Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1 and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10 fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while up-regulating WNT-related genes (WISP2, SFRP2 and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.
Mesenchymal stem cell; adipose-tissue derived stromal cells; pluripotent; multipotent; cell cycle; lineage-commitment; fibroblast; osteogenesis; chondrogenesis; adipogenesis; histone; cyclin; extracellular matrix; CD44; CD73; NT5E; CD90; THY1; CD105; ENG; NES; ACTA2; OCT4; POU5F1; NANOG; KLF4; CCND1; CCNB2; HIST1H3H; HIST1H4A; HIST2H4A; HIST2H4B; E2F1; E2F7; E2F8; HINFP; NPAT; ASPN; ECM2; FMOD; OGN; PODN; WISP2; SFRP2; SFRP4; WNT2; WNT2A; WNT5A; WNT5B; WNT7B; RARRES2; TNNT3; ADH1B; H19; CHI3L1
Emerging evidence indicates that activation of Wnt/β-catenin signaling at the cell surface results in inhibition of glycogen synthase kinase 3β (GSK3β), leading to activation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling. Salinomycin is a novel small molecule inhibitor of LRP6. In the present study, we found that LRP6 overexpression induced mTORC1 signaling activation in cancer cells, and that salinomycin was not only a potent Wnt/β-catenin signaling inhibitor, bus also a strong mTORC1 signaling antagonist in breast and prostate cancer cells. Mechanistically, salinomycin activated GSK3β in cancer cells. Moreover, salinomycin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/β-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity. Our results present novel mechanisms underlying salinomycin-mediated cancer cell death.
salinomycin; LRP6; Wnt signaling; mTORC1 signaling; cancer
Acute myeloid leukemia (AML) is an aggressive hematologic cancer that is characterized by accumulation of immature myeloid cells in the blood and bone marrow. The malignant cells in AML have reduced capacity to mature fully, and often exhibit chromosomal abnormalities, defects in cell signaling, and abnormal cell cycle control. Genetic and epigenetic changes are implicated in the onset and progression of AML. While progress has been made in using genetic and epigenetic changes as prognostic features of AML, these findings have not yet been effectively translated into novel treatment strategies. Disappointingly, rates of recurrence in AML remain high and overall survival is poor. Research strategies should focus on developing a comprehensive landscape of genetic and epigenetic changes in individual patients with AML to expand the clinicians’ therapeutic armamentarium and to individualize and optimize treatment.
Non-coding RNA; DNA Methylation; Histone Modifications; Acute Myeloid Leukemia
Plasminogen activator inhibitor type-1 (PAI-1), a major regulator of the plasmin-dependent pericellular proteolytic cascade, is prominently expressed during the tissue response to injury although the factors that impact PAI-1 induction and their role in the repair process are unclear. Kinetic modeling using established biomarkers of cell cycle transit (c-MYC; cyclin D1; cyclin A) in synchronized human (HaCaT) keratinocytes, and previous cytometric assessments, indicated that PAI-1 transcription occurred early after serum-stimulation of quiescent (G0) cells and prior to G1 entry. It was established previously that differential residence of USF family members (USF1→USF2 switch) at the PE2 region E box (CACGTG) characterized the G0→G1 transition period and the transcriptional status of the PAI-1 gene. A consensus PE2 E box motif (5′-CACGTG-3′) at nucleotides -566 to -561 was required for USF/E box interactions and serum-dependent PAI-1 transcription. Site-directed CG→AT substitution at the two central nucleotides inhibited formation of USF/probe complexes and PAI-1 promoter-driven reporter expression. A dominant-negative USF (A-USF) construct or double-stranded PE2 “decoy” attenuated serum- and TGF-β1-stimulated PAI-1 synthesis. Tet-Off induction of an A-USF insert reduced both PAI-1 and PAI-2 transcripts while increasing the fraction of Ki-67+ cells. Conversely, overexpression of USF2 or adenoviral-delivery of a PAI-1 vector inhibited HaCaT colony expansion indicating that the USF1→USF2 transition and subsequent PAI-1 transcription are critical events in the epithelial go-or-grow response. Collectively, these data suggest that USF2, and its target gene PAI-1, regulate serum-stimulated keratinocyte growth, and likely the cadence of cell cycle progression in replicatively-competent cells as part of the injury repair program.
