- Email: [email protected]
microRNA and Human Inducible Nitric Oxide Synthase
CHAPTER TWO
microRNA and Human Inducible Nitric Oxide Synthase Zhong Guo, David A. Geller1 Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Regulation of Human iNOS Gene 2.1 Transcriptional 2.2 Posttranscriptional 3. miRNAs Regulation 3.1 miR-939 3.2 other miRNAs 4. Conclusion References
19 20 20 21 21 22 23 25 25
Abstract Regulation of human inducible nitric oxide synthase (iNOS) expression involves both transcriptional and posttranscriptional mechanisms. Human iNOS gene transcription is controlled in a cell type-specific manner by extracellular cytokines. Transcriptional regulation of human iNOS gene involves transcription factors NF-κB, Stat-1, AP-1, C/EBPβ, KLF6, Oct 1, and NRF. Important posttranscriptional mechanisms also regulate human iNOS mRNA stability through RNA binding proteins HuR, TTP, KSRP, and PABP. Recently, there are several miRNAs that were validated to regulate human and rodent iNOS gene expression. Among them, miR-939 and miR-26a were identified to bind with the human iNOS 30 -UTR and exert a translational blockade of human iNOS protein synthesis.
1. INTRODUCTION The expression of inducible nitric oxide synthase (iNOS) can be induced in various cells and tissues following exposure to immunologic and inflammatory stimuli such as cytokines or lipopolysaccharide (LPS) (Kleinert, Pautz, Linker, & Schwarz, 2004). Human iNOS expression in primary human hepatocytes was originally identified by stimulating with a Vitamins and Hormones, Volume 96 ISSN 0083-6729 http://dx.doi.org/10.1016/B978-0-12-800254-4.00002-7
#
2014 Elsevier Inc. All rights reserved.
19
20
Zhong Guo and David A. Geller
cytokine mixture (CM) of TNF-α, IL-1β, IFN-γ, and LPS (Nussler et al., 1992). Subsequently, the human iNOS cDNA was cloned from LPS and cytokine-stimulated primary human hepatocytes (Geller et al., 1993). This cloned gene reveals a 4145-bp cDNA, containing a 3459-bp open reading frame that encodes 1153 amino acids with a 131-kDa molecular mass. The genomic cloning of human iNOS clarified that the completed human iNOS gene consists of 26 exons and 25 introns spanning 37 kb of genomic region and specifically mapped to chromosome 17 at position 17 cen-q11.2 (Chartrain et al., 1994).
2. REGULATION OF HUMAN iNOS GENE 2.1. Transcriptional The expression of human iNOS is controlled in large part by transcriptional mechanisms. The functional promoter region of human iNOS gene can be extended to 16 kb (deVera et al., 1996). The validated binding sites for numerous transcription factors such as AP-1, C/EBP, CREB, GATA, HIF, IRF-1, NF-AT, NF-κB, NF-IL6, Oct-1, PARP1, p53, Sp1, KLF6, STAT-1α, and YY1 (Pautz et al., 2010). There is a classical cytokineinduced enhancer between 5 and 6 kb of human iNOS promoter (Guo, Shao, Du, Park, & Geller, 2007). The human iNOS core promoter contains a TATA box about 30 bp from the transcription start site. Near the TATA box, it also contains binding sites for the transcription factors NF-κB and C/EBP β (Pautz et al., 2010). In human AKN-1, A549, or DLD1 cells, only human iNOS promoter fragments larger than 3.8 kb showed any significant induction with cytokines (deVera et al., 1996; Taylor et al., 1998). We have further shown that TNF-α or IL-1β signal through NF-κB binding to 5.5, 5.8, and 6.1 kb cis-acting DNA elements, while IFN-γ signals through Stat-1 by binding to motifs at 5.2 and 5.8 kb in the human iNOS promoter (Ganster, Taylor, Shao, & Geller, 2001). Another group showed that cytokine-responsiveness required 50 -flanking DNA regions extending to 8 kb and demonstrated CM-inducible activating protein (AP)-1 binding sites at 5.1 and 5.3 kb as well as a functional role for a NF-κB element located at 8.2 kb in the human iNOS promoter (Marks-Konczalik, Chu, & Moss, 1998). Interestingly, NF-κB repressing factor protein binding to a negative response element at 6.7 kb in the human iNOS promoter region was recognized to mediate constitutive silencing of human iNOS transcription (Feng et al., 2002). A far-upstream functional Oct-1 motif was also identified at
microRNA and Human Inducible Nitric Oxide Synthase
21
10.2 kb in the human iNOS promoter that regulates cytokine-induced human iNOS gene transcription (Park, Guo, Shao, Du, & Geller, 2009). The induction of hiNOS expression requires a CM including TNF-α, IL-1β, and IFN-γ (Taylor & Geller, 2000). Activation of NF-κB pathway, JAK2–STAT-1α pathway, and MAPK pathways (p42/44 MAPK, p38MAPK, JNK) are important for hiNOS expression (Ganster & Geller, 2000; Kleinert et al., 2004).
