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. 2010 Feb 6:11:14.
doi: 10.1186/1471-2199-11-14.

Transcription factors Elk-1 and SRF are engaged in IL1-dependent regulation of ZC3H12A expression

Aneta Kasza  1 , Paulina Wyrzykowska, Irena Horwacik, Piotr Tymoszuk, Danuta Mizgalska, Karren Palmer, Hanna Rokita, Andrew D Sharrocks, Jolanta Jura
Affiliations

Transcription factors Elk-1 and SRF are engaged in IL1-dependent regulation of ZC3H12A expression

Aneta Kasza et al. BMC Mol Biol. .

Abstract

Background: MCPIP is a novel CCCH zinc finger protein described as an RNase engaged in the regulation of immune responses. The regulation of expression of the gene coding for MCPIP - ZC3H12A is poorly explored.

Results: Here we report that the proinflammatory cytokine IL-1beta rapidly induces the synthesis of MCPIP in primary monocyte-derived macrophages and HepG2 cells. This up-regulation takes place through the MAP kinase pathway and following activation of the transcription factor Elk-1. Using a ZC3H12A reporter construct we have shown that a ZC3H12A promoter region, stretching from -76 to +60, mediates activation by IL-1beta. This region contains binding sites for Elk-1 and its partner SRF. Chromatin immunoprecipitation analysis confirms in vivo binding of both transcription factors to this region of the ZC3H12A promoter.

Conclusions: We conclude that the transcription factor Elk-1 plays an important role in the activation of ZC3H12A expression in response to IL-1beta stimulation.

