William G. Taylor

Secreted Frizzled-related Protein-1 Binds Directly to Wingless and Is a Biphasic Modulator of Wnt Signaling

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields ∼3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.

Keratinocyte growth factor

Keratinocyte growth factor (KGF) is a member of the heparin‐binding fibroblast growth factor family (FGF‐7) with a distinctive pattern of target‐cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal‐epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr‐2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex sterorid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.

Processing of hepatocyte growth factor to the heterodimeric form is required for biological activity

Hepatocyte growth factor is a plasminogen‐like molecule with diverse biological effects. Although it is synthesized as a single chain polypeptide, it was originally purified as a disulfide‐linked haterodimer which was generated by an internal proteolytic event. Subsequent work indicated that preparations consisting largely of the monomeric form also exhibited potent activity. By using a combination of protease inhibition and site‐directed mutagenesis, we established that conversion of the single chain polypeptide to the hoterodimer occurred during the bioassay and was required ror mitogenic and motogenic activity.

Wnt-3a and Dickkopf-1 Stimulate Neurite Outgrowth in Ewing Tumor Cells via a Frizzled3- and c-Jun N-Terminal Kinase-Dependent Mechanism

ABSTRACT Recombinant Wnt-3a stimulated the rapid formation of elongated processes in Ewing sarcoma family tumor (ESFT) cells that were identified as neurites. The processes stained positively for polymerized actin and microtubules as well as synapsin I and growth-associated protein 43. Inhibition of the Wnt receptor, Frizzled3 (Fzd3), with antiserum or by short interfering RNA (siRNA) markedly reduced neurite extension. Knockdown of Dishevelled-2 (Dvl-2) and Dvl-3 also suppressed neurite outgrowth. Surprisingly, disruption of the Wnt/Fzd/lipoprotein receptor-related protein (LRP) complex and the associated β-catenin signaling by treating cells either with the Wnt antagonist Dickkopf-1 (Dkk1) or LRP5/LRP6 siRNA enhanced neuritogenesis. Neurite outgrowth induced by Dkk1 or with LRP5/LRP6 siRNA was inhibited by secreted Fzd-related protein 1, a Wnt antagonist that binds directly to Wnt. Moreover, Dkk1 stimulation of neurite outgrowth was blocked by Fzd3 siRNA. These results suggested that Dkk1 shifted endogenous Wnt activity from the β-catenin pathway to Fzd3-mediated, noncanonical signaling that is responsible for neurite formation. In particular, c-Jun amino-terminal kinase (JNK) was important for neurite outgrowth stimulated by both Wnt-3a and Dkk1. Our data demonstrate that Fzd3, Dvl, and JNK activity mediate Wnt-dependent neurite outgrowth and that ESFT cell lines will be useful experimental models for the study of Wnt-dependent neurite extension.

Fluorescent light-induced chromosome damage in human IMR-90 fibroblasts role of hydrogen peroxide and related free radicals

Exposure of human fibroblasts (IMR-90) to cool-white fluorescent light causes chromatid breaks and exchanges. This chromatid damage is caused largely by the production of hydrogen peroxide (H2O2) since it can be prevented almost completely by the addition of catalase. In support of this conclusion, exogenous H2O2 is shown to induce chromatid breaks. The clastogenic amounts of H2O2 generated during light exposure are formed within the cell since cells illuminated in saline showed the same extent of damage as cells in culture medium. Addition of selenite to the cultures during light exposure significantly decreases the chromatid damage in a dose-related manner and may be necessary to maintain sufficient activity of glutathione peroxidase. The free hydroxyl radical, . OH, appears to be partially responsible for the light-induced chromatid damage. Of the free-radical scavengers tested, i.e., mannitol, vitamin E, and dimethyl sulfoxide, only mannitol, which scavenges . OH, significantly decreases the light-induced chromatid damage. Thus, both . OH and H2O2 formed within the cell during light exposure are agents that directly or indirectly cause chromatid damage.

DIFFERENTIAL EXPRESSION OF THE KERATINOCYTE GROWTH FACTOR (KGF) AND KGF RECEPTOR GENES IN HUMAN VASCULAR SMOOTH MUSCLE CELLS AND ARTERIES

Keratinocyte growth factor (KGF) is a secreted member of the fibroblast growth factor (FGF) family of heparin‐binding proteins. Studies reported to date indicate that it functions primarily as an important paracrine mediator of epithelial cell growth and differentiation. KGF appears to act via binding to a specific FGF receptor‐2 isoform generated by an alternative splicing mechanism. To determine whether KGF may play a role in vascular smooth muscle cell (SMC) biology, we investigated KGF and KGF receptor gene expression in human SMC cultured in vitro as well as in several human nonatherosclerotic artery and atheroma specimens. KGF mRNA but not KGF receptor mRNA was expressed by SMCs, as determined by Northern blot hybridization analysis or reverse transcription‐polymerase chain reaction assays, respectively. Additional experiments demonstrated that (1) human SMCs produce and secrete mitogenically active KGF and that (2) the cytokine interleukin‐1 increases KGF mRNA and protein levels in human SMCs. We also found that KGF transcripts but not KGF receptor transcripts were expressed in control and atherosclerotic human arteries. Taken together, these results indicate that KGF is unlikely to be involved in SMC growth regulation unless it can function intracellularly or interact with a presently unidentified KGF receptor. J. Cell. Physiol. 173:380–386, 1997. Published 1997 Wiley‐Liss, Inc. This article was prepared by a group of United States government employees and non‐United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

A new culture medium for human skin epithelial cells

SummaryA new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.

Oxygen and light effects on chromosomal aberrations in mouse cells in vitro.

Abstract Decreasing the oxygen concentration in the gas phase from 18% (atmospheric) to 1% decreased the frequency of chromosomal aberrations in mass cultures of cells from adult lung and embryos of two inbred mouse strains. Both the rate of shift from the diploid number and the incidence of abnormal chromosomes were decreased at the lower oxygen level. Similarly, shielding mouse cells from room lights (cool white, fluorescent) during routine fluid renewals reduced the incidence of abnormal chromosomes, particularly minutes and metacentrics. The increased incidence of chromosomal abnormalities on exposure of cells to light and high oxygen presumably results from a photodynamic reaction affecting the DNA or associated proteins of the chromatin fibers.

Biological efficacy of several commercially available peptones for mammalian cells in culture

Abstract The biological efficacy of several commercially available peptones was evaluated for three continuous mammalian cell lines. Peptic peptone, a peptic digest of beef lung, and Bacto-peptone were found to be most suitable.

Chapter 25 Peptones as Serum Substitutes for Mammalian Cells in Culture

A chemically characterized protein-free and reproducible medium is not only desirable but also ultimately necessary in order to study the biosynthetical capacities of cells in vitro … growing mammalian cells in chemically defined media may be a matter of gradually learning to handle a specific cell strain under the more demanding culture conditions of protein-free media rather than a matter of always basically altering the cell strain. — Evans et al. (1964).