Plasticity and stem cells in the vertebrate nervous system

How and when do vertebrate neural precursor cells choose their fates? While some studies suggest a series of commitments on the road to fate choice, many recent experiments indicate that precursor fate choices can often be changed. Additionally, the identification of common gene control mechanisms in precursors suggest that these cells share fundamental properties throughout development.     

Spermatogonial stem cells

The mammalian seminiferous epithelium consists of a highly complex yet well-organized cell population, with germ cells in mitosis and meiosis and postmeiotic cells undergoing transformation to become spermatozoa. To study the factors which control renewal and differentiation of spermatogonial stem cells, animal models are now available which allow for arrest and restart of spermatogonial differentiation. In addition, marked progress has been made in understanding the control of apoptosis and its role in spermatogonia. For the future, spermatogonial stem cell transplantation may have important practical applications.     

Role of cytokines and extracellular matrix in the regulation of haemopoietic stem cells

The understanding of molecular mechanisms regulating the formation, growth and differentiation of haemopoietic stem cells has advanced considerably recently. Particular progress has been made in defining the cytokines, chemokines and extracellular matrix components which retain and maintain primitive haemopoietic cell populations in bone marrow. Furthermore, signal transduction pathways that are critical for haemopoiesis, both in vivo and in vitro, and that are activated by cytokines have also been identified and further characterised. The importance of these processes has, this year, been exemplified by the phenotypes of mice deficient in key signal transduction proteins and the discovery that mutations in the component proteins of some signalling pathways are linked to human diseases. Significant advances in understanding the molecular mechanisms for mobilisation of stem cells from bone marrow have also been made this year; this has potential importance for bone marrow transplantation.     

The tip-top branching ureter

Organ rudiments with their epithelial bud and adjacent mesenchyme look much the same at their initial stage of differentiation. The subsequent branching of the epithelial anlagen determines the final pattern of the organs, but the mesenchyme provides essential signals for epithelial differentiation. Glial cell line derived neurotrophic factor (GDNF) has recently been shown to regulate ureteric branching morphogenesis and is thereby the first defined signalling molecule in the embryonic metanephric kidney. GDNF is expressed by the mesenchyme, binds to the tip of the ureteric bud and functions in both bud induction and bud orientation. The active receptor complex for GDNF includes the receptor tyrosine kinase Ret and a novel class of glycosylphosphatidylinositol-linked receptors, called GDNF family receptor αs.     

The discovery and validation of new drug targets in cancer

Advances in our understanding of the signal transduction pathways involved in cellular growth control have provided several new strategies for cancer therapy. Recent advances now make it possible to develop selective inhibitors targeting genomic instability, the growth, survival, and invasion of the tumor, and its nourishment through the growth of new blood vessels.     

Polarized signaling: basolateral receptor localization in epithelial cells by PDZ-containing proteins

Extracellular signals are normally presented to one surface of epithelial cells and to one end of neurons, and so neuronal and epithelial cell signaling is inherently polarized. Another aspect of signaling polarity is that receptors are often asymmetrically distributed on the surfaces of polarized cells. Recent evidence from studies of Caenorhabditis elegans shows that signaling polarity plays an important role in development. The underlying mesoderm induces the overlying ectoderm to form the vulva, and asymmetric distribution of the signal receptor on the basolateral surface of the epithelium is crucial for this signaling. In neurons, the localization of neurotransmitter receptors and ion channels at synapses allows neurons to be exquisitely sensitive to synaptic inputs. Exciting recent reports suggest that receptor localization to neuronal synapses and the basolateral membrane domains of epithelia may involve a common molecular mechanism involving localization by PDZ-containing proteins.     

Tissue-type plasminogen activator: variants and crystal/solution structures demarcate structural determinants of function

NMR and crystal structures of many components of tissue-type plasminogen activator (t-PA) are now available: the finger-EGF pair and the kringle-2 domain structures have been solved, as have the proteolytic domains of vampire bat PA and human t-PA in two- and single-chain forms. These structures confirm the trypsin-like arrangement of the proteolytic domain of t-PA and show how surface loops near the catalytic centre contribute to the narrow specificity of t-PA. Together with mutational experiments, they identify the Lys 156 sidechain as a cause of the amidolytic activity of single-chain t-PA, as it can provide a substitute salt bridge partner for Asp 194 in the absence of the lle 16 N terminus of the two-chain form. These new findings provide new ideas for the design of PA variants with improved therapeutic properties.     

Distinct functional roles of Akt isoforms for proliferation, survival, migration and EGF-mediated signalling in lung cancer derived disseminated tumor cells

Single disseminated tumor cells (DTC) can be detected in the bone marrow (BM) from 20% to 60% of patients with various tumors including non-small cell lung cancer (NSCLC). Detection of DTC in the BM of NSCLC patients is associated with poor prognosis and may be responsible for metastatic relapse. However, the functional properties of DTC are widely unknown. Here, we performed the first functional analysis of DTC focusing on the activation of the PI3K/Akt signalling pathway and the functional roles of Akt isoforms. In vitro kinase assays revealed a high activity of Akt3 in NSCLC-derived DTC. Proliferation and survival of DTC was reduced by depletion of Akt3 and to a lesser extend by Akt1, but not after depletion of Akt2. The major effect of Akt3 on the proliferation of DTC was associated with an Akt3-mediated regulation of both, cyclin D1 and cyclin D3, whereas Akt1 regulated the expression of cyclin D1 only. In contrast all three Akt isoforms, especially Akt2, were involved in the regulation of migration. Analysis of signalling events downstream of distinct Akt isoforms revealed that expression levels of urokinase-type plasminogen activator and its receptor were decreased after knockdown of Akt1 and Akt3. In addition, EGF-stimulated proliferative and anti-apoptotic signals are mediated by Akt1 and Akt3 in DTC. Finally, by immunofluorescence staining of primary DTC from BM samples of lung cancer patients, pAkt(S473) and Akt3 positive DTC were detected in vivo. Our data demonstrate that Akt1 and notably Akt3 regulate proliferation, survival, migration and EGF-mediated signal transduction in NSCLC-derived DTC.     

