John A. McDonald

Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme

We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2(-/-) embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2(-/-) embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2(-/-) explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, transformation in Has2(-/-) explants mediated by exogenous HA is inhibited by dominant-negative Ras. Collectively, our results demonstrate the importance of HA in mammalian embryogenesis and the pivotal role of Has2 during mammalian development. They also reveal a previously unrecognized pathway for cell migration and invasion that is HA-dependent and involves Ras activation.

Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

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 novoformation 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 UDP-GlcNAc 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 × 105 to 1 × 106 Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 × 105 to ∼2 × 106 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 × 105 to ∼2 × 106 Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 × 106 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.

Characterization and Molecular Evolution of a Vertebrate Hyaluronan Synthase Gene Family

The three mammalian hyaluronan synthase (HAS) genes and the related Xenopus laevis gene,DG42, belong to a larger evolutionarily conserved vertebrate HAS gene family. We have characterized additional vertebrate HAS genes from chicken (chas2 and chas3) andXenopus (xhas2, xhas3, and a uniqueXenopus HAS-related sequence, xHAS-rs). Genomic structure analyses demonstrated that all vertebrate HAS genes share at least one exon-intron boundary, suggesting that they evolved from a common ancestral gene. Furthermore, the Has2 andHas3 genes are identical in structure, suggesting that they arose by a gene duplication event early in vertebrate evolution. Significantly, similarities in the genomic structures of the mouseHas1 and Xenopus DG42 genes strongly suggest that they are orthologues. Northern analyses revealed a similar temporal expression pattern of HAS genes in developing mouse andXenopus embryos. Expression of mouse Has2, Has3, andXenopus Has1 (DG42) led to hyaluronan biosynthesis in transfected mammalian cells. However, only mouse Has2 and Has3 expressing cells formed significant hyaluronan-dependent pericellular coats in culture, implying both functional similarities and differences among vertebrate HAS enzymes. We propose that vertebrate hyaluronan biosynthesis is regulated by a comparatively ancient gene family that has arisen by sequential gene duplication and divergence.

Integrin-linked Protein Kinase Regulates Fibronectin Matrix Assembly, E-cadherin Expression, and Tumorigenicity

Fibronectin (Fn) matrix plays important roles in many biological processes including morphogenesis and tumorigenesis. Recent studies have demonstrated a critical role of integrin cytoplasmic domains in regulating Fn matrix assembly, implying that intracellular integrin-binding proteins may be involved in controlling extracellular Fn matrix assembly. We report here that overexpression of integrin-linked kinase (ILK), a newly identified serine/threonine kinase that binds to the integrin β1 cytoplasmic domain, dramatically stimulated Fn matrix assembly in epithelial cells. The integrin-linked kinase activity is involved in transducing signals leading to the up-regulation of Fn matrix assembly, as overexpression of a kinase-inactive ILK mutant failed to enhance the matrix assembly. Moreover, the increase in Fn matrix assembly induced by ILK overexpression was accompanied by a substantial reduction in the cellular E-cadherin. Finally, we show that ILK-overexpressing epithelial cells readily formed tumors in nude mice, despite forming an extensive Fn matrix. These results identify ILK as an important regulator of pericellular Fn matrix assembly, and suggest a novel critical role of this integrin-linked kinase in cell growth, cell survival, and tumorigenesis.

Assembly of extracellular matrix

A great challenge in understanding how different extracellular matrices assemble is to sort through the vast number of possible interactions between and among matrix molecules. The most profound insights are likely to come from patients with defined defects of matrix molecules and the use of transgenic mice or other experimental technologies that mimic the complexity of the human system.

Identification of a New Biological Function for the Integrin αvβ3: Initiation of Fibronectin Matrix Assembly

Fibronectin matrix assembly is a complex cellular process initiated by specific fibronectin-binding cell surface receptors. Although the integrin α5β1 has been implicated in the assembly of fibrone...

Growth factor and procollagen type I gene expression in human liver disease

BACKGROUND/AIMS Growth factors have been implicated in the pathogenesis of liver fibrosis, a major determinant of the clinical course of chronic liver disease. The aim of this study was to study the relationship of growth factor expression to inflammation and fibrosis in a variety of human liver diseases. METHODS We studied by in situ hybridization the expression of transforming growth factor (TGF) beta 1, platelet-derived growth factor (PDGF) A and PDGF-B, and procollagen type I (pro-I) messenger RNAs (mRNAs) in liver diseases of various etiologies. RESULTS Pro-I mRNA was expressed by mesenchymal cells at sites of inflammation and scarring, where TGF-beta 1 immunoreactivity was often found, and by perisinusoidal cells. TGF-beta 1 and PDGF-A mRNAs were expressed mainly by mononuclear cells and proliferating ductular cells. TGF-beta 1 mRNA was also expressed by perisinusoidal cells. PDGF-A gene expression was more common than that of PDGF-B. Pro-I and TGF-beta 1 expression correlated with both ductular proliferation and tissue inflammation, whereas PDGF-A and PDGF-B only correlated with ductular proliferation. CONCLUSIONS Our data suggest that TGF-beta 1 and PDGF are involved in human liver inflammation and fibrosis. The expression of growth factor mRNAs in proliferating ductular cells may indicate a role for these cells in liver fibrogenesis and may help explain the pathophysiology of conditions such as biliary atresia progressing to fibrosis despite the absence of marked inflammation.

