Publications by authors named "Ernst Pöschl"

36 Publications

Augmentation of and Suppression of Expression in the Pituitary Gland of Female Annexin A5 Null Mouse.

J Endocr Soc 2020 Sep 16;4(9):bvaa096. Epub 2020 Jul 16.

Laboratory of Veterinary Physiology, School of Veterinary Medicine, Kitasato University, Aomori, Japan.

GnRH enhances the expression of annexin A5 (ANXA5) in pituitary gonadotropes, and ANXA5 enhances gonadotropin secretion. However, the impact of ANXA5 regulation on the expression of pituitary hormone genes remains unclear. Here, using quantitative PCR, we demonstrated that ANXA5 deficiency in female mice reduced the expression of and in their pituitary glands. Transcriptome analysis confirmed a specific increase in mRNA expression in addition to lower levels of expression in ANXA5-deficient female pituitary glands. This gene was then found to be a GnRH-inducible immediate early gene, and its increased expression caused protein to accumulate in the nucleus after administration of a GnRH agonist in LβT2 cells, which are an in vitro pituitary gonadotrope model. The increase in ANXA5 protein levels in LβT2 cells clearly suppressed expression. siRNA-mediated inhibition of expression increased expression. The results revealed that GnRH stimulates and sequentially. NR4A3 suppression of may be necessary for later massive secretion of FSH by GnRH in gonadotropes, and would be negatively regulated by ANXA5 to increase FSH secretion.
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http://dx.doi.org/10.1210/jendso/bvaa096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448937PMC
September 2020

Annexin A5 Involvement in Bone Overgrowth at the Enthesis.

J Bone Miner Res 2018 08;33(8):1532-1543

Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama, Japan.

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5 ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5 mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5 mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5 mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5 and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5 mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3453DOI Listing
August 2018

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes.

Nat Commun 2016 11 30;7:13515. Epub 2016 Nov 30.

Institute for Dental Research and Oral Musculoskeletal Biology, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, Cologne 50931, Germany.

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.
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http://dx.doi.org/10.1038/ncomms13515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141367PMC
November 2016

Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis.

Cells Tissues Organs 2016 30;201(4):287-98. Epub 2016 Apr 30.

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.
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http://dx.doi.org/10.1159/000445106DOI Listing
March 2017

Erratum to: Induction of initial steps of angiogenic differentiation and maturation of endothelial cells by pericytes in vitro and the role of collagen IV.

Histochem Cell Biol 2016 May;145(5):527-9

Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

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http://dx.doi.org/10.1007/s00418-016-1429-4DOI Listing
May 2016

miR-126-3p Promotes Matrix-Dependent Perivascular Cell Attachment, Migration and Intercellular Interaction.

Stem Cells 2016 05 9;34(5):1297-309. Epub 2016 Mar 9.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty, University of Cologne, Cologne, Germany.

microRNAs (miRNAs) can regulate the interplay between perivascular cells (PVC) and endothelial cells (EC) during angiogenesis, but the relevant PVC-specific miRNAs are not yet defined. Here, we identified miR-126-3p and miR-146a to be exclusively upregulated in PVC upon interaction with EC, determined their influence on the PVC phenotype and elucidate their molecular mechanisms of action. Specifically the increase of miR-126-3p strongly promoted the motility of PVC on the basement membrane-like composite and stabilized networks of EC. Subsequent miRNA target analysis showed that miR-126-3p inhibits SPRED1 and PLK2 expression, induces ERK1/2 phosphorylation and stimulates TLR3 expression to modulate cell-cell and cell-matrix contacts of PVC. Gain of expression experiments in vivo demonstrated that miR-126-3p stimulates PVC coverage of newly formed vessels and transform immature into mature, less permeable vessels. In conclusion we showed that miR-126-3p regulates matrix-dependent PVC migration and intercellular interaction to modulate vascular integrity. Stem Cells 2016;34:1297-1309.
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http://dx.doi.org/10.1002/stem.2308DOI Listing
May 2016

Induction of initial steps of angiogenic differentiation and maturation of endothelial cells by pericytes in vitro and the role of collagen IV.

Histochem Cell Biol 2016 May 9;145(5):511-25. Epub 2016 Jan 9.

Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

Activation of endothelial cells and recruitment of mural cells define critical steps during the formation of stable vascular elements. Both events are reflected by cocultures of endothelial cells and isolated murine pericyte-like cells and define a versatile platform for the analysis of distinct steps during the angiogenic process in vitro. Isolated pericyte-like cells promote the survival of endothelial cells, induce the assembly of endothelial cells as well as establish direct contacts with forming endothelial alignments. More importantly, they also induce characteristic steps of maturation including the assembly of stable cell-cell junctions, deposition of basement membrane-like matrices and local formation of a central lumen. The presence of pericyte-like cells induces the secretion of extracellular matrices enriched in collagen IV by endothelial cells, which improves endothelial tube formation and provides the adhesive substrate for mural cell recruitment. Collagen-binding integrins contribute differentially to the process, with α1β1 involved in the adhesion of pericyte-like cells to collagen IV and α2β1 mainly involved in endothelial cord formation. These data indicate that pericyte-like cells are essential for the survival of endothelial cells, the efficient formation of endothelial alignments as well as initial steps of maturation of capillary-like structures.
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http://dx.doi.org/10.1007/s00418-015-1398-zDOI Listing
May 2016

Loss of maternal annexin A5 increases the likelihood of placental platelet thrombosis and foetal loss.

Sci Rep 2012 9;2:827. Epub 2012 Nov 9.

Laboratory of Veterinary Physiology, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan.

Antiphospholipid syndrome is associated with an increased risk of thrombosis and pregnancy loss. Annexin A5 (Anxa5) is a candidate autoantigen. It is not known, however, whether endogenous Anxa5 prevents foetal loss during normal pregnancy. We found significant reductions in litter size and foetal weight in Anxa5-null mice (Anxa5-KO). These changes occurred even when only the mother was Anxa5-KO. A small amount of placental fibrin deposition was observed in the decidual tissues, but did not noticeably differ between wild-type and Anxa5-KO mice. However, immunoreactivity for integrin beta 3/CD61, a platelet marker, was demonstrated within thrombi in the arterial canals only in Anxa5-KO mothers. Subcutaneous administration of the anticoagulant heparin to pregnant Anxa5-KO mice significantly reduced pregnancy loss, suggesting that maternal Anxa5 is crucial for maintaining intact placental circulation. Hence, the presence of maternal Anxa5 minimises the risk of thrombosis in the placental circulation and reduces the risk of foetal loss.
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http://dx.doi.org/10.1038/srep00827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494014PMC
May 2013

Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle-mediated mineralization, but lack of collagen X affects hematopoiesis and the Th1/Th2 response.

J Bone Miner Res 2012 Nov;27(11):2399-412

Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany.

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle-mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X-deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) newborns, whereas endochondral ossification and mineralization were not affected in 13-day- and 1-month-old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1-month- and 1-year-old mutants even though the diaphyseal circumference was reduced in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) mice. The percentage of naive immature IgM(+) /IgM(+) B cells and peripheral T-helper cells were increased in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) mutants, and activated splenic T cells isolated from Col10a1(-/-) mice secreted elevated levels of IL-4 and GM-CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process.
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http://dx.doi.org/10.1002/jbmr.1682DOI Listing
November 2012

Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue.

Histochem Cell Biol 2012 Jun 28;137(6):841-7. Epub 2012 Feb 28.

Department of Pharmacology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan.

The β-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-β-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.
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http://dx.doi.org/10.1007/s00418-012-0936-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353101PMC
June 2012

Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair.

Nat Commun 2011 ;2:270

Molecular Imaging and NanoBioTechnology, IECB, UMR-5248 CBMN CNRS-University Bordeaux1-ENITAB, Talence F-33402, France.

Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca(2+) activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.
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http://dx.doi.org/10.1038/ncomms1270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104517PMC
July 2011

Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif.

J Biol Chem 2011 Feb 3;286(7):5708-16. Epub 2010 Dec 3.

Medical Faculty, Center for Biochemistry, University of Cologne, 50931 Cologne, Germany.

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.
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http://dx.doi.org/10.1074/jbc.M110.193086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037683PMC
February 2011

Reversible transdifferentiation of blood vascular endothelial cells to a lymphatic-like phenotype in vitro.

J Cell Sci 2010 Nov 12;123(Pt 21):3808-16. Epub 2010 Oct 12.

Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.

Blood vascular cells and lymphatic endothelial cells (BECs and LECs, respectively) form two separate vascular systems and are functionally distinct cell types or lineages with characteristic gene expression profiles. Interconversion between these cell types has not been reported. Here, we show that in conventional in vitro angiogenesis assays, human BECs of fetal or adult origin show altered gene expression that is indicative of transition to a lymphatic-like phenotype. This change occurs in BECs undergoing tubulogenesis in fibrin, collagen or Matrigel assays, but is independent of tube formation per se, because it is not inhibited by a metalloproteinase inhibitor that blocks tubulogenesis. It is also reversible, since cells removed from 3D tubules revert to a BEC expression profile upon monolayer culture. Induction of the lymphatic-like phenotype is partially inhibited by co-culture of HUVECs with perivascular cells. These data reveal an unexpected plasticity in endothelial phenotype, which is regulated by contact with the ECM environment and/or cues from supporting cells.
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http://dx.doi.org/10.1242/jcs.064279DOI Listing
November 2010

Deficiency of annexins A5 and A6 induces complex changes in the transcriptome of growth plate cartilage but does not inhibit the induction of mineralization.

J Bone Miner Res 2010 Jan;25(1):141-53

Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, and Royal Children's Hospital, Parkville, Victoria, Australia.

Initiation of mineralization during endochondral ossification is a multistep process and has been assumed to correlate with specific interactions of annexins A5 and A6 and collagens. However, skeletal development appears to be normal in mice deficient for either A5 or A6, and the highly conserved structures led to the assumption that A5 and A6 may fulfill redundant functions. We have now generated mice deficient of both proteins. These mice were viable and fertile and showed no obvious abnormalities. Assessment of skeletal elements using histologic, ultrastructural, and peripheral quantitative computed tomographic methods revealed that mineralization and development of the skeleton were not significantly affected in mutant mice. Otherwise, global gene expression analysis showed subtle changes at the transcriptome level of genes involved in cell growth and intermediate metabolism. These results indicate that annexins A5 and A6 may not represent the essential annexins that promote mineralization in vivo.
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http://dx.doi.org/10.1359/jbmr.090710DOI Listing
January 2010

The immune reaction against allogeneic necrotic cells is reduced in Annexin A5 knock out mice whose macrophages display an anti-inflammatory phenotype.

J Cell Mol Med 2009 Jul 9;13(7):1391-9. Epub 2008 Jul 9.

Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen-Nürnberg, Germany.

Proteins of the annexin family bind to phospholipids in a Ca(2+) dependent manner. The exposure of phosphatidylserine (PS) by apoptotic as well as necrotic cells is one major eat-me-signal for macrophages. Annexin A5 (Anx A5) preferentially binds to PS. The availability of Anx A5 knock out (KO) mice allowed us to investigate for the first time if endogenous Anx A5 modulates the immune response towards allogeneic cells. Furthermore, the effect of Anx A5 gene deletion on the phagocytic process as well as on the inflammatory reaction of macrophages was explored. We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells. In vivo phagocytosis experiments revealed that macrophages of Anx A5 KO mice displayed an increased uptake of necrotic cells. Additionally, an increased secretion of the anti-inflammatory cytokine IL-10 of isolated macrophages of Anx A5 KO mice after contact with necrotic cells was observed. Furthermore, the promoter activity of the Anx A5 gene was enhanced after stimulation of macrophages. The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present. These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00395.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496152PMC
July 2009

Expression and thyroid hormone regulation of annexins in the anterior pituitary.

J Endocrinol 2007 Dec;195(3):385-92

Max Planck Institute for Experimental Endocrinology, Feodor-Lynen-Str. 7, D-30625 Hannover, Germany.

Due to their property to bind to phospholipids in a Ca(2)(+)-dependent manner, proteins of the annexin superfamily are involved in many membrane-related events and thus in various forms of physiological and pathological processes. We were therefore interested in analyzing the mRNA expression of the annexins in the severely disorganized pituitaries of the athyroid Pax8(-/-) mice in comparison with that of control animals. In neither condition was mRNA expression of the annexins A3, A7, A8, A9, A11, and A13 detectable. The annexins A2, A4, and A6 were equally expressed in wild-type and Pax8(-/-) mice. Transcript levels of A1 and A10 were highly increased and those of A5 were significantly decreased in the athyroid mutants compared with controls. Treatment of Pax8(-/-) mice with physiological doses of thyroxine for 3 days normalized the mRNA expression of A1, A5, and A10 indicating that the expression of these annexins is directly regulated by thyroid hormone (TH). Since A5 exhibits by far the highest transcript levels of all annexins in the pituitary and its regulation by TH could be also confirmed at the protein level, we analyzed the mRNA expression of pituitary hormones in A5(-/-) mice. In these mutants, only the beta-FSH mRNA expression was found to be significantly reduced, while the mRNA expression levels of the other pituitary hormones were not altered. These results support the concept that annexins might serve important albeit redundant functions as modulators of pituitary hormone secretion.
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http://dx.doi.org/10.1677/JOE-07-0042DOI Listing
December 2007

Distinct acidic clusters and hydrophobic residues in the alternative splice domains X1 and X2 of alpha7 integrins define specificity for laminin isoforms.

J Mol Biol 2007 Aug 2;371(5):1188-203. Epub 2007 Jun 2.

Department of Experimental Medicine I, Nikolaus - Fiebiger Center of Molecular Medicine, University of Erlangen - Nuernberg, 91054 Erlangen, Germany.

The binding specificity of alpha7beta1 integrins for different laminin isoforms is defined by the X1 and X2 splice domains located in the beta-propeller domain of the alpha7 subunit. In order to gain insight into the mechanism of specific laminin-integrin interactions, we defined laminin-binding epitopes of the alpha7X1 and -X2 domains by single amino acid substitutions and domain swapping between X1 and X2. The interaction of mutated, recombinantly prepared alpha7X1beta1 and alpha7X2beta1 heterodimers with various laminin isoforms was studied by surface plasmon resonance and solid phase binding assays. The data show that distinct clusters of surface-exposed acidic residues located in different positions of the X1 and the X2 loops are responsible for the specific recognition of laminins. These residues are conserved between the respective X1 or X2 splice domains of the alpha7 chains of different species, some also in the corresponding X1/X2 splice domains of alpha6 integrin. Interestingly, ligand binding was also modulated by mutating surface-exposed hydrophobic residues (alpha7X1L205, alpha7X2Y208) at positions corresponding to the fibronectin binding synergy site in alpha5beta1 integrin. Mutations in X1 that affected binding to laminin-1 also affected binding to laminin-8 and -10, but not to the same extent, thus allowing conclusions on the specific role of individual surface epitopes in the selective recognition of laminin-1 versus laminins -8 and -10. The role of the identified epitopes was confirmed by molecular dynamics simulations of wild-type integrins and several inactivating mutations. The analysis of laminin isoform interactions with various X1/X2 chimaera lend further support to the key role of negative surface charges and pointed to an essential contribution of the N-terminal TARVEL sequence of the X1 domain for recognition of laminin-8 and -10. In conclusion, specific surface epitopes containing charged and hydrophobic residues are essential for ligand binding and define specific interactions with laminin isoforms.
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http://dx.doi.org/10.1016/j.jmb.2007.05.074DOI Listing
August 2007

Isolated Anxa5+/Sca-1+ perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo.

Exp Cell Res 2007 Jul 8;313(12):2730-43. Epub 2007 May 8.

Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany.

Pericytes are closely associated with endothelial cells, contribute to vascular stability and represent a potential source of mesenchymal progenitor cells. Using the specifically expressed annexin A5-LacZ fusion gene (Anxa5-LacZ), it became possible to isolate perivascular cells (PVC) from mouse tissues. These cells proliferate and can be cultured without undergoing senescence for multiple passages. PVC display phenotypic characteristics of pericytes, as they express pericyte-specific markers (NG2-proteoglycan, desmin, alphaSMA, PDGFR-beta). They also express stem cell marker Sca-1, whereas endothelial (PECAM), hematopoietic (CD45) or myeloid (F4/80, CD11b) lineage markers are not detectable. These characteristics are in common with the pericyte-like cell line 10T1/2. PVC also display a phagocytoic activity higher than 10T1/2 cells. During coculture with endothelial cells both cell types stimulate angiogenic processes indicated by an increased expression of PECAM in endothelial cells and specific deposition of basement membrane proteins. PVC show a significantly increased induction of endothelial specific PECAM expression compared to 10T1/2 cells. Accordingly, in vivo grafts of PVC aggregates onto chorioallantoic membranes of quail embryos recruit endothelial cells, get highly vascularized and deposit basement membrane components. These data demonstrate that isolated Anxa5-LacZ(+) PVC from mouse meninges retain their capacity for differentiation to pericyte-like cells and contribute to angiogenic processes.
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http://dx.doi.org/10.1016/j.yexcr.2007.04.031DOI Listing
July 2007

Modulation of the immune system by dying cells and the phosphatidylserine-ligand annexin A5.