PAI-1; USF; transcription; keratinocytes; gene regulation; expression profiling; SERPINE1
The importance of microRNAs (miRs) in control of gene expression is now clearly recognized. While individual microRNAs are thought to target hundreds of disparate mRNAs via imperfect base pairing, little is known about the characteristics of miR target sites. Here we show that the miRs can be aligned with empirically identified accessible sites in a target RNA (Cytokeratin 19, KRT), and that some of the aligned miRs functionally down-regulate KRT expression post-transcriptionally. We employed an RNase-H-based random library selection protocol to identify accessible sites in KRT RNA. We then aligned the Sanger Institute database collection of human miRs to KRT mRNA, and also aligned them using the web-based MicroInspector program. Most miRs aligned with the accessible sites identified empirically; those not aligned with the empirically identified sites also functioned effectively in RNase-H-based assays. Similar results were obtained with a second target RNA (Mammoglobin). Transient transfection assays established that some of the miRs which aligned with KRT significantly down-regulated it at the protein level, with no effect on RNA level. The functionally effective miRs aligned within the coding region of KRT, whereas a number of miRs which aligned with the 3′-untranslated region did not produce down-regulation.
microRNAs; LIBRARY SELECTION
Intrauterine growth restriction leads to the development of adult onset obesity/metabolic syndrome, diabetes mellitus, cardiovascular disease, hypertension, stroke, dyslipidemia, and non-alcoholic fatty liver disease/steatohepatitis. Continued postnatal growth restriction has been shown to ameliorate many of these sequelae. To further our understanding of the mechanism of how intrauterine and early postnatal growth affects adult health we have employed Affymetrix microarray-based expression profiling to characterize hepatic gene expression of male offspring in a rat model of maternal nutrient restriction in early and late life. At day 21 of life (p21) combined intrauterine and postnatal calorie restriction treatment led to expression changes in circadian, metabolic, and insulin-like growth factor genes as part of a larger transcriptional response that encompasses 144 genes. Independent and controlled experiments at p21 confirm the early life circadian, metabolic, and growth factor perturbations. In contrast to the p21 transcriptional response, at day 450 of life (d450) only seven genes, largely uncharacterized, were differentially expressed. This lack of a transcriptional response identifies non-transcriptional mechanisms mediating the adult sequelae of intrauterine growth restriction. Independent experiments at d450 identify a circadian defect as well as validate expression changes to four of the genes identified by the microarray screen which have a novel association with growth restriction. Emerging from this rich dataset is a portrait of how the liver responds to growth restriction through circadian dysregulation, energy/substrate management, and growth factor modulation.
Intrauterine growth restriction (IUGR); liver; microarray; transcriptome; developmental origins of health and disease; obesity; circadian
The small heat shock protein Hsp27 is a molecular chaperone and an anti-apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti-apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27C137A) and comparing it to wild-type protein (Hsp27WT). Both proteins were multi-subunit oligomers, but subunits of Hsp27WT were disulfide-linked unlike those of Hsp27C137A, which were monomeric. Hsp27C137A was indistinguishable from Hsp27WT with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S-thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27WT. The anti-apoptotic function of Hsp27C137A and Hsp27WT was studied by overexpressing them in CHO cells. No difference in the caspase-3 or -9 activity was observed in staurosporine-treated cells. The rate of apoptosis between Hsp27C137A and Hsp27WT overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild-type protein in preventing caspase-3 and caspase-9 activation and apoptosis induced by 1 mM H2O2 in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti-apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti-apoptotic function against oxidative stress.
