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Macrophage migration inhibitory factor regulates integrin-β1 and cyclin D1 expression via ERK pathway in podocytes
Biomedicine & Pharmacotherapy 124 (2020) 109892
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Macrophage migration inhibitory factor regulates integrin-β1 and cyclin D1 expression via ERK pathway in podocytes
T
Chien-An Chena,b,*, Jer-Ming Changc, Yu-Lin Yangd, Eddy-Essen Changc, Hung-Chun Chenc a
Department of Nephrology, Tainan Sinlau Hospital, Tainan, 701, Taiwan Department of Health Care Administration, Chang Jung Christian University, Tainan, 711, Taiwan c Department of Nephrology, Kaohsiung Medical University, Kaohsiung, 807, Taiwan d Graduate Institute of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, 703, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Macrophage migration inhibitory factor Integrin β1 Podocytes
Aims: Macrophage migration inhibitory factor (MIF) is found to increase in proliferative glomerulonephritis. MIF binds to the MIF receptor (CD74) that activates MAP kinase (ERK and p38). Integrins and cyclinD1 regulate cell proliferation, differentiation and adhesion. This study evaluates whether MIF can regulate integrin-β1/ cyclin D1 expression and cell adhesion of podocytes. Main methods: Expression of integrin-β1 mRNA/protein and cyclin D1 mRNA under stimulation of MIF was evaluated by real-time PCR and Western blotting. MIF receptor (CD74) and MAP kinase under MIF treatment were examined to determine which pathway regulated integrin-β1 and cyclin D1 expression. Cell adhesion was evaluated under MIF treatment and/or anti-integrin-β1 antibody by cell adhesion assay. Key findings: Protein levels of integrin-β1 were up-regulated under MIF treatment in a dosage-dependent manner. CD74 protein levels were not changed after MIF treatment. Integrin-β1 and cyclin D1 mRNA levels were up-regulated after MIF 100 ng/ml treatment. ERK inhibitor U0126 reduced MIF-induced the increase in integrinβ1 mRNA and protein expression following MIF stimulation. However, p38 inhibitor SB 203580 did not inhibit MIF-induced increase in integrin-β1 mRNA and protein expression following MIF stimulation. MIF-induced increase in cyclin D1 mRNA level also was inhibited only by U0126 following MIF stimulation. Podocyte adhesion was increased after MIF treatment, but, anti-integrin-β1 antibody decreased MIF-enhanced podocyte adhesion. Significance: MIF increases integrin-β1 and cyclin D1 expression through the ERK pathway in podocytes, and the up-regulated expression of integrin-β1 increases podocyte adhesion. These results provide further understanding for the role of MIF in developing proliferative glomerulonephritis.
1. Introduction Macrophage migration inhibitory factor (MIF) is a cytokine with pro-inflammatory and immunomodulatory activities, including regulating leukocyte adhesion/infiltration, T-cell activation, cytokine and inflammatory mediator expression, p53 expression, apoptosis, and resident-cell proliferation [1,2]. MIF was originally found to be derived from activated T lymphocytes and monocytes/macrophages [3]. Recent evidences demonstrate that MIF is expressed in many tissues including the kidney where MIF is found in podocytes, mesangial cells, endothelial cells and tubular epithelial cells [1,4,5]. Renal MIF is upregulated in human and experimental glomerulonephritis [4]. Urine excretion of MIF is significantly increased in glomerulonephritis and IgA nephropathy, and correlates with the degree of renal injury [6–8]. MIF receptor, CD74, activates MAP kinase (ERK1/2 and p38 MAPK) in ⁎
a time- and dosage-dependent manner [4,9,10]. In cultured human podocytes, CD 74 is up-regulated by high concentrations of glucose and TNF-α [10]. In clinical and experimental diabetic nephropathy, CD 74 was found to be increased on podocytes [10]. Integrins mediate cells to attach to an extracellular matrix and mediate mechanical and chemical signals from the extracellular matrix to cells or from cells to the extracellular matrix [11]. Integrins regulate cell survival, apoptosis, adhesion, migration, proliferation and differentiation [11]. Integrin-β1 is the major integrin family and expresses on the sole of the foot processes of podocytes [12]. Chen and colleagues demonstrated that integrin-β1-extracellular matrix interaction upregulated cyclin D1 expression in podocytes [13]. Cyclin D1 has a significant role in regulating early-to-mild G1 phase, proliferation/differentiation and survival/ apoptosis [14]. In proliferative glomerulonephritis, MIF increases in glomerulus and
Corresponding author at: Department of Nephrology, Tainan Sinlau Hospital, No. 57, 1 Sec., Dongman Road, Tainan, 701, Taiwan. E-mail address: [email protected] (C.-A. Chen).
https://doi.org/10.1016/j.biopha.2020.109892 Received 23 October 2019; Received in revised form 2 January 2020; Accepted 10 January 2020 0753-3322/ © 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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2.4. Western blot analysis
urine excretion [6,15]. In a rat model of crescentic glomerulonephritis (a form of proliferative glomerulonephritis), MIF expression increases in podocytes, parietal epithelial cells and endothelial cells [16]. In our previous study, we found that mitogens/growth factors in collaboration with integrin-β1 regulated cyclin D1 expression in podocytes [13]. In this study, we find that MIF could regulate integrin-β1 and cyclin D1 expression in podocytes.
2. Materials and methods
Western blot analysis was performed according to our previous report [19]. The following primary antibodies were used: anti-integrin-β1 antibody, rabbit polyclonal anti-ERK antibody, mouse monoclonal antip-ERK antibody, goat polyclonal anti-p38 antibody, mouse monoclonal anti-p-p38 antibody (all from Santa Cruz Biotechnology, CA) and antiCD74 antibody (Abcam, Cambridge, MA, USA). Equality of loading was performed by testing for GAPDH (Sigma). Results are expressed as the ratio (protein of interest/ GAPDH) to correct for sample loading.
2.1. Experimental design
2.5. Cell adhesion assay
To evaluate the effect of MIF on integrin-β1 expression, cells were treated with recombinant mouse MIF (R&D Systems Inc., Minneapolis, MN) with different concentrations (0, 10, 20, 50, 100, and 150 ng/ml) for 24 h, or with MIF (100 ng/ml) for different intervals (0, 12, 24, and 48 h). Expression of integrin-β1 mRNA and protein was analyzed by real-time PCR and Western blotting. To evaluate the effect of MIF on CD74 expression, cells were treated with MIF with different concentrations (0, 10, 20, 40, and 80 ng/ml) for 24 h. Expression of CD74 protein was analyzed by Western blotting. To examine the effect of MIF on cyclin D1 mRNA expression, cells were treated with MIF (100 ng/ ml) for different intervals (0, 6, 12, and 24 h). Expression of cyclin D1 was analyzed by real-time PCR. To evaluate the effect of MIF on the activation of ERK (p-ERK/ERK) and p38 (p-p38/p38), cells were treated with MIF (100 ng/ml) for different intervals (0, 1/6, 1/2, 2, 12, and 24 h). Activation of ERK and p38 was analyzed by Western blotting. To evaluate the effect of ERK and p38 on integrin-β1 and cyclin D1 expression, cells were pretreated with U0126 (10 μM, inhibitor of ERK1/ 2; Calbiochem, San Diego, CA, USA), or SB203580 (10 μM, inhibitor of p38; Calbiochem, San Diego, CA, USA) for 1 h, and then, incubated with MIF 100 ng/ml for 24 h. Expression of integrin-β1 (mRNA and protein) and cyclin D1 (mRNA) were analyzed by real-time PCR and Western blotting. To investigate the effect of MIF (100 ng/ml) and integrin-β1 on cell adhesion, cell adhesion assay was performed following MIF treatment or anti-integrin-β1 antibody (10 μg/ml; Cell signaling Technology, Beverly, Mass, USA) administration to block the interaction between integrin-α3β1 and extracellular matrix in different time intervals.