Autoimmunity 2007 Jun;40(4):254-9

Department of Internal Medicine 3, University Hospital Erlangen, Erlangen. Germany.

Apoptotic cell death and the efficient clearance of dying cells are essential mechanisms to control tissue homeostasis and to eliminate potential autoantigens. Numerous alterations on the surfaces of dying cells define a highly characteristic membrane signature and enable an unequivocal distinction from vital cells. This way, phagocytosis is initiated and signalling events induced which minimize inflammatory reactions. Therefore, the use of proteins interfering with the clearance process may open up new vistas to improve immunization strategies and may help to understand the mechanisms of autoimmune diseases.
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http://dx.doi.org/10.1080/08916930701357331DOI Listing
June 2007

The role of annexin A5 in the modulation of the immune response against dying and dead cells.

Curr Med Chem 2007 ;14(3):271-7

Institute for Clinical Immunology and Exp. Medicine I, Friedrich-Alexander-University of Erlangen-Nuremberg, Glückstrasse 4a and 6, 91054 Erlangen, Germany.

Annexins are characterized by the ability to bind phospholipids of membranes in the presence of Ca2+. Annexin A5 represents a typical member of this protein family and is a natural occurring highly specific ligand for phosphatidylserine (PS). The exposure of PS is one major "eat me" signal for phagocytes of apoptotic and necrotic cells. Apoptotic cells are normally cleared via an anti-inflammatory pathway. In contrast, the uptake and removal of necrotic cells normally involves inflammation and an immune response. Interestingly, the lack of endogenous annexin A5 also leads to a reduced inflammatory potential of necrotic cells. Annexin A5 may interfere in vivo with the immunosuppressive effects of apoptotic cells since it preferentially binds PS with high affinity and inhibits apoptotic cell uptake by macrophages. In this review we focus on how defects in the clearance process can lead to chronic autoimmunity. Furthermore, the role of annexin A5 as important adjuvant for apoptotic cell-based tumour vaccines is discussed. The mechanism of how the immunogenicity of apoptotic cells can be restored by blocking their PS-dependent clearance is outlined in detail. Taken together, annexin A5 is an important modulator of the immune response against PS-exposing particles like apoptotic cells, necrotic cells, and certain viruses.
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http://dx.doi.org/10.2174/092986707779941131DOI Listing
March 2007

BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter.

Histochem Cell Biol 2007 Feb 19;127(2):183-94. Epub 2006 Oct 19.

Department of Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nuremberg, Glueckstr.6, 91054, Erlangen, Germany.

During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing.
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http://dx.doi.org/10.1007/s00418-006-0236-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1779629PMC
February 2007

The influence on the immunomodulatory effects of dying and dead cells of Annexin V.

J Leukoc Biol 2007 Jan 27;81(1):6-14. Epub 2006 Sep 27.

Institute for Clinical Immunology, Department of Internal Medicne 3, Friedrich-Alexander-University of Erlangen-Nuremberg, Glueckstrasse 4a, 91054 Erlangen, Germany.

Apoptotic and necrotic cells expose phosphatidylserine (PS). This membrane modification ensures a swift recognition and uptake by phagocytes of the dying and dead cells. Annexin V (AxV) preferentially binds to anionic phospholipids and thereby, modulates the clearance process. First, we analyzed the influence of AxV on the immunogenicity of apoptotic cells. The addition to apoptotic cells of AxV prior to their injection into mice increased their immunogenicity significantly. Next, we studied the influence of endogenous AxV on the allogeneic reaction against apoptotic and necrotic cells. To preserve heat-labile, short-lived "danger signals," we induced necrosis by mechanical stress. Wild-type mice showed a strong, allogeneic delayed-type hypersensitivity (DTH) reaction. In contrast, AxV-deficient animals showed almost no allogeneic DTH reaction, indicating that endogenous AxV increases the immune response against dead cells. Furthermore, AxV-deficient macrophages had a higher immunosuppressive potential in vitro. Next, we analyzed the influence of AxV on chronic macrophage infection with HIV-1, known to expose PS on its surface. The infectivity in human macrophages of HIV-1 was reduced significantly in the presence of AxV. Finally, we show that AxV also blocked the in vitro uptake by macrophages of primary necrotic cells. Similar to apoptotic cells, necrotic cells generated by heat treatment displayed an anti-inflammatory activity. In contrast, mechanical stress-induced necrotic cells led to a decreased secretion of IL-10, indicating a more inflammatory potential. From the experiments presented above, we conclude that AxV influences the clearance of several PS-exposing particles such as viruses, dying, and dead cells.
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http://dx.doi.org/10.1189/jlb.0306166DOI Listing
January 2007

Twisted gastrulation modulates bone morphogenetic protein-induced collagen II and X expression in chondrocytes in vitro and in vivo.

