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Endocrinology Vol. 147, No. 6 2809-2816
Copyright © 2006 by The Endocrine Society

Mammalian K-ras2 Is a Corticosteroid-Induced Gene in Vivo

Francine E. Brennan and Peter J. Fuller

Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Professor Peter J. Fuller, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: peter.fuller{at}princehenrys.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aldosterone acts via the mineralocorticoid receptor to regulate gene expression. A number of aldosterone-induced genes have been characterized in the distal colon and/or the distal nephron. Using the Xenopus kidney-derived A6 cell line, the K-ras transcript of the K-ras gene was identified as aldosterone induced, with a role in epithelial sodium transport. This study sought to establish whether K-ras expression is also increased in mammalian epithelia in vivo in response to aldosterone. RNA was extracted from the kidney and distal colon of rats treated with aldosterone or dexamethasone. Northern blot analysis and real-time RT-PCR were performed using probes and primers specific for the K-rasA isoform and for total K-ras. The expression of both total K-ras and of the A isoform is induced in the distal colon by aldosterone and by dexamethasone. Given the relative abundances of the two isoforms, this would appear to indicate induction of both isoforms. The time course of the response is consistent with a primary transcriptional response. In contrast to the documented up-regulation in the amphibian kidney, we did not observe regulation by corticosteroids in the kidney. However, regulation in a subpopulation of cells cannot be excluded.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALDOSTERONE ACTS ON the epithelial cells of both the distal colon and the distal nephron to regulate sodium transport. The response is mediated by the mineralocorticoid receptor (MR), a member of the steroid/thyroid/retinoid nuclear receptor family of ligand-dependent transcription factors (1, 2). The MR is also activated by the physiological glucocorticoids, cortisol and corticosterone (in rodents). In vivo, in sodium-transporting epithelia, the MR responds primarily to aldosterone. Cortisol access is precluded by the activity of the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2), which converts cortisol (or corticosterone) to its inactive metabolite, cortisone (or 11-dehydrocorticosterone). This mechanism probably also precludes activation of the glucocorticoid receptor (GR) in these tissues under normal physiological circumstances; however, activation of the GR in these tissues by synthetic glucocorticoids or inhibition of 11ßHSD2 elicits similar responses to those observed with aldosterone (1, 3).

The elucidation of the mechanisms by which aldosterone mediates transepithelial sodium transport has seen considerable progress in the last decade (3). Given that the MR is a transcription factor, attention has focused on identifying genes that are induced by the MR. A number of genes that are regulated by aldosterone in the distal colon and/or distal nephron have been identified and characterized (1, 3, 4, 5). The critical rate-limiting step in aldosterone-mediated sodium transport is the apical amiloride-sensitive epithelial sodium channel (ENaC). The ENaC subunit genes are differentially regulated by corticosteroids in a tissue-specific manner. Expression of the ENaC ß- and {gamma}-subunits is acutely up-regulated in the colon (6, 7), whereas the {alpha}-subunit alone is regulated in the kidney (6). All three subunits are regulated in the lung by glucocorticoids but not by aldosterone (6). Serum and glucocorticoid-regulated kinase-1 (Sgk1) is rapidly up-regulated in both distal colon and nephron by corticosteroids (8, 9). Recent evidence suggests that it acts by inhibiting ENaC ubiquitination and thus subsequent proteosomal degradation (10, 11). The genes encoding the Na.K-ATPase, the energy-dependent pump that is located in the basolateral membrane and mediates sodium efflux, are not acutely regulated by aldosterone (12). The activity of the pump is modulated by members of the FXYD family, one of which is corticosteroid hormone-induced factor (CHIF) (13). CHIF is acutely up-regulated by corticosteroids in the distal colon but not in the kidney (14, 15). GILZ (glucocorticoid-induced leucine-zipper protein) is up-regulated by aldosterone in the distal nephron (16, 17, 18), where it acts to enhance ENaC activity (18). Other genes have been identified as being aldosterone regulated (16, 19, 20); their significance remains to be determined.

