Biomedical subjects

Kay-Dietrich Wagner

Publications and source records attributed to Kay-Dietrich Wagner.

At least 19 recordsLinked to original sources

PubMed2006

The Wilms tumor suppressor Wt1 promotes cell adhesion through transcriptional activation of the alpha4integrin gene.

Cell-matrix interaction through specific adhesion molecules is a critical step during organ development. In addition, down-regulation of cell adhesion receptors may promote tumor invasion and metastasis. We show here that the Wilms tumor suppressor Wt1, which is necessary for normal development of the epicardium, coronary vessels, genitourinary system, and other tissues, activates transcription of the alpha4integrin gene. Binding of the Wt1(-KTS) form, which is transcriptionally active, to the proximal alpha4integrin promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. A reporter construct harboring approximately 1.9 kb of the human alpha4integrin gene promoter was activated significantly by transient co-transfection of a Wt1(-KTS) expression plasmid. Introducing mutations in two identified Wt1(-KTS) binding motifs in the proximal promoter of the alpha4integrin gene abrogated this stimulatory effect. Endogenous alpha4integrin transcripts were increased more than 3-fold in human embryonic kidney 293 cells with stable expression of the Wt1(-KTS) protein. Wt1-overexpressing cells showed augmented adhesion to the alpha4integrin ligand vascular cell adhesion molecule-1 that was abolished upon incubation with an inhibitory alpha4integrin antibody. Double immunofluorescent staining revealed co-localization of Wt1 and alpha4integrin in the developing epicardium of mouse embryos. Cardiac expression of alpha4integrin was reduced significantly in embryos with a homozygous Wt1 defect (Wt1-/-). These findings demonstrate that Wt1 can support cell adhesion through enhanced expression of alpha4integrin. This transcriptional activation of the alpha4integrin gene by Wt1(-KTS) might contribute to normal formation of the epicardium and other tissues in the developing embryo.

Alleles↗

PubMed2006

An inducible mouse model for PAX2-dependent glomerular disease: insights into a complex pathogenesis.

Pax2 is a transcription factor with important functions during kidney development . Ectopic expression of Pax2 in podocytes has been reported in various glomerular diseases , but the functional relevance remains unknown. We developed an inducible mouse model that allows activation of Pax2 specifically in podocytes. Persistent expression of Pax2 did not interfere with the initial differentiation of podocytes, but mice ectopically expressing PAX2 developed end-stage renal failure soon after birth. Similarly, activation of PAX2 in healthy adult animals resulted in renal disease within 3 weeks after podocyte-specific induction of a deleter Cre. PAX2 activation caused repression of the podocyte key regulator molecule Wt1 and consequently a dramatic reduction of nephrin expression. Recruitment of the groucho-related protein TLE4 may be involved in converting Pax2 into a transcriptional repressor of Wt1. Finally, treatment of mice with an angiotensin-converting enzyme (ACE) inhibitor normalized renal function and induced upregulation of the important structural molecule nephrin via a Wt1-independent pathway. Our data demonstrate the functional significance of PAX2 reexpression in mature podocytes for the development of glomerular diseases and suggest that reactivation of PAX genes in terminally differentiated cells leads to a more dedifferentiated phenotype.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2006

Intermediate filament protein nestin is expressed in developing kidney and heart and might be regulated by the Wilms' tumor suppressor Wt1.

Nestin is an intermediate filament protein originally described in neural stem cells and a variety of progenitor cells. More recently, nestin was detected in rat kidney podocytes. We show here that nestin is expressed in a developmentally regulated pattern in the kidney. Nestin was detected by immunohistochemistry in the condensing mesenchyme surrounding the ureter, in developing glomeruli, in podocytes of the adult kidney, and in a podocyte cell line. Nestin shared a striking overlap in expression with the Wilms' tumor suppressor Wt1. Nestin was significantly upregulated in a cell line with inducible Wt1 expression upon induction of Wt1. Cotransfection experiments in human embryonic kidney cells (HEK293) revealed stimulation of a nestin intron 2 enhancer element up to six-fold by the Wt1(-KTS) splice variant. Nestin expression was significantly reduced in an inducible mouse model of glomerular disease. This model is based on podocyte-specific overexpression of Pax2 and associated with a loss of Wt1 expression. Furthermore, also in the developing heart, nestin was found in an overlapping pattern with Wt1 in the epicardium and the forming coronary vessels. Strikingly, in the hearts of Wt1 knockout mice, nestin was barely detectable compared with the hearts of wild-type embryos. Our results show that nestin is expressed at different stages of kidney and cardiac development and suggest that its expression in these organs might be regulated by the Wilms' tumor suppressor Wt1.