J Biol Chem 2006 Oct 10;281(42):31790-800. Epub 2006 Aug 10.

Department of Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nuremberg, 91054 Erlangen, Germany.

Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicated a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. Here we show for the first time by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore we provide evidence that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impaired the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibited BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation.
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http://dx.doi.org/10.1074/jbc.M603419200DOI Listing
October 2006

Differential gene expression in pseudoexfoliation syndrome.

Invest Ophthalmol Vis Sci 2005 Oct;46(10):3742-52

Department of Ophthalmology, University of Erlangen-Nürnberg, Germany.

Purpose: To identify and characterize genes differentially expressed in anterior segment tissues of eyes with pseudoexfoliation (PEX) syndrome and glaucoma.

Methods: Anterior segment tissues (iris, ciliary processes, lens epithelium) were obtained from eight surgically enucleated eyes with PEX-associated open-angle or closed-angle glaucomas and eight age-matched glaucomatous control eyes without PEX. cDNA libraries were generated from three PEX and three control specimens, and their gene expression patterns were compared by means of cDNA subtraction. Differentially expressed clones from the subtracted cDNA libraries were sequenced, and their differential expression was verified by means of RT-PCR, virtual Northern blot analysis, and in situ hybridization with specific RNA probes.

Results: Subtraction of cDNA libraries identified 27 candidate genes for differential expression in PEX tissues, of which 23 genes were confirmed by virtual Northern blot, RT-PCR, and in situ hybridization. One set of genes consistently upregulated in anterior segment tissues from different patients with PEX comprised latent transforming growth factor binding proteins (LTBP-1 and -2), which are structural components of elastic microfibrils, the cross-linking enzyme transglutaminase-2 (TGase-2), tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), A-kinase anchor protein-2 (AKAP-2), apolipoprotein D, and the adenosine receptor-A3 (AdoR-A3). Genes reproducibly downregulated in PEX tissues included TIMP-1, clusterin, microsomal glutathione-S-transferase-1 (mGST-1), and serum amyloid A1. Further transcripts, such as elastase, GST-T1, integrin beta4, and dehydrocholesterol reductase, did not show a consistent differential expression pattern in tissues obtained from different patients. Although fibrillin-1 was not isolated from subtracted cDNA libraries, upregulated expression of this elastic microfibrillar component was also demonstrated by RT-PCR and in situ hybridization.

Conclusions: Differentially expressed genes with a high level of reproducibility in different tissues and different patients with PEX syndrome are mainly related to extracellular matrix metabolism and cellular stress. The underlying pathophysiology of PEX syndrome appears to be associated with an excessive production of elastic microfibril components, enzymatic cross-linking processes, a proteolytic imbalance between matrix metalloproteinases and their inhibitors, and increased cellular and oxidative stress supporting the notion of PEX syndrome as a stress-induced elastic microfibrillopathy.
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http://dx.doi.org/10.1167/iovs.05-0249DOI Listing
October 2005

Dominant mutations of Col4a1 result in basement membrane defects which lead to anterior segment dysgenesis and glomerulopathy.

Hum Mol Genet 2005 Nov 13;14(21):3161-8. Epub 2005 Sep 13.

Molecular Physiology, Centre for Cardiovascular Science, University of Edinburgh, UK.

