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J D Molkentin

Publications and source records attributed to J D Molkentin.

At least 55 records · Page 3Linked to original sources
PubMed1998

A calcineurin-dependent transcriptional pathway for cardiac hypertrophy.

In response to numerous pathologic stimuli, the myocardium undergoes a hypertrophic response characterized by increased myocardial cell size and activation of fetal cardiac genes. We show that cardiac hypertrophy is induced by the calcium-dependent phosphatase calcineurin, which dephosphorylates the transcription factor NF-AT3, enabling it to translocate to the nucleus. NF-AT3 interacts with the cardiac zinc finger transcription factor GATA4, resulting in synergistic activation of cardiac transcription. Transgenic mice that express activated forms of calcineurin or NF-AT3 in the heart develop cardiac hypertrophy and heart failure that mimic human heart disease. Pharmacologic inhibition of calcineurin activity blocks hypertrophy in vivo and in vitro. These results define a novel hypertrophic signaling pathway and suggest pharmacologic approaches to prevent cardiac hypertrophy and heart failure.

Angiotensin II↗
PubMed1998

MEF2B is a component of a smooth muscle-specific complex that binds an A/T-rich element important for smooth muscle myosin heavy chain gene expression.

To understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a smooth muscle-specific marker. In this study, we demonstrate that the SMHC promoter region (-1594 to -1462 base pairs) containing the A/T-rich element can activate the heterologous thymidine kinase promoter in smooth muscle cells, but not in fibroblasts. Mutations of this A/T-rich element decreased SMHC promoter activity significantly. Both gel mobility shift assays and DNase I footprinting revealed that this region binds to specific protein complexes from smooth muscle nuclear extracts, whereas nuclear extracts from skeletal muscle and fibroblasts produced a different binding pattern. We also demonstrate that the protein complex obtained from smooth muscle nuclear extract reacts with MEF2B-specific antibody, but not with antibodies specific to MEF2A, MEF2C, or MEF2D, suggesting that only MEF2B protein binds to the A/T-rich element. Furthermore, MEF2B overexpression in smooth muscle cells up-regulated the SMHC promoter, suggesting that MEF2B is important for SMHC gene regulation. This is the first report demonstrating a role for MEF2 factors in smooth muscle-specific gene expression.

3T3 Cells↗
PubMed1998

Multiple roles for the MyoD basic region in transmission of transcriptional activation signals and interaction with MEF2.

Establishment of skeletal muscle lineages is controlled by the MyoD family of basic helix-loop-helix (bHLH) transcription factors. The ability of these factors to initiate myogenesis is dependent on two conserved amino acid residues, alanine and threonine, in the basic domains of these factors. It has been postulated that these two residues may be responsible for the initiation of myogenesis via interaction with an essential myogenic cofactor. The myogenic bHLH proteins cooperatively activate transcription and myogenesis through protein-protein interactions with members of the myocyte enhancer factor 2 (MEF2) family of MADS domain transcription factors. MyoD-E12 heterodimers interact with MEF2 proteins to synergistically activate myogenesis, while homodimers of E12, which lack the conserved alanine and threonine residues in the basic domain, do not interact with MEF2. We have examined whether the myogenic alanine and threonine in the MyoD basic region are required for interaction with MEF2. Here, we show that substitution of the MyoD basic domain with that of E12 does not prevent interaction with MEF2. Instead, the inability of alanine-threonine mutants of MyoD to initiate myogenesis is due to a failure to transmit transcriptional activation signals provided either from the MyoD or the MEF2 activation domain. This defect in transcriptional transmission can be overcome by substitution of the MyoD or the MEF2 activation domain with the VP16 activation domain. These results demonstrate that myogenic bHLH-MEF2 interaction can be uncoupled from transcriptional activation and support the idea that the myogenic residues in myogenic bHLH proteins are essential for transmission of a transcriptional activation signal.

Animals↗
PubMed1997

Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload.

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the alpha-skeletal actin promoter 1.7-fold whereas the alpha-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1. 6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.

Animals↗
PubMed1997

Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis.

The zinc finger transcription factor GATA4 has been implicated in heart development based on its early expression in precardiogenic splanchnic mesoderm and its ability to activate the expression of a number of cardiac-specific genes. To determine the role of GATA4 in embryogenesis, we generated mice homozygous for a GATA4 null allele. Homozygous GATA4 null mice arrested in development between E7.0 and E9.5 because of severe developmental abnormalities. Mutant embryos most notably lacked a primitive heart tube and foregut and developed partially outside the yolk sac. In the mutants, the two bilaterally symmetric promyocardial primordia failed to migrate ventrally but instead remained lateral and generated two independent heart tubes that contained differentiated cardiomyocytes. We show that these deformities resulted from a general loss in lateral to ventral folding throughout the embryo. GATA4 is most highly expressed within the precardiogenic splanchnic mesoderm at the posterior lip of the anterior intestinal portal, corresponding to the region of the embryo that undergoes ventral fusion. We propose that GATA4 is required for the migration or folding morphogenesis of the precardiogenic splanchnic mesodermal cells at the level of the AIP.

