Biomedical subjects

Jerry B Lingrel

Publications and source records attributed to Jerry B Lingrel.

At least 19 recordsLinked to original sources

PubMed2006

Ouabain inhibits tubuloglomerular feedback in mutant mice with ouabain-sensitive alpha1 Na,K-ATPase.

Initiation of tubuloglomerular feedback (TGF) depends on Na-K-2Cl co-transport in the macula densa (MD), but it is less clear whether Na,K-ATPase is responsible for establishing the inward Na+ gradient. It has been proposed that apical colonic H,K-ATPase, perhaps in concert with the Na/H exchanger (NHE2), may account for MD Na+ exit in these cells. This study evaluated TGF responses by micropuncture in mutant mice with altered ouabain sensitivity of the alpha1 and alpha2 Na,K-ATPase isoforms. TGF responses in alpha1-sensitive/alpha2-resistant mice were inhibited by intravenous ouabain (control stop-flow pressure = 9.7 +/- 0.9 versus 1.6 +/- 0.5 mmHg with intravenous ouabain). Subsequent inclusion of cyclohexyladenosine (10 microM) in the tubule perfusate confirmed the ability of the afferent arteriole to contract in the presence of ouabain. In alpha1-resistant/alpha2-resistant mice, ouabain infusion had no effect on TGF responses. In separate experiments, loop of Henle perfusion with 50 microM ouabain decreased TGF responses (control stop-flow pressure) from 10.5 +/- 1.1 to 3.9 +/- 1.0 mmHg in alpha1-sensitive/alpha2-resistant mice but had no effect in alpha1-resistant/alpha2-resistant mice, and afferent arteriole responsiveness again was confirmed by cyclohexyladenosine. TGF responses in NHE2 and colonic H,K-ATPase knockout mice were not different from those of wild-type mice. These data indicate that TGF requires activity of the alpha1 Na,K-ATPase, presumably in the MD. Furthermore, the data show that neither NHE2 nor colonic H,K-ATPase is essential for initiation of TGF responses.

Adenosine↗

PubMed2006

Kruppel-like factor 2 regulates thymocyte and T-cell migration.

Mammalian Kruppel-like transcription factors are implicated in regulating terminal differentiation of several tissue types. Deficiency in Kruppel-like factor (KLF) 2 (also known as LKLF) leads to a massive loss of the peripheral T-cell pool, suggesting KLF2 regulates T-cell quiescence and survival. Here we show, however, that KLF2 is essential for T-cell trafficking. KLF2-deficient (Klf2-/-) thymocytes show impaired expression of several receptors required for thymocyte emigration and peripheral trafficking, including the sphingosine-1-phosphate (S1P) receptor S1P1, CD62L and beta7 integrin. Furthermore, KLF2 both binds and transactivates the promoter for S1P1--a receptor that is critical for thymocyte egress and recirculation through peripheral lymphoid organs. Our findings suggest that KLF2 serves to license mature T cells for trafficking from the thymus and recirculation through secondary lymphoid tissues.

Adoptive Transfer↗

PubMed2006

Up-regulation of the KLF2 transcription factor by fluid shear stress requires nucleolin.

We have previously characterized the regulation of the KLF2 transcription factor gene by describing an induction complex that binds to and regulates its promoter. In the present study, by using DNA affinity chromatography and mass spectrometry, we have identified nucleolin as an additional protein that binds to a palindromic response region in the KLF2 promoter. The presence of nucleolin on the KLF2 promoter in macrophages was verified by electrophoretic mobility shift assays. Interestingly, in mouse and human endothelial cell lines, electrophoretic mobility shift assays and chromatin immunoprecipitation analyses indicated that nucleolin binds the KLF2 promoter only upon application of fluid shear stress. Pretreatment of the endothelial cells with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked the shear stress-induced binding of nucleolin to the promoter, demonstrating its PI3K-dependent regulation. Additionally, nucleolin exhibited dynamic flow-specific, PI3K-dependent alterations in size. Anti-nucleolin antibodies interacted with a 110-kDa form in static endothelial cells and with several catalytic forms that changed in abundance after the application of shear stress. Immunoprecipitation experiments demonstrated that fluid flow induced the interaction of nucleolin with the p85 regulatory subunit of PI3K. Finally, introduction of small interfering RNAs targeting the nucleolin genetic sequence selectively reduced nucleolin expression and was sufficient to block the induction of KLF2 by shear stress. These data support a general role for nucleolin in gene regulation and identify it as a novel factor involved in regulation of KLF2 expression.

