To begin to dissect the effect of a specific ras mutation on tight junction permeability, we transfected the LLC-PK1 renal epithelial cell line with a construct that contained an activated ras mutation (valine-12). As shown in , the ras-transfected confluent cell sheets exhibited elevated levels of Ras compared with the untransfected and vector control (pBabe) cultures, in which endogenous Ras expression could be observed. The vector control showed expression level similar to LLC-PK1. Expression of Ras in the transfected population was heterogeneous, as shown by immunofluorescence in , with expression levels varying from one cell grouping to another. Expression of Ras in the Ras-A clonal subline was more homogeneous (our unpublished data).
Figure 1. Ras expression in LLC-PK1 cells. LLC-PK1 epithelial cells transfected with either the pBabe vector or that vector containing the activated ras gene were isolated by selection with puromycin and examined for Ras expression using a pan-Ras antibody. Western (more ...)
We first examined growth and morphology properties of the ras
-transfected subline. As shown in , compared with vector control cells (left), confluent cultures of the Ras transfectant (right) showed smaller domes, indicative of a change in transepithelial transport, barrier function, and/or cell adhesion properties. As shown in , the Ras transfectant shows a statistically greater number of domes, which are 50% smaller. The relationship of domes to fluid transport, and barrier function, properties described in , has been discussed previously (Leighton et al., 1970
; Lever, 1979
; Tanner et al., 1983
Figure 2. Morphology of confluent cultures of the pBabe vector control and the Ras-transfected LLC-PK1 cells. Cells were grown to confluence and examined by phase contrast light microscopy. The three-dimensional, fluid-filled domes or hemicysts, which are characteristic (more ...)
Altered dome formation as a result of Ras mutation
Effect of Ras transfection on transepithelial electrical parameters
Also evidenced in the Ras transfectant were occasional multilayered foci of cells (, right, white arrow). In thick section microscopy, this sporadic tendency by the Ras transfectant to multilayer is even better illustrated, and contrasts with the monolayer phenotype of LLC-PK1 and pBabe (). Perhaps in keeping with this tendency to multi-layer, the Ras subline manifested a slightly higher confluent cell density than LLC-PK1 or pBabe (), although growth curves have shown similar rates of cell division (our unpublished data).
Figure 3. Thick section light micrograph of toluidine blue-stained confluent cultures of LLC-PK1 and Ras-transfected cell sheets. Control cell sheets, both LLC-PK1 (top) and pBabe vector controls (indistinguishable from LLC-PK1), manifest single-cell layer-thick (more ...)
Effect of Ras mutation on confluent cell density
In spite of the property of multilayering, the normal morphological polarity of the LLC-PK1 cells (apical microvilli, apically situated desmosomes, and tight junctions) is still in evidence in electron micrographs of the Ras-transfected culture (, bottom right). The only cells whose morphological polarity would be in question would be those in the sublayers of the multilayered foci (, bottom left). Polarity and junctions are, however, clearly evident in the uppermost layer of the multilayered foci.
Figure 4. Transmission electron micrograph cross-sections of LLC-PK1 and Ras-transfected cells from postconfluent cultures seeded on permeable filters. Cells were seeded onto Millicell PCF filters and after 4 d fixed and processed as described in Materials and (more ...)
This morphological polarity of all three cell lines is reflected in a similar electrical polarity (), with all three cell lines manifesting a luminal negative potential difference, indicative of the apical-to basal-lateral Na+
transport demonstrated by LLC-PK1
cells (Misfeldt and Sanders, 1981
). After observing decreased size of domes in the Ras subline, it was unexpected to find that Rt
-transfected cell sheets was significantly (40%) higher than Rt
and pBabe cell sheets (, top, and ). As mentioned above, transmission electron microscopy did, however, show that tight junctions existed between all cell pairs of ras-transfected cells, even those over multilayered sites (). A contribution to this increased Rt
by the sporadic multilayering of cells is a possibility, but it should be noted that multilayering per se does not coincide with increased Rt.
In fact, it has been associated with decreased Rt
(Mullin et al., 1992
). The clonal (and more homogeneously multilayered) Ras-A subline showed an even greater Rt
and lower short circuit current (Iscc
) than that exhibited by the Ras line ().
Figure 5. Ras-transfected LLC-PK1 cell sheets exhibit greater transepithelial [14C]mannitol flux but also greater transepithelial electrical resistance than either control cell sheet. Transepithelial electrical measurements and radiotracer flux studies were performed (more ...)
This increased Rt of the Ras transfectant is in stark contrast to a dramatically increased transepithelial flux of d-mannitol across the cell sheets, an almost sevenfold increase (, bottom). Mannitol is a nonelectrolyte, and Rt is a measurement (in this cell line) of paracellular permeability to Na+ and Cl–. Therefore, it is possible that the paracellular “pore” induced by the Ras overexpression is specific for uncharged solutes. This unusual permeability phenotype seems to be specific for the Ras transfectant because a parallel study with Akt (protein kinase B)-transfected LLC-PK1 cells did not show this peculiarity (our unpublished data). Akt overexpression was observed to increase transepithelial mannitol flux but transepithelial electrical resistance was unchanged, indicating increased paracellular permeability to nonelectrolytes but unaltered permeability to small electrolytes (our unpublished data).
Our main focus in this study was to describe one particular aspect of the nature of the paracellular leak in the Ras-transfected cell sheets. Specifically, we sought to determine how large a solute could now pass across these cell sheets, and whether there was a “ceiling” to where a certain solute was too large to pass. The transepithelial flux of [14C]d-mannitol (Mr 182), [14C]polyethylene glycol (Mr 4000), 14C-methylated dextran (Mr 10,000), and C-methylated dextran (Mr 2,000,000), nonelectrolytes of increasing size, were then compared for each of the three cell lines. Each solute was chosen for its negligible affinity for intracellular uptake and consequent possible transcellular transit. The paracellular pathway was being evaluated in each case. The studies were performed as described in Materials and Methods and include the analyses of the molecular nature of the radioactivity coming across the cell sheet. These analyses are especially necessary for the dextran permeability studies because of our observation that both radiolabeled dextrans were heterogeneous as commercially supplied with regard to molecular weight. For the 10,000-Mr 14C-dextran, only 95% was found to be 10,000-Mr material, the remaining radioactivity being smaller fragment molecules. For the 2,000,000-Mr dextran, an average of only 25% was found to be in fact Mr 2,000,000. These determinations were made by passing the radiolabeled compounds through a Sephadex G25 or Sephacryl 300 column as described in Materials and Methods. All radioactivity coming across the cell sheets was similarly analyzed. The flux of total radioactivity was then corrected by this percentage, to reflect the flux of material of the actual molecular weight in question. The results of the gel filtration analyses were confirmed by centrifugation of the radiolabeled dextran material through Centricon filter units with molecular weight cutoffs of 5000 and 100,000.
Although the radiolabeled d-mannitol and polyethylene glycol were received as pure compounds of a specific molecular weight (Mr 182 and 4000, respectively), we likewise checked the chemical nature of the radioactivity crossing the cell sheets for [14C]mannitol and [14C]polyethylene glycol fluxes by TLC. In the [14C]mannitol flux studies, all radioactivity crossing all cell sheets was, in fact, mannitol. In the [14C]polyethylene glycol flux studies, only 50% of the radioactivity crossing pBabe cell sheets was actually polyethylene glycol, whereas for the (leakier) Ras cell sheets, 97% of the radioactivity coming across was true polyethylene glycol.
shows transepithelial flux results from the untransfected and pBabe and Ras-transfected cell lines comparing 10,000- and 2,000,000-Mr dextrans with mannitol (Mr 182) and PEG (Mr 4000). Compared with the untransfected LLC-PK1 cell sheets, a near sevenfold increase was observed in the transepithelial flux of d-mannitol across the Ras-transfected cell sheets, with no significant difference between the flux across LLC-PK1 versus pBabe. The flux for [14C]mannitol across Ras and Ras-A cell sheets was not significantly different (our unpublished data). The flux of [14C]polyethylene glycol was also increased by sevenfold across Ras cell sheets compared with the pBabe cell sheets. The 10,000-Mr methylated dextran showed the greatest relative increase of all, with the flux across ras-transfected cell sheets being more than ninefold higher than across parental and vector control cell sheets. This indicates that solutes at least as large as Mr 10,000 are able to pass through the tight junctions of ras-transfected cells, depending upon the exact conformation and net charge of the solutes.
Comparisons of the effect of Ras mutation on the transepithelial flux of solutes of increasing molecular size
It needs to be emphasized that “paracellular” transit can be through tight junction strands and lateral intercellular space or through gaping 500-μm holes in the epithelium. Kinetically these routes would seem very similar except for their size exclusion limit.
With the observation that solutes as large as Mr 10,000 can diffuse paracellularly across the ras-transfected cell sheets, the possibility of actual “holes” in the cell sheet needed to be considered. By “holes” we refer to the space that would be created if one or more cells detached from the cell sheet and left actual gaps in the barrier, which would be on the order of many micrometers in width. We saw no indication of this in the microscopy that we performed, but the observation of decreased cell substratum and cell-cell adhesion in the ras-transfected cells made this a distinct possibility. Simply not finding such a hole (a negative result) in a limited number of visually observed microscope sections would not constitute persuasive evidence. We therefore evaluated the transepithelial flux of a 2,000,000-Mr 14C-methylated dextran. As shown in , there was no relative increase in the amount of 2,000,000-Mr radiolabeled material crossing the ras-transfected cell sheet compared with the parental and vector controls. In fact passage of the 2,000,000-Mr material was almost negligible across all three cell sheets. The very low amount of material that did cross all three cell sheets (in roughly equal amounts) could be the result of limited damage of cell sheets in handling and/or a small but finite amount of nonspecific transcytosis. In any event, the ras-transfected cell sheets were no more permeable to material than the two controls. In separate experiments we likewise observed that there was the same order of negligible 2,000,000-Mr 14C-dextran flux across the clonal Ras-A cell sheets. However, as a positive control, we observed that exposure of pBabe cell sheets to the phorbol ester TPA for 3 h at 37°C, resulted in a huge 1000-fold increase in actual 2,000,000-M r 14C-dextran material coming across the cell sheets (our unpublished data).
