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High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway
E XP ER I ME NTAL C E LL RE S E ARCH
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Research Article
High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway Yue Zhanga,1, Hongbo Lib,1, Jun Haob, Yi Zhouc, Wei Liub,n a
Department of Diagnostics, Hebei Medical University, Shijiazhuang 050017, China Department of Pathology, Hebei Medical University, Shijiazhuang 050017, China c Department of Neurology, the Second Hospital of Hebei Medical University, Shijiazhuang 050000, China b
article information
abstract
Article Chronology:
Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in
Received 4 December 2013
the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5),
Received in revised form
who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases,
7 April 2014
has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our
Accepted 16 April 2014
previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how
Keywords: Cdk5 TGF-β1 Podocyte High glucose Diabetic nephropathy
expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. & 2014 Elsevier Inc. All rights reserved.
Introduction Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and the leading cause of end-stage renal
failure [1]. DN is characterized by specific renal morphological and functional alterations. Glomerular visceral epithelial cells, namely podocytes, are terminally differentiated cells overlying the outer aspect of the glomerular basement membrane of renal glomeruli
Abbreviations: Cdk, Cyclin-dependent kinase; DN, Diabetic nephropathy; Egr, Early growth response; ERK, Extracellular signal-regulated kinase; MEK, Mitogen-activated protein kinase/ERK kinase; siRNA, Small interference RNA; TGF-β1, Transforming growth factor-β1. n Correspondence to: Department of Pathology, Hebei Medical University, No.361, Zhongshan East Road, Hebei Province, Shijiazhuang 050017, China. Fax: þ86 0311 86265734. E-mail address: [email protected] (W. Liu). 1 The two authors contributed equally to this study.
http://dx.doi.org/10.1016/j.yexcr.2014.04.014 0014-4827/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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and play a vital role in renal physiology. Among the characteristic findings of DN, podocytes are involved in the development of glomerular hypertrophy, podocytopenia, glomerulosclerosis, and foot process effacement [2,3]. Because podocyte depletion may be a key initiating lesion in these processes, it is considered important to determine the underlying molecular and cellular mechanisms. Cyclin-dependent kinases (Cdks) play essential roles in the regulation of cell division cycle. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that belongs to the family of Cdks. Distinct from other members of the Cdk family, the activation of Cdk5 does not require binding to cyclins, but rather, association with its regulator to perform kinase activity [4]. One major regulating partner for Cdk5 is p35, which was first reported in postmitotic neurons. The crucial role of the Cdk5-p35 complex is to support the development of the central nervous system [5]. However, numerous extraneuronal functions of Cdk5-p35 have been discovered in recent years, in addition to the roles in the central nervous system [6]. For example, in the kidney, the glomerular expression of Cdk5 is limited to podocytes. Moreover, Cdk5 is a key regulator of podocyte differentiation, proliferation and morphology [7]. Recent studies have shown that the absence of p35 confers increased susceptibility of podocytes to apoptosis in disease [8]. Our previous work has shown that the expression of Cdk5 was increased in a time-dependent manner and roscovitine; a Cdk5 inhibitor, significantly ameliorated podocyte injuries in diabetic rats [9]. Furthermore, in cultured podocytes in vitro, high glucose increased the expression of Cdk5, and knockdown of Cdk5 attenuated podocyte apoptosis induced by high glucose stimulation [10]. These findings suggest that Cdk5 plays an important role in multiple mechanisms involved in podocyte injuries of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been elucidated. Transforming growth factor-β1 (TGF-β1) is an important member of a superfamily of multifunctional growth factors involved in many cellular processes including cell proliferation, differentiation, migration, and apoptosis. TGF-β1 has been proposed as a major mediator of matrix accumulation in diabetic kidney and high glucose-exposed mesangial cells and podocytes, leading to the development of DN [11,12]. Moreover, increased expression of TGF-β1 in podocytes coincides with the onset of apoptosis and albuminuria in diabetes [13]. Correspondingly, the increase of the TGF-β1 levels has been recognized as a marker of DN [14,15]. Because TGF-β1 has been shown to act as a mediator of Cdk5 activity in sensory neurons [16], we want to know under the high glucose environment, whether TGF-β1 could act as an upstream regulator of Cdk5 in podocytes, affecting the development and progression of DN. In this study, we investigated that the role of TGF-β1 on Cdk5 expression and activity in high glucose-treated podocytes. We found that high glucose and TGF-β1 up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity. SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity. Furthermore, the ERK1/2-Egr-1 signaling pathway was involved in the regulation of TGF-β1 on p35 and Cdk5 in high glucose-cultured podocytes. More importantly, inhibition of Cdk5 kinase activity was demonstrated to be effective in alleviating podocyte apoptosis induced by high glucose or TGF-β1.
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Materials and methods Antibodies and reagents Primary antibodies recognizing Cdk5 and Egr-1 were purchased from Epitomics Company (CA, USA). Antibody to p35 was purchased from Santa Cruz Biotechnology (CA, USA). Antibodies to TGF-β1, Smad-2, phospho-Smad-2 and β-actin were purchased from Signalway Antibody Company (Maryland, USA). Antibodies against p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204) were obtained from Cell Signaling Technology (MA, USA). Tgfbr1 inhibitor SB431542, the MEK inhibitor U0126 and Cdk5 kinase activity inhibitor roscovitine were purchased from Sigma Aldrich (Dorset, UK). Recombinant murine IFN-γ was purchased from Peprotech Company (NJ, USA).
Conditionally immortalized mouse podocytes in culture Conditionally immortalized mouse podocytes purchased from the Cell Culture Center (PUMC, CAMS, Beijing, China) were cultured as previously described [17]. To induce proliferation, cells were grown on collagen I-coated plastic culture bottles (BD Biosciences, Bedford, MA), at 33 1C in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, USA), 100 U/ml penicillin (Invitrogen, USA), and 100 μg/ml streptomycin (Invitrogen, USA), to which recombinant mouse IFN-γ 10 U/ml (Pepro Tech, USA) was added (growth permissive conditions). To induce quiescence and the differentiated phenotype, podocytes were grown at 37 1C and deprived of IFN-γ (growth restrictive conditions) in DMEM supplemented with 10% FBS, penicillin, and streptomycin. All studies were performed on days 10 to 14 for cells grown under restrictive conditions.
RNA interference analysis Conditionally immortalized mouse podocytes grown in 6-well plates for 24 h in DMEM with 10% FBS were transfected with siRNA against Smad2 ( Smad2 siRNA, sc-38375, Santa Cruz, USA ), Egr-1 (Egr-1 siRNA, sc-35267, Santa Cruz), or control siRNA (sc-37007, Santa Cruz) with Lipofectamine RNAi MAX (Invitrogen, USA) as per the manufacturer's protocol. After 24 h transfection, the cells were treated with high glucose and then were analyzed.
Immunocytochemistry When high glucose treated for 12 h, podocytes were fixed with 4% paraformaldehyde at room temperature for 15 min. After pretreatment with 0.1% Triton X-100 for 10 min at 37 1C, cells were blocked with goat serum for 30 min at 37 1C. Then, the cells were incubated with rabbit anti-Cdk5 (1:200) antibody overnight at 4 1C. After three washes with PBS, cells were incubated with a polymer helper and polyperoxidase-anti-mouse/rabbit IgG at 37 1C for 30 min, and the cells were then stained with diaminobenzidine. A negative control was performed by replacing the primary antibody with PBS buffer.
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Immunofluorescence Indirect immunofluorescence staining was performed according to an established procedure. Briefly, cells cultured on coverslips were washed twice with cold PBS and fixed with cold methanol/ acetone (1:1) for 10 min at 20 1C. Following three extensive washings with PBS, the cells were treated with 0.1% Triton X-100 for 10 min at room temperature, and blocked with 2% normal donkey serum in PBS buffer for 30 min at 37 1C and then incubated with the primary antibody against p35 or TGF-β1 over night at 4 1C. Next day, after incubating with FITC-conjugated secondary antibody for 2 h, the slides were viewed with the Olympus BX63F fluorescence microscope equipped with a digital camera. In each experimental setting, immunofluorescence images were captured with identical light exposure time.
