Activation of the Ca²⁺sensing receptor and the PKC/WNK4 downstream signaling cascade induces incorporation of ZO-2 to tight junctions and its separation from 14-3-3

Abstract

Zonula occludens-2 (ZO-2) is a tight junction (TJ) cytoplasmic protein, whose localization varies according to cell density and Ca2+ in the media. In cells cultured in low calcium (LC), ZO-2 displays a diffuse cytoplasmic distribution, but activation of the Ca2+ sensing receptor (CaSR) with Gd3+ triggers the appearance of ZO-2 at the cell borders. CaSR downstream signaling involves activation of protein kinase C, which phosphorylates and activates with no lysine kinase-4 that phosphorylates ZO-2 inducing its concentration at TJs. In LC, ZO-2 is protected from degradation by association to 14-3-3 proteins. When monolayers are transferred to normal calcium, the complexes ZO-2/14-3-3ζ and ZO-2/14-3-3σ move to the cell borders and dissociate. The 14-3-3 proteins are then degraded in proteosomes, whereas ZO-2 integrates to TJs. From the plasma membrane residual ZO-2 is endocyted and degradaded in lysosomes. The unique region 2 of ZO-2, and S261 located within a nuclear localization signal, are critical for the interaction with 14-3-3 ζ and σ and for the efficient nuclear importation of ZO-2. These results explain the molecular mechanism through which extracellular Ca2+ triggers the appearance of ZO-2 at TJs in epithelial cells and reveal the novel interaction between ZO-2 and 14-3-3 proteins, which is critical for ZO-2 protection and intracellular traffic.

Protective Effects of Baicalin on Peritoneal Tight Junctions in Piglets Challenged with Glaesserella parasuis

“…At the same time, ZO-1 is also the existence of cytoskeleton in PKC signal transduction pathway on cell membrane junction surface [ 42 ]. In the process of tight junction decomposition, ZO-2 is phosphorylated by the atypical PKC serine located at tight junctions and combined with ZO-2 [ 43 , 44 ]. The isotypes of PKC and ZO-1 are co-located on the side of plasma membrane, and ZO-1 may be directly phosphorylated by PKC during tight junction assembly [ 37 ].…”

ZO-2 Is a Master Regulator of Gene Expression, Cell Proliferation, Cytoarchitecture, and Cell Size

“…ZO-2 stability augments with TJ establishment and maturity. Thus, the half-life of ZO-2 in epithelial MDCK cells goes from 7 h in cells cultured in media with low Ca 2+ (1–5 µM) that cannot form TJs, to 19.7 h in cultures with 1.8 mM Ca 2+ [41] and from 8.6 h in sparse cultures to 19.1 h in confluent cultures [3].…”

“…However, if the calcium-switch was done in ZO-2 silenced monolayers, a significant delay occurred in the arrival to the cell borders of ZO-1, occludin, and E-cadherin [17]. Integration of ZO-2 to the TJ is triggered by a signaling cascade that starts at the plasma membrane with the activation of the Ca 2+ -sensing receptor (CaSR) [41], a G-protein coupled receptor [50]. CaSR signals through the Gα q/11 subunit, which activates phospholipase C (PLC).…”

“…The latter induces the release of Ca 2+ from the endoplasmic reticulum, while DAG leads to the activation of nPKCε. This kinase phosphorylates the kinase WNK4 that binds and phosphorylates ZO-2 in serine residues, triggering its disassembly from 14-3-3 and its relocation to TJs [41].…”

E7 oncoprotein from human papillomavirus 16 alters claudins expression and the sealing of epithelial tight junctions

“…A non-cancerous cell line of cervix from a mouse or other mammal was not used as to the best of our knowledge these are not currently commercially available. In addition, from the seminal study of TJs in MDCK cells performed by Cereijido et al (117) in 1978, the cell line has been widely used to investigate the electrical properties of TJs, the permeability of the paracellular pathway (118)(119)(120), the changes in the ultrastructure of TJs visualized in freeze-fracture replicas (38,121), the molecular composition of TJs (46,(122)(123)(124)(125)(126), as well as the response of TJs to a wide variety of factors, including temperature (127), ions (128)(129)(130), signaling cascades (131,132), toxins (133) and growth factors (134,135). Moreover, the role of claudins, in particular claudins -1 (136,137), -2 (46,125,138), -4 (46,125,126) and -10 (33,139) has been extensively studied in MDCK cells.…”