The method was performed as in our previous report [18]. In brief, cells were plated (40,000 per well) onto 96-well plates coated by collagen type I in serum-free medium with or without MIF (100 ng/ml) for 24 and 48 h at 37 °C. For the assay of adhesion to be affected by the interaction between integrin-α3β1and extracellular matrix, the cells treated with MIF ((100 ng/ml) for 48 h were pre-incubated with antianti-integrin-β1 antibody (10 μg/ml) for 30 min at 37 °C. Non-adherent cells were washed away and adherent cells were stained with crystal violet (0.2 %/20 % methanol). Absorbance was measured at 550 nm using a micro-plate reader (Bio-Rad Laboratories, Hercules, CA). 2.6. Statistical analysis Values are expressed as mean ± SEM. Differences between groups were analyzed by ANOVA followed by Bonferroni post hoc testing, and differences were considered to be statistically significant when P < 0.05. 3. Result 3.1. MIF up-regulates integrin-β1 and cyclin D1expression In the study of diabetic nephropathy, the authors showed that highglucose medium or the inflammatory cytokine TNF-α increased the expression of CD74 mRNA and protein coordinately in cultured podocytes [10]. In our previous study, we found that the expression of cyclin D1 mRNA and protein was coordinate in serum-stimulated podocytes after blocking integrin α3β1–ECM interaction [13]. From above results, we chose only to evaluate the expression of CD74 protein and cyclin D1 in this study. In the analysis of dosage effect of MIF on integrin-β1 expression, podocytes were treated with MIF in the dosages of 0, 10, 20, 50, 100, and 150 ng/ml. The integrin-β1 protein expression was analyzed at 24 h after MIF treatment. Fig. 1A showed that integrin-β1 protein expression increased under MIF treatment in a dosage-dependent manner. However, the MIF receptor (CD74) was not increased after MIF treatment in a dosage-independent manner (Fig. 1B). According above results, we chose MIF 100 ng/ml to treat podocytes for evaluating mRNA expression of integrin-β1and cyclin D1 at different time intervals. The integrin-β1 mRNA expression was increased at 12, 24 and 48 h after MIF treatment in a time-dependent manner (Fig. 1C). Then, cyclin D1 mRNA expression was increased at 6, 12 and 24 h after MIF treatment in a time-dependent manner (Fig. 1D). These results show that MIF up-regulates integrin-β1 and cyclin D1expression through a transcriptional pathway in podocytes.
2.2. Primary culture of pododytes Primary podocyte culture was performed as in our previous studies [13,17]. This study was approved by the Animal Care and Use Committee of Kaohsiung Medical University Affidavit of Approval of Animal Use of Protocol Kaohsiung Medical University, IACUC Approval No: 102218. All procedures were according to the principles of the Declaration of Helsinki and the National Institutes of health guide for the care and use of laboratory animals.
2.3. Real-time PCR The mRNA levels of integrin-β1 and cyclin D1 were analyzed by real-time quantitative PCR as in our previous report [18]. The relative mRNA expression levels of the target genes were normalized using glyceraldehydes-3-phosphate dehydrogenase (GAPDH). PCR was performed with the specific sense and antisense primer sets for rat integrin-β1 (forward primer: 5′-GACCTGCCTTGGTGTCTG TGC-3′, reverse primer: 5′-AGCAACCACACCAGCTACAAT-3′), cyclin D1 (forward primer: 5′-AGAAGTGCGAAGAGGAGGTC-3′, reverse primer: 5′-CTTAGAGGCCACGAACATGC-3′) and GAPDH (forward primer: 5′-CAAGTTCAACGGCACAGTCA-3′, reverse primer: 5′-CCCCATTTGAT GTTAGCGGG-3′). Three primer sets were acquired from Protech Technology Enterprise Co., Ltd. (Taiwan).
3.2. MIF regulates integrin-β1 and cyclin D1 expression through ERK To confirm MIF induced phosphorylation of ERK and p38 in podocytes, we evaluated the activation (phosphorylation) of ERK and p38 after MIF treatment by Western blotting. Cells were treated with MIF 100 ng/ml for 0, 1/6, 1/2, 2, 12, and 24 h. Fig. 2A showed that the activation of ERK and p38 was up-regulated from 1/6–24 h. To evaluate the effect of ERK and p38 on integrin-β1 and cyclin D1 expression, cells 2
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Fig. 1. Integrin-β1, MIF receptor (CD74) and cyclin D1 expression after MIF treatment in podocytes. (A) Dose response of integrin-β1 protein expression stimulated by MIF at 24 h. Cells were incubated with MIF at the dosages of 0, 10, 20, 50, 100, and 150 ng/ml for 24 h. After MIF stimulation, integrin-β1 protein expression was significantly increased at MIF 10 ng/ml. The increased integrin-β1 protein expression then was more at MIF 20 ng/ml than at MIF 10 ng/ml. The integrin-β1 protein expression was not different among MIF 20, 50, 100, and 150 ng/m1. *P < 0.05, compared with control (0 h); **P < 0.05, MIF 20 ng/ml compared with MIF 10 ng/ ml. (B) Dose response of CD74 protein expression after MIF stimulation for 24 h. CD74 protein level was not different among MIF 0, 10, 20, 40, and 80 ng/ml. (C) Cells were incubated with MIF 100 ng/ml for 0, 12, 24 and 48 h. Integrin-β1 mRNA expression had a significant and gradual increase at 12, 24 and 48 h. *P < 0.05, 12 h compared with control (0 h); **P < 0.05, 24 h compared with 12 h; ***P < 0.05, 48 h compared with 24 h. (D) Cells were incubated with MIF 100 ng/ml for 0, 6, 12, and 24 h. The cyclin D1 mRNA expression had a significant and gradual increase at 6, 12, and 24 h. *P < 0.05, compared with control. GAPDH was used as a loading control. Data are presented as means ± SED of three different replications. P values < 0.05 are considered to have statistical significance. β1: integrin-β1.
