The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.

Mucification of the cumulus layer around the oocyte is an obligatory process for female fertility. Tumor necrosis factor-induced protein-6 (TNFIP6 or TSG6) has been shown to be specifically expressed during this process. We have generated TNFIP6-deficient mice and tested the ability of their cumulus cells to undergo mucification. Cumulus cell-oocyte complexes fail to expand in TNFIP6-deficient female mice because of the inability of the cumulus cells to assemble their hyaluronan-rich extracellular matrix. The impaired cumulus matrix formation is due to the lack of covalent complexes between hyaluronan and the heavy chains of the inter-α-trypsin inhibitor family. As a consequence, TNFIP6-deficient females are sterile. Cultured TNFIP6-deficient cumulus cell-oocyte complexes also fail to expand when stimulated with dibutyryl cyclic AMP or epidermal growth factor. Recombinant TNFIP6 is able to catalyze the covalent transfer of heavy chains to hyaluronan in a cell-free system, restore the expansion of Tnfip6-null cumulus cell-oocyte complexes in vitro, and rescue the fertility in Tnfip6-null females. These results provide clear evidence that TNFIP6 is a key catalyst in the formation of the cumulus extracellular matrix and indispensable for female fertility.

Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis. In this study, epidermal growth factor (EGF) induced a coat of hyaluronan and a 3-5-fold increase in its rate of synthesis in a rat epidermal keratinocyte cell line that has retained its ability for differentiation. EGF also increased hyaluronan in perinuclear vesicles, suggesting concurrent enhancement in its endocytosis. Cell-associated hyaluronan was most abundant in elongated cells that were stimulated to migrate by EGF, as determined in vitro in a wound healing assay. Large fluctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precursor of hyaluronan, correlated with medium glucose concentrations but not with EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2 mRNA increased 2-3-fold in less than 2 h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has2 mRNA internal standard. The average level of Has2 mRNA increased from ϳ6 copies/ cell in cultures before change of fresh medium, up to ϳ54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of ϳ21 copies/cell at 6 h. The change in the Has2 mRNA levels and the stimulation of hyaluronan synthesis followed a similar temporal pattern, reaching a maximum level at 6 h and declining toward 24 h, a finding in line with a predominantly Has2-dependent hyaluronan synthesis and its transcriptional regulation.Hyaluronan is a large glycosaminoglycan found in the extracellular space of most animal tissues. It forms a loose, highly hydrated, gel-like matrix that contributes to the maintenance of the extracellular space and facilitates nutrient diffusion. Furthermore, hyaluronan is involved in cell proliferation and differentiation, produces an environment favorable for migration (1), and stimulates cell locomotion (2, 3). Elevated tissue levels of hyaluronan occur during embryonic growth of tissues and organs (1), wound healing (4, 5), inflammation (6), and invasion of certain cancers (7-10).In skin epidermis, the narrow extracellular space surrounding keratinocytes contains a high concentration of hyaluronan (11, 12), as do other stratifying squamous epithelia (13, 14). The half-life of labeled epidermal hyaluronan in human skin organ culture is ϳ1 day (15), indicating fast local turnover by keratinocytes. The importance of the strikingly high concentration and turnover of hyaluronan in the multilayered squamous epithelia is not completely understood, but we have hypothesized that the former is necessary to maintain an extracellular space for the nutritional needs of the more superficial cell layers, whereas the latter allows the dramatic modulation of cell shape that occurs during differentiation and for the high migratory potential of keratinocytes that is activated, e.g. in wound healing (16).Unlike other glycosaminoglycans, hyaluronan is synthesized at the inne...