Functional Profiling Reveals Critical Role for miRNA in Differentiation of Human Mesenchymal Stem Cells

BACKGROUND:Mesenchymal stem (MS) cells are excellent candidates for cell-based therapeutic strategies to regenerate injured tissue. Although human MS cells can be isolated from bone marrow and directed to differentiate by means of an osteogenic pathway, the regulation of cell-fate determination is not well understood. Recent reports identify critical roles for microRNAs (miRNAs), regulators of gene expression either by inhibiting the translation or by stimulating the degradation of target mRNAs. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we employed a library of miRNA inhibitors to evaluate the role of miRNAs in early osteogenic differentiation of human MS cells. We discovered that miR-148b, -27a and -489 are essential for the regulation of osteogenesis: miR-27a and miR-489 down-regulate while miR-148b up-regulates differentiation. Modulation of these miRNAs induced osteogenesis in the absence of other external differentiation cues and restored osteogenic potential in high passage number human MS cells. CONCLUSIONS/SIGNIFICANCE:Overall, we have demonstrated the utility of the functional profiling strategy for unraveling complex miRNA pathways. Our findings indicate that miRNAs regulate early osteogenic differentiation in human MS cells: miR-148b, -27a, and -489 were found to play a critical role in osteogenesis

Geophysical signatures of past and present hydration within a young oceanic core complex

Borehole logging at the Atlantis Massif oceanic core complex provides new information on the relationship between the physical properties and the lithospheric hydration of a slow-spread intrusive crustal section. Integrated Ocean Drilling Program Hole U1309D penetrates 1.4 km into the footwall to an exposed detachment fault on the 1.2 Ma flank of the mid-Atlantic Ridge, 30°N. Downhole variations in seismic velocity and resistivity show a strong correspondence to the degree of alteration, a recorder of past seawater circulation. Average velocity and resistivity are lower, and alteration is more pervasive above a fault around 750 m. Deeper, these properties have higher values except in heavily altered ultramafic zones that are several tens of meters thick. Present circulation inferred from temperature mimics this pattern: advective cooling persists above 750 m, but below, conductive cooling dominates except for small excursions within the ultramafic zones. These alteration-related physical property signatures are probably a characteristic of gabbroic cores at oceanic core complexes

Kinome-Wide Functional Genomics Screen Reveals a Novel Mechanism of TNFα-Induced Nuclear Accumulation of the HIF-1α Transcription Factor in Cancer Cells

Hypoxia-inducible factor-1 (HIF-1) and its most important subunit, HIF-1α, plays a central role in tumor progression by regulating genes involved in cancer cell survival, proliferation and metastasis. HIF-1α activity is associated with nuclear accumulation of the transcription factor and regulated by several mechanisms including modulation of protein stability and degradation. Among recent advances are the discoveries that inflammation-induced cytokines and growth factors affect protein accumulation of HIF-1α under normoxia conditions. TNFα, a major pro-inflammatory cytokine that promotes tumorigenesis is known as a stimulator of HIF-1α activity. To improve our understanding of TNFα-mediated regulation of HIF-1α nuclear accumulation we screened a kinase-specific siRNA library using a cell imaging–based HIF-1α-eGFP chimera reporter assay. Interestingly, this systematic analysis determined that depletion of kinases involved in conventional TNFα signaling (IKK/NFκB and JNK pathways) has no detrimental effect on HIF-1α accumulation. On the other hand, depletion of PRKAR2B, ADCK2, TRPM7, and TRIB2 significantly decreases the effect of TNFα on HIF-1α stability in osteosarcoma and prostate cancer cell lines. These newly discovered regulators conveyed their activity through a non-conventional RELB-depended NFκB signaling pathway and regulation of superoxide activity. Taken together our data allow us to conclude that TNFα uses a distinct and complex signaling mechanism to induce accumulation of HIF-1α in cancer cells. In summary, our results illuminate a novel mechanism through which cancer initiation and progression may be promoted by inflammatory cytokines, highlighting new potential avenues for fighting this disease

TNFα-induced nuclear accumulation of the HIF-1α transcription factor.

<p>(A) TNFα induced nuclear accumulation of HIF-1α in LNCaP, DU145, MCF10a and MDA MB231 cancer cell lines. Expression and nuclear accumulation of HIF-1α was determined by immunocytochemistry imaging as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031270#s4" target="_blank">Materials and Methods</a>. (B) TNFα-induced stimulation of HIF-1α-eGFP nuclear accumulation in U2OS osteosarcoma in a concentration dependent manner. (C) TNFα stimulated HIF-1α-eGFP nuclear accumulation in U2OS cells in a time-dependent fashion. All data (Median+/−MAD) normalized to untreated cells. For each panel, data are representative of two independent experiments performed in triplicate *, **: Student's t-test p-value between treated cells and corresponding control group, * - p<0.01, ** - p<0.05.</p

RNAi Screening to identify regulators of TNFα-induced accumulation of HIF-1α.