Codon optimization markedly improves doxycycline regulated gene expression in the mouse heart

Tetracycline regulated gene expression in transgenic animals is potentially a very powerful technique (Furth et al., 1994; Gossen & Bujard 1992). We have utilized this system in an attempt to overcome the perinatal lethality resulting from constitutive transgenic expression in the heart (Valencik & McDonald, Am J Physiol Heart Circ Physiol 280: H361–H367). We found that compound hemizygous animals created by mating selected reverse tetracycline transactivator (rtTA) and transresponder (TR) lines display tightly regulated TR expression in the heart. However, we identified two fundamental problems. First, codon usage bias appeared to severely limit the expression of the rtTA driven by the cardiac α-myosin heavy chain promoter. Second, co-injection of rtTA and TR transgenes led to compound hemizygous animals that exhibited unregulated TR gene expression. Codon optimization of the rtTA construct leads to marked improvement (increasing the average induction from 20-fold to 832-fold) in cardiac myocyte expression. The resulting opt-rtTA lines can be bred to homozygosity, facilitating rapid screening of F0 TR animals for doxycycline regulated transgene expression.

Heparin Inhibits Lung Branching Morphogenesis: Potential Role of Smooth Muscle Cells in Cleft Formation

Lung branching morphogenesis is the process by which the embryonic lung undergoes repetitive branching to form the bronchial tree. This process occurs during the pseudoglandular stage of lung development and requires epithelial-mesenchymal interactions. Coinciding with lung branching morphogenesis is the appearance of parabronchial smooth muscle cells (PSMCs) and the accumulation of extracellular matrices (ECMs) around the developing airways. The authors previously reported in preliminary form that heparin prevents the branching of murine lung explants (Roman et al., Am Rev Respir Dis. 1991; 143:A401); this article corroborates those early observations and expands them by demonstrating that heparin results in disruption of PSMC distribution and abnormal organization of ECMs around the developing airways. These changes were associated with inhibition of lung branching morphogenesis in the absence of effects on cell proliferation. The data provide further support for the role of ECMs in lung branching morphogenesis, and points to PSMCs as potential players in this process.

Integrins Minireview Series

Integrins constitute a large family of ab heterodimeric cell surface, transmembrane proteins that recognize a large number of extracellular ligands through a metal ion-dependent interaction. The prescient term “integrin” reflects their role in integrating cell adhesion and migration with the cytoskeleton (1). Their biological and medical importance is underscored by inherited diseases causing bleeding (Glanzmann’s thromasthenia) and infection (leukocyte adhesion deficiency). Additional important roles for integrins in immune, inflammatory and infectious disease have been revealed by in vitro and gene ablation studies. The minireviews in this and following issues update our understanding of integrins in four general areas: structure and ligand binding (by Edward F. Plow, Thomas A. Haas, Li Zhang, Joseph Loftus, and Jeffrey W. Smith), interactions with the actin cytoskeleton and regulation of ligand binding (by David A. Calderwood, Sanford J. Shattil, and Mark H. Ginsberg), leukocyte integrins (by Estelle S. Harris, Thomas M. McIntyre, Stephen M. Prescott, and Guy A. Zimmerman), and modulation of integrin function by lateral associations with other plasma membrane-spanning molecules (by Anne Woods and John R. Couchman). Early on, it was noted that integrins isolated from cells solubilized with detergents often bound poorly to immobilized ligands. Now, it is clear that binding of integrins to ligands is regulated by intracellular signaling, so-called “inside-out” signaling. This provides a dynamic mechanism for regulating cell adhesion, e.g. during cell adhesion, migration, or platelet aggregation. Ligand binding by integrins is modulated by changes in avidity, e.g. by clustering integrins and increasing interactions with multivalent ligands or by increasing binding affinity by conformational changes as reviewed by Calderwood et al. in the second minireview in this series. Calderwood et al. also provide a comprehensive overview of known interactions between integrin cytoplasmic domains and molecules associated with the actin cytoskeleton. Ligand binding triggers intracellular signaling cascades. Intriguingly, ligand engagement also regulates the response of attached cells to growth factors, a relationship anticipated by Paul Bornstein’s concept of “dynamic reciprocity” between cells and the extracellular matrix (2). In this context, integrins sample the extracellular microenvironment, reporting via intracellular signaling and regulating responses including growth, cellular differentiation, and even death. Integrins do not act alone; indeed, they lack catalytic activity and depend upon an extensive array of extracellular and intracellular partners to localize to membrane microdomains, recruit signaling molecules, and trigger intracellular signaling. The fourth minireview in this series by Woods and Couchman focuses primarily on two classes of membrane-spanning molecules implicated in modulation of integrins, tetraspans, and syndecans, although many other molecules have been implicated. The medical importance of leukocyte integrins and platelet integrins has led to important insights into their chemistry and biology. The development of pharmaceuticals targeting integrins is an important part of many drug discovery portfolios aimed at allergy and inflammatory disease, atherosclerosis, and clotting. In the third minireview of this series Zimmerman and co-workers enlarge on the general theme of integrin structure, ligand binding, and regulation focusing on leukocyte integrins. Collectively, these reviews constitute a good dictionary for the increasingly complex language of integrins.