Lisheng Zhuo

Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice

Hyaluronan (HA) associates with proteins and proteoglycans to form the extracellular HA-rich matrices that significantly affect cellular behaviors. So far, only the heavy chains of the plasma inter-α-trypsin inhibitor (ITI) family, designated as SHAPs (serum-derivedhyaluronan-associated proteins), have been shown to bind covalently to HA. The physiological significance of such a unique covalent complex has been unknown but is of great interest, because HA and the ITI family are abundant in tissues and in plasma, respectively, and the SHAP-HA complex is formed wherever HA meets plasma. We abolished the formation of the SHAP-HA complex in mice by targeting the gene of bikunin, the light chain of the ITI family members, which is essential for their biosynthesis. As a consequence, the cumulus oophorus, an investing structure unique to the oocyte of higher mammals, had a defect in forming the extracellular HA-rich matrix during expansion. The ovulated oocytes were completely devoid of matrix and were unfertilized, leading to severe female infertility. Intraperitoneal administration of ITI, accompanied by the formation of the SHAP-HA complex, fully rescued the defects. We conclude that the SHAP-HA complex is a major component of the HA-rich matrix of the cumulus oophorus and is essential for fertilizationin vivo.

SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-α-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP·HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP·HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IαI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP·HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

Impact of the hyaluronan‐rich tumor microenvironment on cancer initiation and progression

Hyaluronan acts as a microenvironmental stimulus that can influence the malignant phenotype of cancer cells. During cancer progression, hyaluronan assembles an extracellular matrix that is favorable for both the motility and proliferation of cancer cells and the recruitment of inflammatory and bone marrow‐derived progenitor cells. The varied roles of this polysaccharide are regulated via multiple mechanisms involving biosynthesis, degradation, binding with other extracellular molecules, and activation of signaling pathways. Recent animal studies have provided evidence that aberrant biosynthesis of hyaluronan accelerates tumor growth through a diverse repertoire of host–tumor interactions, such as stromal cell recruitment, angiogenesis, lymphangiogenesis, and inflammation. Hyaluronan in the tumor microenvironment thus significantly impacts cancer initiation and progression via stroma–cancer cell interactions. (Cancer Sci 2008; 99: 1720–1725)

Hyaluronan Mediates Ozone-induced Airway Hyperresponsiveness in Mice

Ozone is a common urban environmental air pollutant and significantly contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced bronchoconstriction, remain poorly understood. Hyaluronan was recently shown to play a central role in the response to noninfectious lung injury. Therefore, we hypothesized that hyaluronan contributes to airway hyperreactivity (AHR) after exposure to ambient ozone. Using an established model of ozone-induced airways disease, we characterized the role of hyaluronan in airway hyperresponsiveness. The role of hyaluronan in response to ozone was determined by using therapeutic blockade, genetically modified animals, and direct challenge to hyaluronan. Ozone-exposed mice demonstrate enhanced AHR associated with elevated hyaluronan levels in the lavage fluid. Mice deficient in either CD44 (the major receptor for hyaluronan) or inter-α-trypsin inhibitor (molecule that facilitates hyaluronan binding) show similar elevations in hyaluronan but are protected from ozone-induced AHR. Mice pretreated with hyaluronan-binding peptide are protected from the development of ozone-induced AHR. Overexpression of hyaluronan enhances the airway response to ozone. Intratracheal instillation of endotoxin-free low molecular weight hyaluronan induces AHR dependent on CD44, whereas instillation of high molecular weight hyaluronan protects against ozone-induced AHR. In conclusion, we demonstrate that hyaluronan mediates ozone-induced AHR, which is dependent on the fragment size and both CD44 and inter-α-trypsin inhibitor. These data support the conclusion that pulmonary matrix can contribute to the development of airway hyperresponsiveness.

