Stimulation of hyaluronan biosynthesis by platelet-derived growth factor-BB and transforming growth factor-<i>β</i>1 involves activation of protein kinase C

154

Hyaluronan is a constituent of the extracellular matrix of connective tissue and is actively synthesized during wound healing and tissue repair to provide a framework for ingrowth of blood vessels and fibroblasts. Changes in the serum concentration of hyaluronan are associated with inflammatory and degenerative arthropathies such as rheumatoid arthritis. In addition, hyaluronan has been implicated as an important substrate for migration of adhesion of leukocytes during inflammation. A human hyaluronan synthase (HuHAS1) cDNA was isolated by a functional expression cloning approach. Transfection of CHO cells conferred hyaluronidase-sensitive adhesiveness of a mucosal T cell line via the lymphocyte hyaluronan receptor, CD44, as well as increased hyaluronan levels in the cultures of transfected cells. The HuHAS1 amino acid sequence shows considerable homology to the hasA gene product of Streptococcus pyogenes, a glycosaminoglycan synthetase from Xenopus laevis (DG42), and is the human homolog of a recently described murine hyaluronan synthase.

Section: Resultsmentioning

confidence: 99%

Functional Cloning of the cDNA for a Human Hyaluronan Synthase

Shyjan

Heldin

Butcher

et al. 1996

154

“…In the MCF-7 cells expressing GFP-HAS, hyaluronan synthesis was ϳ30 pg/cell/12 h. For most cell types in culture, the rates of stimulated hyaluronan synthesis range between 2 and 15 pg/cell/12 h (37-40), but quantities as high as 30 and 106 pg/cell/12 h have been reported in foreskin fibroblasts and mesothelial cells, respectively (41,42). Hyaluronan synthesis rates equal or higher than that after Has3 transfections are thus possible through endogenous HAS expression.…”

Section: Discussionmentioning

confidence: 99%

Hyaluronan Synthesis Induces Microvillus-like Cell Surface Protrusions

Kultti

Rilla

Tiihonen

et al. 2006

124

134

Hyaluronan synthases (HASs) are plasma membrane enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space. In cells transfected with green fluorescent protein (GFP)-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3-20-m-long microvillus-type plasma membrane protrusions, which also contained filamentous actin, the hyaluronan receptor CD44, and lipid raft microdomains. Enzymatic activity of HAS was required for the growth of the microvilli, which were not present in cells transfected with other GFP proteins or inactive GFP-Has3 mutants or in cells incubated with exogenous soluble hyaluronan. The microvilli induced by HAS3 were gradually withered by introduction of an inhibitor of hyaluronan synthesis and rapidly retracted by hyaluronidase digestion, whereas they were not affected by competition with hyaluronan oligosaccharides and disruption of the CD44 gene, suggesting independence of hyaluronan receptors. The data bring out the novel concept that the glycocalyx created by dense arrays of hyaluronan chains, tethered to HAS during biosynthesis, can induce and maintain prominent microvilli.Vertebrate cells display slender plasma membrane protrusions like filopodia, microspikes, and microvilli, the formation and maintenance of which are thought to depend on the dynamics of a bundle of actin filaments in the core of these extensions (1). In selected environments, most of the components in the actin polymerization apparatus, when overexpressed or experimentally activated, have been reported to increase the growth of filopodia or microvilli (2-7). This suggests that there is a strong intrinsic potential in cells to display these extensions, ready for implementation by factors even outside the effector and signaling chains directly related to actin. Indeed, at the molecular level, mechanisms that induce the growth of microvilli in vivo are still somewhat obscure (8). We present a novel factor, active hyaluronan synthesis, apparently unrelated to any of the previously described signaling or actin polymerization processes, that triggers extensive microvillus formation in several cell types. Interestingly, the process is primarily driven by the cell surface glycocalyx rather than intracellular systems.Hyaluronan is an ubiquitous pericellular and extracellular matrix glycosaminoglycan important in embryonic development (9), wound healing (10), inflammation (11, 12), mammalian fertilization (13), and cancer (14). It is involved in cell adhesion (15), migration (16 -19), proliferation and epithelial-mesenchymal transition (9), resistance to apoptosis, and multidrug resistance (14). Some of its effects are thought to be mediated by an ability to form a hydrated pericellular matrix, a coat that can modify cellular interactions with other matrix components and neighboring cells, directly regulating processes such as differentiation and migration (20). Some of the biological signals elicited by hyaluronan are dependent on its CD44 receptor on cell su...

