Differential regulation of VEGF by TGF- and hypoxia in rat proximal tubular cell - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:10 |只看该作者 |倒序浏览 |打印

作者：Takahiko Nakagawa,, Hui Y. Lan, Hong J. Zhu, Duk-Hee Kang, George F. Schreiner, and Richard J. Johnson,作者单位：1 Division of Nephrology, Hypertension, and Transplantation, University of Florida, Gainesville, Florida 32610-0224; 2 Division of Nephrology-Medicine, Baylor College of Medicine, Houston, Texas 77030; 3 Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria 305 Australia; 4 Divisi




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      【摘要】( u; H9 c; y" ^- ], \
   VEGF expression by proximal tubular epithelial cells may play a critical role in maintaining peritubular capillary endothelium in renal disease. Two major processes involved in renal injury include hypoxia (from vasoconstriction or vascular injury) and transforming growth factor (TGF)- -dependent fibrosis, both of which are known to stimulate VEGF. Because the TGF- /Smad pathway is activated in hypoxia, we tested the hypothesis that the induction of VEGF in hypoxia could be partially dependent on TGF-. Rat proximal tubular (NRK52E) cells treated with TGF- under normoxic conditions secreted VEGF at 24 h, and this was significantly reduced by blocking Smad activation by overexpressing the inhibitory Smad7 or by blocking p38 and ERK1/2 MAP kinase activation or protein kinase C activation with specific inhibitors. With acute hypoxia, rat proximal tubular cells also express VEGF mRNA and protein as well as TGF-. However, the induction of VEGF occurs before synthesis of TGF- and is not blocked by either a TGF- antagonist, by Smad7 overexpression, or by blockage of ERK1/2, whereas induction is blocked by PKC inhibition or partially blocked by a p38 inhibitor. Finally, the addition of TGF- with hypoxia results in significantly more VEGF expression than either stimulation alone. Thus TGF- and hypoxia act via additive/synergistic but distinct pathways to stimulate VEGF in proximal tubular cells, a finding that may be important in understanding how VEGF is stimulated in renal disease. % I7 ]& K# T: r9 J2 |
      【关键词】 Smad protein kinase C mitogenactivated protein kinase renal angiogenesis vascular endothelial growth factor transforming growth factor1 f% V! W8 [6 G4 L/ E) {
   VEGF IS CONSTITUTIVELY EXPRESSED in podocytes, proximal tubular (S3 segment) cells, and medullary thick ascending limb (mTAL) cells in the juxtamedullary region of the normal kidney ( 11, 13 ). An alteration in VEGF expression has been shown in a variety of renal diseases ( 10, 13 - 15, 17, 37 ). A loss of tubular VEGF has been shown to have a role in renal progression in the remnant kidney model, in aging-associated renal disease, and in a model of chronic thrombotic microangiopathy ( 13, 14, 17 ). In contrast, there is some evidence that an increase in VEGF may contribute to the early hyperfiltration and microalbuminuria in diabetic rats and mice ( 7, 9 ). It is therefore important to better understand the mechanisms regulating renal VEGF expression in normal and disease states.
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Two major mechanisms have been found to stimulate VEGF expression, i.e., hypoxia ( 4, 8, 16, 26 ) and various cytokines, notably transforming growth factor (TGF)- ( 3, 5, 39 ). We have recently found that TGF- -induced VEGF expression in proximal tubular (NRK52E) cells is mediated by activation of Smad3 ( 25 ). Hypoxia has also been reported to stimulate TGF- synthesis in proximal tubular cells ( 28 ) and activate Smad signaling in human umbilical endothelial cells ( 1, 40 ). In addition, it has been also demonstrated that hypoxia-inducible factor (HIF)-1 binds to Smad3, resulting in synergistic effects on VEGF expression ( 30 ). Therefore, Smad3 could play an important role on VEGF regulation under hypoxia. These studies led to the hypothesis that VEGF regulation in response to hypoxia may be partially driven by TGF- in addition to the classic HIF-1 -mediated pathway. In this study, we examined the role of TGF- and its downstream signaling pathways in mediating VEGF regulation in response to acute hypoxia in rat proximal tubular epithelial cells.- o8 ^/ n+ g& E4 t9 X# s
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MATERIALS AND METHODS0 \) B9 h, V. R

