Transcription-Independent Turnover of IκBα during Monocyte Adherence: Implications for a Translational Component Regulating IκBα/MAD-3 mRNA Levels

Abstract

We identified IB␣/MAD-3 as an immediate-early gene in human monocytes that is expressed in response to a variety of signals, including adhesion, lipopolysaccharide, and phorbol myristate acetate. Within 5 min of monocyte adhesion, the level of the IB␣ protein is markedly diminished but is rapidly replaced in a cycloheximide-sensitive manner within 20 min. Accompanying the rapid turnover of the IB␣ protein is simultaneous translocation of NF-B-related transcription factors to nuclei of adhered monocytes. The demonstration that NF-B can regulate IB␣/MAD-3 gene transcription in other cell types suggested that the rapid increase in steady-state IB␣/MAD-3 mRNA levels we observed within 30 min of monocyte adherence would result from NF-B-dependent transcriptional stimulation of the IB␣/MAD-3 gene. Nuclear run-on analyses indicated that, instead, while several immediate-early cytokine genes, such as the interleukin 1␤ (IL-1␤) gene, were transcriptionally activated during monocyte adhesion, the rate of IB␣/MAD-3 gene transcription remained constant. The adherence-dependent increase in IB␣/MAD-3 mRNA levels was also not a consequence of mRNA stabilization events. Interestingly, while increases in both IL-1␤ and IB␣/MAD-3 mRNA levels were detected in nuclei of adherent monocytes, cytoplasmic levels of IL-1␤ mRNA increased during adherence whereas those of IB␣/MAD-3 mRNA did not. Taken together, our data suggest that two interactive mechanisms regulate monocytic IB␣/MAD-3 mRNA levels. We propose that adherent monocytes regulate nuclear processing (or decay) of IB␣/MAD-3 mRNA, thereby increasing mRNA levels without stimulating IB␣/MAD-3 gene transcription. Moreover, since inhibition of protein synthesis leads to accumulation of IB␣/MAD-3 mRNA without stimulating IB␣/MAD-3 gene transcription, we suggest that low cytoplasmic levels of IB␣/MAD-3 mRNA are maintained by a translation-dependent degradation mechanism.Transcription factor NF-B plays a critical role in the regulation of many immediate-early response genes associated with the host response to infection and tissue damage (reviewed in references 25 and 42). Rel/NF-B activity is modulated, in part, by a growing family of regulatory molecules, including IB␣, IB␥, the C-terminal domains of precursors for the p50 and p52 components of NF-B (p105 and p100, respectively), and Bcl-3 (for a recent review, see reference 8). The IB inhibitors function by sequestering Rel family members in the cytoplasm, effectively interfering with their transcription activation potential (reviewed in references 8 and 27). Recently, several groups have reported that cytokine, phorbol myristate acetate, or lipopolysaccharide (LPS) stimulation of a variety of malignant cell lines results in rapid degradation of the IB␣ protein (9,11,17,45,47,60). In each case, loss of IB␣ was associated with simultaneous translocation of p50/65 to the nucleus. This indicates that dissociation of NF-B from IB␣, a prerequisite for B-dependent transcriptional activation, requires proteolytic processing of the IB␣ inh...

Cell adhesion regulates gene expression at translational checkpoints in human myeloid leukocytes

“…Nevertheless, signal-dependent translational control clearly regulates rapid synthesis of UPAR in monocytes adherent to P-selectin (Figs. 3-5) and is likely to be a general mechanism of regulation of critical products in stimulated cells (16,17,45). Additional experiments indicate that other mRNAs (Table 1) are handled by mTOR in adherent monocytes (T.S.M., G.A.Z., unpublished experiments) and in other myeloid leukocytes (S. Lindemann, A.S.W., G.A.Z., unpublished results).…”

Regulation of A + U-rich Element-directed mRNA Turnover Involving Reversible Phosphorylation of AUF1

“…This event rapidly triggers the expression of a battery of cytokines and inflammatory mediators, involving both transcriptional and post-transcriptional mechanisms (15,48). By contrast, THP-1 monocytic leukemia cells grow constitutively in suspension but may become adherent following treatment with phorbol esters.…”

Interleukin-1-induced Nuclear Factor-κB-IκBα Autoregulatory Feedback Loop in Hepatocytes

“…2B), or PMA (Fig. 2C), IB␣ mRNA was detectable in control cells and rapidly depleted at 15 min, perhaps indicating the presence of translation-coupled degradation (24). However, IB␣ mRNA was robustly increased at 60 min, a time point when the peak in IB␣ resynthesis occurs, and gradually decays over the next 5 h. These data indicate that the IB␣ overshoot resynthesis is dependent on the enhanced expression of IB␣ mRNA.…”

“…In other studies, nuclear processing of IB␣ mRNA was proposed to be important in regulating IB␣ protein levels following monocyte adherence (24). In these cells, IB␣ mRNA is predominantly nuclear.…”

“…One potential mechanism, previously described to control IB␣ resynthesis in monocytes, was at the level of mRNA decay in the nucleus (24). To determine the relative distribution of IB␣ RNA, RNA from cytoplasmic and nuclear fractions from control and IL-1␣-stimulated HepG2 cells were analyzed for changes in IB␣ abundance by Northern blot (Fig.…”