Amphibian systems have been used extensively for studies of mineralocorticoid action. The renal epithelium-derived amphibian A6 cell line was used to identify Sgk1 as an aldosterone-induced gene (21). Spindler et al. (22) used RT-PCR differential display to identify genes up-regulated by aldosterone treatment in A6 cells. Two transcripts were identified, one of which was the A splice variant of Xenopus K-ras. This finding was confirmed in vivo for Xenopus, with aldosterone treatment increasing XK-ras mRNA levels in the kidney by 2.5 h (23). Coinjection of XK-rasA and XENaC subunit cRNAs into Xenopus oocytes resulted in increased ENaC-mediated sodium transport relative to injection of ENaC cRNAs alone (24). Subsequent studies in A6 cells have demonstrated the importance of K-rasA in the response to aldosterone (4).

There are three mammalian Ras genes, H-ras, N-ras, and K-ras2 (K-ras1 is a pseudogene), which encode four highly similar monomeric G proteins (25). K-ras2 encodes two proteins that differ in their last 38 amino acids due to alternative splicing. These are designated K-rasA and K-rasB, with levels of the latter predominating in most tissues. The Ras proteins transduce cellular signals to second messenger pathways, and functional differences between the four proteins have recently been described (26, 27, 28).

The well-documented regulation of K-ras by aldosterone in amphibian systems has resulted in a general assumption of universality (29, 30). In addition, there have been studies in cultured mammalian cells (29, 31) that were not derived from primary aldosterone target tissues, but which nevertheless support the amphibian data. It is clearly important to establish whether K-ras is an aldosterone- and/or glucocorticoid-induced gene in mammalian epithelial tissues. The time course and steroid specificity of an observed response would provide an indication as to whether the response is a primary transcriptional one mediated by the MR. We have therefore examined the acute response of K-ras expression to aldosterone and dexamethasone treatment in the rat kidney and distal colon in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental details
Adult male Sprague Dawley rats were bilaterally adrenalectomized 24 h before each experiment and maintained on 0.9% saline and standard rat chow ad libitum. A single dose of aldosterone (50 µg/kg; im injection) or dexamethasone (0.5 mg/kg; ip injection) was administered in 0.2 ml PBS at times ranging between 0.5 and 4 h, before killing the rats by cervical dislocation. Control rats were injected ip with vehicle. Where indicated, cycloheximide was injected at 1 µg/kg ip, 0.5 h before steroid or vehicle as described previously (7, 8, 14). The distal colon and kidney were snap frozen in liquid nitrogen, and total RNA was then isolated. Aldosterone and cycloheximide were purchased from Sigma Aldrich (St. Louis, MO), and dexamethasone sodium phosphate from David Bull Laboratories (Sydney, Australia). The protocols for animal use were approved by the Monash University Animal Ethics Committee according to the National Health and Medical Research Council of Australia Code of Practice for the Care and Use of Animals for Scientific Purposes.

Northern analysis
Denaturing electrophoresis of total RNA from distal colon and kidney was performed using glyoxal/dimethylsulfoxide (7, 8, 14). Northern blot analysis was performed using 32P-labeled antisense RNA probes generated using T7 RNA polymerase and {alpha}-32P-UTP (Amersham Biosciences, Buckinghamshire, UK). Quantitative analysis was performed on phosphorimaged scans using a Storm 860 scanner and ImageQuant 5.1 software (both from Molecular Dynamics Inc., Sunnyvale, CA).

