Vascular smooth muscle Emilin-1 is a regulator of arteriolar myogenic response and blood pressure.

OBJECTIVE: Emilin-1 is a protein of elastic extracellular matrix involved in blood pressure (BP) control by negatively affecting transforming growth factor (TGF)-ß processing. Emilin1 null mice are hypertensive. This study investigates how Emilin-1 deals with vascular mechanisms regulating BP. METHODS AND RESULTS: This study uses a phenotype rescue approach in which Emilin-1 is expressed in either endothelial cells or vascular smooth muscle cells of transgenic animals with the Emilin1(-/-) background. We found that normalization of BP required Emilin-1 expression in smooth muscle cells, whereas expression of the protein in endothelial cells did not modify the hypertensive phenotype of Emilin1(-/-) mice. We also explored the effect of treatment with anti-TGF-ß antibodies on the hypertensive phenotype of Emilin1(-/-) mice, finding that neutralization of TGF-ß in Emilin1 null mice normalized BP quite rapidly (2 weeks). Finally, we evaluated the vasoconstriction response of resistance arteries to perfusion pressure and neurohumoral agents in different transgenic mouse lines. Interestingly, we found that the hypertensive phenotype was coupled with an increased arteriolar myogenic response to perfusion pressure, while the vasoconstriction induced by neurohumoral agents remained unaffected. We further elucidate that, as for the hypertensive phenotype, the increased myogenic response was attributable to increased TGF-ß activity. CONCLUSIONS: Our findings clarify that Emilin-1 produced by vascular smooth muscle cells acts as a main regulator of resting BP levels by controlling the myogenic response in resistance arteries through TGF-ß.

Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI deficiency.

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.

An enhancer required for transcription of the Col6a1 gene in muscle connective tissue is induced by signals released from muscle cells.

Collagen VI is a survival factor for skeletal muscle produced by endomysial cells and localized in connective tissue around muscle fibers. Mutations of its genes (COL6A1, COL6A2 and COL6A3) cause two muscular disorders, Bethlem myopathy and Ullrich disease. Expression of Collagen VI is highly dynamic during development, suggesting that developmental and homeostatic cues of the muscle microenvironment are relevant to confine its expression in this tissue. In face of the large body of work highlighting the relevance for human diseases of the adhesion of muscle cells with their surrounding extracellular matrix, remarkably little is known on how myogenic cells control gene expression in the connective tissue cells that produce such matrix. By expressing promoter-lacZ constructs in transgenic mice, we identify a Col6a1 gene enhancer region that is necessary for activation of transcription in connective tissue cells associated with skeletal muscle. By means of a lacZ transgenic mouse line crossed in metD/D mutant background, in which muscles of limb buds fail to form, we provide evidence that the presence of cells of the myogenic lineage is needed for enhancer activation in mesenchymal cells. Accordingly, lack of myogenic cells in limb buds of metD/D mice reduces Collagen VI deposition in connective tissue. The Col6a1 enhancer characterized here is conserved in mammals and may be relevant in some cases of heritable diseases of Collagen VI.

EMILIN-1 deficiency induces elastogenesis and vascular cell defects.

EMILINs constitute a family of genes of the extracellular matrix with high structural similarity. Four genes have been identified so far in human and mouse. To gain insight into the function of this gene family, EMILIN-1 has been inactivated in the mouse by gene targeting. The homozygous animals were fertile and did not show obvious abnormalities. However, histological and ultrastructural examination revealed alterations of elastic fibers in aorta and skin. Formation of elastic fibers by mutant embryonic fibroblasts in culture was also abnormal. Additional alterations were observed in cell morphology and anchorage of endothelial and smooth muscle cells to elastic lamellae. Considering that EMILIN-1 is adhesive for cells and that the protein binds to elastin and fibulin-5, EMILIN-1 may regulate elastogenesis and vascular cell maintenance by stabilizing molecular interactions between elastic fiber components and by endowing elastic fibers with specific cell adhesion properties.

Overlapping, complementary and site-specific expression pattern of genes of the EMILIN/Multimerin family.

The EDEN gene superfamily comprises genes that contain the EMI domain, a structural motif recently identified in proteins of the extracellular matrix. We report here the detailed expression pattern of genes of the EMILIN/Multimerin family, the most numerous group of EDEN superfamily, during mouse development. In situ hybridization has revealed that the EMILIN/Multimerin genes are particularly expressed in the cardio-vascular system and in mesenchymal cells. In general, the territories of expression of each gene are partially overlapping or complementary with that of other members of the family and, usually, more than one gene of the family is active in different tissues, consistent with the possibility of functional compensation. The analysis is particularly relevant in the interpretation of gene targeting experiments.

Expression of the EMILIN-1 gene during mouse development.

Expression of EMILIN-1, the first member of a newly discovered family of extracellular matrix genes, has been investigated during mouse development. EMILIN-1 mRNA is detectable in morula and blastocyst by RT-PCR. First expression of the gene is found by in situ hybridization in ectoplacental cone in embryos of 6.5 days and in extraembryonic visceral endoderm at 7.5 days. The allantois is also labeled. Staining of ectoplacental cone-derived secondary trophoblast giant cells and spongiotrophoblast is strong up to 11.5 days and then declines. In the embryo, high levels of mRNA are initially expressed in blood vessels, perineural mesenchyme and somites at 8.5 days. Later on, intense labeling is identified in the mesenchymal component of organs anlage (i.e. lung and liver) and different mesenchymal condensations (i.e. limb bud and branchial arches). At late gestation staining is widely distributed in interstitial connective tissue and smooth muscle cell-rich tissues. The data suggest that EMILIN-1 may have a function in placenta formation and initial organogenesis and a later role in interstitial connective tissue.