Molecular Heterogeneity of the SHAP-Hyaluronan Complex ISOLATION AND CHARACTERIZATION OF THE COMPLEX IN SYNOVIAL FLUID FROM PATIENTS WITH RHEUMATOID ARTHRITIS

We previously found that a covalent complex of SHAPs (serum-derived hyaluronan-associated proteins), the heavy chains of inter-α-trypsin inhibitor family molecules, with hyaluronan (HA) is accumulated in synovial fluid of patients with rheumatoid arthritis, and the complex is circulated in patient plasma at high concentrations. How the SHAP-HA complex participates in this disease is unknown. To address this question, it is essential to clarify the structural features of this macromolecule. The SHAP-HA complex purified from synovial fluid of the patients by three sequential CsCl isopycnic centrifugations was heterogeneous in density, and the fractions with different densities had distinct SHAP-to-HA ratios. Agarose gel electrophoresis and column chromatography revealed that there was no apparent difference in the size distribution of HA to which SHAPs were bound between the fractions with different densities. The SHAP-HA complex in the higher density fraction had fewer SHAP molecules per HA chain. Therefore, the difference between the fractions with different densities was due to a heterogeneous population of the SHAP-HA complex, namely the different number of SHAP molecules bound to an HA chain. Based on the SHAP and HA contents of the purified preparations, we estimated that an HA chain with a molecular weight of 2 × 106 has as many as five covalently bound SHAPs, which could give a proteinaceous multivalency to HA. Furthermore, we also found that the SHAP-HA complex tends to form aggregates, judging from the migration and elution profiles in agarose gel electrophoresis and gel filtration, respectively. The multivalent feature of the SHAP-HA complex was also confirmed by the negative staining electron micrographic images of the purified fractions. Taken together, those structural characteristics may underlie the aggregate-forming and extracellular matrix-stabilizing ability of the SHAP-HA complex.

Inhibition of hyaluronan synthesis in breast cancer cells by 4‐methylumbelliferone suppresses tumorigenicity in vitro and metastatic lesions of bone in vivo

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Previous studies demonstrated that inhibition of HA suppressed the tumorigenicity of various malignant tumors including breast cancer. 4‐methylumbelliferone (MU) has been reported to inhibit HA synthesis in several cell types. However, few studies have focused on the effects of HA inhibition in breast cancer cells by MU, nor the effects on bone metastasis. We hypothesized that MU would suppress the progression of bone metastasis via inhibition of HA synthesis. Here, we investigated the effects of MU on HA expression in MDA‐MB‐231 breast cancer cell line in addition to their tumorigenicity in vitro and in vivo. HAS2 mRNA expression was downregulated after 6 and 24 hr treatment with MU. Quantitative analysis of HA revealed that MU significantly inhibited the intracellular and cell surface HA. MU significantly inhibited cell growth and induced apoptosis as determined by cell proliferation and TUNEL assays, respectively. Phosphorylation of Akt was suppressed after 12 and 24 hr treatment with MU. MU treatment also inhibited cell motility as well as cell invasiveness. MU also inhibited cell growth and motility in murine fibroblast cell line NIH3T3. In vivo, administration of MU inhibited the expansion of osteolytic lesions on soft X‐rays in mouse breast cancer xenograft models. HA accumulation in bone metastatic lesions was perturbed peripherally. These data suggest that MU might be a therapeutic candidate for bone metastasis of breast cancer via suppression of HA synthesis and accumulation.

Inhibition of hyaluronan retention by 4-methylumbelliferone suppresses osteosarcoma cells in vitro and lung metastasis in vivo.