Animals↗

PubMed2005

Coronary vessel development requires activation of the TrkB neurotrophin receptor by the Wilms' tumor transcription factor Wt1.

The formation of intramyocardial blood vessels is critical for normal heart development and tissue repair after infarction. We report here expression of the Wilms' tumor gene-1, Wt1, in coronary vessels, which could contribute to the defective cardiac vascularization in Wt1-/- mice. Furthermore, the high-affinity neurotrophin receptor TrkB, which is expressed in the epicardium and subepicardial blood vessels, was nearly absent from Wt1-deficient hearts. Activation of Wt1 in an inducible cell line significantly enhanced TrkB expression. The promoter of NTRK2, the gene encoding TrkB, was stimulated approximately 10-fold by transient cotransfection of a Wt1 expression construct. The critical DNA-binding site for activation of the NTRK2 promoter by Wt1 was delineated by DNase I footprint analysis and electrophoretic mobility shift assay. Transgenic experiments revealed that the identified Wt1 consensus motif in the NTRK2 promoter was necessary to direct expression of a reporter gene to the epicardium and the developing vasculature of embryonic mouse hearts. Finally, mice with a disrupted Ntrk2 gene lacked a significant proportion of their intramyocardial blood vessels. These findings demonstrate that transcriptional activation of the TrkB neurotrophin receptor gene by the Wilms' tumor suppressor Wt1 is a crucial mechanism for normal vascularization of the developing heart.

Animals↗

PubMed2005

A splice variant of the Wilms' tumour suppressor Wt1 is required for normal development of the olfactory system.

Neuronal lineage formation in the developing olfactory epithelium has been extensively studied at the cellular level, but little is known about the genes that control proliferation and differentiation of neuronal progenitor cells. Here, we report that the Wilms' tumour zinc-finger protein, Wt1, is required for normal formation of the olfactory epithelium. Wt1 was detected by immunohistochemistry in the developing olfactory epithelium of wild-type embryos between gestational days E9.5 and E18.5. Embryos with complete lack of Wt1 and embryos with selective ablation of the alternatively spliced Wt1(+KTS) isoform both had thinner olfactory epithelia and fewer neuronal progenitor cells than do normal animals. Mash1 and neurogenin 1, two basic helix-loop-helix transcription factors with critical functions during olfactory neuron development, were reduced in the Wt1(+KTS)-/- mutants compared with the wild-type mice. Stable expression of the Wt1(+KTS) isoform, but not of the Wt1(-KTS) variant, upregulated Mash1 mRNA and protein in vitro. The olfactory epithelia of mouse embryos, which lacked the Wt1(-KTS) protein, appeared normal. However, formation of the neural retina was severely impaired in the Wt1(-KTS)-/- mutants. These findings demonstrate that the Wt1(+KTS) protein, which has been proposed to play a role in mRNA processing, acts upstream of Mash1 to promote the development of the olfactory epithelium. Furthermore, neuron formation depends on distinct functions of alternatively spliced Wt1 products in the embryonic retina and the olfactory epithelium.

Alternative Splicing↗

PubMed2004

The major podocyte protein nephrin is transcriptionally activated by the Wilms' tumor suppressor WT1.