TGF- was ordered from R&D Systems (Minneapolis, MN). A mouse monoclonal antibody (mAb) to Smad2 (Transduction Laboratories, Lexington, KY), phospho-ERK1/2, or phosho-p38 (Cell Signaling, Beverly, MA) and polyclonal Abs to Smad7 (Santa Cruz Biotechnology, Santa Cruz, CA), Smad3 Ab, p-Smad2 Ab, ERK1/2, p38, phospho-MAPKAP2, or MAPKAP2 (Cell Signaling) were used. A specific TGF- inhibitor, NP-40208 (provided by Scios, Sunnyvale, CA), was used in some experiments. NP-40208 is a novel 2,4-disubstituted pteridine that inhibits the intracellular kinase domain of the type I TGF- receptor (T R-I). In an in vitro kinase assay, NP-40208 inhibits the T R-I kinase with an IC 50 of 0.048 µM. When tested at 50 µM, the compound has no effect on type II TGF- receptor kinase (data provided by Scios). PD-98059 (MEK inhibitor), SB-203580 (p38 inhibitor), Ro-318220 (PKC inhibitor), and PMA (PKC stimulator) were purchased from Calbiochem (San Diego, CA)." F4 ?6 J" P7 r- [4 D# T( l' P" c
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Cell culture. The rat proximal tubular epithelial cell line, NRK52E, was cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 60 µg/ml penicillin, and 100 µg/ml streptomycin in 5% CO 2 at 37°C, and the medium was changed every 3 days. Cells were incubated for 24 h with 0.5% FBS before stimulation, such as TGF- (5 ng/ml) or hypoxia (1% O 2 ). To achieve hypoxia, cells were incubated in a GasPak anaerobic culture pouch (BBL Microbiology Systems, Kansas City, MO), as described previously ( 15 ).' u& ^! u5 y2 W8 ~* ~

Establishing doxcyclin-regulated Smad 7-expressing TEC cell lines. The doxycycline (Dox)-regulated Smad7-expressing cell line was established as described previously ( 25 ). Briefly, mouse Smad7 cDNA was subcloned using a tetracycline (Tet)-inducible vector, pTRE (Clontech, Palo Alto, CA). An improved pTet-on vector (Clontech), pEFpurop-Tet-on, in which the gene encoding the "reverse" Tet repressor was subcloned into a pEF-BOS vector, pEFr-PGKpuropAv18, confers puromycin resistance. To obtain Dox (a Tet derivative)-induced Smad7-expressing NRK52E cell lines, pTRE-Smad7 and pEFpurop-Tet-on were cotransfected into NRK52E cells by electroporation, and the stable transfected cells were selected in the presence of puromycin (4 µg/ml). Cells were starved in 0.5% FBS-DMEM with 4 µg/ml Dox before stimulation. Overexpression of Smad7 was confirmed after starvation by Western blotting. At least three experiments were performed throughout the study. Cell viability in each experimental condition was examined by LDH assay with an TOX-7 LDH assay kit (Sigma, St. Louis, MO).

