The N-Terminal Phosphorylation of RB by p38 Bypasses Its Inactivation by CDKs and Prevents Proliferation in Cancer Cells.

Control of the G1/S phase transition by the Retinoblastoma (RB) tumor suppressor is critical for the proliferation of normal cells in tissues, and its inactivation is one of the most fundamental events leading to cancer. Cyclin-dependent kinase (CDK) phosphorylation inactivates RB to promote cell cycle-regulated gene expression. Here we show that, upon stress, the p38 stress-activated protein kinase (SAPK) maximizes cell survival by downregulating E2F gene expression through the targeting of RB. RB undergoes selective phosphorylation by p38 in its N terminus; these phosphorylations render RB insensitive to the inactivation by CDKs. p38 phosphorylation of RB increases its affinity toward the E2F transcription factor, represses gene expression, and delays cell-cycle progression. Remarkably, introduction of a RB phosphomimetic mutant in cancer cells reduces colony formation and decreases their proliferative and tumorigenic potential in mice.

The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress.

The p57(Kip2) cyclin-dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem-cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57(Kip2) is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57(Kip2) at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell-cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell-cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38-mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.

Conditional transgenic mice for studying the role of the glucocorticoid receptor in the renal collecting duct.

The mineralocorticoid receptor (MR) is a major regulator of renal sodium reabsorption and body fluid homeostasis. However, little is known about glucocorticoid receptor (GR)-dependent renal effects. Glucocorticoids may activate both receptors, so it is difficult to distinguish between MR- and GR-mediated effects in vivo. To overcome this complexity, we used a transgenic mouse model allowing conditional GR overexpression (doxycycline inducible TetON system, Hoxb7 promoter) in the renal collecting duct (CD) to identify GR-regulated genes involved in sodium transport in the CD. In microdissected cortical CD, induction of GR expression led (after 2 d of doxycycline) to increased alpha-epithelial sodium channel and glucocorticoid-induced leucine zipper and decreased abundance of with-no-lysine kinase 4 transcripts, without modification of Na,K-ATPase, serum- and glucocorticoid-kinase-1, or MR expression. No changes occurred in the upstream distal and connecting tubules [distal convoluted tubule (DCT), connecting tubule (CNT)]. Sodium excretion was unaltered, but the urinary aldosterone concentration was reduced, suggesting compensation of transitory extracellular volume expansion that subsequently disappeared. At steady state, i.e. after 15 d of doxycycline administration, transcript abundance remained altered in the CD, whereas mirror changes appeared in the DCT and CNT. Plasma aldosterone or glucocorticoids and blood pressure were all unaffected. These experiments show that: 1) GR, in addition to MR, controls epithelial sodium channel- and glucocorticoid-induced leucine zipper expression in vivo in the CD; 2) with-no-lysine kinase 4 is negatively controlled by GR; and 3) the DCT and CNT compensate for these alterations to maintain normal sodium reabsorption and blood pressure. These results suggest that enhanced GR expression may contribute to enhanced sodium retention in some pathological situations.

Implication of chromosome 18 in hypertension by sibling pair and association analyses: putative involvement of the RKHD2 gene.

