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Allograft rejection involves a complex network of multiple immune regulators and effector mechanisms. In the current study, we focused on the role of nuclear factor (NF)-kappaB/Rel. Previous studies had established that deficiency of the p50 NF-kappaB family member prolonged allograft survival only modestly. However, because of its crucial role in signal transduction in inflammatory and immune responses, we hypothesized that other NF-kappaB/Rel family members may produce more profound effects on alloimmunity. Therefore, in addition to p50, we analyzed the role of c-Rel, which is expressed predominantly in lymphocytes. Also, to investigate NF-kappaB activation in T cells, we examined transgenic mice that express a transdominant inhibitor of NF-kappaB [IkappaB(DeltaN)] regulated by a T cell-restricted promoter. Allograft survival was prolonged indefinitely in the c-Rel-deficient and IkappaB(DeltaN)-transgenic recipients. To determine the molecular basis of NF-kappaB modulation of rejection, we analyzed a panel of 58 parameters including effector molecules, chemokines, cytokines, receptors, and cellular markers using hierarchical clustering algorithms and self-organizing maps in p50(-/-), c-Rel(-/-), and IkappaB(DeltaN)-transgenic, experimental groups plus allogeneic-, syngeneic-, and lymphocyte-deficient (alymphoid) control groups. Surprisingly, profiles of gene expression in the c-Rel recipients (which have indefinite graft survival) were similar to the p50(-/-) and allogeneic recipients (which rapidly reject grafts). As expected, gene expression in the IkappaB(DeltaN) recipients (which also have indefinite graft survival) was similar to profiles of nonrejecting syngeneic and alymphoid recipients. Importantly, self-organizing maps identified a small subset of genes including several chemokine receptors and cytokines with expression profiles that correlate with graft survival. Thus, our results demonstrate a crucial role for NF-kappaB in acute allograft rejection, identify different molecular mechanisms of rejection by distinct NF-kappaB family members, and identify a small subset of inducible genes whose inhibition is linked to graft acceptance.
The Rel/NF-kappaB family of transcription factors has been implicated in such diverse cellular processes as proliferation, differentiation, and apoptosis. As each of these processes occurs during post-natal mammary gland morphogenesis, the expression and activity of NF-kappaB factors in the murine mammary gland were examined. Immunohistochemical and immunoblot analyses revealed expression of the p105/p50 and RelA subunits of NF-kappaB, as well as the major inhibitor, IkappaBalpha, in the mammary epithelium during pregnancy, lactation, and involution. Electrophoretic mobility shift assay (EMSA) demonstrated that DNA-binding complexes containing p50 and RelA were abundant during pregnancy and involution, but not during lactation. Activity of an NF-kappaB-dependent luciferase reporter in transgenic mice was highest during pregnancy, decreased to near undetectable levels during lactation, and was elevated during involution. This highly regulated pattern of activity was consistent with the modulated expression of p105/p50, RelA, and IkappaBalpha.
Activation of T lymphocytes by Ags or cytokines results in translocation of the transcription factors NF-kappa B, AP-1, NFAT, and STAT from the cytoplasm into the nucleus. The first step in the nuclear import process is recognition of a nuclear localization sequence (NLS) within the karyophilic protein by a cytoplasmic receptor such as the importin (karyopherin)-alpha subunit. The NLSs of NF-kappa B, AP-1, and NFAT differ and the NLS of STAT1 has not yet been identified. Herein we demonstrate that the inducible nuclear import of NF-kappa B, AP-1, NFAT, and STAT1 in Jurkat T lymphocytes is significantly inhibited by a cell-permeable peptide carrying the NLS of the NF-kappa B p50 subunit. NLS peptide-mediated disruption of the nuclear import of these transcription factors results in inhibition of I kappa B alpha and IL-2 gene expression, processes dependent on NF-kappa B or the combination of NF-kappa B, AP-1, and NFAT. Further, we show that inhibitory NLS peptide interacts in vitro with a cytoplasmic NLS receptor complex comprised of the Rch1/importin (karyopherin)-beta heterodimer expressed in Jurkat T cells. Taken together, these data indicate that the inducible nuclear import of NF-kappa B, AP-1, NFAT, and STAT1 in Jurkat T cells can be regulated by NLS peptide delivered noninvasively to the cytoplasm of Jurkat T cells to target members of the importin (karyopherin)-alpha beta NLS receptor complex.
Normal adult human dermal fibroblasts grown in a three-dimensional collagen lattice increase mRNA level of collagen receptor integrin subunit alpha2 (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239- 249.) and DNA binding activity of a nuclear transcription factor, NF-kappaB (Xu, J., and R.A.F. Clark. 1997. J. Cell Biol. 136:473-483.). Here we present evidence that the collagen lattice induced the nuclear translocation of p50, one member of NF-kappaB family, and the degradation of an NF-kappaB inhibitor protein, IkappaB-alpha. The inhibition of NF-kappaB activity by SN50, a peptide inhibitor targeted at nuclear translocation of NF-kappaB, significantly reduced the induction of integrin alpha2 mRNA and protein by the collagen lattice. A region located between -549 and -351 bp in the promoter of integrin alpha2 gene conferred the inducibility by three-dimensional collagen lattice. The presence of either SN50 or IkappaB-alpha32, 36, a stable mutant of IkappaB-alpha, abrogated this inducibility, indicating that the activation of integrin alpha2 gene expression was possibly mediated by NF-kappaB through this region. Although there were three DNA-protein binding complexes forming in this region that are sensitive to the inhibition of NF-kappaB nuclear translocation, NF-kappaB was not directly present in the binding complexes. Therefore, an indirect regulatory mechanism by NF-kappaB in integrin alpha2 gene expression induced by three-dimensional collagen lattice is suggested. The involvement of NF-kappaB in reorganization and contraction of three-dimensional collagen lattice, a process that requires the presence of abundant integrin alpha2beta1, was also examined. The inhibition of NF-kappaB activity by SN50 greatly blocked the contraction, suggesting its critical role in not only the induction of integrin alpha2 gene expression by three-dimensional collagen lattice, but also alpha2beta1-mediated tissue-remodeling process.