Monocyte adherence results in the rapid transcriptional activation and mRNA stabilization of numerous mediators of inflammation and tissue repair. While the enhancer and promoter elements associated with transcriptional activation have been studied, mechanisms linking adhesion, mRNA stabilization, and translation are unknown. GRO␣ and interleukin-1␤ (IL-1␤) mRNAs are highly labile in nonadhered monocytes but stabilize rapidly after adherence. GRO␣ and IL-1␤ transcripts both contain A؉U-rich elements (AREs) in the 3 untranslated region (UTR) which have been directly associated with rapid mRNA turnover. To determine if the GRO␣ ARE region was recognized by factors associated with mRNA degradation, we carried out mobility gel shift analyses using a series of RNA probes encompassing the entire GRO␣ transcript. Stable complexes were formed only with the proximal 3 UTR which contained the ARE region. The two slower-moving complexes were rapidly depleted following monocyte adherence but not direct integrin engagement. Deadherence reactivated the two largest ARE-binding complexes and destabilized IL-1␤ transcripts. Antibody supershift studies demonstrated that both of these ARE RNA-binding complexes contained AUF1. The formation of these complexes and the accelerated mRNA turnover are phosphorylation-dependent events, as both are induced in adherent monocytes by the tyrosine kinase inhibitor genistein and the p38 MAP kinase inhibitor of IL-1␤ translation, SK&F 86002. These results demonstrate that cell adhesion and deadhesion rapidly and reversibly modify both cytokine mRNA stability and the RNA-binding complexes associated with AUF1.

Monocyte adhesion resulted in rapid tyrosine phosphorylation and subsequent cytokine mRNA induction. The objective of this study was to determine the role of specific tyrosine phosphorylation events, particularly those involving members of the MAP kinase family, in regulating adhesioninduced cytokine expression. Using nuclear run-on analyses, we demonstrated that on adhesion, monocytes rapidly transcriptionally activated numerous cytokine mRNAs, coincident with the activation of the transcription factors NF-B and AP-1. Both an inhibitor of tyrosine phosphorylation, genistein, and the cytoplasmic tyrosine phosphatase PTP1B, were unable to prevent adhesion-mediated transcriptional activation. However, both blocked adhesion-induced ERK and JNK but not p38 kinase activation and at the same time decreased the stability of interleukin-1␤ (IL-1␤) and IL-8 transcripts. In addition, whereas adhesive events occurred in the presence of genistein and PTP1B, monocyte spreading was markedly inhibited. Our results suggest that the majority of protein phosphorylation events are associated with adhesioninduced cytokine expression through transcript stabilization and cytoskeletal organization. A minority of protein phosphorylation events, not sensitive to genistein or PTP1B exposure, may be instrumental in regulating transcription. Thus the spectrum of protein tyrosine kinases required for transcription appear distinct from those involved in maintaining the stability of some cytokine mRNAs and the integrity of the cytoskeleton to which mRNA destined for translation must be associated. J. Leukoc. Biol. 67: 216-225; 2000.

SummaryMonocyte extravasation initiates reorganization of the cytoskeleton (CSK) and adhesion-dependent cytokine gene transcription. The actin CSK is thought to be crucial for compartmentalization and translation of mRNA, many of which contain AU-rich (ARE) instability motifs in the 3′ untranslated region. We investigated regulation of adhesion-induced IL-1β expression by the monocyte CSK. In serum-free adherent monocytes, the induced IL-1β mRNA was stable and did not coextract with actin filaments. In contrast, in cells adherent in autologous serum, IL-1β transcripts were unstable, coextracted with actin filaments and were associated with only transient activation of the mitogen-activated protein kinases (MAPK). Under both conditions of adherence, the ARE-binding protein AUF1/hnRNP D was readily extracted in the cytosolic fraction. Electro-injection with AUF1/ hnRNP D modified the actin CSK and, surprisingly, stabilized IL-1β transcripts. These data suggest that the control of mRNA degradation is linked with changes in the CSK. Mitogen-activated protein kinase activation or alterations in the availability of mRNA degradation factors may mediate these effects.

Summary Differences in IL-1b mRNA expression, stability and translation between non-adherent monocytes and those stimulated by adherence suggest that cytokine regulation is coupled to the function and assembly of cytoskeletal structures. In situ hybridization studies were performed to visualize expression and positioning of IL-1b mRNA in adherently cultivated monocytes. IL-1b mRNA expression was heterogeneous with high transcript levels found in spread or polarized cells. Transcripts were compartmentalized to the perinuclear region in spread cells, and partially redistributed with polarization. In contrast to mRNA distribution in other motile cell populations, IL-1b mRNA did not localize to the distal or proximal actin cytoskeleton. Perinuclear confinement of transcripts required intact actin microfilaments. Treatment with cytoskeleton disruption and detergent extraction suggested that most non-translated IL-1b mRNA was associated with intermediate filaments. In monocytes stimulated by LPS, IL-1b, but not IL-1Ra transcripts were redistributed and partially associated, yet not bound to actin microfilaments. The present study demonstrates that IL-1b mRNA expression and localization in adherent monocytes is interrelated with the cytoskeletal rearrangement upon adherence, spreading and polarization.