Hsp27; CHAPERONE; APOPTOSIS; CYSTEINE
We previously described a color-coded imaging model that can quantify the length of nascent blood vessels using Gelfoam® implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice. In ND-GFP mice, nascent blood vessels are labeled with GFP. We report here that osteosarcoma cells promote angiogenesis in the Gelfoam® angiogenesis assay in ND-GFP mice. Gelfoam® was initially transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation of Gelfoam®, skin flaps were made and human 143B osteosarcoma cell expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in cytoplasm was injected into the transplanted Gelfoam®. The control group mice had only implanted Gelfoam®. Skin flaps were made at days 14, 21, and 28 after transplantation of the Gelfoam® to allow imaging of vascularization in the Gelfoam® using a variable-magnification small animal imaging system and confocal fluorescence microscopy. ND-GFP expressing nascent blood vessels penetrated and spread into the Gelfoam® in a time-dependent manner in both control and osteosarcoma-implanted mice. ND-GFP expressing blood vessels in the Gelfoam® of the osteosarcoma-implanted mice were associated with the cancer cells and larger and longer than in the Gelfoam®-only implanted mice (p<0.01). The results presented in this report demonstrate the strong angiogenesis induction by osteosarcoma cells and suggest this is a potential therapeutic target for this disease.
green fluorescent protein; red fluorescent protein; osteosarcoma; angiogenesis; Gelfoam®; nestin; transgenic nude mouse; confocal microscopy
Cell–cell contacts mediated by cadherins are known to inhibit the small Rho-GTPase RhoA. We here show that in epithelial cells the disruption of these cell–cell contacts as mediated by a calcium switch leads to actin re-organization and the activation of RhoA. We identified the serine/threonine kinase protein kinase D1 (PKD1) as a downstream target for RhoA in this pathway. After disruption of cell–cell contacts, PKD1 relayed RhoA activation to the induction of the transcription factor NF-κB. We found that a signaling complex composed of the kinases ROCK, novel protein kinase C (nPKC), and Src family kinases (SFKs) is upstream of PKD1 and crucial for RhoA-mediated NF-κB activation. In conclusion, our data suggest a previously undescribed signaling pathway of how RhoA is activated by loss of cell–cell adhesions and by which it mediates the activation of NF-κB. We propose that this pathway is of relevance for epithelial tumor cell biology, where loss of cell–cell contacts has been implicated in regulating cell survival and motility.
Rho; NF-κB; PKD; Src
The 16.5 kb human mitochondrial genome encodes for 13 polypeptides, 22 tRNAs and 2 rRNAs involved in oxidative phosphorylation. Mitochondrial DNA (mtDNA), unlike its nuclear counterpart, is not packaged into nucleosomes and is more prone to the adverse effects of reactive oxygen species (ROS) generated during oxidative phosphorylation. The past few decades have witnessed an increase in the number of proteins observed to translocate to the mitochondria for the purposes of mitochondrial genome maintenance. The mtDNA damage produced by ROS, if not properly repaired, leads to instability and can ultimately manifest in mitochondrial dysfunction and disease. The base excision repair (BER) pathway is employed for the removal and consequently the repair of deaminated, oxidized, and alkylated DNA bases. Specialized enzymes called DNA glycosylases, which locate and cleave the damaged base, catalyze the first step of this highly coordinated repair pathway. This review focuses on members of the four human BER DNA glycosylase superfamilies and their subcellular localization in the mitochondria and/or the nucleus, as well as summarizes their structural features, biochemical properties, and functional role in the excision of damaged bases.
