Ulrike Novak

Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation.

Interaction of thrombopoietin (TPO) with its receptor, c‐Mpl, triggers cell growth and differentiation responses controlling primitive haemopoietic cell production and megakaryocytopoiesis. To examine the important receptor domains and signal transduction pathways involved in these cellular responses, c‐Mpl cytoplasmic domain truncation and tyrosine substitution mutants were generated. In the myelomonocytic leukaemia cell lines WEHI3B‐D+ and M1, ectopic expression of the wild‐type c‐Mpl receptor induced TPO‐dependent cellular differentiation characterized by increased cell migration through agar and acquisition of the morphology and molecular markers of macrophages. Consistent with the concept that proliferative and differentiation signals emanate from distinct receptor domains, the C‐terminal 33 amino acids of c‐Mpl were dispensable for a proliferative response in Ba/F3 cells but proved critical for WEHI3B‐D+ and M1 differentiation. Finer mapping revealed that substitution of Tyr599 by phenylalanine within this c‐Mpl domain was sufficient to abolish the normal differentiation response. Moreover, in contrast to the normal c‐Mpl receptor, this same mplY599F mutant was also incapable of stimulating TPO‐dependent Shc phosphorylation, the association of Shc with Grb2 or c‐Mpl and of inducing c‐fos expression. Thus activation of components of the Ras signalling cascade, initiated by interaction of Shc with c‐Mpl Tyr599, may play a decisive role in specific differentiation signals emanating from the c‐Mpl receptor.

EGF receptor modifies cellular responses to hyaluronan in glioblastoma cell lines.

Cell contact with the extracellular matrix component, hyaluronan, plays a pivotal role in glioma cell invasion and proliferation. Although it is well established that glioma cells can bind hyaluronan to their surface via the expression of CD44, the cellular responses following ligand-receptor interaction remain poorly understood. Given that a large proportion of human high grade gliomas over express the epidermal growth factor receptor (EGFR) and ErbB2, this study aimed to investigate whether an interaction exists between CD44 and these receptor tyrosine kinases. Here we present evidence that CD44 co-immunoprecipitates with EGFR and ErbB2 in the glioma cell lines U87MG and SMA560. Hyaluronan treatment mediated the rapid and transient phosphorylation of extracellular signal regulated kinases 1 and 2 (ERK1 and ERK2) in glioma cell lines. This response to hyaluronan was augmented by the co-expression of EGFR. EGFR also differentially modified the hyaluronan induced expression of a number of genes associated with cellular invasion and proliferation. Northern blot analysis demonstrated that genes encoding urokinase type plasminogen activator (uPA), urokinase type plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinases (TIMP-1) and c- myc were up-regulated in response to hyaluronan. Furthermore, zymographic analysis revealed increased levels of uPA in the conditioned medium of hyaluronan stimulated cells. These results indicate a novel functional relationship between CD44 and EGFR in glioma cell lines. The capacity of CD44 to form stable complexes with receptor tyrosine kinases may provide a versatile system for the regulation of cellular invasion and proliferation that allows hyaluronan to activate signal transduction pathways and modulate gene expression via an EGFR-dependent manner. These findings provide new insights into the mode by which hyaluronan regulates the malignant phenotype and also suggest a role for EGFR-CD44 interactions in glial tumorigenesis.

The Carboxyl-terminal Domains of gp130-related Cytokine Receptors Are Necessary for Suppressing Embryonic Stem Cell Differentiation INVOLVEMENT OF STAT3

Cell type-specific responses to the leukemia inhibitory factor (LIF)/interleukin 6 cytokine family are mediated by dimerization of the LIF receptor α-chain (LIFRα) with the signal transducer gp130 or of two gp130 molecules followed by activation of the JAK/STAT and Ras/mitogen-activated protein kinase cascades. In order to dissect the contribution of gp130 and LIFRα individually, chimeric molecules consisting of the extracellular domain of the granulocyte colony stimulating factor receptor (GCSF-R) and various mutant forms of the cytoplasmic domains of gp130 or LIFRα were expressed in embryonic stem (ES) cells to test for suppression of differentiation, or in a factor-dependent plasma cytoma cell line to assess for induction of proliferation. Carboxyl-terminal domains downstream of the phosphatase (SHP2)-binding sites were dispensable for mitogen-activated protein kinase activation and the transduction of proliferative signals. Moreover, carboxyl-terminal truncation mutants which lacked intact Box 3 homology domains showed decreased STAT3 activation, failed to induce Hck kinase activity and suppress ES cell differentiation. Moreover, STAT3 antisense oligonucleotides impaired LIF-dependent inhibition of differentiation. Substitution of the tyrosine residue within the Box 3 region of the GSCF-R abolished receptor-mediated suppression of differentiation without affecting the transduction of proliferative signals. Thus, distinct cytoplasmic domains within the LIFRα, gp130, and GCSF-R transduce proliferative and differentiation suppressing signals.