Probes
Probes were cloned from total RNA from the distal colon by RT-PCR. Oligo deoxythymidine (dT) was used to prime cDNA synthesis by avian myeloblastosis virus-reverse transcriptase (Roche Diagnostics GmbH, Mannheim, Germany), and PCR was then performed using primers described in Fig. 1Go and Table 1Go. Amplicons were cloned into SmaI-digested pGEM-4Z (Promega Corp., Madison, WI), and confirmed by sequencing. A 424-bp probe spanning exons 1–3 for detection of both transcripts was amplified using primers 1 and 2. A 371-bp amplicon extending from exon 2 to exon 4B was generated using primers 3 and 4, cloned and sequenced to confirm the presence of the 124bp exon 4A. This exon was subcloned within a 139-bp fragment using restriction sites at 9 bp (MnlI at nucleotide 441) and 6 bp (HincII at nucleotide 456) outside the exon boundaries. See Fig. 1Go and Table 1Go; nucleotide positions refer to GenBank accession no. U09793.


Figure 1
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  		FIG. 1. Diagram of the K-ras2 gene arrangement showing the alternative splicing which gives rise to the two transcripts, A and B. The location of the primer sequences used, which are given in Table 1Go, and the probes and PCR amplicons are indicated.

 

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  		TABLE 1. Oligonucleotide primers for probe generation and RT-PCR

 
Analysis of relative transcript expression
Total RNA samples from distal colon and kidney from four rats were reverse transcribed using oligo dT and avian myeloblastosis virus-reverse transcriptase. PCR was then performed using primers 3 and 4 (Table 1Go) for 20, 22, 24, 26, and 28 cycles. The reactions were electrophoresed through a 2% agarose gel in the presence of ethidium bromide. The intensity of UV light-generated fluorescence of the two bands amplified in each sample was analyzed using Quantity One version 4.0.2 software (Bio-Rad Laboratories, Hercules, CA) and plotted against the number of cycles to confirm that the amplification of the reactions was linear. The gel was Southern blotted and probed with 32P-labeled antisense RNA complementary to exons 1–3 (Table 1Go). Quantitative analysis was performed on the resultant autoradiograph film using a GS-710 Imaging Densitometer (Bio-Rad Laboratories) and Quantity One software. The ratio of pixel intensity values was obtained for each sample, and the mean value for each cycle number was determined.

