Knock Out of CGN and CGNL1 in MDCK Cells Affects Claudin-2 but Has a Minor Impact on Tight Junction Barrier Function

Cingulin (CGN) and paracingulin (CGNL1) are cytoplasmic proteins of tight junctions (TJs), where they play a role in tethering ZO-1 to the actomyosin and microtubule cytoskeletons. The role of CGN and CGNL1 in the barrier function of epithelia is not completely understood. Here, we analyzed the effect of the knock out (KO) of either CGN or CGNL1 or both on the paracellular permeability of monolayers of kidney epithelial (MDCK) cells. KO cells displayed a modest but significant increase in the transepithelial resistance (TER) of monolayers both in the steady state and during junction assembly by the calcium switch, whereas the permeability of the monolayers to 3 kDa dextran was not affected. The permeability to sodium was slightly but significantly decreased in KO cells. This phenotype correlated with slightly increased mRNA levels of claudin-2, slightly decreased protein levels of claudin-2, and reduced junctional accumulation of claudin-2, which was rescued by CGN or CGNL1 but not by ZO-1 overexpression. These results confirm previous observations indicating that CGN and CGNL1 are dispensable for the barrier function of epithelia and suggest that the increase in the TER in clonal lines of MDCK cells KO for CGN, CGNL1, or both is due to reduced protein expression and junctional accumulation of the sodium pore-forming claudin, claudin-2.</p

RAP and LRP-1 silencing by siRNA decreased the level and density of lysosome in MCF-7R cells.

MCF-7S, MCF-7R cells that incubated with or without 500 nM RAP for 12 hours were used in this experiment. The endocytic organelles were isolated by density gradient centrifugation as detailed in Materials and Methods. sucrose gradient was analysed using invertase enzyme assay as described in Materials and Methods. Detection of P-gp and Lamp-1 were evaluated by Western-blot in all collected aliquots (A-D). The intensity of the bands was quantified by densitometry using quantity one program. Student’s t-test was used for the statistical significance of different values. ** p < 0.01, *** p < 0.001 for MCF-7R cells versus MCF-7S cells, ## p < 0.01, ### p < 0.001 for MCF-7R treated cells versus MCF-7R untreated cells (E,F,G).</p

RAP and LRP-1 silencing by siRNA promoted the caspase-7 activation in Dox-treated MCF-7R cells.

MCF-7S, MCF-7R, scRNA-MCF-7R and siRNA-MCF-7R cells were incubated with 5 μM Dox with or without 500 nM RAP for 12 hours. A, Procaspase-7 and cleaved caspase-7 were detected by Western blotting. β-actin antibody was used as a control. The results were represented by three independent experiments. Quantity one software was used to quantify the intensity of the bands. Student’s t-test was used for the statistical significance of different values. *** pB, Caspase-7 activity was measured by caspACE assay kit. The results obtained from three independent experiments were represented with standard deviation (S.D.). Student’s t-test was used for the statistical significance of different values. ** p<0.01 and **** p<0.0001 compared to control cells. ¥¥¥ p<0.001 compared to Dox-treated cells.</p

Cingulin and paracingulin tether myosins-2 to junctions to mechanoregulate the plasma membrane

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.</p

Schematic diagram of P-gp internalization by LRP-1 receptor and its implication in MCF-7 cells drug resistance.

Schematic diagram of P-gp internalization by LRP-1 receptor and its implication in MCF-7 cells drug resistance.</p

RAP and LRP-1 silencing by siRNA promoted the ERK1/2 phosphorylation in Dox-treated MCF-7R cells.

A, MCF-7S cells were incubated with 1 μM Dox for 0.5, 1 and 2 hours. Detection of pERK, ERK, pAKT, AKT, Phospho-p38 and p-38 were evaluated by Western-blot. A representative blot of three independent experiments was shown. The intensity of the bands was quantified by densitometry using quantity one program. The ratio was calculated with densitometry value of the pERK/ densitometry value of ERK. Student’s t-test was used for the statistical significance of different values. *** p B, MCF-7S and MCF-7R cells were incubated with 1 μM Dox with or without 500 nM RAP for 0.5, 1 and 2 hours. Detection of pERK and ERK were evaluated by Western-blot. A representative blot of three independent experiments was shown. The intensity of the bands was quantified by densitometry using quantity one program. The ratio was calculated with densitometry value of the pERK/ densitometry value of ERK. Student’s t-test was used for the statistical significance of different values.° NS, ** p C, MCF-7S cells were incubated with or without 500 nM RAP for 0.5, 1 and 2 hours. Detection of pERK and ERK were evaluated by Western-blot. A representative blot of three independent experiments was shown. The intensity of the bands was quantified by densitometry using quantity one program. The ratio was calculated with densitometry value of the pERK/ densitometry value of ERK. Student’s t-test was used for the statistical significance of different values.° NS, RAP treated cells versus untreated cells.</p

RAP sensitized MCF-7R cells to Dox cytotoxic effects and reduced IC₅₀.

A and B, MCF-7S and MCF-7R cells were treated with different Dox concentrations (from 0 to 10 μM) with or without 500 nM RAP. After 48 and 72h, cell viability was measured by UptiBlue Viable Cell Counting Assay. The results were presented as percentage of control and represented with standard deviation (S.D.) of at least three independent experiments. Student’s t-test was used for the statistical significance of different values.° NS, *** pC, MCF-7R cells were pretreated with Verapamil (5 μM) for 6h and incubated with Dox (1 μM). After 48 and 72h, cell viability was measured using UptiBlue Viable Cell Assay. The results obtained from three independent experiments (% of control) were represented with standard deviation (S.D.). Student’s t-test was used for the statistical significance of different values. **** pD, summary table of IC50 and RI (resistance Index). IC50 represents the mean half maximal inhibitory concentration. RI was assessed using the quotient of the IC50 values (IC50 MCF-7R/IC50 MCF-7) in each treatment conditions. Student’s t-test was used for the statistical significance of different values. *** p<0.001 for RAP-MCF-7R cells compared to untreated MCF-7R cells.</p

Overexpression of P-gp and LRP-1 in MCF-7R.

MCF-7S and MCF-7R cells were cultured for 24 hours. Detection of P-gp, Lamp-1 and LRP-1/β chain was evaluated by Western-blot. β-actin antibody was used as a control. The results were represented by three independent experiments. Quantity one software was used to quantify the intensity of the bands. Student’s t-test was used for the statistical significance of different values. *** p<0.001 for MCF-7R cells compared to MCF-7S cells. The ratio was calculated with densitometry value of the protein of interest/ densitometry value of the β-actin.</p

ERK1/2 inhibition reduced Dox cytotoxic effects on MCF-7 cells.

MCF-7S and MCF-7R cells were incubated with 1 μM Dox with or without 500 nM RAP and 1 μM U0126 48 hours. A, Cell viability was measured using UptiBlue Viable Cell Assay. B, Caspase-7 activity was measured by caspACE assay kit. The results obtained from three independent experiments (% of control) were represented with standard deviation (S.D.). Student’s t-test was used for the statistical significance of different values. *** p<0.001 compared to Dox-treated cells and ¥¥¥ p<0.001, ¥¥¥¥ p<0.0001 compared to Dox- and RAP-treated cells.° NS, U0126-treated cells versus Dox-treated cells.</p