Overexpression of hyaluronan synthase-2 reduces the tumorigenic potential of glioma cells lacking hyaluronidase activity.

OBJECTIVE The interactions of CD44 with hyaluronan are thought to be crucial for tumor cell attachment to the extracellular matrix, migration, and invasion. For migration to occur, however, the interactions between hyaluronan and cell surface receptors need to be transient. Hyaluronidases may facilitate the degradation of hyaluronan bound to the cell surface and thus reduce the interactions of the cells with the matrix, whereas the overproduction of hyaluronan in the absence of hyaluronidase activity may prevent cells from proliferating or invading normal surrounding tissue. METHODS We analyzed the effects in vitro and in vivo of hyaluronan synthase-2 (HAS2) overexpression on a murine glioma cell line that is deficient in hyaluronidase activity. In addition, we evaluated the expression levels of HAS and hyaluronidase genes in human glioma cell lines and in glioma specimens. RESULTS Increased hyaluronan synthesis had no effect on the in vitro proliferation of the cells but diminished their in vivo growth rate. Several human glioma cell lines were found to overexpress hyaluronan synthases, but they did so in conjunction with hyaluronidase Hyal2 and MGEA5 expression. Similarly, all glioblastomas multiforme expressed hyaluronidases MGEA5 and Hyal2. CONCLUSION The data suggest that an increased synthesis of hyaluronan by astrocytoma cells is only promoting tumor cell growth in vivo if the cells express hyaluronidases as well.

Formation of STAT5-containing DNA Binding Complexes in Response to Colony-stimulating Factor-1 and Platelet-derived Growth Factor

Colony-stimulating factor (CSF-1) activates several members belonging to the STAT (signal transducers and activators of transcription) family of transcription factors. We investigated the DNA binding complexes activated by CSF-1 in several cell lines and compared them with complexes activated by platelet-derived growth factor and interleukin 3. Our results indicate that the SIF-A complex activated by CSF-1 and platelet-derived growth factor may contain STAT3/STAT5 heterodimers binding to the high affinity SIF binding site, m67. In addition, both growth factors activate one or several STAT5-containing protein complexes binding to the prolactin-inducible element, PIE. The formation of these complexes was cell type and growth factor specific. Interleukin 3 activated only PIE binding complexes containing STAT5A and STAT5B and did not activate m67 binding complexes. It appears, therefore, that STAT5 cannot bind to m67 as a homodimer, but it can bind if it is dimerized with STAT3, whereas it can bind to the PIE element without being either complexed with STAT3 or any other known STAT protein, possibly as a homodimer or as STAT5A/STAT5B heterodimer. However, in addition, STAT5 may heterodimerize with other proteins and form novel PIE binding complexes.

DISTINCT ROLES FOR LEUKEMIA INHIBITORY FACTOR RECEPTOR ALPHA -CHAIN AND GP130 IN CELL TYPE-SPECIFIC SIGNAL TRANSDUCTION

Leukemia inhibitory factor (LIF) induces a variety of disparate biological responses in different cell types. These responses are thought to be mediated through the functional LIF receptor (LIFR), consisting of a heterodimeric complex of LIFR α-chain (LIFRα) and gp130. The present study investigated the relative capacity of the cytoplasmic domains of each receptor subunit to signal particular responses in several cell types. To monitor the signaling potential of LIFRα and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and cytoplasmic regions of either LIFRα or gp130. Both chimeric receptors and the full-length GCSFR in expressed in M1 myeloid leukemic cells to measure differentiation induction, in embryonic stem cells to measure differentiation inhibition, and in Ba/F3 cells to measure cell proliferation. Our results demonstrated that whereas GCSFR-gp130 receptor homodimer mediated a GCSF-induced signal in all three cell types, the GCSFR-LIFRα receptor homodimer was only functional in embryonic stem cells. These findings suggest that the signaling potential of gp130 and LIFRα cytoplasmic domains may differ depending upon the tissue and cellular response initiated.

