Janine N. Post

Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction

The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.

Synthesis and bioconjugation of gold nanoparticles as potential molecular probes for light-based imaging techniques

We have synthesized and characterized gold nanoparticles (spheres and rods) with optical extinction bands within the “optical imaging window.” The intense plasmon resonant driven absorption and scattering peaks of these nanoparticles make them suitable as contrast agents for optical imaging techniques. Further, we have conjugated these gold nanoparticles to a mouse monoclonal antibody specific to HER2 overexpressing SKBR3 breast carcinoma cells. The bioconjugation protocol uses noncovalent modes of binding based on a combination of electrostatic and hydrophobic interactions of the antibody and the gold surface. We discuss various aspects of the synthesis and bioconjugation protocols and the characterization results of the functionalized nanoparticles. Some proposed applications of these potential molecular probes in the field of biomedical imaging are also discussed.

ERK nuclear translocation is dimerization-independent but controlled by the rate of phosphorylation

Upon activation, ERKs translocate from the cytoplasm to the nucleus. This process is required for the induction of many cellular responses, yet the molecular mechanisms that regulate ERK nuclear translocation are not fully understood. We have used a mouse embryo fibroblast ERK1-knock-out cell line expressing green fluorescent protein (GFP)-tagged ERK1 to probe the spatio-temporal regulation of ERK1. Real time fluorescence microscopy and fluorescence correlation spectroscopy revealed that ERK1 nuclear accumulation increased upon serum stimulation, but the mobility of the protein in the nucleus and cytoplasm remained unchanged. Dimerization of ERK has been proposed as a requirement for nuclear translocation. However, ERK1-Δ4, the mutant shown consistently to be dimerization-deficient in vitro, accumulated in the nucleus to the same level as wild type (WT), indicating that dimerization of ERK1 is not required for nuclear entry and retention. Consistent with this finding, energy migration Förster resonance energy transfer and fluorescence correlation spectroscopy measurements in living cells did not detect dimerization of GFP-ERK1-WT upon activation. In contrast, the kinetics of nuclear accumulation and phosphorylation of GFP-ERK1-Δ4 were slower than that of GFP-ERK1-WT. These results indicate that the differential shuttling behavior of the mutant is a consequence of delayed phosphorylation of ERK by MEK rather than dimerization. Our data demonstrate for the first time that a delay in cytoplasmic activation of ERK is directly translated into a delay in nuclear translocation.

One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD‐paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single‐photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two‐photon fs pulses in the range of 780–840 nm. Viability in whole‐mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two‐photon activation of paGFP and the use of a paGFP‐fusion protein in investigations of whole organisms.

Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate

Significance Multipotent cells, such as mesenchymal stromal cells (MSCs), have the capacity to differentiate into cartilage-forming cells. Chondrocytes derived from MSCs obtain an epiphyseal cartilage-like phenotype, which turns into bone upon implantation via endochondral ossification. Here, we report that the chondrogenic fate of MSCs can be metabolically programmed by low oxygen tension to acquire an articular chondrocyte-like phenotype via mechanisms that resemble natural development. Our study identifies metabolic programming of stem cells by oxygen tension as a powerful tool to control cell fate, which may have broad applications for the way in which stem cells are now prepared for clinical use. Actively steering the chondrogenic differentiation of mesenchymal stromal cells (MSCs) into either permanent cartilage or hypertrophic cartilage destined to be replaced by bone has not yet been possible. During limb development, the developing long bone is exposed to a concentration gradient of oxygen, with lower oxygen tension in the region destined to become articular cartilage and higher oxygen tension in transient hypertrophic cartilage. Here, we prove that metabolic programming of MSCs by oxygen tension directs chondrogenesis into either permanent or transient hyaline cartilage. Human MSCs chondrogenically differentiated in vitro under hypoxia (2.5% O2) produced more hyaline cartilage, which expressed typical articular cartilage biomarkers, including established inhibitors of hypertrophic differentiation. In contrast, normoxia (21% O2) prevented the expression of these inhibitors and was associated with increased hypertrophic differentiation. Interestingly, gene network analysis revealed that oxygen tension resulted in metabolic programming of the MSCs directing chondrogenesis into articular- or epiphyseal cartilage-like tissue. This differentiation program resembled the embryological development of these distinct types of hyaline cartilage. Remarkably, the distinct cartilage phenotypes were preserved upon implantation in mice. Hypoxia-preconditioned implants remained cartilaginous, whereas normoxia-preconditioned implants readily underwent calcification, vascular invasion, and subsequent endochondral ossification. In conclusion, metabolic programming of MSCs by oxygen tension provides a simple yet effective mechanism by which to direct the chondrogenic differentiation program into either permanent articular-like cartilage or hypertrophic cartilage that is destined to become endochondral bone.

Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.

