Epigenetic regulation of megakaryocytic and erythroid differentiation by PHF2 histone demethylase.

Plant homeodomain finger 2 (PHF2) is a JmjC family histone demethylase that demethylates H3K9me2, a repressive gene marker. PHF2 was found to play a role in the differentiation of several tissue types such as osteoblast and adipocyte differentiation. We report here that PHF2 plays a role in the epigenetic regulation of megakaryocytic (MK) and erythroid differentiation. We investigated PHF2 expression during MK and erythroid differentiation in K562 and human CD34+ progenitor (hCD34+ ) cells. Our data demonstrate that PHF2 expression is down-regulated during megakaryopoiesis and erythropoiesis. PHF2 has a negative role in MK and erythroid differentiation of K562 cells; knockdown of PHF2 promotes MK and erythroid differentiation of hCD34+ cells. Similarly, we found that p53 expression is also down-regulated during MK and erythroid differentiation, which parallels PHF2 expression. PHF2 binds to the p53 promoter and regulates the expression of p53 by demethylating H3K9me2 in the promoter region of p53. Taken together, our data show that PHF2 is a negative epigenetic regulator of MK and erythroid differentiation, and that one of the pathways through which PHF2 affects MK and erythroid differentiation is via regulation of p53 expression.

New role of LRP5, associated with nonsyndromic autosomal-recessive hereditary hearing loss.

Human hearing loss is a common neurosensory disorder about which many basic research and clinically relevant questions are unresolved. At least 50% of hearing loss are due to a genetic etiology. Although hundreds of genes have been reported, there are still hundreds of related deafness genes to be found. Clinical, genetic, and functional investigations were performed to identify the causative mutation in a distinctive Chinese family with postlingual nonsyndromic sensorineural hearing loss. Whole-exome sequencing (WES) identified lipoprotein receptor-related protein 5 (LRP5), a member of the low-density lipoprotein receptor family, as the causative gene in this family. In the zebrafish model, lrp5 downregulation using morpholinos led to significant abnormalities in zebrafish inner ear and lateral line neuromasts and contributed, to some extent, to disabilities in hearing and balance. Rescue experiments showed that LRP5 mutation is associated with hearing loss. Knocking down lrp5 in zebrafish results in reduced expression of several genes linked to Wnt signaling pathway and decreased cell proliferation when compared with those in wild-type zebrafish. In conclusion, the LRP5 mutation influences cell proliferation through the Wnt signaling pathway, thereby reducing the number of supporting cells and hair cells and leading to nonsyndromic hearing loss in this Chinese family.

Endogenous tissue factor pathway inhibitor in vascular smooth muscle cells inhibits arterial thrombosis.

Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor-mediated coagulation. TFPI is expressed by endothelial and smooth muscle cells in the vasculature. Endothelium-derived TFPI has been reported to play a regulatory role in arterial thrombosis. However, the role of endogenous TFPI in vascular smooth muscle cells (VSMCs) in thrombosis and vascular disease development has yet to be elucidated. In this TFPIFlox mice crossbred with Sma-Cre mice were utilized to establish TFPI conditional knockout mice and to examine the effects of VSMC-directed TFPI deletion on development, hemostasis, and thrombosis. The mice with deleted TFPI in VSMCs (TFPISma) reproduced viable offspring. Plasma TFPI concentration was reduced 7.2% in the TFPISma mice compared with TFPIFlox littermate controls. Plasma TFPI concentration was also detected in the TFPITie2 (mice deleted TFPI in endothelial cells and cells of hematopoietic origin) mice. Plasma TFPI concentration of the TFPITie2 mice was 80.4% lower (P < 0.001) than that of the TFPIFlox mice. No difference in hemostatic measures (PT, APTT, and tail bleeding) was observed between TFPISma and TFPIFlox mice. However, TFPISma mice had increased ferric chloride-induced arterial thrombosis compared with TFPIFlox littermate controls. Taken together, these data indicated that endogenous TFPI from VSMCs inhibited ferric chloride-induced arterial thrombosis without causing hemostatic effects.

Conditional knockout of TFPI-1 in VSMCs of mice accelerates atherosclerosis by enhancing AMOT/YAP pathway.

BACKGROUND: Tissue factor pathway inhibitor-1 (TFPI-1) has multiple functions and its precise role and molecular mechanism during the development of atherosclerosis are not clear. OBJECTIVES: To determine the effect and molecular mechanism of TFPI-1 deficiency in vascular smooth muscle cells (VSMCs) in atherosclerosis in the apolipoprotein E knockout (ApoE-/-) mouse. METHODS AND RESULTS: A mouse model with a conditional knockout of TFPI-1 in VSMCs in an atherosclerosis-prone background (ApoE-/-) was generated. Mice were fed a high fat diet for 18weeks and were then euthanized. Arterial trees and aortas were stained with Sudan IV and were labeled via immunohistochemistry. Cell proliferation and migration of VSMCs in atherosclerotic plaques were assessed. More atherosclerotic lesions and higher levels of proliferation and migration of VSMCs were observed in TFPI-1fl/fl/Sma-Cre+ApoE-/-mice. An interaction between TFPI-1 and angiomotin (AMOT) was identified in human VSMCs by mass spectrometry, immunoprecipitation and co-localization analyses. Signal pathway changes were detected by Western blot analysis, and the expression levels of target genes were determined by real-time PCR. Decreased phosphorylation of AMOT and Yes-associated protein 1 (YAP) in TFPI-1fl/fl/Sma-Cre+ApoE-/- mice resulted in increased expression levels of snail family zinc finger 2 (SLUG) and connective tissue growth factor (CTGF), which are target genes of the Hippo signaling pathway that have been verified as atherosclerosis candidate genes. CONCLUSION: Deficiency in TFPI-1 in the VSMCs of ApoE-/- mice accelerated the development of atherosclerosis by promoting the proliferation and migration of VSMCs which may be caused by the decreased phosphorylation of AMOT and YAP. SIGNIFICANCE: TFPI-1 has been found to has an anticoagulant activity, induce cell apoptosis and prevent cell proliferation. For the first time, we constructed a line of conditional knockout mice in which the TPFI-1 gene is deleted in VSMCs. We found that TFPI-1 deficiency clearly promoted the development of atherosclerosis when these mice were crossed into an ApoE-/-background. One notable feature of atherosclerosis is the proliferation and migration of smooth muscle cells. Previous reports involved TFPI-1 do not completely explain the proliferation and migration of VSMCs because heterozygous TF deficient (TF±) mice bred in an ApoE-/- background did not show diminished atherosclerosis compared to TF+/+ mice bred in the same background. Our results first confirmed that TFPI-1 interacts with AMOT, which led to a decrease in the phosphorylation of YAP and further increased the genes expression of the proliferation and migration involved. Our results further confirmed that atherosclerosis was a localized disease.