2.2. Posttranscriptional RNA binding proteins (RNA-BP) HuR, TTP, and KSRP have been shown to be involved in the posttranscriptional mechanisms of human iNOS regulation (Pautz et al., 2010). Cytokine induction can result in in vitro binding of HuR to the iNOS mRNA and thus leads to iNOS mRNA stabilization (Linker et al., 2005). The KH-type splicing regulatory protein (KSRP) showed the destabilizing effect on human iNOS mRNA expression as a human iNOS 30 -UTR binding protein (Linker et al., 2005). The zinc-finger protein tristetraprolin (TTP) regulates iNOS mRNA expression indirect via an interaction with KSRP. Cytokine induction can enhance TTP–KSRP interaction and this results in reduced in vitro binding of KSRP to the human iNOS mRNA (Linker et al., 2005). The cytosolic poly (A)-binding protein (PABP) is recently identified to bind to the 50 -UTR and 30 -UTR of hiNOS mRNA and to posttranscriptionally enhance human iNOS mRNA stability (Casper et al., 2013). Besides HuR, TTP, KSRP, and PABP, other RNA-BP such as hnRNP A1, PTB, and TIAR can also increase human iNOS expression by enhancement of iNOS mRNA stability. Noteworthy, the ARE/poly (U)-binding/ degradation factor 1 (AUF1, also named hnRNP D), seems to regulate human iNOS expression by modulation of iNOS mRNA stability, whereas all four isoforms of the ARE/poly (U)-binding/degradation factor 1 (AUF1) negatively regulate human iNOS expression by destabilization of the mRNA (Pautz et al., 2010).
3. miRNAs REGULATION microRNAs (miRNA) are short (21) nucleotides that are complementary to 30 -UTR mRNA sequences and have been widely reported to regulate human gene expression (Zeng, Yi, & Cullen, 2003; John et al., 2004).
22
Zhong Guo and David A. Geller
In primary human hepatocytes, cytokines induce human iNOS mRNA, protein, and NO synthesis. In contrast, primary human macrophages as well as certain human tumor cell lines demonstrate iNOS mRNA following cytokine stimulation, yet human iNOS protein is difficult to detect (Chan et al., 2005). Likewise, in primary human cardiac myocytes, human iNOS mRNA was readily seen after cytokine stimulation, but iNOS protein and NO synthesis were not identified (Luss et al., 1997). These observations are consistent with a translational blockade and suggest the possible role of miRNA in exerting negative posttranscriptional regulation.
3.1. miR-939 miR-939 was originally cloned from human cervical cancer cells (Lui, Pourmand, Patterson, & Fire, 2007). It has two mature forms: hsa-miR939-5p and hsa-miR-939-3p. It is located at 8q24.3 genomic locus and is also imbedded in cleavage and polyadenylation specific factor 1 (CPSF1) gene. One group reported that miR-939 regulates the replication of H1N1 influenza virus in MDCK cells (Liu, Song, & Huang, 2010). We identified adjacent miR-939 binding sites in the hiNOS 30 -UTR and further proved that miR-939 binds in vitro and in vivo to exert a translational blockade of cytokine-stimulated hiNOS protein expression in primary human hepatocytes (Guo et al., 2012). The hiNOS 30 -UTR confers posttranscriptional repression of basal and cytokine-induced hiNOS transcriptional activity in an orientationdependent manner. miRNA-939 decreases cytokine-induced hiNOS protein expression, but does not affect hiNOS mRNA levels or hiNOS mRNA stability. The hiNOS 30 -UTR contains two functional miR-939 binding sites at +99 and +112 base pairs that are critical for miR-939 mediated translational blockade. Cytokines can induce endogenous miR-939 expression and consequently, miR-939/hiNOS mRNA complex in human hepatocytes. Therefore, we proposed a model regarding the functional role of miR-939 in the posttranscriptional regulation of the iNOS gene in human hepatocytes (Fig. 2.1). Human primary hepatocytes can be stimulated by a combination of cytokines (TNF-α, IL-1β, and IFN-γ) to strongly express human iNOS mRNA. The cytokines activate specific transcription factors NF-κB, Stat-1, AP1, and C/EBPβ, which functionally interact with their corresponding cis-acting DNA binding sites to drive human iNOS transcription. Meanwhile, the same cytokines also increase miR-939 levels
23
microRNA and Human Inducible Nitric Oxide Synthase
miRNA-939 represses human iNOS gene translation TNF α, IL 1β, IFN γ, LPS
NFκB,AP 1,Stats
hiNOS Gene
?
hiNOS mRNA
miR-939, ?
hiNOS Protein
NO
HiNOS 3¢-UTR (1 – 496 bp)
hiNOS promoter
hiNOS gene
+99 112 bp AAA 939 939 miRNA binding sites
Figure 2.1 Proposed model of human iNOS gene regulation in human hepatocytes with miR-939 exerting a translational blockade for iNOS protein synthesis by binding to the iNOS 30 -UTR.
which bind to two specific miR-939 binding sites in the human iNOS 30 -UTR leading to translational inhibition of CM-induced human iNOS protein expression. Binding to both sites is likely required for maximal translational repression, because mutation of either binding site partially abrogates the inhibitory effect of exogenous miR-939 on luciferase activity. Cytokine induction of a negative miRNA regulator of human iNOS expression would theoretically serve to protect the host against untoward consequences of prolonged human iNOS overexpression in a check-and-balance system.