Convergence of p53 and TGF-beta signaling networks.

p53 is a protein with many talents. One of the most fundamental is the ability to act as essential growth checkpoint that protects cells against cellular transformation. p53 does so through the induction of genes leading to growth arrest or apoptosis. Most of the studies focusing on the mechanisms of p53 activity have been performed in cultured cells upon treatment with well-established p53-activating inputs, such as high doses of radiations, DNA-damaging drugs and activated oncogenes. However, how the tumor suppressive functions of p53 become concerted with the extracellular cues arriving at the cell surface during tissue homeostasis, remains largely unknown. Intriguingly, two recent papers have shed new light into this unexplored field, indicating that p53 plays a key role in TGF-beta-induced growth arrest and, unexpectedly, in the developmental effects of TGF-beta in early embryos. Here we review and comment on these findings and on their implications for cancer biology.

Lentiviral-mediated RNAi in vivo silencing of Col6a1, a gene with complex tissue specific expression pattern.

RNA interference (RNAi) through the use of lentiviral vectors is a valuable technique to induce loss of function mutations in mammals. Although very promising, the method has found only limited application and its general applicability remains to be established. Here we analyze how different factors influence RNAi mediated silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue specific expression. Our results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of insertion of the provirus. Although low copy number may produce efficient interference with low frequency, the general trend is that the number of integrated proviral copies determines the level of silencing and the severity of phenotypic traits. The site of insertion not only determines the overall intensity of expression of the small interfering RNA (siRNA), but also introduces slight variability of silencing in different organs. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline-inducible production of siRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of siRNA synthesis was not complete. The data support the application of lentiviral vectors used here in transgenesis.

Emilin genes are duplicated and dynamically expressed during zebrafish embryonic development.

Emilins are a family of extracellular matrix proteins with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure with the human ortholog. In situ hybridization revealed that expression of zebrafish emilin genes is regulated in a spatiotemporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of certain emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in key morphogenetic processes.

Emilin1 links TGF-beta maturation to blood pressure homeostasis.

TGF-beta proteins are main regulators of blood vessel development and maintenance. Here, we report an unprecedented link between TGF-beta signaling and arterial hypertension based on the analysis of mice mutant for Emilin1, a cysteine-rich secreted glycoprotein expressed in the vascular tree. Emilin1 knockout animals display increased blood pressure, increased peripheral vascular resistance, and reduced vessel size. Mechanistically, we found that Emilin1 inhibits TGF-beta signaling by binding specifically to the proTGF-beta precursor and preventing its maturation by furin convertases in the extracellular space. In support of these findings, genetic inactivation of Emilin1 causes increased TGF-beta signaling in the vascular wall. Strikingly, high blood pressure observed in Emilin1 mutants is rescued to normal levels upon inactivation of a single TGF-beta1 allele. This study highlights the importance of modulation of TGF-beta availability in the pathogenesis of hypertension.

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.

SREBP contributes to induction of collagen VI transcription by serum starvation.

Collagen VI is a main extracellular matrix protein whose mutation is linked to myopathic diseases. In myoblasts and other cell types, collagen VI gene transcription peaks during cell-cycle exit that precedes differentiation, upon serum withdrawal or confluence. To get insight into this transcriptional regulation, we characterized a growth arrest responsive region (GARR) in the Col6a1 promoter responsible for this effect. In this work, we identify sterol regulatory element binding protein (SREBP) as a GARR binding protein and provide evidence that SREBP contributes to induction of Col6a1 transcription in serum free conditions. Furthermore, our data unveil a previously unexpected link between extracellular matrix production and LDL signaling.

Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors.

Wntbeta-catenin signaling plays key roles in several developmental and pathological processes. Domains of Wnt expression have been extensively investigated in the mouse, but the tissues receiving the signal remain largely unidentified. To define which cells respond to activated beta-catenin during mammalian development, we generated the beta-catenin-activated transgene driving expression of nuclear beta-galactosidase reporter (BAT-gal) transgenic mice, expressing the lacZ gene under the control of beta-cateninT cell factor responsive elements. Reporter gene activity is found in known organizing centers, such as the midhindbrain border and the limb apical ectodermal ridge. Moreover, BAT-gal expression identifies novel sites of Wnt signaling, like notochord, endothelia, and areas of the adult brain, revealing an unsuspected dynamic pattern of beta-catenin transcriptional activity. Expression of the transgene was analyzed in mutant backgrounds. In lipoprotein receptor-related protein 6-null homozygous mice, which lack a Wnt coreceptor, BAT-gal staining is absent in mutant tissues, indicating that BAT-gal mice are bona fide in vivo indicators of Wntbeta-catenin signaling. Analyses of BAT-gal expression in the adenomatous polyposis coli (multiple intestinal neoplasia+) background revealed betacatenin transcriptional activity in intestinal adenomas but surprisingly not in normal crypt cells. In summary, BAT-gal mice unveil the entire complexity of Wntbeta-catenin signaling in mammals and have broad application potentials for the identification of Wnt-responsive cell populations in development and disease.