Steve Benson

Peroxisome proliferator-activated receptor (PPAR)-γ expression in human vascular smooth muscle cells: inhibition of growth, migration, and c-fos expression by the peroxisome proliferator-activated receptor (PPAR)-γ activator troglitazone

This study was conducted to determine whether cultured human coronary artery and aorta vascular smooth muscle (VSM) cells express the nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma); whether the thiazolidinedione troglitazone, a ligand for PPARgamma, would inhibit c-fos expression by these cells; and whether troglitazone would inhibit proliferation and migration induced in these cells by mitogenic growth factors. Using immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, we show that both human aorta and coronary artery VSM cell lines expressed PPARgamma protein and mRNA for both PPARgamma isoforms, PPARgamma1 and PPARgamma2. Immunocytochemical staining localized the PPARgamma protein primarily within the nucleus. Troglitazone inhibited basic fibroblast growth factor and platelet-derived growth factor-BB induced DNA synthesis in a dose-dependent manner and downregulated the growth-factor-induced expression of c-fos. Troglitazone also inhibited the migration of coronary artery VSM cells along a platelet-derived growth factor-BB concentration gradient. These findings demonstrate for the first time the expression and nuclear localization of PPARgamma in human coronary artery and aorta VSM cells. The data also suggest that the downregulation of c-fos expression, growth-factor-induced proliferation, and migration by VSM may, in part, be mediated by activation of the PPARgamma receptor.

Primary mesenchyme cells of the sea urchin embryo require an autonomously produced, nonfibrillar collagen for spiculogenesis

A collagen molecule in the sea urchin embryo was characterized by analysis of a 2.7-kb cDNA clone. This clone, Spcoll, was obtained by screening a gastrula stage Strongylocentrotus purpuratus cDNA library with a 237-bp genomic clone encoding a collagen-like sequence previously isolated by Venkatesan et al. (1986). DNA sequence analysis of the cDNA clone demonstrated the nonfibrillar nature of the encoded molecule--13 interruptions of the Gly-X-Y repeat motif were found in the 85-kDa open reading frame. The mRNA of approximately 9 kb accumulated specifically in mesenchyme cells of the embryo through development to the pluteus larva. Polyclonal antibodies generated against a Spcoll-beta-galactosidase fusion protein were utilized to identify and localize the native Spcoll. This collagen molecule of approximately 210 kDa was deposited into the blastocoel by the primary mesenchyme cells. When primary mesenchyme cells were cultured in vitro, Spcoll was secreted into the media and accumulated at sites of cell-substrate interaction. Addition of anti-Spcoll antibodies to primary mesenchyme cell cultures selectively inhibited spiculogenesis, whereas other antibodies had no inhibitory effect. Since collagen is not a component of the organic matrix of spicules (Benson et al., 1986), these results suggest that the autonomous production of Spcoll by differentiating mesenchyme cells in turn influences the point in differentiation at which these cell initiate biomineralization.

The synthesis and secretion of collagen by cultured sea urchin micromeres

Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.

Expression of an embryonic spicule matrix gene in calcified tissues of adult sea urchins

The sea urchin spicule is composed of CaCO3 associated with an organic matrix containing at least 10 proteins. We have previously shown that one of these proteins, a 50-kDa glycoprotein (SM 50), is encoded by a gene which is only transcribed in cells of the micromere-mesenchyme lineage. In this report we examined RNA from five adult tissues: ovary, coelomocytes, intestine, tube feet, and spine for the presence of SM 50 transcripts and protein. Only RNA from cells of spine and tube feet hybridized to a SM 50-specific probe on Northern blots. These same two tissues undergo biomineralization and contain a protein which is immunologically related to the SM 50 protein. The restricted accumulation of SM 50 transcripts and protein suggests that the differentiation of biomineralizing mesenchyme cells in embryonic and adult tissue may utilize the same genetic program.

Analysis of competence in cultured sea urchin micromeres

Sea urchin embryo micromeres form the primary mesenchyme, the skeleton-producing cells of the embryo. Almost nothing is known about nature and timing of the embryonic cues which induce or initiate spicule formation by these cells. A related question concerns the competence of the micromeres to respond to the cues. To examine competence in this system we have exposed cultured sea urchin micromeres to an inducing medium containing horse serum for various periods of time and have identified a period when micromeres are competent to respond to serum and form spicules. This window, between 30 and 50 h after fertilization, corresponds to the time when mesenchyme cells in vivo are aggregating and beginning to form the syncytium in which the spicule will be deposited. The loss of competence after 50 h is not due to impaired cell health since protein synthesis at this time is not significantly different from controls. Likewise the accumulation of a spicule matrix mRNA (SM 50) and a cell surface glycoprotein (msp 130), both indices of micromere/mesenchyme differentiation, still occurs in cells that have lost competence to respond to serum by forming spicules. These experiments demonstrate that the acquisition and loss of competence in these cells are regulated developmental events and establish an in vitro system for the identification of the molecular basis for inductive signal recognition and signal transduction.

Developmental characterization of the gene for laminin α-chain in sea urchin embryos

Abstract We describe the isolation and characterization of a cDNA clone encoding a region of the carboxy terminal globular domain (G domain) of the α-1 chain of laminin from the sea urchin, Strongylocentrotus purpuratus. Sequence analysis indicates that the 1.3 kb cDNA (spLAM-α) encodes the complete G2 and G3 subdomains of sea urchin a-laminin. The 11 kb spLAM-α mRNA is present in the egg and declines slightly in abundance during development to the pluteus larva. The spLAM-α gene is also expressed in a variety of adult tissues. Whole mount in situ hybridization of gastrula stage embryos indicates that ectodermal and endodermal epithelia and mesenchyme cells contain the spLAM-α mRNA. Immunoprecipitation experiments using an antibody made to a recombinant fusion protein indicates spLAM-α protein is synthesized continuously from fertilization as a 420 kDa protein which accumulates from low levels in the egg to elevated levels in the pluteus larva. Light and electron microscopy identify spLAM-α as a component of the basal lamina. Blastocoelic microinjection of an antibody to recombinant spLAM-α perturbs gastrulation and skeleton formation by primary mesenchyme cells suggesting an important role for laminin in endodermal and mesodermal morphogenesis.

Prolyl hydroxylase activity during sea urchin development

Prolyl hydroxylase activity appears at the blastula stage of development in the sea urchin Strongylocentrotus purpuratus and increases over 7-fold by the prism larva stage. The enzyme requires ascorbate, ferrous ions, and α-ketoglutarate for maximum activity and is inhibited by α,α′-dipyridyl. The significance of prolyl hydroxylase activity in embryonic collagen metabolism and morphogenesis is discussed.