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

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

Targeting interleukin-1β protects from aortic aneurysms induced by disrupted transforming growth factor β signaling

Aortic aneurysms are life-threatening conditions with effective treatments mainly limited to emergency surgery or trans-arterial endovascular stent grafts, thus calling for the identification of specific molecular targets. Genetic studies have highlighted controversial roles of transforming growth factor β (TGF-β) signaling in aneurysm development. Here, we report on aneurysms developing in adult mice after smooth muscle cell (SMC)-specific inactivation of Smad4, an intracellular transducer of TGF-β. The results revealed that Smad4 inhibition activated interleukin-1β (IL-1β) in SMCs. This danger signal later recruited innate immunity in the adventitia through chemokine (C-C motif) ligand 2 (CCL2) and modified the mechanical properties of the aortic wall, thus favoring vessel dilation. SMC-specific Smad4 deletion in Il1r1- or Ccr2-null mice resulted in milder aortic pathology. A chronic treatment with anti-IL-1β antibody effectively hampered aneurysm development. These findings identify a mechanistic target for controlling the progression of aneurysms with compromised TGF-β signaling, such as those driven by SMAD4 mutations

Extracellular Collagen VI Has Prosurvival and Autophagy Instructive Properties in Mouse Fibroblasts

Collagen VI (ColVI) is an abundant and distinctive extracellular matrix protein secreted by fibroblasts in different tissues. Human diseases linked to mutations on ColVI genes are primarily affecting skeletal muscle due to non-cell autonomous myofiber defects. To date, it is not known whether and how fibroblast homeostasis is affected by ColVI deficiency, a critical missing information as this may strengthen the use of patients’ fibroblasts for preclinical purposes. Here, we established primary and immortalized fibroblast cultures from ColVI null (Col6a1-/-) mice, the animal model of ColVI-related diseases. We found that, under nutrient-stringent condition, lack of ColVI affects fibroblast survival, leading to increased apoptosis. Moreover, Col6a1-/- fibroblasts display defects in the autophagy/lysosome machinery, with impaired clearance of autophagosomes and failure of Parkin-dependent mitophagy. Col6a1-/- fibroblasts also show an increased activation of the Akt/mTOR pathway, compatible with the autophagy impairment, and adhesion onto purified ColVI elicits a major effect on the autophagic flux. Our findings reveal that ColVI ablation in fibroblasts impacts on autophagy regulation and cell survival, thus pointing at the new concept that this cell type may contribute to the pathological features of ColVI-related diseases

Electrospun Structures Made of a Hydrolyzed Keratin-Based Biomaterial for Development of in vitro Tissue Models

The aim of this study is the analysis and characterization of a hydrolyzed keratin-based biomaterial and its processing using electrospinning technology to develop in vitro tissue models. This biomaterial, extracted from poultry feathers, was mixed with type A porcine gelatin and cross-linked with γ-glycidyloxy-propyl-trimethoxy-silane (GPTMS) to be casted initially in the form of film and characterized in terms of swelling, contact angle, mechanical properties, and surface charge density. After these chemical-physical characterizations, electrospun nanofibers structures were manufactured and their mechanical properties were evaluated. Finally, cell response was analyzed by testing the efficacy of keratin-based structures in sustaining cell vitality and proliferation over 4 days of human epithelial, rat neuronal and human primary skin fibroblast cells

Persistent Dystrophin Protein Restoration 90 Days after a Course of Intraperitoneally Administered Naked 2′OMePS AON and ZM2 NP-AON Complexes in mdx Mice

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2 -O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules

multimerin 2 maintains vascular stability and permeability

Abstract Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2−/− (Mmrn2−/−) mice. Although Mmrn2−/− mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2−/− mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2−/− vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2−/− mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy

F-actin dynamics regulates mammalian organ growth and cell fate maintenance.