(inhibiting the activation of integrin-α3β1 blocking the interaction between integrin-β1 and extracellular matrix) decreased MIF-enhanced podocyte adhesion at 48 h (Fig. 3B). These results reveal that MIF enhances podocyte adhesion through up-regulation of integrin-β1 expression and the activation of integrin-α3β1.
were pretreated with U0126 (an inhibitor of ERK1/2), or SB203580 (an inhibitor of p38). Fig. 2B showed that U0126 greatly reduced MIF-induced increase in integrin-β1 protein expression at 24 h following MIF stimulation. SB203580 did not inhibit MIF-induced increase in integrinβ1 protein expression at 24 h following MIF stimulation. U0126 reduced MIF-induced increase in integrin-β1 mRNA expression at 24 h following MIF stimulation (Fig. 2C). However, SB203580 did not reduce MIF-induced increase in integrin-β1 mRNA expression (Fig. 2C). Fig. 2D showed that U0126 greatly reduced MIF-induced increase in cyclin D1 mRNA expression at 24 h following MIF stimulation. SB203580 did not inhibit MIF-induced increase in cyclin D1 mRNA expression at 24 h following MIF stimulation. These results indicate that the up-regulation of integrin-β1 and cyclin D1 expression under MIF stimulation was through the ERK pathway in podocytes.
4. Discussion There was a marked up-regulation of MIF mRNA and protein expression in severe proliferative glomerulonephritis including crescentic glomerulonephritis, mensangiocapillary proliferative glomerulonephritis, and lupus nephritis in human [15]. The up-regulation of MIF expression by glomerular endothelial cells and glomerular epithelial cells (podocytes) in crescentic glomerulonephritis and mensangiocapillary proliferative glomerulonephritis was associated with prominent macrophage accumulation, contributing to the development of glomerular hypercellularity, focal segmental lesions and crescent formation [15]. In patients with mesangioproliferative IgA nephropathy and rapid progressive glomerulonephritis, MIF mRNA expression was increased up to fivefold in microdissected human glomeruli [20]. They also found that podocytes up-regulated their MIF expression and
3.3. MIF promotes podocyte adhesion through integrin-β1 To evaluate the effect of MIF and integrin-β1 on podocyte adhesion, podocytes incubated with MIF and/or anti-integrin-β1 antibody were analyzed by cell adhesion assay. Fig. 3A showed that MIF increased podocyte adhesion at 48 h. However, anti-integrin-β1 antibody 3
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Fig. 2. ERK and p38 regulated integrin-β1 and cyclin D1 expression after MIF stimulation in podocytes. (A) Cells were treated with MIF (100 ng/ml) for 0, 1/6, 1/2, 2, 12 and 24 h. ERK activation increased from 1/6 h–24 h and was the highest at 24 h. p38 activation increased from 1/6 h–24 h and was the highest at 1/6 h. *P < 0.05, compared with control (0 h). In (B), (C) and (D), cells were pretreated with U0126 (10 μM, inhibitor of MEK1/2), or SB203580 (10 μM, inhibitor of p38) for 1 h, and then incubated with MIF 100 ng/ml for 24 h. (B) U0126, but not SB203580, reduced MIF-induced increase in integrin-β1 protein expression at 24 h following MIF stimulation. *P < 0.05, MIF compared with control; **P < 0.05, MIF+U0126 compared with MIF. (C) U0126, but not SB203580, reduced MIFinduced increase in integrin-β1 mRNA expression at 24 h following MIF stimulation. *P < 0.05, MIF compared with control; **P < 0.05, MIF+U0126 compared with MIF. (D) U0126 reduced MIF-induced increase in cyclin D1 mRNA expression at 24 h following MIF stimulation. However, SB203580 did not reduce the MIFinduced increase in cyclin D1 mRNA expression at 24 h following MIF stimulation. U0126 also inhibited only cyclin D1 mRNA expression compared with control. *P < 0.05, MIF compared with control; **P < 0.05, MIF+U0126 compared with MIF; ***P < 0.05, U0126 only compared with control. GAPDH was used as a loading control. Data are presented as means ± SED of three different replications. P value < 0.05 is considered to represent statistical significance. C: normal control groups. β1: integrin-β1.
secretion upon stress both in vitro and in vivo [20]. Urine MIF concentration also increases in patients with proliferative glomerulonephritis, with the greatest increase in patients with crescentic
glomerulonephritis [6]. In an animal transgenetic model of MIF in podocytes, podocytes underwent characteristic changes including cell flattening, contracted foot processes and attenuated synaptopodin [21]. Fig. 3. MIF increased podocyte adhesion through integrin-β1. Podocytes incubated with MIF (100 ng/ml) or anti-integrin-β1 antibody (inhibiting the activation of integrin-α3β1 by the blockade of the interaction between integrin-β1 and extracellular matrix) were analyzed by cell adhesion assay. (A) MIF increased podocyte adhesion at 48 h after MIF treatment. *P < 0.05, 48 h compared with control (0 h). (B) Anti-integrin-β1 antibody inhibited MIFinduced increase in podocyte adhesion at 48 h. *P < 0.05, compared with control; **P < 0.05, MIF+anti-integrin-β1 antibody compared with MIF. Data are presented as means ± SED of three different replications. P value < 0.05 is considered to indicate statistical significance. C: normal control groups; Ab: anti-integrin-β1 antibody. 4
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found that blocking integrin-extracellular matrix interaction and ERK inhibitor (U0126) reduced cyclin D1 mRNA and protein expression [13]. The blocking integrin-extracellular matrix interaction also reduced ERK activation [13]. In the current study, ERK inhibitor (U0126) also only decreased cyclin D1 mRNA expression compared with normal control. This is because integrin-extracellular matrix interaction activates ERK, which promotes cyclin D1 expression. These results suggest that increased cell adhesion through up-regulation of integrins induced by MIF may promote subsequent MIF secretion and cell cycle activation, which may lead to cell proliferation and differentiation/de-defferentiation. This may explain the mechanism of MIF in proliferative glomerulonephritis. In a rat model of diabetes mellitus, the MIF receptor (CD74) was increased in podocytes [10]. Knockdown of CD74 expression by RNAi inhibits proliferation and induces apoptosis in DU-145 prostate cancer cells, and knockdown of MIF expression by RNAi increases CD74 mRNA and protein in DU-145 prostate cancer cells [31]. We found that CD74 was expressed in podocytes and was not up-regulated by MIF. These results suggest that a coordinated regulation of CD74 expression results in receptor regulation. This study had a limitation that integrin-β1 and cyclin D1 was not evaluated in animal models of proliferative glomerulonephritis. In the further study, we will evaluate whether the expression of MIF, integrinβ1 and cyclin D1 is increased coordinately in animal model and human of proliferative glomerulonephritis.
Fig. 4. Schematic model of proposed mechanism of MIF-induced integrin-β1 and cyclin D1 expression.
