👤 I Martin Sheldon

Preventing postpartum uterine disease depends on the ability of endometrial cells to tolerate the presence of the bacteria that invade the uterus after parturition. Postpartum uterine disease and endometrial pathology in cattle are most associated with the pathogen Trueperella pyogenes. Trueperella pyogenes secretes a cholesterol-dependent cytolysin, pyolysin, which causes cytolysis by forming pores in the plasma membrane of endometrial stromal cells. The aim of the present study was to identify cell-intrinsic pathways that increase bovine endometrial stromal cell tolerance to pyolysin. Pyolysin caused dose-dependent cytolysis of bovine endometrial stromal cells and leakage of lactate dehydrogenase into supernatants. Cell tolerance to pyolysin was increased by inhibitors that target the mevalonate and cholesterol synthesis pathway, but not the mitogen-activated protein kinase, cell cycle, or metabolic pathways. Cellular cholesterol was reduced and cell tolerance to pyolysin was increased by supplying the mevalonate-derived isoprenoid farnesyl pyrophosphate, or by inhibiting farnesyl-diphosphate farnesyltransferase 1 or geranylgeranyl diphosphate synthase 1 to increase the abundance of farnesyl pyrophosphate. Supplying the mevalonate-derived isoprenoid geranylgeranyl pyrophosphate also increased cell tolerance to pyolysin, but independent of changes in cellular cholesterol. However, geranylgeranyl pyrophosphate inhibits nuclear receptor subfamily 1 group H receptors (NR1H, also known as liver X receptors), and reducing the expression of the genes encoding NR1H3 or NR1H2 increased stromal cell tolerance to pyolysin. In conclusion, mevalonate-derived isoprenoids increased bovine endometrial stromal cell tolerance to pyolysin, which was associated with reducing cellular cholesterol and inhibiting NR1H receptors. Show less

Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-β receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology. Show less

Postsynaptic density (PSD)-93 and PSD-95 are the major membrane-associated guanylate kinases (MAGUKs) at excitatory synapses of the brain linking the N-methyl-d-aspartate receptor (NMDAR) with neuronal nitric oxide synthase (nNOS), which contributes to cell death after neonatal hypoxia-ischemia (HI). We investigated whether deletion of PSD-93 would dissociate the NMDAR from nNOS and be neuroprotective. Postnatal day 7 wild-type (+/+), heterozygous (+/-), and homozygous (-/-) PSD-93 knockout mice were subjected to HI by permanent ligation of the right carotid artery, followed by exposure to 8% O2/92% N2 for 1 hour. Brains were scored 5 days later for damage with cresyl violet and iron stains. Western blot and coimmunoprecipitation were used to determine the expression and association of the major PSD proteins. There was no significant difference between PSD-93 (-/-) and (+/+) mice in mortality or degree of brain injury. In the absence of PSD-93, PSD-95 still interacted with NR2B and nNOS. Under physiological conditions, PSD-95, nNOS, NR2A, and NR2B were unaltered in the (-/-) pups. However, at 24 hours after HI, protein expression of PSD-95, nNOS, and NR2A but not NR2B was markedly higher in the (-/-) than in the (+/+) pups. In (+/+) pups, HI resulted in decreased expression of NR2A but not NR2B in cortex and decreased NR2A and NR2B expression in hippocampus, but this reduction was not observed in (-/-) pups. PSD-93 is not essential for baseline synaptic function but may participate in regulation of NMDAR-associated signaling pathways after HI injury. Deletion of PSD-93 alone does not provide neuroprotection after neonatal HI, possibly a result, in part, of upregulation of PSD-95. MAGUKs may substitute for one another, allowing normal NMDAR function in the postnatal period. Show less