Met acts through Abl to regulate p53 transcriptional outcomes and cell survival in the developing liver.

BACKGROUND & AIMS: Genetic studies indicate that distinct signaling modulators are each necessary but not individually sufficient for embryonic hepatocyte survival in vivo. Nevertheless, how signaling players are interconnected into functional circuits and how they coordinate the balance of cell survival and death in developing livers are still major unresolved issues. In the present study, we examined the modulation of the p53 pathway by HGF/Met in embryonic livers. METHODS: We combined pharmacological and genetic approaches to biochemically and functionally evaluate p53 pathway modulation in primary embryonic hepatocytes and in developing livers. RT-PCR arrays were applied to investigate the selectivity of p53 transcriptional response triggered by Met. RESULTS: Met recruits p53 to regulate the liver developmental program, by qualitatively modulating its transcriptional properties: turning on the Mdm2 survival gene, while keeping death and cell-cycle arrest genes Pmaip1 and p21 silent. We investigated the mechanism leading to p53 regulation by Met and found that Abl and p38MAPK are required for p53 phosphorylation on S(389), Mdm2 upregulation, and hepatocyte survival. Alteration of this signaling mechanism switches p53 properties, leading to p53-dependent cell death in embryonic livers. RT-PCR array studies affirmed the ability of the Met-Abl-p53 axis to modulate the expression of distinct genes that can be regulated by p53. CONCLUSIONS: A signaling circuit involving Abl and p38MAPK is required downstream of Met for the survival of embryonic hepatocytes, via qualitative regulation of the p53 transcriptional response, by switching its proapoptotic into survival properties.

SGK1-dependent upregulation of connective tissue growth factor by angiotensin II.

Angiotensin II has previously been shown to trigger fibrosis, an effect involving connective tissue growth factor (CTGF). The signaling pathways linking angiotensin II to CTGF formation are, however, incompletely understood. A gene highly expressed in fibrosing tissue is the serum- and glucocorticoid-inducible kinase SGK1. The present study explored whether SGK1 is transcriptionally regulated by angiotensin II and participates in the angiotensin II-dependent regulation of CTGF expression. To this end, experiments have been performed in human kidney fibroblasts and mouse lung fibroblasts from gene-targeted mice lacking SGK1 (sgk1-/-) and their wild-type littermates (sgk1+/+). In human renal fibroblasts, SGK1 and CTGF protein expression were enhanced by angiotensin II (10 nM) within 4 h. In sgk1+/+ mouse fibroblasts, SGK1 transcript levels were significantly increased after 4 h of angiotensin II treatment. Angiotensin II stimulated both transcript and protein abundance of CTGF in fibroblasts from sgk1+/+ mice, effects significantly blunted in fibroblasts of sgk1-/- mice. In conclusion, angiotensin II stimulates the expression of SGK1, which is in turn required for the stimulating effect of angiotensin II on the expression of CTGF. Thus, SGK1 presumably contributes to the profibrotic effect of angiotensin II.

Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse.

In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approximately 20% of PDK1 (pdk1(hm)) were compared with their wild-type littermates (pdk1(wt)). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse (DeltapH(NHE)). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1(hm) and pdk1(wt) mice. DeltapH(NHE) was in pdk1(hm) mice, only 30 +/- 6% of the value obtained in pdk1(wt) mice. Conversely, DeltapH(NHE) was 32 +/- 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1(hm) and pdk1(wt) mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 microM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.

Renal Ca2+ handling in sgk1 knockout mice.

Coexpression studies in Xenopus oocytes revealed the ability of the serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate the renal epithelial Ca(2+) channel TRPV5. SGK1 increases the abundance of the channel protein in the plasma membrane, an effect requiring the participation of the Na(+)/H(+) exchanger regulating factor 2 (NHERF2). The present study was performed to explore the role of SGK1 in the regulation of renal Ca(2+) handling in vivo. To this end, TRPV5, calbindin D-28K abundance, and renal Ca(2+) excretion were analyzed in gene-targeted mice lacking functional SGK1 (sgk1( -/- )) and their age- and sex-matched littermates (sgk1( +/+ )). Immunohistochemistry revealed lower abundance of TRPV5 and calbindin D-28K protein in sgk1( -/- ) mice than in sgk1( +/+ ) mice, both fed with control diet. Feeding the mice a Ca(2+)-deficient diet marked ly increased TRPV5 protein abundance in both genotypes. Renal Ca(2+) excretion under control diet was significantly lower in sgk1 ( -/- ) than in sgk1( +/+ ) mice. The Ca(2+)-deficient diet decreased renal excretion of Ca(2+) to the same levels in both phenotypes. Furosemide increased fractional Ca(2+) excretion and dissipated the difference between phenotypes. We conclude that lack of SGK1 may lead to decrease in TRPV5 abundance in connecting tubules but does not abrogate TRPV5 regulation. The decrease in abundance of TRPV5 in connecting tubules of sgk1( -/- ) mice is presumably compensated for by enhanced Ca(2+) reabsorption in upstream nephron segments such as the loop of Henle, which may indirectly result from impaired SGK1-dependent Na(+) reabsorption in the aldosterone-sensitive distal part of the nephron, salt loss, and enhanced Na(+) (and Ca(2+)) reabsorption in those upstream nephron segments.

SGK1-dependent cardiac CTGF formation and fibrosis following DOCA treatment.

