Tissue extravazation of monocytes involves adhesive and motile steps which are dependent on integrin engagement and cytoskeletal reorganization leading to a generalized activation of transcription factors within 5 minutes of adherence. A hallmark of this robust response is the stabilization of most cytokine transcripts and the lack of corresponding translation in the absence of a second signal. In contrast, engagement of integrins on non-adhered monocytes results in equivalent levels of transcription, but these transcripts are unstable and are efficiently translated. Profound and rapid cytoskeletal changes accompany monocyte adherence to ECM components or plastic highlighted by the appearance of lamellipodia which are associated with stable IL-1 transcripts. In contrast, engagement of integrins on non-adhered cells results in internalization of the integrins and disaggregation of the existing cytoskeleton. It has been established that lamellipodia are the site of rapid reorganization of actin under the direct control of the GTPase, Rac. The 3'UTR of most cytokines contain repetitions of the basic AUUUA motif that is intimately involved in splicing and nuclear export of transcripts, stabilization of cytokine mRNA's, and may also be involved in regulating the translatability of these genes. Several distinct proteins have been shown to bind to these AU-rich elements (ARE's), including the hRNP's and a 37 kD protein, AUF1 which is able to initiate RNA degradation in vitro when associated with polyribosomes. We have used an ARE probe from the 3'UTR of GRO to assess the number and activity of proteins constitutively present in monocytes. Our data demonstrates that a single protein, AUF1, appears to predominant. Following adherence, there is a rapid loss of AUF1 binding activity consistent with transcript stabilization and a lack of translation. Conversely, AUF1 activity is retained following direct integrin engagement that leads to translation and transcript instability. In addition, deadherence results in a rapid return of AUF1 activity and IL-1 transcript instability. It is our hypothesis that the mRNA stabilization and translational blockade are regulated by a Rac-dependent organization of the cytoskeleton and subsequent phosphorylation of AUF1. To test our hypothesis, we will:
AIM1. Determine the basic parameters regulating AUF1 expression in monocytes and THP-1 cells.
AIM2. Determine if - AUF1 is the critical factor regulating monokine mRNA stability and if transcript stability is controlled by a GTP-ase-dependent phosphorylation pathway.
AIM3. Examine the hypothesis that the principle role of AUF1 is to facilitate translation.
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