GREM1, FRZB and DKK1 mRNA levels correlate with osteoarthritis and are regulated by osteoarthritis-associated factors

IntroductionOsteoarthritis is, at least in a subset of patients, associated with hypertrophic differentiation of articular chondrocytes. Recently, we identified the bone morphogenetic protein (BMP) and wingless-type MMTV integration site (WNT) signaling antagonists Gremlin 1 (GREM1), frizzled-related protein (FRZB) and dickkopf 1 homolog (Xenopus laevis) (DKK1) as articular cartilage’s natural brakes of hypertrophic differentiation. In this study, we investigated whether factors implicated in osteoarthritis or regulation of chondrocyte hypertrophy influence GREM1, FRZB and DKK1 expression levels.MethodsGREM1, FRZB and DKK1 mRNA levels were studied in articular cartilage from healthy preadolescents and healthy adults as well as in preserved and degrading osteoarthritic cartilage from the same osteoarthritic joint by quantitative PCR. Subsequently, we exposed human articular chondrocytes to WNT, BMP, IL-1β, Indian hedgehog, parathyroid hormone-related peptide, mechanical loading, different medium tonicities or distinct oxygen levels and investigated GREM1, FRZB and DKK1 expression levels using a time-course analysis.ResultsGREM1, FRZB and DKK1 mRNA expression were strongly decreased in osteoarthritis. Moreover, this downregulation is stronger in degrading cartilage compared with macroscopically preserved cartilage from the same osteoarthritic joint. WNT, BMP, IL-1β signaling and mechanical loading regulated GREM1, FRZB and DKK1 mRNA levels. Indian hedgehog, parathyroid hormone-related peptide and tonicity influenced the mRNA levels of at least one antagonist, while oxygen levels did not demonstrate any statistically significant effect. Interestingly, BMP and WNT signaling upregulated the expression of each other’s antagonists.ConclusionsTogether, the current study demonstrates an inverse correlation between osteoarthritis and GREM1, FRZB and DKK1 gene expression in cartilage and provides insight into the underlying transcriptional regulation. Furthermore, we show that BMP and WNT signaling are linked in a negative feedback loop, which might prove essential in articular cartilage homeostasis by balancing BMP and WNT activity.

TCF4 is a pro-catabolic and apoptotic factor in human articular chondrocytes by potentiating NF-κB signaling

Background: TCF/LEF transcription factors are downstream effectors of Wnt/β-catenin signaling. Results: TCF4 stimulates MMP expression and apoptosis in human articular chondrocytes by potentiating NF-κB signaling. Conclusion: Increased TCF4 expression may contribute to cartilage degeneration in osteoarthritis. Significance: Study of Wnt signaling transcription factors opens a new window for the treatment of degenerative cartilage disease. T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors are downstream effectors of Wnt/β-catenin signaling, which has been implicated in the development and progression of osteoarthritis (OA). This study aimed to investigate the role of TCF/LEF transcription factors in human articular chondrocytes. Primary human osteoarthritic cartilage predominantly expressed TCF4 and to a lesser extent, LEF1 and TCF3 mRNA. Overexpression of TCF4, but not of TCF3 or LEF1, induced MMP-1, -3, and -13 expression and generic MMP activity in human chondrocytes. This was due to potentiating NF-κB signaling by a protein-protein interaction between TCF4 and NF-κB p65 activating established NF-κB target genes such as MMPs and IL-6. LEF1 competed with TCF4 for binding to NF-κB p65. IκB-α was able to counteract the effect of TCF4 on NF-κB target gene expression. Finally, we showed that TCF4 mRNA expression was elevated in OA cartilage compared with healthy cartilage and induced chondrocyte apoptosis at least partly through activating caspase 3/7. Our findings suggest that increased TCF4 expression may contribute to cartilage degeneration in OA by augmenting NF-κB signaling.

WNT Signaling and Cartilage: Of Mice and Men

In adult articular cartilage, the extracellular matrix is maintained by a balance between the degradation and the synthesis of matrix components. Chondrocytes that sparsely reside in the matrix and rarely proliferate are the key cellular mediators for cartilage homeostasis. There are indications for the involvement of the WNT signaling pathway in maintaining articular cartilage. Various WNTs are involved in the subsequent stages of chondrocyte differentiation during development, and deregulation of WNT signaling was observed in cartilage degeneration. Even though gene expression and protein synthesis can be activated upon injury, articular cartilage has a limited ability of self-repair and efforts to regenerate articular cartilage have so far not been successful. Because WNT signaling was found to be involved in the development and maintenance of cartilage as well as in the degeneration of cartilage, interfering with this pathway might contribute to improving cartilage regeneration. However, most of the studies on elucidating the role of WNT signaling in these processes were conducted using in vitro or in vivo animal models. Discrepancies have been found in the role of WNT signaling between chondrocytes of mouse and human origin, and extrapolation of results from mouse models to the human situation remains a challenge. Elucidation of detailed WNT signaling functions will provide knowledge to improve cartilage regeneration.