The subcellular compartmentalization of TGFbeta-RII and the dynamics of endosomal formation during the signaling events

Abstract

We previously showed that intraperitoneal administration of Freund's adjuvant treatment resulted in acute peritonitis and TGF-beta was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF-beta signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF-beta signaling. Using immunocytochemical approach, we could detect TbetaRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TbetaRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system. Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TbetaRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF-beta signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions.