures are expressed as mean ± SEM. All
experiments were performed as a minimum of three replicates. Statistical significance was evaluatedby application of the Student’s t-test or one-way ANOVA with
Bonferroni’s multiple comparison test. A p value of less than 0.05 was considered to represent statistically significant difference between test populations.
IV 結果 Results:
YC-1-inhibited cell growth, BrdU incorporation and induced G1 arrest for mesangial cells
Treatment of mesangial cells with YC-1 in the concentration range of 1 ~ 30µM resulted in a time- and dose-dependent inhibition of cell growth for cultured cells (Figure 1A). The synthesis of DNA by PDGF-stimulated mesangial cells was also inhibited by a similar concentration of YC-1 as revealed by the BrdU-incorporation assay (Figure 1B). We then analyzed the effect of YC-1 upon cell-cycle profiles by flow cytometry. Mesangial cells commenced entry into S phase from G1 phase during the period of from eight-to-twelve hours subsequent to PDGF stimulation. The cell number in S phase reached its greatest level 18 hours subsequent to PDGF stimulation. Figure 2 reveals representative cell-cycle profiles for cells stained with propidium iodide both prior to and 18 hours following PDGF stimulation. The mesangial cells treated with YC-1 exhibited a decreased fraction of S-phase cells and an increased accumulation of G1-phase cells. Such a result indicated that treatment with YC-1 inhibited cellular growth through cell-cycle arrest between G1 and S phases within mesangial cells.
The levels of LDH in the cultured-cell supernatant did not increase following four-days of incubation for the YC-1-treated groups compared to the vehicle group (vehicle, 107.4 ± 3.5; YC-1 [1µM], 109 ± 5.0; YC-1 [5µM], 111.1 ± 6.2; YC-1 [10µM], 107.0 ± 5.1; YC-1 [20µM], 114.0 ± 4.4; YC-1 [30µM], 117.0 ± 6.8 IU/L). The proportion (percentage) of detached cells in the supernatant was less than 5% for all groups and did not differ between YC-1-treated cells and control cells. The
proportion of membrane-damaged adherent cells stained with trypan blue was less than 1% for all groups and did not differ between cells featuring YC-1 treatment and those that did not. Therefore, on the basis of such results, the antiproliferative effect of YC-1 upon cultured mesangial cells was not able to be attributed to YC-1’s cytotoxicity.
YC-1 suppressed the expression of cyclin D1 protein and mRNA, but had no effect upon protein expression of cyclin E, p21waf1, p27kip1, CDK2 and CDK4
For mammalian cells, the major cyclin/CDK complexes involved in the G1-S cell-cycle transition are cyclin D1/CDK4 and Cyclin E/CDK2 (Sherr, 1993). P21waf1 and p27kip1 are the two cyclin-dependent kinase inhibitors for these two major
of these cell-cycle proteins for mesangial cells. Following PDGF stimulation, cyclin D1 protein expression achieved its greatest level approximately six-to-nine hours later. Such expression was inhibited by the simultaneous administration of YC-1. Treatment with YC-1 simultaneously with PDGF stimulation, however, did not affect the protein expression of cyclin E, p21waf1, p27kip1, CDK2 or CDK4 on the same period compared to vehicle treated cells (Figure 3A). Similar such effects of YC-1 upon the protein expression for the above-mentioned cell-cycle proteins were also observed beyond nine hours, and extending to 24 hours subsequent to growth-factor stimulation (data not shown). Northern-blot analysis revealed that the mRNA expression of cyclin D1 was also inhibited by treatment with YC-1 which meant that the inhibition of protein expression proceeded through the down-regulation of mRNA expression (Figure 3B). YC-1 did not increase intracellular GMP concentration in mesangial cells and
inhibition of cyclin D1 expression by YC-1 was not reversed by inhibitors of sGC, PKG, or PKA
YC-1, a soluble guanylate cyclase activator, has been demonstrated to be able to increase cGMP synthesis for a number of different cell types. YC-1 is also a weak phosphodiesterase inhibitor (Galle et al., 1999) which has been shown to be able to increase cAMP levels in neutrophil (Hwang et al., 2003) and possibly also cGMP levels through inhibition of phosphodiesterase activity. To investigate whether the inhibition of cyclin D1 protein expression was related to the activation of the
cGMP/cyclic GMP-dependent protein kinase (PKG) -signaling pathway, we measured the intracellular cGMP level after stimulation with various concentration of YC-1 and treated cultured mesangial cells with ODQ, a sGC inhibitor, in order to counteract the activation of sGC by YC-1. KT5823, a PKG inhibitor, was used to block the
downstream action of cGMP once it was up-regulated by YC-1. One hour after PDGF stimulation the intracellular cGMP concentration did not increase in mesangial cells which were stimulated with the concentration of 1, 10, or 30 µM of YC-1 (Figure 4A). Similar effect was also observed 15 minute or two hours after PDGF stimulation in mesangial cells. Besides, the inhibition of cyclin D1 protein expression by YC-1 was also not reversed by the co-treatment with either ODQ (30µM) or KT5823 (1µM). The application of either ODQ or KT5823 to cultured cells, did not, by themselves, elicit any effect upon the expression of cyclin D1 protein (Figure 4B). These indicated that the suppressive effect of YC-1 upon cyclin D1 protein expression was not related to cGMP/PKG-signaling pathway. The addition of H89, in the concentration of 1 µM adequate to inhibit PKA activity in mesangial cells (Lin et al., 2003), also did not reverse the inhibitory effect upon cyclin D1 expression by YC-1 (Figure 4C), suggesting that the cyclin D1 expression inhibition by YC-1 also did not proceed through the cAMP/PKA-signaling pathway.