Western blotting The different groups of cells were harvested and homogenized in ice-cold homogenization buffer (1% Nonidet P-40, 0.1% Triton X-100, 30 mM sodium phosphate, pH 7.4, containing 1 mM sodium orthovanadate, 100 mM NaCl, 2.5 mM Tris–HCl, pH 7.5, and protease inhibitors), followed by centrifugation at 12,000g for 20 min at 4 1C. The supernatant was collected to characterize the relative levels of protein expression by Western blot assays. The protein concentrations were quantified with the Bio-Rad protein colorimetric assay (Bio-Rad, USA). Whole cell extracts (100 μg of protein/lane) were loaded, separated by 10% SDS-PAGE, and transferred to PVDF membranes (Millipore, MA). After blocking with 5% skimmed milk in Trisbuffered saline/Tween buffer (TBST buffer) for 2 h, the membranes were incubated overnight at 4 1C with primary antibody described above. Subsequently, the membranes were incubated with goat anti-rabbit or mouse IgG horseradish peroxidase conjugate, and then the results were detected by an Odyssey FC detection system using an enhanced chemiluminescence system. The intensity of the bands was measured using LabWorks 4.5.
RNA isolation and real-time PCR Total RNA was extracted with TriZol Reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Complementary DNA was synthesized from the total RNA (0.5 μg) using the PrimeScriptTM RT regent Kit following the instructions provided by the manufacturer (Takara Biotechnology, Dalian, China). Subsequently, the cDNA was subjected to quantitative RT-PCR (qRT-PCR) using a Power SYBR Green PCR Master Mix (Takara Biotechnology, Dalian, China). Each real-time PCR reaction consisted of 2 μl diluted RT product, 10 μl SYBR Green PCR Master Mix (2 ) and 250 nM forward and reverse primers in a total volume of 20 μl. Reactions were carried out on a 7500 qRT-PCR System (Applied Biosystems) for 40 cycles (95 oC for 15 s, 60 1C for 45 s) after initial 10 min incubation at 95 1C. The primers used for real-time PCR were as follows: p35 forward primer (F): 50 -GCC CTT CCT GGT AGA GAG CTG-30 and p35 reverse primer (R): 50 -GTG TGA AAT AGT GTG GGT CGG C-30 ; Cdk5 F: 50 -TAG GCT CTC TGA ACC CCA GT-30 , Cdk5 R: 50 -ATC CCA CAC CCG ACT CTT C-30 ; Tgfbr1 F: 50 -TGC ATT GCA CTT ATG CTG ATG GT-30 , Tgfbr1 R: 50 -ACC TGA TCC AGA CCC TGA TGT T-30 ; TGF-β1 F: 50 -CAC CTG CAA GAC CAT CGA CAT-30 , TGF-β1 R: 50 -GAG CCT TAG TTT GGA CAG GAT CTG-30 ;
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Egr-1 F: 50 -CCC TTC CAG GGT CTG GAG AAC CGT-30 , Egr-1 R: 50 -GGG GTA CTT GCG CAT GCG GCT GGG-30 . The mRNA levels were standardized by using the following primers to 18S: 18S F: 50 -CAT TCG AAC GTC TGC CCT ATC-30 and 18S R: 50 -CCT GCT GCC TTC CTT GGA-30 .
Immunoprecipitation and Cdk5 kinase activity assay The treated cells were lyzed in 10 mM HEPES, pH 7.5 (or 7.4), containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor mixture. The lysates were centrifuged at 12,000 rpm for 15 min. For Cdk5 immunoprecipitation, the supernatant was incubated with 50 μl protein A agarose (Santa Cruz Biotechnology, USA) and 2 μg anti-Cdk5 antibody for 4 h at 4 1C. The immune complex was separated by centrifugation at 4000 rpm. Kinase assay was performed with an ADP-GloTM kinase assay kit (Promega, USA) according with the manufacturer's protocol. Briefly, immunoprecipitates were washed three times with lysis buffer and then once with 1 reaction buffer containing 40 mM Tris–HCl, pH 7.5, 20 mM MgCl2, 0.1 μg/μl BSA and 0.5 mM DTT. Kinase assays were performed in the same buffer by addition of 250 μM ATP and 5 μg substrate histone H1. Then the mixture was incubated at room temperature for 10 min. Next, 25 μl ADP-GloTM reagents were added to terminate the reaction and deplete the remaining ATP. After 50 μl kinase detection reagents were added into the mixture to incubate for 30 min, the results were recorded by measuring the luminescence with a plate-reading luminometer. Data are shown as relative light units (RLU) that directly correlate to the amount of ADP produced.
Terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) staining Apoptotic cells were identified using the TUNEL technique according to the manufacturer's instructions (Roche Applied Science, Hangzhou, China). For quantification of TUNEL-positive (apoptotic) cells, a minimum of 200 cells were counted per group, and the percentage of the positively labeled cells was calculated.
Annexin V and propidium iodide staining assay Apoptotic rate was detected by an Annexin V/PI apoptosis detection kit according to manufacturer's protocol (MultiSciences Biotech, Hangzhou, China). Briefly, the cell pellet was resuspended in 1 binding buffer followed by incubation with 5 μl of Annexin V (conjugated with FITC) and 10 μl of PI in dark for 5 min. Cell fluorescence was then analyzed by a flow cytometer (Epics-XLII, Becman Coulter). This test discriminates intact cells (Annexin V /PI ), early apoptotic cells (Annexin Vþ/PI ) and late apoptotic cells (Annexin Vþ/PIþ).
Statistical analysis All values are expressed as mean7SD. Statistical analyses were performed using the SPSS 17.0 program. One-way ANOVA was applied to compare the means between groups. Po0.05 was considered to be statistically significant.
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Results High glucose up-regulated the expression of Cdk5 and p35 in podocytes with a concomitant increase in Cdk5 kinase activity In order to examine the effects of high glucose on the expression levels of Cdk5 and its co-activator p35, podocytes were cultured
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in DMEM medium containing 5.6 mM glucose (control group), 5.6 mM glucose plus 24.4 mM mannitol (M group, as an osmotic control) or 30 mM glucose (HG group) for 12 h. As expected, mannitol had no effect on the protein levels of Cdk5 and p35. However, the expression of Cdk5 and p35 was significantly increased in high glucose-treated podocytes, compared with the control group or the mannitol group (Fig. 1A and B). Next, we examined that the time course of high glucose (30 mM)-mediated
Fig. 1 – Effects of high glucose on expression of Cdk5 and p35 and Cdk5 kinase activity. Podocytes were incubated with 5.6 mM glucose (control group), 5.6 mM glucoseþ24.4 mM mannitol (M group) or 30 mM glucose (HG group) for 12 h. (A) Protein representative photographs of Cdk5 and p35. (B) Protein levels of Cdk5 and p35. (C) Cdk5 positive expression was detected using immunocytochemical staining at different time points (3, 6 and 12 h). (200 ) (D) The p35 positive expression was detected using immunofluorescence staining at different time points (3, 6 and 12 h). (400 ) (E) Protein representative photographs of Cdk5 and p35 at different time points (3, 6 and 12 h). (F) Protein levels of Cdk5 and p35. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. (G) The relative mRNA levels of Cdk5 and p35 detected by real-time PCR. (H) Cdk5 kinase activity. Data are expressed as mean7SD. npo0.05 vs. control group; # po0.05 vs. M group. Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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increase of Cdk5 expression. As shown in Fig. 1C, the positive expressions of Cdk5, detected by immunocytochemical staining at different time points (3, 6 and 12 h), were both significantly increased at 3 h after high glucose treatment and remained elevated until 12 h. The positive performance of p35 detected by immunofluorescence showed the same trend with Cdk5 (Fig. 1D). Similarly, the protein and mRNA levels of Cdk5 and p35, examined by Western blot or real-time PCR analysis, were also increased with a time-dependent manner following high glucose treatment (Fig. 1E–G). Furthermore, we assayed the Cdk5 kinase activity using histone H1 as substrate. Compared with the control group, high glucose-treated podocytes also showed an increased Cdk5 kinase activity with a time-dependent manner (Fig. 1H). Together, these results indicated that high glucose up-regulated the expression of Cdk5 and p35 in podocytes, resulted in increased Cdk5 kinase activity.
