The CREB family of activators is required for endochondral bone development

Development ◽  
2001 ◽  
Vol 128 (4) ◽  
pp. 541-550
Author(s):  
F. Long ◽  
E. Schipani ◽  
H. Asahara ◽  
H. Kronenberg ◽  
M. Montminy
Keyword(s):  
Transgenic Mice ◽  
Bone Development ◽  
Dominant Negative ◽  
Endochondral Bone Formation ◽  
Wild Type ◽  
Growth Plate Chondrocytes ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone ◽  
Proliferative Zone ◽  
Distinct Signaling Pathway

We have evaluated the importance of the CREB family of transcriptional activators for endochondral bone formation by expressing a potent dominant negative CREB inhibitor (A-CREB) in growth plate chondrocytes of transgenic mice. A-CREB transgenic mice exhibited short-limbed dwarfism and died minutes after birth, apparently due to respiratory failure from a diminished rib cage circumference. Consistent with the robust Ser133 phosphorylation and, hence, activation of CREB in chondrocytes within the proliferative zone of wild-type cartilage during development, chondrocytes in A-CREB mutant cartilage exhibited a profound decrease in proliferative index and a delay in hypertrophy. Correspondingly, the expression of certain signaling molecules in cartilage, most notably the Indian hedgehog (Ihh) receptor patched (Ptch), was lower in A-CREB expressing versus wild-type chondrocytes. CREB appears to promote Ptch expression in proliferating chondrocytes via an Ihh-independent pathway; phospho-CREB levels were comparable in cartilage from Ihh(−/−) and wild-type mice. These results demonstrate the presence of a distinct signaling pathway in developing bone that potentiates Ihh signaling and regulates chondrocyte proliferation, at least in part, via the CREB family of activators.

Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation

Development ◽  
2001 ◽  
Vol 128 (24) ◽  
pp. 5099-5108 ◽  
Author(s):  
Fanxin Long ◽  
Xiaoyan M. Zhang ◽  
Seth Karp ◽  
Yingzi Yang ◽  
Andrew P. McMahon
Keyword(s):  
Cyclin D1 ◽  
Hedgehog Signaling ◽  
Genetic Manipulation ◽  
Bone Development ◽  
Transmembrane Protein ◽  
Wild Type ◽  
Chondrocyte Proliferation ◽  
Direct Role ◽  
Endochondral Bone ◽  
Proliferation And Differentiation

Indian hedgehog (Ihh), one of the three mammalian hedgehog (Hh) proteins, coordinates proliferation and differentiation of chondrocytes during endochondral bone development. Smoothened (Smo) is a transmembrane protein that transduces all Hh signals. In order to discern the direct versus indirect roles of Ihh in cartilage development, we have used the Cre-loxP approach to remove Smo activity specifically in chondrocytes. Animals generated by this means develop shorter long bones when compared to wild-type littermates. In contrast to Ihh mutants (Ihhn/Ihhn), chondrocyte differentiation proceeds normally. However, like Ihhn/Ihhn mice, proliferation of chondrocytes is reduced by about 50%, supporting a direct role for Ihh in the regulation of chondrocyte proliferation. Moreover, by overexpressing either Ihh or a constitutively active Smo allele (Smo*) specifically in the cartilage using the bigenic UAS-Gal4 system, we demonstrate that activation of the Ihh signaling pathway is sufficient to promote chondrocyte proliferation. Finally, expression of cyclin D1 is markedly downregulated when either Ihh or Smo activity is removed from chondrocytes, indicating that Ihh regulates chondrocyte proliferation at least in part by modulating the transcription of cyclin D1. Taken together, the present study establishes Ihh as a key mitogen in the endochondral skeleton.

Analysis of Hoxd-13 and Hoxd-11 misexpression in chick limb buds reveals that Hox genes affect both bone condensation and growth

Development ◽  
1997 ◽  
Vol 124 (3) ◽  
pp. 627-636 ◽  
Author(s):  
D.J. Goff ◽  
C.J. Tabin
Keyword(s):  
Growth Phase ◽  
Target Genes ◽  
Hox Genes ◽  
Bone Development ◽  
Tritiated Thymidine ◽  
Retroviral Vectors ◽  
Dominant Negative ◽  
Thymidine Uptake ◽  
Growth Promoters ◽  
Proliferative Zone