YC-1 activated the p38 MAPK but had no effect upon the ERK1/2 MAPK or PI3-Akt-GSK3β-signaling pathway
In the regulation signaling transduction pathways, cyclin D1 synthesis is positively regulated by ERK 1/2 MAPK (Lavoie et al., 1996) and negatively regulated by p38 MAPK pathway (Lavoie et al., 1996 and Page and Li, 2001). The PI3K-Akt-GSK3β pathway is the inhibitory-signaling process related to the destruction of the cyclin D1 protein (Cross et al., 1995 and Diehl et al., 1997). In order to investigate which signaling pathway is involved in the cyclin D1 suppression by YC-1, we evaluated the active form (phosphorylated form) of these signaling proteins by Western blotting. Within a one-hour period subsequent to PDGF stimulation of mesangial cells, a prominent increase in the intra-cellular level of the phosphorylated form of p38 MAPK was observed for YC-1-treated mesangial cells compared to the vehicle treated cells. The level of the phosphorylated form of ERK1/2 MAPK, Akt and GSK3β present did not alter as a consequence of the treatment of such cells with YC-1. Such an observation suggests that YC-1 probably affects only the activity of the p38 MAPK-signaling pathway, and not the ERK1/2 MAPK and PI3K/Akt pathways (Figure 5A).
Inhibition of cyclin D1 expression and cyclin D1/CDK4 kinase activity by YC-1 were partially reversed by the p38 MAPK inhibitor SB203580
The inhibition of both mRNA and protein expression for cyclin D1 as elicited by mesangial-cell exposure to YC-1, could be partially reversed by the co-treatment of the cells with SB203580 (10µM), a p38 MAPK inhibitor in a concentration known to inhibit p38 MAPK kinase activity in mesangial cells ( Inui et al., 2000). When
mesangial cells were treated with only SB203580 (10µM), the level of mRNA and protein expression for cyclin D1 did not change (Figure 5B). Immunoprecipitation of the cyclin D1/CDK4 complex in the cell lysate was performed with mouse anti-CDK4 antibody and the level of kinase activity was evaluated in order to determine the ability of cyclin D1/CDK4 complex to phosphorylate the Rb fusion protein fragment in-vitro. We found that treatment of mesangial cells with YC-1 inhibited the ability of the cyclin D1/CDK4 complex to phosphorylate the Rb protein fragment at the
serine-780 site which has long been known to be the target of cyclinD1/CDK4 kinase activity (Kitagawa et al., 1996). The suppression of such phosphorylation by YC-1 was also partially reversed by co-treatment with SB203580 (Figure 5C). Such a result suggested that YC-1 inhibited cyclin D1 expression and related kinase activity, at least partially, through activation of the p38 MAPK-signaling transduction pathway. V 討論
In this study, we have demonstrated that YC-1, within the concentration range of 1 ~ 30µM, is able to inhibit the proliferation of cultured mesangial cells. The inhibitory effect of such YC-1 exposure proceeded via the inhibition of DNA synthesis and the arrest of the cell-cycle transition from G1 to S phase. The suppression of cyclin D1 synthesis and related CDK4 kinase activity resulted in this cell-cycle arrest. Such findings suggest that the p38 MAPK pathway might play some role in the signaling transduction process leading to the inhibition of DNA synthesis and cell proliferation.
