Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K m values for the two substrates UDPGlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 ؋ 10 5 to 1 ؋ 10 6 Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 ؋ 10 5 to ϳ2 ؋ 10 6 Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 ؋ 10 5 to ϳ2 ؋ 10 6 Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 ؋ 10 6 Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions. Hyaluronan (HA)1 is a major component of most extracellular matrices, particularly in tissues with rapid cell proliferation and cell migration (1). The interaction of HA with various HA-binding proteins and cell-surface receptors plays important roles in regulating fundamental cell behaviors such as cell adhesion, migration, and differentiation (2, 3). Thus, HA has been greatly implicated in morphogenesis, regeneration, wound healing, tumor invasion, and cancer metastasis (4 -6). In addition, HA is an important structural molecule required for the maintenance of various aspects of tissue architecture and function. The physical and biological properties of HA appear to be affected by many factors including HA concentration and chain length. Indeed, high molecular weight HA at high concentrations suppresses endothelial cell growth, whereas low molecular weight HA stimulated cell growth leading to induction of angiogenesis (7). In addition, viscosity of the HA gel and the ability to hydrate large amounts of water were shown to be dependent on the molecular size of the HA chain.HA is a high molecular weight linear polymer composed of GlcUA ␤-1,3-GlcNAc ␤-1,4 disaccharide units and is synthesized by HA synthase at the inner face of the plasma membrane (8). Although a great deal of effort has been made to elucidate the mechanism of HA biosynthesis in mammalian cells, it has remained unclear due to difficulty in biochemical isolation of the active enzyme (9 -11). Recently, three distinct yet highly conserved genes encoding mammali...

High-voltage-activated Ca2+ channels are essential for diverse biological processes. They are composed of four or five subunits, including alpha1, alpha2-delta, beta and gamma (ref. 1). Their expression and function are critically dependent on the beta-subunit, which transports alpha1 to the surface membrane and regulates diverse channel properties. It is believed that the beta-subunit interacts with alpha1 primarily through the beta-interaction domain (BID), which binds directly to the alpha-interaction domain (AID) of alpha1; however, the molecular mechanism of the alpha1-beta interaction is largely unclear. Here we report the crystal structures of the conserved core region of beta3, alone and in complex with AID, and of beta4 alone. The structures show that the beta-subunit core contains two interacting domains: a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain. The AID binds to a hydrophobic groove in the GK domain through extensive interactions, conferring extremely high affinity between alpha1 and beta-subunits. The BID is essential both for the structural integrity of and for bridging the SH3 and GK domains, but it does not participate directly in binding alpha1. The presence of multiple protein-interacting modules in the beta-subunit opens a new dimension to its function as a multi-functional protein.

Modulation of voltage-gated Ca2ϩ channels via G-protein-coupled receptors is a prime mechanism regulating neurotransmitter release and synaptic plasticity. Despite extensive studies, the molecular mechanism underlying G q/11 -mediated modulation remains unclear. We found cloned and native N-type Ca 2ϩ channels to be regulated by phosphotidylinositol 4,5-bisphosphate (PIP 2 ). In inside-out oocyte patches, PIP 2 greatly attenuated or reversed the observed rundown of expressed channels. In sympathetic neurons, muscarinic M 1 ACh receptor suppression of the Ca 2ϩ current (I Ca ) was temporally correlated with PIP 2 hydrolysis, blunted by PIP 2 in whole-cell pipettes, attenuated by expression of PIP 2 -sequestering proteins, and became irreversible when PIP 2 synthesis was blocked. We also probed mechanisms of receptor specificity. Although bradykinin also induced PIP 2 hydrolysis, it did not inhibit I Ca . However, bradykinin receptors became nearly as effective as M 1 receptors when PIP 2 synthesis, IP 3 receptors, or the activity of neuronal Ca 2ϩ sensor-1 were blocked, suggesting that bradykinin receptor-induced intracellular Ca 2ϩ increases stimulate PIP 2 synthesis, compensating for PIP 2 hydrolysis. We suggest that differential use of PIP 2 signals underlies specificity of G q/11 -coupled receptor actions on the channels.

We have attempted to regenerate bone in a significant osseous defect with minimal invasiveness and good plasticity, and to provide a clinical alternative to autogenous bone grafts. Platelet-rich plasma (PRP) may enhance the formation of new bone and is nontoxic, nonimmunoreactive, and accelerates existing wound-healing pathways. We have used a combination of PRP as an autologous scaffold with in vitro-expanded mesenchymal stem cells (MSCs) to increase osteogenesis, compared with using the scaffold alone or autogenous particulate cancellous bone and marrow (PCBM). The newly formed bones were evaluated by radiography, histology, and histomorphometric analysis in the defects at 2, 4, and 8 weeks. According to the histological observations, the dog MSCs (dMSCs)/PRP group had well-formed mature bone and neovascularization compared with the control (defect only), PRP, and PCBM groups at 2 and 4 weeks. Histometrically, at 8 weeks newly formed bone areas were 18.3 +/- 4.84% (control), 29.2 +/- 5.47% (PRP), 61.4 +/- 3.38% (PCBM), and 67.3 +/- 2.06% (dMSCs/PRP). There were significant differences between the PCBM, dMSCs/PRP, and control groups. These results demonstrate that the dMSCs/PRP mixture is useful as a osteogenic bone substitute.

Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell-cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.H yaluronan (HA) is a linear polysaccharide composed of a, and is widely present as a major component of extracellular matrix in most vertebrate tissues (1, 2). HA is synthesized by three HA synthase isoforms: HAS1, HAS2, and HAS3 (3, 4). The three HAS genes have distinct expression patterns during mouse development (5), and their products have significantly different enzymatic properties and roles in the formation of the HA matrix (6).HA synthesis and matrix formation may have a role in the development, progression and pathogenesis of cancer. For example, the rate of HA synthesis is enormously increased when oncogenic viruses transform fibroblasts (7,8), and elevated levels of HA are associated with the hyperproliferative and malignant phenotypes in melanomas and various carcinomas (9-11).Studies done with cultured cancer cells showed that overproduction of HA enhances their anchorage-independent growth, tumorigenicity, and metastatic potential (12, 13), suggesting an important role for HA in tumor growth and malignant progression. However, it was unclear whether the overproduction could induce a malignant transformation of nontransformed cells.We overexpressed the three HAS isoforms to test the ability of HA to transform nontransformed cells. We also investigated whether the three HAS isoforms are functionally distinct in nontransformed cells. Materials and MethodsCell Culture and Transfection. 3Y1 1-B6 cells were obtained from Riken Cell Bank (Tsukuba, Japan). The 3Y1 cells were cultured in DMEM containing 10% FCS and 2 mM L-glutamine (growth medium). Stable transfectants were established as previously described (6) and were routinely cultured in ...