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

Abstract

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.

Figure 1

Cav-1 inhibition with shRNA. 293T cells were transfected with the lentiviral packaging plasmids (pSPAX2, pMD2G, and Cav-1 shRNA), and the viral particles (Cav-1 shRNA) were collected. The virus titer of lentivector was 2 × 10⁸ TU/mL, as assessed by green fluorescence under a fluorescence microscope using the whole dilution method. Cav-1 expression in HBEC-5i was silenced by transduction with Cav-1 shRNA and Ctr shRNA lentiviral vectors. The Cav-1 inhibition rate of the stable monoclonal cell lines was 85.7%, as detected by qRT-PCR. Data are representative of three independent experiments. ^∗∗∗P < 0.001 versus control.

Figure 2

Cell viability assay. Effects of puromycin at 0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 μg/mL for 24 h on the viability of HBEC-5i cells were assessed by a CCK8 assay. Cell viability was affected over 1 μg/mL of puromycin (a). Cav-1 expression in HBEC-5i cells was silenced by transduction with lentiviral vectors of Cav-1 shRNA. Effects of HIV-1 Tat at 0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 μg/mL for 24 h on the viability of HBEC-5i cells silenced with Cav-1 shRNA were also assessed by a CCK8 assay. Cell viability was not affected by 1 μg/mL HIV-1 Tat for 24 h (b). Data are representative of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus control.

Figure 3

Role of Cav-1 shRNA in HIV-1 Tat-induced changes of occludin. Cav-1 expression in HBEC-5i cells silenced with Cav-1 shRNA or Ctr shRNA was exposed to HIV-1 Tat for 24 h. Occludin protein (a) and mRNA (b) levels were detected by Western blot or qRT-PCR, respectively. Occludin protein and mRNA levels in Ctr shRNA were decreased following HIV-1 Tat exposure but were upregulated in the Cav-1 shRNA group. Data are representative of three independent experiments. ^∗∗P < 0.01 compared to Ctr shRNA. ^##P < 0.01 compared to Ctr shRNA+HIV-1 Tat.

Figure 4

Role of Cav-1 shRNA in HIV-1 Tat-induced changes in RAGE and LRP-1. HBEC-5i with Cav-1 silencing by shRNA were exposed to HIV-1 Tat for 24 h. RAGE and LRP-1 protein and mRNA levels were detected by Western blot (a, c) and qRT-PCR (b, d). Compared with the Ctr shRNA, RAGE protein and mRNA levels were increased with HIV-1 Tat exposure but were downregulated in the Cav-1 shRNA group. Compared with the Ctr shRNA, LRP-1 protein and mRNA levels were downregulated by HIV-1 Tat exposure but were upregulated in the Cav-1 shRNA group. Data are representative of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01 compared to Ctr shRNA. ^##P < 0.01 compared to Ctr shRNA+HIV-1 Tat.

Figure 5

Role of Cav-1 shRNA in HIV-1 Tat-induced changes of RhoA. The RhoA protein levels were detected by Western blot. Compared with the blank control, HIV-1 Tat treatment increased the protein levels of RhoA with the longer exposure time (a). Compared with the Ctr shRNA, the RhoA protein level was upregulated following HIV-1 Tat exposure but was downregulated in the Cav-1 shRNA group (b). Data are representative of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 compared to Ctr shRNA. ^##P < 0.01 compared to Ctr shRNA+HIV-1 Tat.