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Biomedical subjects

M D Schneider

Publications and source records attributed to M D Schneider.

At least 19 recordsLinked to original sources
PubMed2005

Overlapping roles of pocket proteins in the myocardium are unmasked by germ line deletion of p130 plus heart-specific deletion of Rb.

The pocket protein family of tumor suppressors, and Rb specifically, have been implicated as controlling terminal differentiation in many tissues, including the heart. To establish the biological functions of Rb in the heart and overcome the early lethality caused by germ line deletion of Rb, we used a Cre/loxP system to create conditional, heart-specific Rb-deficient mice. Mice that are deficient in Rb exclusively in cardiac myocytes (CRbL/L) are born with the expected Mendelian distribution, and the adult mice displayed no change in heart size, myocyte cell cycle distribution, myocyte apoptosis, or mechanical function. Since both Rb and p130 are expressed in the adult myocardium, we created double-knockout mice (CRbL/L p130-/-) to determine it these proteins have a shared role in regulating cardiac myocyte cell cycle progression. Adult CRbL/L p130-/- mice demonstrated a threefold increase in the heart weight-to-body weight ratio and showed increased numbers of bromodeoxyuridine- and phosphorylated histone H3-positive nuclei, consistent with persistent myocyte cycling. Likewise, the combined deletion of Rb plus p130 up-regulated myocardial expression of Myc, E2F-1, and G1 cyclin-dependent kinase activities, synergistically. Thus, Rb and p130 have overlapping functional roles in vivo to suppress cell cycle activators, including Myc, and maintain quiescence in postnatal cardiac muscle.

Animals↗
PubMed2001

Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival.

Cardiac muscle regeneration after injury is limited by "irreversible" cell cycle exit. Telomere shortening is one postulated basis for replicative senescence, via down-regulation of telomerase reverse transcriptase (TERT); telomere dysfunction also is associated with greater sensitivity to apoptosis. Forced expression of TERT in cardiac muscle in mice was sufficient to rescue telomerase activity and telomere length. Initially, the ventricle was hypercellular, with increased myocyte density and DNA synthesis. By 12 wk, cell cycling subsided; instead, cell enlargement (hypertrophy) was seen, without fibrosis or impaired function. Likewise, viral delivery of TERT was sufficient for hypertrophy in cultured cardiac myocytes. The TERT virus and transgene also conferred protection from apoptosis, in vitro and in vivo. Hyperplasia, hypertrophy, and survival all required active TERT and were not seen with a catalytically inactive mutation. Thus, TERT can delay cell cycle exit in cardiac muscle, induce hypertrophy in postmitotic cells, and promote cardiac myocyte survival.

Animals↗
PubMed2001

Inducible gene targeting in postnatal myocardium by cardiac-specific expression of a hormone-activated Cre fusion protein.

Cardiac-restricted expression of Cre recombinase can provoke lineage-specific gene excision in the myocardium. However, confounding early lethality may still preclude using loss-of-function models to study the postnatal heart. Here, we have tested whether inducible, heart-specific recombination can be triggered after birth by transgenic expression of a Cre fusion protein that incorporates a mutated progesterone receptor ligand binding domain (PR1) that is activated by the synthetic antiprogestin, RU486, but not by endogenous steroid hormones. CrePR1 driven by the alpha-myosin heavy chain (alphaMHC) promoter was expressed specifically in heart. Translocation of CrePR1 from cytoplasm to nuclei in ventricular myocytes was induced by RU486. To establish whether this approach can mediate cardiac-specific, drug-dependent excision between loxP sites in vivo, we mated alphaMHC-CrePR1 mice with a ubiquitously expressed (ROSA26) Cre reporter line. Offspring harboring alphaMHC-CrePR1 and/or the floxed allele were injected with RU486 versus vehicle, and the prevalence of beta-galactosidase (beta-gal)-positive cells was determined, indicative of Cre-mediated excision. Little or no baseline recombination was seen 1 week after birth. Cardiac-restricted, RU486-inducible recombination was demonstrated in bigenic mice at age 3 and 6 weeks, using each of 3 independent CrePR1 lines. Recombination in the absence of ligand paralleled the levels of CrePR1 protein expression and was more evident at 6 weeks. Thus, conditional, posttranslational activation of a Cre fusion protein can bypass potential embryonic and perinatal effects on the heart and permits inducible recombination in cardiac muscle. High levels of the chimeric Cre protein, in particular, were associated with progressive recombination in the absence of drug.

Animals↗
PubMed2001

Transforming growth factor-beta receptor-associated protein 1 is a Smad4 chaperone.

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through unique cell membrane receptor serine-threonine kinases to activate downstream targets. TRAP1 is a previously described 96-kDa cytoplasmic protein shown to bind to TGF-beta receptors and suggested to play a role in TGF-beta signaling. We now fully characterize the binding properties of TRAP1, and show that it associates strongly with inactive heteromeric TGF-beta and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-beta signaling in functional assays, deletion constructs of TRAP1 inhibit TGF-beta signaling and diminish the interaction of Smad4 with Smad2. These are the first data to identify a specific molecular chaperone for Smad4, suggesting a model in which TRAP1 brings Smad4 into the vicinity of the receptor complex and facilitates its transfer to the receptor-activated Smad proteins.