High glucose activated the TGF-β1 signaling pathway in podocytes To confirm previous reports that TGF-β1 signaling pathway is responsive to high glucose in podocytes, we treated the mouse
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podocytes for 12 h with 30 mM glucose. Using immunofluorescence staining, the TGF-β1 positive expression showed an obvious increase after getting exposed to 30 mM glucose (Fig. 2A). Similarly, the protein levels of TGF-β1 and the ratio of phosphorylated and total Smad2 were significantly elevated in high glucosetreated podocytes (Fig. 2B–D). Furthermore, the mRNA levels of TGF-β1 and Tgfbr1 were also notably increased in podocytes when 30 mM glucose treatment, respectively five times and eight times higher than that of the control or the mannitol group (Fig. 2E). These results demonstrated that high glucose promoted the activation of TGF-β1 signaling pathway.
TGF-β1 up-regulated the expression of Cdk5 and p35 and Cdk5 kinase activity in podocytes In order to examine the direct effects of TGF-β1 on Cdk5 expression and activity, the cultured podocytes were treated with different concentrations of TGF-β1 (1, 5, 10 ng/ml) for 12 h. As shown in Fig. 3A–C, compared with the control group, the protein and mRNA expressions of Cdk5 and p35 were significantly increased with a concentration-dependent manner in TGF-β1-treated podocytes. Moreover, the Cdk5 kinase activity also showed the same increased
Fig. 2 – Effects of high glucose on TGF-β1 signaling pathway. Podocytes were incubated with 5.6 mM glucose (control group), 5.6 mM glucoseþ24.4 mM mannitol (M group) or 30 mM glucose (HG group) for 12 h. (A) The TGF-β1 positive expression was detected using immunofluorescence staining. (200 ) (B) Protein representative photographs of TGF-β1, Smad2 and p-Smad2. (C) Protein levels of TGF-β1. (D) The ratio of p-Smad2 and Smad2. (E) The relative mRNA levels of TGF-β1 and Tgfbr1 detected by real-time PCR. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. M group. Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Fig. 3 – Effects of TGF-β1 on expression of Cdk5 and p35 and Cdk5 kinase activity. Podocytes were treated with different concentrations of TGF-β1 (1, 5, 10 ng/ml) for 12 h. (A) Protein representative photographs of Cdk5 and p35. (B) Protein levels of Cdk5 and p35. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. (C) The relative mRNA levels of Cdk5 and p35 detected by real-time PCR. (D) Cdk5 kinase activity. Data are expressed as mean7SD. npo0.05 vs. control group.
trend (Fig. 3D), suggesting that TGF-β1 can directly up-regulate the expression of Cdk5 and p35, and Cdk5 kinase activity.
SB431542 inhibited the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-cultured podocytes To evaluate the effect of TGF-β1 on Cdk5 expression and kinase activity under high glucose environment, SB431542 (the Tgfbr1 inhibitor) was used to treat podocytes. As shown in Fig. 4A, the mRNA level of Tgfbr1 induced by high glucose was markedly inhibited by SB431542. Moreover, SB431542 treatment decreased the protein level of phospho-Smad2 (Fig. 4B and C), which demonstrated that SB431542 effectively played its role. More importantly, the increases of Cdk5 and p35 protein and mRNA levels induced by high glucose were also significantly prevented by SB431542 in a dose-dependent manner from 0.1 μM to 10 μM (Fig. 4D–F). Similarly, SB431542 treatment strongly blocked the increases of Cdk5 kinase activity (Fig. 4G), indicating that under the high glucose environment, TGF-β1 plays a direct and important role in elevating Cdk5 expression and activity in podocytes.
High glucose increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway in podocytes Egr-1 is a member of a family of zinc-finger transactivators and has been known as a regulator of the p35 promoter [18]. To identify the molecular mechanism underlying TGF-β1-mediated regulation of Cdk5 activity and evaluate if high glucose activate the Egr-1 expression, SB431542 (10 μM) and U0126 (20 μM) were
used to treat podocytes. U0126 is a specific MEK inhibitor for inhibiting ERK1/2 activation induced by TGF-β1. By Western blot analysis, the ratio of phosphorylated and total ERK1/2 and the protein levels of Egr-1 were significantly increased when exposed to 30 mM glucose, whereas SB431542 or U0126 strongly decreased these increases (Fig. 5A–C). The protein expression of Cdk5 and p35 induced by high glucose was also reversed by SB431542 or U0126 (Fig. 5D and E). Consistent with the protein expression results, the increased mRNA levels of Egr-1, Cdk5 and p35 induced by high glucose were all inhibited by SB431542 or U0126 treatment (Fig. 5F). In order to explore whether Smad2 is involved in the regulation of TGF-β1 on ERK1/2, Smad2 was inhibited using siRNA to evaluate the total- and phospho-ERK1/2 expression. As shown in Fig. 5G and H, the ratio of phosphorylated and total ERK1/2 had no obvious change between high glucose and Smad2 siRNA treated podocytes. From these results, we conclude that TGF-β1 activate ERK1/2 pathway through its receptor in high glucosecultured podocytes, which is independent of Smad2 pathway and necessary for induction of Egr-1.
Inhibition of Egr-1 decreases Cdk5 kinase activity by lowering the expression of p35 in high glucose-cultured podocytes To confirm whether Egr-1 is essential for high glucose-induced p35 expression in podocytes, we knocked down Egr-1 levels by siRNA-mediated gene silencing to evaluate the p35 expression.
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Fig. 4 – Effects of SB431542 on expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-cultured podocytes. Podocytes were incubated with 5.6 mM glucose (control group), 30 mM glucose (HG group) or 30 mM glucoseþ10 μM SB431542 (HGþSB431542 group) for 12 h. (A) The relative mRNA levels of Tgfbr1 detected by real-time PCR. (B) Protein representative photographs of Smad2 and p-Smad2. (C) The ratio of p-Smad2 and Smad2. (D) Protein representative photographs of Cdk5 and p35 with different concentrations of SB431542 (0.1, 1 and 10 μM). (E) Protein levels of Cdk5 and p35. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. (F) The relative mRNA levels of Cdk5 and p35 detected by real-time PCR. (G) Cdk5 kinase activity. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. HG group.
A scrambled siRNA was used as a control and downregulation of Egr-1 was confirmed by Western blot and real-time PCR analysis (Fig. 6A–C). Fig. 7A and B shows that the increased expression of p35 in high glucose-cultured podocytes was markedly attenuated by using Egr-1 siRNA, but the increased protein level of Cdk5 induced by high glucose was not influenced by Egr-1 siRNA. Furthermore, adding Egr-1 siRNA into podocytes cultured by high glucose also significantly decreased the Cdk5 kinase activity (Fig. 7C). These results suggested that Egr-1 activation mediated the induction of p35 in response to high glucose, with a concomitant change in Cdk5 kinase activity.
Inhibition of Cdk5 kinase activity alleviated podocyte apoptosis induced by high glucose or TGF-β1 Roscovitine is a purine analog that inhibits CDKs with a high specificity for CDK5 [19]. To investigate the relationship between Cdk5 and podocyte apoptosis, roscovitine (50 μmol/L) was used to treated high glucose or TGF-β1 cultured podocytes. As shown in
Fig. 8A and B, either high glucose or TGF-β1 induced observable increases of TUNEL-positive (apoptotic) cells, and roscovitine or SB431542 effectively attenuated these increases. Similarly, the apoptotic rates examined by Annexin V/PI for flow cytometry analysis were also significantly elevated in high glucose or TGF-β1 cultured podocytes. Treated by roscovitine or SB431542, the apoptotic rates were effectively inhibited, although it was not completely abolished (Fig. 8C and D).
Discussion Two decades ago, a new member of cyclin-dependent kinase (Cdk) family, Cdk5, was discovered. Initially, Cdk5 kinase activity was predominantly found in postmitotic neurons, and played an important role in regulating neuronal migration, actin and microtubule remodeling, axonal guidance and synaptic plasticity during the nervous system development [20]. Recently, Cdk5 has been observed in multiple non-neuronal cells and tissues, e.g.,
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Fig. 5 – High glucose increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. Podocytes were incubated with normal glucose (control group), 30 mM glucose (HG group), 30 mM glucose plus SB431542 (HGþSB431542 group) and 30 mM glucose plus U0126 (HGþU0126 group) for 12 h. (A) Protein representative photographs of ERK1/2, p-ERK1/2 and Egr-1. (B) The ratio of p-ERK1/2 and ERK1/2. (C) Protein levels of Egr-1. (D) Protein representative photographs of Cdk5 and p35. (E) Protein levels of Cdk5 and p35. (F) The relative mRNA levels of Egr-1, p35 and Cdk5 detected by real-time PCR. (G) Protein representative photographs of ERK1/2 and p-ERK1/2. (H) The ratio of p-ERK1/2 and ERK1/2. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. HG group.
