Physical and functional antagonism between tumor suppressor protein p53 and fortilin, an anti-apoptotic protein.

Tumor suppressor protein p53, our most critical defense against tumorigenesis, can be made powerless by mechanisms such as mutations and inhibitors. Fortilin, a 172-amino acid polypeptide with potent anti-apoptotic activity, is up-regulated in many human malignancies. However, the exact mechanism by which fortilin exerts its anti-apoptotic activity remains unknown. Here we present significant insight. Fortilin binds specifically to the sequence-specific DNA binding domain of p53. The interaction of fortilin with p53 blocks p53-induced transcriptional activation of Bax. In addition, fortilin, but not a double point mutant of fortilin lacking p53 binding, inhibits p53-dependent apoptosis. Furthermore, cells with wild-type p53 and fortilin, but not cells with wild-type p53 and the double point mutant of fortilin lacking p53 binding, fail to induce Bax gene and apoptosis, leading to the formation of large tumor in athymic mice. Our results suggest that fortilin is a novel p53-interacting molecule and p53 inhibitor and that it is a logical molecular target in cancer therapy.

Embryonic lethality of fortilin-null mutant mice by BMP-pathway overactivation.

BACKGROUND: Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. METHODS AND RESULTS: In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin(+/-) mice have the ability to survive and exhibit normal growth, while fortilin(-/-) mice are embryonically lethal around the 3.5 days post-coital (dpc). Cultured blastocysts from fortilin(+/-) embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin(-/-) embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin(+/-) embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilin's message severely compromises the formation of neural tissue, even in the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. CONCLUSION: We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. GENERAL SIGNIFICANCE: Abnormalities in the fortilin gene may be associated with early pregnancy loss.

The novel angiotensin II type 1 receptor (AT1R)-associated protein ATRAP downregulates AT1R and ameliorates cardiomyocyte hypertrophy.

Activation of angiotensin II (Ang II) type 1 receptor (AT1R) signaling is reported to play an important role in cardiac hypertrophy. We previously cloned a novel molecule interacting with the AT1R, which we named ATRAP (for Ang II type 1 receptor-associated protein). Here, we report that overexpression of ATRAP significantly decreases the number of AT1R on the surface of cardiomyocytes, and also decreases the degree of p38 mitogen-activated protein kinase phosphorylation, the activity of the c-fos promoter and protein synthesis upon Ang II treatment. These results indicate that ATRAP significantly promotes downregulation of the AT1R and further attenuates certain Ang II-mediated hypertrophic responses in cardiomyocytes.

Nuclear receptor LXRalpha is involved in cAMP-mediated human renin gene expression.

The cAMP-signaling pathway plays a crucial role in the regulation of the renin gene, but the mechanism involved remains poorly understood. We have focused our studies of renin gene regulation on the unique cAMP responsive element (huREN/CNRE, -135 to -107) in the human renin promoter. We have cloned a protein that binds to this unique CNRE and demonstrated that this protein is liver X receptor-alpha (LXRalpha), a transcriptional factor of the nuclear receptor family. Transient expression of LXRalpha in human renin-producing Calu-6 cells increased cAMP inducibility of human renin promoter. Similarly, LXRalpha-stably transfected Calu-6 cells exhibited increased cAMP inducibility of renin promoter as well as the endogenous renin gene. Site-directed mutation of huREN/CNRE, which disrupted LXRalpha binding, decreased cAMP-induced transcriptional activity of human renin promoter. Furthermore, we demonstrated that the binding of LXRalpha derived from human juxtaglomerular cells, the main production site of renin in the kidney, to the huREN/CNRE in vivo. These results suggest that LXRalpha plays an important role in the cAMP-mediated regulation of human renin gene transcription by binding to CNRE.

Differential induction of protein kinase C isoforms at the cardiac hypertrophy stage and congestive heart failure stage in Dahl salt-sensitive rats.