Activins↗
PubMed2001

Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43.

Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1+/-0.13 versus 1.66+/-0.06; P:<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.

Animals↗
PubMed2001

Requirement for TGFbeta receptor signaling during terminal lens fiber differentiation.

Several families of growth factors have been identified as regulators of cell fate in the developing lens. Members of the fibroblast growth factor family are potent inducers of lens fiber differentiation. Members of the transforming growth factor beta (TGFbeta) family, particularly bone morphogenetic proteins, have also been implicated in various stages of lens and ocular development, including lens induction and lens placode formation. However, at later stages of lens development, TGFbeta family members have been shown to induce pathological changes in lens epithelial cells similar to those seen in forms of human subcapsular cataract. Previous studies have shown that type I and type II TGFbeta receptors, in addition to being expressed in the epithelium, are also expressed in patterns consistent with a role in lens fiber differentiation. In this study we have investigated the consequences of disrupting TGFbeta signaling during lens fiber differentiation by using the mouse alphaA-crystallin promoter to overexpress mutant (kinase deficient), dominant-negative forms of either type I or type II TGFbeta receptors in the lens fibers of transgenic mice. Mice expressing these transgenes had pronounced bilateral nuclear cataracts. The phenotype was characterized by attenuated lens fiber elongation in the cortex and disruption of fiber differentiation, culminating in fiber cell apoptosis and degeneration in the lens nucleus. Inhibition of TGFbeta signaling resulted in altered expression patterns of the fiber-specific proteins, alpha-crystallin, filensin, phakinin and MIP. In addition, in an in vitro assay of cell migration, explanted lens cells from transgenic mice showed impaired migration on laminin and a lack of actin filament assembly, compared with cells from wild-type mice. These results indicate that TGFbeta signaling is a key event during fiber differentiation and is required for completion of terminal differentiation.

Actins↗
PubMed2000

TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice.

The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

Activating Transcription Factor 6↗
PubMed2000

A novel Rb- and p300-binding protein inhibits transactivation by MyoD.

The retinoblastoma protein (Rb) regulates both the cell cycle and tissue-specific transcription, by modulating the activity of factors that associate with its A-B and C pockets. In skeletal muscle, Rb has been reported to regulate irreversible cell cycle exit and muscle-specific transcription. To identify factors interacting with Rb in muscle cells, we utilized the yeast two-hybrid system, using the A-B and C pockets of Rb as bait. A novel protein we have designated E1A-like inhibitor of differentiation 1 (EID-1), was the predominant Rb-binding clone isolated. It is preferentially expressed in adult cardiac and skeletal muscle and encodes a 187-amino-acid protein, with a classic Rb-binding motif (LXCXE) in its C terminus. Overexpression of EID-1 in skeletal muscle inhibited tissue-specific transcription. Repression of skeletal muscle-restricted genes was mediated by a block to transactivation by MyoD independent of G(1) exit and, surprisingly, was potentiated by a mutation that prevents EID-1 binding to Rb. Inhibition of MyoD may be explained by EID-1's ability to bind and inhibit p300's histone acetylase activity, an essential MyoD coactivator. Thus, EID-1 binds both Rb and p300 and is a novel repressor of MyoD function.

Acetyltransferases↗
PubMed2000

Genetic dissection of cardiac growth control pathways.

Cardiac muscle cells exhibit two related but distinct modes of growth that are highly regulated during development and disease. Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle irreversibly soon after birth, following which the predominant form of growth shifts from hyperplastic to hypertrophic. Much research has focused on identifying the candidate mitogens, hypertrophic agonists, and signaling pathways that mediate these processes in isolated cells. What drives the proliferative growth of embryonic myocardium in vivo and the mechanisms by which adult cardiac myocytes hypertrophy in vivo are less clear. Efforts to answer these questions have benefited from rapid progress made in techniques to manipulate the murine genome. Complementary technologies for gain- and loss-of-function now permit a mutational analysis of these growth control pathways in vivo in the intact heart. These studies have confirmed the importance of suspected pathways, have implicated unexpected pathways as well, and have led to new paradigms for the control of cardiac growth.

Animals↗
PubMed1999

E1A can provoke G1 exit that is refractory to p21 and independent of activating cdk2.