“…In addition to putative HA binding sites, numerous consensus sequences for phosphorylation by PKC and cAMP-dependent kinases were identified within the predicted intracellular loop of the molecule. This is significant, as mammalian HA biosynthesis has been shown to be dependent on activation by PKC (8,9), and suggests that the PKC dependence may partly involve direct activation of Has2 through phosphorylation. Experiments to investigate the role of potential PKC phosphorylation sites on Has2 function are currently starting.…”

Section: Fig 6 Southern Analysis Of Mouse Has2mentioning

confidence: 99%

Molecular Cloning and Characterization of a Putative Mouse Hyaluronan Synthase

Spicer

Augustine

McDonald

1996

157

104

We report the isolation of a novel mouse gene which encodes a putative hyaluronan synthase. The cDNA was identified using degenerate reverse transcriptase-polymerase chain reaction. Degenerate primers were designed based upon an alignment of the amino acid sequences of Streptococcus pyogenes HasA, Xenopus laevis DG42, and Rhizobium meliloti NodC. A mouse embryo cDNA library was screened with the resultant polymerase chain reaction product, and multiple cDNA clones spanning 3 kilobase pairs (kb) were isolated. The open reading frame predicted a 63-kDa protein with several transmembrane sequences, multiple consensus phosphorylation sites, and four putative hyaluronan binding motifs. The amino acid sequence displayed 55% identity to mouse HAS, 56% identity to Xenopus DG42, and 21% identity to Streptococcus HasA. Northern analysis identified transcripts of 4.8 kb and 3.2 kb, which were expressed highly in the developing mouse embryo and at lower levels in adult mouse heart, brain, spleen, lung, and skeletal muscle. Transfection experiments demonstrated that mouse Has2 could direct hyaluronan coat biosynthesis in transfected COS cells, as evidenced by a classical particle exclusion assay. These results suggest that mammalian HA synthase activity is regulated by at least two related genes. Accordingly, we propose the name Has2 for this gene.Hyaluronan (HA 1 ) is a linear unbranched polymer made up of repeating disaccharide units of D-glucuronic acid(␤133)Nacetylglucosamine(␤134). More than 60 years after the isolation of hyaluronan from the vitreous humor (1), its synthetic pathway remains incompletely characterized. HA is synthesized as a free, linear polymer at the inner face of the plasma membrane of eukaryotic cells and is subsequently extruded to the outside of the cell (2-7). HA biosynthesis in mammalian cells may be regulated in part through signaling cascades (8, 9).Certain bacteria can synthesize an HA polymer that is identical to the polymer synthesized by mammalian cells (10 -12). Indeed, investigation of HA biosynthesis in the Group A Streptococcus, Streptococcus pyogenes, has recently led to the identification and cloning of several genes that encode enzymes critical for HA biosynthesis in this bacterium (11). However, until very recently, no genes have been identified that encode enzymes with similar activities in mammalian cells.Degenerate reverse transcriptase-PCR has been a useful tool in the identification and cloning of many genes and gene families (13-15). This approach relies upon conserved sequences deduced from alignments of related gene or protein sequences. The hasA gene of S. pyogenes encodes hyaluronan synthase in this bacterium (11). Sequence analysis predicts that this protein is a membrane protein with a large intracellular loop encoding the active site of the enzyme (11). Similarly, in mammalian cells, the HA synthase has been localized to the plasma membrane, with the active site on the inner face of the membrane (4, 5). Data base searches have identified the Rhizobium sp. nodulation fa...