Project description:Mutations in repulsive guidance molecule c (RGMc) / hemojuvelin (HJV) cause juvenile hemochromatosis, an aggravated iron overload disorder that presents early in life. Patients with juvenile hemochromatosis, and RGMc knockout mice, have diminished expression of the key iron-regulatory peptide, hepcidin. This suggests that RGMc plays a critical role in the regulation of iron homeostasis; however the mechanisms of RGMc actions are unknown. Recent studies have shown that RGMc directly binds to the growth factors, bone morphogenetic protein 2 and 6 (BMP2 and BMP6), and it is possible that this interaction regulates aspects of iron metabolism. We used microarrays to examine the effects of RGMc on BMP2- and BMP6-mediated gene expression. In our experimental model we treated Hep3B liver cells that had been serum starved for 16 hours as follows: (1) un-treated, (2) BMP2, (3) BMP2 + 10-fold molar excess of Noggin (a potent BMP2 inhibitor), (4) BMP2 + 20-fold molar excess of RGMc, (5) BMP6, (6) BMP6 + 10-fold molar excess of Noggin, (7) BMP6 + 20-fold molar excess of RGMc. Binding was allowed to proceed for BMP and Noggin or RGMc for 3 hr at 20°C prior to treatment of Hep3B cells. Treatment time was 4 hr at which cells were collected for RNA isolation.
Project description:Mutations in repulsive guidance molecule c (RGMc) / hemojuvelin (HJV) cause juvenile hemochromatosis, an aggravated iron overload disorder that presents early in life. Patients with juvenile hemochromatosis, and RGMc knockout mice, have diminished expression of the key iron-regulatory peptide, hepcidin. This suggests that RGMc plays a critical role in the regulation of iron homeostasis; however the mechanisms of RGMc actions are unknown. Recent studies have shown that RGMc directly binds to the growth factors, bone morphogenetic protein 2 and 6 (BMP2 and BMP6), and it is possible that this interaction regulates aspects of iron metabolism. We used microarrays to examine the effects of RGMc on BMP2- and BMP6-mediated gene expression.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Human pulmonary arterial endothelial cells (PAECs) and blood outgrowth endothelial cells (BOECs) from healthy subjects were stimulated with BMP9, BMP2 or BMP6. and assessed for changes in gene transcription by microarray. Unlike BMP2 and BMP6, which had negligible impacts on gene expression, BMP9 induced the differential regulation of 1883 genes (adjusted P value < 0.05), including several key components of canonical BMP signaling such as ID1, ID2 and BMPR2.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression. Two-condition experiment, Normoxic MSCs vs. Hypoxic MSCs.
Project description:Microvascular endothelial cells (EC) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific EC control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal EC (LSEC) are uniquely differentiated to fulfil important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSEC. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSEC using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver, and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is non-redundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ, but also for the homeostasis of the whole organism.
Project description:Iron induces hepcidin by activating bone morphogenetic protein (BMP)6-SMAD signaling. Liver endothelial cells (LECs) produce BMP6, but the molecular mechanisms are incompletely understood. To address this, we performed proteomics and RNA-sequencing on LECs from iron-adequate and iron-loaded mice. Gene set enrichment analysis identified transcription factors activated by high iron, including Nrf-2, which was previously reported to contribute to BMP6 regulation, and proto-oncogene c-Jun (encoded by Jun). Jun knockdown blocked Bmp6, but not Nrf-2 pathway, induction by iron in LEC cultures. Moreover, chromatin immunoprecipitation of mouse livers showed iron-dependent c-Jun binding to predicted sites in Bmp6 regulatory regions. Finally, c-Jun inhibitor blunted induction of Bmp6 and hepcidin, but not Nrf-2 activity, in iron-loaded mice. However, Bmp6 expression and iron parameters were unchanged in endothelial Jun knockout mice. Our data suggest that c-Jun participates in iron-mediated BMP6 regulation independent of Nrf-2, though the mechanisms may be redundant and/or multifactorial.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs. Two-condition experiment, KP MSCs vs. 3A6 MSCs.