Background:Hyaluronan (HA) plays crucial roles in the tumourigenicity of many types of malignant tumours. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis. Several studies have shown its inhibitory effects on malignant tumours; however, none have focused on its effects on osteosarcoma.Methods:We investigated the effects of MU on HA accumulation and tumourigenicity of highly metastatic murine osteosarcoma cells (LM8) that have HA-rich cell-associated matrix, and human osteosarcoma cell lines (MG-63 and HOS).Results:In vitro, MU inhibited HA retention, thereby reducing the formation of functional cell-associated matrices, and also inhibited cell proliferation, migration, and invasion. Akt phosphorylation was suppressed by MU (1.0 mM). In vivo, although MU showed only a mild inhibitory effect on the growth of the primary tumour, it markedly inhibited (75% reduction) the development of lung metastasis. Hyaluronan retention in the periphery of the primary tumour was markedly suppressed by MU.Conclusion:These findings suggested that MU suppressed HA retention and cell-associated matrix formation in osteosarcoma cells, resulting in a reduction of tumourigenicity, including lung metastasis. 4-Methylumbelliferone is a promising therapeutic agent targeting both primary tumours and distant metastasis of osteosarcoma, possibly via suppression of HA retention.

Structure and function of inter-alpha-trypsin inhibitor heavy chains.

Inter-α-trypsin inhibitor family proteins are mainly detected in plasma and urine and comprise the common light chain bikunin and at least 6 closely related heavy chains. The bikunin moiety exhibits protease inhibitory activity and has been studied extensively; however, the heavy chains have been largely overlooked. Recent studies clearly indicate that the heavy chain moieties have important biological functions either in association with or independent of bikunin. Because the heavy chains comprise the main part of the protein structure of this family, it is important to understand their functions. This review summarizes the domain structural features of heavy chains, the heavy chain-interacting molecules identified thus far, and the association of heavy chains with diseases to encourage the discovery of novel heavy chains-interacting molecules and to gain a deeper insight into their functions.

Inter-α-Trypsin Inhibitor Attenuates Complement Activation and Complement-Induced Lung Injury

Complement activation is a central component of inflammation and sepsis and can lead to significant tissue injury. Complement factors are serum proteins that work through a cascade of proteolytic reactions to amplify proinflammatory signals. Inter-α-trypsin inhibitor (IaI) is an abundant serum protease inhibitor that contains potential complement-binding domains, and has been shown to improve survival in animal sepsis models. We hypothesized that IaI can bind complement and inhibit complement activation, thus ameliorating complement-dependent inflammation. We evaluated this hypothesis with in vitro complement activation assays and in vivo in a murine model of complement-dependent lung injury. We found that IaI inhibited complement activation through the classical and alternative pathways, inhibited complement-dependent phagocytosis in vitro, and reduced complement-dependent lung injury in vivo. This novel function of IaI provides a mechanistic explanation for its observed salutary effects in sepsis and opens new possibilities for its use as a treatment agent in inflammatory diseases.

A physiological function of serum proteoglycan bikunin: the chondroitin sulfate moiety plays a central role.

Bikunin is a small chondroitin sulfate proteoglycan that occurs in blood as the light chain of inter-α-trypsin inhibitor (ITI) family members. The relatively short chondroitin sulfate chain of bikunin shows a characteristic pattern of sulfation in both the linkage region and the chondroitin sulfate backbone. To the internal N-acetylgalactosamines in the lower sulfated portion near the non-reducing end, up to two “side” proteins could bind covalently via a unique ester bond to form “core protein-glycosaminoglycan-side protein” complexes, the ITI family. ITI molecules are synthesized in hepatocytes, and then secreted into circulation at high concentrations. In the presence of yet unidentified factors, the side proteins are transferred from chondroitin sulfate to hyaluronan by a transesterification reaction to form what has been described as the Serum-derived Hyaluronan-Associated Protein (SHAP)-hyaluronan complex. The formation of this complex is required for the stabilization of the extracellular matrix of fibroblasts, mesothelial cells, and cumuli oophori. When the gene for bikunin is inactivated, female mice exhibit severe infertility as a consequence of a defect of the side protein precursor in forming a complex with the hyaluronan in cumulus oophorus before ovulation. Therefore, the chondroitin sulfate moiety of bikunin is essential for presenting SHAP to hyaluronan, which is indispensable for ovulation and fertilization in mammals. Published in 2003.