Distinct patterns of intracerebral hemorrhage-induced alterations in NF-jB subunit, iNOS, and COX-2 expression Xiurong Zhao, Yujian Zhang, Roger Strong, Jie Zhang, James C. Grotta and Jaroslaw Aronowski Department of Neurology, Stroke Program, University of Texas–Houston Medical School, Houston, Texas, USA Abstract Transcription factor nuclear factor-jB (NF-jB), plays a key role in regulating inflammation in brain pathologies. The goal of this study was to characterize temporal changes in NF-jB activation, NF-jB subunit expression, and expression of selected NF-jB-regulated gene products [inducible form of nitric oxide synthase (iNOS) and cyclooxygenase-2], at the transcriptional and translational level in the brain after intracerebral hemorrhage (ICH). Employing the intrastriatal injection of autologous blood in rats to model ICH, we dem- onstrated, using NF-jB–DNA binding assay, a robust and prolonged NF-jB activation, starting as early as 15 min after the onset of ICH. Consequently, we demonstrated that the mRNA and protein for p50, p52, p65, c-Rel, and RelB NF-jB subunits, as well as IjBa were all up-regulated, with a time course ranging from minutes to days following ICH, depending on the subunit. Using reverse transcription-polymerase chain reaction to analyze mRNA and immunoblotting to analyze protein in ICH-affected tissue, we found robust induction of iNOS at both mRNA and protein levels that followed a time- course similar to changes in p65, p52, and RelB mRNA. Oddly, in contrast to iNOS, cyclooxygenase-2 mRNA and protein following an early transient increase demonstrated significant reduction in response to ICH. In summary, NF-jB activation occurs within minutes and persists for at least a week in response to ICH. This reaction utilizes different NF-jB regulatory subunits and is associated with the expression of selected target genes. Keywords: cyclooxygenase-2, intracerebral hemorrhage, nuclear factor-jB, p65, RelB, IjBa, inducible form of nitric oxide synthase. J. Neurochem. (2007) 101, 652–663. Intracerebral hemorrhage (ICH) is a common form of stroke with significant mortality and morbidity. The evidence for an inflammatory response after ICH has been reported both in humans and in experimental rat models of hemorrhagic stroke (Garcia et al. 1994; Hickenbottom et al. 1999; Del Bigio et al. 2000; Gong et al. 2000; Hua et al. 2000; Al- Senani et al. 2001; Mayne et al. 2001; Castillo et al. 2002; Felberg et al. 2002; Huang et al. 2002; Wang et al. 2003; Wagner et al. 2004; Aronowski and Hall 2005). The preponderance of inflammatory responses in the brain is coordinated and regulated by the transcription factor, nuclear factor-jB (NF-jB) (Baeuerle and Henkel 1994; Barnes and Karin 1997). To further understand the potential implications of NF-jB activation, it is imperative to first characterize the nature of NF-jB activation, including NF-jB subunit expression and gene product changes after ICH. Nuclear factor-jB exists as a dimer composed primarily of the (NF-jB1) p50 and (RelA) p65 subunits, as well as other members of the NF-jB/Rel family, including RelB, c-Rel, and (NF-jB2) p52 (Verma 2004). In a resting cell, inactive NF-jB is sequestered in the cytoplasm by the inhibitory jB proteins (IjBs) to prevent its translocation to the nucleus. Phosphorylation of IjB by IjB kinase mediates its ubiqui- tination and proteasomal degradation leading to a liberation and translocation of NF-jB to the nucleus (Baeuerle 1998). In the nucleus, NF-jB binds to a conserved DNA consensus sequence, termed NF-jB response element, in the promoter of target gene. This interaction modulates transcription of many genes (Baeuerle 1998). The NF-jB subunits differ in their abilities to activate transcription. Among the NF-jB family, only p65, RelB, and c-Rel were found to contain Received August 8, 2006; revised manuscript received October 25, 2006; accepted October 25, 2006. Address correspondence and reprint requests to: Jaroslaw Aronowski, Department of Neurology, Stroke Program, University of Texas–Hous- ton Medical School, Rm 7.044, Houston, Texas 77030, USA. E-mail: j.aronowski@uth.tmc.edu Abbreviations used: AREs, AU-reach elements; cDNA, comple- mentary DNA; COX-2, cyclooxygenase-2; DTT, dithiothreitol; ICH, intracerebral hemorrhage; IjB, inhibitory jB; iNOS, inducible form of nitric oxide synthase; NF-jB, nuclear factor-jB; NO, nitric oxide; PBS, phosphate-buffered saline; RT, reverse transcription; RT-PCR, reverse transcription-polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBST, Tris-Buffered Saline. Journal of Neurochemistry , 2007, 101, 652–663 doi:10.1111/j.1471-4159.2006.04414.x 652 Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 652–663 Ó 2007 The Authors