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ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex.

Zhang Q, Yang Z, Jia Z, Liu C, Guo C, Lu H, Chen P, Ma K, Wang W, Zhou C - Mol. Cancer (2014)

Bottom Line: Recently, ISL-1 has been found in some types of human cancers.Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples.Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, 38 Xueyuan Road, 100191 Beijing, China. wwp@bjmu.edu.cn.

ABSTRACT

Background: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear.

Methods and results: The expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL.

Conclusions: Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

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p-STAT3/p-c-Jun/ ISL-1 forms a transcriptional complex and binds directly to ISL-1 promoter. (A) Consensus binding sites for p-STAT3 and p-c-Jun on the ISL-1 promoter were analyzed by Matinspector software. (B) The luciferase activity of ISL-1-luc was analyzed by luciferase reporter assay in Ly3 cells after treated with IL-6 (4 ng/ml), STATTIC (6 μM), Anisomycin (15 ng/ml) or SP600125 (10 μM) for 24 h. (C) ChIP assay was performed with anti-p-STAT3 Ab (left panel) or anti-p-c-Jun Ab (right panel) for immunoprecipitation using chromatin harvested from Ly3 cells. The DNA extractions were amplified using the primers that cover the p-STAT3 (primers 2) or p-c-Jun (primers 4) binding sites, or control primers (primers 1, 3) on the ISL-1 promoter by real-time PCR with normal IgG as a control. (D) Co-IP assay was performed in Ly3 for the transcriptional complex recruited on the ISL-1 promoter. (E) ChIP-re-IP assay was performed first with anti-ISL-1 Ab or rabbit IgG Ab and then with anti-p-STAT3, anti-p-c-Jun or IgG Abs for immunoprecipitation using chromatin harvested from Ly3 cells. (F) The transcriptional activity of ISL-1 on ISL-1-luc was analyzed in Ly3 cells by luciferase reporter assay. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01 vs. the control).
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Figure 8: p-STAT3/p-c-Jun/ ISL-1 forms a transcriptional complex and binds directly to ISL-1 promoter. (A) Consensus binding sites for p-STAT3 and p-c-Jun on the ISL-1 promoter were analyzed by Matinspector software. (B) The luciferase activity of ISL-1-luc was analyzed by luciferase reporter assay in Ly3 cells after treated with IL-6 (4 ng/ml), STATTIC (6 μM), Anisomycin (15 ng/ml) or SP600125 (10 μM) for 24 h. (C) ChIP assay was performed with anti-p-STAT3 Ab (left panel) or anti-p-c-Jun Ab (right panel) for immunoprecipitation using chromatin harvested from Ly3 cells. The DNA extractions were amplified using the primers that cover the p-STAT3 (primers 2) or p-c-Jun (primers 4) binding sites, or control primers (primers 1, 3) on the ISL-1 promoter by real-time PCR with normal IgG as a control. (D) Co-IP assay was performed in Ly3 for the transcriptional complex recruited on the ISL-1 promoter. (E) ChIP-re-IP assay was performed first with anti-ISL-1 Ab or rabbit IgG Ab and then with anti-p-STAT3, anti-p-c-Jun or IgG Abs for immunoprecipitation using chromatin harvested from Ly3 cells. (F) The transcriptional activity of ISL-1 on ISL-1-luc was analyzed in Ly3 cells by luciferase reporter assay. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01 vs. the control).

Mentions: The above results have shown that p-STAT3 and p-c-Jun could increase the expression level of ISL-1 to promote the proliferation of NHL cells. However, it is unknown how p-STAT3 and p-c-Jun control ISL-1 expression. Bioinformatic analysis showed that the core transcriptional regulatory region of ISL-1 (-1000 ~ ATG)[30] contains conserved p-STAT3 and p-c-Jun binding sites (Figure 8A). Luciferase assay with ISL-1-luc, a ISL-1 luciferase reporter construct containing the binding site of c-Jun and STAT3, was performed in Ly3 cells treated with IL-6/STATTIC (JAK/STAT signaling activator/inhibitor) or Anisomycin/SP600125 (JNK signaling activator/inhibitor), respectively. As shown in Figure 8B, ISL-1-luc activity was increased in Anisomycin or IL-6 treated cells. Whereas a significant decrease of ISL-1-luc activity could be observed after SP600125 or STATTIC treatment. These results further demonstrate that both JNK and JAK/STAT signaling pathways are able to activate the ISL-1 transcription effectively.To confirm whether p-STAT3 and p-c-Jun bind to the ISL-1 regulatory region, a set of primers covering the ISL-1 promoter region between -994 and -216 were designed for real-time PCR in ChIP assay (Figure 8A). The ChIP analysis showed that p-STAT3 was recruited to the region of ISL-1 promoter covered by primer 2 (-790 ~ -630) by approximately 12 folds (Figure 8C left panel), and p-c-Jun was recruited to the region of ISL-1 promoter covered by primer 4 (-397 ~ -216) by about 6 folds (Figure 8C right panel), respectively, as compared with primer 1 (-994 ~ -772) as the control. Interestingly, we also observed magnificent enrichment of p-STAT3 at the p-c-Jun binding region (primer 4 covered region, Figure 8C left panel), p-c-Jun at the p-STAT3 binding region (primer 2 covered region, Figure 8C right panel), and both p-STAT3 and p-c-Jun at the primer 3 (-613 ~ -373) covered region (Figure 8C left and right panel). Therefore, we suppose that p-STAT3 possibly cooperate with p-c-Jun and synergistically regulate ISL-1 expression in NHL cells.


ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex.