RNAse protection assay. Riboprobes were prepared as previously described ( 25 ). Briefly, rat VEGF (327 bp) was subcloned into Bluescript SK (Stratagene, La Jolla, CA). After linearization, an antisense riboprobe was synthesized with T7 polymerase in the presence of - 32 P-labeled UTP. Total RNA was isolated with TRIzol (Invitrogen). Three micrograms of total RNA samples were hybridized for 30 min at 90°C with a mixture of [ 32 P]UTP-labeled riboprobes of rat VEGF and the housekeeping gene (L32; 1 x 10 5 counts/min for each probe), and an RNAse protection assay was performed as described previously ( 25 ). The protected hybridized RNA was denatured at 85°C and electrophoresed on 10% polyacrylamide gels. The gels were transferred to Whatman filter paper, dried, and exposed to Kodak X-Omat film overnight at -70°C.  ?) {9 \; Z9 Y7 i
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ELISA for VEGF or TGF- 1 protein in cell culture supernatants. NRK52E cells were grown in six-well plates, and their supernatants were analyzed for TGF- using a Quantikine human TGF- 1 ELISA kit and for VEGF using a Quantikine mouse VEGF ELISA kit (R&D Systems), both of which cross-react with rat TGF- and VEGF ( 39 ), respectively. Results are expressed as picograms VEGF or TGF- per total cell protein [µg; the latter was measured by Bio-Rad protein assay (Bio-Rad, Richmond, CA)].
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Western blotting. Cells were washed in PBS and lysed in 100 µl of cell lysis buffer (cell signaling) for 30 min on ice. Samples were centrifuged at 14,000 g for 5 min to pellet cell debris. To isolate nuclear proteins, cells from 100-mm flasks were resuspended in 100 µl of 10 mM Tris·HCl, 2 mM MgCl 2, 5 mM KCl, 10% glycerol, 1 mM EDTA, and 1 mM dithiothreitol. NP-40 (1%) was added, and cells were placed on ice and then vortexed. Lysates were centrifuged (700 g, 4°C), and nuclear were pellets resuspended in 30 µl of 20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol before sonication. After determination of the protein concentration using the Bio-Rad protein assay (Bio-Rad), either 30 µg of whole cell protein samples or 10 µg of nuclear protein were mixed with sample buffer (Invitrogen), boiled, resolved on NuPAGE Bis-Tris Gel (4-12%) gel, and transferred to nitrocellulose membranes by electroblotting. Membranes were blocked with 5% (wt/vol) BSA in Tris-buffered saline with 0.1% Triton (TBST) for 60 min at room temperature. Each primary antibody was incubated at 4°C overnight. After being washed with TBST, membranes were rocked with a secondary antibody [anti-mouse IgG or anti-rabbit IgG, HRP-linked antibody (Cell Signaling)] for 60 min at room temperature. The blot was then developed using an ECL detection kit (Amersham International) to produce a chemiluminescence signal, which was captured on X-ray film. Positive immunoreactive bands were quantified by densitometry.* E/ u0 }- C  ]
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Role of PKC. Fifty micrograms of cell protein were used for a PKC assay (PepTag assay for nonradioactive detection of PKC, Promega, Madison, MI) according to the manufacturer's protocol. Ro-318220 (5 µM) or PMA (100 nM) was used to inhibit or stimulate PKC activation, respectively.8 K2 ?/ J+ |1 E- @+ `8 [" ^) ]0 k

Role of MAPK. To inhibit MAPK, a MEK inhibitor (PD-98059; 20-10 µM) or p38 inhibitor (SB-203589; 40-5 µM) was incubated 1 h before stimulation. To examine p38 kinase activity, phosphorylation of MAPK-activated protein kinase 2 (MAPKAP-2), which is a substrate of p38 ( 19 ), was examined.) c! l! E2 y* x2 Q/ X
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Statistical analysis. All values presented are expressed as means ± SD. Analysis of variance followed by a Bonferroni correction was used in all instances. Significance was defined as P
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RESULTS

Time-dependent effect of hypoxia on VEGF mRNA expression and TGF- synthesis. The effects of acute hypoxia on the expression of VEGF mRNA were studied by RNase protection assay in NRK52E cells. The VEGF RNA probe could detect three signals (VEGF 165, VEGF 189 VEGF 121, VEGF 189) ( Fig. 1 A ). VEGF mRNA was induced as early as 8 h after exposure to hypoxia and increased over the 24-h time period ( Fig. 1 A ). The increase in VEGF mRNA was paralleled by an increase in VEGF protein in the supernatant of the culture media (2.39 ± 0.63 vs. 0.32 ± 0.03; VEGF vs. control at 8 h, P ) I/ v8 a$ u9 ^5 O
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Fig. 1. Time course of VEGF mRNA expression was measured by RNAse protection assay (RPA). L32 was used as a control ( A ). B : time course of transforming growth factor (TGF)- protein synthesis under hypoxia or normoxia. Protein in supernatant of culture media was measured by ELISA. Total protein was corrected by total cell protein.5 O5 @, {# c" m3 @, Z
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We next examined the effect of acute hypoxia on TGF- expression. Acute hypoxia also stimulated TGF- expression, which reached significance at 24 h ( Fig. 1 B ). This observation is consistent with a prior report ( 27, 28 ). Several experiments were then performed to determine whether the acute hypoxia-induced expression of VEGF involved the TGF- pathway.