E1A can evoke G1 exit in cardiac myocytes and other cell types by displacing E2F transcription factors from tumor suppressor "pocket" proteins and by a less well-characterized p300-dependent pathway. Bypassing pocket proteins (through overexpression of E2F-1) reproduces the effect of inactivating pocket proteins (through E1A binding); however, pocket proteins associate with a number of molecular targets apart from E2F. Hence, pocket protein binding by E1A might engage mechanisms for cell cycle reentry beyond those induced by E2F-1. To test this hypothesis, we used adenoviral gene transfer to express various E2F-1 and E1A proteins in neonatal rat cardiac myocytes that are already refractory to mitogenic serum, in the absence or presence of several complementary cell cycle inhibitors-p16, p21, or dominant-negative cyclin-dependent kinase-2 (Cdk2). Rb binding by E2F-1 was neither necessary nor sufficient for G1 exit, whereas DNA binding was required; thus, exogenous E2F-1 did not merely function by competing for the Rb "pocket." E2F-1-induced G1 exit was blocked by the "universal" Cdk inhibitor p21 but not by p16, a specific inhibitor of Cdk4/6; p21 was permissive for E2F-1 induction of cyclins E and A, but prevented their stimulation of Cdk2 kinase activity. In addition, E2F-1-induced G1 exit was blocked by dominant-negative Cdk2. Forced expression of cyclin E induced endogenous Cdk2 activity but not G1 exit. Thus, E2F-1-induced Cdk2 function was necessary, although not sufficient, to trigger DNA synthesis in cardiac muscle cells. In contrast, pocket protein-binding forms of E1A induced G1 exit that was resistant to inhibition by p21, whereas G1 exit via the E1A p300 pathway was sensitive to inhibition by p21. Both E1A pathways-via pocket proteins and via p300-upregulated cyclins E and A and Cdk2 activity, consistent with a role for Cdk2 in G1 exit induced by E1A. However, p21 blocked Cdk2 kinase activity induced by both E1A pathways equally. Thus, E1A can cause G1 exit without an increase in Cdk2 activity, if the pocket protein-binding domain is intact. E1A also overrides p21 in U2OS cells, provided the pocket protein-binding domain is intact; thus, this novel function of E1A is not exclusive to cardiac muscle cells. In summary, E1A binding to pocket proteins has effects beyond those produced by E2F-1 alone and can drive S-phase entry that is resistant to p21 and independent of an increase in Cdk2 function. This suggests the potential involvement of other endogenous Rb-binding proteins or of alternative E1A targets.

Adenovirus E1A Proteins↗
PubMed1998

A constitutive mutation of ALK5 disrupts cardiac looping and morphogenesis in mice.

TGF beta family members are implicated in cardiac organogenesis, growth control, and positional information, including the direction of cardiac looping. However, genetic analysis of TGF beta signaling in mice has been confounded, in some cases, by noncardiac and generalized defects. Hence, deciphering TGF beta function in myocardium would benefit from cardiac-restricted mutations. We developed a constitutively activated type I receptor, ALK5L193A,P194A,T204D, and directed it to embryonic myocardium in transgenic mice. Expression of the activated ALK5 gene arrests looping morphogenesis and causes a linear, dilated, hypoplastic heart tube, despite normal expression of Nkx2.5 and dHAND, cardiogenic transcription factors whose absence provokes a similar phenotype. Ventricular hypoplasia was associated with precocious induction of the cyclin-dependent kinase inhibitor, p21. Thus, an ALK5-sensitive pathway mediates looping, perhaps through control of cardiac myocyte proliferation.

Activin Receptors, Type I↗
PubMed1998

A novel protein distinguishes between quiescent and activated forms of the type I transforming growth factor beta receptor.

Transforming growth factor beta (TGFbeta) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFbeta receptor (TbetaR-II) phosphorylating type I TGFbeta receptor (TbetaR-I). Because the failure of interaction cloning, thus far, to identify bona fide TbetaR-I substrates might reasonably have been due to the use of inactive TbetaR-I as bait, we sought to identify molecules that interact specifically with active TbetaR-I, employing the triple mutation L193A,P194A,T204D in a yeast two-hybrid system. The Leu-Pro substitutions prevent interaction with FK506-binding protein 12 (FKBP12), whose putative function in TGFbeta signaling we have previously disproved; the charge substitution at Thr204 constitutively activates TbetaR-I. Unlike previous screens using wild-type TbetaR-I, where FKBP12 predominated, none of the resulting colonies encoded FKBP12. A novel protein was identified, TbetaR-I-associated protein-1 (TRAP-1), that interacts in yeast specifically with mutationally activated TbetaR-I, but not wild-type TbetaR-I, TbetaR-II, or irrelevant proteins. In mammalian cells, TRAP-1 was co-precipitated only by mutationally activated TbetaR-I and ligand-activated TbetaR-I, but not wild-type TbetaR-I in the absence of TGFbeta. The partial TRAP-1 protein that specifically binds these mutationally and ligand-activated forms of TbetaR-I can inhibit signaling by the native receptor after stimulation with TGFbeta or by the constitutively activated receptor mutation, as measured by a TGFbeta-dependent reporter gene. Thus, TRAP-1 can distinguish activated forms of the receptor from wild-type receptor in the absence of TGFbeta and may potentially have a functional role in TGFbeta signaling.

Activin Receptors, Type I↗