Members of the type IV collagen family are essential components of all basement membranes (BMs) and define structural stability as well as tissue-specific functions. The major isoform, alpha1.alpha1.alpha2(IV), contributes to the formation of many BMs and its deficiency causes embryonic lethality in mouse. We have identified an allelic series of three ENU induced dominant mouse mutants with missense mutations in the gene Col4a1 encoding the alpha1(IV) subunit chain. Two severe alleles (Bru and Svc) have mutations affecting the conserved glycine residues in the Gly-Xaa-Yaa collagen repeat. Bru heterozygous mice display defects similar to Axenfeld-Rieger anomaly, including iris defects, corneal opacity, vacuolar cataracts, significant iris/corneal adhesions, buphthalmos and optic nerve cupping, a sign indicative of glaucoma. Kidneys of Bru mice have peripheral glomerulopathy characterized by hypertrophy and hyperplasia of the parietal epithelium of Bowman's capsule. A milder allele (Raw) contains a mutation in the Yaa residue of the collagen repeat and was identified by a silvery appearance of the retinal arterioles. All phenotypes are associated with BM defects that affect the eye, kidney and other tissues. This allelic series shows that mutations affecting the collagen domain cause dominant negative effects on the expression and function of the major collagen IV isoform alpha1(IV), and pathological effects vary with the individual mutations.
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http://dx.doi.org/10.1093/hmg/ddi348DOI Listing
November 2005

Perivascular cells expressing annexin A5 define a novel mesenchymal stem cell-like population with the capacity to differentiate into multiple mesenchymal lineages.

Development 2005 Jun 27;132(11):2657-68. Epub 2005 Apr 27.

Department of Cell and Matrix Biology, MCRI, 3052 Parkville Victoria, Australia.

The annexin A5 gene (Anxa5) was recently found to be expressed in the developing and adult vascular system as well as the skeletal system. In this paper, the expression of an Anxa5-lacZ fusion gene was used to define the onset of expression in the vasculature and to characterize these Anxa5-lacZ-expressing vasculature-associated cells. After blastocyst implantation, Anxa5-lacZ-positive cells were first detected in extra-embryonic tissues and in angioblast progenitors forming the primary vascular plexus. Later, expression is highly restricted to perivascular cells in most blood vessels resembling pericytes or vascular smooth muscle cells. Viable Anxa5-lacZ+ perivascular cells were isolated from embryos as well as adult brain meninges by specific staining with fluorescent X-gal substrates and cell-sorting. These purified lacZ+ cells specifically express known markers of pericytes, but also markers characteristic for stem cell populations. In vitro and in vivo differentiation experiments show that this cell pool expresses early markers of chondrogenesis, is capable of forming a calcified matrix and differentiates into adipocytes. Hence, Anxa5 expression in perivascular cells from mouse defines a novel population of cells with a distinct developmental potential.
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http://dx.doi.org/10.1242/dev.01846DOI Listing
June 2005

The NC1 domain of human collagen IV is necessary to initiate triple helix formation.

Biochem Biophys Res Commun 2004 Dec;325(1):276-80

Osteoarticular and Arthritis Research, Department of Pathology, University of Erlangen-Nürnberg, Germany.

Type IV collagen is a heterotrimeric molecule, which contains the N-terminal 7S, a central triple-helical domain, and the globular C-terminal NC1 domain. A zipper-like mechanism of triple helix formation, starting from the C-terminus, has been proposed for most collagens but for collagen type IV there has only been indirect evidence so far. In this study we expressed trimeric human collagen type IV to compare the effects of different structural variants on the formation of collagen IV molecules. Our data show that the NC1 but not 7S domain is essential for the chain association and initiation of triple helix formation. This strongly suggests an N-to-C terminal mechanism of triple helix formation. Additionally, we could show that the human alpha2(IV) chain can form chimeric alpha1.alpha1.alpha2(IV) heterotrimers with mouse subunits when expressed in PF-HR9 cells.
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http://dx.doi.org/10.1016/j.bbrc.2004.10.034DOI Listing
December 2004

Inhibition of phosphatidylserine recognition heightens the immunogenicity of irradiated lymphoma cells in vivo.

J Exp Med 2004 Nov 25;200(9):1157-65. Epub 2004 Oct 25.

Clinical Immunology Unit, Cancer Immunotherapy and Gene Therapy Program, H. San Raffaele Institute, DIBIT 3A1, via Olgettina 58, 20132 Milano, Italy.