Involvement of TGF- /Smad pathway in the expression of VEGF under hypoxia. Because TGF- acts primarily through the Smad pathway and because Smad signaling can also be activated by hypoxia in endothelial cells ( 1, 40 ), we examined the role of Smads on VEGF expression in response to hypoxia. We did not observe any evidence for Smad3 activation in NRK52E cells at multiple time points up to 4 h after exposure to hypoxia (data not shown). To confirm that early stimulation of VEGF was not dependent on activation of Smads, we overexpressed the inhibitory Smad, Smad7, in NRK52E cells using a Dox-inducible expression system. The overexpression of Smad7 by Dox had no effect on basal expression of Smad3 in nuclear extracts, whereas it could block the translocation of Smad3 into the nuclei in response to TGF- ( Fig. 2 A ). However, overexpression of Smad7 under hypoxic conditions ( Fig. 2 B ) did not prevent the induction of VEGF mRNA expression in response to hypoxia ( Fig. 2, C and E ). In contrast, VEGF expression (mRNA and protein) in response to TGF- was completely blocked by the overexpression of Smad7 ( Fig. 2, D and E ).& p0 x: Q8 K2 p2 A; t

Fig. 2. Doxycycline (DOX)-induced Smad7 inhibits Smad3 nuclear translocation in response to TGF-. p62 Was used as the control of nuclear protein ( A ). B : DOX induces Smad7 under either normoxia or hypoxia by Western blotting. C : DOX-induced Smad7 did not suppress hypoxia-induced VEGF mRNA expression. D : DOX-Smad7 inhibits VEGF mRNA expression in response to TGF-. DOX-Smad7 also did not suppress VEGF protein synthesis under hypoxia. E : TGF- -induced VEGF was inhibited by DOX-Smad7. F : NP-40288 inhibited VEGF synthesis induced by TGF-, but not hypoxia.

These studies strongly suggested that TGF- was not involved in the induction of VEGF in response to acute hypoxia. Consistent with this observation was the finding that NP-40208, which is a selective T R-I kinase inhibitor, could not inhibit hypoxia-induced VEGF in NRK52E cells, whereas TGF- -induced VEGF was suppressed by NP-40208 ( Fig. 2 F ).3 X8 j' o2 X9 `7 I7 E) f5 P
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Role of ERK, p38, or PKC in regulation of VEGF by hypoxia or TGF-. To further demonstrate that the TGF- - and hypoxia-mediated stimulations of VEGF were acting via distinct pathways, we first examined the activation of MAPKs in response to TGF- or hypoxia over a 4-h period. Interestingly, ERK1/2 and p38 showed a different response to these stimuli. While both hypoxia and TGF- activated p38 at 60 and 240 min, ERK1/2 activation was not observed in response to hypoxia, whereas prominent activation occurred with TGF- stimulation ( Fig. 3 A ). Next, the blocking effect of PD-98059 (10-20 µM) for ERK1/2 or SB-203589 (10-40 µM) for p38 was confirmed by ERK1/2 phosphorylation or MAPKAP-2 phosphorylation, the latter a substrate for p38 ( 19 ), respectively ( Fig. 3 B ). In response to hypoxia as well as TGF-, 10-40 µM SB-203589 blocked MAPKAP-2 phosphorylation. TGF- -induced ERK1/2 activation was also inhibited by 10 µM PD-98059 ( Fig. 3 B ). Finally, VEGF expression induced by TGF- was totally blocked by either PD-98059 (10 µM) or SB-203589 (10 µM) ( Fig. 3 C ). On the other hand, the blocking of p38 kinase with SB-203589 partially reduced hypoxia-induced VEGF protein synthesis, whereas blocking ERK1/2 with PD-98059 showed no blocking effect ( Fig. 3 D ).
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Fig. 3. Hypoxia (Hypo) activates p38 at 60 and 240 min, but not ERK1/2 ( A, top ). Bottom : TGF- (TGF) activates both ERK1/2 and p38 at 60 and 240 min. con, Control. B : 10 µM PD-98059 (PD10) inhibits ERK1/2 activation in response to TGF- ( top ); middle : 10 µM SB-203589 (SB10) inhibits MAPK-activated protein kinase 2 (MAPKAP-2) phosphorylation induced by TGF-; bottom : 10-40 µM SB-203589 blocks phosphorylation of MAPKAP-2 under hypoxia. C : TGF- -induced VEGF protein synthesis is blocked by PD-98059 (PD) as well as SB-203589 ( a P