3.2. other miRNAs Besides miR-939, miR-146a was also reported to regulate human iNOS gene (Table 2.1). However, unlike direct binding of miR-939 to the human iNOS 30 -UTR, miR-146a was shown to be indirectly involved in the regulation of iNOS gene expression. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS, and ion channel, TRPV1) in human glial cells (Li et al., 2011). Recently, miR-26a was identified to suppress the expression of iNOS protein at the posttranscriptional level by interacting with 30 -UTR in cancer cells (Zhu et al., 2013). There are at least four miRNAs that have been validated to regulate rodent iNOS genes (Table 2.1). Dai et al. reported that miR-146a, a negative regulator of Toll-like receptor (TLR) signaling, was decreased in
24
Zhong Guo and David A. Geller
Table 2.1 Summary of miRNAs in regulating iNOS genes in different species Specie miRNA Regulation Mechanism References
Human miR-939
Down
Translational blockage by binding 30 -UTR
Guo et al. (2012)
miR-146a Down
Modulating inflammatory cytokines
Li et al. (2011)
miR-26a
Translational blockage by binding 30 -UTR
Zhu et al. (2013)
Negative regulator of Toll-like receptor (TLR) signaling
Dai et al. (2008)
Down
Mouse miR-146a Down
Rat
miR-155
Up
Inhibition of cytokine signal (SOCS)-1
Wang et al. (2009)
miR-27b
Up
Targeting KSRP to increase iNOS mRNA stability
Zhou, Gong, Eischeid, and Chen (2012)
miR-34b
Down
Its inhibitor prevents IL-1β Abouheif et al. induced iNOS gene (2010)
freshly isolated splenic lymphocytes from estrogen-treated mice. Increasing the activity of miR-146a significantly inhibited LPS-induced IFN-γ and iNOS expression in mouse splenic lymphocytes. Enhancing the activity of miR-146a also inhibited the expression of LPS-induced iNOS and NO (Dai et al., 2008). Additionally, a recent report indicates that miR155 expression was increased in MKP-1-deficient macrophages compared with wild-type macrophages. Transfect on of miR-155 attenuated the expression of Suppressor of Cytokine Signal (SOCS)-1 and subsequently enhanced the expression of iNOS (Wang et al., 2009). miR-27b regulates the stabilization of iNOS mRNA through targeting KSRP (Zhou et al., 2012). The expression of miR-34a was significantly upregulated by IL-1β. Silencing of miR-34a significantly prevented IL-1β-induced downregulation of Col2a1, as well as IL-1β-induced upregulation of rat iNOS gene (Abouheif et al., 2010). These studies indicate that specific miRNAs (miR-155, miR-146a, or miR-34) can indirectly up- or downregulate iNOS expression by altering upstream signal transduction pathways that subsequently effect rodent iNOS expression while miR-27b can suppress KSRP and affect iNOS mRNA stability.
microRNA and Human Inducible Nitric Oxide Synthase
25
4. CONCLUSION Transcriptional and posttranscriptional regulations are two major mechanisms for hiNOS gene regulation. Pathways in human iNOS expression vary in different cells and tissues. Transcription factors such as NF-κB, AP 1, and STAT-1α functionally binding to their corresponding elements in hiNOS promoter are necessary for human iNOS transcription in most human cells. However, a quite complex network of RNA-BP (AUF1, HuR, KSRP, PTB, TIAR, TTP, and PABP) is involved in the posttranscriptional regulation of human iNOS expression. Moreover, recent data also indicates that regulation of human iNOS expression is also controlled by miRNAs such as miR-939, miR-146a, and miR-26a.
REFERENCES Abouheif, M. M., Nakasa, T., Shibuya, H., Niimoto, T., Kongcharoensombat, W., & Ochi, M. (2010). Silencing microRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro. Rheumatology, 49(11), 2054–2060. Casper, I., Nowag, S., Koch, K., Hubrich, T., Bollmann, F., Henke, J., et al. (2013). Posttranscriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(A)-binding protein (PABP). Nitric Oxide, 33, 6–17. Chan, G. C., Fish, J. E., Mawji, I. A., Leung, D. D., Rachlis, A. C., & Marsden, P. A. (2005). Epigenetic basis for the transcriptional hyporesponsiveness of the human inducible nitric oxide synthase gene in vascular endothelial cells. The Journal of Immunology, 175, 3846–3861. Chartrain, N., Geller, D. A., Koty, P. P., Sitrin, N. F., Nussler, A. K., Hoffman, E. P., et al. (1994). Molecular cloning, structure, and chromosomal mapping of the human inducible nitric oxide synthase gene. The Journal of Biological Chemistry, 269, 6765–6772. Dai, R., Phillips, R. A., Zhang, Y., Khan, D., Crasta, O., & Ahmed, S. A. (2008). Suppression of LPS-induced Interferon-gamma and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: A novel mechanism of immune modulation. Blood, 112(12), 4591–4597. deVera, M. E., Shapiro, R. A., Nussler, A. K., Mudgett, J. S., Simmons, R. L., Morris, S. M., et al. (1996). Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: Initial analysis of the human NOS2 promoter. Proceedings of the National Academy of Sciences of the United States of America, 93, 1054–1059. Feng, X., Guo, Z., Nourbakhsh, M., Hauser, H., Ganster, R., Shao, L., et al. (2002). Identification of a negative response element in the human inducible nitric oxide synthase (hiNOS) promoter: The role of NF-κB repressing factor (NRF) in basal repression of the hiNOS gene. Proceedings of the National Academy of Sciences of the United States of America, 99, 14212–14217. Ganster, R. W., & Geller, D. A. (2000). Nitric oxide. San Diego, CA: Academic Press. Ganster, R. W., Taylor, B. S., Shao, L., & Geller, D. A. (2001). Complex regulation of human iNOS gene transcription by Stat 1 and NF-kB. Proceedings of the National Academy of Sciences of the United States of America, 98, 8638–8643. Geller, D. A., Lowenstein, C. J., Shapiro, R. A., Nussler, A. K., Di Silvio, M., Wang, S. C., et al. (1993). Molecular cloning and expression of inducible nitric oxide synthase from
26
Zhong Guo and David A. Geller
human hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 90, 3491–3495. Guo, Z., Shao, L., Du, Q., Park, K. S., & Geller, D. A. (2007). Identification of a classic cytokine-induced enhancer upstream in the human iNOS promoter. FASEB Journal, 21(2), 535–542. Guo, Z., Shao, L., Zheng, L., Du, Q., Li, P., John, B., et al. (2012). miRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 109(15), 5826–5831. John, B., Enright, A. J., Aravin, A., Tuschl, T., Sander, C., & Marks, D. S. (2004). Human microRNA targets. PLoS Biology, 2(11), e363. Kleinert, H., Pautz, A., Linker, K., & Schwarz, P. M. (2004). Regulation of the expression of inducible nitric oxide synthase. European Journal of Pharmacology, 500255–266. Li, X., Gibson, G., Kim, J. S., Kroin, J., Xu, S., van Wijnen, A. J., et al. (2011). MicroRNA146a is linked to pain-related pathophysiology of osteoarthritis. Gene, 480, 34–41. Linker, K., Pautz, A., Fechir, M., Hubrich, T., Greeve, J., & Kleinert, H. (2005). Involvement of KSRP in the post-transcriptional regulation of human iNOS expressioncomplex interplay of KSRP with TTP and HuR. Nucleic Acids Research, 33, 4813–4827. Liu, H., Song, L., & Huang, W. (2010). MiR26a and miR939 regulate the replication of H1N1 influenza virus in MDCK cell. Wei Sheng Wu Xue Bao, 50, 1399–1405. Lui, W. O., Pourmand, N., Patterson, B. K., & Fire, A. (2007). Patterns of known and novel small RNAs in human cervical cancer. Cancer Research, 67, 6031–6043. Luss, H., Li, R., Shapiro, R. A., Tzeng, E., McGowan, F. X., Yoneyama, T., et al. (1997). Dedifferentiated human ventricular cardiac myocytes express inducible nitric oxide synthase mRNA but not protein in response to IL-1, TNF, IFNg, & LPS. Journal of Molecular and Cellular Cardiology, 29, 1153–1165. Marks-Konczalik, J., Chu, S. C., & Moss, J. (1998). Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites. The Journal of Biological Chemistry, 273, 22201–22208. Nussler, A. K., DiSilvio, M., Billiar, T. R., Hoffman, R. A., Geller, D. A., Selby, R., et al. (1992). Stimulation of the nitric oxide pathway in human hepatocytes by cytokines and endotoxin. The Journal of Experimental Medicine, 176, 261–264. Park, K. S., Guo, Z., Shao, L., Du, Q., & Geller, D. A. (2009). A far-upstream Oct-1 motif regulates cytokine-induced transcription of the human inducible nitric oxide synthase gene. Journal of Molecular Biology, 390, 595–603. Pautz, A., Art, J., Hahn, S., Nowag, S., Voss, C., & Kleinert, H. (2010). Regulation of the expression of inducible nitric oxide synthase. Nitric Oxide, 23, 75–93. Taylor, B. S., de Vera, M. E., Ganster, R. W., Wang, Q., Shapiro, R. A., Billiar, T. R., et al. (1998). A novel NF-κB enhancer element regulates cytokine induction of the human inducible nitric oxide synthase gene promoter. The Journal of Biological Chemistry, 273, 15148–15156. Taylor, B. S., & Geller, D. A. (2000). Molecular regulation of the human inducible nitric oxide synthase (iNOS) gene (review). Shock, 13, 413–424. Wang, X., Zhao, Q., Matta, R., Meng, X., Liu, X., Liu, C. G., et al. (2009). Inducible nitricoxide synthase expression is regulated by mitogen-activated protein kinase phosphatase1. The Journal of Biological Chemistry, 284, 27123–27134. Zeng, Y., Yi, R., & Cullen, B. R. (2003). miRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proceedings of the National Academy of Sciences of the United States of America, 100, 9779–9784.
microRNA and Human Inducible Nitric Oxide Synthase
27
Zhou, R., Gong, A. Y., Eischeid, A. N., & Chen, X. M. (2012). miR-27b targets KSRP to coordinate TLR4-mediated epithelial defense against Cryptosporidium parvum infection. PLoS Pathogens, 8, e1002702. Zhu, H., Vishwamitra, D., Curry, C. V., Manshouri, R., Diao, L., Khan, A., et al. (2013). NPM-ALK up-regulates iNOS expression through a STAT3/microRNA-26adependent mechanism. The Journal of Pathology, 230, 82–94.
