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• W2005431362 abstract "Article15 October 2002free access Signaling pathways and late-onset gene induction associated with renal mesangial cell hypertrophy Sandro Goruppi Sandro Goruppi Diabetes Research Laboratory, Department of Medicine, Massachusetts General Hospital, Charlestown, MA, 02129 USA Present address: Molecular Cardiology Research Institute, New England Medical Center, Department of Medicine, Tufts University School of Medicine, Boston, MA, 02111 USA Search for more papers by this author Joseph V. Bonventre Joseph V. Bonventre Renal Unit, Department of Medicine, Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author John M. Kyriakis Corresponding Author John M. Kyriakis Present address: Molecular Cardiology Research Institute, New England Medical Center, Department of Medicine, Tufts University School of Medicine, Boston, MA, 02111 USA Search for more papers by this author Sandro Goruppi Sandro Goruppi Diabetes Research Laboratory, Department of Medicine, Massachusetts General Hospital, Charlestown, MA, 02129 USA Present address: Molecular Cardiology Research Institute, New England Medical Center, Department of Medicine, Tufts University School of Medicine, Boston, MA, 02111 USA Search for more papers by this author Joseph V. Bonventre Joseph V. Bonventre Renal Unit, Department of Medicine, Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author John M. Kyriakis Corresponding Author John M. Kyriakis Present address: Molecular Cardiology Research Institute, New England Medical Center, Department of Medicine, Tufts University School of Medicine, Boston, MA, 02111 USA Search for more papers by this author Author Information Sandro Goruppi1,2, Joseph V. Bonventre3 and John M. Kyriakis 2 1Diabetes Research Laboratory, Department of Medicine, Massachusetts General Hospital, Charlestown, MA, 02129 USA 2Present address: Molecular Cardiology Research Institute, New England Medical Center, Department of Medicine, Tufts University School of Medicine, Boston, MA, 02111 USA 3Renal Unit, Department of Medicine, Harvard Medical School, Charlestown, MA, 02129 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5427-5436https://doi.org/10.1093/emboj/cdf535 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In chronic diseases such as diabetes mellitus, continuous stress stimuli trigger a persistent, self-reinforcing reprogramming of cellular function and gene expression that culminates in the pathological state. Late-onset, stable changes in gene expression hold the key to understanding the molecular basis of chronic diseases. Renal failure is a common, but poorly understood complication of diabetes. Diabetic nephropathy begins with mesangial cell hypertrophy and hyperplasia, combined with excess matrix deposition. The vasoactive peptide endothelin promotes the mesangial cell hypertophy characteristic of diabetic nephropathy. In this study, we examined the signaling pathways and changes in gene expression required for endothelin-induced mesangial cell hypertrophy. Transcriptional profiling identified seven genes induced with slow kinetics by endothelin. Of these, p8, which encodes a small basic helix–loop–helix protein, was most strongly and stably induced. p8 is also induced in diabetic kidney. Mesangial cell hypertrophy and p8 induction both require activation of the ERK, JNK/SAPK and PI-3-K pathways. Small interfering RNA (siRNA)-mediated RNA interference indicates that p8 is required for endothelin-induced hypertrophy. Thus, p8 is a novel marker for diabetic renal hypertrophy. Introduction With the rising incidence of obesity in the developed world has come an epidemic of type 2 diabetes mellitus. It is currently estimated that in the USA alone, 16–17 million individuals have type 2 diabetes, and the number of adults with type 2 diabetes increased by 49% between 1991 and 2000 (Mokdad et al., 2001). Type 2 diabetes is a complex disease caused by multiple genetic and environmental factors. A substantial fraction of the morbidity and mortality due to diabetes can be attributed to the pathophysiology of diabetic complications. In the absence of a clear genetic understanding