<p>(A) Representative data set from a kinome-wide screen of 788 siRNAs. Data (Median+/−MAD) are representative of three individual transfections and are normalized to control siRNA. (B) Selected siRNA hit candidates were tested in separate experiments. siRNAs targeting <i>ADCK2, RIOK2, PRKAR2B, TRIB2, TRIO and TRPM7</i> were confirmed as hit candidates and subjected to further analysis. All data normalized to cells transfected with control siRNA NTC1. (C) Effect of selected siRNA hit candidates on HIF-1α accumulation in LNCaP prostate cancer cell line. Data (Median+/−MAD) are representative of three independent experiments performed in triplicate. All data normalized to TNFα-treated cells. *, **: Student's t-test p-value between treated cells and corresponding control group, * - p<0.01, ** - p<0.05.</p

Validation of siRNAs targeting selected hit candidates.

<p>(A) Selected siRNAs decrease TNFα-mediated HIF-1α accumulation in a concentration dependent maner. (B) siRNA pools and individual siRNA molecules produced comparable effects on HIF-1α accumulation. (C) siRNA pools and individual siRNA molecules for the selected hit candidates provide effective target gene depletion. U2OS cells were incubated with TNFα for 24 hr. All data normalized to cells transfected with control siRNA NTC1. Data are representative of two independent experiments, three (A,B,Median+/−MAD) or two (C, Mean+/−STDEV) individual transfections each.</p

TNFα-induced regulation of HIF-1α is governed by intracellular superoxide accumulation.

<p>(A) While addition of hydrogen peroxide (100 µM, 3 hr incubation) can stimulate HIF-1α accumulation, it diminishes such accumulation in TNFα-treated cells (21 hr incubation with TNFα only followed by 3 hr incubation in the presence of both TNFα and hydrogen peroxide). Data (Median+/−MAD) normalized to cells transfected with control siRNA NTC1 and treated with DMSO. (B) Superoxide scavengers rescue HIF-1α accumulation in cells transfected with siRNAs targeting <i>ADCK2</i> and <i>TRIB2</i> but not <i>PRKAR2B</i> or <i>TRPM7</i>. Cells were treated with TNFα similar to (A) and incubated with Tiron (0.3 mM), 4-hydroxy-TEMPO (0.1 mM) or DMSO (vehicle control) for 3 hr. Data (Median+/−MAD) normalized to cells transfected with control siRNA NTC1 and treated with DMSO. For each panel data are representative of two independent experiments performed in quadruplicate. *, **: Student's t-test p-value between treated cells and corresponding control group, * - p<0.01, ** - p<0.05.</p

TNFα induces accumulation of HIF-1α through a non-conventional NFκB-dependent pathway.

<p>(A) Depletion of NFκB1, RELB and cREL but not NFκB2 and RELA interfere with HIF-1α accumulation in TNFα-treated cells. (B) Depletion of ADCK2 and PRKAR2B prevents accumulation of RELB in the nuclei of TNFα-treated cells. (C) Effect of depletion of RELB is rescued by ROS scavengers. Cells were treated similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031270#pone-0031270-g004" target="_blank">Fig. 4C</a>. (D) All data (Median+/−MAD) is normalized to cells transfected with control siRNA NTC1 treated with TNFα (10 ng/ml, 24 hr). The data are representative of two independent experiments performed in triplicate. *, **: Student's t-test p-value between treated cells and corresponding control group, * - p<0.01, ** - p<0.05.</p

Evaluating the Utility of Bi-functional Degrader Molecules for Selective Inhibition of PDE4 In Acrodysostosis Type2

Background: Acrodysostosis Type 2 (ACRDYS2) is a rare autosomal dominant skeletal dysplasia associated with intellectual disability and gain-of-function mutations in the phosphodiesterase type 4D gene (PDE4D) which, in turn, leads to a paucity of intracellular cAMP due to increased PDE4D activity. This increased PDE4 activity may be due to a greater existence of a mutant monomeric form of PDE4D. To date, the clinical use of PDE4 inhibitors in ACRDYS2 has been hampered by the possibility of off-target effects as there are 25 different PDE4 isoforms in humans. Aim: To explore the utility of novel bi-functional PDE4 degrader compounds in ACRDYS2. Methods: As PDE4 inhibitors act on all PDE4 isoforms as their catalytic sites are almost identical, a novel PDE4 bi-functional degrader was created by targeting the E3 ligase Cereblon to PDE4. To ensure selectivity, a PDE4 inhibitor was used to deliver the PROTAC complex and the ability of this complex to silence wild type and ACRDYS2 mutants of PDE4 isoforms was studied in over-expression studies using transiently transfected human embryonic kidney cells (HEK). Results: The bi-functional degraders did not affect over-expressed, dimerised PDE4 longforms as they are sterically hindered from reaching their docking site within the PDE4 structure, however they degraded short-forms and mutated long-forms that cannot dimerise. The PDE4 degraders were effective at 10nM and worked well after 6 hours. Many of the ACRDYS2 mutations occur in the regions of the PDE4D enzyme that act as protein-protein interaction docking domains, which are vital for dimer formation. We have shown that our PDE4 degraders can target certain Acrodysostosis PDE4D mutants (e.g. T577A, PDE4D4), showing significant reductions in PDE4D protein expression via enhanced degradation. This degradation is channelled via the ubiquitin-proteosome system as it can be reversed using a proteosome inhibitor (Bortezomib). PDE4 proteolysis was induced by low nanomolar concentrations of the PDE4 degraders and was long lasting (no compensatory increases in PDE4 protein expression observed 96 hours after removal of degraders). Conclusion: Our innovative approach represents an exciting novel therapeutic strategy for ACRDYS2 and possibly other cAMP-related developmental conditions