NPHS1 encodes the structural protein nephrin, which has a crucial role in the filtration barrier of the glomerular podocyte. Mutations or deregulation of NPHS1 are associated with a variety of renal diseases, including the Finnish type congenital nephrotic syndrome. This study analyzed a potential regulation of nephrin by the Wilms' tumor protein, Wt1. Using an inducible U2OS osteosarcoma cell line, it is shown that upon Wt1 induction, endogenous nephrin mRNA becomes highly upregulated. Co-transfection studies demonstrate that Wt1 can activate the nephrin promoter >10-fold. DNase footprinting and mutation analysis identify a Wt1 responsive element in the nephrin promoter, which is required for the binding of Wt1 protein. Mutations or deletion of this Wt1 responsive element completely abolished transactivation of the nephrin promoter by Wt1. Moreover, transgenic analysis demonstrates the requirement of the identified binding site to direct podocyte-specific expression of a reporter gene in transgenic mice, thus confirming the importance of this site for the regulation of nephrin in vivo. Finally, it is shown that nephrin expression is lowest in kidneys of mice that lack specifically the Wt1(-KTS) splice variant, but in comparison with wild-type littermates, it is also reduced in animals with disruption of the Wt1(+KTS) splice variant. Taken together, these data identify nephrin as a direct transcriptional target for Wt1 and underline the importance of Wt1 as a key regulator in podocyte function.

Animals↗

PubMed2003

Oxygen-regulated expression of the Wilms' tumor suppressor Wt1 involves hypoxia-inducible factor-1 (HIF-1).

The Wilms' tumor gene Wt1 is unique among tumor suppressors because of its requirement for the development of certain organs. We recently described de novo expression of Wt1 in myocardial blood vessels of ischemic rat hearts. The purpose of this study was to analyze the mechanism(s) of hypoxic/ischemic induction of Wt1. We show here that Wt1 mRNA and protein is up-regulated in the heart and kidneys of rats exposed to normobaric hypoxia (8% O2). Ectopic Wt1 immunoreactivity was detected in renal tubules of hypoxic rats, which also expressed the antiapoptotic protein Bcl-2 and contained significantly fewer TUNEL-positive cells than in normoxic kidneys. Wt1 expression was enhanced in the osteosarcoma line U-2OS and in Reh lymphoblast cells that were grown either at 1% O2 or in the presence of CoCl2 and desferrioxamine, respectively. The promoter of the Wt1 gene was capable of mediating expression of a luciferase reporter in response to hypoxia. We identified a hypoxia-responsive element in the Wt1 sequence that bound to hypoxia-inducible factor-1 (HIF-1) and was required for activation of the Wt1 promoter by CoCl2 and HIF-1. These findings demonstrate that Wt1 expression can be stimulated by hypoxia, which involves activation of the Wt1 promoter by HIF-1.

Animals↗

PubMed2003

The complex life of WT1.

The Wilms' tumour gene, WT1, encodes a zinc-finger transcription factor that is inactivated in a subset of Wilms' tumours. Mutation analysis in human patients and genetic experiments in mice have revealed that WT1 has a role much wider than just tumour suppression. Alternative splicing, RNA editing, and the use of alternative translation initiation sites generate a multitude of isoforms, which seem to have overlapping but also distinct functions during embryonic development and the maintenance of organ function. Recently, mouse strains lacking the WT1(-KTS) or WT1(+KTS) splice variants of exon 9 were generated. More severe defects of kidneys and gonads are found in mice lacking the WT1(-KTS) variant. Animals lacking the WT1(+KTS) variant show disturbed podocyte function and male-to-female sex reversal. Alternative splicing of exon 5, however, might not modify WT1 function dramatically. Recently, it was also described that reduction of WT1 levels in the kidney results in glomerulosclerosis and upregulation of WT1 in the heart might contribute to neovascularization after infarction.

Alternative Splicing↗

PubMed2003

Mechanically induced potentials in atrial fibroblasts from rat hearts are sensitive to hypoxia/reoxygenation.