Animals↗
PubMed1996

Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors.

Members of the MyoD family of muscle-specific basic helix-loop-helix (bHLH) proteins function within a genetic pathway to control skeletal muscle development. Mutational analyses of these factors suggested that their DNA binding domains mediated interaction with a coregulator required for activation of muscle-specific transcription. Members of the myocyte enhancer binding factor 2 (MEF2) family of MADS-box proteins are expressed at high levels in muscle and neural cells and at lower levels in several other cell types. MEF2 factors are unable to activate muscle gene expression alone, but they potentiate the transcriptional activity of myogenic bHLH proteins. This potentiation appears to be mediated by direct interactions between the DNA binding domains of these different types of transcription factors. Biochemical and genetic evidence suggests that MEF2 factors are the coregulators for myogenic bHLH proteins. The presence of MEF2 and cell-specific bHLH proteins in other cell types raises the possibility that these proteins may also cooperate to regulate other programs of cell-specific gene expression. We present a model to account for such cooperative interactions.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of the MADS-Box transcription factor MEF2C enhances its DNA binding activity.

Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors activate muscle gene expression by binding an A/T-rich DNA sequence in the control regions of muscle-specific genes. There are four MEF2 factors in vertebrates, MEF2A-D, which share homology in an amino-terminal MADS domain and an adjacent region known as the MEF2 domain, that together mediate DNA binding and dimerization. We show that serine 59 located between the MADS and MEF2 domains of MEF2C is phosphorylated in vivo and can be phosphorylated in vitro by casein kinase-II (CKII). Phosphorylation of this site enhanced the DNA binding and transcriptional activity of MEF2C by increasing its DNA binding activity 5-fold. In vivo 32P labeling experiments showed that serine 59 is the only phosphorylation site in the MADS and MEF2 domains. Mutagenesis of this serine to an aspartic acid resulted in an increase in DNA binding and transcriptional activity of MEF2C comparable to that observed when this site was phosphorylated, suggesting that phosphorylation augments DNA binding activity by introducing negative charge. This phosphorylation site, which corresponds to a CKII recognition site, is conserved in all known MEF2 factors in organisms ranging from flies to humans, consistent with its importance for the functions of MEF2C.

Amino Acid Sequence↗
PubMed1996

Alpha-myosin heavy chain gene regulation: delineation and characterization of the cardiac muscle-specific enhancer and muscle-specific promoter.

The alpha-myosin heavy chain (alpha-MHC) gene encodes a cardiac muscle-specific protein involved in active force generation. The mechanism responsible for restricting expression of this gene to the heart should provide clues for the identification of transcriptional regulatory events involved in the induction and maintenance of the cardiac cell lineage. In this report we dissect the alpha-MHC regulatory region to identify the components necessary for directing high levels of cardiac muscle-restricted expression. Deletion, site-specific mutant and heterologous promoter constructs were assayed for expression after injection into adult rat heart and skeletal muscle or transfection into non-muscle cells. These studies indicated that sequences from -344 to -156 directed high levels of cardiac-muscle specific expression from a heterologous promoter that was independent of position and orientation. This region includes a previously uncharacterized CArG box, alpha-MHC sequences from -86 to +16 promoted activity from two heterologous enhancers in a muscle-specific fashion. Mutational analysis of an E-box and a CArG box within the promoter revealed that they act as negative and positive regulatory elements, respectively. Based on competitive binding and supershift electrophoretic mobility shift assays, serum response factor was shown interact with the CArG boxes found in the promoter and enhancer. Similar experiments demonstrated that the E-box bound to a factor immunologically related to upstream stimulatory factor. Together, these results identify two distinct regions with different regulatory function that are critical for the tissue restricted expression of the alpha-MHC gene.

Animals↗
PubMed1996

Defining the regulatory networks for muscle development.

The formation of skeletal muscle during vertebrate embryogenesis requires commitment of mesodermal precursor cells to the skeletal muscle lineage, withdrawal of myoblasts from the cell cycle, and transcriptional activation of dozens of muscle structural genes. The myogenic basic helix-loop-helix (bHLH) factors - MyoD, myogenin, Myf5, and MRF4 - act at multiple points in the myogenic lineage to establish myoblast identity and to control terminal differentiation. Recent studies have begun to define the inductive mechanisms that regulate myogenic bHLH gene expression and muscle cell determination in the embryo. Myogenic bHLH factors interact with components of the cell cycle machinery to control withdrawal from the cell cycle and act combinatorially with other transcription factors to induce skeletal muscle transcription. Elucidation of these aspects of the myogenic program is leading to a detailed understanding of the regulatory circuits controlling muscle development.