This study aims to test the implication of regions on chromosomes 9, 17, and 18 in essential hypertension (EH) by combining sibling-pair linkage analysis and case-control association studies. The selection of these chromosomal regions is based on previous evidence of their implication in EH or in related phenotypes by comparative genomics in several rat models and from genome-wide linkage studies in humans. For the affected sibling-pair linkage analysis, 27 microsatellite markers were genotyped in 56 pedigrees from Spain with hypertensive sibling pairs. Linkage analysis showed significant excess allele sharing at the D18S474 marker on 18q21.1, as shown by maximum likelihood of allele sharing methods (logarithm of odds=3.24; P=0.00011) and nonparametric linkage calculations (nonparametric linkage=3.32; P=0.00044). On the contrary, no significant results with any of the markers analyzed on chromosomes 9 and 17 were obtained. We further focused on the Ring finger and KH domain containing 2 (RKHD2) gene located 6 Kb distal from D18S474 and performed a case-control association study based on linkage disequilibrium in 112 hypertensive patients and 156 control subjects. We selected 2 RKHD2-tagged single nucleotide polymorphisms, rs1941958 and rs1893379, covering, in terms of linkage disequilibrium, the entire gene, and observed a significant overrepresentation of the rs1941958G-rs1893379T RKHD2 haplotype in the group of hypertensive patients in comparison with controls (2P=0.0004; odds ratio: 2.32). We also detected epistatic effects between the 2 RKHD2 single nucleotide polymorphisms (2P=0.002; odds ratio: 2.48). Our data confirm the implication of chromosome 18 in EH and support a contribution of RKHD2 to the genetic susceptibility of this complex phenotype.

Aldosterone and tight junctions: modulation of claudin-4 phosphorylation in renal collecting duct cells.

Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.

12-Lipoxygenase metabolism in mouse distal convoluted tubule cells.

BACKGROUND: Several lines of evidence point to the 12-lipoxygenase (12-LOX) family as important mediators in hypertension, diabetes, and other cardiovascular diseases. The kidney has been a main focus for research of the role of this pathway in several disease models. While most of the studies have focused on mesangial or vascular cells, less is known about 12-LOX regulation at the renal tubular level. The aim of the study was to characterize the expression and regulation by hormones of the family of 12-LOX in mouse distal convoluted tubule at the molecular level. METHODS: An immortalized mouse distal convoluted tubule (mDCT) cell line was used. mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively, while 12(S)-HETE production was evaluated by enzyme-linked immunosorbent assay (ELISA). Cells were challenged with aldosterone, angiotensin II, 8Br-cAMP, and vasopressin. RESULTS: We showed that both platelet (P) and leukocyte (L)-type 12-LOX are expressed in the mDCT cell line, as well as in distal tubules of human kidneys. The production of 12(S)-HETE by mDCT cells was increased in response to cAMP (by two-fold) and by vasopressin (by 1.5-fold). In contrast, neither aldosterone nor angiotensin II exerted appreciable effects on 12(S)-HETE production. The mRNA and protein levels of P-12LOX and L-12LOX were not changed by the different hormones, suggesting that they may act by modulating enzyme activity. We further have demonstrated that this mDCT cell line also expressed the recently cloned 12(R)-LOX. CONCLUSION: mDCT cells show an active 12-LOX metabolism that appears to be modulated by cAMP and vasopressin.

Association of TGF-beta1 polymorphisms with chronic renal disease.

BACKGROUND: Transforming growth factor beta1 (TGF-beta1) plays an important role in tissue fibrosis and has been found to participate in cardiovascular disease (CVD). This study aimed to evaluate the association of TGF-beta1 polymorphisms with chronic renal disease (CRD), and its progression to dialysis in a retrospective longitudinal study of an end-stage renal disease (ESRD) cohort. METHODS: The Arg/Pro (codon 25) and Leu/Pro (codon 10) polymorphisms were genotyped in 104 ESRD patients aged 64 +/- 14 yrs (mean +/- SD), 62 males, and in 104 matched controls. RESULTS: The genotype distribution of Leu10Pro and Arg25Pro polymorphisms was different between patients and controls: Leu/Leu, Leu/Pro, Pro/Pro: 0.35, 0.50, 0.15 vs. 0.30, 0.24, 0.46 (p=0.001) and Arg/Arg, Arg/Pro, Pro/Pro: 0.79, 0.21, 0 vs. 0.87, 0.10, 0.03 (p=0.019). Similarly, haplotypes constructed with the combination of both polymorphisms were different among groups. There were no differences in CRD progression rate among genotypes. Codon 10 Leu allele was associated with the presence of clinical CVD in the ESRD patients (Leu/Leu, Leu/Pro, Pro/Pro: with CVD 0.49, 0.49, 0.02 vs. without CVD 0.27, 0.51, 0.22 (p=0.01). Combined polymorphism haplotypes were also significantly different between ESRD patients with and without CVD. This association was independent from other risk factors. CONCLUSIONS: TGF-beta1 polymorphisms are associated with ESRD, particularly in patients with associated clinical CVD, and could be useful as genetic markers of CRD and higher cardiovascular risk.

