u-0126 and 1-(2-4-dichlorobenzyl)indazole-3-carbohydrazide

Recent studies have shown that transforming growth factor (TGF)-beta3 regulates blood-testis barrier (BTB) dynamics in vivo, plausibly by determining the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB site via the p38 MAP kinase signaling pathway. Since BTB is composed of coexisting TJs and basal ectoplasmic specializations [ES, a testis-specific adherens junction (AJ) type] in the seminiferous epithelium of the rat testis, we sought to examine if TGF-beta3 would also regulate anchoring junction dynamics. Using an in vivo model in which rats were treated with AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell AJs without affecting the integrity of TJs at the BTB, it was noted that the event of germ cell loss from the epithelium was associated with a transient surge in TGF-beta3. Furthermore, it was also associated with a surge in the protein levels of Ras, p-ERK, and the intrinsic activity of ERK, illustrating TGF-beta3 apparently regulates Sertoli-germ cell ES function via the Ras/MEK/ERK signaling pathway. Indeed, pretreatment of rats with TbetaRII/Fc chimera, a TGF-beta antagonist, or U0126, a specific MEK inhibitor, could significantly delay and partially block the disruptive effects of AF-2364 in depleting germ cells from the epithelium. While the protein levels of the cadherin/catenin complex were significantly induced during AF-2364-mediated germ cell loss, perhaps being used to retain germ cells in the epithelium, this increase failed to reverse the loss of adhesion function between Sertoli and germ cells because of a loss of protein-protein interactions between cadherins and catenins. Collectively, these results illustrate that the testis has a novel mechanism in place in which an agent that primarily disrupts TJs can induce secondary loss of AJ function, leading to germ cell loss from the seminiferous epithelium. Yet an agent that selectively disrupts AJs (e.g., AF-2364) can limit its effects exclusively at the Sertoli-germ cell adhesive site without perturbing the Sertoli-Sertoli TJs.

Topics: Animals; beta Catenin; Butadienes; Cadherins; Cytoskeletal Proteins; DNA-Binding Proteins; ets-Domain Protein Elk-1; Extracellular Matrix; Gene Expression Regulation, Developmental; Hydrazines; Image Processing, Computer-Assisted; Immunoblotting; Immunohistochemistry; Immunoprecipitation; Indazoles; Kinetics; Male; MAP Kinase Signaling System; Membrane Proteins; Mice; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 3; Models, Biological; Nitriles; Occludin; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins; ras Proteins; Rats; Rats, Sprague-Dawley; Seminiferous Epithelium; Sertoli Cells; Signal Transduction; Testis; Tight Junctions; Time Factors; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta3; Up-Regulation