Hyaluronan is a component of the extracellular matrix, which affects tissue homeostasis. In this study, we investigated the regulatory mechanisms of one of the hyaluronan-synthesizing enzymes, HAS2. Ectopic expression of Flag-and 6myc-HAS2 in COS-1 cells followed by immunoprecipitation and immunoblotting revealed homodimers; after co-transfection with Flag-HAS3, also heterodimers were seen. Furthermore, the expressed HAS2 was ubiquitinated. We identified one acceptor site for ubiquitin on lysine residue 190. Mutation of this residue led to inactivation of the enzymatic activity of HAS2. Interestingly, K190R-mutated HAS2 formed dimers with wt HAS2 and quenched the activity of wt HAS2, thus demonstrating a functional role of the dimeric configuration.Hyaluronan is abundantly found in tissues throughout the body and has key roles in tissue organization and homeostasis (1). Abnormal accumulation of hyaluronan in tissues and serum is associated with the progression of many diseases, such as inflammatory diseases and cancer (2-5). The biosynthesis of hyaluronan is tightly regulated by growth factors and cytokines as well as other stimuli that promote wound healing, inflammation, or transformation. External regulatory signals, such as platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-, and phorbol 12-myristate 13-acetate (PMA), regulate both the size (6) and amount of the produced hyaluronan (7-14).The hyaluronan-synthesizing enzymes (HAS) 4 were cloned and characterized first in the bacterium Streptococcus pyogenes, and then in mammals, where three different HAS isoforms were characterized, HAS1, HAS2, and HAS3 (15, 16). Studies by us and other laboratories (6, 11) revealed that growth factormediated increase of hyaluronan synthesis is often due to induction of the HAS2 gene in fibroblasts, whereas external signals have been shown to predominantly regulate HAS1 and HAS3 transcripts in synoviocytes and keratinocytes, respectively (12,14,17). In addition to the regulation of HASs at the transcriptional level (6,11,13,14), there is evidence that the activity of HAS isoforms is regulated by phosphorylation by protein kinase C (PKC), protein kinase A (PKA), calcium-dependent protein kinase, and extracellular signal-regulated kinase (10, 18 -20). Prediction from their amino acid sequences suggests that HASs are multi-pass membrane proteins (15) that occur in the plasma membrane, but there are also evidence that much of the HAS enzymes reside at intracellular localizations including perinuclear membrane, endoplasmic reticulum (ER)-Golgi pathway, and endocytic vesicles (21-23).Ubiquitination of proteins affects their stability, activity, interaction with other proteins as well as subcellular localization and trafficking (24, 25). Modification of a protein with K48-linked poly-Ub chains can trigger the proteasomal degradation of the protein. The functional consequences of protein ubiquitination by K63-linked poly-Ub chains include activation of proteins or alteration of their trafficking. Protein modi...
Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12-or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.
Cell strains derived from corneal stroma and sclera fibroblast cells can be established using HPV16 E6/E7 immortalized genes of the same origin. The phenotypic cell characteristics did not change after transfection, immortalization, or successive passages in culture.
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