Real-time PCR
The primers used are described in Fig. 1Go and Table 1Go. Primers 5 and 6 were used to amplify a 187-bp amplicon from exons 2–3 for total K-ras mRNA detection, and primers 7 and 8 were used to amplify one of 155 bp from exons 3–4A. In addition, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment of 300 bp extending from exons 2–4 was amplified as a normalization control. The system used was the LightCycler with the FastStart DNA Master SYBR Green I kit, both from Roche Diagnostics GmbH. Reactions were optimized for cDNA dilution, primer concentration, MgCl2 concentration, and annealing temperature to ensure a single product was produced for detection by SYBR Green I fluorescence. The melt curves, plotted as the change in fluorescence vs. temperature, all showed fluorescence change over approximately 3.5–4 C. Peak values occurred at 81.6, 83.1, and 87.0 C for the K-rasA, total K-ras, and GAPDH reactions, respectively. Melt curve analysis was used to determine the temperature at which fluorescence was to be recorded for each reaction protocol; primer-dimers were not able to be eliminated for the K-rasA reaction only, so data were collected above their melting temperature. Total RNA was reverse transcribed using oligo dT primer, and the cDNA was then amplified on the same or the next day. cDNA stored overnight before amplification was held at 4 C to minimize degradation caused by freezing-thawing. All samples were amplified using both K-ras primer pairs and the GAPDH primers. Melt curves were always studied for uniformity between samples and lack of evidence of contaminant products before accepting reaction sets for analysis. PCR efficiency (E) was determined for each reaction set using serial dilutions of reverse-transcribed RNA from the same tissue type. All plots of fluorescence vs. cycle number for the serially diluted samples had regression coefficients of –1.00, with mean squared error values below 0.06. E values were determined from these plots as 10–1/gradient (32). The average E values obtained for each primer pair were 1.88 for K-rasA, 1.87 for total K-ras and 1.96 for GAPDH. K-ras expression was determined using crossing points (CP) i.e. cycle numbers at equal fluorescence (i.e. equal product) above background level as ECP, and normalized using this value obtained for GAPDH expression on the same day (32). Determination of expression using CP values gives inverse relative differences, i.e. higher expression results in lower CP values. The values obtained were therefore inversed, then standardized to the mean control level. Statistical analysis was performed using ANOVA followed by the Games-Howell test for significance of data sets with heterologous variances.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A search of the gene databases for rat K-ras sequences yielded only a B isoform cDNA sequence (GenBank accession no. U09793). The primers used were designed from this sequence. A K-ras cRNA probe for detection of both isoforms was amplified using primers 1 and 2 (Fig. 1Go and Table 1Go). This hybridized to transcripts of approximately 5.2 and 2.4 kb on a Northern blot of multiple tissue RNAs (Fig. 2Go). The sizes of these transcripts were consistent with previous reports (33, 34). The levels were generally low, with more prominent expression being observed in the gastrointestinal tissues, particularly the distal colon. The two isoforms differ by 124 bp (Fig. 1Go) and were therefore not distinguishable. It was not possible to create a K-rasB-specific probe because both transcripts contain exon 4B (Fig. 1Go). A probe for specific detection of K-rasA transcripts was created by cloning exon 4A. Total RNA from rat colon was amplified using primers 3 and 4 (Table 1Go). This yielded a 371-bp amplicon containing 124 bp between exons 3 and 4B, which was identical in sequence to the murine K-ras 4A exon (GenBank accession no. X02455). The exon 4A cDNA was subcloned as described in Materials and Methods. Both the total K-ras and K-rasA probes detected the approximately 2.4-bp transcript on Northern blots of colonic (see Fig. 4Go) and kidney RNA (data not shown). The larger transcript, presumably a primary/partially processed one, was usually not observed. The K-rasA probe hybridized to a transcript of very low abundance on Northern blots of colonic RNA. The abundance of the transcript was such that it could not be reliably detected in kidney RNA by Northern blot hybridization. This prompted examination of tissue-specific isoform expression.


Figure 2
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  		FIG. 2. K-ras mRNA expression in a range of tissues. A Northern blot was probed with a cRNA complementary to exons 1–3. The positions of the 28S and 18S ribosomal bands and molecular weight markers are indicated. The transcripts detected, of sizes approximately 5.2 kb and 2.4 kb, are indicated. The blot was reprobed with GAPDH (lower panel) to confirm the presence and integrity of the RNA loaded in each lane. The tissues examined are corpus of stomach (C), duodenum (D), ileum (I), distal colon (DC), kidney (K), liver (Li), spleen (S), and lung (Lu).

 

Figure 4
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  		FIG. 4. K-ras mRNA levels observed on probed blots of colonic RNA. A, Total K-ras and K-rasA mRNA levels in adrenal-intact and adrenalectomized (adrex) rats. B, Total K-ras and K-rasA mRNA levels at 1, 2, and 4 h after a single dose of aldosterone vs. control levels. C. Total K-ras and K-rasA mRNA levels at 1, 2, and 4 h after a single dose of dexamethasone vs. control levels.

 
To establish the relative abundance of the A and B transcripts of K-ras, total RNA from colon and kidney was subjected to RT-PCR with oligo dT followed by primers spanning exon 4A (primers 3 and 4). This yielded two amplicons of 381 and 257 bp, according to presence and absence, respectively, of exon 4A (Fig. 3Go). Densitometric analysis of reactions halted in the linear phase demonstrated that the B isoform was predominant in both kidney and colon. However, the ratio of B to A was found to differ in the two tissues, being approximately 3 in the colon and approximately 7 in the kidney.