Zhang Q, Yang Z, Jia Z, Liu C, Guo C, Lu H, Chen P, Ma K, Wang W, Zhou C - Mol. Cancer (2014)

p-STAT3/p-c-Jun/ ISL-1 forms a transcriptional complex and binds directly to ISL-1 promoter. (A) Consensus binding sites for p-STAT3 and p-c-Jun on the ISL-1 promoter were analyzed by Matinspector software. (B) The luciferase activity of ISL-1-luc was analyzed by luciferase reporter assay in Ly3 cells after treated with IL-6 (4 ng/ml), STATTIC (6 μM), Anisomycin (15 ng/ml) or SP600125 (10 μM) for 24 h. (C) ChIP assay was performed with anti-p-STAT3 Ab (left panel) or anti-p-c-Jun Ab (right panel) for immunoprecipitation using chromatin harvested from Ly3 cells. The DNA extractions were amplified using the primers that cover the p-STAT3 (primers 2) or p-c-Jun (primers 4) binding sites, or control primers (primers 1, 3) on the ISL-1 promoter by real-time PCR with normal IgG as a control. (D) Co-IP assay was performed in Ly3 for the transcriptional complex recruited on the ISL-1 promoter. (E) ChIP-re-IP assay was performed first with anti-ISL-1 Ab or rabbit IgG Ab and then with anti-p-STAT3, anti-p-c-Jun or IgG Abs for immunoprecipitation using chromatin harvested from Ly3 cells. (F) The transcriptional activity of ISL-1 on ISL-1-luc was analyzed in Ly3 cells by luciferase reporter assay. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01 vs. the control).
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Figure 8: p-STAT3/p-c-Jun/ ISL-1 forms a transcriptional complex and binds directly to ISL-1 promoter. (A) Consensus binding sites for p-STAT3 and p-c-Jun on the ISL-1 promoter were analyzed by Matinspector software. (B) The luciferase activity of ISL-1-luc was analyzed by luciferase reporter assay in Ly3 cells after treated with IL-6 (4 ng/ml), STATTIC (6 μM), Anisomycin (15 ng/ml) or SP600125 (10 μM) for 24 h. (C) ChIP assay was performed with anti-p-STAT3 Ab (left panel) or anti-p-c-Jun Ab (right panel) for immunoprecipitation using chromatin harvested from Ly3 cells. The DNA extractions were amplified using the primers that cover the p-STAT3 (primers 2) or p-c-Jun (primers 4) binding sites, or control primers (primers 1, 3) on the ISL-1 promoter by real-time PCR with normal IgG as a control. (D) Co-IP assay was performed in Ly3 for the transcriptional complex recruited on the ISL-1 promoter. (E) ChIP-re-IP assay was performed first with anti-ISL-1 Ab or rabbit IgG Ab and then with anti-p-STAT3, anti-p-c-Jun or IgG Abs for immunoprecipitation using chromatin harvested from Ly3 cells. (F) The transcriptional activity of ISL-1 on ISL-1-luc was analyzed in Ly3 cells by luciferase reporter assay. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01 vs. the control).
Mentions: The above results have shown that p-STAT3 and p-c-Jun could increase the expression level of ISL-1 to promote the proliferation of NHL cells. However, it is unknown how p-STAT3 and p-c-Jun control ISL-1 expression. Bioinformatic analysis showed that the core transcriptional regulatory region of ISL-1 (-1000 ~ ATG)[30] contains conserved p-STAT3 and p-c-Jun binding sites (Figure 8A). Luciferase assay with ISL-1-luc, a ISL-1 luciferase reporter construct containing the binding site of c-Jun and STAT3, was performed in Ly3 cells treated with IL-6/STATTIC (JAK/STAT signaling activator/inhibitor) or Anisomycin/SP600125 (JNK signaling activator/inhibitor), respectively. As shown in Figure 8B, ISL-1-luc activity was increased in Anisomycin or IL-6 treated cells. Whereas a significant decrease of ISL-1-luc activity could be observed after SP600125 or STATTIC treatment. These results further demonstrate that both JNK and JAK/STAT signaling pathways are able to activate the ISL-1 transcription effectively.To confirm whether p-STAT3 and p-c-Jun bind to the ISL-1 regulatory region, a set of primers covering the ISL-1 promoter region between -994 and -216 were designed for real-time PCR in ChIP assay (Figure 8A). The ChIP analysis showed that p-STAT3 was recruited to the region of ISL-1 promoter covered by primer 2 (-790 ~ -630) by approximately 12 folds (Figure 8C left panel), and p-c-Jun was recruited to the region of ISL-1 promoter covered by primer 4 (-397 ~ -216) by about 6 folds (Figure 8C right panel), respectively, as compared with primer 1 (-994 ~ -772) as the control. Interestingly, we also observed magnificent enrichment of p-STAT3 at the p-c-Jun binding region (primer 4 covered region, Figure 8C left panel), p-c-Jun at the p-STAT3 binding region (primer 2 covered region, Figure 8C right panel), and both p-STAT3 and p-c-Jun at the primer 3 (-613 ~ -373) covered region (Figure 8C left and right panel). Therefore, we suppose that p-STAT3 possibly cooperate with p-c-Jun and synergistically regulate ISL-1 expression in NHL cells.

Bottom Line: Recently, ISL-1 has been found in some types of human cancers.Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples.Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, 38 Xueyuan Road, 100191 Beijing, China. wwp@bjmu.edu.cn.

ABSTRACT

Background: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear.

Methods and results: The expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL.

Conclusions: Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

Show MeSH
Related in: MedlinePlus