Bardet-Biedl syndrome obesity: BBS4 regulates cellular ER stress in early adipogenesis

Background: Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy, presenting with early obesity onset. The etiology of BBS obesity involves both central and peripheral defects, through mechanisms mostly yet to be deciphered. We previously showed BBS4 expression in adipogenesis, peaking at day 3 of differentiation. Obesity is characterized by cellular stress which promotes pathological consequences. Aims: We set out to test a possible role of BBS4 in adipocyte endoplasmic reticulum (ER) stress-induced unfolding protein response (UPR). Methods: BBS4 silenced (SiBBS4) and overexpressing (OEBBS4) pre-adipocyte murine cell lines were subjected to ER-stress induction (Tunicamycin, TM) during adipogenesis. ER-stress UPR was analyzed at the transcript, protein and biochemical levels (microscopy, immunocytochemistry, western blotting, quantitative RT-PCR and X-box binding protein 1 (XBP-1) splicing). Results: In silico analysis showed that BBS4 harbors an ER localization sequences indicative of ER localization. We verified BBS4's ER localization in adipocytes by immunocytochemistry and cellular protein fractionation. Furthermore, we demonstrated that BBS4 expression is significantly up-regulated by ER-stress, as indicated by protein and transcript levels. SiBBS4 adipocytes exhibited swollen ER typical to ER-stress and significant XBP-1 down-regulation at day 3 of differentiation. Following ER-stress, SiBBS4 adipocytes exhibited XBP-1 ER retention, failure to translocate to the nucleus and depletion of the nuclear active cleaved ATF6α. BBS4 did not alter ATF6α processing by S1P and S2P in the Golgi. Notably, SiBBS4 cells demonstrated significant reduction in the downstream activated phospho-IRE1α, independent of ER-stress. Conclusions: At day 3 of adipogenesis, coinciding with the timing of its peak expression, BBS4 is localized to the ER and is involved in the ER stress response and trafficking. BBS4 depletion results in swollen ER with impaired intracellular nucleus translocation of XBP-1 and ATF6α. Thus, BBS4 affects the ER stress response in early adipogenesis, altering ER stress responsiveness and the adipocyte ER phenotype.