The H3K27M mutation alters stem cell growth, epigenetic regulation, and differentiation potential.

BACKGROUND: Neurodevelopmental disorders increase brain tumor risk, suggesting that normal brain development may have protective properties. Mutations in epigenetic regulators are common in pediatric brain tumors, highlighting a potentially central role for disrupted epigenetic regulation of normal brain development in tumorigenesis. For example, lysine 27 to methionine mutation (H3K27M) in the H3F3A gene occurs frequently in Diffuse Intrinsic Pontine Gliomas (DIPGs), the most aggressive pediatric glioma. As H3K27M mutation is necessary but insufficient to cause DIPGs, it is accompanied by additional mutations in tumors. However, how H3K27M alone increases vulnerability to DIPG tumorigenesis remains unclear. RESULTS: Here, we used human embryonic stem cell models with this mutation, in the absence of other DIPG contributory mutations, to investigate how H3K27M alters cellular proliferation and differentiation. We found that H3K27M increased stem cell proliferation and stem cell properties. It interfered with differentiation, promoting anomalous mesodermal and ectodermal gene expression during both multi-lineage and germ layer-specific cell specification, and blocking normal differentiation into neuroectoderm. H3K27M mutant clones exhibited transcriptomic diversity relative to the more homogeneous wildtype population, suggesting reduced fidelity of gene regulation, with aberrant expression of genes involved in stem cell regulation, differentiation, and tumorigenesis. These phenomena were associated with global loss of H3K27me3 and concordant loss of DNA methylation at specific genes in H3K27M-expressing cells. CONCLUSIONS: Together, these data suggest that H3K27M mutation disrupts normal differentiation, maintaining a partially differentiated state with elevated clonogenicity during early development. This disrupted response to early developmental cues could promote tissue properties that enable acquisition of additional mutations that cooperate with H3K27M mutation in genesis of DMG/DIPG. Therefore, this work demonstrates for the first time that H3K27M mutation confers vulnerability to gliomagenesis through persistent clonogenicity and aberrant differentiation and defines associated alterations of histone and DNA methylation.

Macrophage secretion of miR-106b-5p causes renin-dependent hypertension.

Myeloid cells are known mediators of hypertension, but their role in initiating renin-induced hypertension has not been studied. Vitamin D deficiency causes pro-inflammatory macrophage infiltration in metabolic tissues and is linked to renin-mediated hypertension. We tested the hypothesis that impaired vitamin D signaling in macrophages causes hypertension using conditional knockout of the myeloid vitamin D receptor in mice (KODMAC). These mice develop renin-dependent hypertension due to macrophage infiltration of the vasculature and direct activation of renal juxtaglomerular (JG) cell renin production. Induction of endoplasmic reticulum stress in knockout macrophages increases miR-106b-5p secretion, which stimulates JG cell renin production via repression of transcription factors E2f1 and Pde3b. Moreover, in wild-type recipient mice of KODMAC/miR106b-/- bone marrow, knockout of miR-106b-5p prevents the hypertension and JG cell renin production induced by KODMAC macrophages, suggesting myeloid-specific, miR-106b-5p-dependent effects. These findings confirm macrophage miR-106b-5p secretion from impaired vitamin D receptor signaling causes inflammation-induced hypertension.

Ca(2+)-activated but not G protein-mediated inositol phosphate responses in rat neonatal cardiomyocytes involve inositol 1,4, 5-trisphosphate generation.

Inositol phosphate (InsP) responses to receptor activation are assumed to involve phospholipase C cleavage of phosphatidylinositol 4,5-bisphosphate to generate Ins(1,4,5)P(3). However, in [(3)H]inositol-labeled rat neonatal cardiomyocytes (NCM) both initial and sustained [(3)H]InsP responses to alpha(1)-adrenergic receptor stimulation with norepinephrine (100 microM) were insensitive to the phosphatidylinositol 4,5-bisphosphate-binding agent neomycin (5 mM). Introduction of 300 microM unlabeled Ins(1,4, 5)P(3) into guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)-stimulated, permeabilized [(3)H]inositol-labeled NCM increased [(3)H]Ins(1,4,5)P(3) slightly but did not significantly reduce levels of its metabolites [(3)H]Ins(1,4)P(2) and [(3)H]Ins(4)P, suggesting that these [(3)H]InsPs are not formed principally from [(3)H]Ins(1,4,5)P(3). In contrast, the calcium ionophore A23187 (10 microM) provoked [(3)H]InsP responses in intact NCM which were sensitive to neomycin, and elevation of free calcium in permeabilized NCM led to [(3)H]InsP responses characterized by marked increases in [(3)H]Ins(1,4,5)P(3) (2.9 +/- 0.2% of total [(3)H]InsPs after 20 min of high Ca(2+) treatment in comparison to 0. 21 +/- 0.05% of total [(3)H]InsPs accumulated after 20 min of GTPgammaS stimulation). These data provide evidence that Ins(1,4, 5)P(3) generation is not a major contributor to G protein-coupled InsP responses in NCM, but that substantial Ins(1,4,5)P(3) generation occurs under conditions of Ca(2+) overload. Thus in NCM, Ca(2+)-induced Ins(1,4,5)P(3) generation has the potential to worsen Ca(2+) overload and thereby aggravate Ca(2+)-induced electrophysiological perturbations.

