RESUMO
Kaposi's sarcoma (KS) is a malignancy associated to conditions of immune system impairment such as HIV-1 infection and post-transplantation therapy. Here we report that HIV-1-Tat protein, at concentrations well below those detected in AIDS patients, up-regulates the expression of both CD40 and CD154 on KS cells. This occurred also in the presence of vincristine, that at doses shown to induce apoptosis decreased the expression of both CD40 and CD154 on KS cells. The treatment with a soluble CD40-muIg fusion protein (CD40 fp) that prevents the binding of CD154 with cell surface CD40, as well as the transfection with a vector for soluble CD40 (KS sCD40), decreased the anti-apoptotic effect of Tat. Moreover, Tat-induced motility of KS cells was inhibited by soluble CD40 fp. Tat also enhanced the expression of intracellular proteins known to transduce signals triggered by CD40 engagement, in particular TRAF-3. Tat as well as soluble CD154 (sCD154) prevented vincristine-induced reduction of TRAF-3 in KS cells transfected with a vector for neomycin resistance (KS psv-neo), but not in KS sCD40. Immunoprecipitation studies showed that Tat induced CD40 / TRAF-3 association and that this binding was abrogated upon the incubation with the soluble CD40 fp. These data suggest that Tat activates the CD40-CD154 pathway by enhancing the membrane expression of CD40 and in particular of CD154, and by activating the TRAF-3-dependent signaling pathway of CD40. These findings indicate that the CD40-CD154 pathway mediates the anti-apoptotic and migratory effects of HIV-1- Tat, suggesting the potential therapeutic benefits of blocking CD40 activation in HIV-1-associated KS.
Assuntos
Apoptose/efeitos dos fármacos , Ligante de CD40/biossíntese , Movimento Celular/efeitos dos fármacos , Produtos do Gene tat/farmacologia , Sarcoma de Kaposi/metabolismo , Western Blotting , Antígenos CD40/biossíntese , Antígenos CD40/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/biossíntese , DNA de Neoplasias/isolamento & purificação , HIV-1/metabolismo , Humanos , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Indicadores e Reagentes , Análise de Sequência com Séries de Oligonucleotídeos , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Deposition of atherogenic lipoproteins is associated with various glomerular diseases. In particular, oxidized LDL (oxLDL) may affect mesangial cells and favour the development of glomerulosclerosis. The aim of the present study was to investigate on cultured human mesangial cells (HMC) whether oxLDL induces apoptosis by a mechanism dependent on the inhibition of Akt survival pathway, and whether the engagement of mesangial CD40 by its ligand CD154 inhibits the apoptotic effect of oxLDL. Tunel assays demonstrated that incubation of HMC for 24 h with oxLDL, but not with unmodified LDL, induced a dose-dependent increase in apoptosis of HMC associated with a decrease in Akt phosphorylation. Enzymatic kinase assay showed that also the Akt activity was reduced in a dose-dependent manner by treatment with oxLDL. Stimulation of mesangial CD40 with sCD154 rescued HMC from oxLDL-dependent apoptosis, while two unrelated pharmacological inhibitors of PI3K LY294002 and wortmannin abrogated this anti-apoptotic effect, suggesting an involvement of the PI3K/Akt pathway. Moreover CD40 stimulation maintained an elevated phosphorylation of Akt and preserved its enzymatic activity in the presence of oxLDL. Indeed, CD154 induced a rapid enhancement in Akt enzymatic activity, that was temporarily correlated with the association of CD40 with TRAF3, TRAF6, c-Cbl and the p85 subunit of PI3K. In conclusion, these results suggest that CD40 stimulation protects HMC from toxic effects of oxLDL by promoting PI3K/Akt-dependent cell survival.