Fig. 6 – The expression of Egr-1 was inhibited by siRNA. Podocytes were transfected with a siRNA for Egr-1 and a scrambled siRNA. (A) The relative mRNA levels of Egr-1 detected by real-time PCR. (B) Protein representative photograph of Egr-1. (C) Protein level of Egr-1. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. scrambled siRNA group.
pancreatic β-cells, monocytes, neutrophils, leukocytes, myocytes, epithelial cells, endothelial cells, adipocytes, and others [21]. Cdk5, by phosphorylating a spectrum of proteins, modulates cell maturation, differentiation, migration, and apoptosis in various tissues [22].
In the kidney, the expression of Cdk5 is limited to podocytes. Cellular p35 level, the main activator of Cdk5 kinase activity, has also been detected in the podocytes [8]. Podocytes share many similarities with neurons, as both cell types are considered to be terminally differentiated and highly specialized cells with
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Fig. 7 – Inhibition of Egr-1 by siRNA decreased p35 expression in high glucose-cultured podocytes. Podocytes were transfected with a siRNA for Egr-1, and a scrambled siRNA was used as a control. (A) Protein representative photographs of Egr-1, p35 and Cdk5. (B) Protein levels of Egr-1, p35 and Cdk5. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. (C) Cdk5 kinase activity. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. HG group; and & po0.05 vs. HGþScrambled siRNA group.
Fig. 8 – Inhibition of Cdk5 kinase activity decreased podocyte apoptosis induced by high glucose or TGF-β1. (A) Apoptotic cells were detected by a TUNEL method. Arrows indicated TUNEL-positive (apoptotic) cells. (B) TUNEL-positive cells were counted out of a total of more than 200 cells over 6 random fields. The results were expressed as apoptotic rate (%). (C) Podocytes were stained with Annexin V/PI for flow cytometry analysis. Apoptotic cells were divided into two stages: early apoptotic (Annexin Vþ/PI ) and late apoptotic (Annexin Vþ/PIþ) cells. (D) The total apoptotic rates examined by flow cytometry, including the early and late apoptosis rate, were quantified and shown with a histogram. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. HG group; and &po0.05 vs. TGF-β1 group. complex architectures [23]. The Cdk5/p35 complexes have been demonstrated that regulate the podocyte differentiation, proliferation and morphology [7]. Podocyte is the critical component of
the glomerular filtration barrier, and is required for normal glomerular integrity. A major mechanism underlying the development of glomerulosclerosis and proteinuria in disease is
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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reduced podocyte number resulting from apoptosis, detachment, and the inability to adequately proliferate. Podocyte apoptosis has been extensively reported in diabetic kidney disease [24]. Our previous studies showed that the expression of Cdk5 was elevated either in glomeruli of diabetic rats or in high glucose treated conditionally immortalized mouse podocytes in vitro. Most importantly, the increase of Cdk5 levels was closely correlated with diabetic podocyte injuries and apoptosis [9,10]. In the present study, we further found that, in addition to Cdk5 expression levels, high glucose also up-regulated its co-activator p35 expression with a concomitant increase in Cdk5 kinase activity in a time-dependent manner. Moreover, inhibition of Cdk5 activity prevented podocyte apoptosis induced by high glucose. TGF-β1 is closely associated with the development and progression of DN and the increase of the TGF-β1 levels has been recognized as a marker of DN [14]. Among the features of the diabetic milieu, hyperglycemia, increased non-enzymatic glycation of proteins, de novo synthesis of diacylglycerol and subsequent activation of protein kinase C, increased intracellular glucosamine production, and enhanced renal production of vasoactive agents (angiotensin II, endothelins, thromboxane) have all been shown to increase the expression of TGF-β in cultured renal cells and in animal models of DN [25]. TGF-β1 signals are transduced by transmembrance type I and type II receptors. The Smad proteins are essential components of the intracellular signaling pathway activated by TGF-β1. After binding the TGF-β1, the type II receptor activates the type I serine/threonine kinase receptor. The latter can phosphorylate Smad2 and Smad3, which then forms a complex with Smad4. The Smad complex then moves into the nucleus and interacts with various transcription factors to regulate gene expression. Smad2 is thought to be the signaling arm that primarily mediates the auto-induction of TGFβ1 and its effects on apoptosis and matrix expression [26,27]. In the current study, we found that high glucose up-regulated TGF-β1 expression and Smad2 phosphorylations, which are in line with other studies [28,29]. Most importantly, SB431542, the Tgfbr1 inhibitor, effectively decreased the increases of Cdk5 expression and activity induced by high glucose, suggesting TGF-β1 may be an important and direct regulator of Cdk5 and play a crucial role in mediating the increase of Cdk5 in high glucose-cultured podocytes. In addition to TGF-β1 activation of canonical Smad pathways, recent reports have shown that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the mitogen-activated protein kinases (MAPKs) involved in hyperglycemia-induced TGF-β1 signaling [30]. The ERK1/2 signaling pathway is a major regulator of Cdk5 activity through control of Egr-1 and p35 expression in sensory neurons [16]. Egr-1 is a member of a family of zinc-finger transactivators and was originally identified as one of immediate early genes [31]. The 50 -flanking region of p35 contains Gþ C-rich sequences (73% of GþC content at 1020 to þ90) lacking canonical TATA and CAAT boxes. Several potential sequence motifs for transcription regulatory factors were identified in this region, including Sp1, AP2, MRE, and NGFIA. Four AP2 sites were found in intron 1. It is notable that an identical 17-bp GþC-rich sequence repeated at 500 to 484 and 546 to 530 represents the consensus motif for NGFIA, also known as Egr-1, krox-24, and Zif268 [32]. Takeshi et al. reported that Egr-1 binds directly to the p35 promoter region and upregulates p35
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expression [18]. Cdk5 requires the p35 subunit for its activity. By silencing Egr-1 expression by small interfering RNA, we found Egr-1 can affect Cdk5 kinase activity through regulating p35 expression in high glucose-cultured podocytes. In agreement with our findings, Tong et al. reported that p35 protein levels and Cdk5 activity are reduced in Egr-1 knock-out mouse brains [33]. That is to say Egr-1 can affect Cdk5 kinase activity through regulating p35 expression, but there is no direct effect on Cdk5 protein expression. Several studies have shown that inhibition of Cdk5 can protect neurons from apoptosis. Cdk5 inhibitory peptide, which effectively inhibits Cdk5 kinase activity, reduces neuronal apoptosis induced by β-amyloid [34]. In consistent with the idea that inhibition of Cdk5 produces neuroprotective effects, our results showed that pretreatment of roscovitine prevented podocyte apoptosis induced by high glucose or TGF-β1. Collectively, our results show that in high glucose-cultured podocytes, TGF-β1 can directly regulate Cdk5 kinase activity, and this effect is mediated by ERK1/2 through subsequent activation of Egr-1 and p35 expression. More importantly, inhibition of Cdk5 kinase activity can effectively protect podocytes from death induced by high glucose or TGF-β1. All findings will bring a new insight to our understandings of the role of Cdk5 in diabetic nephropathy. Reducing the p35 expression and Cdk5 activity by inhibiting TGF-β1 signaling pathway will be a plausible strategy for therapy of diabetic renal damage.
Conflict of interest statement The authors declare that there is no conflict of interests associated with this paper.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81302625), the Natural Science Foundation of Hebei Province (No. H2013206139), Excellent Youth Science Fund of Department of Education of Hebei Province of China (Nos. Y2012001 and YQ2013002), Key Project of Hebei Health Department (Nos. 20130140 and 20130461) and Scientific Research Talent Cultivation Project of Hebei Medical University.