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Figures

Figure 1
Figure 1
Regulation of expression of human MCPIP. A, control MOCK cells and B, mIκB cells with disrupted activation of NFκB were treated with IL-1β (15 ng/ml) and PMA (100 nM). To some experimental groups U0126 (10 μM) was added 30 min prior IL-1β stimulation (lanes 5-8 (A and B)). RNA was isolated after 2 h and subjected to northern blot analysis using MCPIP cDNA (top panel) or GAPDH cDNA (middle panel) as probes. Bottom panel - activation of ERK in MOCK and mIkB cells by IL-1β or its inhibition by U0126 was checked by western blot analysis. Cells were stimulated by IL-1β (15 ng/ml) for 30 min (line 2 and 4). To some experimental groups U0126 (10 μM) was added 30 min prior IL-1β stimulation (lanes 3-4). Line 1 - lysate from control cells. C, human macrophages were treated with IL-1β (15 ng/ml) with or without U0126 (10 μM, 30 min prior IL-1β stimulation). Changes in ZC3H12A expression were measured by Real Time PCR. The results are means ± SD of three independent experiments.
Figure 2
Figure 2
Regulation of activation of 136 bp length ZC3H12A promoter fragment. Luciferase activity was measured to examine the regulation of activation of 136 bp length ZC3H12A promoter fragment. HepG2 cells transiently transfected with the luciferase construct containing the fragment of human ZC3H12A promoter located between -76 bp and +60 bp. Increasing amounts of pElk-En (20, 50, 100, 200 ng - lanes 2, 3, 4, 5) or Elk-VP16 (20, 50, 100, 200 ng - lanes 6, 7, 8, 9) were co-transfected to the cells. Lane 1 - control cells without Elk-En or Elk-VP16. Luciferase activity was measured 24 h after transfection. Statistical significance was determined using the Student's test. *P < 0.05, **P < 0.001.
Figure 3
Figure 3
SRF and Elk-1 bind to the ZC3H12A promoter. A, Gel retardation assay with a fragment of ZC3H12A promoter (-95 - +67) containing the WT sequence or a mutated CArG box (ZC3H12A-mut) or a fragment of c-Fos promoter. The DNA was incubated with coreSRF. B and C, Chromatin immunoprecipitation of the transcription factors Elk-1 (B) and SRF (C) bound to the ZC3H12A promoter. HepG2 cells were incubated in 0.5% FCS medium for 12 hrs and then stimulated for 30 min with IL-1β. The antibodies anti-Elk-1 (B) or anti-SRF (C) were used to immunoprecipitate sonicated chromatin. As a negative control non-specific IgG were employed. Eluted DNA served as a template in PCR analysis with oligonucleotides specific for ZC3H12A promoter (top panel B and C) or EGR promoter (bottom panel B and C). 1% of input DNA is shown in lines 5 and 6. The panels show the inverted images of ethidium bromide-stained gels.
Figure 4
Figure 4
IL-1β causes ERK-dependent phosphorylation of Elk-1 on ZC3H12A promoter. A, Activation of Elk-1 after IL-1β stimulation. HepG2 cells were stimulated by IL-1β (15 ng/ml) for 5, 10 and 15 min and the phosphorylation of Elk-1 was analyzed by western blot analysis. To some experimental groups U0126 (10 μM) was added 30 min prior IL-1β stimulation (lanes 5-8). Asterisks indicate unspecific bands. B, Chromatin immunoprecipitation of the phosphorylated Elk-1 bound to the ZC3H12A promoter. HepG2 cells were incubated in 0.5% FCS medium for 12 hrs and then stimulated for 15, 30 or 90 min with IL-1β. The antibodies anti-P-Elk were used to immunoprecipitate sonicated chromatin. As a negative control non-specific IgG were employed. Eluted DNA served as a template in PCR analysis with oligonucleotides specific for ZC3H12A promoter. 1% of input DNA is shown in lines 9-12. The panels show the inverted images of ethidium bromide-stained gels.
Figure 5
Figure 5
Regulation of activation of 136 bp length ZC3H12A promoter fragment. Luciferase activity was measured to examine the regulation of activation of 136 bp length ZC3H12A promoter fragment. HepG2 cells were transiently transfected with the luciferase construct containing the fragment of human ZC3H12A promoter located between -76 bp and +60 bp. 24 hrs after transfection cells were stimulated with IL-1β or/and PMA for 3 hrs. An inhibitor U0126 was added to cells 30 min prior stimulation. Statistical significance was determined using the Student's test. *P < 0.05, **P < 0.001. Bottom panel - activation of ERK by IL-1β or its inhibition by U0126 was checked by western blot analysis Cells were stimulated by IL-1β (15 ng/ml) for 30 min (line 2 and 4). To some experimental groups U0126 (10 μM) was added 30 min prior IL-1β stimulation (lanes 3-4). Line 1 - lysate from control cells.
Figure 6
Figure 6
Mutation of both ets-binding site and CArG box abolished the activation of 136 bp length ZC3H12A promoter fragment. Luciferase activity was measured to examine the regulation of 136 bp length ZC3H12A promoter fragment. HepG2 cells were transiently transfected with the luciferase construct containing the fragment of human ZC3H12A promoter located between -76 bp and +60 bp. Black bars - wild type promoter, striped bars - promoter with the mutation in both ets-binding site and in CArG box. A, 24 h after transfection cells were stimulated with IL-1β and PMA for 3 hrs. B, Cells were co-transfected with increasing amounts of Elk-VP16 (20, 50 ng). Luciferase activity was measured 24 h after transfection. C, Influence of mutation of ets-binding site or CArG box on the activation of 136 bp by Elk-1. Black bars represent wild type promoter, dotted bars represent promoter with the mutation in ets-binding site, light bars represent promoter with the mutation in CArG box. Cells were co-transfected with increasing amounts of Elk-VP16 (10, 20, 50 ng). Luciferase activity was measured 24 h after transfection. Statistical significance was determined using the Student's test. *P < 0.05, **P < 0.001.
Figure 7
Figure 7
Schematic diagram of regulation of the ZC3H12A promoter by IL-1β. IL-1RI - type I IL-1 receptor, IL-1R AcP - IL-1 receptor accessory protein. The main sequences responsible for binding of NFκB are located within the second intron of ZC3H12A promoter [10].
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