microRNA and Human Inducible Nitric Oxide Synthase Zhong Guo, David A. Geller1 Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Regulation of Human iNOS Gene 2.1 Transcriptional 2.2 Posttranscriptional 3. miRNAs Regulation 3.1 miR-939 3.2 other miRNAs 4. Conclusion References
19 20 20 21 21 22 23 25 25
Abstract Regulation of human inducible nitric oxide synthase (iNOS) expression involves both transcriptional and posttranscriptional mechanisms. Human iNOS gene transcription is controlled in a cell type-specific manner by extracellular cytokines. Transcriptional regulation of human iNOS gene involves transcription factors NF-κB, Stat-1, AP-1, C/EBPβ, KLF6, Oct 1, and NRF. Important posttranscriptional mechanisms also regulate human iNOS mRNA stability through RNA binding proteins HuR, TTP, KSRP, and PABP. Recently, there are several miRNAs that were validated to regulate human and rodent iNOS gene expression. Among them, miR-939 and miR-26a were identified to bind with the human iNOS 30 -UTR and exert a translational blockade of human iNOS protein synthesis.
1. INTRODUCTION The expression of inducible nitric oxide synthase (iNOS) can be induced in various cells and tissues following exposure to immunologic and inflammatory stimuli such as cytokines or lipopolysaccharide (LPS) (Kleinert, Pautz, Linker, & Schwarz, 2004). Human iNOS expression in primary human hepatocytes was originally identified by stimulating with a Vitamins and Hormones, Volume 96 ISSN 0083-6729 http://dx.doi.org/10.1016/B978-0-12-800254-4.00002-7
#
2014 Elsevier Inc. All rights reserved.
19
20
Zhong Guo and David A. Geller
cytokine mixture (CM) of TNF-α, IL-1β, IFN-γ, and LPS (Nussler et al., 1992). Subsequently, the human iNOS cDNA was cloned from LPS and cytokine-stimulated primary human hepatocytes (Geller et al., 1993). This cloned gene reveals a 4145-bp cDNA, containing a 3459-bp open reading frame that encodes 1153 amino acids with a 131-kDa molecular mass. The genomic cloning of human iNOS clarified that the completed human iNOS gene consists of 26 exons and 25 introns spanning 37 kb of genomic region and specifically mapped to chromosome 17 at position 17 cen-q11.2 (Chartrain et al., 1994).
2. REGULATION OF HUMAN iNOS GENE 2.1. Transcriptional The expression of human iNOS is controlled in large part by transcriptional mechanisms. The functional promoter region of human iNOS gene can be extended to 16 kb (deVera et al., 1996). The validated binding sites for numerous transcription factors such as AP-1, C/EBP, CREB, GATA, HIF, IRF-1, NF-AT, NF-κB, NF-IL6, Oct-1, PARP1, p53, Sp1, KLF6, STAT-1α, and YY1 (Pautz et al., 2010). There is a classical cytokineinduced enhancer between 5 and 6 kb of human iNOS promoter (Guo, Shao, Du, Park, & Geller, 2007). The human iNOS core promoter contains a TATA box about 30 bp from the transcription start site. Near the TATA box, it also contains binding sites for the transcription factors NF-κB and C/EBP β (Pautz et al., 2010). In human AKN-1, A549, or DLD1 cells, only human iNOS promoter fragments larger than 3.8 kb showed any significant induction with cytokines (deVera et al., 1996; Taylor et al., 1998). We have further shown that TNF-α or IL-1β signal through NF-κB binding to 5.5, 5.8, and 6.1 kb cis-acting DNA elements, while IFN-γ signals through Stat-1 by binding to motifs at 5.2 and 5.8 kb in the human iNOS promoter (Ganster, Taylor, Shao, & Geller, 2001). Another group showed that cytokine-responsiveness required 50 -flanking DNA regions extending to 8 kb and demonstrated CM-inducible activating protein (AP)-1 binding sites at 5.1 and 5.3 kb as well as a functional role for a NF-κB element located at 8.2 kb in the human iNOS promoter (Marks-Konczalik, Chu, & Moss, 1998). Interestingly, NF-κB repressing factor protein binding to a negative response element at 6.7 kb in the human iNOS promoter region was recognized to mediate constitutive silencing of human iNOS transcription (Feng et al., 2002). A far-upstream functional Oct-1 motif was also identified at
microRNA and Human Inducible Nitric Oxide Synthase
21
10.2 kb in the human iNOS promoter that regulates cytokine-induced human iNOS gene transcription (Park, Guo, Shao, Du, & Geller, 2009). The induction of hiNOS expression requires a CM including TNF-α, IL-1β, and IFN-γ (Taylor & Geller, 2000). Activation of NF-κB pathway, JAK2–STAT-1α pathway, and MAPK pathways (p42/44 MAPK, p38MAPK, JNK) are important for hiNOS expression (Ganster & Geller, 2000; Kleinert et al., 2004).
