Arturo Ponce

Tight junctions and apical/basolateral polarity

SummaryPhysiological studies led to devise models of epithelial cells in which the membrane does not have its molecules distributed homogeneously, but polarized towards the apical or towards the basolateral regions. For a while, it was assumed that the TJ, acting as a fence between the two regions, would be responsible for this asymmetry. However, today the information available indicates not only that polarization may proceed independently of the TJ, but that this structure itself may attain its precise location due to a polarization process. Nevertheless, TJs may play a role in restricting to the apical or to the basolateral region those molecules that are free to diffuse in the plane of the cell membrane (e.g., lipids and protein that are not attached to cytoplasmic or extracellular structures).

Ouabain modulates epithelial cell tight junction

Epithelial cells treated with high concentrations of ouabain (e.g., 1 μM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell–cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K+ pumping nor disturb the K+ balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (JDEX). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, JDEX, and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell–cell contacts.

Expression of voltage dependent potassium currents in freshly dissociated rat articular chondrocytes.

The electrophysiological properties of voltage dependent potassium channels from freshly dissociated rat articular chondrocytes were studied. The resting membrane potential (-42.7±2.0 mV) was significantly depolarized by increasing concentrations of external potassium. No change was observed when external chloride concentration was varied. Addition of TEA, 4AP, α-Dendrotoxin and charybdotoxin depolarized resting membrane potential. Whole cell patch clamp studies revealed the presence of outwardly rectifying currents whose kinetic and pharmacological properties suggest the expression of voltage dependent potassium channels. Two kinds of currents were observed under the same experimental conditions. The first one, most frequently observed (80%), starts activating near -50 mV, with V1/2=-18 mV, Gmax=0.30 pS/pF. The second kind was observed in only 10% of cases; It activates near -40 mV, with1/2=+28.35 mV, Gmax=0.28 pS/pF pA/pF and does not inactivates. Inactivating currents were significantly inhibited by TEA (IC50=1.45 mM), 4AP (IC50=0.64 mM), CTX (IC50≈10 nM), α-Dendrotoxin (IC50 < 100 nM) and Margatoxin (IC50=28.5 nM). These results show that rat chondrocytes express voltage dependent potassium currents and suggest a role of voltage-dependent potassium channels in regulating membrane potential of rat chondrocytes.

Polarized Distribution of Chloride Channels in Epithelial Cells

Cl-currents in cultured epithelial cells (MDCK) are inhibited by 4-acetamido-4 ’ -isothiocyano-2,2’-disulfonic acid stilbene; 4,4 ’ -diisothiocyanatostilbene-2,2

Phosphorylation of Zona Occludens-2 by Protein Kinase Cε Regulates Its Nuclear Exportation

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCepsilon. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.

Expression of potassium channels in epithelial cells depends on calcium-activated cell-cell contacts

Harvesting MDCK cells with trypsin-EDTA reduces potassium currents (IK) to a mere 10%, presumably by hydrolysis of K+ channels, but replating at confluence restores them in 12–18 hr, through a process that requires transcription, translation and exocytic fusion of intracellular membrane vesicles to the plasma membrane (Ponce & Cereijido, 1991; Ponce et al., 1991a). In the present work we find that this restoration of IK also requires cell-cell contacts and the presence of 1.8 mm Ca2+. The role of extracellular Ca2+ may be substituted by 2.0 μm TRH, 10 nm PMA or 200 μg/ml DiC8, drags that stimulate the system of phospholipase C (PLC) and protein kinase C (PKC). Conversely, the recovery of IK triggered by Ca-dependent contacts can be blocked by 110 μm neomycin, 2.0 μm H7, and 250 nm staurosporine, inhibitors of PLC and PKC. These results suggest that the expression of new K+ channels depends on Ca2+-activated contacts with neighboring cells and that the information is conveyed through PLC and PKC, a process in keeping with changes in its enzymatic activity and cellular distribution of PKC. Plasma membrane is also reduced and restored upon harvesting and replating, and depends on Ca2+-activated contracts. However, the effects of the chemicals tested on IK differ from the ones they elicit on the recovery of plasma membrane, suggesting that cells can independently regulate their population of K+ channels and the surface of their membrane.

Synthesis of Plasma Membrane and Potassium Channels in Epithelial (MDCK) Cells

Epithelial (Madin-Darby canine kidney) cells suspended by treatment with trypsin-EDTA lose a large percentage of their surface membrane and K+ channels, which are recovered upon replating.

The intracellular fate of zonula occludens 2 is regulated by the phosphorylation of SR repeats and the phosphorylation/O-GlcNAcylation of S257

ZO-2 nuclear import and accumulation in speckles is regulated by phosphorylation of its SR repeats by SRPK1 in a process initiated by EGF activation of AKT. ZO-2 nuclear exportation is favored by O-GlcNAc of S257 at the nucleus, whereas maturation of tight junctions is accompanied by ZO-2 phosphorylation at S257 by PKCζ.

The Role of Swelling-Activated Chloride Currents (I CL,swell ) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

Background/Aims: Articular chondrocytes dwell in an environment that is continuously changing its osmolarity as a consequence of mechanical loading, yet their volume regulation capabilities (RVD) are not fully understood. This work aimed to determine the osmotic sensitivity of freshly isolated rat chondrocytes, their RVD capabilities and to study the properties of any anion currents associated. Methods: Cell volume responses were determined by microscopy. Whole cell patch clamp was used to record ion currents. Results: Chondrocytes showed to be osmotically sensitive and capable of RVD in a size-dependent manner. RVD was accompanied by activation of outwardly rectifying chloride currents, featuring time and voltage independent activation and inactivation at most depolarizing voltage levels, with an anion selectivity sequence of: SCN->I-≅NO3->Br->Cl->F->> Gluconate-> Methanesulphonate-, corresponding to Eisenman’s sequence I. They were also permeable to taurine. These currents were blocked by DIDS, SITS, 9-AC and NPPB and by drugs that block ICl,swell such as fluoxetine, phloretin, DCPIB and tamoxifen. RT-PCR assays show the presence of mRNA for CLC-3 and TMEM16A, that had been proposed as molecular determinants of ICl,swell currents. Conclusions: These findings indicate that freshly isolated rat articular chondrocytes have ICl,swell. The further finding that ICl,swell blockers alter the RVD response suggest that ICl,swell is involved in the RVD response of rat articular chondrocytes.

ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway

The absence of ZO-2 promotes an increase in cell size by two mechanisms: an increase in cyclin D, which extends the time that the cells spend in the G1 phase of the cell cycle, and an accumulation of YAP at the nucleus, which promotes its transcriptional activity, triggering the activation of the mTORC1 complex and its target, S6K1.