Saethre-Chotzen syndrome (SCS) is a human autosomal dominant disorder characterized by premature fusion of cranial sutures caused by mutations of the Twist gene encoding a basic helix-loop-helix (bHLH) transcription factor. We previously showed that Twist haploinsufficiency caused by a Y103X nonsense mutation in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of osteoblast differentiation. Here we show that Twist haploinsufficiency alters osteoblast apoptosis in SCS. Analysis of terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) demonstrated increased osteoblast and osteocyte apoptosis in coronal sutures from two SCS patients with nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins, and one patient with a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding. To assess the mechanisms involved, we studied osteoblast apoptosis in mutant (M-Tw) calvarial cells bearing the Y103X mutation resulting in decreased Twist mRNA and protein levels. M-Tw cells cultured in low serum conditions showed enhanced DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Biochemical analysis showed increased activity of initiator caspases-2 and -8 and downstream effector caspases-3, -6 and -7 in mutant osteoblasts. Caspase-2 was upstream of caspase-8 and effector caspases-3, -6 and -7 because their activities were suppressed by a specific caspase-2 inhibitor. M-Tw osteoblasts also showed increased cytochrome c release from the mitochondria. However, the activity of the downstream effector caspase-9 was not increased due to overexpression of the antagonist protein Hsp70. Detection of differentially expressed genes using cDNA expression array revealed increased Bax and TNFalpha mRNA levels in M-Tw compared to Nl cells, a finding confirmed by RT-PCR and western blot analyses. Neutralization of TNFalpha overexpression using anti-TNFalpha or anti-TNF receptor 1 antibodies abolished the increased activity of caspase-2, caspase-8 and caspases-3, -6 and -7 in M-Tw osteoblasts. These studies provide novel evidence that Twist haploinsufficiency in SCS promotes osteoblast apoptosis by a TNFalpha-caspase-2-caspase-8-caspases-3, -6, -7 cascade, and uncover a molecular mechanism in which Twist plays an anti-apoptotic role in human calvarial osteoblasts.
Actin dynamics is central for cells, and especially for the fast-moving leukocytes. The severing of actin filaments is mainly achieved by cofilin, assisted by Aip1/Wdr1 and coronins. We found that in Wdr1-deficient zebrafish embryos, neutrophils display F-actin cytoplasmic aggregates and a complete spatial uncoupling of phospho-myosin from F-actin. They then undergo an unprecedented gradual disorganization of their nucleus followed by eruptive cell death. Their cofilin is mostly unphosphorylated and associated with F-actin, thus likely outcompeting myosin for F-actin binding. Myosin inhibition reproduces in WT embryos the nuclear instability and eruptive death of neutrophils seen in Wdr1-deficient embryos. Strikingly, depletion of the main coronin of leukocytes, coronin 1A, fully restores the cortical location of F-actin, nuclear integrity, viability, and mobility of Wdr1-deficient neutrophils in vivo. Our study points to an essential role of actomyosin contractility in maintaining the integrity of the nucleus of neutrophils and a new twist in the interplay of cofilin, Wdr1, and coronin in regulating F-actin dynamics.
Most tissues harbor a substantial population of resident macrophages. It is not quite known yet how their quite diverse phenotypes are shaped by the functions that they assume in each tissue. In this study, we elucidate a functional link between the Slc7a7 cationic amino acid transporter and tissue macrophages. We had identified a mutant zebrafish devoid of microglia due to a mutation in the slc7a7 gene. We found that in Slc7a7 deficient larvae, macrophages do enter the retina and brain to become microglia, but then die during the developmental wave of neuronal apoptosis, which triggers intense efferocytic work from them. A similar macrophage demise occurs at other tissues and stages whereby macrophages have to engulf many cell corpses, be it due to developmental or experimentally triggered cell death. We found that slc7a7 is by far the main cationic amino acid transporter gene expressed in macrophages of wild type zebrafish larvae, and that its expression is induced in tissue macrophages within 1-2 hrs upon efferocytosis. Our data altogether indicate that a high level of Slc7a7 is vital not only for microglia but also for any steadily efferocytic tissue macrophages, and that slc7a7 gene induction is one of the adaptive responses that allow them to cope with the catabolism of numerous dead cells without compromising their own viability. P. Herbomel conceived and supervised the project, and obtained the funding. M. Tauzin isolated the cerise mutant, gave it its name, and conducted the initial phenotypic study. D L Demy, M. Carrère and R. Noche conducted most of the subsequent study, with the help of M. Le Bris and C. Baek during their respective Master internship; M. Yousfi contributed the 4C4/Pu.1 double immunostaining images. D L Demy performed the initial mapping of the mutation; I. Leshchiner & W. Goessling identified the slc7a7 mutation via deep sequencing of mutant vs. sibling genome pools. P. Herbomel and D L Demy wrote the paper.
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