Abstract

HIV-1 Tat protein, acting at the cell membrane, stimulates the production by human monocytes of TNF-alpha, a cytokine implicated in both HIV-1 replication and pathogenesis. Here, we analyze, in primary human monocytes, the mechanisms involved in Tat-stimulated calcium mobilization and its relationship with TNF-alpha production. We show that the Tat protein induces a calcium signal by mobilizing calcium from extracellular stores. This calcium signal is totally blocked when cells are stimulated in the presence of DHP receptor inhibitors such as nimodipine or calcicludine, thus suggesting the implication of this L-type calcium channel. By using RT-PCR amplification, Western blot with antibodies directed against the alpha1D subunit, binding assays with specific agonists or antagonists, and inhibition with specific antisense oligonucleotides, we show that DHP receptors are expressed and functional in primary human monocytes. Interestingly, we demonstrate that Tat-induced calcium mobilization is tightly linked to TNF-alpha production, thus indicating that Tat-induced mobilization and TNF-alpha production are entirely mediated by DHP receptors, as shown by their total inhibition by nimodipine, calcicludine, or anti-alpha1D antisense oligonucleotides.