SUMOylation attenuates the transcriptional activity of the NF-κB subunit RelB.

The NF-κB subunit RelB is known to act either as an activator or repressor of NF-κB-dependent gene expression. The RelB-p52 heterodimer, for instance, is the key element of the alternative NF-κB signaling pathway supporting the expression of a subset of NF-κB target genes. By contrast, RelB is crucial for the repression of important pro-inflammatory cytokines like TNFα or interleukin 1ß. Despite accumulating reports describing the functional variability of RelB, the molecular mechanisms underlying these divergent functions are still unknown. One potential explanation could be a functional reprogramming of RelB by different post-translational modifications. Here, we demonstrate that SUMOylation of RelB might be one of these post-translational modifications rendering the function of the NF-κB transcription factor RelB. In vivo SUMOylation analyses using either the UBC9-fusion-directed SUMOylation method or endogenous proteins from Namalwa B cells revealed that RelB is modified by either SUMO1 or SUMO2 attachment at various sites. Functional studies suggest that SUMOylation converts RelB into a transcriptional repressor. For instance, a SUMO1-RelB fusion protein mimicking RelB-SUMOylation displayed a reduced transcriptional activity in comparison to wild type RelB. Consistently, inactivation of specific SUMOylation sites in the central part of RelB augmented the transcription activity of the corresponding RelB mutant. Taken together, our data suggest that SUMOylation might be a potential molecular mechanism involved in reprogramming RelB, thus contributing to its functional diversity.

The Ca2+-dependent phosphatase calcineurin controls the formation of the Carma1-Bcl10-Malt1 complex during T cell receptor-induced NF-kappaB activation.

T cell receptor (TCR) ligation induces increased diacylglycerol and Ca(2+) levels in T cells, and both secondary messengers are crucial for TCR-induced nuclear factor of activated T cells (NF-AT) and NF-κB signaling pathways. One prominent calcium-dependent enzyme involved in the regulation of NF-AT and NF-κB signaling pathways is the protein phosphatase calcineurin. However, in contrast to NF-AT, which is directly dephosphorylated by calcineurin, the molecular basis of the calcium-calcineurin dependence of the TCR-induced NF-κB activity remains largely unknown. Here, we demonstrate that calcineurin regulates TCR-induced NF-κB activity by controlling the formation of a protein complex composed of Carma1, Bcl10, and Malt1 (CBM complex). For instance, increased calcium levels induced by ionomycin or thapsigargin augmented the phorbol 12-myristate 13-acetate-induced formation of the CBM complex and activation of NF-κB, whereas removal of calcium by the calcium chelator EGTA-acetoxymethyl ester (AM) attenuated both processes. Furthermore, inhibition of the calcium-dependent phosphatase calcineurin with the immunosuppressive agent cyclosporin A (CsA) or FK506 as well as siRNA-mediated knockdown of calcineurin A strongly affected the PMA + ionomycin- or anti-CD3 + CD28-induced CBM complex assembly. Mechanistically, the positive effect of calcineurin on the CBM complex formation seems to be linked to a dephosphorylation of Bcl10. For instance, Bcl10 was found to be hyperphosphorylated in Jurkat T cells upon treatment with CsA or EGTA-AM, and calcineurin dephosphorylated Bcl10 in vivo and in vitro. Furthermore, we show here that calcineurin A interacts with the CBM complex. In summary, the evidence provided here argues for a previously unanticipated role of calcineurin in CBM complex formation as a molecular basis of the inhibitory function of CsA or FK506 on TCR-induced NF-κB activity.

Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-kappaB family member RelB.

RelB is the key component of the alternative NF-kappaB (nuclear factor kappaB) signalling pathway. However, RelB exerts also a negative effect via the recruitment of a DNMT1 (DNA methyltransferase 1)-Daxx (death domain-associated protein) complex to NF-kappaB target genes. Importantly, the molecular mechanisms which determine the functions of RelB are still largely unknown. In the present study, we aimed to analyse whether ubiquitination of RelB might be involved in the regulation of RelB. Indeed, RelB is constitutively polyubiquitinated in the B-cell lines Namalwa and 70Z/3. Although a PMA+ionomycin-induced augmentation of RelB polyubiquitination was linked to its proteasomal degradation in B-cells, the constitutive RelB polyubiquitination seems to affect non-proteasomal functions. Consistently, a significant RelB polyubiquitination in HEK (human embryonic kidney)-293 cells correlated with an augmentation of the transcriptional activity of RelB. Yet, neither nuclear localization nor DNA binding was enhanced by RelB polyubiquitination. Interestingly, basal RelB polyubiquitination depends neither on Lys(48) nor on Lys(63) conjugates, but might involve unconventional ubiquitin conjugates. Mapping of the ubiquitination target sites in RelB revealed the existence of various lysine residues, which serve as ubiquitination acceptors. However, only the substitution of Lys(273/274) and Lys(305/308) significantly decreased the basal RelB activity and the ubiquitin-induced augmentation of the RelB activity. Collectively, these results imply a dual role of RelB polyubiquitination for the stability and activity of this transcription factor.

Identification of a 94-bp GC-rich element in the smooth muscle myosin heavy-chain promoter controlling vascular smooth muscle cell-specific gene expression.

The previously described rabbit 2.3-kilobase smooth muscle myosin heavy-chain (SMHCwt) promoter targets gene expression in transgenic animals to vascular smooth muscle cells (SMCs), including coronary arteries. Therefore, SMHCwt is thought to provide a promising tool for human gene therapy. In the present study, we examined tissue specificity and expression levels of wild-type and mutated SMHC promoters within the system of high-capacity adenoviral (hcAd) vectors. SMHCwt and a series of SMHC promoter deletion mutants, a triple promoter as well as a cytomegalovirus-SMHC hybrid promoter driving the enhanced green fluorescence protein (EGFP) reporter gene were transiently transfected into aortic SMCs. Fluorescence intensity was measured by flow cytometric analysis. Consecutively, hcAd vectors were constructed with the SMHCwt and the mutant promoter with the highest fluorescence activity. Levels of EGFP expression were determined after transduction of SMCs derived from human coronary arteries. For analysis of tissue specificity, embryonic stem (ES) cell-derived SMCs (ESdSMHCs) and cardiomyocytes (ESdCMs) were used. In comparison with SMHCwt, only the SMHCdel94 mutant lacking a 94-bp GC-rich element revealed a 1.5-fold increased fluorescence activity. Transduction of primary SMCs of human coronary arteries with hcAd vectors confirmed an increased EGFP expression driven by the SMHCdel94 promoter. In ES-cell-derived embryoid bodies, SMHCwt was exclusively active in transduced ESdSMCs. In contrast, expression of SMHCdel94 was also found in ESdCMs and other nontarget cells of the embryoid body. The tissue-specific rabbit SMHCwt promoter seems to be suitable for adenoviral gene transfer in SMCs of human coronary arteries and deletion of a 94-bp negative cis-acting GC-rich element results in loss of specificity.