of type 2 diabetes, addressing the root causes of diabetic complications is of great significance to the development of more effective treatment approaches. Diabetic nephropathy, a common complication of both type 1and 2 diabetes, is the major cause of end-stage renal failure in the Western world (Fine et al., 1992). In diabetes, hypertrophy and excess extracellular matrix deposition are associated with the progression to renal failure. Hypertrophy is marked by an increase in overall protein synthesis, new gene expression—notably of embryonic and immediate-early genes—and, in some cases, reorganization of the actin cytoskeleton (Bonventre and Force, 1998; Force, 1999; Molkentin, 2000). These changes typically are not accompanied by an increase in cell numbers. The molecular basis of diabetic renal hypertrophy is still an enigma, although diabetic renal hypertrophy and matrix deposition have been linked to pathologic changes in renal mesangial cells. Accordingly, these cells have been studied extensively for clues as to the mechanisms that underlie diabetic renal disease (Fine et al., 1992; Bonventre and Force, 1998). Multiple extracellular stimuli are thought to be involved in the pathophysiology of diabetic mesangial cell hypertrophy. Prominent among these are vasoactive peptides such as endothelin-1 (ET-1) and angiotensin II. The observation that diabetic renal disease often progresses even when glucose homeostasis is well managed suggests that self-reinforcing, long-term changes in gene expression—changes that have yet to be elucidated—may lie at the heart of the molecular events that contribute to this and other chronic conditions. Signal transduction pathways recruited in response to the persistent presence of pro-hypertrophic and other chronic stress agonists may participate in the initiation of pathological hypertrophy and other deleterious effects. Mitogen-activated protein kinases (MAPKs) have been implicated in cardiomyocyte hypertrophy (Wang et al., 1998a,b; Force et al., 1999; Bogoyevitch, 2000; Molkentin, 2000), and activation by hypertrophic stimuli of mesangial cell MAPK subgroups [extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK] has been documented (Bonventre and Force, 1998), but not tracked with changes in gene expression that occur during biological responses such as hypertrophy or matrix deposition. Similarly, the phosphatidylinositol-3′-OH-kinase (PI-3-K) pathway has been implicated in cardiomyocyte hypertrophy (Haq et al., 2000; Antos et al., 2002), but a role for this pathway in diabetic renal hypertrophy has not been established. The ability of MAPKs [through the activator protein-1 (AP-1) and other transcription factors] and downstream effectors of PI-3-K [which also include AP-1, as well as nuclear factor of activated T cell (NFAT) and forkhead (FKH) family transcription factors] to trigger changes in gene expression is thought to underlie a substantial part of the mechanism by which these pathways affect cell function (Boyle et al., 1991; Cross et al., 1995; Rao et al., 1997; Molkentin et al., 1998; Haq et al., 2000; Kops and Burgering, 2000; Kyriakis, 2000; Brazil and Hemmings, 2001; Katso et al., 2001; Kyriakis and Avruch, 2001; Lawlor and Alessi, 2001; Neal and Clipstone, 2001; Scheid and Woodgett, 2001; Antos et al., 2002). The molecular basis by which these various signaling pathways reprogram cells to adapt to chronic stresses, such as those that cause hypertrophy, remains nebulous. In particular, a picture of the more long-term, stable, changes in gene expression that occur as cells progress to a pathologically deranged state is missing. This is important since chronic diseases such as diabetic nephropathy probably involve long-term, systematic changes in gene expression that, in response to chronic stress, essentially condemn cells to a diseased state. Here we report the results of a combined pharmacological, transcriptional profiling and genetic study aimed at beginning to identify renal mesangial cell signaling pathways and transcripts induced by ET-1 under conditions that trigger hypertrophy. We sought to identify either genes induced after the prolonged periods of ET-1 treatment (up to 48 h) needed to trigger hypertrophy, or genes induced rapidly, but stably throughout the 48 h treatment period. Our aim was to implicate ET-1-induced transcripts as necessary to the progress of mesangial cell hypertrophy. One transcript encoding the small basic helix–loop–helix (bHLH) protein p8 (Mallo et al., 1997) was strikingly induced under conditions indistinguishable from those necessary to trigger mesangial cell hypertrophy. Moreover, RNA interference (RNAi) experiments indicate that p8 induction is required for ET-1-stimulated mesangial cell hypertrophy. These findings are significant inasmuch as they identify p8 as a necessary component in the transcriptional program that triggers mesangial cell hypertrophy. In addition, these results indicate that p8 is a novel marker gene for diabetic renal hypertrophy. Given that traditional protein synthesis assays for mesangial cell hypertrophy are cumbersome and inefficient, assaying induction of p8 will streamline the search for novel inhibitors of diabetic renal disease. Finally, our results reveal a striking similarity between the signaling pathways required for renal mesangial cell hypertrophy and cardiac hypertrophy (Molkentin, 2000). Accordingly, p8 may also be important for other hypertrophic pathologies, including those of the heart. Results ET-1 triggers renal mesangial cell hypertrophy Figure 1A is an illustration of the treatment protocol to which we subjected rat renal mesangial cells in these studies. Mesangial cell hypertrophy is defined as a stimulus-dependent increase in cellular protein synthesis ([3H]leucine uptake) without an accompanying increase in DNA synthesis ([3H]thymidine uptake). We routinely observe that ET-1 triggers mesangial cell hypertrophy manifested as an increase in protein synthesis and a decrease in DNA synthesis (Figure 1B). Figure 1.Mesangial cell treatment protocol and ET-1 induction of hypertrophy. (A) ET-1 treatment protocol. The figure describes the time course of treatment for induction of hypertrophy. For certain experiments, as indicated in other figures, cells were harvested at earlier times in order to monitor regulation of signaling pathways or gene expression. (B) The 48 h treatment protocol in (A) is sufficient to trigger mesangial cell hypertrophy, an increase in [3H]leucine uptake and a parallel decrease in [3H]thymidine uptake. Download figure Download PowerPoint Microarray analysis In contrast to cardiomyocyte hypertrophy, few details have emerged concerning gene induction during the progression of renal mesangial cells to hypertrophy. Specifically, no known marker genes have been clearly linked to renal mesangial cell hypertrophy. In order to identify genes induced by ET-1 under conditions that trigger mesangial cell hypertrophy, we performed microarray analysis. Inasmuch as hypertrophy is a delayed process, we were interested in genes whose induction, if rapid, was sustained for a prolonged period of ET-1 treatment. Alternatively, we sought genes induced only after extended ET-1 treatment. Rat renal mesangial cells were treated with vehicle or ET-1 as described above. RNA was collected at 1, 24, 36 and 48 h after treatment; in parallel, progression of the cells to hypertrophy was confirmed as above (Figure 1). A portion of the RNA from the cells treated for 24 and 48 h was reverse transcribed, labeled with 33P and applied to filter microarrays spotted with ∼5500 cDNAs. RNA samples were also subjected simultaneously to northern analysis. Induction values on northern blots were quantitated by phosphoimaging and normalized for gapdh expression. To confirm functional gene induction in the ET-1-treated cells, we analyzed expression of gene 33, a gene induced in response to a variety of proinflammatory and mitogenic stimuli (Messina, 1994; Makkinje et al., 2000). gene 33 expression was stimulated by ET-1, reaching an apparent maximum at 1 h, and declining to baseline by 24 h (Figure 2; Table I). We identified seven genes that obeyed our criteria for late-onset genes. Results for the northern analyses are shown in Figure 2; Table I summarizes the transcriptional profiling for genes detected on both the microarrays and northern