Reactive oxygen species; Base excision repair; DNA glycosylases; Mitochondrial DNA damage and response; Oxidative phosphorylation; Mitochondrial dysfunction
Phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, can modulate 1α, 25 dihydroxyvitamin D3 (1,25(OH)2D3)-induced expression of the 24-hydroxylase (CYP24A1) gene but this has not been studied in differentiated enterocytes, a primary 1,25(OH)2D3 target cell. We found that in differentiated Caco-2 cells, an established model of the mature absorptive epithelial cell, PMA significantly enhanced 1,25(OH)2D3-induced human CYP24A1 (hCYP24A1) mRNA accumulation and hCYP24A1 promoter-luciferase reporter gene activation by 150%. Reporter gene studies further identified the region between −298 to +74 bp in the hCYP24A1 promoter as critical for the PMA enhancing effect and chromatin immunoprecipitation (ChIP) analysis showed that PMA enhanced 1,25(OH)2D3-induced binding of vitamin D receptor to this region. PMA can activate PKC, ERK1/2 and p38 MAP kinases and inhibition of these signaling pathways reduced both 1,25(OH)2D3-induced hCYP24A1 gene transcription and the enhancing effect of PMA. The PMA enhancing effect on 1,25(OH)2D3 action was evident in a minimal promoter with three osteocalcin VDREs and was reduced after mutation of a putative vitamin D stimulatory site in the hCYP24A1 promoter. In contrast, mutation of a Ets binding site in the hCYP24A1 promoter had no impact on 1,25(OH)2D3 action or the PMA enhancing effect. These data suggest that in the differentiated enterocyte PMA-induced activation of several signaling pathways contribute to 1,25(OH)2D3-induced hCYP24A1 gene expression through multiple regulatory motifs within the proximal hCYP24A1 promoter.
Vitamin D; 25-hydroxyvitamin D3 24-hydroxylase; Transcription; Kinases
ARRY-334543 is a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases. We conducted this study to determine whether ARRY-334543 can enhance the efficacy of conventional anticancer drugs through interaction with ABC transporters.
Lung cancer cell line NCI-H460 and its ABCG2-overexpressing NCI-H460/MX20, as well as the ABCG2-, ABCB1-, and ABCC10-overexpressing transfected cell lines were used for the reversal study.
Our results demonstrate that ARRY-334543 (1.0 μM) significantly reversed ABCG2-mediated multidrug resistance (MDR) by directly inhibiting the drug efflux function of ABCG2, resulting in the elevated intracellular accumulation of chemotherapeutic drugs in the ABCG2-overexpressing cell lines. In addition, in isolated membranes, ARRY-334543 stimulated ATPase activity and inhibited photolabeling of ABCG2 with [125I]-iodoarylazidoprazosin in a concentration-dependent manner indicating that this drug directly interacts at the drug-binding pocket of this transporter. ARRY-334543 (1.0 μM) only slightly reversed ABCB1- and partially reversed ABCC10-mediated MDR suggesting that it exhibits high affinity towards ABCG2. Moreover, homology modeling predicted the binding conformation of ARRY-334543 at Arg482 centroid-based grid of ABCG2. However, ARRY-334543 at reversal concentration did not affect the expression level of ABCG2, AKT and ERK1/2 and regulate the re-localization of ABCG2.
We conclude that ARRY-334543 significantly reverses drug resistance mediated by ABCG2.
ARRY-334543; ABCG2; Multidrug resistance; Tyrosine kinase inhibitor; Lung cancer
Human skeletal aging is characterized as a gradual loss of bone mass due to an excess of bone resorption not balanced by new bone formation. Using human marrow cells, we tested the hypothesis that there is an age-dependent increase in osteoclastogenesis due to intrinsic changes in regulatory factors [macrophage-colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG)] and their receptors [c-fms and RANK]. In bone marrow cells (BMCs), c-fms (r=0.61, p=0.006) and RANK expression (r=0.59, p=0.008) were increased with age (27-82 years, n=19). In vitro generation of osteoclasts was increased with age (r=0.89, p=0.007). In enriched marrow stromal cells (MSCs), constitutive expression of RANKL was increased with age (r=0.41, p=0.049) and expression of OPG was inversely correlated with age (r=-0.43, p=0.039). Accordingly, there was an age-related increase in RANKL/OPG (r=0.56, p=0.005). These data indicate an age-related increase in human osteoclastogenesis that is associated with an intrinsic increase in expression of c-fms and RANK in osteoclast progenitors, and, in the supporting MSCs, an increase in pro-osteoclastogenic RANKL expression and a decrease in anti-osteoclastogenic OPG. These findings support the hypothesis that human marrow cells and their products can contribute to skeletal aging by increasing the generation of bone-resorbing osteoclasts. These findings help to explain underlying molecular mechanisms of progressive bone loss with advancing age in humans.
