Hyaluronan Synthesis Induces Microvillus-like Cell Surface Protrusions

Abstract

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...

Hyaluronan-dependent pericellular matrix☆

“…Another study showed that the enzymatic activity of HAS is coupled to plasma membrane residence and the HAS is localized to cell protrusions [30]. In this recent study, HAS overexpression and hyaluronan production was also found to influence the formation of these microvillous protrusions in adenocarcinoma cells and other epithelial cells [31] (Fig. 4).…”

“…The GFP-label imaged in live cells reveals that the coat is actually scaffolded by numerous elongated microvilli, on which Has accumulates (Fig. 5) [31]. Similar thin microvilli embedded in HAcoat have been found in untransfected cells such as those of mesothelium, known to synthesize high amounts of HA (unpublished data).The length of the microvilli can exceed 20 μm and they are extremely thin, almost undetectable in ordinary phase contrast microscopy, and easily destroyed during fixation.…”

“…This occurs even though the cells do not express significant amounts of aggregating proteoglycans. The coat formation is even more pronounced in epithelial cells transfected with GFP-Has2 and GFP-Has3 [30,31] (Fig. 4).…”

Involvement of Hyaluronan in Regulation of Fibroblast Phenotype

“…To investigate the functional consequences of these differences, we studied the effect of the HAS enzyme inhibitor, 4MU, on TGF-␤ 1 -mediated myofibroblastic differentiation in dermal fibroblasts. Depletion of the UDP-glucuronic acid pool by 4MU has been shown to inhibit HA synthesis and pericellular HA coat formation in a number of cell types, including fibroblasts (34,(41)(42)(43)(44). Incubation of dermal fibroblasts with 4MU significantly attenuated both mRNA and protein expression of ␣-sma and resulted in limited cytoskeletal reorganization of filamentous actin.…”

“…It is thought to be formed by newly synthesized HA polymers tethered to the HAS enzymes on the plasma membrane and bound to HA-binding proteins in the matrix. Recent data from Kulti et al (42) has demonstrated that in several cell types, the HA coat can induce and maintain prominent microvilli in cell plasma membranes that rest on filamentous actin. These microvilli were degraded when the pericellular coat was destroyed and were dependent on an intact actin cytoskeleton.…”