In addition, MIF secretion was induced by cell adhesion to extracellular matrix in fibroblasts [22]. Integrins mediate cellular adhesion to extracellular medium and act as regulating cell survival, differentiation, proliferation, migration, tissue remodeling and bidirectional cell signaling [23]. MIF increases integrin αvβ3 expression in human chondrosarcoma cells [24]. In human with rapid progressive glomerulonephritis, Baraldi and associates found that the integrin-β1 and -β3 were up-regulated in crescentic cells [25]. In this study, we found that MIF up-regulates the expression of integrin-β1 mRNA and protein in podocytes, demonstrating that MIF increases integrin-β1 expression through regulation of the transcription pathway. Sanchez-Nino and colleagues have shown that MIF activates ERK and p38 in a time- and dosagedependent manner in podocytes [10]. In this study, only the ERK inhibitor (U0126) reduced MIF-induced integrin-β1 mRNA and protein expression. These results indicate that MIF increases integrin-β1 expression mainly through the ERK pathway. Cyclin D1 has a significant role in cell migration, proliferation, differentiation, survival and apoptosis [14,26]. In this study, we found that MIF up-regulated the expression of cyclin D1 mRNA in podocytes and ERK inhibitor (U0126) reduced MIF-up-regulated cyclin D1 mRNA expression. Swant and colleagues also showed that MIF induced cyclin D1 expression in a Rho-, Rho kinase-, MLC kinase- and ERK-dependent manner in asynchronous NIH 3T3 fibroblasts [27]. In an animal model of membranous nephropathy (passive Heymann nephritis) and human immunodeficiency virus (HIV)-transgenic mice, cyclin D1 proteins were found to increase in podocytes [28]. In the anti-Thy 1.1 nephritis animal model of mesangioproliferative nephritis, the number of cyclin D1positive podocytes was significantly increased [29]. These results indicate that cyclin D1-up-regulated by MIF through ERK may induce podocyte proliferation and differentiation/de-differentiation which may play a significant role in proliferative glomerulonephritis. In this study, podocyte adhesion was increased after MIF treatment, and anti-integrin-β1 antibody then decreased MIF-enhanced podocyte adhesion. Silencing of MIF by siRNA reduces cell adhesion in human lung adenocarcinoma [30]. MIF up-regulates integrin-αvβ3 expression in human chondrosarcoma cells and pretreatment with anti-integrinαvβ3 monoclonal antibody inhibits MIF-induced cell migration [24]. Liao and associates reported that cell adhesion to fibronectin stimulated MIF secretion in quiescent mouse fibroblasts [22]. They also found that adhesion-induced release of MIF subsequently promoted integrin-stimulated activation of MAP kinase, cyclin D1 expression and DNA synthesis [22]. In a study of cyclin D1 expression in podocytes, we
5. Conclusions This study shows that MIF up-regulates integrin-β1 and cyclin D1 expression through an ERK pathway in podocytes (Fig. 4). The upregulated expression of integrin-β1 increases podocyte adhesion which enhances cyclinD1 expression. This increased podocyte adhesion may activate MAP kinase and enhance cyclin D1 expression, which may lead to proliferative glomerulonephritis, especially crescentic glomerulonephritis. These results provide further understanding for the role of MIF in developing proliferative glomerulonephritis and offer useful insights into potential approaches for preventing and treating proliferative glomerular nephropathy. Declaration of Competing Interest All authors declare no conflict of interest. Acknowledgements This work was supported in part by Tainan Sinlau Hospital (1040806) and by the Nephrology Laboratory of Kaohsiung Medical University, Kaohsiung, Taiwan. References [1] A. Kudrin, M. Scott, S. Martin, C.W. Chung, R. Donn, A. McMaster, S. Ellison, D. Ray, K. Ray, M. Binks, Human macrophage migration inhibitory factor: a proven immunomodulatory cytokine? J. Biol. Chem. 281 (2006) 29641–29651. [2] L.L. Santos, E.F. Morand, Macrophage migration inhibitory factor: a key cytokine in RA, SLE and atherosclerosis, Clin. Chim. Acta 399 (2009) 1–7. [3] E.F. Morand, M. Leech, J. Bernhagen, MIF: a new cytokine link between rheumatoid arthritis and atherosclerosis, Nat. Rev. Drug Discov. 5 (2006) 399–410. [4] H.Y. Lan, Role of macrophage migration inhibition factor in kidney disease, Nephron Exp. Nephrol. 109 (2008) e79–83. [5] H.Y. Lan, M. Bacher, N. Yang, W. Mu, D.J. Nikolic-Paterson, C. Metz, A. Meinhardt, R. Bucala, R.C. Atkins, The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat, J. Exp. Med. 185 (1997) 1455–1465. [6] F.G. Brown, D.J. Nikolic-Paterson, P.A. Hill, N.M. Isbel, J. Dowling, C.M. Metz, R.C. Atkins, Urine macrophage migration inhibitory factor reflects the severity of renal injury in human glomerulonephritis, J. Am. Soc. Nephrol. 13 (Suppl 1) (2002) S7–13. [7] J.C. Leung, S.C. Tang, L.Y. Chan, A.W. Tsang, H.Y. Lan, K.N. Lai, Polymeric IgA increases the synthesis of macrophage migration inhibitory factor by human
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mesangial cells in IgA nephropathy, Nephrol. Dial. Transplant 18 (2003) 36–45. [8] K. Matsumoto, K. Kanmatsuse, Urinary levels of macrophage migration inhibitory factor in patients with IgA nephropathy, Nephron 92 (2002) 309–315. [9] L. Leng, C.N. Metz, Y. Fang, J. Xu, S. Donnelly, J. Baugh, T. Delohery, Y. Chen, R.A. Mitchell, R. Bucala, MIF signal transduction initiated by binding to CD74, J. Exp. Med. 197 (2003) 1467–1476. [10] M.D. Sanchez-Nino, A.B. Sanz, P. Ihalmo, M. Lassila, H. Holthofer, S. Mezzano, C. Aros, P.H. Groop, M.A. Saleem, P.W. Mathieson, R. Langham, M. Kretzler, V. Nair, K.V. Lemley, R.G. Nelson, E. Mervaala, D. Mattinzoli, M.P. Rastaldi, M. Ruiz-Ortega, J.L. Martin-Ventura, J. Egido, A. Ortiz, The MIF receptor CD74 in diabetic podocyte injury, J. Am. Soc. Nephrol. 20 (2009) 353–362. [11] R.O. Hynes, Integrins: versatility, modulation, and signaling in cell adhesion, Cell 69 (1992) 11–25. [12] K. Kanasaki, Y. Kanda, K. Palmsten, H. Tanjore, S.B. Lee, V.S. Lebleu, V.H. Gattone Jr., R. Kalluri, Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus, Dev. Biol. 313 (2008) 584–593. [13] C.A. Chen, Y.C. Cheng, J.C. Hwang, J.M. Chang, J.Y. Guh, H.C. Chen, Cyclin D1 expression in podocytes: regulated by mitogens in collaboration with integrin-extracellular matrix interaction through extracellular signal-regulated kinase, Exp. Biol. Med. 237 (2012) 516–523. [14] M. Fu, C. Wang, Z. Li, T. Sakamaki, R.G. Pestell, Minireview: Cyclin D1: normal and abnormal functions, Endocrinology 145 (2004) 5439–5447. [15] H.Y. Lan, N. Yang, D.J. Nikolic-Paterson, X.Q. Yu, W. Mu, N.M. Isbel, C.N. Metz, R. Bucala, R.C. Atkins, Expression of macrophage migration inhibitory factor in human glomerulonephritis, Kidney Int. 57 (2000) 499–509. [16] H.Y. Lan, W. Mu, N. Yang, A. Meinhardt, D.J. Nikolic-Paterson, Y.Y. Ng, M. Bacher, R.C. Atkins, R. Bucala, De Novo renal expression of macrophage migration inhibitory factor during the development of rat crescentic glomerulonephritis, Am. J. Pathol. 