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.     

Image analysis and in vivo imaging as tools for investigation of productivity dynamics in anthocyanin-producing cell cultures of Daucus carota

An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level. An approach was set up in which growth and production of anthocyanins were investigated using a combination of biochemical analysis, image (colour) analysis and in vivo imaging. This novel approach was used to segment the culture in different subpopulations and dissect the productive process in the cell culture grown under two different conditions, known to differ mainly for oxygen supply and mixing intensity (volume of 50 ml or 20 ml in 250 ml flasks). The 20 ml batch cultures gave a higher content and yield of anthocyanins, which depended on a complex balance between events that positively or negatively affected anthocyanin production. A model is proposed in which the different ability of cells to respond to environmental stimuli and stress depends on the different amount of anthocyanins accumulated within cells.     

Dynamics in the cyclic Enterobacterial common antigen as studied by <Superscript>13</Superscript>C NMR relaxation

The motional properties of the cyclic enterobacterial common antigen (cECA), consisting of four trisaccharide repeat units, have been investigated by carbon-13 spin relaxation. R₁, R₂ and NOE relaxation parameters have been determined at three magnetic field strengths. The data were interpreted within the model-free framework to include the possibility of motional anisotropy, and overall as well as local dynamical parameters were fitted separately for each ring carbon. The motional anisotropy was addressed by assuming an axially symmetric diffusion tensor, which was fitted from the overall correlation times for each site in the sugar residues using the previously determined crystal structure. The data were found to be in agreement with an oblate shape of the molecule, and the values for D_iso and were in good agreement with translational diffusion data and an estimate based on calculation of the moment of inertia tensor, respectively. The local dynamics in cECA were found to be residue-dependent. Somewhat lower values for the order parameters, as well as longer local correlation times, were observed for the -linked ManNAcA residue compared to the two -linked residues in the trisaccharide repeat unit.     

The kinetics of PDZ domain-ligand interactions and implications for the binding mechanism

PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A + B --> A.B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex.     

Steady-state and transient kinetic analyses of taurine/alpha-ketoglutarate dioxygenase: effects of oxygen concentration, alternative sulfonates, and active-site variants on the FeIV-oxo intermediate

Taurine/alpha-ketoglutarate (alphaKG) dioxygenase (TauD), an archetype alphaKG-dependent hydroxylase, is a non-heme mononuclear Fe(II) enzyme that couples the oxidative decarboxylation of alphaKG with the conversion of taurine to aminoacetaldehyde and sulfite. The crystal structure of taurine-alphaKG-Fe(II)TauD is known, and spectroscopic studies have kinetically defined the early steps in catalysis and identified a high-spin Fe(IV)-oxo reaction intermediate. The present analysis extends our understanding of TauD catalysis by investigating the steady-state and transient kinetics of wild-type and variant forms of the enzyme with taurine and alternative sulfonates. TauD proteins substituted at residues surrounding the active site were shown to fold properly based on their abilities to form a diagnostic chromophore associated with the anaerobic Fe(II)-alphaKG chelate complex and to generate a tyrosyl radical upon subsequent reaction with oxygen. Steady-state studies of mutant proteins confirmed the importance of His 70 and Arg 270 in binding the sulfonate moiety of taurine and indicated the participation of Asn 95 in recognizing the substrate amine group. The N97A and S158A variants are likely to undergo an increase in hydrophobicity and expansion of the substrate-binding pocket, thus accounting for their decreased K(m) toward pentanesulfonic acid compared to wild-type TauD. Stopped-flow UV-visible spectroscopic examination of the reaction of oxygen with taurine-alphaKG-Fe(II)TauD confirmed a minimal three-step sequence of reactions attributed to Fe(IV)-oxo formation (k(1)), bleaching to the Fe(II) state upon substrate hydroxylation (k(2)), rebinding of excess substrates (k(3)), and indicated that none of the steps exhibit detectable solvent k(H)/k(D) isotope effects. This demonstrates that no protons are involved in the rate-determining step of Fe(IV)-oxo formation, in contrast to heme iron oxygenases. The Fe(IV)-oxo species is likely to be utilized in conversion of the alternative substrates pentanesulfonic acid and 3-N-morpholinopropanesulfonic acid; however, this spectroscopic intermediate was not detected because of the decreased k(1)/k(2) ratio. With taurine, k(1) was shown to depend on the oxygen concentration allowing calculation of a second-order rate constant of 1.58 x 10(5) M(-)(1) s(-)(1) for this irreversible reaction. Stopped-flow analyses of TauD variants provided several insights into how the protein environment influences the rates of Fe(IV)-oxo formation and decay. The Fe(IV)-oxo species was not detected in the N95D or N95A variants because of a reduced k(1)/k(2) ratio, likely related to a decreased substrate-dependent conversion of the six-coordinate to five-coordinate metal site.     

