ABSTRACT
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.
Subject(s)
Collagen Type VI/genetics , Connective Tissue Cells/metabolism , Enhancer Elements, Genetic/genetics , Muscle Cells/metabolism , Transcriptional Activation , Animals , Collagen Type VI/metabolism , Immunohistochemistry , Mice , Mice, Transgenic , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Signal TransductionABSTRACT
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.
Subject(s)
Blood Pressure/physiology , Homeostasis , Membrane Glycoproteins/metabolism , Signal Transduction/physiology , Transforming Growth Factor beta/metabolism , Animals , Arteries/cytology , Arteries/metabolism , Furin/metabolism , Gene Dosage , Genes, Reporter , Humans , Hypertension/etiology , Hypertension/metabolism , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Mice, Transgenic , Nodal Protein , Phenotype , Protein Precursors/metabolism , Protein Structure, Tertiary , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/genetics , Xenopus laevis/embryology , Xenopus laevis/genetics , Xenopus laevis/metabolismABSTRACT
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.