2.2. Posttranscriptional RNA binding proteins (RNA-BP) HuR, TTP, and KSRP have been shown to be involved in the posttranscriptional mechanisms of human iNOS regulation (Pautz et al., 2010). Cytokine induction can result in in vitro binding of HuR to the iNOS mRNA and thus leads to iNOS mRNA stabilization (Linker et al., 2005). The KH-type splicing regulatory protein (KSRP) showed the destabilizing effect on human iNOS mRNA expression as a human iNOS 30 -UTR binding protein (Linker et al., 2005). The zinc-finger protein tristetraprolin (TTP) regulates iNOS mRNA expression indirect via an interaction with KSRP. Cytokine induction can enhance TTP–KSRP interaction and this results in reduced in vitro binding of KSRP to the human iNOS mRNA (Linker et al., 2005). The cytosolic poly (A)-binding protein (PABP) is recently identified to bind to the 50 -UTR and 30 -UTR of hiNOS mRNA and to posttranscriptionally enhance human iNOS mRNA stability (Casper et al., 2013). Besides HuR, TTP, KSRP, and PABP, other RNA-BP such as hnRNP A1, PTB, and TIAR can also increase human iNOS expression by enhancement of iNOS mRNA stability. Noteworthy, the ARE/poly (U)-binding/ degradation factor 1 (AUF1, also named hnRNP D), seems to regulate human iNOS expression by modulation of iNOS mRNA stability, whereas all four isoforms of the ARE/poly (U)-binding/degradation factor 1 (AUF1) negatively regulate human iNOS expression by destabilization of the mRNA (Pautz et al., 2010).
3. miRNAs REGULATION microRNAs (miRNA) are short (21) nucleotides that are complementary to 30 -UTR mRNA sequences and have been widely reported to regulate human gene expression (Zeng, Yi, & Cullen, 2003; John et al., 2004).
22
Zhong Guo and David A. Geller
In primary human hepatocytes, cytokines induce human iNOS mRNA, protein, and NO synthesis. In contrast, primary human macrophages as well as certain human tumor cell lines demonstrate iNOS mRNA following cytokine stimulation, yet human iNOS protein is difficult to detect (Chan et al., 2005). Likewise, in primary human cardiac myocytes, human iNOS mRNA was readily seen after cytokine stimulation, but iNOS protein and NO synthesis were not identified (Luss et al., 1997). These observations are consistent with a translational blockade and suggest the possible role of miRNA in exerting negative posttranscriptional regulation.
3.1. miR-939 miR-939 was originally cloned from human cervical cancer cells (Lui, Pourmand, Patterson, & Fire, 2007). It has two mature forms: hsa-miR939-5p and hsa-miR-939-3p. It is located at 8q24.3 genomic locus and is also imbedded in cleavage and polyadenylation specific factor 1 (CPSF1) gene. One group reported that miR-939 regulates the replication of H1N1 influenza virus in MDCK cells (Liu, Song, & Huang, 2010). We identified adjacent miR-939 binding sites in the hiNOS 30 -UTR and further proved that miR-939 binds in vitro and in vivo to exert a translational blockade of cytokine-stimulated hiNOS protein expression in primary human hepatocytes (Guo et al., 2012). The hiNOS 30 -UTR confers posttranscriptional repression of basal and cytokine-induced hiNOS transcriptional activity in an orientationdependent manner. miRNA-939 decreases cytokine-induced hiNOS protein expression, but does not affect hiNOS mRNA levels or hiNOS mRNA stability. The hiNOS 30 -UTR contains two functional miR-939 binding sites at +99 and +112 base pairs that are critical for miR-939 mediated translational blockade. Cytokines can induce endogenous miR-939 expression and consequently, miR-939/hiNOS mRNA complex in human hepatocytes. Therefore, we proposed a model regarding the functional role of miR-939 in the posttranscriptional regulation of the iNOS gene in human hepatocytes (Fig. 2.1). Human primary hepatocytes can be stimulated by a combination of cytokines (TNF-α, IL-1β, and IFN-γ) to strongly express human iNOS mRNA. The cytokines activate specific transcription factors NF-κB, Stat-1, AP1, and C/EBPβ, which functionally interact with their corresponding cis-acting DNA binding sites to drive human iNOS transcription. Meanwhile, the same cytokines also increase miR-939 levels
23
microRNA and Human Inducible Nitric Oxide Synthase
miRNA-939 represses human iNOS gene translation TNF α, IL 1β, IFN γ, LPS
NFκB,AP 1,Stats
hiNOS Gene
?
hiNOS mRNA
miR-939, ?
hiNOS Protein
NO
HiNOS 3¢-UTR (1 – 496 bp)
hiNOS promoter
hiNOS gene
+99 112 bp AAA 939 939 miRNA binding sites
Figure 2.1 Proposed model of human iNOS gene regulation in human hepatocytes with miR-939 exerting a translational blockade for iNOS protein synthesis by binding to the iNOS 30 -UTR.
which bind to two specific miR-939 binding sites in the human iNOS 30 -UTR leading to translational inhibition of CM-induced human iNOS protein expression. Binding to both sites is likely required for maximal translational repression, because mutation of either binding site partially abrogates the inhibitory effect of exogenous miR-939 on luciferase activity. Cytokine induction of a negative miRNA regulator of human iNOS expression would theoretically serve to protect the host against untoward consequences of prolonged human iNOS overexpression in a check-and-balance system.