blots as ET-1 late-onset-inducible. Induced with slow kinetics in response to ET-1 is a set of genes associated with stress and inflammation [copper/zinc-containing superoxide dismutase (Cu/Zn-sod), bag2], signaling [ADP ribosylation factor-6 (arf6), phospholipase-Cβ4 (PLCβ4), p116 rho-interacting protein (p116rip), myr5] and/or diabetes [cystatin C, platelet-derived growth factor A chain (PDGF-A)] (Silver et al., 1989; Bhandari and Abboud, 1993; Jiang et al., 1994; Hosler and Brown, 1995; Gebbink et al., 1997; Hall, 1998; Post et al., 1998; Takayama et al., 1999; Mostov et al., 2000; Song et al., 2001; Mussap et al., 2002) (Figure 2; Table I). Figure 2.Early- and late-onset gene induction in mesangial cells treated with ET-1. Mesangial cells were treated with ET-1 for the indicated times. RNA was subjected to microarray analysis, and genes whose induction was confirmed on the arrays were subjected to further analysis by northern blotting. Induction is quantitated in Table I. Download figure Download PowerPoint Table 1. Time course of induction of various genes by ET-1 in rat renal mesangial cells Function Protein name Fold induction at indicated time post ET-1 treatment 1 h 24 h 36 h 48 h Signaling PDGF A chain 0.8 1.2 ND 2.0 p116rip 0.7 1.5 ND 2.1 Gene 33 6.0 1.1 1.1 1.1 myr5 1.4 ND ND 2.5 Phospholipase C-β4 0.9 ND ND 1.9 bag2 5.5 ND ND 1.3 Nuclear p8 2.4 3.0 8.0 5.1 Enzymes Cu/Zn superoxide dismutase (SOD) 2.2 2.1 2.2 1.9 ADP ribosylation factor-6 1.6 1.3 2.1 1.6 ECM Cystatin C 0.6 1.1 ND 2.2 Gene induction was detected initially by microarray and confirmed by northern blot analysis. Fold inductions shown were determined by northern blot analysis. ND, not determined. By far, the gene most strikingly and stably induced by ET-1 is p8. p8 encodes a small bHLH nuclear protein up-regulated in pancreatitis and implicated in V12-ras-dependent tumorigenesis and regulation of paired box transcription factors of the Pax family (Mallo et al., 1997; Hoffmeister et al., 2002; Vasseur et al., 2002b). During the course of ET-1 treatment, p8 is induced significantly at 1 h (3.3-fold), with induction continuing out to 24 h (4-fold), reaching a maximum of 6- to 10-fold induction at 36 h treatment (Figure 2; Table I). Most notably, p8 induction remains elevated at least 7-fold above basal after 48 h of ET-1 treatment. ET-1 activates the mesangial cell ERK, JNK/SAPK and p38 MAPK pathways as well as the PI-3-K pathway To establish a clear picture of the signaling pathways recruited by ET-1, we assessed the activation of various MAPK pathways and the PI-3-K pathway during the course of ET-1-induced mesangial cell hypertrophy. MAPKs are recruited as part of three tiered MAPK kinase kinase (MAP3K)→MAPK kinase (MKK)→MAPK core pathways. MAPKs require concomitant tyrosine and threonine phosphorylation for activity (Kyriakis and Avruch, 2001). Using antibodies specific for the phosphorylated, active forms of ERK1/2, JNK/SAPK and p38, as well as their corresponding MKKs (MEK1/2, MKK4, and MKKs 3 and 6, respectively), we find that all three of these MAPK pathways are activated in response to ET-1 (Figure 3A–D). ERK activation is comparable with that incurred by epidermal growth factor (EGF), used as positive control for cell activation and as an indicator of the reliability of the phospho-antibodies. In contrast, JNK/SAPK and p38 pathway activation, while significant, is more modest than that stimulated by hyperosmotic shock (500 mM sorbitol, like EGF used as positive control) (Figure 3A and C). In all cases, MAPK pathway activation by ET-1 is maximal after 5–10 min and declines to basal by 60 min (Figure 3A and C). After 60 min, JNK/SAPK and p38 activation remain at basal levels for the remainder of the 48 h treatment protocol (Figure 3A and B). ERK pathway activity, however, rises modestly for a second time, reaching a submaximal plateau by 48 h (Figure 3B and D), a point in time when the cells are clearly hypertrophic (Figure 1). ERK activity is also elevated after 24 h of EGF stimulation (Figure 3B). Figure 3.Activation of mesangial cell ERK, JNK/SAPK and p38 MAPK pathways by ET-1. (A) Cells were treated with the indicated agonists for the indicated