Silencing BRE expression in human umbilical cord perivascular (HUCPV) progenitor cells accelerates osteogenic and chondrogenic differentiation.

BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing BRE expression, using BRE-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of BRE-silenced cells. BRE-silencing negatively regulated OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and components of the TGF-ß/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that BRE-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.

Promyelocytic leukemia (PML) protein plays important roles in regulating cell adhesion, morphology, proliferation and migration.

PML protein plays important roles in regulating cellular homeostasis. It forms PML nuclear bodies (PML-NBs) that act like nuclear relay stations and participate in many cellular functions. In this study, we have examined the proteome of mouse embryonic fibroblasts (MEFs) derived from normal (PML(+/+)) and PML knockout (PML(-/-)) mice. The aim was to identify proteins that were differentially expressed when MEFs were incapable of producing PML. Using comparative proteomics, total protein were extracted from PML(-/-) and PML(+/+) MEFs, resolved by two dimensional electrophoresis (2-DE) gels and the differentially expressed proteins identified by LC-ESI-MS/MS. Nine proteins (PML, NDRG1, CACYBP, CFL1, RSU1, TRIO, CTRO, ANXA4 and UBE2M) were determined to be down-regulated in PML(-/-) MEFs. In contrast, ten proteins (CIAPIN1, FAM50A, SUMO2 HSPB1 NSFL1C, PCBP2, YWHAG, STMN1, TPD52L2 and PDAP1) were found up-regulated. Many of these differentially expressed proteins play crucial roles in cell adhesion, migration, morphology and cytokinesis. The protein profiles explain why PML(-/-) and PML(+/+) MEFs were morphologically different. In addition, we demonstrated PML(-/-) MEFs were less adhesive, proliferated more extensively and migrated significantly slower than PML(+/+) MEFs. NDRG1, a protein that was down-regulated in PML(-/-) MEFs, was selected for further investigation. We determined that silencing NDRG1expression in PML(+/+) MEFs increased cell proliferation and inhibited PML expression. Since NDRG expression was suppressed in PML(-/-) MEFs, this may explain why these cells proliferate more extensively than PML(+/+) MEFs. Furthermore, silencing NDRG1expression also impaired TGF-ß1 signaling by inhibiting SMAD3 phosphorylation.

Cardiogenol C can induce Mouse Hair Bulge Progenitor Cells to Transdifferentiate into Cardiomyocyte-like Cells.

BACKGROUND: Hair bulge progenitor cells (HBPCs) are multipotent stem cells derived from the bulge region of mice vibrissal hairs. The purified HBPCs express CD34, K15 and K14 surface markers. It has been reported that HBPCs could be readily induced to transdifferentiate into adipocytes and osteocytes. However, the ability of HBPCs to transdifferentiate into cardiomyocytes has not yet been investigated. METHODOLOGY/PRINCIPAL FINDINGS: The cardiomyogenic potential of HBPCs was investigated using a small cell-permeable molecule called Cardiogenol C. We established that Cardiogenol C could induce HBPCs to express transcription factors GATA4, Nkx2.5 and Tbx5, which are early specific markers for pre-cardiomyogenic cells. In prolonged cultures, the Cardiogenol C-treated HBPCs can also express muscle proteins, cardiac-specific troponin I and sarcomeric myosin heavy chain. However, we did not observe the ability of these cells to functionally contract. Hence, we called these cells cardiomyocyte-like cells rather than cardiomyocytes. We tried to remedy this deficiency by pre-treating HBPCs with Valproic acid first before exposing them to Cardiogenol C. This pretreatment inhibited, rather than improved, the effectiveness of Cardiogenol C in reprogramming the HBPCs. We used comparative proteomics to determine how Cardiogenol C worked by identifying proteins that were differentially expressed. We identified proteins that were involved in promoting cell differentiation, cardiomyocyte development and for the normal function of striated muscles. From those differentially expressed proteins, we further propose that Cardiogenol C might exert its effect by activating the Wnt signaling pathway through the suppression of Kremen1. In addition, by up-regulating the expression of chromatin remodeling proteins, SIK1 and Smarce1 would initiate cardiac differentiation. CONCLUSIONS/SIGNIFICANCE: In conclusion, our CD34+/K15+ HBPCs could be induced to transdifferentiate into cardiomyocyte-like cells using a small molecule called Cardiogenol C. The process involves activation of the Wnt signaling pathway and altered expression of several key chromatin remodeling proteins. The finding is clinically significant as HBPCs offer a readily accessible and autologous source of progenitor cells for cell-based therapy of heart disease, which is one of major killers in developed countries.

A bifunctional locus (BIO3-BIO1) required for biotin biosynthesis in Arabidopsis.

We identify here the Arabidopsis (Arabidopsis thaliana) gene encoding the third enzyme in the biotin biosynthetic pathway, dethiobiotin synthetase (BIO3; At5g57600). This gene is positioned immediately upstream of BIO1, which is known to be associated with the second reaction in the pathway. Reverse genetic analysis demonstrates that bio3 insertion mutants have a similar phenotype to the bio1 and bio2 auxotrophs identified using forward genetic screens for arrested embryos rescued on enriched nutrient medium. Unexpectedly, bio3 and bio1 mutants define a single genetic complementation group. Reverse transcription-polymerase chain reaction analysis demonstrates that separate BIO3 and BIO1 transcripts and two different types of chimeric BIO3-BIO1 transcripts are produced. Consistent with genetic data, one of the fused transcripts is monocistronic and encodes a bifunctional fusion protein. A splice variant is bicistronic, with distinct but overlapping reading frames. The dual functionality of the monocistronic transcript was confirmed by complementing the orthologous auxotrophs of Escherichia coli (bioD and bioA). BIO3-BIO1 transcripts from other plants provide further evidence for differential splicing, existence of a fusion protein, and localization of both enzymatic reactions to mitochondria. In contrast to most biosynthetic enzymes in eukaryotes, which are encoded by genes dispersed throughout the genome, biotin biosynthesis in Arabidopsis provides an intriguing example of a bifunctional locus that catalyzes two sequential reactions in the same metabolic pathway. This complex locus exhibits several unusual features that distinguish it from biotin operons in bacteria and from other genes known to encode bifunctional enzymes in plants.