Molecular details of cAMP generation in mammalian cells: a tale of two systems

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.     

Switching addictions between HER2 and FGFR2 in HER2-positive breast tumor cells: FGFR2 as a potential target for salvage after lapatinib failure

Agents that target HER2 have improved the prognosis of patients with HER2-amplified breast cancers. However, patients who initially respond to such targeted therapy eventually develop resistance to the treatment. We have established a line of lapatinib-resistant breast cancer cells (UACC812/LR) by chronic exposure of HER2-amplified and lapatinib-sensitive UACC812 cells to the drug. The mechanism by which UACC812/LR acquired resistance to lapatinib was explored using comprehensive gene hybridization. The FGFR2 gene in UACC812/LR was highly amplified, accompanied by overexpression of FGFR2 and reduced expression of HER2, and a cell proliferation assay showed that the IC₅₀ of PD173074, a small-molecule inhibitor of FGFR tyrosine kinase, was 10,000 times lower in UACC812/LR than in the parent cells. PD173074 decreased the phosphorylation of FGFR2 and substantially induced apoptosis in UACC812/LR, but not in the parent cells. FGFR2 appeared to be a pivotal molecule for the survival of UACC812/LR as they became independent of the HER2 pathway, suggesting that a switch of addiction from the HER2 to the FGFR2 pathway enabled cancer cells to become resistant to HER2-targeted therapy. The present study is the first to implicate FGFR in the development of resistance to lapatinib in cancer, and suggests that FGFR-targeted therapy might become a promising salvage strategy after lapatinib failure in patients with HER2-positive breast cancer.     

IGF2 derived from SH-SY5Y neuroblastoma cells induces the osteoclastogenesis of human monocytic precursors

The insulin-like growth factors 2 (IGF2) is a peptide hormone that binds to the insulin-like growth factor 1 receptor (IGF1R) and is abundantly stored in bone. IGF1R is deeply involved in the pathogenesis of many cancers that growth within bone and is also involved in osteoclast biology. Among different cell lines representative of osteolytic tumors, we found a very high expression of IGF2 in SH-SY5Y cells derived from neuroblastoma (NB). We previously showed that NB cells induce an osteolytic process through the Osteoprotegerin/RANKL/RANK and the canonical Wnt pathway system. Here, we hypothesized that NB promotes osteoclastogenesis also via IGF2. First, we demonstrated the presence of IGF1R on the osteoclast basolateral membrane, and we observed a cyclic IGF1R activation along with the differentiation process, also when induced by SH-SY5Y. Moreover, we found that IGF2 mRNA expression in SH-SY5Y cells was further increased when co-cultured with mesenchymal stromal cells, suggesting that IGF2 is important for NB interaction with the bone microenvironment. Finally, the treatment of SH-SY5Y cells with an anti-IGF2 siRNA or the addition of anti-IGF1R molecules impaired NB-induced osteoclastogenesis, even though the chemoattraction of monocytes by NB cells was unaffected. Our findings suggest that in IGF2-producing osteolytic tumors IGF1R is a good candidate for targeted therapies in combination with conventional drugs.     

Targeting CD9 produces stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis: a novel antiangiogenic therapy

The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis, and appears to be an effective and safe antiangiogenic approach. These results shed light on the biological roles of CD9 and may lead to novel antiangiogenic therapies.     

miR-27a suppresses triglyceride accumulation and affects gene mRNA expression associated with fat metabolism in dairy goat mammary gland epithelial cells

MicroRNAs (miRNAs), a well-defined group of small RNAs containing about 22 nucleotides, participate in various biological metabolic processes. miR-27a is a miRNA that is known to regulate fat synthesis and differentiation in preadipocyte cells. However, little is known regarding the role that miR-27a plays in regulating goat milk fat synthesis. In this study, we determined the miR-27a expression profile in goat mammary gland and found that miR-27a expression was correlated with the lactation cycle. Additionally, prolactin promoted miR-27a expression in goat mammary gland epithelial cells. Further functional analysis showed that over-expression of miR-27a down-regulated triglyceride accumulation and decreased the ratio of unsaturated/saturated fatty acid in mammary gland epithelial cells. miR-27a also significantly affected mRNA expression related to milk fat metabolism. Specifically, over-expression of miR-27a reduced gene mRNA expression associated with triglyceride synthesis by suppressing PPARγ protein levels. This study provides the first experimental evidence that miR-27a regulates triglyceride synthesis in goat mammary gland epithelial cells and improves our understanding about the importance of miRNAs in milk fat synthesis.     

Proteases involved in cartilage matrix degradation in osteoarthritis

Osteoarthritis is a common joint disease for which there are currently no disease-modifying drugs available. Degradation of the cartilage extracellular matrix is a central feature of the disease and is widely thought to be mediated by proteinases that degrade structural components of the matrix, primarily aggrecan and collagen. Studies on transgenic mice have confirmed the central role of Adamalysin with Thrombospondin Motifs 5 (ADAMTS-5) in aggrecan degradation, and the collagenolytic matrix metalloproteinase MMP-13 in collagen degradation. This review discusses recent advances in current understanding of the mechanisms regulating expression of these key enzymes, as well as reviewing the roles of other proteinases in cartilage destruction. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.