Strategies to enhance the immunogenicity of tumors are urgently needed. Although vaccination with irradiated dying lymphoma cells recruits a tumor-specific immune response, its efficiency as immunogen is poor. Annexin V (AxV) binds with high affinity to phosphatidylserine on the surface of apoptotic and necrotic cells and thereby impairs their uptake by macrophages. Here, we report that AxV preferentially targets irradiated lymphoma cells to CD8+ dendritic cells for in vivo clearance, elicits the release of proinflammatory cytokines and dramatically enhances the protection elicited against the tumor. The response was endowed with both memory, because protected animals rejected living lymphoma cells after 72 d, and specificity, because vaccinated animals failed to reject unrelated neoplasms. Finally, AxV-coupled irradiated cells induced the regression of growing tumors. These data indicate that endogenous adjuvants that bind to dying tumor cells can be exploited to target tumors for immune rejection.
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http://dx.doi.org/10.1084/jem.20040327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211859PMC
November 2004

A highly conserved enhancer in mammalian type X collagen genes drives high levels of tissue-specific expression in hypertrophic cartilage in vitro and in vivo.

Matrix Biol 2004 Aug;23(5):309-22

Department of Experimental Medicine I, Nikolaus-Fiebiger-Center of Molecular Medicine, University of Erlangen-Nuremberg, Glueckstr.6, D-91054, Germany.

Previously we have identified a cis-acting regulatory domain in the human type X collagen gene upstream of the transcription start site which acts as a strong enhancer in hypertrophic, but not in resting chondrocytes. Here we show that this enhancer is highly conserved also in the murine and bovine Col10a1 genes, but not found in the known promoter sequences of chicken Col10a1. It contains a functionally active AP-1 site (TPA Responsive Element, TRE) which is essential for the high transcriptional activity of the COL10A1 enhancer in transiently transfected hypertrophic chondrocytes. Gel-shift experiments with nuclear extracts of hypertrophic chondrocytes revealed FosB and Fra-1 as candidates regulating AP-1 factors binding to the TRE site. In fact, coexpression of FosB and Fra-1 in reporter gene assays greatly stimulated transcriptional activity of enhancer bearing reporter genes. Quantitative analysis of AP-1 factor mRNA levels in distinct fractions of fetal bovine epiphyseal chondrocytes by real-time PCR confirmed significant levels of FosB and Fra-1 mRNA besides other AP-1 factors in hypertrophic chondrocytes. A key role of the enhancer element in regulating tissue-specific expression of the Col10a1 gene was shown by establishing transgenic mouse lines with a reporter gene containing a 4.6 kb murine Col10a1 promoter fragment which included the enhancer, exon 1, part of exon 2 and the first intron. Reporter gene expression was seen exclusively in hypertrophic cartilages in the growth plates of long bones, ribs, vertebrae, sternum and mandibles of 17.5-18.5 dpc embryos, confirming that the 4.6 kb promoter is able to drive specific expression of Col10a1 in hypertrophic cartilage. These established transgenic lines should facilitate the genetic analysis of regulatory pathways of chondrocyte maturation and Col10a1 gene expression in the future.
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http://dx.doi.org/10.1016/j.matbio.2004.05.010DOI Listing
August 2004

Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development.

Development 2004 Apr 3;131(7):1619-28. Epub 2004 Mar 3.

Department of Experimental Medicine I, University Erlangen-Nürnberg, 91054 Erlangen, Germany.

Basement membranes are specialized extracellular matrices consisting of tissue-specific organizations of multiple matrix molecules and serve as structural barriers as well as substrates for cellular interactions. The network of collagen IV is thought to define the scaffold integrating other components such as, laminins, nidogens or perlecan, into highly organized supramolecular architectures. To analyze the functional roles of the major collagen IV isoform alpha1(IV)(2)alpha2(IV) for basement membrane assembly and embryonic development, we generated a null allele of the Col4a1/2 locus in mice, thereby ablating both alpha-chains. Unexpectedly, embryos developed up to E9.5 at the expected Mendelian ratio and showed a variable degree of growth retardation. Basement membrane proteins were deposited and assembled at expected sites in mutant embryos, indicating that this isoform is dispensable for matrix deposition and assembly during early development. However, lethality occurred between E10.5-E11.5, because of structural deficiencies in the basement membranes and finally by failure of the integrity of Reichert's membrane. These data demonstrate for the first time that collagen IV is fundamental for the maintenance of integrity and function of basement membranes under conditions of increasing mechanical demands, but dispensable for deposition and initial assembly of components. Taken together with other basement membrane protein knockouts, these data suggest that laminin is sufficient for basement membrane-like matrices during early development, but at later stages the specific composition of components including collagen IV defines integrity, stability and functionality.
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http://dx.doi.org/10.1242/dev.01037DOI Listing
April 2004