Figure 3
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  		FIG. 3. Relative levels of K-rasA and K-rasB transcripts. Linear phase PCR (20–28 cycles) of cDNA prepared from distal colon and kidney RNA was performed with primers 3 and 4 (Fig. 1Go and Table 1Go). The sizes of the amplicons resulting from the two K-ras transcripts are given. Densitometric analysis was performed on autoradiographs of Southern blots hybridized with an exon 1–3 probe (Fig. 1Go) to determine B:A transcript ratios. The section of the autoradiograph scan shown was the result at 24 cycles (n = 4; mean ± SEM).

 
Northern blot hybridization showed no significant alteration in colonic total K-ras (i.e. both transcripts) and K-rasA levels after adrenalectomy (Fig. 4AGo). The response of K-rasA and total K-ras to a single dose of aldosterone or dexamethasone was examined in distal colon by Northern blot analysis (Fig. 4Go, B and C). A small but significant increase was seen for both probes at 2 h in response to aldosterone and at 4 h in response to dexamethasone (data not shown). K-ras transcript levels in the kidney were too low for examination by this method. Although the results obtained strongly indicated that K-ras was indeed regulated by corticosteroids in the distal colon, the low signal to noise ratio resulting from the low abundance of the transcripts, particularly K-rasA, lead us to the use of real-time PCR to quantitate the response in both distal colon and kidney.

Due to the high sequence homology at the exon 3–4A and 3–4B splice points, it was not possible to create a K-rasB-specific amplification reaction. Thus, total K-ras and K-rasA mRNA levels were again measured. The results for the distal colon (Fig. 5Go) are consistent with the results obtained by Northern blot analysis. K-rasA levels were significantly increased at 2 h after a single dose of either aldosterone or dexamethasone. Similarly, total K-ras mRNA levels were increased at 2 h in response to aldosterone and at 1, 2, and 4 h after a single dose of dexamethasone. When the same analysis was applied to kidney RNA, no significant increases in total K-ras and K-rasA transcript levels were observed in response to either dexamethasone or aldosterone (Fig. 6Go).


Figure 5
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  		FIG. 5. Real-time PCR analysis of K-ras mRNA levels in the distal colon after corticosteroid treatment. A, K-rasA mRNA response to aldosterone; B, K-rasA mRNA response to dexamethasone; C, total K-ras mRNA response to aldosterone; D, total K-ras mRNA response to dexamethasone. The K-ras mRNA levels have been normalized to the GAPDH mRNA level determined in parallel in the samples (n = 4–12; mean ± SEM; *, P < 0.05; **, P < 0.01).

 

Figure 6
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  		FIG. 6. Real-time PCR analysis of K-ras mRNA levels in the kidney after corticosteroid treatment. A, K-rasA mRNA response to aldosterone; B, K-rasA mRNA response to dexamethasone; C, total K-ras mRNA response to aldosterone; D, total K-ras mRNA response to dexamethasone. The K-ras mRNA levels have been normalized to the GAPDH mRNA level determined in parallel in the samples (n = 4–8; mean ± SEM). No statistically significant difference was found between the controls and any of the treated groups.

 
The rapid response of K-ras to aldosterone in the distal colon is consistent with a primary transcriptional response. To examine this possibility, the effect of protein synthesis inhibition by cycloheximide on the response to aldosterone was studied. However, this did not provide further evidence of primary regulation because cycloheximide alone increased K-ras mRNA levels (data not shown). This is in contrast to the results obtained previously for other induced genes including Sgk1 (8), which show superinduction of the response to aldosterone by cycloheximide but no effect of cycloheximide alone.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Ras proteins, K-, H-, and N-Ras, are monomeric guanine nucleotide-binding proteins of molecular mass 21 kDa that play a central role in the regulation of cellular functions, particularly growth, differentiation, and apoptosis (25). These functions are largely mediated by cell membrane receptors. The three ras genes are very highly conserved from yeast to human and are also highly similar to each other. All have four coding exons, with two isoforms of K-ras arising through alternative splicing of exon 4A or 4B (Fig. 1Go). The Ras proteins undergo a series of posttranslational modifications of the C terminus in the endoplasmic reticulum. These modifications facilitate the membrane anchoring required for recruitment of Ras proteins to receptor signaling complexes (35). Indeed, it is becoming clear that differences in these modifications lead to their localization for signal transduction in different cellular compartments (28, 36, 37).

