Cell Type-specific Transcription of the α1(VI) Collagen Gene ROLE OF THE AP1 BINDING SITE AND OF THE CORE PROMOTER

Analysis of the chromatin of different cell types has identified four DNase I-hypersensitive sites in the 5′-flanking region of the α1(VI) collagen gene, mapping at −4.6, −4.4, −2.5, and −0.1 kilobase (kb) from the RNA start site. The site at −2.5 kb was independent from, whereas the other three sites could be related to, α1(VI) mRNA expression. The site at −0.1 kb was present in cells expressing (NIH3T3 and C2C12) but absent in cells not expressing (EL4) the mRNA; the remaining two sites were apparently related with high levels of mRNA. DNase I footprinting and gel-shift assays with NIH3T3 and C2C12 nuclear extracts have located a binding site for transcription factor AP1 (activator protein 1) between nucleotides −104 and −73. When nuclear extracts from EL4 lymphocytes were used, the AP1 site-containing sequence was bound by proteins not related to AP1. The existence of the hypersensitive site at −0.1 kb may be related to the binding of AP1 and of additional factors to the core promoter (Piccolo, S., Bonaldo, P., Vitale, P., Volpin, D., and Bressan, G. M. (1995) J. Biol. Chem. 270, 19583–19590). The function of the AP1 binding site and of the core promoter in the transcriptional regulation of the Col6a1gene was investigated by expressing several promoter-reporter gene constructs in transgenic mice and in cell cultures. The results indicate that regulation of transcription of the Col6a1gene by different cis-acting elements (core promoter, AP1 binding site and enhancers) is not completely modular, but the final output depends on the specific interactions among the three elements in a defined cell type

Analysis of Regulatory Regions of Emilin1 Gene and Their Combinatorial Contribution to Tissue-specific Transcription

The location of regions that regulate transcription of the murine Emilin1 gene was investigated in a DNA fragment of 16.8 kb, including the entire gene and about 8.7 and 0.6 kb of 5'- and 3'-flanking sequences, respectively. The 8.7-kb segment contains the 5'-end of the putative 2310015E02Rik gene and the sequence that separates it from Emilin1, whereas the 0.6-kb fragment covers the region between Emilin1 and Ketohexokinase genes. Sequence comparison between species identified several conserved regions in the 5'-flanking sequence. Most of them contained chromatin DNase I-hypersensitive sites, which were located at about -950 (HS1), -3100 (HS2), -4750 (HS3), and -5150 (HS4) in cells expressing Emilin1 mRNA. Emilin1 transcription initiates at multiple sites, the major of which correspond to two Initiator sequences. Promoter assays suggest that core promoter activity was mainly dependent on Initiator1 and on Sp1-binding sites close to the Initiators. Moreover, one important regulatory region was contained between -1 and -169 bp and a second one between -630 bp and -1.1 kb. The latter harbors a putative binding site for transcription factor AP1 matching the location of HS1. The function of different regions was studied by expressing lacZ constructs in transgenic mice. The results show that the 16.8-kb segment contains regulatory sequences driving high level transcription in all the tissues where Emilin1 is expressed. Moreover, the data suggest that transcription in different tissues is achieved through combinatorial cooperation between various regions, rather than being dependent on a single cis-activating region specific for each tissue

Emilin-1 controls arterial blood pressure by regulating contractility of vascular smooth muscle cells

Emilin-1 is a protein of the elastic extracellular matrix (ECM) expressed in interstitial connective tissue and in the cardiovascular system. Emilin1 null mice display hypotrophic remodeling of the wall of conductance arteries and increased blood pressure. The protein regulates the bioavailability of TGF-b by inhibiting proteolysis of the proTGF-b precursor to LAP/TGF-b, a complex from which the growth factor can be subsequently released for receptor binding. In the absence of Emilin-1, the amount of active TGF-b is increased. As Emilin-1 is expressed in blood vessels starting from early stages of embryonic development to adulthood, a key question concerning the function of the protein is whether the Emilin1-/- phenotype is the result of a developmental defect or the function of the protein is required for the regulation of blood pressure and arterial structure also in the adult. The conditional gene targeting procedure chosen to inactivate the Emilin1 gene in smooth muscle cells (SMCs) of adult mice included the use of floxed Emilin1 and CreERT2 (a tamoxifen inducible Cre recombinase) under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. Tamoxifen administration induced activity of Cre specifically in vascular and visceral SMCs, as revealed by X-gal staining of tissues from animals with the Rosa26R mutation. When Emilin1flox/flox mice carrying the Smmhc-CreERT2 transgene were given tamoxifen for 7 days, Emilin-1 disappeared completely in 10-12 days from start of treatment. In the same time, blood pressure increased of about 20 mmHg, a level that was stably maintained thereafter. The myogenic response of second branch meseteric arteries, evaluated using a pressure myograph, was found to be increased in Emilin1-/- mice. Additional experiments with aorta and mesenteric artery SMC cultures from control and mutant mice showed that lack of Emilin-1  enhanced phosphorylation of myosin light chain 20 when cells were stimulated with the a1-adrenergic receptor agonists phenylephrine or with angiotensin II. Moreover, basal cytosolic Ca2+ levels and calcium transients induced by stimulation with phenylephrine and angiotensin II were increased in SMCs from Emilin1-/- mutants. The data suggest that Emilin-1 expression is continuously required for regulation of blood pressure and that the increase of TGF-b activity induced by diminished Emilin-1  stimulates, likely through alteration of intracellular calcium homeostasis, contractility of vascular SMC to mechanical and chemical stimuli with ensuing hypertension