149 (1996) 1119–1127. [17] C.A. Chen, J.C. Tsai, P.W. Su, Y.H. Lai, H.C. Chen, Signaling and regulatory mechanisms of integrinalpha3beta1 on the apoptosis of cultured rat podocytes, J. Lab. Clin. Med. 147 (2006) 274–280. [18] C.A. Chen, J.M. Chang, E.E. Chang, H.C. Chen, Y.L. Yang, TGF-beta1 modulates podocyte migration by regulating the expression of integrin-beta1 and -beta3 through different signaling pathways, Biomed. Pharmacother. 105 (2018) 974–980. [19] C.A. Chen, J.M. Chang, E.E. Chang, H.C. Chen, Y.L. Yang, Crosstalk between transforming growth factor-beta1 and endoplasmic reticulum stress regulates alpha-
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Macrophage migration inhibitory factor regulates integrin-β1 and cyclin D1 expression via ERK pathway in podocytes
T
Chien-An Chena,b,*, Jer-Ming Changc, Yu-Lin Yangd, Eddy-Essen Changc, Hung-Chun Chenc a
Department of Nephrology, Tainan Sinlau Hospital, Tainan, 701, Taiwan Department of Health Care Administration, Chang Jung Christian University, Tainan, 711, Taiwan c Department of Nephrology, Kaohsiung Medical University, Kaohsiung, 807, Taiwan d Graduate Institute of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, 703, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Macrophage migration inhibitory factor Integrin β1 Podocytes
Aims: Macrophage migration inhibitory factor (MIF) is found to increase in proliferative glomerulonephritis. MIF binds to the MIF receptor (CD74) that activates MAP kinase (ERK and p38). Integrins and cyclinD1 regulate cell proliferation, differentiation and adhesion. This study evaluates whether MIF can regulate integrin-β1/ cyclin D1 expression and cell adhesion of podocytes. Main methods: Expression of integrin-β1 mRNA/protein and cyclin D1 mRNA under stimulation of MIF was evaluated by real-time PCR and Western blotting. MIF receptor (CD74) and MAP kinase under MIF treatment were examined to determine which pathway regulated integrin-β1 and cyclin D1 expression. Cell adhesion was evaluated under MIF treatment and/or anti-integrin-β1 antibody by cell adhesion assay. Key findings: Protein levels of integrin-β1 were up-regulated under MIF treatment in a dosage-dependent manner. CD74 protein levels were not changed after MIF treatment. Integrin-β1 and cyclin D1 mRNA levels were up-regulated after MIF 100 ng/ml treatment. ERK inhibitor U0126 reduced MIF-induced the increase in integrinβ1 mRNA and protein expression following MIF stimulation. However, p38 inhibitor SB 203580 did not inhibit MIF-induced increase in integrin-β1 mRNA and protein expression following MIF stimulation. MIF-induced increase in cyclin D1 mRNA level also was inhibited only by U0126 following MIF stimulation. Podocyte adhesion was increased after MIF treatment, but, anti-integrin-β1 antibody decreased MIF-enhanced podocyte adhesion. Significance: MIF increases integrin-β1 and cyclin D1 expression through the ERK pathway in podocytes, and the up-regulated expression of integrin-β1 increases podocyte adhesion. These results provide further understanding for the role of MIF in developing proliferative glomerulonephritis.
1. Introduction Macrophage migration inhibitory factor (MIF) is a cytokine with pro-inflammatory and immunomodulatory activities, including regulating leukocyte adhesion/infiltration, T-cell activation, cytokine and inflammatory mediator expression, p53 expression, apoptosis, and resident-cell proliferation [1,2]. MIF was originally found to be derived from activated T lymphocytes and monocytes/macrophages [3]. Recent evidences demonstrate that MIF is expressed in many tissues including the kidney where MIF is found in podocytes, mesangial cells, endothelial cells and tubular epithelial cells [1,4,5]. Renal MIF is upregulated in human and experimental glomerulonephritis [4]. Urine excretion of MIF is significantly increased in glomerulonephritis and IgA nephropathy, and correlates with the degree of renal injury [6–8]. MIF receptor, CD74, activates MAP kinase (ERK1/2 and p38 MAPK) in ⁎
a time- and dosage-dependent manner [4,9,10]. In cultured human podocytes, CD 74 is up-regulated by high concentrations of glucose and TNF-α [10]. In clinical and experimental diabetic nephropathy, CD 74 was found to be increased on podocytes [10]. Integrins mediate cells to attach to an extracellular matrix and mediate mechanical and chemical signals from the extracellular matrix to cells or from cells to the extracellular matrix [11]. Integrins regulate cell survival, apoptosis, adhesion, migration, proliferation and differentiation [11]. Integrin-β1 is the major integrin family and expresses on the sole of the foot processes of podocytes [12]. Chen and colleagues demonstrated that integrin-β1-extracellular matrix interaction upregulated cyclin D1 expression in podocytes [13]. Cyclin D1 has a significant role in regulating early-to-mild G1 phase, proliferation/differentiation and survival/ apoptosis [14]. In proliferative glomerulonephritis, MIF increases in glomerulus and
Corresponding author at: Department of Nephrology, Tainan Sinlau Hospital, No. 57, 1 Sec., Dongman Road, Tainan, 701, Taiwan. E-mail address: [email protected] (C.-A. Chen).
https://doi.org/10.1016/j.biopha.2020.109892 Received 23 October 2019; Received in revised form 2 January 2020; Accepted 10 January 2020 0753-3322/ © 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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2.4. Western blot analysis
urine excretion [6,15]. In a rat model of crescentic glomerulonephritis (a form of proliferative glomerulonephritis), MIF expression increases in podocytes, parietal epithelial cells and endothelial cells [16]. In our previous study, we found that mitogens/growth factors in collaboration with integrin-β1 regulated cyclin D1 expression in podocytes [13]. In this study, we find that MIF could regulate integrin-β1 and cyclin D1 expression in podocytes.