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Research Article
High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway Yue Zhanga,1, Hongbo Lib,1, Jun Haob, Yi Zhouc, Wei Liub,n a
Department of Diagnostics, Hebei Medical University, Shijiazhuang 050017, China Department of Pathology, Hebei Medical University, Shijiazhuang 050017, China c Department of Neurology, the Second Hospital of Hebei Medical University, Shijiazhuang 050000, China b
article information
abstract
Article Chronology:
Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in
Received 4 December 2013
the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5),
Received in revised form
who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases,
7 April 2014
has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our
Accepted 16 April 2014
previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how
Keywords: Cdk5 TGF-β1 Podocyte High glucose Diabetic nephropathy
expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. & 2014 Elsevier Inc. All rights reserved.
Introduction Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and the leading cause of end-stage renal
failure [1]. DN is characterized by specific renal morphological and functional alterations. Glomerular visceral epithelial cells, namely podocytes, are terminally differentiated cells overlying the outer aspect of the glomerular basement membrane of renal glomeruli
Abbreviations: Cdk, Cyclin-dependent kinase; DN, Diabetic nephropathy; Egr, Early growth response; ERK, Extracellular signal-regulated kinase; MEK, Mitogen-activated protein kinase/ERK kinase; siRNA, Small interference RNA; TGF-β1, Transforming growth factor-β1. n Correspondence to: Department of Pathology, Hebei Medical University, No.361, Zhongshan East Road, Hebei Province, Shijiazhuang 050017, China. Fax: þ86 0311 86265734. E-mail address: [email protected] (W. Liu). 1 The two authors contributed equally to this study.
http://dx.doi.org/10.1016/j.yexcr.2014.04.014 0014-4827/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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and play a vital role in renal physiology. Among the characteristic findings of DN, podocytes are involved in the development of glomerular hypertrophy, podocytopenia, glomerulosclerosis, and foot process effacement [2,3]. Because podocyte depletion may be a key initiating lesion in these processes, it is considered important to determine the underlying molecular and cellular mechanisms. Cyclin-dependent kinases (Cdks) play essential roles in the regulation of cell division cycle. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that belongs to the family of Cdks. Distinct from other members of the Cdk family, the activation of Cdk5 does not require binding to cyclins, but rather, association with its regulator to perform kinase activity [4]. One major regulating partner for Cdk5 is p35, which was first reported in postmitotic neurons. The crucial role of the Cdk5-p35 complex is to support the development of the central nervous system [5]. However, numerous extraneuronal functions of Cdk5-p35 have been discovered in recent years, in addition to the roles in the central nervous system [6]. For example, in the kidney, the glomerular expression of Cdk5 is limited to podocytes. Moreover, Cdk5 is a key regulator of podocyte differentiation, proliferation and morphology [7]. Recent studies have shown that the absence of p35 confers increased susceptibility of podocytes to apoptosis in disease [8]. Our previous work has shown that the expression of Cdk5 was increased in a time-dependent manner and roscovitine; a Cdk5 inhibitor, significantly ameliorated podocyte injuries in diabetic rats [9]. Furthermore, in cultured podocytes in vitro, high glucose increased the expression of Cdk5, and knockdown of Cdk5 attenuated podocyte apoptosis induced by high glucose stimulation [10]. These findings suggest that Cdk5 plays an important role in multiple mechanisms involved in podocyte injuries of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been elucidated. Transforming growth factor-β1 (TGF-β1) is an important member of a superfamily of multifunctional growth factors involved in many cellular processes including cell proliferation, differentiation, migration, and apoptosis. TGF-β1 has been proposed as a major mediator of matrix accumulation in diabetic kidney and high glucose-exposed mesangial cells and podocytes, leading to the development of DN [11,12]. Moreover, increased expression of TGF-β1 in podocytes coincides with the onset of apoptosis and albuminuria in diabetes [13]. Correspondingly, the increase of the TGF-β1 levels has been recognized as a marker of DN [14,15]. Because TGF-β1 has been shown to act as a mediator of Cdk5 activity in sensory neurons [16], we want to know under the high glucose environment, whether TGF-β1 could act as an upstream regulator of Cdk5 in podocytes, affecting the development and progression of DN. In this study, we investigated that the role of TGF-β1 on Cdk5 expression and activity in high glucose-treated podocytes. We found that high glucose and TGF-β1 up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity. SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity. Furthermore, the ERK1/2-Egr-1 signaling pathway was involved in the regulation of TGF-β1 on p35 and Cdk5 in high glucose-cultured podocytes. More importantly, inhibition of Cdk5 kinase activity was demonstrated to be effective in alleviating podocyte apoptosis induced by high glucose or TGF-β1.
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Materials and methods Antibodies and reagents Primary antibodies recognizing Cdk5 and Egr-1 were purchased from Epitomics Company (CA, USA). Antibody to p35 was purchased from Santa Cruz Biotechnology (CA, USA). Antibodies to TGF-β1, Smad-2, phospho-Smad-2 and β-actin were purchased from Signalway Antibody Company (Maryland, USA). Antibodies against p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204) were obtained from Cell Signaling Technology (MA, USA). Tgfbr1 inhibitor SB431542, the MEK inhibitor U0126 and Cdk5 kinase activity inhibitor roscovitine were purchased from Sigma Aldrich (Dorset, UK). Recombinant murine IFN-γ was purchased from Peprotech Company (NJ, USA).
Conditionally immortalized mouse podocytes in culture Conditionally immortalized mouse podocytes purchased from the Cell Culture Center (PUMC, CAMS, Beijing, China) were cultured as previously described [17]. To induce proliferation, cells were grown on collagen I-coated plastic culture bottles (BD Biosciences, Bedford, MA), at 33 1C in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, USA), 100 U/ml penicillin (Invitrogen, USA), and 100 μg/ml streptomycin (Invitrogen, USA), to which recombinant mouse IFN-γ 10 U/ml (Pepro Tech, USA) was added (growth permissive conditions). To induce quiescence and the differentiated phenotype, podocytes were grown at 37 1C and deprived of IFN-γ (growth restrictive conditions) in DMEM supplemented with 10% FBS, penicillin, and streptomycin. All studies were performed on days 10 to 14 for cells grown under restrictive conditions.
RNA interference analysis Conditionally immortalized mouse podocytes grown in 6-well plates for 24 h in DMEM with 10% FBS were transfected with siRNA against Smad2 ( Smad2 siRNA, sc-38375, Santa Cruz, USA ), Egr-1 (Egr-1 siRNA, sc-35267, Santa Cruz), or control siRNA (sc-37007, Santa Cruz) with Lipofectamine RNAi MAX (Invitrogen, USA) as per the manufacturer's protocol. After 24 h transfection, the cells were treated with high glucose and then were analyzed.
Immunocytochemistry When high glucose treated for 12 h, podocytes were fixed with 4% paraformaldehyde at room temperature for 15 min. After pretreatment with 0.1% Triton X-100 for 10 min at 37 1C, cells were blocked with goat serum for 30 min at 37 1C. Then, the cells were incubated with rabbit anti-Cdk5 (1:200) antibody overnight at 4 1C. After three washes with PBS, cells were incubated with a polymer helper and polyperoxidase-anti-mouse/rabbit IgG at 37 1C for 30 min, and the cells were then stained with diaminobenzidine. A negative control was performed by replacing the primary antibody with PBS buffer.
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Immunofluorescence Indirect immunofluorescence staining was performed according to an established procedure. Briefly, cells cultured on coverslips were washed twice with cold PBS and fixed with cold methanol/ acetone (1:1) for 10 min at 20 1C. Following three extensive washings with PBS, the cells were treated with 0.1% Triton X-100 for 10 min at room temperature, and blocked with 2% normal donkey serum in PBS buffer for 30 min at 37 1C and then incubated with the primary antibody against p35 or TGF-β1 over night at 4 1C. Next day, after incubating with FITC-conjugated secondary antibody for 2 h, the slides were viewed with the Olympus BX63F fluorescence microscope equipped with a digital camera. In each experimental setting, immunofluorescence images were captured with identical light exposure time.