Protein kinase C-related kinase targets nuclear localization signals in a subset of class IIa histone deacetylases

Class IIa histone deacetylases (HDACs) -4, -5, -7 and -9 undergo signal-dependent nuclear export upon phosphorylation of conserved serine residues that are targets for 14-3-3 binding. Little is known of other mechanisms for regulating the subcellular distribution of class IIa HDACs. Using a biochemical purification strategy, we identified protein kinase C-related kinase-2 (PRK2) as an HDAC5-interacting protein. PRK2 and the related kinase, PRK1, phosphorylate HDAC5 at a threonine residue (Thr-292) positioned within the nuclear localization signal (NLS) of the protein. HDAC7 and HDAC9 contain analogous sites that are phosphorylated by PRK, while HDAC4 harbors a non-phosphorylatable alanine residue at this position. We provide evidence to suggest that the unique phospho-acceptor cooperates with the 14-3-3 target sites to impair HDAC nuclear import.

Structured summary

MINT-7710106:HDAC5 (uniprotkb:Q9UQL6) physically interacts (MI:0915) with PRK2 (uniprotkb:Q16513) by pull down (MI:0096)     

Nerve growth factor and its precursor differentially regulate hair cycle progression in mice.

Nerve growth factor (NGF) promotes proliferation via its high affinity receptor (TrkA). Its precursor proNGF promotes apoptosis via the pan-neurotrophin-receptor p75. Recently, we have identified NGF and p75 as important hair growth terminators. However, if proNGF is involved or if NGF can also promote hair growth via TrkA is unclear. By RT-PCR we found that NGF/proNGF mRNA levels peak during early anagen in murine back skin, whereas NGF/proNGF protein levels peak during catagen, indicating high turnover in early anagen and protein accumulation in catagen. By immunohistochemistry, NGF and TrkA are found in the proliferating compartments of the epidermis and hair follicle throughout the cycle. In contrast, strong proNGF is found in the highly differentiated inner root sheath and adjacent to the p75+ regressing epithelial strand in catagen. Commercial 7S NGF, which contains both NGF and proNGF, promotes anagen development in organ-cultured early anagen mouse skin, whereas it promotes catagen development in late anagen skin. Together, our findings suggest an anagen-promoting or anagen-supporting role for NGF/TrkA, and a catagen-promoting role for proNGF/p75 interactions. This has important implications for the future design of specific neurotrophin receptor ligands as novel pharmaceuticals in the modification of tissue remodeling processes such as hair growth or wound healing.     

Identification and analysis of differentially expressed cDNAs during nonself-competitive interaction between Phlebiopsis gigantea and Heterobasidion parviporum

The molecular factors regulating interspecific interaction between the saprotrophic biocontrol fungus Phlebiopsis gigantea and the conifer pathogen Heterobasidion parviporum were investigated. We constructed cDNA libraries and used expressed sequence tag analysis for the identification and characterization of genes expressed during the self and nonself-hyphal interaction. cDNA clones from either the pathogen or biocontrol agent were arrayed on nylon membrane filters and differentially screened with cDNA probes made from mycelia forming the barrage zone during nonself-interactions, mycelia growing outside the barrage zones or monocultures. BlastX analysis of the differentially expressed clones led to the identification of genes with diverse functions, including those with potential as virulence factors, such as hydrophobins. Because of the high sequence conservation (r2 = 0.81) between P. gigantea and H. parviporum, a selected number of genes from either fungus were used to monitor the expression profile under varying interaction conditions by virtual northern blot. The results are discussed with respect to the potential role of the induced genes during the nonself-competitive interaction for space and nutrients between P. gigantea and H. parviporum.