Hox genes are important regulators of limb pattern in vertebrate development. Misexpression of Hox genes in chicks using retroviral vectors provides an opportunity to analyze gain-of-function phenotypes and to assess their modes of action. Here we report the misexpression phenotype for Hoxd-13 and compare it to the misexpression phenotype of Hoxd-11. Hoxd-13 misexpression in the hindlimb results in a shortening of the long bones, including the femur, the tibia, the fibula and the tarsometatarsals. Mutations in an alanine repeat region in the N-terminus of Hoxd-13 have recently been implicated in human synpolydactyly (Muragaki, Y., Mundlos, S., Upton, J. and Olsen, B. R. (1996) Science 272, 548–551). N-terminal truncations of Hoxd-13 which lack this repeat were constructed and were found to produce a similar, although slightly milder, misexpression phenotype than the full-length Hoxd-13. The stage of bone development regulated by Hox genes has not previously been examined. The changes in bone lengths caused by Hoxd-13 misexpression are late phenotypes that first become apparent during the growth phase of the bones. Analysis of tritiated thymidine uptake by the tibia and fibula demonstrates that Hox genes can pattern the limb skeleton by regulating the rates of cell division in the proliferative zone of growing cartilage. Hoxd-11, in contrast to Hoxd-13, acts both at the initial cartilage condensation phase in the foot and during the later growth phase in the lower leg. Ectopic Hoxd-13 appears to act in a dominant negative manner in regions where it is not normally expressed. We propose a model in which all Hox genes are growth promoters, regulating the expression of the same target genes, with some Hox genes being more effective promoters of growth than other Hox genes. According to this model, the overall rate of growth in a given region is the result of the combined action of all of the Hox genes expressed in that region competing for the same target genes.

Impairment of TGF-β signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice

2003 ◽  
Vol 284 (3) ◽  
pp. G525-G535 ◽  
Author(s):  
Christoph Schramm ◽  
Martina Protschka ◽  
Heinz H. Köhler ◽  
Jürgen Podlech ◽  
Matthias J. Reddehase ◽  
...  
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Autoimmune Hepatitis ◽  
Mononuclear Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Histological Score ◽  
Tgf Β1 ◽  
Increased Susceptibility ◽  
Experimental Autoimmune

In autoimmune hepatitis, strong TGF-β1 expression is found in the inflamed liver. TGF-β overexpression may be part of a regulatory immune response attempting to suppress autoreactive T cells. To test this hypothesis, we determined whether impairment of TGF-β signaling in T cells leads to increased susceptibility to experimental autoimmune hepatitis (EAH). Transgenic mice of strain FVB/N were generated expressing a dominant-negative TGF-β type II receptor in T cells under the control of the human CD2 promoter/locus control region. On induction of EAH, transgenic mice showed markedly increased portal and periportal leukocytic infiltrations with hepatocellular necroses compared with wild-type mice (median histological score = 1.8 ± 0.26 vs. 0.75 ± 0.09 in wild-type mice; P < 0.01). Increased IFN-γ production (118 vs. 45 ng/ml) and less IL-4 production (341 vs. 1,256 pg/ml) by mononuclear cells isolated from transgenic livers was seen. Impairment of TGF-β signaling in T cells therefore leads to increased susceptibility to EAH in mice. This suggests an important role for TGF-β in immune homeostasis in the liver and may teleologically explain TGF-β upregulation in response to T cell-mediated liver injury.

scholarly journals BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development

2011 ◽  
Vol 124 (20) ◽  
pp. 3428-3440 ◽  
Author(s):  
B. Shu ◽  
M. Zhang ◽  
R. Xie ◽  
M. Wang ◽  
H. Jin ◽  
...  
Keyword(s):  
Bone Development ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone

EXT1 regulates chondrocyte proliferation and differentiation during endochondral bone development

Bone ◽  
2005 ◽  
Vol 36 (3) ◽  
pp. 379-386 ◽  
Author(s):  
Matthew J. Hilton ◽  
Laura Gutiérrez ◽  
Daniel A. Martinez ◽  
Dan E. Wells
Keyword(s):  
Bone Development ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone ◽  
Proliferation And Differentiation

scholarly journals Isolation of Glomerular Podocytes by Cationic Colloidal Silica-coated Ferromagnetic Nanoparticles

2016 ◽  
Vol 9 (1) ◽  
pp. 67-87
Author(s):  
Andreas Blutke
Keyword(s):  
Transgenic Mice ◽  
Transgenic Animals ◽  
Dominant Negative ◽  
Ferromagnetic Nanoparticles ◽  
Wild Type ◽  
Isolated Cells ◽  
Specific Expression ◽  
Specific Antibodies ◽  
Isolated Glomeruli ◽  
Fluorescent Markers

Background: Podocyte homeostasis plays a crucial role for the maintenance of physiological glomerular function and podocyte injury is regarded as a major determinant of development and progression of renal disease. Objective: Investigation of podocytes requires appropriate methods for their isolation. Previously reported methods use podocyte specific antibodies or transgenic mice with podocyte specific expression of fluorescent markers for isolation of podocytes by magnetic or fluorescence activated cell sorting. Method: Here, a novel, antibody-free method for isolation of podocyte protein and RNA from mouse glomeruli is described. Preparations of isolated glomeruli were added to a suspension of cationic silica-coated colloidal ferromagnetic nanoparticles. The nanoparticles bound to the negatively charged cell surfaces of podocytes residing on the outer surface of the isolated glomeruli. After enzymatic and mechanical dissociation of glomerular cells, nanoparticle-coated podocytes were isolated in a magnetic field. The method was tested in adult wild-type mice without renal lesions and in mice of two nephropathy models (Growth hormone (GH)-transgenic mice and transgenic mice expressing a dominant negative receptor for the glucose dependent insulinotropic polypeptide, GIPRdn) displaying albuminuria, glomerular hypertrophy and evidence for a reduced negative cell surface charge of podocytes. Results: The isolated cells displayed typical morphological and ultrastructural properties of podocytes. On average, 182,000 ± 37,000 cells were counted in the podocyte isolates harvested from ~10,000-12,000 glomeruli per mouse. On the average, the purity of podocyte isolates of these mice accounted for ~63 ± 18 % and the podocyte isolates displayed high mRNA and protein expression abundances of podocyte markers (nephrin and WT1), whereas the expression of endothelial (Cd31) and mesangial markers (Serpinb7) was significantly decreased in podocyte isolates, as compared to samples of isolated glomeruli. The numbers of cells isolated from GH- transgenic and GIPRdn-transgenic mice were not markedly different from that of wild-type mice. Conclusion: The described method represents an alternative for podocyte isolation, particularly in experiments where podocyte specific antibodies or transgenic animals with podocyte specific expression of fluorescent markers are not applicable.

scholarly journals Chondrocytes Play a Major Role in the Stimulation of Bone Growth by Thyroid Hormone

Endocrinology ◽  
2014 ◽  
Vol 155 (8) ◽  
pp. 3123-3135 ◽  
Author(s):  
Clémence Desjardin ◽  
Cyril Charles ◽  
Catherine Benoist-Lasselin ◽  
Julie Riviere ◽  
Mailys Gilles ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Bone Growth ◽  
Cell Types ◽  
Postnatal Growth ◽  
Dominant Negative ◽  
Skeletal Growth ◽  
Endochondral Bone Formation ◽  
Endochondral Bone ◽  
Phenotype Analysis ◽  
Visible Effect

Thyroid hormone (T3) is required for postnatal skeletal growth. It exerts its effect by binding to nuclear receptors, TRs including TRα1 and TRβ1, which are present in most cell types. These cell types include chondrocytes and osteoblasts, the interactions of which are known to regulate endochondral bone formation. In order to analyze the respective functions of T3 stimulation in chondrocytes and osteoblasts during postnatal growth, we use Cre/loxP recombination to express a dominant-negative TRα1L400R mutant receptor in a cell-specific manner. Phenotype analysis revealed that inhibiting T3 response in chondrocytes is sufficient to reproduce the defects observed in hypothyroid mice, not only for cartilage maturation, but also for ossification and mineralization. TRα1L400R in chondrocytes also results in skull deformation. In the meantime, TRα1L400R expression in mature osteoblasts has no visible effect. Transcriptome analysis identifies a number of changes in gene expression induced by TRα1L400R in cartilage. These changes suggest that T3 normally cross talks with several other signaling pathways to promote chondrocytes proliferation, differentiation, and skeletal growth.

Attenuation of Transient Focal Cerebral Ischemic Injury in Transgenic Mice Expressing a Mutant ICE Inhibitory Protein

1997 ◽  
Vol 17 (4) ◽  
pp. 370-375 ◽  
Author(s):  
Hideaki Hara ◽  
Klaus Fink ◽  
Matthias Endres ◽  
Robert M. Friedlander ◽  
Valeria Gagliardini ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Transgenic Mice ◽  
Artery Occlusion ◽  
Dominant Negative ◽  
Neurological Deficits ◽  
Wild Type ◽  
Brain Swelling ◽  
Inhibitory Protein ◽  
Endogenous Expression ◽  
Dominant Negative Mutation

We used transgenic mice expressing a dominant negative mutation of interleukin-1β converting enzyme (ICE) (C285G) in a model of transient focal ischemia in order to investigate the role of ICE in ischemic brain damage. Transgenic mutant ICE mice (n = 11) and wild-type littermates (n = 9) were subjected to 3 h of middle cerebral artery occlusion followed by 24 h of reperfusion. Cerebral infarcts and brain swelling were reduced by 44% and 46%, respectively. Neurological deficits were also significantly reduced. Regional CBF, blood pressure, core temperature, and heart rate did not differ between groups when measured for up to 1 h after reperfusion. Increases in immunoreactive IL-1β levels, observed in ischemic wild-type brain at 30 min after reperfusion, were 77% lower in the mutant strain, indicating that proIL-1β cleavage is inhibited in the mutants. DNA fragmentation was reduced in the mutants 6 and 24 h after reperfusion. Hence, endogenous expression of an ICE inhibitor confers resistance to cerebral ischemia and brain swelling. Our results indicate that down-regulation of ICE expression might provide a useful therapeutic target in cerebral ischemia.

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