3.2. other miRNAs Besides miR-939, miR-146a was also reported to regulate human iNOS gene (Table 2.1). However, unlike direct binding of miR-939 to the human iNOS 30 -UTR, miR-146a was shown to be indirectly involved in the regulation of iNOS gene expression. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS, and ion channel, TRPV1) in human glial cells (Li et al., 2011). Recently, miR-26a was identified to suppress the expression of iNOS protein at the posttranscriptional level by interacting with 30 -UTR in cancer cells (Zhu et al., 2013). There are at least four miRNAs that have been validated to regulate rodent iNOS genes (Table 2.1). Dai et al. reported that miR-146a, a negative regulator of Toll-like receptor (TLR) signaling, was decreased in
24
Zhong Guo and David A. Geller
Table 2.1 Summary of miRNAs in regulating iNOS genes in different species Specie miRNA Regulation Mechanism References
Human miR-939
Down
Translational blockage by binding 30 -UTR
Guo et al. (2012)
miR-146a Down
Modulating inflammatory cytokines
Li et al. (2011)
miR-26a
Translational blockage by binding 30 -UTR
Zhu et al. (2013)
Negative regulator of Toll-like receptor (TLR) signaling
Dai et al. (2008)
Down
Mouse miR-146a Down
Rat
miR-155
Up
Inhibition of cytokine signal (SOCS)-1
Wang et al. (2009)
miR-27b
Up
Targeting KSRP to increase iNOS mRNA stability
Zhou, Gong, Eischeid, and Chen (2012)
miR-34b
Down
Its inhibitor prevents IL-1β Abouheif et al. induced iNOS gene (2010)
freshly isolated splenic lymphocytes from estrogen-treated mice. Increasing the activity of miR-146a significantly inhibited LPS-induced IFN-γ and iNOS expression in mouse splenic lymphocytes. Enhancing the activity of miR-146a also inhibited the expression of LPS-induced iNOS and NO (Dai et al., 2008). Additionally, a recent report indicates that miR155 expression was increased in MKP-1-deficient macrophages compared with wild-type macrophages. Transfect on of miR-155 attenuated the expression of Suppressor of Cytokine Signal (SOCS)-1 and subsequently enhanced the expression of iNOS (Wang et al., 2009). miR-27b regulates the stabilization of iNOS mRNA through targeting KSRP (Zhou et al., 2012). The expression of miR-34a was significantly upregulated by IL-1β. Silencing of miR-34a significantly prevented IL-1β-induced downregulation of Col2a1, as well as IL-1β-induced upregulation of rat iNOS gene (Abouheif et al., 2010). These studies indicate that specific miRNAs (miR-155, miR-146a, or miR-34) can indirectly up- or downregulate iNOS expression by altering upstream signal transduction pathways that subsequently effect rodent iNOS expression while miR-27b can suppress KSRP and affect iNOS mRNA stability.
microRNA and Human Inducible Nitric Oxide Synthase
25
4. CONCLUSION Transcriptional and posttranscriptional regulations are two major mechanisms for hiNOS gene regulation. Pathways in human iNOS expression vary in different cells and tissues. Transcription factors such as NF-κB, AP 1, and STAT-1α functionally binding to their corresponding elements in hiNOS promoter are necessary for human iNOS transcription in most human cells. However, a quite complex network of RNA-BP (AUF1, HuR, KSRP, PTB, TIAR, TTP, and PABP) is involved in the posttranscriptional regulation of human iNOS expression. Moreover, recent data also indicates that regulation of human iNOS expression is also controlled by miRNAs such as miR-939, miR-146a, and miR-26a.
REFERENCES Abouheif, M. M., Nakasa, T., Shibuya, H., Niimoto, T., Kongcharoensombat, W., & Ochi, M. (2010). Silencing microRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro. Rheumatology, 49(11), 2054–2060. Casper, I., Nowag, S., Koch, K., Hubrich, T., Bollmann, F., Henke, J., et al. (2013). Posttranscriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(A)-binding protein (PABP). Nitric Oxide, 33, 6–17. Chan, G. C., Fish, J. E., Mawji, I. A., Leung, D. D., Rachlis, A. C., & Marsden, P. A. (2005). Epigenetic basis for the transcriptional hyporesponsiveness of the human inducible nitric oxide synthase gene in vascular endothelial cells. The Journal of Immunology, 175, 3846–3861. Chartrain, N., Geller, D. A., Koty, P. P., Sitrin, N. F., Nussler, A. K., Hoffman, E. P., et al. (1994). Molecular cloning, structure, and chromosomal mapping of the human inducible nitric oxide synthase gene. The Journal of Biological Chemistry, 269, 6765–6772. Dai, R., Phillips, R. A., Zhang, Y., Khan, D., Crasta, O., & Ahmed, S. A. (2008). Suppression of LPS-induced Interferon-gamma and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: A novel mechanism of immune modulation. Blood, 112(12), 4591–4597. deVera, M. E., Shapiro, R. A., Nussler, A. K., Mudgett, J. S., Simmons, R. L., Morris, S. M., et al. (1996). Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: Initial analysis of the human NOS2 promoter. Proceedings of the National Academy of Sciences of the United States of America, 93, 1054–1059. Feng, X., Guo, Z., Nourbakhsh, M., Hauser, H., Ganster, R., Shao, L., et al. (2002). Identification of a negative response element in the human inducible nitric oxide synthase (hiNOS) promoter: The role of NF-κB repressing factor (NRF) in basal repression of the hiNOS gene. Proceedings of the National Academy of Sciences of the United States of America, 99, 14212–14217. Ganster, R. W., & Geller, D. A. (2000). Nitric oxide. San Diego, CA: Academic Press. Ganster, R. W., Taylor, B. S., Shao, L., & Geller, D. A. (2001). Complex regulation of human iNOS gene transcription by Stat 1 and NF-kB. Proceedings of the National Academy of Sciences of the United States of America, 98, 8638–8643. Geller, D. A., Lowenstein, C. J., Shapiro, R. A., Nussler, A. K., Di Silvio, M., Wang, S. C., et al. (1993). Molecular cloning and expression of inducible nitric oxide synthase from
26
Zhong Guo and David A. Geller
human hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 90, 3491–3495. Guo, Z., Shao, L., Du, Q., Park, K. S., & Geller, D. A. (2007). Identification of a classic cytokine-induced enhancer upstream in the human iNOS promoter. FASEB Journal, 21(2), 535–542. Guo, Z., Shao, L., Zheng, L., Du, Q., Li, P., John, B., et al. (2012). miRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 109(15), 5826–5831. John, B., Enright, A. J., Aravin, A., Tuschl, T., Sander, C., & Marks, D. S. (2004). Human microRNA targets. PLoS Biology, 2(11), e363. Kleinert, H., Pautz, A., Linker, K., & Schwarz, P. M. (2004). Regulation of the expression of inducible nitric oxide synthase. European Journal of Pharmacology, 500255–266. Li, X., Gibson, G., Kim, J. S., Kroin, J., Xu, S., van Wijnen, A. J., et al. (2011). MicroRNA146a is linked to pain-related pathophysiology of osteoarthritis. Gene, 480, 34–41. Linker, K., Pautz, A., Fechir, M., Hubrich, T., Greeve, J., & Kleinert, H. (2005). Involvement of KSRP in the post-transcriptional regulation of human iNOS expressioncomplex interplay of KSRP with TTP and HuR. Nucleic Acids Research, 33, 4813–4827. Liu, H., Song, L., & Huang, W. (2010). MiR26a and miR939 regulate the replication of H1N1 influenza virus in MDCK cell. Wei Sheng Wu Xue Bao, 50, 1399–1405. Lui, W. O., Pourmand, N., Patterson, B. K., & Fire, A. (2007). Patterns of known and novel small RNAs in human cervical cancer. Cancer Research, 67, 6031–6043. Luss, H., Li, R., Shapiro, R. A., Tzeng, E., McGowan, F. X., Yoneyama, T., et al. (1997). Dedifferentiated human ventricular cardiac myocytes express inducible nitric oxide synthase mRNA but not protein in response to IL-1, TNF, IFNg, & LPS. Journal of Molecular and Cellular Cardiology, 29, 1153–1165. Marks-Konczalik, J., Chu, S. C., & Moss, J. (1998). Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites. The Journal of Biological Chemistry, 273, 22201–22208. Nussler, A. K., DiSilvio, M., Billiar, T. R., Hoffman, R. A., Geller, D. A., Selby, R., et al. (1992). Stimulation of the nitric oxide pathway in human hepatocytes by cytokines and endotoxin. The Journal of Experimental Medicine, 176, 261–264. Park, K. S., Guo, Z., Shao, L., Du, Q., & Geller, D. A. (2009). A far-upstream Oct-1 motif regulates cytokine-induced transcription of the human inducible nitric oxide synthase gene. Journal of Molecular Biology, 390, 595–603. Pautz, A., Art, J., Hahn, S., Nowag, S., Voss, C., & Kleinert, H. (2010). Regulation of the expression of inducible nitric oxide synthase. Nitric Oxide, 23, 75–93. Taylor, B. S., de Vera, M. E., Ganster, R. W., Wang, Q., Shapiro, R. A., Billiar, T. R., et al. (1998). A novel NF-κB enhancer element regulates cytokine induction of the human inducible nitric oxide synthase gene promoter. The Journal of Biological Chemistry, 273, 15148–15156. Taylor, B. S., & Geller, D. A. (2000). Molecular regulation of the human inducible nitric oxide synthase (iNOS) gene (review). Shock, 13, 413–424. Wang, X., Zhao, Q., Matta, R., Meng, X., Liu, X., Liu, C. G., et al. (2009). Inducible nitricoxide synthase expression is regulated by mitogen-activated protein kinase phosphatase1. The Journal of Biological Chemistry, 284, 27123–27134. Zeng, Y., Yi, R., & Cullen, B. R. (2003). miRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proceedings of the National Academy of Sciences of the United States of America, 100, 9779–9784.
microRNA and Human Inducible Nitric Oxide Synthase
27
Zhou, R., Gong, A. Y., Eischeid, A. N., & Chen, X. M. (2012). miR-27b targets KSRP to coordinate TLR4-mediated epithelial defense against Cryptosporidium parvum infection. PLoS Pathogens, 8, e1002702. Zhu, H., Vishwamitra, D., Curry, C. V., Manshouri, R., Diao, L., Khan, A., et al. (2013). NPM-ALK up-regulates iNOS expression through a STAT3/microRNA-26adependent mechanism. The Journal of Pathology, 230, 82–94.