2. Materials and methods
Western blot analysis was performed according to our previous report [19]. The following primary antibodies were used: anti-integrin-β1 antibody, rabbit polyclonal anti-ERK antibody, mouse monoclonal antip-ERK antibody, goat polyclonal anti-p38 antibody, mouse monoclonal anti-p-p38 antibody (all from Santa Cruz Biotechnology, CA) and antiCD74 antibody (Abcam, Cambridge, MA, USA). Equality of loading was performed by testing for GAPDH (Sigma). Results are expressed as the ratio (protein of interest/ GAPDH) to correct for sample loading.
2.1. Experimental design
2.5. Cell adhesion assay
To evaluate the effect of MIF on integrin-β1 expression, cells were treated with recombinant mouse MIF (R&D Systems Inc., Minneapolis, MN) with different concentrations (0, 10, 20, 50, 100, and 150 ng/ml) for 24 h, or with MIF (100 ng/ml) for different intervals (0, 12, 24, and 48 h). Expression of integrin-β1 mRNA and protein was analyzed by real-time PCR and Western blotting. To evaluate the effect of MIF on CD74 expression, cells were treated with MIF with different concentrations (0, 10, 20, 40, and 80 ng/ml) for 24 h. Expression of CD74 protein was analyzed by Western blotting. To examine the effect of MIF on cyclin D1 mRNA expression, cells were treated with MIF (100 ng/ ml) for different intervals (0, 6, 12, and 24 h). Expression of cyclin D1 was analyzed by real-time PCR. To evaluate the effect of MIF on the activation of ERK (p-ERK/ERK) and p38 (p-p38/p38), cells were treated with MIF (100 ng/ml) for different intervals (0, 1/6, 1/2, 2, 12, and 24 h). Activation of ERK and p38 was analyzed by Western blotting. To evaluate the effect of ERK and p38 on integrin-β1 and cyclin D1 expression, cells were pretreated with U0126 (10 μM, inhibitor of ERK1/ 2; Calbiochem, San Diego, CA, USA), or SB203580 (10 μM, inhibitor of p38; Calbiochem, San Diego, CA, USA) for 1 h, and then, incubated with MIF 100 ng/ml for 24 h. Expression of integrin-β1 (mRNA and protein) and cyclin D1 (mRNA) were analyzed by real-time PCR and Western blotting. To investigate the effect of MIF (100 ng/ml) and integrin-β1 on cell adhesion, cell adhesion assay was performed following MIF treatment or anti-integrin-β1 antibody (10 μg/ml; Cell signaling Technology, Beverly, Mass, USA) administration to block the interaction between integrin-α3β1 and extracellular matrix in different time intervals.
The method was performed as in our previous report [18]. In brief, cells were plated (40,000 per well) onto 96-well plates coated by collagen type I in serum-free medium with or without MIF (100 ng/ml) for 24 and 48 h at 37 °C. For the assay of adhesion to be affected by the interaction between integrin-α3β1and extracellular matrix, the cells treated with MIF ((100 ng/ml) for 48 h were pre-incubated with antianti-integrin-β1 antibody (10 μg/ml) for 30 min at 37 °C. Non-adherent cells were washed away and adherent cells were stained with crystal violet (0.2 %/20 % methanol). Absorbance was measured at 550 nm using a micro-plate reader (Bio-Rad Laboratories, Hercules, CA). 2.6. Statistical analysis Values are expressed as mean ± SEM. Differences between groups were analyzed by ANOVA followed by Bonferroni post hoc testing, and differences were considered to be statistically significant when P < 0.05. 3. Result 3.1. MIF up-regulates integrin-β1 and cyclin D1expression In the study of diabetic nephropathy, the authors showed that highglucose medium or the inflammatory cytokine TNF-α increased the expression of CD74 mRNA and protein coordinately in cultured podocytes [10]. In our previous study, we found that the expression of cyclin D1 mRNA and protein was coordinate in serum-stimulated podocytes after blocking integrin α3β1–ECM interaction [13]. From above results, we chose only to evaluate the expression of CD74 protein and cyclin D1 in this study. In the analysis of dosage effect of MIF on integrin-β1 expression, podocytes were treated with MIF in the dosages of 0, 10, 20, 50, 100, and 150 ng/ml. The integrin-β1 protein expression was analyzed at 24 h after MIF treatment. Fig. 1A showed that integrin-β1 protein expression increased under MIF treatment in a dosage-dependent manner. However, the MIF receptor (CD74) was not increased after MIF treatment in a dosage-independent manner (Fig. 1B). According above results, we chose MIF 100 ng/ml to treat podocytes for evaluating mRNA expression of integrin-β1and cyclin D1 at different time intervals. The integrin-β1 mRNA expression was increased at 12, 24 and 48 h after MIF treatment in a time-dependent manner (Fig. 1C). Then, cyclin D1 mRNA expression was increased at 6, 12 and 24 h after MIF treatment in a time-dependent manner (Fig. 1D). These results show that MIF up-regulates integrin-β1 and cyclin D1expression through a transcriptional pathway in podocytes.
2.2. Primary culture of pododytes Primary podocyte culture was performed as in our previous studies [13,17]. This study was approved by the Animal Care and Use Committee of Kaohsiung Medical University Affidavit of Approval of Animal Use of Protocol Kaohsiung Medical University, IACUC Approval No: 102218. All procedures were according to the principles of the Declaration of Helsinki and the National Institutes of health guide for the care and use of laboratory animals.
2.3. Real-time PCR The mRNA levels of integrin-β1 and cyclin D1 were analyzed by real-time quantitative PCR as in our previous report [18]. The relative mRNA expression levels of the target genes were normalized using glyceraldehydes-3-phosphate dehydrogenase (GAPDH). PCR was performed with the specific sense and antisense primer sets for rat integrin-β1 (forward primer: 5′-GACCTGCCTTGGTGTCTG TGC-3′, reverse primer: 5′-AGCAACCACACCAGCTACAAT-3′), cyclin D1 (forward primer: 5′-AGAAGTGCGAAGAGGAGGTC-3′, reverse primer: 5′-CTTAGAGGCCACGAACATGC-3′) and GAPDH (forward primer: 5′-CAAGTTCAACGGCACAGTCA-3′, reverse primer: 5′-CCCCATTTGAT GTTAGCGGG-3′). Three primer sets were acquired from Protech Technology Enterprise Co., Ltd. (Taiwan).
