[Show abstract][Hide abstract] ABSTRACT: The fibroblast integrin α11β1 is a key receptor for fibrillar collagens. To study the potential function of α11 in vivo, we
generated a null allele of the α11 gene. Integrin α11−/− mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors.
Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary
defect in the incisor ligament leads to halted tooth eruption. α11β1-defective embryonic fibroblasts displayed severe defects
in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract
collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed
disturbed MMP13 and MMP14 synthesis in α11−/− cells. We show that α11β1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that α11β1 integrin
is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate
the forces needed for axial tooth movement. Our data show a unique role for α11β1 integrin during tooth eruption.
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.
No preview · Article · Dec 2006 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: During early mouse embryogenesis, each laminin (Lm) chain of the first described Lm, a heterotrimer of alpha1, beta1, and gamma1 chains (Lm-1), is essential for basement membrane (BM) assembly, which is required for pregastrulation development. Individual domains may have other functions, not necessarily structural. The cell binding C terminus of Lm alpha1 chain contains five Lm globular (LG) domains. In vitro, alpha1LG1-3 domains bind integrins, and alpha1LG4 binds dystroglycan, heparin, and sulfatides. A prevailing hypothesis is that alpha1LG4 is crucial as a structural domain for BM assembly, whereas integrin-binding sites conduct signaling. The in vivo role of alpha1LG4-5 (also called E3) has not been studied. Mice lacking alpha1LG4-5 were therefore made. Null embryos implanted, but presumptive epiblast cells failed to polarize and did not survive past day 6.5. BM components including truncated Lm alpha1 were detected in Reichert's membrane. Surprisingly, embryonic BM assembly between visceral endoderm and stem cells was normal in null embryos and in embryoid bodies of alpha1LG4-5-null embryonic stem cells. Yet, stem cells could not develop into polarized epiblast cells. Thus, alpha1LG4-5 provides vital signals for the conversion of stem cells to polarized epithelium.
Full-text · Article · Mar 2005 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The ribosomal protein S19 (RPS19) is located in the small (40S) subunit and is one of 79 ribosomal proteins. The gene encoding
RPS19 is mutated in approximately 25% of patients with Diamond-Blackfan anemia, which is a rare congenital erythroblastopenia.
Affected individuals present with decreased numbers or the absence of erythroid precursors in the bone marrow, and associated
malformations of various organs are common. We produced C57BL/6J mice with a targeted disruption of murine Rps19 to study its role in erythropoiesis and development. Mice homozygous for the disrupted Rps19 were not identified as early as the blastocyst stage, indicating a lethal effect. In contrast, mice heterozygous for the
disrupted Rps19 allele have normal growth and organ development, including that of the hematopoietic system. Our findings indicate that zygotes
which are Rps19−/− do not form blastocysts, whereas one normal Rps19 allele in C57BL/6J mice is sufficient to maintain normal ribosomal and possibly extraribosomal functions.
Full-text · Article · Jun 2004 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Mast cell activation, or neurogenic inflammation, is known to induce lowering of interstitial fluid pressure (P(if)) and plasma protein extravasation (PPE) in several tissues from both rats and mice. To examine a possible role of connective tissue mast cells (CTMCs) in these inflammatory responses, we used mice with dysfunctional CTMCs due to lack of the N-deacetylase/N-sulfotransferase-2 enzyme (NDST-2(-/-)). P(if) and PPE were measured after challenge with compound 48/80 (C48/80), and P(if) alone was measured after treatment either with capsaicin, substance P (SP), or calcitonin gene-related peptide (CGRP). Measurements of P(if) in anesthetized (fentanyl/fluanison and midazolam, 1:1) mice were performed in paw skin with glass capillaries connected to a servo-controlled counterpressure system. PPE was measured with microdialysis by using hollow plasmapheresis fibers (cutoff at 3,000 kDa) placed subcutaneously on the back. Intravenous administration of C48/80 lowered P(if) significantly (P < 0.05) in NDST-2(-/-) mice (-1.67 +/- 0.42 mmHg) compared with vehicle (-0.57 +/- 0.17 mmHg) but the lowering was significantly (P < 0.05) less compared with that of the NDST-2(+/+) mice (-2.31 +/- 0.47 mmHg). PPE was increased 300% after treatment with C48/80 in NDST-2(+/+) mice, whereas there was no increase in PPE in NDST-2(-/-) mice. Capsaicin, SP, and CGRP lowered P(if) significantly (P < 0.05) compared with vehicle and to the same extent in both NDST-2(+/+) and NDST-2(-/-) mice. We can conclude that although NDST-2(-/-) mice demonstrate an altered response in P(if) after mast cell activation, there was no similar alteration after neurogenic inflammation. Therefore, we suggest that neurogenic inflammation in mouse skin is not exclusively dependent on intact CTMCs.
[Show abstract][Hide abstract] ABSTRACT: The glycosaminoglycan, heparan sulfate (HS), binds proteins to modulate signaling events in embryogenesis. All identified protein-binding HS epitopes contain l-iduronic acid (IdoA). We report that targeted disruption of the murine d-glucuronyl C5-epimerase gene results in a structurally altered HS lacking IdoA. The corresponding phenotype is lethal, with renal agenesis, lung defects, and skeletal malformations. Unexpectedly, major organ systems, including the brain, liver, gastrointestinal tract, skin, and heart, appeared normal. We find that IdoA units are essential for normal kidney, lung, and skeletal development, albeit with different requirement for 2-O-sulfation. By contrast, major early developmental events known to critically depend on heparan sulfate apparently proceed normally even in the absence of IdoA.
No preview · Article · Sep 2003 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The biosynthesis of heparan sulfate, present on the cell surface and in the basal lamina surrounding cells, is a multistep process in which each step is mediated by a specific enzyme. The initial modification of the precursor polysaccharide, N-deacetylation followed by N-sulfation of selected N-acetyl-D-glucosamine residues, is catalyzed by the enzyme glucosaminyl N-deacetylase/N-sulfotransferase (NDST). This event is a key step that regulates the overall sulfate content of the polysaccharide. Here, we report on the effects of NDST deficiency on Ca2+ kinetics in myotubes from NDST-1- and NDST-2-deficient mice, indicating a novel role for heparan sulfate in skeletal muscle physiology. Immunostaining for specific heparan sulfate epitopes showed major changes in the heparan sulfate composition in skeletal muscle tissue derived from NDST-1-/- mice and NDST-/- cultured myotubes. Biochemical analysis indicates a relative decrease in both N-sulfation and 2-O-sulfation of skeletal muscle heparan sulfate. The core protein of heparan sulfate proteoglycan perlecan was not affected, as judged by immunohistochemistry. Also, acetylcholine receptor clustering and the occurrence of other ion channels involved in excitation-contraction coupling were not altered. In NDST-2-/- mice and heterozygous mice no changes in heparan sulfate composition were observed. Using high-speed UV confocal laser scanning microscopy, aberrant Ca2+ kinetics were observed in NDST-1-/- myotubes, but not in NDST-2-/- or heterozygous myotubes. Electrically induced Ca2+ spikes had significantly lower amplitudes, and a reduced removal rate of cytosolic Ca2+, indicating the importance of heparan sulfate in muscle Ca2+ kinetics.
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.
No preview · Article · Jan 2003 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene forN-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2−/− mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases,
including chymases, tryptases, and carboxypeptidase A. In this work we have addressed the possible functional consequences
of the lack of sulfated heparin. Peritoneal cells were harvested from normal and NDST-2−/− mice. After culturing the cells, conditioned media were collected and were subjected to SDS-polyacrylamide gel electrophoresis
under reducing conditions. Several differences in the protein patterns were observed, including the presence of large amounts
of a ∼250-kDa protein in medium from NDST-2−/− mice that was absent in normal controls. Peptide microsequencing revealed identity of this protein with fibronectin. Western
blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent
in cultures from NDST-2−/− animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2+/+ mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. This report thus indicates a biological function
for chymase/heparin proteoglycan complexes in fibronectin turnover.
Preview · Article · Feb 2001 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfate is a sulfated polysaccharide present on most cell surfaces and in the extracellular matrix. In vivo functions of heparan sulfate can be studied in mouse strains lacking enzymes involved in the biosynthesis of heparan sulfate.
Glucosaminyl N-deacetylase/N-sulfotransferase (NDST) catalyzes the first modifying step in the biosynthesis of the polysaccharide. This bifunctional enzyme
occurs in several isoforms. We here report that targeted gene disruption of NDST-1 in the mouse results in a structural alteration
of heparan sulfate in most basement membranes as revealed by immunohistochemical staining of fetal tissue sections using antibodies
raised against heparan sulfate. Biochemical analysis of heparan sulfate purified from fibroblast cultures, lung, and liver
of NDST-1-deficient embryos demonstrated a dramatic reduction in N-sulfate content. Most NDST-1-deficient embryos survive until birth; however, they turn out to be cyanotic and die neonatally
in a condition resembling respiratory distress syndrome. In addition, a minor proportion of NDST-1-deficient embryos die during
the embryonic period. The cause of the embryonic lethality is still obscure, but incompletely penetrant defects of the skull
and the eyes have been observed.
Preview · Article · Aug 2000 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The distribution of laminin alpha1 chain in adult mouse tissue was determined by immunofluorescence using monoclonal antibody 200, reacting with the globular carboxyterminus E3 fragment of alpha1 chain. Strong reactivity was noted only in a few tissues. Reactivity was restricted to epithelial basement membranes. Expression was noted in several epithelial basement membranes of the urinary tract, and male and female reproductive organs. In addition, expression was seen in some parts of the nervous system. Expression was seen in pia mater which surrounds the brain, and in the extracellular matrices covering the vitreous chamber and the lens of the eye. Staining was seen in the adrenal gland cortex, with strongest staining in the zona glomerulosa. Staining was negative in all other studied epithelial basement membranes, such as the lung (trachea or lung epithelium), epidermis, and all parts of the gastrointestinal tract (liver, gut) except for weak staining in the ventricle and Brunner's glands. No expression was seen in basement membranes of fat, Schwann, or endothelial cells in any studied parts of the body. Both small- and large-size vessel walls were negative both in endothelial basement membranes and blood vessel walls, with the exception of some larger brain blood vessels in locations where epithelial cells have invaginated. Neither smooth muscle, myocardium or striated muscle expressed alpha1 chain. We conclude that alpha1-containing heterotrimers including laminin-1 (alpha1beta1gamma1) have a very restricted tissue distribution.
[Show abstract][Hide abstract] ABSTRACT: Heparin is a sulphated polysaccharide, synthesized exclusively by connective-tissue-type mast cells and stored in the secretory granules in complex with histamine and various mast-cell proteases. Although heparin has long been used as an antithrombotic drug, endogenous heparin is not present in the blood, so it cannot have a physiological role in regulating blood coagulation. The biosynthesis of heparin involves a series of enzymatic reactions, including sulphation at various positions. The initial modification step, catalysed by the enzyme glucosaminyl N-deacetylase/N-sulphotransferase-2, NDST-2, is essential for the subsequent reactions. Here we report that mice carrying a targeted disruption of the gene encoding NDST-2 are unable to synthesize sulphated heparin. These NDST-2-deficient mice are viable and fertile but have fewer connective-tissue-type mast cells; these cells have an altered morphology and contain severely reduced amounts of histamine and mast-cell proteases. Our results indicate that one site of physiological action for heparin could be inside connective-tissue-type mast cells, where its absence results in severe defects in the secretory granules.
[Show abstract][Hide abstract] ABSTRACT: Subcutaneous injection of β1 integrin-deficient embryonic stem cells in mice causes the formation of teratomas although they occur with a lower frequency and are smaller than wild-type cells. Immunofluorescence analysis of these deficient tumors indicates a disorganized deposition of several basement membrane proteins. This was confirmed by electron microscopy which demonstrated frequent gaps in cell-associated basement membranes or loss of close contacts to the cells. Further aberrant features were multilaminar structures and amorphous deposits, indicating a strong impairment of correct basement membrane assembly. Quantitative radioimmunoassays were used to determine the levels of specific proteins in successive tissue extracts with neutral buffer in the absence and presence of EDTA and with 6 M guanidine. This demonstrated a more than 90% decrease in the content of laminin-1 (α1β1γ1) and a 70% decrease in nidogen in the β1 integrin-deficient teratomas. No significant changes were detected for other matrix proteins (perlecan, fibronectin, fibulins). This selective change impaired the formation of laminin-nidogen complex and enhanced nidogen degradation. Northern blots also demonstrated a distinctly reduced expression of laminin α1, β1, and γ1 chains. Similar reductions were also observed in cultured embryonic stem cells prior to any differentiation. No or only smaller changes were observed for laminin α2 and β2 chain, nidogen, and perlecan mRNA. These data emphasize a distinct role of β1 integrins in the correct assembly of basement membranes which may occur through direct ligand binding and/or regulatory events at the transcriptional level.
No preview · Article · Feb 1998 · Experimental Cell Research
[Show abstract][Hide abstract] ABSTRACT: Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives
of all primitive germ layers. To study the role of β1 integrin during teratoma formation, we compared teratomas induced
by normal and β1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, β1-null ES cells
either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis
of β1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived
stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas,
diffusely deposited in β1-null teratomas. Basement membranes were present but with irregular shape and detached from the
Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell–derived endothelial
cells. In contrast, β1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore,
endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although
β1- deficient endothelial cells were absent in teratomas, β1-null ES cells could differentiate in vitro into endothelial
cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in β1-null embryoid
bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive
branching of blood vessels in normal embryoid bodies, it had no effect in β1-null embryoid bodies.
No preview · Article · Oct 1997 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of β1 integrin during teratoma formation, we compared teratomas induced by normal and β1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, β1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of β1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in β1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface.
Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell–derived endothelial cells. In contrast, β1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although β1- deficient endothelial cells were absent in teratomas, β1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in β1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in β1-null embryoid bodies.
Full-text · Article · Oct 1997 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Transgenic technology has developed at breakneck speed in the past years. The establishment of embryonic stem cells and the finding that they can serve as bridge between genetic manipulations in vitro and biological analysis in vivo enabled the systematic creation of mouse strains with defined genetic alterations. This review lists the strategies which can be used to alter the genetic makeup of mice and summarizes some of the results which have been obtained in genetically altered mice of immunological interest.
No preview · Article · May 1995 · International Archives of Allergy and Immunology