With the prospect that ras transfection was in fact generating a molecular “pore” to allow the passage of small- and moderate-sized nonelectrolytes through the tight junctional barrier, we examined Ras-related changes in the abundance of several tight junctional proteins. We focused on transmembrane barrier proteins (occludin and various claudins) rather than intracellular tight junctional associated proteins (e.g., ZO-1 and cingulin). Cells were fractionated into membrane-bound (triton soluble) and cytoskeletal (triton-insoluble) pools because certain claudins were barely detectable in whole cell lysates (our unpublished data). shows Western blot analyses comparing occludin and six claudins in cytosolic (S) as well as membrane-bound (M) and cytoskeletal (C) fractions. Tight junctional protein abundance patterns fell into four categories. Occludin, claudin-1, and claudin-4 showed increased levels in the membrane-associated (Triton-soluble) cell fraction in the Ras transfectant in two separate cell passages of each cell line. For this group, only occludin was detectable in the cytosolic fraction, with claudins-1 and -4 being undetectable. The second category includes only claudin-2, whose abundance was greatly decreased in the M and C fractions in the Ras transfectant relative to the parental (L) and vector (P) controls. The third category includes claudins-3 and -5, whose abundance did not noticeably change in the Ras transfectant. In the fourth category, claudin-7 was unique in several respects. It was the only claudin that was readily detectable in the S fraction and may have manifested phosphoprotein bands, which were distinctly different in the M and C fractions. The abundance of the lower band was significantly greater than the upper band in the M fractions, but much less of the protein was detected in the C fractions. However in both cases, the Ras transfectant (R) showed more claudin-7 protein than the LLC-PK1 (L) or pBabe (P) controls.
Figure 6. Western blot analysis of integral tight junction proteins in three distinct subcellular fractions of the LLC-PK1 (L), pBabe-LLC-PK1 (P), and Ras-LLC-PK1 (R) cell lines. Cytosolic (S), soluble (M), and Triton-insoluble Triton(C) fractions were isolated (more ...)
Concerning these relative changes in expression of the various claudins as a result of Ras mutation, one may need to consider that the Ras subline contains a mixed population of cells, one of which may not possess actual tight junctions. As a result of the Ras mutation, multilayering of cells in distinct foci became evident across the Ras-transfected cell sheet (Figures , , ). As was true for multilayered foci in LLC-PK1 cell sheets exposed chronically to phorbol esters, the cells of the underlayers do not uniformly manifest morphological polarity nor does one observe tight junctional electron density in these cells. It is therefore possible that the decrease in claudin-2 seen in the Western immunoblot of is in part due to this mixed cell population contributing to the total protein loaded onto the gel. However, it should be mentioned that one does not see a decrease in other claudins in the Ras subline and in fact occludin and claudins-1 and -4 show increased levels in the Ras-transfected mixed population.
To verify the uniformity of the changes in claudin expression as a result of Ras overexpression in the LLC-PK1 cells, we examined the cells by immunofluorescence for claudin-1, -2, and -5. Transfected cells were grown to confluence on membrane filters to establish tight junctions without dome formation and then stained and visualized by immunofluorescence microscopy (). A comparison between the vector and ras-transfected cells shows that claudin-1 clearly increases and claudin-2 declines as predicted by the Western blot data (). We also show claudin-5, which does not change appreciably in its expression pattern, regardless of the status of Ras abundance in the epithelial cells.
Figure 7. Claudin expression in ras-transfected LLC-PK1 cells by immunofluorescence analysis. LLC-PK1 cells transfected with either empty vector (pBabe) or that vector containing Ras were grown to confluence on 0.4-μm filters and stained by immunofluorescence (more ...)
Because Ras overexpression seems to alter the expression of specific tight junction claudin molecules, which in turn has a drastic effect on the permeability through those tight junctions, we asked whether this might be mediated through changes in the ERK–MAP kinase pathway, a downstream mediator of activated Ras. Cultures of the transfected cells were grown to confluence, growth arrested by serum deprivation, and stimulated for 1 h with 10% serum. Western blot analysis of whole cell lysates for ERK-2 and phospho-ERK-1/2 is shown in . Our results show a dramatic increase in phospho-ERK-2 and a moderate increase in ERK-2 protein in the Ras-transfectant compared with the vector and parental controls. These results suggest that the striking physiological changes that we observed in the junctional permeability and select tight junction proteins as a result of Ras overexpression in the epithelial cells, may be initially triggered by changes in the MAP kinase pathway. Our future efforts will focus on this pathway as a potential mechanism for our observations.
Figure 8. Increased levels of phosphorylated ERK-2 as a result of Ras transfection. Cultures of LLC-PK1, pBabe, Ras, and Ras-A were grown to confluence in 75-cm2 culture flasks and harvested for total cell lysates as described in text. Immunoblots were probed with (more ...)