Aging; Osteoclast; Marrow Stromal Cell; RANKL; OPG
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. This article is protected by copyright. All rights reserved
Human pluripotent stem cells; Hemogenic endothelial cells; Hematopoietic stem cells
In addition to osteoblast lineage cells, the TNF-like factor receptor activator of NF-κB ligand (RANKL) is expressed in both B and T cells and may play a role in bone resorption. Rankl gene (Tnfsf11) expression in mouse T cells is mediated through multiple distal elements marked by increased transcription factor occupancy, histone tail acetylation, and RNA polymerase II recruitment. Little is known, however, of the regulation of human TNFSF11 in T cells. Accordingly, we examined the consequence of T cell activation on the expression of this factor both in Jurkat cells and in primary human T cells. We then explored the mechanism of this regulation by scanning over 400 kb of DNA surrounding the TNFSF11 locus for regulatory enhancers using ChIP-chip analysis. Histone H3/H4 acetylation enrichment identified putative regulatory regions located between −170 and −220 kb upstream of the human TNFSF11 TSS that we designated the human T cell control region (hTCCR). This region showed high sequence conservation with the mouse TCCR. Inhibition of MEK1/2 by U0126 resulted in decreased RANKL expression suggesting that stimulation through MEK1/2 was a prerequisite. ChIP-chip analysis also revealed that c-FOS was recruited to the hTCCR as well. Importantly, both the human TNFSF11 D5a/b (RLD5a/b) enhancer and segments of the hTCCR mediated robust inducible reporter activity following TCR activation. Finally, SNPs implicated in diseases characterized by dysregulated BMD co-localized to the hTCCR region. We conclude that the hTCCR region contains a cell-selective set of enhancers that plays an integral role in the transcriptional regulation of the TNFSF11 gene in human T cells.
ChIP-CHIP ANALYSIS; T CELL RANKL; DISTAL ENHANCERS; EPIGENETIC MARKS; C-FOS
The epithelial lining of the respiratory system originates from a small group of progenitor cells in the ventral foregut endoderm of the early embryo. Research in the last decade has revealed a number of paracrine signaling pathways that are critical for the development of these respiratory progenitors. In the post genomic era the challenge now is to figure out at the genome wide level how these different signaling pathways and their downstream transcription factors interact in a complex “gene regulatory network” (GRN) to orchestrate early lung development. In this prospective we review our growing understanding of the GRN governing lung specification. We discuss key gaps in our knowledge and describe emerging opportunities that will soon provide an unprecedented understanding of lung development and accelerate our ability to apply this knowledge to regenerative medicine.
lung development; gene regulatory network; Nkx2.1; respiratory epithelium; specification
Active glutamine utilization is critical for tumor cell proliferation. Glutaminolysis represents the first and rate-limiting step of glutamine utilization and is catalyzed by glutaminase (GLS). Activation of ErbB2 is one of the major causes of breast cancers, the second most common cause of death for women in many countries. However, it remains unclear whether ErbB2 signaling affects glutaminase expression in breast cancer cells. In this study, we show that MCF10A-NeuT cell line has higher GLS1 expression at both mRNA and protein levels than its parental line MCF10A, and knockdown of ErbB2 decreases GLS1 expression in MCF10A-NeuT cells. We further show that in these cells, ErbB2-mediated upregulation of GLS1 is not correlated to c-Myc expression. Moreover, activation of neither PI3K-Akt nor MAPK pathway is sufficient to upregulate GLS1 expression. Interestingly, inhibition of NF-κB blocks ErbB2-stimulated GLS1 expression, whereas stimulation of NF-κB is sufficient to enhance GLS1 levels in MCF10A cells, suggesting a PI3K-Akt-independent activation of NF-κB upregulates GLS1 in ErbB2-positive breast cancer cells. Finally, knockdown or inhibition of GLS1 significantly decreased the proliferation of breast cancer cells with high GLS1 levels. Taken together, our data indicate that ErbB2 activation promotes GLS1 expression via a PI3K-Akt-independent NF-κB pathway in breast cancer cells, identifying another oncogenic signaling pathway which stimulates GLS1 expression, and thus promoting glutamine utilization in cancer cells. These findings, if validated by in vivo model, may facilitate the identification of novel biochemical targets for cancer prevention and therapy.