At the tissue level, the expression of the Ras proteins is cell specific. In the human kidney, H-, N-, and K-Ras are all expressed in proximal tubule cells and collecting duct cells, whereas only H- and K-Ras are evident in interstial and mesangial cells. In all cases, expression occurs at different relative abundances (38, 39). Expression of H- and K-Ras but not N-Ras has been reported in human colonic epithelial cells (40); however, the observed K-Ras expression was extremely low. These studies have all measured total K-Ras expression rather than individual isoforms. Both K-Ras proteins have been detected in epithelia of small and large intestine, kidney, and lung of the mouse. All showed higher levels of the K-RasB isoform (41).

One family of primary effector proteins activated by Ras are the Raf proteins (Raf-1, A-Raf, and B-Raf). Voice et al. (26) demonstrated that the four human Ras proteins differ in their ability to activate Raf-1, induce transformation and stimulate cell motility, suggesting that they have significantly different roles in vivo. K-RasB activates Raf-1 over 2-fold more efficiently than K-RasA and much more efficiently than H-Ras does (26). H-Ras activates phosphatidylinositol-3 kinase (PI-3 kinase) more efficiently than K-Ras (presumably B) (42).

Spindler et al. (22) identified the Xenopus K-rasA transcript as being up-regulated by aldosterone at 1 h in the Xenopus kidney-derived A6 epithelial cell line. In a subsequent study, they demonstrated increased total XK-ras mRNA in the Xenopus kidney 2.5 h after a single injection of aldosterone in vivo; in addition, the response was not blocked by cycloheximide in A6 cells (23). They also reported that constitutively active XK-RasA increased the activity of the epithelial sodium channels in Xenopus oocytes (24). An important caveat for these studies is that the Xenopus MR is not present in A6 cells, where aldosterone acts via the GR (43).

In view of these detailed studies of the regulation of K-rasA expression in amphibian epithelial systems, we thought it important to establish whether the same was true of two well-characterized aldosterone-responsive mammalian epithelial tissues, the distal colon and the kidney. The present studies demonstrate that K-ras is also regulated in mammalian distal colon in vivo by aldosterone. The observed response parallels the up-regulation of the expression of other aldosterone-responsive genes in this tissue including Sgk1, CHIF, ß-ENaC, and {gamma}-ENaC (7, 8, 14). In contrast to the clear effect of endogenous corticosteroids on Sgk1 (8) and CHIF (14) expression, basal K-ras expression does not appear to significantly involve corticosteroids in the distal colon because adrenalectomy produced no such clearly observable effect. We were not able to detect a response in the kidney, in contrast to the marked increase observed in Xenopus kidney in response to aldosterone (23).

The similarity of the sequences at the splice regions for exon 3 with both exons 4A and 4B is such that it was not possible to devise a PCR-based strategy that would yield a K-rasB-specific amplicon. Butz et al. (44) have recently used this system to develop a novel assay for splice variants; however, they also did not employ a K-rasB-specific assay. When the two transcripts were amplified in the same PCR (Fig. 3Go), the B isoform was predominant in both tissues, as has been reported previously (34, 41, 44, 45). There are some discrepancies between reported mRNA ratios, which may reflect tissue sampling and/or species differences. Human (44) and mouse (41, 45) colon have been reported to express both K-ras transcripts at similar levels. The higher B:A ratio found in the present study may be due to the use of distal, rather than total, colon. More significantly, kidney B:A relative expression in the mouse has been reported to be approximately equal (41), ≥ 2 (45) and approximately 13 (34). The ratio of approximately 7 found here for rat kidney is within this large range, which is presumably due to the difficulty of accurately measuring low expression levels.