Amino Acid Sequence↗

PubMed2006

Physiological role of the alpha1- and alpha2-isoforms of the Na+-K+-ATPase and biological significance of their cardiac glycoside binding site.

An interesting feature of Na+-K+-ATPase is that it contains four isoforms of the catalytic alpha-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the alpha1- and alpha2-isoforms. While knockout mice demonstrated the importance of the alpha1- and alpha2-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the Na+-K+-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the Na+-K+-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant alpha2-isoform of Na+-K+-ATPase. Interestingly, animals with the ouabain-sensitive alpha1-isoform and a ouabain-resistant alpha2-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the Na+-K+-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH.

Animals↗

PubMed2005

The highly conserved cardiac glycoside binding site of Na,K-ATPase plays a role in blood pressure regulation.

The Na,K-ATPase contains a binding site for cardiac glycosides, such as ouabain, digoxin, and digitoxin, which is highly conserved among species ranging from Drosophila to humans. Although advantage has been taken of this site to treat congestive heart failure with drugs such as digoxin, it is unknown whether this site has a natural function in vivo. Here we show that this site plays an important role in the regulation of blood pressure, and it specifically mediates adrenocorticotropic hormone (ACTH)-induced hypertension in mice. We used genetically engineered mice in which the Na,K-ATPase alpha2 isoform, which is normally sensitive to cardiac glycosides, was made resistant to these compounds. Chronic administration of ACTH caused hypertension in WT mice but not in mice with an ouabain-resistant alpha2 isoform of Na,K-ATPase. This finding demonstrates that the cardiac glycoside binding site of the Na,K-ATPase plays an important role in blood pressure regulation, most likely by responding to a naturally occurring ligand. Because the alpha1 isoform is sensitive to cardiac glycosides in humans, we developed mice in which the naturally occurring ouabain-resistant alpha1 isoform was made ouabain-sensitive. Mice with the ouabain-sensitive "human-like" alpha1 isoform and an ouabain-resistant alpha2 isoform developed ACTH-induced hypertension to greater extent than WT animals. This result indicates that the cardiac glycoside binding site of the alpha1 isoform can also mediate ACTH-induced hypertension. Taken together these results demonstrate that the cardiac glycoside binding site of the alpha isoforms of the Na,K-ATPase have a physiological function and supports the hypothesis for a role of the endogenous cardiac glycosides.

Adrenocorticotropic Hormone↗

PubMed2005

Human Kruppel-like factor 5 is a target of the E3 ubiquitin ligase WWP1 for proteolysis in epithelial cells.

The transcription factor KLF5 plays an important role in human carcinogenesis. In epithelial cells, the KLF5 protein is tightly regulated by the ubiquitin-proteasome pathway. To better understand the mechanisms for the regulation of KLF5 protein, we identified and characterized an E3 ubiquitin ligase for KLF5, i.e. WWP1. We found that WWP1 formed a protein complex with KLF5 in vivo and in vitro. Furthermore, WWP1 mediated the ubiquitination and degradation of KLF5, and the catalytic cysteine residue of WWP1 is essential for its function. A PY motif in a transactivation domain of KLF5 is necessary for its interaction with WWP1. Finally, WWP1 was amplified and overexpressed in some cancer cell lines from the prostate and breast, which negatively regulated the function of KLF5 in gene regulation. These findings not only established WWP1 as an E3 ubiquitin ligase for KLF5, they also further implicated the KLF5 pathway in human carcinogenesis.

Amino Acid Motifs↗

PubMed2005

Sodium pump alpha2 subunits control myogenic tone and blood pressure in mice.

A key question in hypertension is: How is long-term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain-like compound (EOLC) and induces salt-dependent hypertension mediated by Na(+)/Ca(2)(+) exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na(+) pump alpha1 (alpha1(+/-)) or alpha2 (alpha2(+/-)) catalytic subunits. Both low-dose ouabain (1-100 nm; inhibits only alpha2) and high-dose ouabain (> or =1 microm; inhibits alpha1) elevate myocyte Ca(2)(+) and constrict arteries from alpha1(+/-), as well as alpha2(+/-) and wild-type mice. Nevertheless, only mice with reduced alpha2 Na(+) pump activity (alpha2(+/-)), and not alpha1 (alpha1(+/-)), have elevated blood pressure. Also, isolated, pressurized arteries from alpha2(+/-), but not alpha1(+/-), have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB-R7943) normalize myogenic tone in ouabain-treated arteries. Only the NCX blockers normalize the elevated myogenic tone in alpha2(+/-) arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt-dependent and ouabain-induced hypertension. Thus, chronically reduced alpha2 activity (alpha2(+/-) or chronic ouabain) apparently regulates myogenic tone and long-term blood pressure whereas reduced alpha1 activity (alpha1(+/-)) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt-dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing alpha2 Na(+) pump activity and promoting Ca(2)(+) entry via NCX in myocytes.

Animals↗

PubMed2005

Krüppel-like factor 2, a novel immediate-early transcriptional factor, regulates IL-2 expression in T lymphocyte activation.

Ag presentation to T lymphocytes and subsequent activation are characterized by a cascade of signaling events, some of which result in the transcriptional activation of a diverse set of genes. An important example is the induction of the IL-2 gene, which is a critical event in the escalation of T cell activation. Previous studies have found that expression of Krüppel-like factor 2 (KLF2), a zinc finger transcription factor, is extinguished after T cell activation. However, the biological role of KLF2 during T cell activation is still unknown. In this study we found that KLF2 protein degradation is delayed, and KLF2 expression is up-regulated during the early stage of T cell activation in primary T cells. Within a few hours, this process is reversed, and KLF2 expression is turned off. Next, we found that the expression of KLF2 significantly increases IL-2 production 4-fold in activated T cells, resulting from activation of the IL-2 promoter. By narrowing down the 2.0-kb IL-2 promoter region, we found that the KLF2 responsive element in the IL-2 promoter is a CACCC element, the KLF consensus binding motif. Moreover, KLF2 binds to this promoter in vivo under different conditions. Our studies show that KLF2 regulates IL-2 promoter activity in the earliest stages of T cell activation, indicating that KLF2 may act as a novel immediate-early transcriptional factor to maximally prime T cell activation.

Base Sequence↗

PubMed2005

The KLF2 transcription factor does not affect the formation of preadipocytes but inhibits their differentiation into adipocytes.

Kruppel-like transcription factor 2 (KLF2), a critical gene for mouse embryogenesis, was recently identified as an inhibitor of adipogenesis. However, it is still unknown whether KLF2 is a natural repressor of adipocyte differentiation and if KLF2 affects the formation of preadipocytes. It may also be important for preadipocyte formation, as KLF2 is crucial for lung development and blood vessel formation. In this study, we show that differentiation of preadipocytes not only results in a concomitant decrease in the levels of KLF2 protein but also significantly reduces KLF2 promoter activity. We have generated tet-responsive lines of 3T3L1 that express physiological levels of KLF2 and show that reexpression of KLF2 prevents preadipocyte differentiation, thereby confirming the inhibition of adipogenesis by KLF2, partially via the restoration of Pref-1. In addition, we studied the contribution of KLF2-negative cells to the formation and subsequent differentiation of preadipocytes. We demonstrate that embryoid bodies derived from KLF2(-)(/)(-) ES cells can differentiate into adipocytes as evidenced by the accumulation of lipids and expression of several biochemical markers. Moreover, mouse embryonic fibroblasts (MEFs) derived from KLF2(-)(/)(-) mouse embryos differentiate efficiently into adipocytes. Interestingly, quantification of lipid accumulation in MEFs indicated that KLF2(-)(/)(-) cells are more prone to differentiate at the early stage of the process, suggesting that KLF2 is a natural repressor of differentiation in vivo. Taken together, these studies demonstrate that KLF2 does not affect the commitment of multipotent stem cells into the preadipocytic lineage but rather maintains their preadipocyte state and thereby negatively regulates their transition into adipocytes.

Adipocytes↗

PubMed2005

KLF2 is essential for primitive erythropoiesis and regulates the human and murine embryonic beta-like globin genes in vivo.

The Krüppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krüppel-like factor (EKLF or KLF1) positively regulates the beta-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2-/- mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the beta-like globin genes in KLF2 knockout mice. Our results show that KLF2-/- mice have a significant reduction of murine embryonic Ey- and beta h1-globin but not zeta-globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult beta(maj)- and beta(min)-globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic epsilon-globin gene but not the adult beta-globin gene, suggesting that this developmental-stage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2-/- embryos have a significantly increased number of primitive erythroid cells undergoing apoptotic cell death.

Animals↗

PubMed2005

Kruppel-like factor 2 transcriptional regulation involves heterogeneous nuclear ribonucleoproteins and acetyltransferases.

Kruppel-like factor 2 (KLF2) is expressed in several cell types, and knockout animals have shown that KLF2 gene regulation is involved in multiple biological processes. These include maintaining T-cells in the quiescent state, preventing preadipocytes from differentiating into mature adipocytes, stabilizing blood vessel walls through endothelial cell function, and advancing the later stages of lung development. Defining the regulation of KLF2 expression is important to understand these diverse functions. Promoter analysis of KLF2 has revealed that a region between -138 and -111 base pairs is required for its transcription, and this nucleotide sequence occurs in a region that is highly conserved in evolution. The present study was carried out to identify transcription factors that bind to this region of the KLF2 promoter. Nuclear factors were enriched by DNA affinity chromatography using the conserved nucleotide sequence of the KLF2 promoter. Mass spectrometry analysis of the proteins eluted from the affinity matrix identified several proteins, including glucose regulated protein-78 kDa (GRP-78), heterogeneous nuclear ribonucleoprotein (hnRNP)-U, hnRNP-D, CArG binding factor (CBF), P300/CBP associated factor (PCAF), cAMP response element binding protein (CREB) and SWI/SNF. The binding of these proteins to the highly conserved region of the KLF2 promoter element was tested by electrophoretic mobility supershift assays and chromatin immunoprecipitation analysis. These procedures confirmed that hnRNP-U, hnRNP-D, PCAF, and P-300 bind to the KLF2 promoter. Transactivation experiments demonstrated that these proteins are important for regulating KLF2 transcription. Of special interest is the role of hnRNPs in the transcription of the KLF2 gene.

Acetyltransferases↗

PubMed2005

Induction of KLF2 by fluid shear stress requires a novel promoter element activated by a phosphatidylinositol 3-kinase-dependent chromatin-remodeling pathway.

Fluid shear stress maintains vascular homeostasis by influencing endothelial gene expression. One mechanism by which shear stress achieves this is through the induction of transcription factors including Krüppel-like factor 2(KLF2). We have previously reported that a 62-bp region of the KLF2 promoter is responsible for its shear stress-induced expression via the binding of nuclear factors. In this study, we find that the 62-bp shear stress response region contains a 30-bp tripartite palindrome motif. Electrophoretic mobility supershift and chromatin immunoprecipitation assays demonstrate that PCAF (P-300/cAMP-response element-binding protein-binding protein-associated factor)) and heterogeneous nuclear ribonucleoprotein D bind this region as components of the shear stress regulatory complex. We have also characterized a PI3K-dependent/Akt-independent pathway responsible for shear stress-induced KLF2 nuclear binding, promoter activation, and mRNA expression. Furthermore, the shear stress response region of the KLF2 promoter was specifically immunoprecipitated by antibodies against acetylated histones H3 and H4 in shear-stressed but not static hemangioendothelioma cells. The acetylation of these histones was blocked by PI3K inhibition. Finally, we have found that KLF2 increases endothelial nitric-oxide synthase expression in murine endothelial cultures, an effect that is also blocked by PI3K inhibition. These results define the DNA regulatory element, signal transduction pathway, and molecular mechanism activating the flow-dependent expression of a vital endothelial transcription factor.

Acetyltransferases↗

PubMed2005

Decreased expression of both the alpha1- and alpha2-subunits of the Na-K-ATPase reduces maximal alveolar epithelial fluid clearance.

Impaired epithelial sodium channel function predisposes to delayed resorption of pulmonary edema and more severe experimental lung injury, whereas even a small fraction of the normal Na-K-ATPase activity is thought to be sufficient to maintain normal ion transport. However, direct proof is lacking. Therefore, we studied baseline and cAMP stimulated alveolar fluid clearance (AFC) in mice with a 50% decrease in lung protein expression of the alpha(1)- and/or alpha(2)-subunit of the Na-K-ATPase. There was no difference in basal and stimulated AFC in alpha(1)(+/-) or alpha(2)(+/-) mice compared with wild-type littermates. Also, the compound heterozygous mice (alpha(1)(+/-)/alpha(2)(+/-)) had normal basal AFC. However, the combined alpha(1)(+/-)/alpha(2)(+/-) mice showed a significant decrease in cAMP-stimulated AFC compared with wild-type littermates (11.1 +/- 1.0 vs. 14.9 +/- 1.8%/30 min, P < 0.001). When exposed to 96 h of >95% hyperoxia, the decrease in stimulated AFC in the alpha(1)(+/-)/alpha(2)(+/-) mice was not associated with more lung edema compared with wild-type littermates (lung wet-to-dry weight ratio 6.6 +/- 0.9 vs. 5.9 +/- 1.1, respectively; P = not significant). Thus a 50% decrease in protein expression of the alpha(1)- or alpha(2)-subunits of the Na-K-ATPase does not impair basal or stimulated AFC. However, a 50% protein reduction in both the alpha(1)- and alpha(2)-subunits of the Na-K-ATPase produces a submaximal stimulated AFC, suggesting a synergistic role for alpha(1)- and alpha(2)-subunits in cAMP-dependent alveolar epithelial fluid clearance.

Animals↗

PubMed2005

Relative abundance of alpha2 Na(+) pump isoform influences Na(+)-Ca(2+) exchanger currents and Ca(2+) transients in mouse ventricular myocytes.

In the mouse, genetic reduction in the Na(+), K(+)-ATPase alpha1 or alpha2 isoforms results in different functional phenotypes: heterozygous alpha2 isolated hearts are hypercontractile, whereas heterozygous alpha1 hearts are hypocontractile. We examined Na(+)/Ca(2+) exchange (NCX) currents in voltage clamped myocytes (pipette [Na(+)]=15 mM) induced by abrupt removal of extracellular Na(+). In wild-type (WT) myocytes, peak exchanger currents were 0.59+/-0.04 pA/pF (mean+/-S.E.M., n=10). In alpha1(+/-) myocytes (alpha2 isoform increased by 54%), NCX current was reduced to 0.33+/-0.05 (n=9, P<0.001) indicating a lower subsarcolemmal [Na(+)]. In alpha2(+/-) myocytes (alpha2 isoform reduced by 54%), the NCX current was increased to 0.89+/-0.11 (n=8, P=0.03). The peak sarcolemmal Na(+) pump currents activated by abrupt increase in [K(+)](o) to 4 mM in voltage clamped myocytes in which the Na(+) pump had been completely inhibited for 5 min by exposure to 0 [K(+)](o) were similar in alpha1(+/-) (0.86+/-0.12, n=10) and alpha2(+/-) myocytes (0.94+/-0.08 pA/pF, n=16), and were slightly but insignificantly reduced relative to WT (1.03+/-0.05, n=24). The fluo-3 [Ca(2+)](i) transient (F/F(o)) in WT myocytes paced at 0.5 Hz was 2.18+/-0.09, n=34, was increased in alpha2(+/-) myocytes (F/F(o)=2.56+/-0.14, n=24, P=0.02), and was decreased in alpha1(+/-) myocytes (F/F(o)=1.93+/-0.08, n=28, P<0.05). Thus the alpha2 isoform rather than the alpha1 appears to influence Na(+)/Ca(2+) exchanger currents [Ca(2+)](i) transients, and contractility. This finding is consistent with the proposal that alpha2 isoform of the Na pump preferentially alters [Na(+)] in a subsarcolemmal micro-domain adjacent to Na(+)/Ca(2+) exchanger molecules and SR Ca(2+) release sites.

Animals↗

PubMed2005

Genetic profiling reveals global changes in multiple biological pathways in the hearts of Na, K-ATPase alpha 1 isoform haploinsufficient mice.

The Na,K-ATPase transports three sodium ions out of the cell and two potassium ions into the cell using ATP hydrolysis for energy. The ion gradient formed by the Na,K-ATPase contributes to the resting membrane potential, maintains cellular excitability and is important for glucose and amino acid uptake in the cell. The alpha1 catalytic isoform is expressed in virtually all cell types. We have previously examined cardiac physiology of mice lacking one copy of the alpha1 isoform gene of the Na,K-ATPase. The observation of reduced cardiac contractility in the alpha1 heterozygous mice was unexpected since mice heterozygous for the alpha2 isoform displayed enhanced cardiac contractility similar to what would be observed with cardiac glycoside treatment. We further examined hearts from alpha1 heterozygous mice to identify genomic responses to reduced Na,K-ATPase capacity. Using microarray analyses, we identified groups of genes whose expressions were perturbed in the alpha1 heterozygous hearts compared to wild-type. Known functional relationships of these genes suggest that multiple biological pathways are altered by alpha1 hemizygosity including activation of the renin-angiotensin system, changes in genes of energy metabolism and transport and elevated brain natriuretic peptide. This suggests that Na,K-ATPase alpha1 isoform activity may be required in numerous cellular processes.

Aldosterone↗

PubMed2004

KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1.

Kruppel-like factor 2 (KLF2) is a member of the KLF family of zinc-finger transcription factors and is involved in maintaining T-cell quiescence, regulating preadipocyte differentiation, endothelial cell function and lung development. We used a tetracycline-inducible system in Jurkat T leukemia cells to study the biological role of KLF2 in cellular growth and differentiation. Our results show that expression of KLF2 inhibits cell growth in autonomously proliferating Jurkat cells. Further, 3H-thymidine uptake assays indicate that KLF2 inhibits DNA synthesis in these cells. Moreover, both activation and inhibitory domains are required for KLF2 to suppress Jurkat cell proliferation. In addition, KLF2 upregulates p21WAF1/CIP1 expression. Additionally, we found that KLF2 upregulates p21WAF1/CIP1 promoter activity in Jurkat, HepG2 and SW480 cells. Our analysis shows that the potential KLF2 responsive elements are located between -124 and -60 of the p21WAF1/CIP1 promoter. The sole CACCC site, a sequence recognized by KLF2, in this region is not the element responsive to KLF2. Finally, we determined that the Sp1-3-binding site is the functional responsive element of KLF2 in the p21WAF1/CIP1 promoter, and we conclude that KLF2 directly regulates p21WAF1/CIP1 expression.

Cell Division↗

PubMed2004

The alpha 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart.

The primary objective of this study was to examine the functional role of the Na,K-ATPase alpha 1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the alpha 1 or alpha 2 isoform gene exhibit opposite phenotypes. Hearts from alpha 2(+/-) animals are hypercontractile, whereas those of the alpha 1(+/-) animals are hypocontractile. The cardiac phenotype of the alpha 1(+/-) animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive alpha 1 isoform and a ouabain-resistant alpha 2 isoform of the Na,K-ATPase, and we analyzed cardiac contractility following selective inhibition of the alpha1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the alpha 1 isoform without affecting the activity of the alpha 2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified alpha 1 and alpha 2 isoforms. Pretreatment with 10 microm KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the alpha1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The alpha 1 isoform co-immunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the alpha 1 isoform regulates cardiac contractility, and that both the alpha 1 and alpha 2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart.

Animals↗

PubMed2004

The alpha2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro.

Although ouabain is known to induce hypertension, the mechanism of how this cardiac glycoside affects blood pressure is uncertain. The present study demonstrates that the alpha2-isoform of the Na-K-ATPase mediates the pressor effects of ouabain in mice. To accomplish this, we analyzed the effect of ouabain on blood pressure in wild-type mice, where the alpha2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive alpha2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the alpha2-isoform of Na-K-ATPase. As the alpha1-isoform is naturally resistant to ouabain in rodents, it will not be inhibited by ouabain in either genotype. Whereas prolonged administration of ouabain increased levels of ouabain in serum from both wild-type and targeted animals, hypertension developed only in wild-type mice. In addition, bolus intravenous infusion of ouabain increased the systolic, mean arterial, and left ventricular blood pressure in only wild-type anesthetized mice. In vitro, ouabain increased vascular tone and thereby phenylephrine-induced contraction of the aorta in intact and endothelium-denuded wild-type mice but in alpha2-resistant mice. Ouabain also increased the magnitude of the spontaneous contractions of portal vein and the basal tone of the intact aorta from only wild-type mice. The increase in aortic basal tone was dependent on the presence of endothelium. Our studies also demonstrate that the alpha2-isoform of Na-K-ATPase mediates the ouabain-induced increase in vascular contractility. This could play a role in the development and maintenance of ouabain-induced hypertension.

Anesthesia↗