breast cancer; ErbB2; glutaminase; glutamine; GLS1; nuclear factor κ B
In mammals, formation of new nephrons ends perinatally due to consumption of mesenchymal progenitor cells. Premature depletion of progenitors due to prematurity or postnatal loss of nephrons due to injury causes chronic kidney disease and hypertension. Intensive efforts are currently invested in designing regenerative strategies to form new nephron progenitors from pluripotent cells, which upon further differentiation provide a potential source of new nephrons. To know if reprogramed renal cells can maintain their identity and fate requires knowledge of the epigenetic states of native nephron progenitors and their progeny. In this article, we summarize current knowledge and gaps in the epigenomic landscape of the developing kidney. We now know that Pax2/PTIP/H3K4 methyltransferase activity provides the initial epigenetic specification signal to the metanephric mesenchyme. During nephrogenesis, the cap mesenchyme housing nephron progenitors is enriched in bivalent chromatin marks; as tubulogenesis proceeds, the tubular epithelium acquires H3K79me2. The latter mark is uniquely induced during epithelial differentiation. Analysis of histone landscapes in clonal metanephric mesenchyme cell lines and in Wilms tumor and normal fetal kidney has revealed that promoters of poised nephrogenesis genes carry bivalent histone signatures in progenitors. Differentiation or stimulation of Wnt signaling promotes resolution of bivalency; this does not occur in Wilms tumor cells consistent with their developmental arrest. The use of small cell number ChIP- Seq should facilitate the characterization of the chromatin landscape of the metanephric mesenchyme and various nephron compartments during nephrogenesis. Only then we will know if stem and somatic cell reprogramming into kidney progenitors recapitulates normal development.
HIV-1 gene transcription is controlled by the cooperation of viral and host factors which bind to specific DNA sequences within the viral promoter spanning the long terminal repeat, LTR. Previously we showed that the St. John's Wort DING phosphatase, p27SJ, suppresses HIV-1 gene transcription by binding to the viral protein Tat and preventing its nuclear import. Here, we describe the inhibitory effect of p27SJ on the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). This inhibition leads to the suppression of the association of RNAPII with the LTR. Inhibition of binding of RNAPII to LTR by p27SJ resulted in the suppression of LTR transcription elongation and a decrease in LTR transcriptional activity. Another form of the St. John's Wort DING phosphatase, p38SJ, also suppressed binding of RNAPII to the LTR, reduced transcription elongation and was even more powerful than p27SJ in inhibiting the transcriptional activity of the LTR. Our data suggest a possible mechanism by which the p27SJ/p38SJ DING phosphatase can regulate HIV-1 LTR expression by inhibiting phosphorylation of the CTD of RNAPII and suppressing LTR transcription elongation.
p27SJ; DING proteins; RNA Polymerase II CTD; HIV-I
The Bcl-2-associated athanogene, BAG, protein family through their BAG domain associates with the heat shock protein 70 (HSP-70) and modulates its chaperone activity. One member of this family, BAG3, appears to play an important role in protein homeostasis, as its expression promotes cell survival by preventing polyubiquitination of Hsp-70 client proteins. Expression of BAG3 is enhanced by a variety of stress-inducing agents. Here we describe a role for BAG3 to modulate transcription of its own promoter through a positive feedback loop involving its 5′-UTR sequence. Activation of the BAG3 promoter is mediated by the BAG domain and is independent of BAG3 association with the UTR sequence. Autoactivation of the BAG3 gene is observed in several cultures of human glial cells including gliomas, but not in several other non-glial cell lines such as He La and others. Results from cell fractionation and immunocytochemistry showed BAG3 in the cytoplasm as well as the nuclei of glial cells. These observations suggest that BAG3 gene expression is controlled by its own product and that this may be critical for the biological activity of BAG3 in some cell types.
BAG3; 5′-UTR; positive feedback; transcription regulation; Bcl-2 regulatory protein
Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFβ1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells.
