The mitochondrial calcium uniporter regulates procoagulant platelet formation.

Essentials Mitochondrial hyperpolarization enhances the conversion of platelets to a procoagulant phenotype. Mitochondrial calcium uniporter (MCU) function is essential in procoagulant platelet formation. Mitochondrial calcium uniporter deletion does not impact other aspects of platelet activation. Ablation of MCU results in the emergence of a permeability transition pore-independent pathway. SUMMARY: Background Procoagulant platelets comprise a phenotypically distinct subpopulation of activated platelets with high-level phosphatidylserine externalization. When initiated by co-stimulation with thrombin and a glycoprotein VI (GPVI) agonist, the transition to the procoagulant phenotype is mediated by extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation. Objectives The intracellular mechanisms coordinating these distinct cytoplasmic and mitochondrial processes remain unclear. The mitochondrial calcium uniporter (MCU) protein is a central component of the transmembrane ion channel that allows the passage of Ca2+ from the cytosol into the mitochondrial matrix. Here we investigate the role of the MCU in the regulation of procoagulant platelet formation. Results Procoagulant platelet formation was directly correlated with pre-stimulatory mitochondrial transmembrane potential, a key determinant of calcium flux from the cytoplasm to the mitochondria. The role of MCU in the regulation of procoagulant platelet formation was investigated using MCU null platelets. Procoagulant platelet formation in MCU null platelets was significantly decreased coincident with decreased mPTP formation. In contrast, neither granule release nor initial integrin activation was altered in response to stimulation. In the genomic absence of MCU, developmental induction of an alternative intracellular pathway partially rescued procoagulant platelet formation. Conclusion These results identify a key role for the mitochondrial calcium uptake channel in the regulation of mPTP-mediated procoagulant platelet formation and suggest a novel pharmacologic target for procoagulant-platelet-related pathologies.

The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression.

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.

cis-Acting sequences that mediate induction of beta-myosin heavy chain gene expression during left ventricular hypertrophy due to aortic constriction.

BACKGROUND: Marked alterations in the expression of specific genes occur during the development of cardiac hypertrophy in vivo. Little is known, however, about the cis-acting elements that mediate these changes in response to clinically relevant hypertrophic stimuli, such as hemodynamic overload, in intact adult animals. METHODS AND RESULTS: The left ventricular expression of a directly injected reporter gene driven by 3542 bp of rat beta-myosin heavy chain (beta-MHC) promoter was increased 3.0-fold by aortic constriction (P<.005), an increment similar to the 3.2-fold increase in the level of the endogenous beta-MHC mRNA in the same left ventricles. Subsequent analysis identified a 107-bp beta-MHC promoter sequence (-303/-197) sufficient to convert a heterologous neutral promoter to one that is activated by aortic constriction. These sequences contain two M-CAT elements, which have previously been demonstrated to mediate inducible expression during alpha1-adrenergic-stimulated hypertrophy in cultured neonatal cardiac myocytes, and a GATA element. Although simultaneous mutation of both M-CAT elements markedly decreased the basal transcriptional activity of an injected 333-bp beta-MHC promoter, it had no effect on aortic constriction-stimulated transcription (3.5-fold increase, P<.005 for both wild type and mutant). In contrast, mutation of the GATA motif markedly attenuated aortic constriction-stimulated transcription (1.6-fold, P=NS) without affecting the basal transcriptional activity. This GATA site can interact with in vitro translated GATA-4 and compete with an established GATA site for GATA-4 binding activity in nuclear extracts from aortic constricted hearts. CONCLUSIONS: Basal and aortic constriction-stimulated transcription of the beta-MHC gene is mediated, at least in part, through different mechanisms. A GATA element within beta-MHC sequences -303/-197 plays a role in the transcriptional activation of this gene by aortic constriction.

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.

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.

Amino acid changes in the peptide binding site have structural consequences at the surface of class I glycoproteins.

Structural changes on the surface of the class I Ag binding domain resulting from point mutations localized inside the Ag binding cleft of the H-2Kb and Kf glycoproteins were revealed using mAb. Both the loss and gain of antibody binding sites found among naturally occurring K glycoproteins resulted from single amino acid substitutions at a variety of different positions buried within the Ag binding groove. Each of the amino acid replacements analyzed represented naturally occurring diversity known to exist among the functional class I Ag-presenting molecules of the mouse. The binding of the affected mAb was not significantly altered in Kb molecules expressed by transfected T2 cells. Because T2 cells have been shown to express Kb molecules that are either largely devoid of bound peptides or bind a vastly different set of low affinity peptides, it is unlikely that the detected structural changes were caused by alterations in the spectrum of peptides bound by the class I variant glycoproteins. Similarly, a class I point mutant, Kb-97R, that also has been shown previously to bind a very different set of peptides in comparison to the parental Kb molecule also displays normal antibody binding properties. We conclude from these studies that structural diversity within the Ag binding cleft indirectly influences the external surface of the Ag-presenting domain of the class I H chain. Significantly, this surface is the interface between the T cell receptor and MHC molecules and may make contributions to the fine specificity of allorecognition.

Expression of the alpha-myosin heavy chain gene in the heart is regulated in part by an E-box-dependent mechanism.

Proximal regulatory element B (PRE-B), located from positions -318 to -284 in the alpha-myosin heavy chain (MHC) promoter, stimulated expression from an otherwise weak alpha-MHC promoter fragment in primary rat neonatal cardiomyocytes but not in the C2C12 myogenic cell line. PRE-B interacted with alpha-MHC binding factor 2 (BF-2), a protein found in nuclear extracts from several neonatal rat tissues and cell types including cardiomyocytes. BF-2 DNA binding activity was greatly reduced in adult versus neonatal tissues. Methylation interference footprints indicated that BF-2 bound to an element that included an E-box consensus sequence. Site-directed mutations in the BF-2-binding site, that abolish BF-2 binding, reduced expression from the full-length alpha-MHC promoter by 70%. A BF-2-like protein interacts within the HF-1a element of the myosin light chain-2 (MLC-2) promoter suggesting that one of the proteins that regulates the alpha-MHC and MLC-2 genes is identical or closely related. Analysis of binding by competition gel shift experiments indicated that both BF-2 and HF-1a are E-box-binding proteins. The alpha-MHC and MLC-2 genes encode contractile proteins which are precursors of myosin. Regulation by the same transcription factor might indicate that the expression of alpha-MHC and MLC-2 is coordinately controlled.