Cyclin D1 is a key regulatorfor early G1-phase progression in mesangial cells
(Terada et al., 1998 and Lang et al., 2000). The inhibition of cyclin D1 by YC-1 was first seen at around three hours post-exposure, and was observed to be greatest at around six-to-nine hours post-stimulation. It has previously been reported that cyclin D1 forms complexes with CDK4 and then stimulates kinase activity within mesangial cells (Terada et al., 1998). We have demonstrated that the decrease in the intracellular cyclin D1 level elicited by YC-1 stimulation of mesangial cells resulted in an overall decrease in the kinase activity of cyclin D1/CDK4 present in the whole-cell lysate six hours subsequent to stimulation. Cyclin D-dependent kinase executes this critical function during middle-to-late G1 phase (Baldin et al., 1993 and Quelle et al., 1993). In our study, mesangial cells began to enter cell-cycle S phase from G1 phase at around 8~12 hours subsequent to PDGF stimulation. The specific time point at which cyclin D1 levels were suppressed by YC-1 was noted to be prior to the onset of the G1/S transition. Therefore, it would appear quite reasonable to conclude that the inhibition of cyclinD1/CDK kinase activity by YC-1 led to cellular growth arrest in G1/S phase. This finding appears to contrast an earlier observation by other workers that YC-1-induced cell-cycle G1/S arrest for endothelial cells via the augmentation of p21waf1 and p27kip1 protein levels, 18 hours subsequent to growth stimulation (Hsu et al., 2003). The average protein level of these CDKIs did not appear to change as a consequence of treatment with YC-1 in our study.
Increasing intracellular cGMP level or activating the PKG pathway could inhibit the proliferation of mesangial cells. In 2000, Pandey et al. reported that activation of PKG by natriuretic peptide receptor A could result in inhibition of the proliferation of mesangial cells (Pandey et al., 2000). A previous study has revealed that cGMP was able to delay the normal cell-cycle transition through the suppression of cyclin D1 and CDK4 activation within vascular smooth-muscle cells (Fukumoto et al., 1999). Herein, we considered it was necessary to attempt to evaluate whether YC-1 inhibited cyclin D1 synthesis through the activation of some form of cGMP/PKG-signaling system. However, in this study YC-1 failed to increase intracellular cGMP level significantly in mesangial cells. The reason was unknown. One of the hypothesis is that YC-1 (within the concentration of 1 ~30 µM) may only facilitate cGMP production under
the co-stimulation with other sGC activator (such as nitric oxide) in mesangial cell. This need further study to uncover the truth. To exclude the influence by minor change of intracellular cGMP, we further blocked the possible effect of YC-1 upon sGC by co-treatment of cells with ODQ, a sGC inhibitor, and blocked the activity of PKG with KT5823 to block the downstream-effect machinery cGMP pathway, once activated by intracellular cGMP. Both of these consecutive treatments failed to reverse the inhibition of cyclin D1 expression by YC-1. We therefore conclude that the suppression of cyclin D1 expression by YC-1 in our study was independent of the cGMP/PKG-signaling pathway. In the 2003 study of Hsu et al., the growth inhibitory effect of YC-1 upon human umbilical endothelial cells was also not related to
cGMP-pathway activation (Hsu et al., 2003).