3.2. MIF regulates integrin-β1 and cyclin D1 expression through ERK To confirm MIF induced phosphorylation of ERK and p38 in podocytes, we evaluated the activation (phosphorylation) of ERK and p38 after MIF treatment by Western blotting. Cells were treated with MIF 100 ng/ml for 0, 1/6, 1/2, 2, 12, and 24 h. Fig. 2A showed that the activation of ERK and p38 was up-regulated from 1/6–24 h. To evaluate the effect of ERK and p38 on integrin-β1 and cyclin D1 expression, cells 2
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Fig. 1. Integrin-β1, MIF receptor (CD74) and cyclin D1 expression after MIF treatment in podocytes. (A) Dose response of integrin-β1 protein expression stimulated by MIF at 24 h. Cells were incubated with MIF at the dosages of 0, 10, 20, 50, 100, and 150 ng/ml for 24 h. After MIF stimulation, integrin-β1 protein expression was significantly increased at MIF 10 ng/ml. The increased integrin-β1 protein expression then was more at MIF 20 ng/ml than at MIF 10 ng/ml. The integrin-β1 protein expression was not different among MIF 20, 50, 100, and 150 ng/m1. *P < 0.05, compared with control (0 h); **P < 0.05, MIF 20 ng/ml compared with MIF 10 ng/ ml. (B) Dose response of CD74 protein expression after MIF stimulation for 24 h. CD74 protein level was not different among MIF 0, 10, 20, 40, and 80 ng/ml. (C) Cells were incubated with MIF 100 ng/ml for 0, 12, 24 and 48 h. Integrin-β1 mRNA expression had a significant and gradual increase at 12, 24 and 48 h. *P < 0.05, 12 h compared with control (0 h); **P < 0.05, 24 h compared with 12 h; ***P < 0.05, 48 h compared with 24 h. (D) Cells were incubated with MIF 100 ng/ml for 0, 6, 12, and 24 h. The cyclin D1 mRNA expression had a significant and gradual increase at 6, 12, and 24 h. *P < 0.05, compared with control. GAPDH was used as a loading control. Data are presented as means ± SED of three different replications. P values < 0.05 are considered to have statistical significance. β1: integrin-β1.
(inhibiting the activation of integrin-α3β1 blocking the interaction between integrin-β1 and extracellular matrix) decreased MIF-enhanced podocyte adhesion at 48 h (Fig. 3B). These results reveal that MIF enhances podocyte adhesion through up-regulation of integrin-β1 expression and the activation of integrin-α3β1.
were pretreated with U0126 (an inhibitor of ERK1/2), or SB203580 (an inhibitor of p38). Fig. 2B showed that U0126 greatly reduced MIF-induced increase in integrin-β1 protein expression at 24 h following MIF stimulation. SB203580 did not inhibit MIF-induced increase in integrinβ1 protein expression at 24 h following MIF stimulation. U0126 reduced MIF-induced increase in integrin-β1 mRNA expression at 24 h following MIF stimulation (Fig. 2C). However, SB203580 did not reduce MIF-induced increase in integrin-β1 mRNA expression (Fig. 2C). Fig. 2D showed that U0126 greatly reduced MIF-induced increase in cyclin D1 mRNA expression at 24 h following MIF stimulation. SB203580 did not inhibit MIF-induced increase in cyclin D1 mRNA expression at 24 h following MIF stimulation. These results indicate that the up-regulation of integrin-β1 and cyclin D1 expression under MIF stimulation was through the ERK pathway in podocytes.
4. Discussion There was a marked up-regulation of MIF mRNA and protein expression in severe proliferative glomerulonephritis including crescentic glomerulonephritis, mensangiocapillary proliferative glomerulonephritis, and lupus nephritis in human [15]. The up-regulation of MIF expression by glomerular endothelial cells and glomerular epithelial cells (podocytes) in crescentic glomerulonephritis and mensangiocapillary proliferative glomerulonephritis was associated with prominent macrophage accumulation, contributing to the development of glomerular hypercellularity, focal segmental lesions and crescent formation [15]. In patients with mesangioproliferative IgA nephropathy and rapid progressive glomerulonephritis, MIF mRNA expression was increased up to fivefold in microdissected human glomeruli [20]. They also found that podocytes up-regulated their MIF expression and
3.3. MIF promotes podocyte adhesion through integrin-β1 To evaluate the effect of MIF and integrin-β1 on podocyte adhesion, podocytes incubated with MIF and/or anti-integrin-β1 antibody were analyzed by cell adhesion assay. Fig. 3A showed that MIF increased podocyte adhesion at 48 h. However, anti-integrin-β1 antibody 3
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Fig. 2. ERK and p38 regulated integrin-β1 and cyclin D1 expression after MIF stimulation in podocytes. (A) Cells were treated with MIF (100 ng/ml) for 0, 1/6, 1/2, 2, 12 and 24 h. ERK activation increased from 1/6 h–24 h and was the highest at 24 h. p38 activation increased from 1/6 h–24 h and was the highest at 1/6 h. *P < 0.05, compared with control (0 h). In (B), (C) and (D), cells were pretreated with U0126 (10 μM, inhibitor of MEK1/2), or SB203580 (10 μM, inhibitor of p38) for 1 h, and then incubated with MIF 100 ng/ml for 24 h. (B) U0126, but not SB203580, reduced MIF-induced increase in integrin-β1 protein expression at 24 h following MIF stimulation. *P < 0.05, MIF compared with control; **P < 0.05, MIF+U0126 compared with MIF. (C) U0126, but not SB203580, reduced MIFinduced increase in integrin-β1 mRNA expression at 24 h following MIF stimulation. *P < 0.05, MIF compared with control; **P < 0.05, MIF+U0126 compared with MIF. (D) U0126 reduced MIF-induced increase in cyclin D1 mRNA expression at 24 h following MIF stimulation. However, SB203580 did not reduce the MIFinduced increase in cyclin D1 mRNA expression at 24 h following MIF stimulation. U0126 also inhibited only cyclin D1 mRNA expression compared with control. *P < 0.05, MIF compared with control; **P < 0.05, MIF+U0126 compared with MIF; ***P < 0.05, U0126 only compared with control. GAPDH was used as a loading control. Data are presented as means ± SED of three different replications. P value < 0.05 is considered to represent statistical significance. C: normal control groups. β1: integrin-β1.