times. Crude extracts were prepared and subjected to SDS–PAGE and immunoblotting with phospho-MAPK-specific antibodies (top three panels) or total MAPK blots (bottom three panels). (B) The same as (A), except that cells were treated for longer time periods. (C) Activation of MKKs by ET-1. Cells were treated with the indicated stimuli as in (A). Extracts were prepared and subjected to SDS–PAGE and immunoblotting with the indicated phospho-MKK-specific antibodies (top three panels) or, as a loading control, with an antibody to smooth muscle actin (SMA, bottom panel). (D) The same as (C), except that blots were probed with anti-phospho-MEK only. (E) Activation of CREB by ET-1. Mesangial cells were treated with the indicated agonists for the indicated times, and crude extracts were subjected to SDS–PAGE and immunoblotting with phospho-CREB/ATF-1-specific antibody. Download figure Download PowerPoint The transcription factor cAMP response element-binding protein (CREB) functions in diverse physiological processes, including the control of cellular metabolism and mitogen-dependent cell survival (Mayr and Montminy, 2001). In response to mitogens and stresses, CREB is phosphorylated at Ser133 and activated in a cAMP-independent, ERK- and p38 MAPK-dependent manner (Deak et al., 1998). We observe that ET-1 stimulates CREB Ser133 phosphorylation, as well as phosphorylation of the related transcription factor activating transcription factor-1 (ATF1), consistent with the idea of functionally relevant MAPK pathway activation (Figure 3E). PI-3-K and its effectors are essential to the control of cell survival, cell size, proliferation and metabolism (Katso et al., 2001). It is of note that PI-3-K effectors have been implicated in cardiac hypertrophy (Molkentin et al., 1998; Haq et al., 2000; Antos et al., 2002). We also observe that the rat renal mesangial cell PI-3-K pathway is activated by ET-1. Recruitment of PI-3-K leads to 3′-phosphoinositide-dependent phosphorylation at Ser308 and Ser473, and consequent activation of protein kinase B (PKB) (Alessi et al., 1996, 1997a,b). ET-1 treatment triggers the rapid phosphorylation and activation of mesangial cell PKB as detected on immunoblots probed with antibodies directed towards PKB phosphorylated at Ser473 (Figure 4). Activated PKB directly phosphorylates and inhibits two key effectors: glycogen synthase kinase-3 (at Ser9 for GSK3β and Ser21 for GSK3α) and transcription factors of the FKH family (Cross et al., 1995; Kops and Burgering, 2000; Brazil and Hemmings, 2001; Scheid and Woodgett, 2001). We find that ET-1 triggers phosphorylation of GSK3β at Ser9, GSK3α at Ser21, and of the FKH transcription factors AFX (at Ser193) and FKHR (at Ser256) (Figure 4). Stimulus-induced inhibition of GSK3β has been linked to the development of cardiomyocyte hypertrophy via relief of GSK3-mediated inhibition of NFATs (Haq et al., 2000; Antos et al., 2002). Figure 4.Activation of the mesangial cell PI-3-K pathway by ET-1. Primary mesangial cells were treated with the indicated agonists for the indicated times. Crude extracts were subjected to SDS–PAGE and immunoblotting with phospho-PKB, phospho-GSK3, phospho-FKHR or, as a loading control, SMA as previously reported (Goruppi et al., 2001). Download figure Download PowerPoint ET-1 induction of renal mesangial cell hypertrophy requires ERK, JNK/SAPK and PI-3-K, but not p38 MAPK The signal transduction mechanisms that control renal mesangial cell hypertrophy are not known. Studies of cardiac hypertrophy indicate a role for MAPKs of the ERK, JNK/SAPK and p38 groups (Choukroun et al., 1998, 1999; Wang et al., 1998a,b; Force, 1999). In addition, PI-3-K and its effectors—most notably, GSK3 and NFATs—have been implicated in cardiomyocyte hypertrophy (Molkentin et al., 1998; Haq et al., 2000; Molkentin, 2000; Antos et al., 2002). Mesangial cell ERK, JNK/SAPK, p38 and PI-3-K are activated substantially by ET-1 under conditions associated with the induction of hypertrophy (Figures 1, 3 and 4). In recent years, a suite of extremely selective, cell-permeant inhibitors of protein kinases has been developed (Davies et al., 2000). Using these specific reagents, we find that