From the Northern-blotting results, the mRNA expression of cyclin D1 proved to be lower for YC-1-treated cells than it was for their untreated controls. The quantity of intracellular message RNA is one of the principal determinants of the extent of protein expression; in effect, a lower level of mRNA expression will lead to a lower level of protein expression and conversely so. Therefore, it could be concluded that YC-1 inhibits cyclin D1 protein expression through the mechanism of eliciting a reduction to mRNA expression. We did observe that treatment of cultured mesangial cells with YC-1 was able to increase the intra-cellular level of the phosphorylated form of p38 MAPK. By co-treatment of such cells with SB203580, a p38 MAPK inhibitor, the cyclin D1 protein and mRNA expression inhibited by cell exposure to YC-1 was able to be partially reversed. The analogous observation was also made for the kinase activity of the cyclin D1/CDK4 complex. Such a result suggests that there was a possible role for YC-1 as regards inhibiting the cyclin D1 expression of mesangial cells through the activation of p38 MAPK. Further, a number of recent studies have reported that activation of the p38 MAPK-signaling pathway has led to the suppression of cyclin D1 mRNA expression (Lavoie et al., 1996 and Page et al., 2001), however, the reversal of cyclin D1 suppression in YC-1 treated cells by SB203580 did not appear to be complete in our study. Thus, we speculate that there might be another possible mechanism for the inhibition of cyclin D1 expression by YC-1. The ERK1/2 MAPK pathway was the stimulatory signaling mechanism for cyclin D1 mRNA synthesis (Cheng et al., 1998, Peeper et al., 1997 and Atktas et al., 1997) whilst the PI3K-Akt-GSK3β pathway was the mechanism capable of stabilizing cyclin D1 protein expression (Cross et al., 1995 and Diehl et al., 1997). The collective activities of these two pathways, which were represented by the phosphorylated form of the kinase protein as shown in Western blotting, were not affected by the exposure of cultured mesangial cells to YC-1. It is possible that YC-1 also inhibits cyclin D1 expression through another mechanism, such as YC-1’s influence upon protein kinase
C signaling transduction, such a possibility clearly warranting further investigation. It was interesting to recognize that YC-1 inhibits cyclin D1 expression through activation of p38 MAPK as we shown here. Browning et al reporting in 1999 that YC-1 was able to activate p38 MAPK within cultured human neutrophils (Browning et al., 1999). From these authors’ study, the stimulation of
lipopolysaccharide-activated p38 MAPK occurred through nitric oxide-dependent cGMP activation, and, such activation was able to be blocked with KT5823
(Browning et al., 1999). In 2000, Browning’s group subsequently confirmed that the activation of p38MAPK within human fibroblasts by nitric oxide and a cGMP analogue required the presence of PKG (Browning et al., 2000). Whether YC-1 activates p38 MAPK through activation of PKG or not will remain as an interesting point of speculation. From the results of our study, the intracellular cGMP level was not elevated and blocking the activity of PKG with KT5823 did not correspondingly block the inhibition of cyclin D1 expression elicited by the exposure of mesangial cells to YC-1. The addition of H89, a PKA inhibitor, to the culture also failed to block inhibition of cyclin D1 by YC-1. Thus it remains difficult to say that the cGMP-PKG- or cAMP-PKA-signaling pathway played any substantial role in the activation of p38 MAPK which subsequently led to cyclin D1 suppression for cultured mesangial cells. Therefore, there must remain the possibility that YC-1 activates P38 MAPK via another different mechanism.
The proliferation of mesangial cells is one of the most-important pathological changes that arise for many different examples of proliferative glomerulonephritis (Peace et al., 1991). Although YC-1 was observed to have been able to inhibit the proliferation of mesangial cells, it is possible that YC-1 may demonstrate other biological effects within cells, such as eliciting p38 MAPK activation independently of cGMP- or cAMP-signaling pathways, as appeared to have been the case in this study,. Such in-vitro effects might be associated with rather unexpected
pharmacological effects in vivo. Before extending the results of such investigation to any clinical application for human disease, it is necessary to disclose the possible range of pharmacological effects of YC-1, including its influence upon inflammation, matrix synthesis and vascular hemodynamic change in vivo. The underlying effect and mechanism of YC-1 upon signaling transduction for pathways other than the cGMP pathway should also be evaluated.
Acknowledgments:
This work was supported by grants from the National Science Council (92-2314-B-002-363), National Taiwan University Hospital Yulin branch (NTUHYLS007), the Mrs. Hsiu-Chin Lee Kidney Research Fund and Ta-Tung
Research Fund, Taipei, Taiwan. We also would like to thank Huang Kuo-Don for his technical support for this study.
