cAMP perturbs inter-Sertoli tight junction permeability barrier in vitro via its effect on proteasome-sensitive ubiquitination of occludin.
Throughout spermatogenesis, inter-Sertoli tight junctions (TJs) that constitute the blood-testis barrier must be disassembled and reassembled to permit the timely movement of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism and the participating molecules that regulate the bioavailability of TJ proteins are entirely unknown. Using Sertoli cell culture, it was shown that there was an increase in occludin level, concomitant with a reduction of an E3 ubiquitin ligase, Itch, at the time when inter-Sertoli TJs were assembled. By co-immunoprecipitation, occludin was shown to associate with Itch at the TJs. A novel interaction between Itch and UBC4 (an ubiquitin-conjugating enzyme) was identified. When TJs were disrupted by dibutyryl-cAMP (db-cAMP), an increase in protein levels of Itch and UBC4 along with a significant reduction in endogenous occludin was detected. These results seemingly suggest that the interaction of Itch and UBC4 on occludin is potentially involved in regulating Sertoli TJ dynamics. Addition of a proteasome inhibitor, MG-132, into Sertoli cells cultured with db-cAMP blocked the db-cAMP-induced occludin loss in vitro. Accumulations of ubiquitin-conjugated and Itch-conjugated occludin were detected in Sertoli cells cultured in the presence of both MG-132 and db-cAMP. These results suggest that MG-132 prevented db-cAMP-induced TJ disruption by altering the rate of occludin degradation. Taken collectively, the results reported herein support the notion that db-cAMP-induced TJ disruption was mediated by an induction of Itch protein expression, which in turn triggered the ubiquitination of occludin resulting in TJ disruption.
Lui
Wing Yee
WY
Department of Zoology, The University of Hong Kong, Pokfulam, Hong Kong.
Lee
Will M
WM
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D015536:Down-Regulation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D062793:Occludin; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D012708:Sertoli Cells / Q000187:drug effects / Q000378:metabolism*; D013091:Spermatogenesis / Q000502:physiology; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; C568987:Ocln protein, rat; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D003994:Bucladesine; D000242:Cyclic AMP; C471637:Itch protein, mouse; D044767:Ubiquitin-Protein Ligases; D020930:MAP Kinase Kinase Kinases; C414538:MAP kinase kinase kinase ubc4; D046988:Proteasome Endopeptidase Complex