In vitro characterization of aldosterone and cAMP effects in mouse distal convoluted tubule cells.

The distal nephron plays a capital role in the fine regulation of sodium reabsorption. Compared with the cortical collecting duct, much less information is available on the hormonal regulation of sodium transporter genes in the distal convoluted tubule (DCT), where the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is the major entry pathway for Na(+). The purpose of this study was to characterize the in vitro effects of aldosterone (Aldo; 1 microM) and cAMP (8-BrcAMP; 0.5 mM) on mouse DCT (mDCT) by using an immortalized mDCT cell line. Western blot analysis and semiquantitative RT-PCR were performed to analyze the expression of genes involved in sodium transport. The mDCTcell line expressed the 11 beta-hydroxysteroid dehydrogenase type 2 gene and both the mineralocorticoid and glucocorticoid receptor genes, suggesting Aldo responsiveness. In this sense, we found that mDCT cells expressed the amiloride-sensitive Na(+) channel (ENaC) and responded to Aldo by upregulating the alpha-subunit protein. Similarly, alpha(1) Na(+)-K(+)-ATPase protein was upregulated by Aldo and 8-BrcAMP. In addition, the Aldo intermediate gene sgk1 mRNA was increased in response to both Aldo and 8-BrcAMP, and the transcription factor HNF-3 alpha mRNA was induced by 8-BrcAMP. With respect to NCC regulation, although Aldo induced NCC protein levels in mice in vivo, neither Aldo nor 8-BrcAMP significantly induced the NCC mRNA or protein levels in mDCT cells. These results suggest that in mDCT, Aldo and cAMP modulate some downstream mediators and effectors in vitro but do not influence the expression of NCC in this cell model.

Association between the A1166C polymorphism of the angiotensin II receptor type 1 and progression of chronic renal insufficiency.

BACKGROUND: In some studies genetic variation in the renin-angiotensin-aldosterone system (RAAS) has been associated with hypertension and rapid progression of renal insufficiency to end-stage renal disease (ESRD). Most of these studies do not take into account covariables influencing progression. We studied retrospectively the role of angiotensinogen (AGT) M235T, angiotensin converting enzyme (ACE) insertion/deletion (I/D), angiotensin II type 1 receptor (AT1R) A1166C, aldosterone syntase (CYP11B2) -344C/T and intron 2 W/C polymorphisms in conjunction with clinical and biochemical covariables on the rate of progression of renal insufficiency in a group of patients with ESRD of various etiologies. METHODS: Genotyping was performed by polymerase chain reaction (PCR) in 104 ESRD patients (62 males and 42 females), aged 64 +/- 14 years (mean +/- SD) with mean initial serum creatinine of 2.6 +/- 1.1 mg/dL and a mean time to reach ESRD of 52 +/- 38 months. RESULTS: The univariate analysis showed that there was a significant difference in the values of the slopes among the AT1R A1166C polymorphism genotypes: AA -4.87 +/- 0.22, AC -5.09 +/- 0.65 and CC -5.52 +/- 0.66 (p<0.05). None of the remainder polymorphisms showed significant association with progression. Stepwise multiple regression analysis including all the clinical, biochemical and genetic variables showed that only systolic blood pressure (SBP), serum PTHi and AT1R genotype were independently associated with the rate of progression, excluding the other variables from the model. CONCLUSIONS: These results indicate that susceptibility to faster progression to ESRD is associated with the AT1R A1166C polymorphism. This association remains significant after adjustment for relevant covariates, highlighting the importance of analyzing genetic risk factors in the context of clinical and biochemical variables.

