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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 4, 2004 as doi:10.1096/fj.03-1053fje. |
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* Department of Internal Medicine, University of Torino, Torino, Italy; and
Department of Internal Medicine, University of Genoa, Genoa, Italy
1Correspondence: Department of Internal Medicine, University of Torino, Corso Dogliotti 14, Torino, Italy. E-mail: mariafelice.brizzi{at}unito.it
SPECIFIC AIMS
The aim of the present study was: 1) to investigate the molecular events associated with acute and chronic exposure of mesangial cells (MC) to hyperglycemia or to TGF-ß; 2) to analyze cell cycle events as well as the expression of p21waf after high glucose (HG), Amadori adducts, AGE, and TGF-ß treatments; and 3) to evaluate the activation of the transcriptional factor STAT5 and its possible role in regulating either the expression of the cell cycle associated protein p21waf or the production of collagen.
PRINCIPAL FINDINGS
1. AGE and TGF-ß, but not HG or Amadori adducts, increase p21waf expression and regulate cell cycle progression in MC
We first evaluated the effect of HG (25 mM), Amadori adducts, AGE and TGF-ß on the cell cycle machinery. We found that AGE and TGF-ß, unlike HG and Amadori adducts, increased the expression of p21waf. On the contrary, cyclin D1 expression was increased in response to HG and Amadori adducts but not to AGE and TGF-ß. Consistent with the increase of p21waf, flow cytometric analysis for DNA content demonstrated that the cell population in the S phase decreased after AGE and TGF-ß treatment (9% and 21% for AGE and TGF-ß compared with 28% and 30% for untreated or albumin-treated cells, respectively). This effect was associated with an increase (65% and 62%, respectively) in the percentage of cells in G0/G1. Although addition of the neutralizing anti-TGF-ß antibody prevented the effect of TGF-ß, it only partially reversed the effect of AGE (G0/G1 52% and 59%, respectively). The blocking anti-RAGE antibody was used. Consistent with the failure of HG and Amadori adducts to affect the expression of p21waf, no change in the proportion of cells in the different phases of the cell cycle was observed when MC were challenged with either stimuli.
2. AGE, independent of TGF-ß, triggers STAT5 activation and p21waf expression but not collagen production
Kinetics of STAT5 activation demonstrated that whereas AGE induced a rapid and transient STAT5 activation, the effect of TGF-ß treatment was more persistent (Fig. 1
A). To assess the role of STAT5 on AGE- and TGF-ß-mediated p21waf expression, EMSA analysis was performed using a p21SIE2 sequence. Nuclear extracts from stimulated MC were able to form a p21SIE2 complex containing STAT5 as shown by supershift experiments (Fig. 1B
). The role of RAGE in AGE-induced STAT5 activation was demonstrated by a blocking anti-RAGE antibody (Fig. 1C
). In agreement with the effect of the anti-TGF-ß antibody on AGE-mediated cell cycle events, activation of STAT5 (Fig. 1C
) and the expression of p21waf (Fig. 1D
) were abrogated by TGF-ß blockade in TGF-ß- but not in AGE-treated MC. Figure 1E
also shows that, unlike long-term, short-term HG treatment does not lead to STAT5 activation. No effect on STAT5 activation was detected after either short or long-term treatment with Amadori adducts. Finally, AGE binding to RAGE induced collagen production and unlike for p21waf expression, this response was strictly controlled by the release of TGF-ß (Fig. 1F
).
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3. AGE and TGF-ß-mediated p21waf expression and G1 arrest depends on STAT5 activation
The role of STAT5 in regulating p21waf expression was also evaluated in MC transfected with an inactive form (
STAT5) or with a constitutive activated form of STAT5 (1*6 STAT5). MC expressing empty vectors retained the ability to increase p21waf expression in response to AGE and TGF-ß, but not in response to HG or Amadori adducts. By contrast, when either AGE or TGF-ß were used to treat MC expressing the STAT5 construct, no increase in p21waf expression was detected. When the same experiments were performed in MC expressing the constitutive activated STAT5 protein, p21waf was highly expressed and its level could not be further increased by any treatment. Flow cytometry analysis of MC expressing the empty vectors showed that AGE and TGF-ß treatment increased the percentage of cells in G0/G1. By contrast, as much as 60% of MC expressing the activated STAT5 protein and a high level of p21waf were in G0/G1. This percentage could not be further increased by different treatments, including serum. We consistently found a reduction in the percentage of cells in G0/G1 and a corresponding increase in the percentage of cells in the S phase (
50%) in MC expressing the STAT5 construct.