secretion upon stress both in vitro and in vivo [20]. Urine MIF concentration also increases in patients with proliferative glomerulonephritis, with the greatest increase in patients with crescentic
glomerulonephritis [6]. In an animal transgenetic model of MIF in podocytes, podocytes underwent characteristic changes including cell flattening, contracted foot processes and attenuated synaptopodin [21]. Fig. 3. MIF increased podocyte adhesion through integrin-β1. Podocytes incubated with MIF (100 ng/ml) or anti-integrin-β1 antibody (inhibiting the activation of integrin-α3β1 by the blockade of the interaction between integrin-β1 and extracellular matrix) were analyzed by cell adhesion assay. (A) MIF increased podocyte adhesion at 48 h after MIF treatment. *P < 0.05, 48 h compared with control (0 h). (B) Anti-integrin-β1 antibody inhibited MIFinduced increase in podocyte adhesion at 48 h. *P < 0.05, compared with control; **P < 0.05, MIF+anti-integrin-β1 antibody compared with MIF. Data are presented as means ± SED of three different replications. P value < 0.05 is considered to indicate statistical significance. C: normal control groups; Ab: anti-integrin-β1 antibody. 4
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found that blocking integrin-extracellular matrix interaction and ERK inhibitor (U0126) reduced cyclin D1 mRNA and protein expression [13]. The blocking integrin-extracellular matrix interaction also reduced ERK activation [13]. In the current study, ERK inhibitor (U0126) also only decreased cyclin D1 mRNA expression compared with normal control. This is because integrin-extracellular matrix interaction activates ERK, which promotes cyclin D1 expression. These results suggest that increased cell adhesion through up-regulation of integrins induced by MIF may promote subsequent MIF secretion and cell cycle activation, which may lead to cell proliferation and differentiation/de-defferentiation. This may explain the mechanism of MIF in proliferative glomerulonephritis. In a rat model of diabetes mellitus, the MIF receptor (CD74) was increased in podocytes [10]. Knockdown of CD74 expression by RNAi inhibits proliferation and induces apoptosis in DU-145 prostate cancer cells, and knockdown of MIF expression by RNAi increases CD74 mRNA and protein in DU-145 prostate cancer cells [31]. We found that CD74 was expressed in podocytes and was not up-regulated by MIF. These results suggest that a coordinated regulation of CD74 expression results in receptor regulation. This study had a limitation that integrin-β1 and cyclin D1 was not evaluated in animal models of proliferative glomerulonephritis. In the further study, we will evaluate whether the expression of MIF, integrinβ1 and cyclin D1 is increased coordinately in animal model and human of proliferative glomerulonephritis.
Fig. 4. Schematic model of proposed mechanism of MIF-induced integrin-β1 and cyclin D1 expression.
In addition, MIF secretion was induced by cell adhesion to extracellular matrix in fibroblasts [22]. Integrins mediate cellular adhesion to extracellular medium and act as regulating cell survival, differentiation, proliferation, migration, tissue remodeling and bidirectional cell signaling [23]. MIF increases integrin αvβ3 expression in human chondrosarcoma cells [24]. In human with rapid progressive glomerulonephritis, Baraldi and associates found that the integrin-β1 and -β3 were up-regulated in crescentic cells [25]. In this study, we found that MIF up-regulates the expression of integrin-β1 mRNA and protein in podocytes, demonstrating that MIF increases integrin-β1 expression through regulation of the transcription pathway. Sanchez-Nino and colleagues have shown that MIF activates ERK and p38 in a time- and dosagedependent manner in podocytes [10]. In this study, only the ERK inhibitor (U0126) reduced MIF-induced integrin-β1 mRNA and protein expression. These results indicate that MIF increases integrin-β1 expression mainly through the ERK pathway. Cyclin D1 has a significant role in cell migration, proliferation, differentiation, survival and apoptosis [14,26]. In this study, we found that MIF up-regulated the expression of cyclin D1 mRNA in podocytes and ERK inhibitor (U0126) reduced MIF-up-regulated cyclin D1 mRNA expression. Swant and colleagues also showed that MIF induced cyclin D1 expression in a Rho-, Rho kinase-, MLC kinase- and ERK-dependent manner in asynchronous NIH 3T3 fibroblasts [27]. In an animal model of membranous nephropathy (passive Heymann nephritis) and human immunodeficiency virus (HIV)-transgenic mice, cyclin D1 proteins were found to increase in podocytes [28]. In the anti-Thy 1.1 nephritis animal model of mesangioproliferative nephritis, the number of cyclin D1positive podocytes was significantly increased [29]. These results indicate that cyclin D1-up-regulated by MIF through ERK may induce podocyte proliferation and differentiation/de-differentiation which may play a significant role in proliferative glomerulonephritis. In this study, podocyte adhesion was increased after MIF treatment, and anti-integrin-β1 antibody then decreased MIF-enhanced podocyte adhesion. Silencing of MIF by siRNA reduces cell adhesion in human lung adenocarcinoma [30]. MIF up-regulates integrin-αvβ3 expression in human chondrosarcoma cells and pretreatment with anti-integrinαvβ3 monoclonal antibody inhibits MIF-induced cell migration [24]. Liao and associates reported that cell adhesion to fibronectin stimulated MIF secretion in quiescent mouse fibroblasts [22]. They also found that adhesion-induced release of MIF subsequently promoted integrin-stimulated activation of MAP kinase, cyclin D1 expression and DNA synthesis [22]. In a study of cyclin D1 expression in podocytes, we
5. Conclusions This study shows that MIF up-regulates integrin-β1 and cyclin D1 expression through an ERK pathway in podocytes (Fig. 4). The upregulated expression of integrin-β1 increases podocyte adhesion which enhances cyclinD1 expression. This increased podocyte adhesion may activate MAP kinase and enhance cyclin D1 expression, which may lead to proliferative glomerulonephritis, especially crescentic glomerulonephritis. These results provide further understanding for the role of MIF in developing proliferative glomerulonephritis and offer useful insights into potential approaches for preventing and treating proliferative glomerular nephropathy. Declaration of Competing Interest All authors declare no conflict of interest. Acknowledgements This work was supported in part by Tainan Sinlau Hospital (1040806) and by the Nephrology Laboratory of Kaohsiung Medical University, Kaohsiung, Taiwan. References [1] A. Kudrin, M. Scott, S. Martin, C.W. Chung, R. Donn, A. McMaster, S. Ellison, D. Ray, K. Ray, M. Binks, Human macrophage migration inhibitory factor: a proven immunomodulatory cytokine? J. Biol. Chem. 281 (2006) 29641–29651. [2] L.L. Santos, E.F. Morand, Macrophage migration inhibitory factor: a key cytokine in RA, SLE and atherosclerosis, Clin. Chim. Acta 399 (2009) 1–7. [3] E.F. Morand, M. Leech, J. Bernhagen, MIF: a new cytokine link between rheumatoid arthritis and atherosclerosis, Nat. Rev. Drug Discov. 5 (2006) 399–410. [4] H.Y. Lan, Role of macrophage migration inhibition factor in kidney disease, Nephron Exp. Nephrol. 109 (2008) e79–83. [5] H.Y. Lan, M. Bacher, N. Yang, W. Mu, D.J. Nikolic-Paterson, C. Metz, A. Meinhardt, R. Bucala, R.C. Atkins, The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat, J. Exp. Med. 185 (1997) 1455–1465. [6] F.G. Brown, D.J. Nikolic-Paterson, P.A. Hill, N.M. Isbel, J. Dowling, C.M. Metz, R.C. Atkins, Urine macrophage migration inhibitory factor reflects the severity of renal injury in human glomerulonephritis, J. Am. Soc. Nephrol. 13 (Suppl 1) (2002) S7–13. [7] J.C. Leung, S.C. Tang, L.Y. Chan, A.W. Tsang, H.Y. Lan, K.N. Lai, Polymeric IgA increases the synthesis of macrophage migration inhibitory factor by human
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