Caspase 8 is absent or low in many ex vivo gliomas

Better treatments are required urgently for patients with malignant glioma, which currently is incurable. Death ligands, such as tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL), may offer promise for the treatment high‐grade glioma if such ligands induce apoptotic signaling in vivo in glioma cells. Caspase 8 is required for death ligand signaling, and its levels may influence the sensitivity of glioma cells to death ligands. It also may act as a tumor suppressor protein. The authors analyzed caspase 8 expression levels in ex vivo glioma specimens and explored potential mechanisms of its regulation.

ADAM proteins in the brain

ADAM proteins are a family of metalloproteinases with a disintegrin domain. They have proteolytic as well as adhesive functions and can be involved in cell fusion events. Some ADAM proteins are expressed in a highly tissue restricted fashion, whereas others are expressed quite ubiquitously. In the brain, ADAM proteins have a role in neural development, axon guidance and inflammatory responses. Although there may be some functional overlap, homozygous deletion of some ADAM genes in mice can have fatal consequences. The expression and possible role of ADAM proteins in the brain will be discussed.

ADAM22, Expressed in Normal Brain but not in High-Grade Gliomas, Inhibits Cellular Proliferation via the Disintegrin Domain

OBJECTIVE:To study the expression and function of the brain-specific proteinase deficient disintegrins, ADAM11 and ADAM22 (a disintegrin and metalloproteinase). METHODS:Specimens of low- and high-grade gliomas and normal brain were analyzed for ADAM11 and ADAM22 expression using Western blotting. The effects of overexpression of ADAM11 and ADAM22 in glioma cells on growth were analyzed using bromodeoxyuridine incorporation linked to immunocytochemistry. Similarly analyzed were the effects on cell proliferation of bacterially expressed glutathione S-transferase fusion proteins with the disintegrin domain of ADAM11 and ADAM22. RESULTS:ADAM22 is expressed in normal brain and some low-grade gliomas, but not in high-grade gliomas, whereas ADAM11 is expressed in all low- and high-grade gliomas. In vitro, ADAM22 inhibits cellular proliferation of glioma derived astrocytes. The growth inhibition appears to be mediated by interactions between the disintegrin domain of ADAM22 and specific integrins expressed on the cell surface. This growth inhibition can be avoided by over-expression of integrin linked kinase. CONCLUSION:ADAM22, a brain-specific cell surface protein, mediates growth inhibition using an integrin dependent pathway. It is expressed in normal brain but not in high-grade gliomas. A related protein, ADAM11, has only a minor effect on cell growth, and its expression is unchanged in low- and high-grade gliomas.

Cytokine modulation of plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocytes. Down-regulation by tumor necrosis factor α and up-regulation by transforming growth factor-β and basic fibroblast growth factor

Recombinant human cytokines were examined for their effects on plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocyte monolayer cultures. Cartilage and chondrocytes were cultured with and without added cytokines and the conditioned media assayed for PAI-1 by a specific enzyme-linked immunosorbent assay, and mRNA levels determined by Northern blot analysis. Tumor necrosis factor alpha (TNF alpha) reduced, and transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) increased, the levels of PAI-1 antigen and mRNA in the culture fluids and cell extracts, respectively. The effects of TNF alpha and TGF-beta on PAI-1 antigen levels were both time- and concentration-dependent; optimum doses being 10-100 pM TNF alpha and 0.4-0.8 nM TGF-beta, with each cytokine exerting its effect on PAI-1 antigen levels within 8 h of addition to culture. TNF alpha (and interleukin-1 alpha) also countered the effects of TGF-beta and bFGF. The anti-inflammatory drugs, indomethacin and dexamethasone, did not appear to modulate PAI-1 levels in cultures of cartilage tissue. The inhibition of PAI-1 levels by cytokines and reagents which stimulate cartilage resorption (i.e., TNF alpha, interleukin-1 alpha, retinoic acid) and enhancement by cytokines which counter it (i.e., TGF-beta, bFGF) further implicate plasminogen activator in the mechanism(s) of cartilage degradation in diseases such as arthritis.