Inositol 1,4,5-trisphosphate and reperfusion arrhythmias.

1. The present review focuses on the role of the Ca2+-releasing second messenger inositol 1,4,5-trisphosphate (IP3) in initiating arrhythmias during early reperfusion following a period of myocardial ischaemia. 2. Evidence for an arrhythmogenic action of IP3 was provided by studies showing a correlation between the extent of the increase in IP3 and the incidence of arrhythmias in early reperfusion. In addition, phospholipase C inhibitors selective for thrombin receptor stimulation were anti-arrhythmic only when arrhythmias were thrombin initiated. 3. Mechanisms by which IP3 could initiate arrhythmias are discussed, with particular emphasis on the role of slow and unscheduled Ca2+ release. 4. The reperfusion-induced IP3 and arrhythmogenic responses can be initiated through either alpha1-adrenoceptors or thrombin receptors, but endothelin receptor stimulation was ineffective. Further studies have provided evidence that the noradrenaline-mediated response was mediated by alpha1A-receptors, while the alpha1B-adrenoceptor subtype appeared to be protective. 5. Reperfusion-induced IP3 responses could be inhibited by procedures known to reduce the incidence of arrhythmias under these conditions, including preconditioning, inhibiting Na+/H+ exchange or by dietary supplementation with n-3 polyunsaturated fatty acids. 6. Inositol 1,4,5-trisphosphate generation in cardiomyocytes can be facilitated by raising intracellular Ca2+ and it seems likely that the rise in Ca2+ in ischaemia and reperfusion is responsible for the generation of IP3, which will, in turn, further exacerbate Ca2+ overload.

Evidence for selective coupling of alpha 1-adrenergic receptors to phospholipase C-beta 1 in rat neonatal cardiomyocytes.

Activation of phospholipase C (PLC) in neonatal rat cardiomyocytes (NCM) generates primarily inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) in response to rises in intracellular Ca(2+), or inositol 1,4-bisphosphate (Ins(1,4)P(2)) in response to norepinephrine (NE) (Matkovich, S. J. and Woodcock, E. A. (2000) J. Biol. Chem. 275, 10845-10850). To examine the PLC subtype mediating the alpha(1)-adrenergic receptor response, PLC-beta(1) and PLC-beta(3) were overexpressed in NCM using adenoviral infection (Ad-PLC-beta(1) NCM and Ad-PLC-beta(3) NCM, respectively) and PLC responses assessed from [(3)H]inositol phosphate (InsP) generation in the presence of 10 mm LiCl. The [(3)H]InsP response to NE (100 microm) was enhanced in Ad-PLC-beta(1) NCM relative to cells infected with blank virus (Ad-MX NCM), but was reduced in Ad-PLC-beta(3) NCM. In contrast, the [(3)H]InsP response to ATP (100 microm) was not elevated in Ad-PLC-beta(1) NCM, and was enhanced rather than diminished in Ad-PLC-beta(3) NCM, showing that effects of the two PLC-beta isoforms were specific for particular receptor types. PLC-delta(1) overexpression selectively reduced NE-induced [(3)H]InsP responses, without affecting the ATP stimulation. The reduced NE response was associated with a selective loss of PLC-beta(1) expression in Ad-PLC-delta(1) NCM. alpha(1)-Adrenergic receptor activation caused phosphorylation of PLC-beta(1) but not PLC-beta(3), whereas stimulation by ATP induced phosphorylation of PLC-beta(3) but not PLC-beta(1.) Taken together, these studies provide evidence that NE-stimulated InsP generation in NCM is primarily mediated by PLC-beta(1), despite the presence of both PLC-beta(1) and PLC-beta(3) isoforms.