BACKGROUND & AIMS: In vitro, several data indicate that cell function can be regulated by the mechanical properties of cells and of the microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by transducing forces into biochemical signals that instruct cell behavior. Among these, the transcriptional coactivators YAP/TAZ recently emerged as key factors mediating multiple responses to actomyosin contractility. However, whether mechanical cues regulate adult liver tissue homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. METHODS & RESULTS: Here we show that the F-actin capping protein CAPZ is a critical negative regulator of actomyosin contractility and mechanotransduction. Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased cellular traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small geometry; in vivo, inactivation of Capzb in the adult mouse liver induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. CONCLUSIONS: These results indicate a previously unrecognized role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated hepatocyte state and to regulate organ size. More in general, it indicates for the first time a physiological role of mechanotransduction in maintaining tissue homeostasis in mammals. LAY SUMMARY: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. A recent advancement in our understanding of these phenomena has been the identification of YAP and TAZ as key factors mediating the biological responses of cells to mechanical signals in vitro. However, whether the mechanical properties of cells and/or the mechanical regulation of YAP/TAZ are relevant for mammalian tissue physiology remains unknown. Here we challenge this issue by genetic inactivation of CAPZ, a protein that regulates the cytoskeleton, i.e. the cells' scaffold by which they sense mechanical cues. We found that inactivation of CAPZ alters cells' and liver tissue's mechanical properties, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals for the maintenance of adult liver homeostasis.This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) Investigator Grant 15307, WCR (Worldwide Cancer Research) Grant 15-1192, CARIPARO Eccellenza Program 2017 and University of Padua BIRD Grant to SD, AIRC ‘Hard ROCK Café’ Fellowship to GS, Marie Sklodowska-Curie Individual Fellowship (796547) to AG, AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ 10016 to SB, UK Medical Research Council and Sackler Foundation Doctoral Training Grant RG70550 to ACL, UK Medical Research Council Career Development Award G1100312/1 and an Isaac Newton Trust Research Grant 17.24(p) to KF

F-actin dynamics regulates mammalian organ growth and cell fate maintenance.

BACKGROUND & AIMS: In vitro, several data indicate that cell function can be regulated by the mechanical properties of cells and of the microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by transducing forces into biochemical signals that instruct cell behavior. Among these, the transcriptional coactivators YAP/TAZ recently emerged as key factors mediating multiple responses to actomyosin contractility. However, whether mechanical cues regulate adult liver tissue homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. METHODS & RESULTS: Here we show that the F-actin capping protein CAPZ is a critical negative regulator of actomyosin contractility and mechanotransduction. Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased cellular traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small geometry; in vivo, inactivation of Capzb in the adult mouse liver induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. CONCLUSIONS: These results indicate a previously unrecognized role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated hepatocyte state and to regulate organ size. More in general, it indicates for the first time a physiological role of mechanotransduction in maintaining tissue homeostasis in mammals. LAY SUMMARY: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. A recent advancement in our understanding of these phenomena has been the identification of YAP and TAZ as key factors mediating the biological responses of cells to mechanical signals in vitro. However, whether the mechanical properties of cells and/or the mechanical regulation of YAP/TAZ are relevant for mammalian tissue physiology remains unknown. Here we challenge this issue by genetic inactivation of CAPZ, a protein that regulates the cytoskeleton, i.e. the cells' scaffold by which they sense mechanical cues. We found that inactivation of CAPZ alters cells' and liver tissue's mechanical properties, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals for the maintenance of adult liver homeostasis.This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) Investigator Grant 15307, WCR (Worldwide Cancer Research) Grant 15-1192, CARIPARO Eccellenza Program 2017 and University of Padua BIRD Grant to SD, AIRC ‘Hard ROCK Café’ Fellowship to GS, Marie Sklodowska-Curie Individual Fellowship (796547) to AG, AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ 10016 to SB, UK Medical Research Council and Sackler Foundation Doctoral Training Grant RG70550 to ACL, UK Medical Research Council Career Development Award G1100312/1 and an Isaac Newton Trust Research Grant 17.24(p) to KF

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