HIV-1 Tat protein alter the tight junction integrity and function of retinal pigment epithelium: an in vitro study.
BACKGROUND
How HIV-1 enter into the eyes remains obscure. We postulated that HIV-1 Tat protein can alter the expression of specific tight-junction proteins and disturb the blood retinal barrier, and contributes to HIV trafficking into the eyes. This study is to determine the effects of HIV-1 Tat proteins on the barrier function and tight-junction protein expression of retinal pigment epithelial cell (RPE).


METHODS
A human RPE cell line (D407) cultured on microporous filter-supports was used. After treating with HIV-1 Tat protein, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudins were determined by real-time polymerase chain reaction, immunofluorescence, and Western blot analysis. Activation of ERK1/2 was detected by Western blot analysis with specific antiphospho protein antibodies. NF-kappaB DNA binding activity was determined by transcription factor assay. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in barrier destruction of RPE cells exposed to HIV-1 Tat.


RESULTS
Treating cultured human retinal pigment epithelial cells with 100 nM Tat for 24 hours increased the permeability and decreased the TER of the epithelial monolayer. HIV-1 Tat also disrupted and downregulated the tight-junction proteins claudin-1, claudin-3, and claudin-4 in these cells, whereas claudin-2 was upregulated, and the expression of occludin was unaffected. HIV-1 Tat protein also induced activation of ERK1/2 and NF-kappaB. HIV-1 Tat protein induced barrier destruction, changes in expression of TJs, and activation of ERK1/2 and NF-kappaB were abrogated by inhibitor of ERK1/2 and NF-kappaB.


CONCLUSIONS
HIV-1 Tat protein causes increases in the paracellular permeability of RPE cells in vitro concomitant with changes in expression of certain transmembrane proteins associated with the tight junction. The effects of HIV-1 Tat on barrier function of the RPE may be mediated by ERK MAPK and NF-kappaB activation, which may represent potential targets for novel therapeutic approaches for the retinopathy induced by HIV infection.
Key Laboratory of Ophthalmology, Ministry of Education; Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, PR China. larkling@163.com
Ling
L
Bai
Zhenping
Z
Zhang
Hui
H
Zhang
Xiumei
X
Li
Qiurong
Q
Yu
Haotian
H
Lin
Wenhui
W
Yang
D002460:Cell Line; D002463:Cell Membrane Permeability; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D015497:HIV-1; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D062793:Occludin; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology; D019108:Tight Junctions / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005419:Flavonoids; D008565:Membrane Proteins; D016328:NF-kappa B; C568985:OCLN protein, human; D062793:Occludin; D013859:Thiocarbamates; D054322:tat Gene Products, Human Immunodeficiency Virus; C066229:prolinedithiocarbamate; D011392:Proline; D048049:Extracellular Signal-Regulated MAP Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one