G-protein beta3-subunit gene variant, blood pressure and erythrocyte sodium/lithium countertransport in essential hypertension.

Recently, a C825T polymorphism in the gene coding for the beta3 subunit of G proteins (GNB3) has been described in cells from patients with essential hypertension and enhanced Na+/H+ exchange activity. This study aims to evaluate the association between the 825T allele and activity of erythrocyte sodium/lithium countertransport (Na+/Li+ CT) and other sodium transport systems in red blood cells from patients with essential hypertension. A group of 77 patients (36 male, 41 female; aged 51.7 +/- 1.1 years) was studied. The maximal rates (Vmax) of Na+/Li+ CT, Na+/K+/Cl- cotransport and Na+K+ ATPase were evaluated in erythrocytes from all the patients. They were genotyped for the C825T polymorphism by a polymerase chain reaction (PCR) method, followed by digestion with BseDI. Body mass index (BMI) was higher in CT+TT patients than in CC patients (28.9 +/- 0.5 vs. 27.0 +/- 0.7 kg/m2; P=0.023). Hypertensives with the T allele (CT+TT genotypes) showed significantly higher systolic blood pressure (BP) values (156.9 +/- 2.1 vs. 148.9 +/- 2.8 mmHg; P=0.024), whereas differences in diastolic BP did not reach statistical significance (96.4 +/- 1.0 vs. 94.0 +/- 1.1 mmHg; P=0.120). No differences in the Vmax of Na+/Li+ CT between the genotypes was seen (CC: 236 +/- 19 and CT+TT 277 +/- 23 mmol/L cells per h; P=0.221). Similarly, no differences were detected in the Vmax of erythrocyte Na+/K+/Cl- cotransport and Na+K+ ATPase among the genotypes. There was no appreciable association between the G-protein beta3-subunit C825T polymorphism and erythrocyte Na+/Li+ CT and other sodium transport systems in the hypertensive patient sample studied; however, those with the T allele were more obese and had more severe systolic hypertension.

Association of the G protein beta3 subunit T allele with insulin resistance in essential hypertension.

A polymorphism (C825T) in the gene encoding the G protein beta3 subunit (GNB3) has recently been associated with hypertension and obesity in several populations. The aim of the study was to analyse the relationship between this polymorphism and insulin sensitivity, an hypothesised unifying factor for hypertension and obesity. One hundred thirty unrelated patients with essential hypertension, 70 female and 60 male, aged 58 +/- 1 years with systolic blood pressure of 173 +/- 2 mm Hg and diastolic blood pressure of 105 +/- 1 mm Hg, were genotyped for the GNB3 polymorphism by PCR and restriction digestion with BseDI, and classified in two groups according to the genotypes CC and CT + TT. Body mass index (BMI) was significantly higher in patients with the T allele as compared with patients without the T allele (29.3 +/- 0.4 vs. 26.7 +/- 0.6 kg/m2, p<0.001). On the contrary, there were no differences in the level of systolic or diastolic blood pressure among the genotypes. Insulin sensitivity was measured in a subgroup of 35 patients by means of an euglycemic hyperinsulinemic clamp test. In this subgroup, patients with the T allele displayed lower insulin sensitivity index (1.6 +/- 0.3 vs. 2.7 +/- 0.3 mg/kg/min, p = 0.022), higher fasting serum insulin (121 +/- 16 vs. 77 +/- 11 pmol/L, p = 0.032), higher serum glucose 120 min after 75 g load (9.8 +/- 1.2 vs. 7.0 +/- 0.5 mmol/L, p = 0.038), and higher glycosilated haemoglobin (5.7 +/- 0.4 vs. 4.7 +/- 0.2%; p = 0.042) as compared with patients without the T allele. A regression analysis showed that the association between the T allele and insulin sensitivity was independent of BMI (beta coefficient -0.386, p = 0.022). These results suggest a relationship between the 825T allele of GNB3 and insulin resistance in the essential hypertensive patients studied, which seems to be independent of BMI.