Western blotting The different groups of cells were harvested and homogenized in ice-cold homogenization buffer (1% Nonidet P-40, 0.1% Triton X-100, 30 mM sodium phosphate, pH 7.4, containing 1 mM sodium orthovanadate, 100 mM NaCl, 2.5 mM Tris–HCl, pH 7.5, and protease inhibitors), followed by centrifugation at 12,000g for 20 min at 4 1C. The supernatant was collected to characterize the relative levels of protein expression by Western blot assays. The protein concentrations were quantified with the Bio-Rad protein colorimetric assay (Bio-Rad, USA). Whole cell extracts (100 μg of protein/lane) were loaded, separated by 10% SDS-PAGE, and transferred to PVDF membranes (Millipore, MA). After blocking with 5% skimmed milk in Trisbuffered saline/Tween buffer (TBST buffer) for 2 h, the membranes were incubated overnight at 4 1C with primary antibody described above. Subsequently, the membranes were incubated with goat anti-rabbit or mouse IgG horseradish peroxidase conjugate, and then the results were detected by an Odyssey FC detection system using an enhanced chemiluminescence system. The intensity of the bands was measured using LabWorks 4.5.
RNA isolation and real-time PCR Total RNA was extracted with TriZol Reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Complementary DNA was synthesized from the total RNA (0.5 μg) using the PrimeScriptTM RT regent Kit following the instructions provided by the manufacturer (Takara Biotechnology, Dalian, China). Subsequently, the cDNA was subjected to quantitative RT-PCR (qRT-PCR) using a Power SYBR Green PCR Master Mix (Takara Biotechnology, Dalian, China). Each real-time PCR reaction consisted of 2 μl diluted RT product, 10 μl SYBR Green PCR Master Mix (2 ) and 250 nM forward and reverse primers in a total volume of 20 μl. Reactions were carried out on a 7500 qRT-PCR System (Applied Biosystems) for 40 cycles (95 oC for 15 s, 60 1C for 45 s) after initial 10 min incubation at 95 1C. The primers used for real-time PCR were as follows: p35 forward primer (F): 50 -GCC CTT CCT GGT AGA GAG CTG-30 and p35 reverse primer (R): 50 -GTG TGA AAT AGT GTG GGT CGG C-30 ; Cdk5 F: 50 -TAG GCT CTC TGA ACC CCA GT-30 , Cdk5 R: 50 -ATC CCA CAC CCG ACT CTT C-30 ; Tgfbr1 F: 50 -TGC ATT GCA CTT ATG CTG ATG GT-30 , Tgfbr1 R: 50 -ACC TGA TCC AGA CCC TGA TGT T-30 ; TGF-β1 F: 50 -CAC CTG CAA GAC CAT CGA CAT-30 , TGF-β1 R: 50 -GAG CCT TAG TTT GGA CAG GAT CTG-30 ;
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Egr-1 F: 50 -CCC TTC CAG GGT CTG GAG AAC CGT-30 , Egr-1 R: 50 -GGG GTA CTT GCG CAT GCG GCT GGG-30 . The mRNA levels were standardized by using the following primers to 18S: 18S F: 50 -CAT TCG AAC GTC TGC CCT ATC-30 and 18S R: 50 -CCT GCT GCC TTC CTT GGA-30 .
Immunoprecipitation and Cdk5 kinase activity assay The treated cells were lyzed in 10 mM HEPES, pH 7.5 (or 7.4), containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor mixture. The lysates were centrifuged at 12,000 rpm for 15 min. For Cdk5 immunoprecipitation, the supernatant was incubated with 50 μl protein A agarose (Santa Cruz Biotechnology, USA) and 2 μg anti-Cdk5 antibody for 4 h at 4 1C. The immune complex was separated by centrifugation at 4000 rpm. Kinase assay was performed with an ADP-GloTM kinase assay kit (Promega, USA) according with the manufacturer's protocol. Briefly, immunoprecipitates were washed three times with lysis buffer and then once with 1 reaction buffer containing 40 mM Tris–HCl, pH 7.5, 20 mM MgCl2, 0.1 μg/μl BSA and 0.5 mM DTT. Kinase assays were performed in the same buffer by addition of 250 μM ATP and 5 μg substrate histone H1. Then the mixture was incubated at room temperature for 10 min. Next, 25 μl ADP-GloTM reagents were added to terminate the reaction and deplete the remaining ATP. After 50 μl kinase detection reagents were added into the mixture to incubate for 30 min, the results were recorded by measuring the luminescence with a plate-reading luminometer. Data are shown as relative light units (RLU) that directly correlate to the amount of ADP produced.
Terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) staining Apoptotic cells were identified using the TUNEL technique according to the manufacturer's instructions (Roche Applied Science, Hangzhou, China). For quantification of TUNEL-positive (apoptotic) cells, a minimum of 200 cells were counted per group, and the percentage of the positively labeled cells was calculated.
Annexin V and propidium iodide staining assay Apoptotic rate was detected by an Annexin V/PI apoptosis detection kit according to manufacturer's protocol (MultiSciences Biotech, Hangzhou, China). Briefly, the cell pellet was resuspended in 1 binding buffer followed by incubation with 5 μl of Annexin V (conjugated with FITC) and 10 μl of PI in dark for 5 min. Cell fluorescence was then analyzed by a flow cytometer (Epics-XLII, Becman Coulter). This test discriminates intact cells (Annexin V /PI ), early apoptotic cells (Annexin Vþ/PI ) and late apoptotic cells (Annexin Vþ/PIþ).
Statistical analysis All values are expressed as mean7SD. Statistical analyses were performed using the SPSS 17.0 program. One-way ANOVA was applied to compare the means between groups. Po0.05 was considered to be statistically significant.
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Results High glucose up-regulated the expression of Cdk5 and p35 in podocytes with a concomitant increase in Cdk5 kinase activity In order to examine the effects of high glucose on the expression levels of Cdk5 and its co-activator p35, podocytes were cultured
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in DMEM medium containing 5.6 mM glucose (control group), 5.6 mM glucose plus 24.4 mM mannitol (M group, as an osmotic control) or 30 mM glucose (HG group) for 12 h. As expected, mannitol had no effect on the protein levels of Cdk5 and p35. However, the expression of Cdk5 and p35 was significantly increased in high glucose-treated podocytes, compared with the control group or the mannitol group (Fig. 1A and B). Next, we examined that the time course of high glucose (30 mM)-mediated
Fig. 1 – Effects of high glucose on expression of Cdk5 and p35 and Cdk5 kinase activity. Podocytes were incubated with 5.6 mM glucose (control group), 5.6 mM glucoseþ24.4 mM mannitol (M group) or 30 mM glucose (HG group) for 12 h. (A) Protein representative photographs of Cdk5 and p35. (B) Protein levels of Cdk5 and p35. (C) Cdk5 positive expression was detected using immunocytochemical staining at different time points (3, 6 and 12 h). (200 ) (D) The p35 positive expression was detected using immunofluorescence staining at different time points (3, 6 and 12 h). (400 ) (E) Protein representative photographs of Cdk5 and p35 at different time points (3, 6 and 12 h). (F) Protein levels of Cdk5 and p35. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. (G) The relative mRNA levels of Cdk5 and p35 detected by real-time PCR. (H) Cdk5 kinase activity. Data are expressed as mean7SD. npo0.05 vs. control group; # po0.05 vs. M group. Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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increase of Cdk5 expression. As shown in Fig. 1C, the positive expressions of Cdk5, detected by immunocytochemical staining at different time points (3, 6 and 12 h), were both significantly increased at 3 h after high glucose treatment and remained elevated until 12 h. The positive performance of p35 detected by immunofluorescence showed the same trend with Cdk5 (Fig. 1D). Similarly, the protein and mRNA levels of Cdk5 and p35, examined by Western blot or real-time PCR analysis, were also increased with a time-dependent manner following high glucose treatment (Fig. 1E–G). Furthermore, we assayed the Cdk5 kinase activity using histone H1 as substrate. Compared with the control group, high glucose-treated podocytes also showed an increased Cdk5 kinase activity with a time-dependent manner (Fig. 1H). Together, these results indicated that high glucose up-regulated the expression of Cdk5 and p35 in podocytes, resulted in increased Cdk5 kinase activity.