In contrast, PKC was activated at 1 h under both hypoxia and with TGF- stimulation ( Fig. 4 A ), and blocking PKC with the inhibitor Ro-318220 (5 µM) suppressed VEGF expression under both conditions ( Fig. 4, B and C ). Moreover, PMA (100 nM), a stimulator of PKC, enhanced VEGF expression ( Fig. 4 D ).
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Fig. 4. Role of PKC on VEGF expression. PKC is activated by hypoxia and TGF- at 1 h ( A ). Ro-318220 (Ro; 5 µM) abolishes VEGF protein synthesis under hypoxia ( B ) as well as in response to TGF- ( C ) at 24 h. PMA (100 nM) induces VEGF protein synthesis at24 h ( D ).5 J# }! O0 J: v5 Y, @  }
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Additive/synergistic effect of TGF- on acute hypoxia-induced VEGF expression. These studies suggested that acute hypoxia and TGF- act via distinct pathways to stimulate VEGF expression. We next determined whether these pathways were additive. As shown in Fig. 5 A, an exogenous TGF- dose dependently increased VEGF mRNA expression under hypoxia. Furthermore, the increase in VEGF protein in response to both acute hypoxia and TGF- was additive/synergistic and significantly greater than with either stimulus alone ( Fig. 5 B ).

Fig. 5. Synergistic effect as well as dose dependency of TGF- was seen on VEGF mRNA expression under hypoxia ( A ). Synergistic effect of TGF- with hypoxia on VEGF protein synthesis at 24 h is also shown ( B )., x7 t% G0 X2 v  L! J

DISCUSSION3 N6 S8 T8 M& y& P( u( u

In this study, we demonstrate that TGF- and acute hypoxia act distinctly, in parallel, and in an additive/synergistic manner to stimulate VEGF expression in proximal tubular cells. Specifically, we found that TGF- -mediated VEGF expression was dependent on Smad3 activation, MAPK (ERK1/2 and p38), and PKC activation, whereas acute hypoxia-mediated VEGF expression was independent of ERK1/2, Smad signaling, or TGF-, although it was dependent of PKC and partially dependent on p38. We further showed that the addition of TGF- to hypoxia potentiated the induction of VEGF mRNA and protein, suggesting an additive/synergistic effect. Hence, these studies emphasize the ability to these two major mediator systems to independently regulate VEGF.

