Role of Cav-1 in HIV-1 Tat-Induced Dysfunction of Tight Junctions and Aβ-Transferring Proteins

Abstract EN

Objective.

To evaluate the role of caveolin-1 (Cav-1) in HIV-1 Tat-induced dysfunction of tight junction and amyloid β-peptide- (Aβ-) transferring proteins.

Methods.

A Cav-1 shRNA interference target sequence was cloned into the lentiviral vector pHBLV-U6-Scramble-ZsGreen-Puro and verified by double enzyme digestion and DNA sequencing.

Human cerebral microvascular endothelium (HBEC-5i) cells were transduced with viral particles made in 293T cells by transfection with lentiviral packaging plasmids.

HBEC-5i cells transduced with Cav-1 shRNA or Ctr shRNA were exposed to HIV-1 Tat for 24 h, and the protein and mRNA levels of the tight junction protein occludin, Aβ-transferring protein, receptor for advanced glycation end products (RAGE), low-density lipoprotein receptor-related protein- (LRP-) 1, and RhoA were evaluated with Western blot and real-time reverse transcription polymerase chain reaction (qRT-PCR) assays, respectively.

Results.

After sequencing, an RNA interference recombinant lentivirus expressing a vector targeting Cav-1 was successfully established.

The recombined lentiviral particles were made by using 293T cells to package the recombined lentiviral vector.

A stable monoclonal cell line with strong GFP expression was acquired with a Cav-1 knockdown rate of 85.7%.

The occludin protein and mRNA levels in the Ctr shRNA group were decreased with HIV-1 Tat exposure but were upregulated in the Cav-1 shRNA group.

The HIV-1 Tat-induced alterations of RAGE and LRP-1 protein and mRNA levels in the Ctr shRNA group were attenuated in the Cav-1 shRNA group.

The RhoA protein levels in the Ctr shRNA group were upregulated by HIV-1 Tat exposure but were downregulated in the Cav-1 shRNA group.

Conclusion.

These results show that HIV-1 Tat-induced downregulation of occludin and LRP-1 and upregulation of RAGE and RhoA may result in the accumulation of Aβ in the brain.

Silencing the Cav-1 gene with shRNA plays a key role in the protection against HIV-1 Tat-induced dysfunction of the blood-brain barrier and Aβ accumulation.