High glucose activated the TGF-β1 signaling pathway in podocytes To confirm previous reports that TGF-β1 signaling pathway is responsive to high glucose in podocytes, we treated the mouse
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podocytes for 12 h with 30 mM glucose. Using immunofluorescence staining, the TGF-β1 positive expression showed an obvious increase after getting exposed to 30 mM glucose (Fig. 2A). Similarly, the protein levels of TGF-β1 and the ratio of phosphorylated and total Smad2 were significantly elevated in high glucosetreated podocytes (Fig. 2B–D). Furthermore, the mRNA levels of TGF-β1 and Tgfbr1 were also notably increased in podocytes when 30 mM glucose treatment, respectively five times and eight times higher than that of the control or the mannitol group (Fig. 2E). These results demonstrated that high glucose promoted the activation of TGF-β1 signaling pathway.
TGF-β1 up-regulated the expression of Cdk5 and p35 and Cdk5 kinase activity in podocytes In order to examine the direct effects of TGF-β1 on Cdk5 expression and activity, the cultured podocytes were treated with different concentrations of TGF-β1 (1, 5, 10 ng/ml) for 12 h. As shown in Fig. 3A–C, compared with the control group, the protein and mRNA expressions of Cdk5 and p35 were significantly increased with a concentration-dependent manner in TGF-β1-treated podocytes. Moreover, the Cdk5 kinase activity also showed the same increased
Fig. 2 – Effects of high glucose on TGF-β1 signaling pathway. Podocytes were incubated with 5.6 mM glucose (control group), 5.6 mM glucoseþ24.4 mM mannitol (M group) or 30 mM glucose (HG group) for 12 h. (A) The TGF-β1 positive expression was detected using immunofluorescence staining. (200 ) (B) Protein representative photographs of TGF-β1, Smad2 and p-Smad2. (C) Protein levels of TGF-β1. (D) The ratio of p-Smad2 and Smad2. (E) The relative mRNA levels of TGF-β1 and Tgfbr1 detected by real-time PCR. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. M group. Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Fig. 3 – Effects of TGF-β1 on expression of Cdk5 and p35 and Cdk5 kinase activity. Podocytes were treated with different concentrations of TGF-β1 (1, 5, 10 ng/ml) for 12 h. (A) Protein representative photographs of Cdk5 and p35. (B) Protein levels of Cdk5 and p35. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. (C) The relative mRNA levels of Cdk5 and p35 detected by real-time PCR. (D) Cdk5 kinase activity. Data are expressed as mean7SD. npo0.05 vs. control group.
trend (Fig. 3D), suggesting that TGF-β1 can directly up-regulate the expression of Cdk5 and p35, and Cdk5 kinase activity.
SB431542 inhibited the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-cultured podocytes To evaluate the effect of TGF-β1 on Cdk5 expression and kinase activity under high glucose environment, SB431542 (the Tgfbr1 inhibitor) was used to treat podocytes. As shown in Fig. 4A, the mRNA level of Tgfbr1 induced by high glucose was markedly inhibited by SB431542. Moreover, SB431542 treatment decreased the protein level of phospho-Smad2 (Fig. 4B and C), which demonstrated that SB431542 effectively played its role. More importantly, the increases of Cdk5 and p35 protein and mRNA levels induced by high glucose were also significantly prevented by SB431542 in a dose-dependent manner from 0.1 μM to 10 μM (Fig. 4D–F). Similarly, SB431542 treatment strongly blocked the increases of Cdk5 kinase activity (Fig. 4G), indicating that under the high glucose environment, TGF-β1 plays a direct and important role in elevating Cdk5 expression and activity in podocytes.
High glucose increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway in podocytes Egr-1 is a member of a family of zinc-finger transactivators and has been known as a regulator of the p35 promoter [18]. To identify the molecular mechanism underlying TGF-β1-mediated regulation of Cdk5 activity and evaluate if high glucose activate the Egr-1 expression, SB431542 (10 μM) and U0126 (20 μM) were
used to treat podocytes. U0126 is a specific MEK inhibitor for inhibiting ERK1/2 activation induced by TGF-β1. By Western blot analysis, the ratio of phosphorylated and total ERK1/2 and the protein levels of Egr-1 were significantly increased when exposed to 30 mM glucose, whereas SB431542 or U0126 strongly decreased these increases (Fig. 5A–C). The protein expression of Cdk5 and p35 induced by high glucose was also reversed by SB431542 or U0126 (Fig. 5D and E). Consistent with the protein expression results, the increased mRNA levels of Egr-1, Cdk5 and p35 induced by high glucose were all inhibited by SB431542 or U0126 treatment (Fig. 5F). In order to explore whether Smad2 is involved in the regulation of TGF-β1 on ERK1/2, Smad2 was inhibited using siRNA to evaluate the total- and phospho-ERK1/2 expression. As shown in Fig. 5G and H, the ratio of phosphorylated and total ERK1/2 had no obvious change between high glucose and Smad2 siRNA treated podocytes. From these results, we conclude that TGF-β1 activate ERK1/2 pathway through its receptor in high glucosecultured podocytes, which is independent of Smad2 pathway and necessary for induction of Egr-1.
Inhibition of Egr-1 decreases Cdk5 kinase activity by lowering the expression of p35 in high glucose-cultured podocytes To confirm whether Egr-1 is essential for high glucose-induced p35 expression in podocytes, we knocked down Egr-1 levels by siRNA-mediated gene silencing to evaluate the p35 expression.
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Fig. 4 – Effects of SB431542 on expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-cultured podocytes. Podocytes were incubated with 5.6 mM glucose (control group), 30 mM glucose (HG group) or 30 mM glucoseþ10 μM SB431542 (HGþSB431542 group) for 12 h. (A) The relative mRNA levels of Tgfbr1 detected by real-time PCR. (B) Protein representative photographs of Smad2 and p-Smad2. (C) The ratio of p-Smad2 and Smad2. (D) Protein representative photographs of Cdk5 and p35 with different concentrations of SB431542 (0.1, 1 and 10 μM). (E) Protein levels of Cdk5 and p35. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. (F) The relative mRNA levels of Cdk5 and p35 detected by real-time PCR. (G) Cdk5 kinase activity. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. HG group.
A scrambled siRNA was used as a control and downregulation of Egr-1 was confirmed by Western blot and real-time PCR analysis (Fig. 6A–C). Fig. 7A and B shows that the increased expression of p35 in high glucose-cultured podocytes was markedly attenuated by using Egr-1 siRNA, but the increased protein level of Cdk5 induced by high glucose was not influenced by Egr-1 siRNA. Furthermore, adding Egr-1 siRNA into podocytes cultured by high glucose also significantly decreased the Cdk5 kinase activity (Fig. 7C). These results suggested that Egr-1 activation mediated the induction of p35 in response to high glucose, with a concomitant change in Cdk5 kinase activity.
Inhibition of Cdk5 kinase activity alleviated podocyte apoptosis induced by high glucose or TGF-β1 Roscovitine is a purine analog that inhibits CDKs with a high specificity for CDK5 [19]. To investigate the relationship between Cdk5 and podocyte apoptosis, roscovitine (50 μmol/L) was used to treated high glucose or TGF-β1 cultured podocytes. As shown in
Fig. 8A and B, either high glucose or TGF-β1 induced observable increases of TUNEL-positive (apoptotic) cells, and roscovitine or SB431542 effectively attenuated these increases. Similarly, the apoptotic rates examined by Annexin V/PI for flow cytometry analysis were also significantly elevated in high glucose or TGF-β1 cultured podocytes. Treated by roscovitine or SB431542, the apoptotic rates were effectively inhibited, although it was not completely abolished (Fig. 8C and D).
Discussion Two decades ago, a new member of cyclin-dependent kinase (Cdk) family, Cdk5, was discovered. Initially, Cdk5 kinase activity was predominantly found in postmitotic neurons, and played an important role in regulating neuronal migration, actin and microtubule remodeling, axonal guidance and synaptic plasticity during the nervous system development [20]. Recently, Cdk5 has been observed in multiple non-neuronal cells and tissues, e.g.,
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Fig. 5 – High glucose increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. Podocytes were incubated with normal glucose (control group), 30 mM glucose (HG group), 30 mM glucose plus SB431542 (HGþSB431542 group) and 30 mM glucose plus U0126 (HGþU0126 group) for 12 h. (A) Protein representative photographs of ERK1/2, p-ERK1/2 and Egr-1. (B) The ratio of p-ERK1/2 and ERK1/2. (C) Protein levels of Egr-1. (D) Protein representative photographs of Cdk5 and p35. (E) Protein levels of Cdk5 and p35. (F) The relative mRNA levels of Egr-1, p35 and Cdk5 detected by real-time PCR. (G) Protein representative photographs of ERK1/2 and p-ERK1/2. (H) The ratio of p-ERK1/2 and ERK1/2. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. HG group.
