Partial cloning and sequencing of chick fibrillin-1 cDNA.

The recent identification of numerous matrix genes and gene products has allowed a detailed examination of their roles in development. Two of these extracellular matrix proteins, fibrillin-1 and fibrillin-2, are components of the elastin-associated microfibrils. Given what is known about the distribution of the fibrillins in normal tissues and the abnormalities that result when mutations occur, a basic hypothesis has emerged: fibrillin-1 is primarily responsible for load bearing and providing structural integrity, whereas fibrillin-2 may be a director of elastogenesis. Nevertheless, examination of phenotypes in disorders caused by mutations in fibrillin-1 or fibrillin-2 suggests some common functions. To better understand these similar and diverse roles, it would be helpful to examine these proteins during chick development. To accomplish this goal, it is first necessary to characterize the chick homologs of the known fibrillins. In this study, the partial chick FBN1 cDNA was identified by polymerase chain reaction-aided cloning as a first step toward elucidating these goals. Sequence analysis indicated that there is striking conservation between chick and mammalian fibrillin-1 at the DNA and protein levels. Antisense and sense riboprobes were synthesized and used in in situ hybridization in stage 14 chick embryos and high levels of FBN1 transcripts were observed in the heart.

Homocysteine signal cascade: production of phospholipids, activation of protein kinase C, and the induction of c-fos and c-myb in smooth muscle cells.

Hyperhomocysteinemia has been recognized as an independent risk factor for cerebral, coronary, and peripheral atherosclerosis. To examine the contribution of homocysteine (H[cys]) in the pathogenesis of vascular diseases, we sought to determine whether the H[cys] effect on vascular smooth muscle (VSMC) proliferation is mediated by a specific receptor/transporter or is due to an interaction with growth factors or cytokines. We show that H[cys] induced c-fos and c-myb and increased DNA synthesis and cell proliferation 12-fold in neural crest-derived VSMC (N-VSMC). The H[cys] effect on N-VSMC proliferation is inhibited by Mk-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, a glutamate-gated calcium ion channel receptor, and CGS 19755, a competitive antagonist of NMDA-type glutamate receptor. H[cys] stimulates the synthesis of mass amounts of sn-1,2 diacylglycerol, and activates protein kinase C translocation from the nucleus and cytoplasm to cell membranes. Furthermore, protein kinase C inhibitors block the growth effect mediated by H[cys]. These findings indicate that H[cys]-mediated responses are coupled to diacylglycerol-dependent protein kinase C activation. Our results suggest that homocysteine activates a receptor/transporter-like factor in neural crest derived smooth muscle.

Differential response of mesoderm- and neural crest-derived smooth muscle to TGF-beta1: regulation of c-myb and alpha1 (I) procollagen genes.

Previously, we demonstrated that avian vascular smooth muscle cells (VSMC) derived from embryonic abdominal and thoracic aorta grow differently in the presence of transforming growth factor beta (TGF-beta1) and platelet-derived growth factor (PDGF-BB) (Wrenn et al., In Vitro Cell. Dev. Biol. 29, 73-78, 1992). The thoracic VSMC (N-VSMC) are derived from neural crest, and therefore differentiate from ectoderm; the abdominal VSMC (M-VSMC) are derived from mesoderm. The present study was designed to identify factors that mediate the differential responses of the VSMC to TGF-beta1. We found that TGF-beta1 increased DNA synthesis by approximately sevenfold in N-VSMC. Levels of both alpha1 (I) procollagen and c-myb mRNAs were markedly induced in N-VSMC treated with TGF-beta1. Chimeric plasmids containing up to 3.5 kb of alpha1 (I) procollagen 5' flanking DNA were induced to equivalent levels as procollagen mRNA in N-VSMC. However, TGF-beta1 increased DNA synthesis by threefold in M-VSMC; there was no effect on alpha1 (I) procollagen expression, and c-myb was not expressed, as demonstrated by immunohistochemistry staining and RNA analyses. Antisense c-myb oligodeoxynucleotides blocked the TGF-beta1 induction of alpha1 (I) procollagen and the growth of N-VSMC. The increase in DNA synthesis by M- and N-VSMC was correlated with the secretion of PDGF-AA, and staurosporine and antibodies directed against PDGF-AA suppressed DNA synthesis. Our results demonstrate that TGF-beta1 activity and c-myb expression modulate the expression of alpha1 (I) collagen and cell proliferation in neural crest-derived smooth muscle. The regulation of these events by TGF-beta1 may be important during morphogenesis of blood vessels and vascular diseases.

Embryonic lineage of vascular smooth muscle cells determines responses to collagen matrices and integrin receptor expression.