Membrane potential changes of atrial fibroblasts in response to mechanical stress have been considered to modulate the rhythmic electrical activity of healthy hearts. Our recent findings suggest that cardiac arrhythmia after infarction is related to enhanced susceptibility of the fibroblasts to physical stretch. In this study, we analysed the effect of hypoxia/reoxygenation, which are major components of tissue ischemia/reperfusion, on the membrane potential of atrial fibroblasts. Intracellular microelectrode recordings were performed together with isometric force measurements on isometrically contracting right atrial tissue preparations from adult rats. Lowering the oxygen tension in the perfusate from 80 kPa to 3.5 kPa reduced active force development and decreased the resting membrane potential of the cardiac fibroblasts from -23+/-5 mV to -5+/-2 mV ( n=35). Application of gadolinium (40 microM) to inhibit non-selective cation channels prevented hypoxia-induced membrane depolarization of the fibroblasts. Reoxygenation of the myocardial tissue resulted in a transient increase of the resting membrane potential to maximally -60+/-8 mV. These findings indicate that transmembrane currents in atrial fibroblasts are sensitive to changes in tissue oxygenation. In conclusion, altered electro-mechanical function of the ischemic heart may possibly involve changes of the membrane potential of the cardiac fibroblasts.

Action Potentials↗

PubMed2003

The Wilms' tumor suppressor Wt1 encodes a transcriptional activator of the class IV POU-domain factor Pou4f2 (Brn-3b).

The Wilms' tumor gene Wt1 encodes a zinc finger protein, which is required for normal formation of the genitourinary system and mesothelial tissues. Our recent findings indicate that Wt1 also plays a critical role in the development of ganglion cells in the vertebrate retina. Here we show that the POU-domain factor Pou4f2 (formerly Brn-3b), which is necessary for retinal ganglion cell survival, is up-regulated in human embryonic kidney (HEK)293 cells with stable Wt1 expression. Consistent with our previous observations of increased Pou4f2 mRNA in stably Wt1-transfeced HEK293 cells [EMBO J. 21 (2002) 1398], endogenous Pou4f2 was also elevated at the protein level in the HEK293 transfectants as well as in U2OS osteosarcoma cells that expressed an inducible Wt1 isoform. Transient co-transfection of a Wt1 expression construct activated a Pou4f2 promoter-reporter construct approximately 4-fold. Stimulation of the Pou4f2 promoter required a Wt1 binding element that was similar to a degenerative consensus site previously identified in other Wt1 responsive genes. Double-immunofluorescent labeling revealed co-expression of Pou4f2 and Wt1 in glomerular podocytes of adult kidney and in developing retinal ganglion cells of mouse embryos. Pou4f2 immunoreactivity was absent from the retinas of Wt1(-/-) embryos. In conclusion, we identified Pou4f2 as a novel downstream target gene of Wt1. Co-localization of both proteins in glomerular podocytes of the kidney and in developing retinal ganglion cells suggests a role for Wt1-Pou4f2 interaction in these tissues.

Animals↗

PubMed2003

Characterization of stretch-activated ion currents in isolated atrial myocytes from human hearts.

To explore further the mechanisms that may underlie cardiac arrhythmia, we analysed stretch-activated ion currents in human atrial myocytes. Longitudinal stretch of freshly isolated atrial myocytes prolonged the duration of action potentials, depolarized the resting membrane potential and caused extra action potentials. Under voltage-clamp conditions, the amplitude of stretch-induced transmembrane currents increased reversibly with the intensity of stretch. Stretch-activated currents ( I(SAC)) had a reversal potential of 0 mV and were insensitive to substitution of Cl(-) with aspartate ions in the extracellular fluid. I(SAC) was suppressed by 5 micro M gadolinium (Gd(3+)). Furthermore, mechanical stretch decreased transmembrane ion fluxes through L-type calcium channels (I(Ca,L)). This reduction of I(Ca,L) was inhibited by dialysing the cells for 5 min with 5 mM BAPTA prior to application of stretch. In contrast, both BAPTA and removal of Ca(2+) from the extracellular bathing solution had no significant effect on stretch activation of I(SAC). These findings suggest that non-selective cation channels in human atrial myocytes are sensitive to mechanical stimulation. We propose that activation of transmembrane influx of cations, preferentially Na(+), by local stretch may play a role in cardiac arrhythmia.

Action Potentials↗

PubMed2003

1,25-dihydroxyvitamin D3-induced apoptosis of retinoblastoma cells is associated with reciprocal changes of Bcl-2 and bax.

The active vitamin D metabolite 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and related substances have previously been tested in tissue culture and animal models of retinoblastoma for their use as anti-tumor drugs. However, despite of the potential therapeutic value, the molecular mechanisms through which 1,25-(OH)(2)D(3) inhibits the growth of retinoblastoma cells are incompletely understood. To elucidate possible signalling pathways for the anti-proliferative action of vitamin D compounds in retinal tumor cells, we analyzed the effect of 1,25-(OH)(2)D(3) and its synthetic analogue KH1060 on the growth of human retinoblastoma-derived Y79 cells. Vitamin D receptor (VDR) mRNA was detected by reverse transcription PCR in Y79 cells and in tissue specimens of human retinoblastoma. VDR transcripts were confirmed at the protein level by strong immunostaining of solid retinal tumors for VDR. Incubation with 1,25-(OH)(2)D(3) and KH1060 (10(-10)-10(-6)moll(-1)) decreased the number of Y79 cells in a timely and dose-dependent manner. Treatment with 1,25-(OH)(2)D(3) (10(-10)moll(-1)) for 24 hr caused cell cycle arrest in the G0/1 phase. Apoptosis of Y79 cells in response to 1,25-(OH)(2)D(3) was demonstrated by the means of TdT-dUTP terminal nick-end labelling (TUNEL), annexin V staining, and detection of DNA fragmentation on agarose gels. 1,25-(OH)(2)D(3)-induced programmed death of Y79 cells was accompanied by a concentration-dependent increase in Bax protein and a reduction in Bcl-2 content. These findings suggest that 1,25-(OH)(2)D(3) inhibits the growth of retinoblastoma cells by causing cell cycle arrest and apoptosis. 1,25-(OH)(2)D(3)-induced programmed death of retinoblastoma cells appears to involve reciprocal changes in Bcl-2 and Bax proteins.

Analysis of Variance↗

PubMed2003

The long-lasting impact of postnatal neuropeptide Y.

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Aging↗

PubMed2002

Transgenic overexpression of the sarcoplasmic reticulum Ca2+ATPase improves reticular Ca2+ handling in normal and diabetic rat hearts.

Slowed relaxation in diabetic cardiomyopathy (CM) is partially related to diminished expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase SERCA2a. To evaluate the impact of SERCA2a overexpression on SR Ca2+ handling in diabetic CM, we 1) generated transgenic rats harboring a human cytomegalovirus enhancer/chicken beta-actin promotor-controlled rat SERCA2 transgene (SERCA2-TGR), 2) characterized their SR phenotype, and 3) examined whether transgene expression may rescue SR Ca2+ transport in streptozotocin-induced diabetes. The transgene was expressed in all heart chambers. Compared to wild-type (WT) rats, a heterozygous line exhibited increased SERCA2 mRNA (1.5-fold), SERCA2 protein (+26%) and SR Ca2+ uptake (+37%). Phospholamban expression was not altered. In SERCA2-TGR, contraction amplitude (+48%) and rates of contraction (+34%) and relaxation (+35%) of isolated papillary muscles (PM) were increased (P2+ uptake and SERCA2 protein of SERCA2-TGR were 1.3-fold higher (P2+ uptake, accelerates relaxation and compensates, in part, for depressed Ca2+ uptake in diabetic CM. Therefore, SERCA2 expression might constitute an important therapeutic target to rescue cardiac SR Ca2+ handling in diabetes.

Animals↗

PubMed2002

The Wilms' tumor suppressor Wt1 is expressed in the coronary vasculature after myocardial infarction.

Expression of the Wilms' tumor gene Wt1 in the epicardium is critical for normal heart development. Mouse embryos with inactivated Wt1 gene have extremely thin ventricles, which can result in heart failure and death. Here, we demonstrate that Wt1 can be activated in adult hearts by local ischemia. Wt1 mRNA was increased more than twofold in the left ventricular myocardium of rats between 1 day and 9 wk after infarction. Wt1 expression was localized by means of mRNA in situ hybridization and immunohistochemistry to vascular endothelial and vascular smooth muscle cells in the border zone of the infarcted tissue. A strikingly similar distribution was seen for vascular endothelial growth factor and two different cell proliferation markers in the coronary vessels of the ischemic heart. No Wt1 could be detected in the vasculature of the noninfarcted right ventricles. Wt1 expression in the coronary vessels of the ischemic heart was mimicked by exposure of rats to normobaric hypoxia (8% O2) and 0.1% CO, respectively. These findings demonstrate that Wt1 is expressed in the vasculature of the heart in response to local ischemia and hypoxia. They suggest that Wt1 has a role in the growth of coronary vessels after myocardial infarction.

Animals↗

PubMed2002

The Wilms' tumor gene Wt1 is required for normal development of the retina.

The Wilms' tumor gene Wt1 is known for its important functions during genitourinary and mesothelial formation. Here we show that Wt1 is necessary for neuronal development in the vertebrate retina. Mouse embryos with targeted disruption of Wt1 exhibit remarkably thinner retinas than age-matched wild-type animals. A large fraction of retinal ganglion cells is lost by apoptosis, and the growth of optic nerve fibers is severely disturbed. Strikingly, expression of the class IV POU-domain transcription factor Pou4f2 (formerly Brn-3b), which is critical for the survival of most retinal ganglion cells, is lost in Wt1(-/-) retinas. Forced expression of Wt1 in cultured cells causes an up-regulation of Pou4f2 mRNA. Moreover, the Wt1(-KTS) splice variant can activate a reporter construct carrying 5'-regulatory sequences of the human POU4F2. The lack of Pou4f2 and the ocular defects in Wt1(-/-) embryos are rescued by transgenic expression of a 280 kb yeast artificial chromosome carrying the human WT1 gene. Taken together, our findings demonstrate a continuous requirement for Wt1 in normal retina formation with a critical role in Pou4f2-dependent ganglion cell differentiation.

Animals↗

PubMed2002

Time-dependent changes of the susceptibility of cardiac contractile function to hypoxia-reoxygenation after myocardial infarction in rats.

In this study we analyzed the susceptibility of contractile function of the myocardium to hypoxia-reoxygenation after infarction. For this purpose, the contractility of isolated papillary muscles from rats was studied at high oxygen tension (pO2 80 kPa) and during hypoxia (pO2 3 kPa) with subsequent reoxygenation at variable intervals between 15 h and 9 weeks after permanent ligation of the left coronary artery. Hypoxic exposure reduced the contractile performance of the preparations to a similar extent in both groups. Notably, the contractility and, in particular, the relaxation rates recovered more completely from hypoxia in the hypertrophied myocardium of rats with coronary artery ligation than in sham-operated (SO) animals. The recovery of contractile function was improved maximally between 6 and 9 weeks after myocardial infarction (MI). The lower sensitivity of the (post)ischemic myocardium to hypoxia-reoxygenation correlated with enhanced left ventricular glutathione peroxidase (GSH-Px) activity (15 h to 9 weeks post-MI) and 2-3-fold increased expression levels (15 h to 6 weeks post-MI) of the 72 kDa heat shock protein (HSP72) in the papillary muscles. These findings suggest that the greater antioxidant potential and, possibly, stimulation of HSPs contribute to the sustained tolerance of the myocardium to hypoxia-reoxygenation injury after infarction.

Animals↗

PubMed2002

A possible role for atrial fibroblasts in postinfarction bradycardia.

Atrial fibroblasts are considered to modulate the contractile activity of the heart in response to mechanical stretch. In this study we examined whether atrial fibroblasts are possibly involved in bradyarrhythmia, which is a severe complication after myocardial infarction. For this purpose, transmembrane electrical potentials were recorded in cardiac fibroblasts near the sinoatrial node from sham-operated rats and from rats with myocardial infarction. Twenty days after infarction due to coronary artery ligation, the right atrial tissue weights and the sensitivity of the fibroblast membrane potential to mechanical stretch correlated positively with the infarct size. Cardiac growth was enhanced, but the stretch sensitivity and the resting membrane potential of the atrial fibroblasts declined between 8 and 30 days after infarction. The frequency of spontaneous atrial contractions was significantly reduced 8 days after myocardial infarction and recovered in parallel with the membrane potential of the fibroblasts. These findings suggest that changes in the susceptibility of atrial fibroblasts to mechanical stretch may contribute to bradyarrhythmia during postinfarct remodeling of the heart.

Animals↗