[Genetic polymorphisms of the renin-angiotensin system and essential hypertension]. / Polimorfismos genéticos del sistema renina-angiotensina e hipertensión arterial esencial.

BACKGROUND: The renin-angiotensin system (RAS) is known to regulate the blood pressure (BP). Several RAS polymorphisms have been associated with essential hypertension (EH), but there is uncertainty about this association. We examined whether the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene, and the M235T polymorphism of the angiotensinogen (AGT) gene were associated with EH in a sample of Spanish hipertensive patients. PATIENTS AND METHOD: We studied 1,204 patients with EH (BP > 140/90 mmHg): 668 males, aged 50.8 (13.6) years with systolic BP 151.7 (19.1) and diastolic BP 94.3 (13) mmHg [mean (SD)] and 536 females, aged 52.4 (13.9) years with systolic BP 155.1 (19.8) and diastolic BP 94.5 (12.3) mmHg. As a control group, 367 men and 280 women with no family history of cardiovascular disease who had a normal blood pressure were included. Polymorphisms were determined by PCR amplification of genomic DNA, followed by enzyme digestion for the AGT gene polymorphism. RESULTS: The genotype distribution and allele frequencies of the two RAS polymorphisms were similar in hypertensive and control subjects. Similarly, there were no differences in BP level with regard to the genotype in male or female patients. In addition, we did not find any compound effect of the I/D ACE gene and M235T AGT gene polymorphisms on BP levels in hypertensive subjects. CONCLUSIONS: This study suggests that in the population studied, the contribution of the ACE I/D polymorphism and the AGT M235T polymorphism in the development of EH is less important than previously estimated.

G-protein ß3-subunit gene variant, blood pressure and erythrocyte sodium/lithium countertransport in essential hypertension.

Recently, a C825T polymorphism in the gene coding for the ß3 subunit of G proteins (GNB3) has been described in cells from patients with essential hypertension and enhanced Na+/H+ exchange activity. This study aims to evaluate the association between the 825T allele and activity of erythrocyte sodium/lithium countertransport (Na+/Li+ CT) and other sodium transport systems in red blood cells from patients with essential hypertension. A group of 77 patients (36 male, 41 female; aged 51.7 ± 1.1 years) was studied. The maximal rates (Vmax) of Na+/Li+ CT, Na+/K+/Cl-cotransport and Na+K+ ATPase were evaluated in erythrocytes from all the patients. They were genotyped for the C825T polymorphism by a polymerase chain reaction (PCR) method, followed by digestion with BseDI. Body mass index (BMI) was higher in CT+TT patients than in CC patients (28.9 ± 0.5 vs. 27.0 ± 0.7 kg/m2; P=0.023). Hypertensives with the T allele (CT+TT genotypes) showed significantly higher systolic blood pressure (BP) values (156.9 ± 2.1 vs. 148.9 ± 2.8 mmHg; P=0.024), whereas differences in diastolic BP did not reach statistical significance (96.4 ± 1.0 vs. 94.0 ± 1.1 mmHg; P=0.120). No differences in the Vmax of Na+/Li+ CT between the genotypes was seen (CC: 236 ± 19 and CT+TT 277 ± 23 mmol/L cells per h; P=0.221). Similarly, no differences were detected in the Vmax of erythrocyte Na+/K+/Cl-cotransport and Na+K+ ATPase among the genotypes. There was no appreciable association between the G-protein ß3-subunit C825T polymorphism and erythrocyte Na+/Li+ CT and other sodium transport systems in the hypertensive patient sample studied; however, those with the T allele were more obese and had more severe systolic hypertension.