ET-1-induced mesangial cell hypertrophy requires activation of ERK, JNK/SAPK and PI-3-K. While p38 activation is observed in response to ET-1, we do not find that activation of p38α or β is necessary for hypertrophy. U0126 is a highly specific inhibitor of the activation of MEK1/2 by the MAP3K Raf-1 (Davies et al., 2000). We find that, in parallel with inhibition of ET-1 activation of ERK, U0126 reverses the ET-1-induced increase in mesangial cell protein synthesis (Figure 5). Thus, ERK activity is necessary for optimal ET-1-induced mesangial cell hypertrophy. Similar but more striking results were obtained with LY294002, a highly specific synthetic inhibitor of PI-3-K (Davies et al., 2000), indicating a role for PI-3-K in ET-1-induced mesangial cell hypertrophy (Figure 5A). SP600125 is a high affinity anthrapyrazolone inhibitor of the JNKs/SAPKs. SP600125 does not inhibit ERK or p38 MAPK in vivo (Bennett et al., 2001; Figure 5B). SP600125 also reverses ET-1-induced mesangial cell hypertrophy (Figure 5A). Thus, as in cardiomyocytes, JNK/SAPK is an important element in ET-1-induced mesangial cell hypertrophy. In contrast, SB203580, a selective inhibitor of the α and β (but not the γ and δ) isoforms of p38 MAPK (Davies et al., 2000; Figure 5B), fails to prevent mesangial cell hypertrophy in response to ET-1, in spite of blocking the activation of p38 (Figure 5). Figure 5.ERK, JNK/SAPK and PI-3-K, but not p38, are required for ET-1-induced mesangial cell hypertrophy. PP2B is also required for optimal ET-1-induced hypertrophy. (A) Mesangial cells were pre- treated with the indicated inhibitor compounds for 30 min, at which time vehicle or ET-1 was added and the incubation continued for an additional 48 h. Hypertrophy was measured as in Materials and methods. (B) Efficacy of the indicated inhibitors. A portion of the cells in (A) was lysed and the lysates subjected to analysis with phospho-specific antibodies. (C) FK506 blocks mesangial cell hypertrophy. Mesangial cells were pre-treated with 100 nM FK506 and treated with ET-1 as in (A). ET-1 triggers a significant increase in leucine incorporation (indicated by *, as determined by t-test; P < 0.05, n = 3); however, FK506 pre-treatment reduces leucine incorporation to insignificant levels (indicated by **, as determined by t-test; P > 0.05, n = 3). Download figure Download PowerPoint ET-1 stimulates Ca2+ uptake (Bonventre and Force, 1998). FK506 is a potent Ca2+/calmodulin-dependent inhibitor of PP2B (Schreiber and Crabtree, 1992). ET-1 elicits cardiomyocyte hypertrophy in part through an NFATs-dependent mechanism. ET-1 inhibits GSK3, and activates PP2B, reversing GSK3's inhibitory NFAT phosphorylation (Molkentin et al., 1998; Haq et al., 2000; Antos et al., 2002). We find that FK506 reverses ET-1-induced mesangial cell hypertrophy (Figure 5C). Taken together with the results in Figure 4 indicating that ET-1 triggers an inhibitory phosphorylation of mesangial cell GSK3, FK506 inhibition of mesangial cell hypertrophy is consistent with the idea that NFATs or other PP2B effectors are important to ET-1-induced hypertrophy. ET-1 induction of p8 and hypertrophy requires the same signaling pathways Despite the significant clinical importance of renal mesangial cell hypertrophy to the pathogenesis of diabetic nephropathy, little is known of the molecular mechanisms underlying this process. Accordingly, we have begun to seek transcripts induced by ET-1 in a manner indistinguishable from that of hypertrophy, and to determine if these transcripts are also induced in diabetic kidney. To this end, we treated mesangial cells with ET-1 under conditions sufficient to trigger hypertrophy. Some of the cells were pre-treated with specific inhibitors of kinase pathways. Our findings (Figure 6) indicate that p8 induction is regulated in a manner indistinguishable from that of hypertrophy. Thus, we find that the strong ET-1 induction of p8 is reversed by the PI-3-K inhibitor LY294002, the strongest inhibitor of hypertrophy (Figures 5A and 6A). Inhibition of p8 induction is also observed with the ERK pathway inhibitor U0126 and, to a lesser degree, by the JNK/SAPK inhibitor SP600125. FK506 treatment also