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ABSTRACT: To examine the effects of glucosamine (GlcN) and some N-acylated (GlcNAcyl) derivatives on the proliferation and proteoglycan (PG) synthesis of bovine articular chondrocyte (BAC); and to expand these results to human articular chondrocytes (HAC) and study the modulation of gene regulation by these compounds.
The compounds tested were: glucose (Glc), GlcN.HCl, N-acetyl GlcN (GlcNAc), and N-butyryl GlcN, (GlcNBu). GlcNBu was synthesized from GlcN and butyric anhydride. For the chondrocyte cultures, both anchorage-dependent (AD) and an anchorage-independent (AI) system (alginate beads) were evaluated. Following the various additions, BAC were assessed for total cell number, DNA, or total PG synthesis at different times. Utilizing similar conditions, human cDNA microarrays were performed for the HAC after harvesting total RNA.
For AD cultures, the addition of GlcN.HCl (0.1-5.0 mM) to BAC or HAC cultures inhibited cell proliferation and total PG synthesis in a dose-dependent manner. For AI cultures, the inhibitory effects of GlcN.HCl on cell proliferation were less prominent, and PG synthesis increased slightly more for the GlcNAcyl than the GlcN additions. In the AD system, the addition of GlcNAc did not result in the inhibitory effect of GlcN.HCl, while GlcNBu addition resulted in an increase in BAC proliferation and PG synthesis that could not be explained by the Bu moiety alone. For the HAC, additions of 0.1 mM GlcNBu resulted in upregulation of a large number of genes, with only a few downregulated, while GlcN addition resulted in no upregulation and one downregulated gene, for preset stringency criteria.
Addition of GlcNBu to BAC or HAC to AD cultures generally stimulated cell proliferation and PG synthesis, while addition of GlcN resulted in inhibition of these indicators. The inhibitory effects of the GlcN molecule appear to be related to the unsubstituted amino group. Additions of GlcNBu, but not GlcN, to HAC resulted in upregulation in the expression of a large number of genes.
The Journal of Rheumatology 10/2005; 32(9):1775-86. · 3.69 Impact Factor
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ABSTRACT: Stat3 (signal transducer and activator of transcription-3) activity is required for transformation by a number of oncogenes, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. Although in most instances Stat3 is growth-promoting, the impact of cell density on Stat3 activation status and the biological importance of Stat3 during growth arrest have not been characterized. Previous results indicated that cell density alters tyrosine phosphorylation levels of cultured cells. Since signalling through Stat3 is determined by a key phosphorylation at tyr705, we examined the effects of cell density upon Stat3 activity in normal breast epithelial cells, breast carcinoma lines and normal mouse fibroblasts. Intriguingly, the results revealed a dramatic increase in Stat3, tyr705 phosphorylation and activity with cell density, which gradually declined at later stages. This activation was dependent upon cell-cell contact, since it was eliminated if cell adhesion was disrupted through calcium chelation, while it was reinstated through cell aggregation. Furthermore, this activation was suppressed following inhibition of JAKs (Janus kinases) but not inhibition of Fer, IGF1-R, or kinases of the c-Src family. On the other hand, constitutively active Stat3 in carcinoma lines, known to harbor activated Src, was blocked by pharmacological inhibitors of Src as well as JAKs. These results point to the existence of two distinct pathways of Stat3 activation in breast carcinomas, based on Src dependence. More importantly, our results suggest that Stat3 activity is upregulated during the confluence-mediated growth arrest by a signalling mechanism that requires JAKs.
Oncogene 05/2004; 23(15):2600-16. · 6.37 Impact Factor
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ABSTRACT: Relatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A-mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A-independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell-derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.
Blood 03/2004; 103(3):912-20. · 9.90 Impact Factor
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Peter Greer
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ABSTRACT: Fps/Fes and Fer are the only known members of a distinct subfamily of the non-receptor protein-tyrosine kinase family. Recent studies indicate that these kinases have roles in regulating cytoskeletal rearrangements and inside out signalling that accompany receptor ligand, cell matrix and cell cell interactions. Genetic analysis using transgenic mouse models also implicates these kinases in the regulation of inflammation and innate immunity.
Nature Reviews Molecular Cell Biology 05/2002; 3(4):278-89. · 39.12 Impact Factor