Several protein kinase C (PKC) isoforms may play important roles in cellular signaling pathways. Recent reports have suggested that PKC plays critical isoform-specific roles in the development of cardiac hypertrophy and heart failure. The purpose of the present study was to examine the expression profiles of PKC isoforms in models of cardiac hypertrophy and heart failure. We examined the cardiac expression of individual PKC isoforms at the cardiac hypertrophy stage and the heart failure stage in Dahl salt-sensitive rats by Western blot analysis. The levels of all PKC isoforms increased at the cardiac hypertrophy stage and the heart failure stage, but the pattern of increase differed among PKC isoforms at the heart failure stage. The expressions of PKCalpha, beta, and delta increased at the cardiac hypertrophy stage and remained elevated at the heart failure stage. On the other hand, the expression of PKCepsilon and atypical PKCs (aPKCs) increased at the cardiac hypertrophy stage, but this increase tended to decline at the congestive heart failure stage. These results suggest that there are two groups of PKC isoforms. Several reports have shown that PKCalpha, beta, and delta are involved in the development of cardiac hypertrophy and heart failure, and that PKCepsilon plays a role in the physiological hypertrophic responses and cardioprotective actions. These facts suggest that all PKC isoforms (PKCalpha, beta, delta, epsilon, and aPKCs) expressed in the heart may have similar functions at the cardiac hypertrophy stage, but that two groups of PKC isoforms (PKCalpha, beta, delta, and PKCepsilon, aPKCs) have different functions at the congestive heart failure stage.

The physiology and pathophysiology of a novel angiotensin receptor-binding protein ATRAP/Agtrap.

The Ang II type 1 receptor (AT1R)-associated protein (ATRAP/Agtrap) is a molecule specifically interacting with the carboxyl- terminal domain of AT1R. The results of in vitro studies showed that ATRAP suppresses Ang II-mediated pathological responses in cardiovascular cells by promoting AT1R internalization. With respect to the tissue distribution and regulation of ATRAP expression in vivo, ATRAP is broadly expressed in many tissues as is AT1R. Accumulating evidence indicates that a tissue-specific regulatory balancing of ATRAP and AT1R expression may be involved in the modulation of AT1R signaling at local tissue sites and also in the pathophysiology of hypertension and its associated end-organ injury. Furthermore, the activation of ATRAP in transgenic-models inhibited inflammatory vascular remodeling and cardiac hypertrophy in response to Ang II stimulation. These results suggest the clinical potential benefit of an ATRAP activation strategy in the treatment of hypertension and related organ injury.

Expression of MAK-V/Hunk in renal distal tubules and its possible involvement in proliferative suppression.

MAK-V/Hunk is an SNF1-related serine/threonine kinase which was previously shown to be highly expressed in the mammary gland and central nervous system. In this study, we found MAK-V/Hunk is abundantly and specifically expressed in the thick ascending limbs and distal convoluted tubules (DCT) of the kidney from the embryonic stage to the adult stage. We demonstrated that dietary salt depletion significantly enhances renal MAK-V/Hunk mRNA levels compared with a normal-salt diet. To analyze the possible renal cellular function of this kinase, we employed mouse distal convoluted tubule (mDCT) cells. The results of reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that MAK-V/Hunk is expressed endogenously in mDCT cells. Overexpression of MAK-V/Hunk by adenoviral gene transfer significantly inhibited the ANG II-induced stimulation of c-fos gene transcription and suppressed the ANG II-mediated increases in transforming growth factor-beta production into the medium. This phenomenon was accompanied by inhibition of ANG II-induced activation of BrdU incorporation. On the other hand, the MAK-V/Hunk knockdown by siRNA activated the ANG II-induced c-fos gene expression. In the consecutive sections stained for MAK-V/Hunk and AT(1) receptor, MAK-V/Hunk-immunopositive distal tubules expressed the AT(1) receptor. This is the first report on the intrarenal localization of MAK-V/Hunk and its cellular function in renal tubular cells.

Analysis of factors that affect short-term blood pressure variability in patients with chronic renal failure.

Recent reports suggest the relationship of short-term blood pressure (BP) variability to cardiovascular target organ damage. In this study, short-term BP variability was assessed as the standard deviation of daytime and nighttime BP in 36 hospitalized patients with chronic renal failure (CRF) who underwent ambulatory BP monitoring. Positive correlations were observed between body mass index (BMI) and daytime systolic and diastolic BP variability, BMI and nighttime diastolic BP variability, cholesterol and daytime systolic BP variability, cholesterol and nighttime systolic and diastolic BP variability, nocturnal decline in BP and nighttime diastolic BP variability, and plasma concentration of norepinephrine (p-NE) and nighttime systolic BP variability. In multivariate linear regression analyses, BMI showed the strongest association with daytime and nighttime diastolic BP variability (p < .005 and p < .05). On the other hand, cholesterol and p-NE were the primary determinants of daytime and nighttime systolic BP variability, respectively (p < .01 and p < .0005). Interestingly, CRF patients with ischemic heart disease (IHD) had significantly increased daytime systolic and diastolic BP variability and nighttime systolic BP variability (p < .05 or less). Furthermore, logistic regression analysis demonstrated that nighttime systolic BP variability was an independent risk factor of IHD in patients with CRF (odds ratio 1.50 [95% confidence interval 1.01 to 2.25]; p < .05). Taken together, short-term BP variability is suggested to be affected by BMI, cholesterol, and p-NE in CRF patients. Furthermore, sympathetic nerve overactivity may be involved in cardiovascular complications in CRF patients through the increase in nighttime systolic BP variability.

Alterations in renal endothelial nitric oxide synthase expression by salt diet in angiotensin type-1a receptor gene knockout mice.

The effects of altered dietary salt intake and/or hydralazine-induced hypotension on renal endothelial nitric oxide synthase (eNOS) expression were determined in angiotensin type-1a receptor gene knockout (At1a-/-) and wild-type (At1a+/+) mice. In At1a-/- mice, the levels of renal cortical eNOS mRNA and protein were 5 times and 3.5 times higher, respectively, in the high-salt (4% NaCl) group than in the low-salt group (0.3% NaCl). Systemic BP of the high-salt group (105 +/- 4.4 mmHg) was significantly higher than that of the low-salt group (77.0 +/- 4.7 mmHg). When hydralazine was administered to the mutant mice fed a high-salt diet, BP was reduced to 72.5 +/- 1.3 mmHg, with decreases in the levels of renal eNOS mRNA and protein expression to about half of those found in nontreated group. Consistent with the results for eNOS mRNA and protein expression, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and eNOS immunoreactivity localized in the endothelium of the renal vasculature changed parallel with the amount of salt intake. In contrast to mutant mice, At1a+/+ mice did not show any changes in renal eNOS expression during the manipulation of salt intake and/or hydralazine-induced hypotension. These results suggest that At1a receptor-mediated inputs play critical roles in maintaining renal vascular eNOS expression and activity during changes in salt-water balance and systemic BP.

Acute renal failure due to cholesterol crystal embolism treated with LDL apheresis followed by corticosteroid and candesartan.

Cholesterol crystal embolism (CCE) is caused by the shedding of cholesterol crystals into the bloodstream, and it has been recently recognized as a serious complication after vascular procedures. Our case of CCE, which was diagnosed by skin and renal biopsies, occurred in a patient with hypertension and diabetes mellitus, 3 months after coronary angiography, with the development of renal failure and blue toes. After low-density lipoprotein apheresis (LDL-A), the skin lesions, including livedo reticularis and pain from the acrocyanotic toes, dramatically improved, with partial recovery of renal function. Following the administration of low-dose corticosteroid and candesartan--an angiotensin II type 1 receptor antagonist (ARB)--the eosinophilia disappeared and renal function improved gradually with a decrease in urinary protein excretion. Therefore, a combination therapy of LDL-A, low-dose corticosteroid, and an ARB is a possible treatment for CCE, although the possibility of spontaneous recovery of renal function cannot be eliminated for this patient.