Perlecan/HSPG2; Prostate Cancer; TNF-α; TGFβ; Cytokines; Gene Expression
Eph receptor (Eph)-ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4– ephrinB2 regulates cardiovascular development. To assess the role of EphB4–ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5+ cardiac progenitor cells and in α-MHC+ cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5-GFP+ cardiac progenitor cells, but not in α-MHC-GFP+ cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL-RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4–ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full-length EphB4 or intracellular domain-truncated EphB4 in EphB4-null ES cells was established to investigate the role of EphB4-forward signaling in ES cells. Interestingly, while full-length EphB4 was able to restore the cardiac lineage development in EphB4-null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor-kinase-dependent EphB4-forward signaling plays a crucial role in the development of cardiac progenitor cells.
EMBRYONIC STEM (ES) CELLS; CARDIOMYOCYTES; EphB4; ephrinB2; CARDIAC PROGENITOR CELLS; Nkx 2.5; α-MHC
WNT signaling pathways play important roles in the behavior of human bone marrow stromal cells. Although WNT expression has been examined in human bone marrow stromal cells (hMSCs) with limited numbers of subjects or from commercial sources, there are conflicting results on WNT gene expression in hMSCs. Furthermore, the effects of age and gender on WNT expression in hMSCs are largely unknown. In this study, we evaluated RNA expression of all the WNT genes in hMSCs from 19 subjects, 12 women and 7 men, aged from 36 to 85 years. Analysis of WNT gene expression in young and old groups indicated that WNT7B and 14 were expressed significantly higher in the young group. WNT2 and WNT13 showed a trend of higher expression in young group. WNT7B, 13, and 14 were inversely correlated with age. Further analysis for gender-specific difference indicated that WNT16 was expressed significantly higer in men than in women. WNT11 showed a trend of higher expression in hMSCs from women. For the hMSCs from women, WNT13 was inversely correlated with age and WNT4 was positively correlated with age. For the hMSCs from men, WNT7B and WNT14 were inversely correlated with age. These data indicated that most of the age-related WNT genes belong to the canonical WNT signaling pathway. Further, there are gender-specific differences in the expression of WNT4, 7B, 13, 14, and 16 in hMSCs. Age and gender account for many of the sample-to-sample variations in WNT gene expression in human marrow stromal cells.
WNT; human marrow stromal cells; age; gender; expression profiling
Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of arterial atherosclerosis and venous neointimal hyperplasia. We examined the effects of PDGF isoforms on smooth muscle cells (SMCs) from arterial and venous origins in order to further understand the differential responsiveness of these vasculatures to proliferative stimuli. Serum-starved human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Whereas, proliferation of arterial SMCs was strongly stimulated by PDGF-AA, venous SMCs showed no proliferative response to PDGF-AA, but instead demonstrated a significantly greater proliferative response to PDGF-BB than arterial SMCs. Part of this difference could be attributed to differences in PDGF receptors expression. There was a 2.5-fold higher (P <0.05) density of PDGF receptor-α (PDGF-Rα) and a 6.6-fold lower (P <0.05) density of PDGF-Rβ expressed on arterial compared to venous SMCs. Concomitant with an increased proliferative response to PDGF-AA in arterial SMCs was a marked PDGF-Rα activation, enhanced phosphorylation of ERK1/2 and Akt, a transient activation of c-Jun NH2-terminal kinase (JNK), and a significant reduction in expression of the cell-cycle inhibitor p27kip1. This pattern of signaling pathway changes was not observed in venous SMCs. No phosphorylation of PDGF-Rα was detected after venous SMC exposure to PDGF-AA, but there was enhanced phosphorylation of ERK1/2 and Akt in venous SMCs, similar to that seen in the arterial SMCs. PDGF-BB stimulation of venous SMC resulted in PDGF-Rβ activation as well as transactivation of epidermal growth factor receptor (EGF-R); transactivation of EGF-R was not observed in arterial SMCs. These results may provide an explanation for the differential susceptibility to proliferative vascular diseases of arteries and veins.
PLATELET-DERIVED GROWTH FACTOR (PDGF); SIGNALING PATHWAYS; CELL PROLIFERATION; ARTERIAL AND VENOUS SMC