Fig. 6 – The expression of Egr-1 was inhibited by siRNA. Podocytes were transfected with a siRNA for Egr-1 and a scrambled siRNA. (A) The relative mRNA levels of Egr-1 detected by real-time PCR. (B) Protein representative photograph of Egr-1. (C) Protein level of Egr-1. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. scrambled siRNA group.
pancreatic β-cells, monocytes, neutrophils, leukocytes, myocytes, epithelial cells, endothelial cells, adipocytes, and others [21]. Cdk5, by phosphorylating a spectrum of proteins, modulates cell maturation, differentiation, migration, and apoptosis in various tissues [22].
In the kidney, the expression of Cdk5 is limited to podocytes. Cellular p35 level, the main activator of Cdk5 kinase activity, has also been detected in the podocytes [8]. Podocytes share many similarities with neurons, as both cell types are considered to be terminally differentiated and highly specialized cells with
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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Fig. 7 – Inhibition of Egr-1 by siRNA decreased p35 expression in high glucose-cultured podocytes. Podocytes were transfected with a siRNA for Egr-1, and a scrambled siRNA was used as a control. (A) Protein representative photographs of Egr-1, p35 and Cdk5. (B) Protein levels of Egr-1, p35 and Cdk5. The level of each protein was quantified by densitometric analysis and normalized to the level of β-actin. (C) Cdk5 kinase activity. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. HG group; and & po0.05 vs. HGþScrambled siRNA group.
Fig. 8 – Inhibition of Cdk5 kinase activity decreased podocyte apoptosis induced by high glucose or TGF-β1. (A) Apoptotic cells were detected by a TUNEL method. Arrows indicated TUNEL-positive (apoptotic) cells. (B) TUNEL-positive cells were counted out of a total of more than 200 cells over 6 random fields. The results were expressed as apoptotic rate (%). (C) Podocytes were stained with Annexin V/PI for flow cytometry analysis. Apoptotic cells were divided into two stages: early apoptotic (Annexin Vþ/PI ) and late apoptotic (Annexin Vþ/PIþ) cells. (D) The total apoptotic rates examined by flow cytometry, including the early and late apoptosis rate, were quantified and shown with a histogram. Data are expressed as mean7SD. npo0.05 vs. control group; #po0.05 vs. HG group; and &po0.05 vs. TGF-β1 group. complex architectures [23]. The Cdk5/p35 complexes have been demonstrated that regulate the podocyte differentiation, proliferation and morphology [7]. Podocyte is the critical component of
the glomerular filtration barrier, and is required for normal glomerular integrity. A major mechanism underlying the development of glomerulosclerosis and proteinuria in disease is
Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014
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reduced podocyte number resulting from apoptosis, detachment, and the inability to adequately proliferate. Podocyte apoptosis has been extensively reported in diabetic kidney disease [24]. Our previous studies showed that the expression of Cdk5 was elevated either in glomeruli of diabetic rats or in high glucose treated conditionally immortalized mouse podocytes in vitro. Most importantly, the increase of Cdk5 levels was closely correlated with diabetic podocyte injuries and apoptosis [9,10]. In the present study, we further found that, in addition to Cdk5 expression levels, high glucose also up-regulated its co-activator p35 expression with a concomitant increase in Cdk5 kinase activity in a time-dependent manner. Moreover, inhibition of Cdk5 activity prevented podocyte apoptosis induced by high glucose. TGF-β1 is closely associated with the development and progression of DN and the increase of the TGF-β1 levels has been recognized as a marker of DN [14]. Among the features of the diabetic milieu, hyperglycemia, increased non-enzymatic glycation of proteins, de novo synthesis of diacylglycerol and subsequent activation of protein kinase C, increased intracellular glucosamine production, and enhanced renal production of vasoactive agents (angiotensin II, endothelins, thromboxane) have all been shown to increase the expression of TGF-β in cultured renal cells and in animal models of DN [25]. TGF-β1 signals are transduced by transmembrance type I and type II receptors. The Smad proteins are essential components of the intracellular signaling pathway activated by TGF-β1. After binding the TGF-β1, the type II receptor activates the type I serine/threonine kinase receptor. The latter can phosphorylate Smad2 and Smad3, which then forms a complex with Smad4. The Smad complex then moves into the nucleus and interacts with various transcription factors to regulate gene expression. Smad2 is thought to be the signaling arm that primarily mediates the auto-induction of TGFβ1 and its effects on apoptosis and matrix expression [26,27]. In the current study, we found that high glucose up-regulated TGF-β1 expression and Smad2 phosphorylations, which are in line with other studies [28,29]. Most importantly, SB431542, the Tgfbr1 inhibitor, effectively decreased the increases of Cdk5 expression and activity induced by high glucose, suggesting TGF-β1 may be an important and direct regulator of Cdk5 and play a crucial role in mediating the increase of Cdk5 in high glucose-cultured podocytes. In addition to TGF-β1 activation of canonical Smad pathways, recent reports have shown that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the mitogen-activated protein kinases (MAPKs) involved in hyperglycemia-induced TGF-β1 signaling [30]. The ERK1/2 signaling pathway is a major regulator of Cdk5 activity through control of Egr-1 and p35 expression in sensory neurons [16]. Egr-1 is a member of a family of zinc-finger transactivators and was originally identified as one of immediate early genes [31]. The 50 -flanking region of p35 contains Gþ C-rich sequences (73% of GþC content at 1020 to þ90) lacking canonical TATA and CAAT boxes. Several potential sequence motifs for transcription regulatory factors were identified in this region, including Sp1, AP2, MRE, and NGFIA. Four AP2 sites were found in intron 1. It is notable that an identical 17-bp GþC-rich sequence repeated at 500 to 484 and 546 to 530 represents the consensus motif for NGFIA, also known as Egr-1, krox-24, and Zif268 [32]. Takeshi et al. reported that Egr-1 binds directly to the p35 promoter region and upregulates p35
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expression [18]. Cdk5 requires the p35 subunit for its activity. By silencing Egr-1 expression by small interfering RNA, we found Egr-1 can affect Cdk5 kinase activity through regulating p35 expression in high glucose-cultured podocytes. In agreement with our findings, Tong et al. reported that p35 protein levels and Cdk5 activity are reduced in Egr-1 knock-out mouse brains [33]. That is to say Egr-1 can affect Cdk5 kinase activity through regulating p35 expression, but there is no direct effect on Cdk5 protein expression. Several studies have shown that inhibition of Cdk5 can protect neurons from apoptosis. Cdk5 inhibitory peptide, which effectively inhibits Cdk5 kinase activity, reduces neuronal apoptosis induced by β-amyloid [34]. In consistent with the idea that inhibition of Cdk5 produces neuroprotective effects, our results showed that pretreatment of roscovitine prevented podocyte apoptosis induced by high glucose or TGF-β1. Collectively, our results show that in high glucose-cultured podocytes, TGF-β1 can directly regulate Cdk5 kinase activity, and this effect is mediated by ERK1/2 through subsequent activation of Egr-1 and p35 expression. More importantly, inhibition of Cdk5 kinase activity can effectively protect podocytes from death induced by high glucose or TGF-β1. All findings will bring a new insight to our understandings of the role of Cdk5 in diabetic nephropathy. Reducing the p35 expression and Cdk5 activity by inhibiting TGF-β1 signaling pathway will be a plausible strategy for therapy of diabetic renal damage.
Conflict of interest statement The authors declare that there is no conflict of interests associated with this paper.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81302625), the Natural Science Foundation of Hebei Province (No. H2013206139), Excellent Youth Science Fund of Department of Education of Hebei Province of China (Nos. Y2012001 and YQ2013002), Key Project of Hebei Health Department (Nos. 20130140 and 20130461) and Scientific Research Talent Cultivation Project of Hebei Medical University.
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Please cite this article as: Y. Zhang, et al., High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.014