Although we hypothesized that hypoxia-induced TGF- contributes to VEGF expression, our study demonstrated that this was not the case because 1 ) the VEGF expression preceded TGF- synthesis; 2 ) blocking TGF- signaling by overexpressing Smad7, the inhibitory Smad, was not able to block hypoxia-induced VEGF expression, whereas it was effective at blocking TGF- -induced VEGF expression; and 3 ) direct blockade of TGF- with a novel T R-I kinase inhibitor did not inhibit VEGF synthesis in response to hypoxia. Furthermore, we have found that Smad3 is critical for the stimulation of VEGF by TGF- ( 25 ); however, we found no evidence that either of the receptor Smads (Smad2/3) was activated acutely by hypoxia (data not shown). Finally, we found distinct differences in the role of MAPKs in mediating VEGF expression in response to hypoxia and TGF-. Specifically, ERK1/2 was found to be critical only in response to TGF-, whereas p38 phosphorylation has a role in mediating VEGF expression in either condition. In contrast, we found that PKC was activated by both hypoxia and TGF- and that blocking this kinase prevented VEGF upregulation in response to either stimuli.& E' ~4 E* P3 e9 @8 Y
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The roles of ERK1/2 and p38 under hypoxia are complicated, which depend on the specific cell types ( 2, 23, 24, 34, 38, 39 ). In the current study using NRK52E cells, ERK1/2 activation was not observed in response to hypoxia, whereas activation of p38 could readily be demonstrated. These data are consistent with a previous observation using microglia ( 29 ). In contrast, ERK1/2 activation has been shown to be critical in hypoxia-induced VEGF expression in HepG2 cells ( 23 ). In terms of p38, several studies have reported its critical role under hypoxia ( 34 ), which is compatible with our data. On the other hand, cytokine or growth factor regulation of VEGF has been found to be mediated by ERK1/2 in retinal epithelial cells ( 24 ), by p38 in vascular smooth muscle cells ( 39 ), and by PKC in glioma cells ( 38 ), consistent with our results. To our knowledge, the role of MAPKs in VEGF regulation of renal proximal tubular cells in response to either hypoxia or TGF- has not been previously examined. The demonstration of different roles of ERK/p38 in VEGF expression under these two conditions may be important in the mechanisms regulating VEGF expression in disease states.

An important new observation was that TGF- and acute hypoxia not only activate distinct pathways to stimulate VEGF expression but they also act synergistically/additively. Specifically, the addition of TGF- to cells exposed to acute hypoxia resulted in significantly more VEGF mRNA and protein than either stimulus alone. Importantly, our studies were limited to the effects of acute hypoxia, because in our system it was not possible to maintain live cells for extended periods. However, it is important to recognize that with chronic hypoxia the contributory role of TGF- /Smad in the maintenance of VEGF expression could be important.2 a3 h3 J" R% C+ d' U; ~! @) N- U

Furthermore, in several cell types one can demonstrate a role for TGF- in the regulation of VEGF in response to acute hypoxia. Thus the association of HIF-1 with Smad3 has been demonstrated in the regulation of several hypoxia-induced genes, including VEGF ( 30 ), erythropoietin ( 32 ), and endoglin ( 31 ). In addition, HIF-1 has been shown to regulate TGF- gene expression in trophoblasts in response to acute hypoxia ( 33 ), which in turn enhanced HIF-1 DNA-binding activities ( 35 ). Thus these data suggest that TGF- /Smad signaling may be a pathway for enhancing VEGF expression in certain cell types in response to hypoxia. However, in proximal tubular cells the acute hypoxia-induced regulation of VEGF does not depend on TGF- /Smad signaling, a factor that may be important in understanding the process of renal injury and repair.; T* Y3 Q+ e$ q, I# `8 t& [

The observation that both TGF- and hypoxia stimulate VEGF independently and synergistically may be important in diseases such as cyclosporine nephropathy. Indeed, in acute models of cyclosporine nephropathy, the intense renal vasoconstriction is associated with intrarenal hypoxia ( 21 ), TGF- synthesis ( 36 ), and an acute increase in VEGF synthesis ( 37 ). Other diseases, particularly models of glomerular disease and diabetes, are also associated with acute increase in VEGF ( 6, 7, 9, 10 ). The VEGF produced likely has an important role in maintaining local capillary integrity and health and has a protective role in models of chronic renal disease ( 12, 14 ). However, marked increases in VEGF may also have a role in the angiogenesis associated with inflammation ( 18, 20, 22 ), and in diabetes may contribute to the early hyperfiltration, proteinuria, and renal hypertrophy that accompany the early phase of the disease ( 6, 7 ). Thus VEGF may have a complex role in renal disease depending on its site and timing of expression. The primary observation in this study, that both hypoxia and TGF- work independently but synergistically to regulate VEGF expression in renal proximal tubular cells, further improves our understanding of VEGF regulation and hence may shed further light on the role of VEGF in the kidney.  G) X7 L& T# S2 R0 \" }" x
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GRANTS

This study was supported by a National Kidney Foundation fellowship (to T. Nakagawa), National Institutes of Health Grants RO1-DK-52121 and HL-68607, and George O'Brien Center Grant 1P50  -DK-064233-01.
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