Developmental studies have demonstrated that the vascular smooth muscle cells (VSMC) present within the elastic arteries are differentiated from two definitive origins, the neural crest and the mesoderm. Cells from these distinct progenitors differ in their ability to determine long-range spatial order of the extracellular matrix, in proliferative responses, and in the expression of critical proteins. The present study utilizes collagen gel contraction assays and the analysis of integrin receptor subunit expression to evaluate cell-matrix interactions. In the presence of serum and transforming growth factor-beta 1 (TGF) or TGF-beta 1 alone, VSMC isolated from the abdominal aorta (AA-VSMC) were found to contract collagen matrices to a significantly greater extent than VSMC from the thoracic aorta (TA-VSMC). However, in TA-VSMC, beta 1 integrin and gel contraction were stimulated only in the presence of serum factors. Metabolic labeling and immunoprecipitation of integrin subunits revealed that TGF-beta 1 induced beta 1 and alpha 5 integrin subunits in AA-VSMC four-and ninefold, respectively. AA-VSMC gel contraction stimulated by serum and TGF-beta 1 alone was inhibited with anti-beta 1 integrin antibody by 70 and 100%, respectively. However, the beta 1 integrin-specific antibody inhibited serum-induced TA-VSMC gel contraction by 25%. The data suggest that vascular smooth muscle cell ontogeny is an important determinant of cell function, phenotype, and response to growth factors such as TGF-beta 1.

Expression of elastin, smooth muscle alpha-actin, and c-jun as a function of the embryonic lineage of vascular smooth muscle cells.

In the avian embryo, vascular smooth muscle cells (VSMC) in the aortic arch (elastic) arteries originate in the neural crest, whereas other VSMC develop from local mesoderm. These two lineages have been shown previously to be significantly different in the timing and expression of the smooth muscle phenotype and in their respective abilities to produce an orderly elastic matrix. Two differing kinds of VSMC also have been shown in mammals. In the experimental absence of neural crest (NC) in the avian embryo, the matrix is spatially disordered. The molecular basis of the difference between the normal NC-VSMC and the surrogate mesodermal (MDM)-VSMC has not previously been investigated. In this study the expression of vascular smooth muscle alpha-actin, tropoelastin, c-fos and c-jun were examined via immunoblotting, immunohistochemistry, Northern blot, and/or transcription run-on assays. Control avian VSMC of NC origin were compared with experimental MDM-derived VSMC that populate the cardiac outflow after surgical ablation of the NC. The results show that, when they are grown under identical conditions in vitro or freshly removed from an embryonic vessel, surrogate MDM-VSMC express about 10 times more alpha-actin and tropoelastin than the normal NC-VSMC; and MDM-VSMC express up to 15 times more c-jun, whereas c-fos was not different. These results show profound heterogeneity in the regulation of VSMC-specific genes that is based in the embryonic lineage of the cells.

Characterization of progesterone biosynthesis in a transformed granulosa cell line.

The study of regulation of steroidogenesis in primary cultures of rat granulosa cells is difficult because the cells do not undergo more than one cell doubling in culture. Furthermore, there is size and steroidogenic heterogeneity in granulosa cells and it is difficult to obtain pure, functionally defined populations. Hence, it is advantageous to develop a homogeneous population of granulosa cells. In this report we describe the characterization of one such cell line (Rao-gcl-29) developed from diethylstilbestrol treated immature rat granulosa cells by transformation with SV40 T antigen. In this cell line cyclic AMP analogs induce high levels of progesterone biosynthesis, though there was no effect on estradiol biosynthesis. Also, FSH and hCG have no effect on progesterone biosynthesis. In the presence of FBS medium (20% fetal bovine serum in DMEM/F-12) and enriched medium (10% fetal bovine serum, 10% horse serum and 2% UltraSer G in DMEM/F-12 medium), 1 mM cAMP analogs induce high levels of progesterone biosynthesis up to 96 h. Ultrastructural features of the cell line resemble those of primary granulosa cells, in addition to forming gap junctions. Cyclic AMP analogs also induced cytochrome P450scc mRNA in this cell line by 48 h, and this effect is apparent by 24 h. Thus, this cell line could be useful in understanding the molecular mechanisms of regulation of cytochrome P450scc gene regulation.

Glucocorticoid receptors in human fibroblasts derived from normal dermis and keloid tissue.

Hydrocortisone stimulates proliferation and System A amino acid transport in cultured human fibroblasts, while decreasing production of collagen. Fibroblasts isolated from keloid tissue have an unusual glucocorticoid response; they are hyporesponsive with regard to proliferation and collagen production but hyperresponsive with regard to the induction of System A amino acid transport (Russell, J. D., Russell, S. B., and Trupin, K. M. (1978) J. Cell. Physiol. 97, 221-229; Russell, S. B., Russell, J. D., and Trupin, J. S. (1982) J. Biol. Chem. 256, 9525-9531). We show here that despite these differences, the glucocorticoid receptors of keloid cells do not differ from those of normal dermal fibroblasts in steroid specificity, dissociation constant (Kd), total number of binding sites (Bmax), or nuclear binding of glucocorticoid-receptor complexes. A single glucocorticoid binding species of molecular weight 93,000 was found in both cell types. A monolayer assay for glucocorticoid receptor binding is described which facilitates analysis of multiple strains of cultured cells. This assay gives the same specificity and dissociation constants as the conventional cytosol assay, but it is more sensitive. The magnitude of induction of System A amino acid transport was found to be directly proportional to glucocorticoid receptor occupancy in both keloid-derived and normal fibroblasts. This induction requires serum, which can be replaced with 1 nM insulin.