4. Collagen production is independent of STAT5 signaling
Collagen production was also evaluated in MC expressing both STAT5 mutants and in p21–/– fibroblasts. Since all stimuli induced collagen production in MC expressing the STAT5 mutants, we concluded that STAT5 was specifically involved in the control of cell cycle progression. We evaluated the involvement of p21waf in AGE or TGF-ß-induced G1 arrest and on collagen production. We found that p21–/– fibroblasts failed to undergo growth arrest in response to different stimuli, including HG and Amadori adducts, but retained their ability to produce collagen.
5. STAT5 is activated and p21waf is expressed in diabetic kidney biopsies
As shown in Fig. 2
, the expression of the activated STAT5 and of p21waf could not be detected in kidney biopsies from nondiabetic subjects. By contrast, activated STAT5 and p21waf were coexpressed by fewer cells in kidney sections from microalbuminuric patients than in those from macroalbuminuric patients (15%±3 vs. 45%±4, P<0.05).
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CONCLUSIONS
Besides the direct and acute tissue damage exerted by hyperglycemia, some tissue dysfunctions are induced by AGE in diabetic patients. Data support the notion that AGE-mediated effects depend mainly on their ability to induce the release of several soluble mediators, which include TGF-ß. Like TGF-ß and hyperglycemia, AGE can elicit profound effects on cell growth mainly by affecting the progression through the late G1. Moreover, increased expression of cyclin D1 has been reported in response to HG. Accordingly, we demonstrated that HG and Amadori adducts induced the expression of cyclin D1 without affecting the expression of p21waf and its associated cell-cycle events. Conversely, TGF-ß or AGE treatment led to an increased expression of p21waf and inhibition of the progression through the cell cycle. These findings suggest that different intracellular signaling mechanisms regulate HG- or AGE- and TGF-ß-induced biological response and that acute hyperglycemia per se could affect MC independent of TGF-ß.
AGE and TGF-ß effects on MC biology are quite similar, but whether AGE can affect MC responses directly or indirectly remains to be elucidated. We found that neutralization of TGF-ß abrogated AGE- and TGF-ß-mediated collagen production and the effect of TGF-ß on p21waf expression and the cell cycle events, while it was unable to completely inhibit those mediated by RAGE engagement. Thus, we first demonstrated that AGE, by activating RAGE, can affect cell cycle progression independent of TGF-ß (Fig. 3
), and suggest a molecular mechanism for the limited therapeutic effect of the anti-TGF-ß antibody in vivo.
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We report that STAT5 DNA binding activity is required for AGE- and TGF-ß-mediated p21waf expression and G1 arrest, but not for collagen production, thus identifying STAT5 as an alternative transcriptional factor other than p53 controlling p21waf. The observation that, unlike short-term, long-term HG stimulation led to STAT5 activation raises the possibility that soluble mediators released in response to HG contribute directly to the constitutive activation of STAT5.
Our results provide evidence that besides activating the transcriptional activity of Smads, TGF-ß can regulate the expression of its target genes by a STAT5-mediated pathway and that p21waf plays a crucial role in mediating AGE and TGF-ß-induced inhibition of cell cycle progression. Our data (summarized in Fig. 3
) identify a novel signaling pathway activated independent of TGF-ß and AGE that, by converging on p21waf expression, can contribute to the progression of diabetic nephropathy. The potential in vivo role of these molecular mechanisms is sustained by the immunofluorescence analysis on kidney biopsies showing a positive immunostaining for both activated STAT5 and p21waf in MC and an increase of their expression from early to advanced diabetic nephropathy. Although adherence to the therapeutic recommendations significantly decreased diabetes-associated microvascular complications, conventional therapies and experimental use of anti-TGF-ß antibody did not halt the progression of nephropathy. RAGE engagement by AGE could represent the TGF-ß-independent mechanism that contributes to the maintenance or progression of diabetic nephropathy. Moreover, the demonstration that the STAT5 signaling pathway is involved in this pathological setting may provide a rational for novel therapeutic strategies that, combining anti-TGF-ß antibodies and specific inhibitors of the STAT pathway, might control the progression of diabetic nephropathy.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1053fje; doi: 10.1096/fj.03-1053fje
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