Asin, Susana

Abstract Alveolar macrophages play a crucial role in initiating the inflammatory response in allergic asthma through the cross-linking of the low affinity IgE receptors (FcεRIIb or CD23) by IgE-allergen immunocomplexes. We have previously shown that CD23 cross-linking in monocytes and U937 cells targets IκBα, leading to the activation of the transcription factor NF-κB. We demonstrate in this paper that CD23-initiated signaling in U937 cells leads to hyperphosphorylation of IκBα at Ser32/Ser36 residues. Overexpression of a dominant-negative IκBα transgene containing mutations at Ser32/Ser36 completely inhibits degradation of IκBα, NF-κB activation, and gene transcription that follows CD23 cross-linking. Investigation of the second messengers mediating the CD23-dependent activation of NFκB demonstrates that IκB kinases (IKKs) but not p90rsk are selectively activated following CD23 cross-linking and mediates the phosphorylation of IκBα. Cotransfection experiments with an IKKβ negative dominant completely inhibit CD23 induced NFκB activation. Furthermore, the activation of tyrosine kinase(s) by CD23 is required for the induction of IKK activity, IκBα degradation, and NF-κB nuclear translocation. Taken together, our results show that CD23 cross-linking in the monocytic lineage induces tyrosine kinase activation followed by activation of IKK, which phosphorylates IκBα at the N-terminal domain (Ser32/Ser36), inducing its degradation, NF-κB activation and gene transcription.

ABSTRACTHuman monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-κB in the nucleus secondary to increased IκBα, IκBβ, and IκBɛ degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IκB, we have sought to identify the regulatory domains of IκBα targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IκBα, we determined that persistent HIV infection of these cells targets the NH2but not the COOH terminus of IκBα. Further analysis demonstrated that phosphorylation at S32and S36is necessary for HIV-dependent IκBα degradation and NF-κB activation. Of the putative N-terminal IκBα kinases, we demonstrated that the Iκκ complex, but not p90rsk, is activated by HIV infection and mediates HIV-dependent NF-κB activation. Analysis of viral replication in cells that constitutively express IκBα negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-κB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.

ABSTRACTLatently human immunodeficiency virus (HIV)-infected memory CD4+T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCθ, PKCθA148E, and PKCα, PKCαA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4+T cells and that both PKCθA148E and PKCαA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCα and PKCθ with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCα or PKCθ. Since suppression of PKCα and PKCθ together has no greater inhibitory effect on HIV reactivation than inhibition of PKCα alone, our data confirm that PKCα and PKCθ act in sequence. The requirement for PKCα and PKCθ for prostratin-induced HIV reactivation and the ability of selective PKCα or PKCθ agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.

ABSTRACTThe role of NF-κB in the reactivation of human immunodeficiency virus (HIV) from latency in CD4 T lymphocytes is well documented. However, its role in driving HIV transcription in human macrophages, which contain a constitutive nuclear pool of NF-κB, is less well understood. In this study we have investigated the role that the constitutive pool of NF-κB and the NF-κBcis-acting motifs of the HIV long terminal repeat (LTR) play in regulating HIV transcription in human monocytic cells and primary macrophages. Inhibition of the constitutive nuclear pool of NF-κB (RelA and RelB) in the promonocytic U937 cell line using dominant-negative IκBα significantly decreases HIV replication. Moreover, it is demonstrated that in the differentiated monocytic cell line THP1, which contains a constitutive nuclear pool of NF-κB (RelB),an HIV provirus containing mutations of the κBcis-acting sites in the LTR is transcriptionally impaired. Reduction of the constitutive pool of NF-κB in human macrophages by an adenovirus vector expressing a dominant-negative IκBα also reduces HIV transcription. Lastly, mutation of the NF-κBcis-acting sites in the LTR of an R5 HIV provirus completely abrogates the first cycle of HIV transcription. These studies indicate that thecis-acting NF-κB motifs of the HIV LTR are critical in initiating HIV transcription in human macrophages and suggest that the constitutive nuclear pool of NF-κB is important in regulating HIV transcription in these cells.