Animals↗
PubMed1996

Mutational analysis of the DNA binding, dimerization, and transcriptional activation domains of MEF2C.

There are four members of the myocyte enhancer factor 2 (MEF2) family of transcription factors in vertebrates, MEF2A, -B, -C, and -D, which have homology within a MADS box at their amino termini and an adjacent motif known as the MEF2 domain. These factors activate muscle gene expression by binding as homo- and heterodimers to an A/T-rich DNA sequence in the control regions of muscle-specific genes. To understand the mechanisms of muscle gene activation of MEF2 factors, we generated a series of deletion and site-directed mutants of MEF2C. These mutants demonstrated that the MADS and MEF2 domains mediate DNA binding and dimerization, whereas the carboxyl terminus is required for transcriptional activation. Amino acids that are essential for MEF2 site-dependent transcription but which do not affect DNA binding were also identified in the MEF2 domain. This type of positive-control mutant demonstrates that the transcription activation domain of MEF2C, although separate from the MEF2 domain, is dependent on this domain for transcriptional activation through the MEF2 site. MEF2 mutants that are defective for DNA binding act as dominant negative mutants and can inhibit activation of MEF2-dependent genes by wild-type MEF2C.

Amino Acid Sequence↗
PubMed1996

MEF2B is a potent transactivator expressed in early myogenic lineages.

There are four members of the myocyte enhancer binding factor 2 (MEF2) family of transcription factors, MEF2A, -B, -C, and -D, that have homology within an amino-terminal MADS box and an adjacent MEF2 domain that together mediate dimerization and DNA binding. MEF2A, -C, and -D have previously been shown to bind an A/T-rich DNA sequence in the control regions of numerous muscle-specific genes, whereas MEF2B was reported to be unable to bind this sequence unless the carboxyl terminus was deleted. To further define the functions of MEF2B, we analyzed its DNA binding and transcriptional activities. In contrast to previous studies, our results show that MEF2B binds the same DNA sequence as other members of the MEF2 family and acts as a strong transactivator through that sequence. Transcriptional activation by MEF2B is dependent on the carboxyl terminus, which contains two conserved sequence motifs found in all vertebrate MEF2 factors. During mouse embryogenesis, MEF2B transcripts are expressed in the developing cardiac and skeletal muscle lineages in a temporospatial pattern distinct from but overlapping with those of the other Mef2 genes. The mouse Mef2b gene maps to chromosome 8 and is unlinked to other Mef2 genes; its intron-exon organization is similar to that of the other vertebrate Mef2 genes and the single Drosophila Mef2 gene, consistent with the notion that these different Mef2 genes evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1995

Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins.

Members of the myocyte enhancer factor-2 (MEF2) family of MADS domain transcription factors cannot induce myogenesis in transfected fibroblasts, but when coexpressed with the myogenic basic-helix-loop-helix (bHLH) proteins MyoD or myogenin they dramatically increase the extent of myogenic conversion above that seen with either myogenic bHLH factor alone. This cooperativity required direct interactions between the DNA-binding domains of MEF2 and the myogenic bHLH factors, but only one of the factors needed a transactivation domain, and only one of the factors needed to be bound to DNA. These interactions allow either factor to activate transcription through the other's binding site and reveal a novel mechanism for indirect activation of gene expression via protein-protein interactions between the DNA-binding domains of heterologous classes of transcription factors.

Amino Acid Sequence↗
PubMed1995

A series of mutations in the D-MEF2 transcription factor reveal multiple functions in larval and adult myogenesis in Drosophila.

The D-mef2 gene encodes a MADS domain transcription factor expressed in differentiated muscles and their precursors in the Drosophila embryo. Embryos deficient for D-MEF2 protein due to a deletion of upstream transcriptional control sequences fail to form muscle, suggesting that the gene is required for muscle cell differentiation. To directly demonstrate a role for D-mef2 in embryonic myogenesis, we isolated gene mutants containing EMS-induced point mutations, characterized the effects of these mutations on D-MEF2 protein stability and nuclear localization, and analyzed the resulting muscle phenotypes. Our results show that in the somatic muscle lineage, D-mef2 is required for both the formation and patterning of body wall muscle. In the absence of somatic myogenesis, there is extensive apoptosis among the myoblast cell population. In contrast, in the cardiac muscle lineage, morphogenesis of the dorsal vessel occurs normally but the three myosin subunit genes are not expressed. Mutant embryos also exhibit an abnormal midgut morphology, which correlates with the absence of alpha PS2 integrin gene expression and muscle-specific enhancer function, suggesting that D-mef2 regulates the inflated locus which encodes this integrin subunit. D-MEF2 is also expressed in adepithelial cells and rare D-mef2 transheterozygous mutant adults fail to fly, consistent with defects observed in the indirect flight muscles. These results demonstrate that the D-mef2 gene has multiple functions in myogenesis and tissue morphogenesis during Drosophila development.