Northern blot analysis suggested regulation of K-ras by aldosterone in the colon but the low abundance of the transcripts, particularly A, precluded accurate analysis. Real-time PCR revealed that both K-rasA and total K-ras mRNA levels were increased at 2 h in response to aldosterone and dexamethasone in the distal colon. The increase in total K-ras mRNA levels in response to aldosterone cannot be explained by the observed increase in K-rasA levels alone. When the relative abundance of the A and B isoforms is taken into account, the increase in K-rasB mRNA levels appears at least equivalent to that of K-rasA levels. Curiously, K-rasB does not appear to have been examined in previous studies of K-ras induction by aldosterone (4, 22, 23, 24).

The time course of the response is consistent with a primary transcriptional response, as has been observed for Sgk1 and CHIF (8, 14, 15); the response to administration of the protein synthesis inhibitor, cycloheximide, did not assist the clarification of this point. Not only is the response of Sgk1 and CHIF to aldosterone not inhibited, but it is enhanced (superinduced) in the presence of cycloheximide, whereas cycloheximide alone has no effect. In contrast, cycloheximide alone increased K-ras levels, rendering the response to combined cycloheximide and aldosterone treatment difficult to interpret (data not shown). The effect of cycloheximide is consistent with the involvement of a short half-life protein in K-ras mRNA turnover, as might be expected for regulation of a gene involved in rapid responses to extracellular signals. Therefore, although the observed apparent increase in both isoforms in the distal colon is consistent with a transcriptional response, as reported in A6 cells (23), this remains to be formally demonstrated.

As also observed for Sgk1, CHIF, and the ß- and {gamma}ENaC subunit genes in the distal colon (7, 8, 14), expression was induced by both aldosterone and dexamethasone treatment. At the dose used, dexamethasone is unlikely to be significantly activating the MR. Indeed, both Sgk1 and CHIF are up-regulated by the GR-specific agonist RU28362 (8, 14). These findings are consistent with the concept that, in these epithelial tissues, specificity is largely conferred in vivo at a prereceptor level by the action of 11ß-HSD2 (46, 47).

The human, rat, and murine K-ras genes contain putative glucocorticoid response elements within the first intron and the first coding exon (48). Hendron and Stockand (30) have reported that this region of the murine K-ras gene confers GR-responsiveness on a heterologous promoter. We would speculate that the aldosterone-induced increase in K-ras levels observed in vivo is mediated by the interaction of the steroid-bound MR with this glucocorticoid response element. Such a mechanism of regulation would argue for coordinated up-regulation of transcripts for both isoforms, assuming that both isoforms are found within the same cell rather than resulting from a tissue- or cell-specific pattern of splicing. Steroid receptor-mediated regulation of RNA splicing has been recently described (49); however, in this case, the increase in both transcripts argues for regulation at the level of transcription rather than differential splicing.

We were not able to detect a response in the kidney. The low abundance of K-ras may not allow a possible response in a small number of MR-responsive cells to be detected. It may also be that it is not regulated, as reported by Verrey et al. (50) for the distal nephron. However, Terada et al. (51) reported a 2-fold increase in K-RasA protein levels at 1 h in response to aldosterone in isolated rat mesangial cells. Although Sgk1 is regulated by aldosterone in both kidney and colon, other genes show differential regulation. CHIF, ßENaC, and {gamma}ENaC are regulated in the colon only by aldosterone, whereas {alpha}ENaC is regulated only in the kidney. Thus, it appears that aspects of the mechanism of aldosterone-mediated sodium flux differ between these primary target tissues.