blocks ET-1 induction of p8, suggesting that PP2B activity is important to both ET-1-stimulated hypertrophy and p8 expression. The extent to which these inhibitors prevent p8 induction coincides well with the degree to which these drugs block mesangial cell hypertrophy (Figure 6A). SB203580, which fails to prevent ET-1-induced mesangial cell hypertrophy (Figure 5A), also fails to suppress ET-1 induction of mesangial cell p8 (Figure 6A). Induction of Cu/Zn-sod and cystatin C by ET-1 also tracks a priori with hypertrophy, insofar as expression of these genes is inhibited by LY294002, U0126 and SP600125; however, the sensitivity of these genes to the different drugs differs from that of p8 and hypertrophy. For example, SP600125 is a relatively stronger inhibitor of Cu/Zn-sod and cystatin C induction than it is an inhibitor of p8 induction and hypertrophy (Figures 5 and 6A). The rapid induction of bag2 by ET-1 after 1 h does not appear to be under the same regulation as that of p8. Thus, neither LY294002, U0126 nor SP600125 prevents bag2 induction (Figure 6A). Instead, SB203580 strongly suppresses ET-1 induction of bag2, indicating that ET-1-stimulated bag2 expression, unlike mesangial cell hypertrophy, requires p38α and/or β. The consistent robustness of p8 induction (Figures 2 and 6A; Table I) as well as the induction of p8 in diabetic nephropathy and by high glucose (Figures 6B and 7) makes p8 an excellent candidate marker for diabetic hypertrophy. Figure 6.Regulation of mesangial cell gene expression by ET-1 requires distinct signaling pathways. Regulation of p8 expression is indistinguishable from that of hypertrophy. (A) Suppression of gene induction by specific protein kinase pathway inhibitors. Mesangial cells were pre-treated with the indicated inhibitors and then treated with ET-1 for 24 h (upper panel) or for 1 h (lower panel). Expression was detected by northern blotting, and gapdh expression was used as a loading control. (B) High glucose induces p8 expression to a degree commensurate with ET-1. ET-1 and glucose are not synergistic. Serum-starved cells were treated with high glucose (30 mM), ET-1 or both for 24 h. p8 expression was monitored by northern blotting. Download figure Download PowerPoint Figure 7.Induction of diabetes in rats stimulates expression of p8. As indicated in the figure, rats were made diabetic with the injection of STZ, subjected to unilateral nephrectomy, or both. RNA was prepared from the kidneys at the indicated times. For nephrectomized animals, RNA was prepared from the remaining kidney at the indicated times. Control animals were injected with water. Northern blotting was performed and quantitated as described in Materials and methods. Ethidium bromide staining of 28S RNA served as a loading control. NP indicates nephropathy in diabetic animals as occurs 5 weeks after STZ administration. Download figure Download PowerPoint The results shown in Figures 2, 5 and 6A are consistent with the hypothesis that p8 is a marker transcript for diabetic mesangial cell hypertrophy. To provide further support for this idea, we sought to determine if p8 was induced by high glucose (which on its own is known to trigger mesangial cell responses reminiscent of diabetes; Fine et al., 1992) and during the progression of diabetic kidney disease in rats. In Figure 6B, mesangial cells were treated with high glucose (30 mM) or with ET-1 for 24 h. High glucose alone is sufficient to elicit elevated mesangial cell p8 expression. The degree of p8 induction is comparable to that incurred by ET-1, and ET-1 does not synergize with glucose to superinduce p8 (Figure 6B). We also subjected rats to treatment with the diabetogenic genotoxin streptozotocin (STZ), unilateral nephrectomy or both (Figure 7). Induction of p8 and Cu/Zn-sod was then determined. Unilateral nephrectomy alone does not induce p8 or Cu/Zn-sod. In contrast, at 23 h post STZ treatment, when the rats exhibit glucosuria, prominent induction of p8 is observed. Lesser induction of Cu/Zn-sod is also observed. At 5 weeks post-STZ (NP in Figure 7), the rats are clearly nephropathic, with frank proteinuria and renal hypertrophy. At this time, expression o" @default.