The mineralocorticoids aldosterone and deoxycorticosterone acetate (DOCA) stimulate renal tubular salt reabsorption, increase salt appetite, induce extracellular volume expansion, and elevate blood pressure. Cardiac effects of mineralocorticoids include stimulation of matrix protein deposition leading to cardiac fibrosis, which is at least partially due to the direct action of the hormones on cardiac cells. The signaling mechanisms mediating mineralocorticoid-induced cardiac fibrosis have so far remained elusive. Mineralocorticoids have been shown to upregulate the serum- and glucocorticoid-inducible kinase 1 (SGK1), which participates in the effects of mineralocorticoids on renal tubular Na+ reabsorption and salt appetite. To explore the involvement of SGK1 in the pathogenesis of mineralocorticoid-induced cardiac fibrosis, SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+) were implanted a 21-day-release 50-mg DOCA pellet and supplied with 1% NaCl in drinking water for 18 days. This DOCA/high-salt treatment increased blood pressure in both genotypes but led to significant cardiac fibrosis only in sgk1+/+ but not in sgk1-/- mice. According to real-time polymerase chain reaction and Western blotting, DOCA/high-salt treatment enhanced transcript levels and protein expression of cardiac connective tissue growth factor (CTGF) only in sgk1+/+ but not in sgk1-/- mice. Furthermore, DOCA (10 microM) upregulated CTGF expression and enhanced CTGF promoter activity in lung fibroblasts isolated from sgk1+/+ but not from sgk1-/- mice, an effect involving spironolactone-sensitive mineralocorticoid receptors and activation of nuclear factor-kappaB (NFkappaB). Our results suggest that SGK1 plays a decisive role in mineralocorticoid-induced CTGF expression and cardiac fibrosis.

DOCA-induced phosphorylation of glycogen synthase kinase 3beta.

Mineralocorticoid excess leads to cardiac fibrosis, a leading cause of morbidity and mortality. Cardiac hypertrophy and fibrosis are inhibited by the glycogen synthase kinase GSK3 which itself is a target of protein kinase B (PKB) and the serum and glucocorticoid inducible kinase SGK1. Phosphorylation of GSK3 by PKB or SGK1 inhibits GSK3 activity and should thus favour the development of cardiac hypertrophy and fibrosis. As SGK1 is transcriptionally upregulated by mineralocorticoids and has been recently shown to play an important role in the pathogenesis of mineralocorticoid-induced cardiac fibrosis, the present study explored whether mineralocorticoid excess had any effect on the phosphorylation status of the a and beta isoforms of GSK3. Western blotting using an antibody specific for the PKB/SGK1 consensus phosphorylation site in GSK3a/beta (serine 21 and 9 respectively) revealed an increase in GSK3a/beta phosphorylation in human embryonic kidney 293 (HEK293) cells overexpressing wild type SGK1, constitutively active SGK1, but not catalytically inactive SGK1. The effect of SGK1 was mimicked by PKB and SGK3. Furthermore, DOCA/high salt treatment of wild type mice induced a robust increase in cardiac GSK3beta phosphorylation and, to a much lesser extent, GSK3a phosphorylation. However, under this treatment GSK3beta phosphorylation was apparent even in mice lacking functional SGK1, indicating that the phosphorylation of GSK3beta was not exclusively mediated by this kinase. Despite similar cardiac GSK3beta phosphorylation cardiac fibrosis following DOCA/high salt treatment was significantly blunted in SGK1 knockout mice. In conclusion, mineralocorticoid excess leads to phosphorylation and thus inactivation of GSK3beta, an effect not only due to upregulation of SGK1 but as well due to activation of additional kinases. The inactivation of GSK3 may play a permissive role in the stimulation of cardiac fibrosis but may by itself not be sufficient to trigger cardiac fibrosis.

Intestinal function of gene-targeted mice lacking serum- and glucocorticoid-inducible kinase 1.

In vitro experiments have revealed the ability of serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate intestinal Na(+)-coupled glucose cotransporter 1 (SGLT1) and intestinal Na(+)/H(+) exchanger 3 (NHE3). The present study explored the contribution of SGK1 to the regulation of intestinal transport in vivo. SGK1 transcript levels were determined by real-time PCR and glucose-induced currents (I(g)) reflecting SGLT1 activity by Ussing chamber experiments. BCECF fluorescence was utilized for the determination of Na(+)-dependent pH recovery from an ammonium pulse (DeltapH(NHE)) reflecting NHE activity. As a result, intestinal SGK1 transcript levels were significantly enhanced by a 4-day treatment with 10 microg.mg body wt(-1).day(-1) dexamethasone (Dex). I(g) was, under control conditions, virtually identical in sgk1 knockout mice (sgk1(-/-)) and their wild type littermates (sgk1(+/+)). A 4-day treatment with Dex, however, increased I(g) approximately threefold in sgk1(+/+) mice but not in sgk1(-/-) mice. DeltapH(NHE) was similar in sgk1(-/-) and sgk1(+/+) mice before treatment. Dex increased DeltapH(NHE) approximately threefold in sgk1(+/+) mice and approximately twofold in sgk1(-/-)mice, an effect significantly blunted in the presence of the specific NHE3 blocker S-3226 (10 microM). According to Western blot analysis, Dex significantly enhanced SGLT1 and NHE3 protein abundance in brush-border membranes of sgk1(+/+) mice but not of sgk1(-/-)mice. In conclusion, basic functions of SGLT1 and NHE3 in the intestine do not require stimulation by SGK1. However, the effects of glucocorticoids on SGLT1 are fully, and on NHE3 partially, dependent on SGK1.