RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

Abstract

Although ras mutations have been shown to affect epithelial architecture and polarity, their role in altering tight junctions remains unclear. Transfection of a valine-12 mutated ras construct into LLC-PK 1 renal epithelia produces leakiness of tight junctions to certain types of solutes. Transepithelial permeability of D-mannitol increases sixfold but transepithelial electrical resistance increases >40%. This indicates decreased paracellular permeability to NaCl but increased permeability to nonelectrolytes. Permeability increases to D-mannitol (M r 182), polyethylene glycol (M r 4000), and 10,000-M r methylated dextran but not to 2,000,000-M r methylated dextran. This implies a "ceiling" on the size of solutes that can cross a ras-mutated epithelial barrier and therefore that the increased permeability is not due to loss of cells or junctions. Although the abundance of claudin-2 declined to undetectable levels in the ras-overexpressing cells compared with vector controls, levels of occludin and claudins 1, 4, and 7 increased. The abundance of claudins-3 and -5 remained unchanged. An increase in extracellular signal-regulated kinase-2 phosphorylation suggests that the downstream effects on the tight junction may be due to changes in the mitogen-activated protein kinase signaling pathway. These selective changes in permeability may influence tumorigenesis by the types of solutes now able to cross the epithelial barrier. INTRODUCTIONMutations in the three closely related ras genes, H-ras, K-ras, and N-ras, are among the most common mutations found in human cancer, reaching 50% in some types of tumors, such as colorectal carcinoma (Forrester et al., 1987;Andreyev et al., 1997Andreyev et al., , 1998Andreyev et al., , 2001). Vogelstein and colleagues have shown that mutations in K-ras occur early in the development of colon carcinomas (Vogelstein et al., 1988). This dominantly acting mutation in ras results in its constitutive activation and the subsequent triggering of its signaling pathway by influencing GTPase activity. It is clear that mutations in ras contribute both to the recurrence of the disease and to decreased survival (Andreyev et al., 1998). It is less clear, however, what the consequences of ras mutations are during the early stages of tumorigenesis. In this regard, one important issue is the effect of Ras activation on epithelial barrier function, perhaps the most basic of all epithelial functions, and a function shared by all epithelial tissues. Ras can exert fundamental effects on epithelial barrier function through the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase pathway or through direct binding to the tight junction-associated protein, AF-6, which binds to ZO-1, which in turn links the tight junction to the actin cytoskeleton. Activated Ras is known to inhibit interaction between the tight junction-associated proteins, AF-6 and ZO-1, with resultant perturbation of intercellular junctions (Yamamoto et al., 1997). Modulation of tight junctions by G proteins in ge...

The impact of CLAUDIN-1 on follicular thyroid carcinoma aggressiveness

“…It can thus be suggested that the elevated pathogenic character of RASV12 transfected FTC-133 cells involves the increase of CLAUDIN-1 protein expression. This increase in CLAUDIN-1 expression after RASV12 transfection was also observed in normal renal epithelial cells, whereby in these normal cells CLAUDIN-1 was localized in the cell membrane (Mullin et al 2005). As a side note, we tried CLAUDIN-1 downregulation by CLAUDIN-1 siRNA transfection of RASV12 transfected FTC-133 cells, which resulted in cell death within a few hours, potentially due to the augmented stress of cells, and irrespective if cells were transfected either with control siRNA or CLAUDIN-1 siRNA transfection mixture (unpublished observation).…”

Insulin-Like Growth Factor-I Produced by Seminal Vesicles: Relationship to Intraepithelial Basal Cell Hyperplasia in the Prostate

“…The penetration of factors such as IGF-I into these areas may signify a local loss of epithelial barrier function, and it has been suggested that a local breach in the epithelial barrier may be an early event in tumor formation (22). We have shown that oncogenic conversion of epithelial cells produces profound changes in epithelial barrier function (23), and it has been reported that seminal fluid can alter epithelial barrier function in vitro (24,25). This supports the idea that factors from the seminal fluid enter the basal compartment in the prostate where these factors can influence basal cell proliferation.…”

Role of Extracellular Signal-Regulated Kinase (ERK) in Regulation of Intestinal Epithelial Tight Junctions

“… ERK has a variable effect on the integrity of epithelial tight junctions; it can disrupt tight junction integrity in some cases 121,122,125,126 whereas, it can also protect tight junction integrity in other cases. 94,123 The mechanistic basis for this paradoxical ERK activity in tight junction regulation remains unclear.…”

“…124 Another study showed that a mutation in Ras (a signaling molecule upstream of ERK) is associated with increase in extra cellular signal-regulated kinase-2 phosphorylation and produces leakiness of tight junctions to certain types of solutes. 125 Similarly, Raf-1-another signaling molecule upstream of ERK has been implicated in causing disruption of epithelial TJs via downregulation of occludin. 126 Another study demonstrated that downregulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.…”

“…Some of the previous studies that showed the disruptive effect of MAPK pathway on tight junctions used under-differentiated cells or the cell types that do not differentiate. 121,122,125 On the other hand, highly differentiated cells were used when a protective response of MAPK pathway activation was observed on tight junction. 94 In the present study, we were able to demonstrate these opposing effects of ERK in the same cell type but at different stage of differentiation.…”