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• W2005431362 date "2002-10-15" @default.
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• W2005431362 title "Signaling pathways and late-onset gene induction associated with renal mesangial cell hypertrophy" @default.
• W2005431362 cites W1599894891 @default.
• W2005431362 cites W1611419331 @default.
• W2005431362 cites W1726025741 @default.
• W2005431362 cites W1888445200 @default.
• W2005431362 cites W1908445283 @default.
• W2005431362 cites W1949053129 @default.
• W2005431362 cites W1967943295 @default.
• W2005431362 cites W1969971113 @default.
• W2005431362 cites W1988373895 @default.
• W2005431362 cites W1999163644 @default.
• W2005431362 cites W2001333805 @default.
• W2005431362 cites W2008421466 @default.
• W2005431362 cites W2015143075 @default.
• W2005431362 cites W2019829774 @default.
• W2005431362 cites W2020931266 @default.
• W2005431362 cites W2041798988 @default.
• W2005431362 cites W2045562658 @default.
• W2005431362 cites W2048404789 @default.
• W2005431362 cites W2052750950 @default.
• W2005431362 cites W2054865582 @default.
• W2005431362 cites W2058138221 @default.
• W2005431362 cites W2065547075 @default.
• W2005431362 cites W2067057343 @default.
• W2005431362 cites W2068836851 @default.
• W2005431362 cites W2069813140 @default.
• W2005431362 cites W2074187803 @default.
• W2005431362 cites W2084040587 @default.
• W2005431362 cites W2086285660 @default.
• W2005431362 cites W2088359631 @default.
• W2005431362 cites W2090895117 @default.
• W2005431362 cites W2096791952 @default.
• W2005431362 cites W2096931514 @default.
• W2005431362 cites W2105014609 @default.
• W2005431362 cites W2106436159 @default.
• W2005431362 cites W2108777244 @default.
• W2005431362 cites W2119376722 @default.
• W2005431362 cites W2122057254 @default.
• W2005431362 cites W2123481720 @default.
• W2005431362 cites W2126440278 @default.
• W2005431362 cites W2131162745 @default.
• W2005431362 cites W2137952321 @default.
• W2005431362 cites W2140707292 @default.
• W2005431362 cites W2142262231 @default.
• W2005431362 cites W2143562096 @default.
• W2005431362 cites W2144634347 @default.
• W2005431362 cites W2146157811 @default.
• W2005431362 cites W2147019740 @default.
• W2005431362 cites W2151584690 @default.
• W2005431362 cites W2152993806 @default.
• W2005431362 cites W2154608911 @default.
• W2005431362 cites W2155591792 @default.
• W2005431362 cites W2156433522 @default.
• W2005431362 cites W2158482235 @default.
• W2005431362 cites W2162346310 @default.
• W2005431362 cites W2168784370 @default.
• W2005431362 cites W2170693414 @default.
• W2005431362 cites W2329628902 @default.
• W2005431362 cites W2405568852 @default.
• W2005431362 cites W2411961303 @default.
• W2005431362 cites W2415091092 @default.
• W2005431362 cites W627595417 @default.
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