The group of Stockand (4) has extensively characterized the role of Ras in the sodium flux induced by aldosterone in A6 cells. This group (52) reported that aldosterone treatment of A6 cells increased Ras protein levels 2- to 8-fold within 4 h, and also increased plasma membrane-associated Ras and MAPK activity (30). The increase in sodium flux was mediated by an increase in sodium channel activity. More recently the same group (53) has reported activation by Ras of both the MAPK and PI-3 kinase pathways in response to aldosterone. Blockade of ERK1/2 activity in A6 cells had little effect on sodium transport, whereas it was significantly decreased by PI-3 kinase inhibition. In contrast, Michlig et al. (54) found that, in both ex vivo murine microdissected cortical collecting duct cells and cultured cortical collecting duct principal cells, ERK1/2 activity was necessary for sodium transport. The ERK1/2 activity was not altered by aldosterone treatment. This highlights the caution that must be applied to drawing general conclusions about regulation across systems/species. PI-3 kinase acts via 3-phosphoinositide-dependent protein kinase 1 to activate Sgk1. It has also been shown to have more direct effects on ENaC to increase open probability. Pochynyuk et al. (55) have presented evidence for direct Sgk1-independent interaction of phospholipid products of PI-3 kinase activity with ENaC. This has been demonstrated to occur as a result of aldosterone action (56).

The model presented by Tong et al. (53), based on evidence gained from amphibian systems, proposes that K-RasA activates both PI-3 kinase and Raf-1, with the former effecting inactivation of the latter via Akt. Relative levels of activated Raf-1 and PI-3 kinase determine the induction of Raf-Akt interaction (57). If levels of both isoforms of K-Ras are increased by aldosterone, as the results presented here for mRNA levels in the distal colon suggest, this cross talk would be highly significant in regulating the stages of the cellular response. K-RasB activates PI-3 kinase weakly (42) but activates Raf-1 approximately 2-fold more efficiently than K-RasA (26). The relative capacity of K-RasA to activate PI-3 kinase is not known, but the two proteins have been shown to interact in A6 cells (54). The aldosterone-induced gene GILZ has recently been shown to inhibit phospho-ERK levels (18). GILZ has been demonstrated to bind Raf-1 and inhibit its activation (58). Thus, GILZ may complement activated Ras in promoting PI-3 kinase signaling at the expense of activation of the Raf-ERK pathway, as reported in A6 cells (53), favoring a differentiated (i.e. activating vectorial sodium transport) rather than a proliferative (ERK mediated) response.

Although regulation of K-ras by aldosterone was observed in the distal colon in this study, it was not found in the kidney, which is in contrast to reports from amphibian systems. However, corticosteroid-regulated expression may occur in a subpopulation of cells in the mammalian kidney. The response in the mammalian distal colon parallels that of other corticosteroid-induced genes in that tissue. Although the in vivo significance of this finding remains to be determined, it would seem likely that K-Ras plays a role in mediating the aldosterone-induced PI-3 kinase-dependent phosphorylation of Sgk1 and increase in ENaC activity, in the distal colon at least.


   Acknowledgments
 
The authors thank Claudette Thiedeman and Sue Panckridge for help in preparation of the manuscript.


   Footnotes
 
This work was supported by a project grant (169001) and a Research Fellowship (to P.J.F.) from the National Health and Medical Research Council of Australia.

Disclosure Statement: F.E.B. has nothing to declare. P.J.F. has received consulting fees from Merck, lecture fees from Novartis and was in receipt of a previous research grant from Pfizer; none of these activities relate to the present study.

First Published Online March 16, 2006

Abbreviations: CHIF, Corticosteroid hormone-induced factor; dT, deoxythymidine; ENaC, epithelial sodium channel; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GILZ, glucocorticoid-induced leucine-zipper protein; GR, glucocorticoid receptor; 11ßHSD2, 11ß-hydroxysteroid dehydrogenase type 2; MR, mineralocorticoid receptor; PI-3 kinase, phosphatidylinositol-3 kinase; Sgk1, serum and glucocorticoid-regulated kinase-1.

Received November 22, 2005.

Accepted for publication March 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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