The elastin associated glycoprotein Gp 115: synthesis and secretion by cells in colture

Synthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents

Isolation and characterization of a 115,000-dalton matrix-associated glycoprotein from chick aorta.

Chick aortas were extracted sequentially with phosphate-buffered saline, 6 M guanidine HCl, and 6 M guanidine HCl containing dithioerythritol. The proteins present in the guanidine HCl + dithioerythritol extract were separated by DEAE-cellulose chromatography, and the fractions recovered were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five major glycoprotein components with apparent Mr = 205,000, 195,000, 150,000, 135,000, and 115,000 (gp 115) were identified. gp 115 was further studied since it was the only noncollagenous protein based on amino acid analysis. The protein was purified to homogeneity by preparative electrophoresis. Its amino acid composition was characterized by a high content of glutamic acid and arginine and a relatively high content of leucine, glycine, and alanine. The concentration of gp 115 in the guanidine HCl + dithioerythritol extract was about 15-fold that in the guanidine and saline extracts. Overall, about 80% of the protein was solubilized with guanidine HCl + dithioerythritol, suggesting that most of it formed large aggregates stabilized by disulfide bonds in vivo. Immunofluorescence studies with specific antibodies showed that gp 115 formed an extracellular fibrillar network in the aorta wall. One-dimensional finger printing with Staphylococcus aureus V8 protease and immunological studies indicated that the protein was unrelated to fibronectin and laminin. The data led us to conclude that gp 115 is a novel extracellular component of chick aorta

Mapping of binding sites for monoclonal antibodies to chick tropoelastin by recombinant DNA techniques.

A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV)n. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin

Glycoprotein 115, a glycoprotein isolated from chick blood vessels, is widely distributed in connective tissue.

An extracellular glycoprotein (gp 115) with an apparent Mr = 115,000 isolated from chick aortas (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin, 1983, J. Biol. Chem., 258:13262-13267), was used to immunize mice. The antisera were shown to specifically recognize gp 115 by numerous criteria: a major band around Mr = 115,000 plus minor bands of lower Mr were visible by immunoblotting on aorta extracts, and a similar pattern was observed with a monoclonal antibody; no cross-reactivity was detected by radioimmunobinding with other extracellular proteins, namely, fibronectin, laminin, and collagen types I, III, IV, V, and VI. Antigen distribution on frozen tissue sections from newborn chicks was investigated by using affinity-purified antibody. Strong immunoreactivity was always found in blood vessels. In the digestive tract, the fluorescent staining was localized both at the level of muscular layers and in the stromal matrix of the villi. Within skeletal muscle and myocardium, staining was associated with large connective tissue bundles and the matrix around each muscle fiber. Intense fluorescence was observed in the kidney, in smooth muscle cells rich areas of parabronchi, and within the portal space and along liver sinusoids. The antigen was not detected at the epidermal-dermal junction; immunoreactivity in the dermis was present as a diffuse fibrillar pattern. That the antigen detected by immunofluorescence in the various organs was indeed gp 115 was demonstrated by immunoblotting analysis: as in aorta extracts, a major band around Mr = 115,000 was detected in several tissues. Antibody-reacting material was also incorporated into the extracellular matrix produced by embryo smooth muscle cells grown in vitro and was organized as a meshwork of fine fibrils

Identification of a TGF-beta responsive element in the human elastin promoter.

In a previous report (Marigo, V., Volpin, D., and Bressan, G. M. (1993) Biochim. Biophys. Acta 1172, 31-36) it was shown that the elastin promoter contains a region mediating transcriptional activation by TGF-beta in aorta cells, but not in tendon fibroblasts from chick embryos. In this paper we have identified the sequence responsible for this effect by a combination of CAT assays with mutant constructs, DNase I footprinting and electrophoretic mobility shift assays. This TGF-beta responsive element binds different nuclear proteins in chick embryo aorta and tendon cells. Whereas association of the aorta protein(s) to the element is necessary for TGF-beta activation, binding of the tendon protein(s) has apparently no effect on promoter stimulation by the cytokine

Ultrastructural immuno-localization of tropoelastin in the chick eye.

Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma- of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties