Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor and endoglin, a TGF-β co-receptor, play an essential role in... Show moreGenetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for anti-angiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti hALK1-antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting, but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF-VEGFR and the BMP9-ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously is an attractive strategy to overcome resistance to anti-angiogenesis therapy. Show less
Zhou, F.F.; Zhang, X.F.; Dam, H. van; Dijke, P. ten; Huang, H.Z.; Zhang, L. 2012
HLA-DQ2 and HLA-DQ8 are strongly predisposing haplotypes for type 1 diabetes (T1D). Yet, HLA-DQ2/8 heterozygous individuals have a synergistically increased risk compared to HLA-DQ2 or HLA-DQ8... Show moreHLA-DQ2 and HLA-DQ8 are strongly predisposing haplotypes for type 1 diabetes (T1D). Yet, HLA-DQ2/8 heterozygous individuals have a synergistically increased risk compared to HLA-DQ2 or HLA-DQ8 homozygote subjects that may result from the presence of a transdimer formed between the α-chain of HLA-DQ2 (DQA1*0501) and the β-chain of HLA-DQ8 (DQB1*0302). We generated cells exclusively expressing this transdimer (HLA-DQ8trans), characterized its peptide binding repertoire and defined a unique transdimer specific peptide-binding motif that was found to be distinct from those of HLA-DQ2 and HLA-DQ8. This motif predicts an array of peptides of islet autoantigens as candidate T cell epitopes, many of which selectively bind to the HLA-transdimer, whereas others bind to both HLA-DQ8 and transdimer with similar affinity. Our findings provide a molecular basis for the association between HLA-DQ transdimers and T1D and set the stage for rational testing of potential diabetogenic peptide epitopes. Show less
Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of... Show moreSeveral missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF. Show less
Transforming growth factor-beta (TGF beta) binding to its receptor leads to intracellular phosphorylation of Smad2 and Smad3, which oligomerize with Smad4. These complexes accumulate in the nucleus... Show moreTransforming growth factor-beta (TGF beta) binding to its receptor leads to intracellular phosphorylation of Smad2 and Smad3, which oligomerize with Smad4. These complexes accumulate in the nucleus and induce gene transcription. Here we describe mitogen-and stress-activated kinase 1 (MSK1) as an antagonist of TGF beta-induced cell death. Induction of MSK1 activity by TGF beta depends on Smad4 and p38 MAPK activation. Knockdown of GADD45, a Smad4-induced upstream regulator of p38 MAPK prevents TGF beta-induced p38 and MSK1 activity. MSK1 functionally regulates pro-apoptotic BH3-only BCL2 proteins, as MSK1 knockdown reduces Bad phosphorylation and enhances Noxa and Bim expression, leading to enhanced TGF beta-induced caspase-3 activity and cell death. This finding suggests that MSK1 represents a pro-survival pathway bifurcating downstream of p38 and antagonizes the established pro-apoptotic p38 MAPK function. Furthermore, EGF could reverse all the effects observed after MSK1 knockdown. Monitoring the status of MSK1 activity in cancer promises new therapeutic targets as inactivating both MSK1 and EGF signaling may (re)-sensitize cells to TGF beta-induced cell death. Show less
Zaferani, A.; Vives, R.R.; Pol, P. van der; Hakvoort, J.J.; Navis, G.J.; Goor, H. van; ... ; Born, J. van den 2011
Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying... Show moreSclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins. Show less
Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate... Show morePlasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl) thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED50 values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates. Show less
![The Vital Role of Polymerase zeta and REV1 in Mutagenic, but Not Correct, DNA Synthesis across Benzo[a]pyrene-dG and Recruitment of Polymerase zeta by REV1 to Replication-stalled Site](/sites/all/modules/custom/islandora_solr_search/images/defaultimg.png?solr_nav%5Bid%5D=496fa459960250abd7fe&solr_nav%5Bpage%5D=3&solr_nav%5Boffset%5D=4)