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Figure 2 A
B
Legends:
Figure 1: Effect of YC-1 upon the growth and BrdU incorporation of cultured rat mesangial cells. A) The same numbers of mesangial cells stimulated with 10% serum were cultured with various concentrations of YC-1. Cell number was determined at the indicated time points. The proliferation of mesangial cells was inhibited by treatment with YC-1 in a dose- and time-dependent manner. (*, p < 0.05 versus vehicle [DMSO] treated on days 2, 3, and 4) B) Mesangial cells were stimulated with PDGF (20ng/ml) following deprivation of growth stimulation and simultaneous treatment with YC-1 at a variety of concentrations. Cellular incorporation of BrdU was determined 18 hours subsequent to PDGF stimulation. A greater dose of YC-1 exhibited a more-pronounced level of suppression of BrdU incorporation. (*, p < 0.05 versus vehicle treated)
Figure 2: Effect of YC-1 upon mesangial-cell cell-cycle profile. Mesangial cells were collected and cell-cycle analyses performed with propidium iodide and flow
cytometry. The proportion (percentage) of cells in each phase of the cell cycle is presented as the mean (± SEM). A higher proportion of mesangial cells in G1 phase were found prior to PDGF stimulation. Eighteen hours subsequent to PDGF
stimulation, a significantly greater number of mesangial cells had entered into S phase from G1 phase than was the case prior to such stimulation. The transition between the two phases was inhibited by the administration of YC-1 (30µM). There were fewer cells in S phase and more cells in G1 phase for the YC-1-treated group than was the case for the group devoid of YC-1 treatment. (*, p < 0.05 versus growth-arrested; **, p < 0.05 versus vehicle treated)
Figure 3: Effect of YC-1 on the expression of cyclin D1, cyclin E, CDK2, CDK4, p21waf1, p27kip1 within nine hours from growth stimulation. A) Subsequent to stimulation with PDGF, protein expression for cyclin D1 increased, this increment being inhibited by the treatment of cells with YC-1 (30µM). The protein expression of cyclin E, CDK2, CDK4, p21waf1, p27Kip1 were not different between the cells treated with YC-1 and vehicle on the same time point, although the p21waf1 protein amount changed after PDGF stimulation. B) The mRNA expression of cyclin D1 was also inhibited by YC-1 (30µM). C) Densitometric analysis of cyclin D1 expression treated with YC-1 or vehicle on various time points in Western/Northern blotting assays shown in figure 4A and figure 4B. The number below each lane indicates the time point subsequent to PDGF stimulation. (*, p < 0.05 versus vehicle treated on the same time point)
Figure 4: A) Intracellular cGMP was measured one hour after PDGF stimulation. The cGMP level was not different significantly between YC-1 treated cells or vehicle
treated cells. (p > 0.05 for all YC-1 treated cells versus vehicle treated cells) B) Cell lysate was collected before and six hours subsequent to PDGF stimulation. Protein expression of cyclin D1 was inhibited by YC-1 (30µM) stimulation. This inhibition was not reversed by co-treatment of cells with ODQ (30µM) or KT5823 (1µM). Neither ODQ nor KT5823 themselves affected the expression of cyclin D1. C) The inhibition of cyclin D1 by YC-1 was also not reversed by the co-treatment of cells with H89 (1µM). (*, p < 0.05 versus vehicle treated)
Figure 5: A) The effect of YC-1 (30µM) upon the phosphorylation of relevant signaling proteins within six hours following PDGF stimulation, including ERK1/2, p38 MAPK, Akt, and GSK3β, with only the activation of p38 MAPK being apparent. For each signaling protein, the upper lane represented the phosphorylated form of the protein and the lower lane represented the total form. The number below each lane indicates the time point following PDGF stimulation (*, p < 0.05 versus vehicle treated at the same time point). B) Cell lysate was collected both prior to and six hours following PDGF stimulation. Protein expression of cyclin D1 was inhibited by cellular exposure to YC-1 (30µM). This inhibition was partially reversed by
co-treatment of cells with SB203580 (10µM), a p38 MAPK inhibitor. SB203580 itself did not significantly alter the expression of cyclin D1. Analogous changes to the expression of mRNA were also seen as a consequence of the treatment of mesangial cells with these drugs. (α, p < 0.05 versus vehicle treated; β, p < 0.05 versus YC-1 treated only; γ, p < 0.05 versus SB203580 treated only; *, p < 0.01 versus vehicle treated only; **, p < 0.05 versus YC-1 treated only; ***, p < 0.01 versus SB203580 treated only) C) Cyclin D1/CDK4 complex in cell lysate was immunoprecipitated with anti-CDK4 antibody. The kinase activity of the precipitated complex was evaluated by its ability to phosphorylate the Rb fusion protein fragment at the serine 780 site in vitro. For YC-1 treated cells, the kinase activity was lower than for the controls, and this inhibition was able to be partially reversed by co-treatment with SB203580 (10µM). SB203580 did not affect the kinase activity of cyclin D1/CDK4 complex. (*, p < 0.05 versus vehicle treated; **, p < 0.05 versus YC-1 treated only; ***, p =0.09 versus SB203580 treated only)
VII 成果評量
本研究計畫教預定時間晚三個月完成, 但已將計畫預定項目完成, 並進一步探討 訊息傳遞的機轉, 已寫成論文並已投稿. 預期不久將來可知是否被期刊接受. 本 研究對研究藥物對細胞增生影響有進一步的了解, 有助於臨床藥物的發展, 計畫 的進行也對助理研究人員訓練大有幫助.