Animals↗
PubMed1994

Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene.

The alpha-myosin heavy-chain (alpha-MHC) gene is the major structural protein in the adult rodent myocardium. Its expression is restricted to the heart by a complex interplay of trans-acting factors and their cis-acting sites. However, to date, the factors that have been shown to regulate expression of this gene have also been found in skeletal muscle cells. Recently, transcription factor GATA-4, which has a tissue distribution limited to the heart and endodermally derived tissues, was identified. We recently found two putative GATA-binding sites within the proximal enhancer of the alpha-MHC gene, suggesting that GATA-4 might regulate its expression. In this study, we establish that GATA-4 interacts with the alpha-MHC GATA sites to stimulate cardiac muscle-specific expression. Mutation of the GATA-4-binding sites either individually or together decreased activity by 50 and 88% in the adult myocardium, respectively. GATA-4-dependent enhancement of activity from a heterologous promoter was mediated through the alpha-MHC GATA sites. Coinjection of an alpha-MHC promoter construct with a GATA-4 expression vector permitted ectopic expression in skeletal muscle but not in fibroblasts. Thus, the lack of alpha-MHC expression in skeletal muscle correlates with a lack of GATA-4. GATA-4 DNA binding activity was significantly up-regulated in triiodothyronine- or retinoic acid-treated cardiomyocytes. Putative GATA-4-binding sites are also found in the regulatory regions of other cardiac muscle-expressed structural genes. This indicates a mechanism whereby triiodothyronine and retinoic acid can exert coordinate control of the cardiac phenotype through a trans-acting regulatory factor.

Animals↗
PubMed1994

An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha-cardiac myosin heavy-chain gene in vivo.

Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy.

Age Factors↗
PubMed1993

Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac myosin heavy chain gene expression in vitro and in vivo.

A myocyte-specific enhancer-binding factor (MEF-2) DNA binding site was identified in the rat alpha-myosin heavy chain (MHC) gene adjacent to the E-box binding site for alpha-MHC binding factor-2 (BF-2). Mutation of the MEF-2 site, within the context of the full-length promoter, reduced activity by 85 and 80% in neonatal cardiomyocytes and the adult heart, respectively. Mutation of the BF-2 site reduced activity approximately 70% in both models. A MEF-2/BF-2 double mutant gave significantly less activity than the BF-2 mutant but not the MEF-2 mutant, suggesting the possibility that BF-2 and MEF-2 interact. Mutations in MEF-2, which decreased functional activity, also abolished MEF-2 DNA binding activity. MEF-2 DNA binding activity was present in the developing heart, reached a peak in the late fetal and early neonatal stages, and then declined to low levels in the adult heart. The adult levels were sufficient to support alpha-MHC gene expression. MEF-2 activity was increased 2-3-fold in the adult heart subjected to a pressure or volume overload. Two working models are proposed as possible explanations of the antithetic relationship between MEF-2 levels and alpha-MHC gene expression.

Animals↗
PubMed1993

The bovine mannose 6-phosphate/insulin-like growth factor II receptor. The role of arginine residues in mannose 6-phosphate binding.

The extracytoplasmic region of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) consists of 15 homologous repeating domains, each of which is approximately 147 residues in length. The receptor contains two high affinity mannose 6-phosphate (Man-6-P) binding sites and our recent studies (Westlund, B., Dahms, N. M., and Kornfeld, S. (1991) J. Biol. Chem. 266, 23233-23239) have localized these two binding sites to domains 1-3 and 7-11. To further define the location of the Man-6-P binding sites and to determine the role of specific arginine residues in Man-6-P binding, site-directed mutagenesis was utilized to create truncated soluble forms of the M6P/IGF-II receptor in conjunction with either conservative (Lys) or nonconservative (Ala) replacement of arginine residues. These mutants were expressed transiently in COS-1 cells and assayed for their ability to bind phosphomannosyl residues by affinity chromatography. Analysis of the ligand binding activity of carboxyl-terminal truncated forms of the receptor's extracytoplasmic region demonstrated that the second Man-6-P binding site is contained within domains 7-9. Substitution of Arg435 in domain 3 of the amino-terminal binding site and Arg1334 in domain 9 of the second binding site results in a dramatic loss of ligand binding activity. However, substitutions at positions 435 and/or 1334 did not affect the secretion, glycosylation, or immunoreactivity of these truncated proteins. Taken together, these results indicate that Arg435 and Arg1334 are essential components of the M6P/IGF-II receptor's high affinity Man-6-P binding sites.

Amino Acid Sequence↗