Cooperation between p27 and p107 during Endochondral Ossification Suggests a Genetic Pathway Controlled by p27 and p130

ABSTRACT Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can promote differentiation. However, which members of these gene families, which cell type they interact in, and what they do to promote differentiation in that cell type during mouse development are largely unknown. To identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic similarities between p107 −/− p27 D51/D51 and p107 −/− p130 −/− mice and the cells derived from them suggest that p27 and p130 act in an analogous pathway during chondrocyte maturation.

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Selective Deficiency of the “Bone-related”Runx2-II Unexpectedly Preserves Osteoblast-mediated Skeletogenesis

Journal of Biological Chemistry ◽

10.1074/jbc.m401109200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 20307-20313 ◽

Cited By ~ 66

Author(s):

Zhou-Sheng Xiao ◽

Anita B. Hjelmeland ◽

L. D. Quarles

Keyword(s):

Bone Formation ◽

Ex Vivo ◽

Mesenchymal Cells ◽

Endochondral Bone Formation ◽

Endochondral Bone ◽

Chondrocyte Maturation ◽

Intramembranous Bone ◽

Exon 1 ◽

Metaphyseal Bone ◽

Deficient Mice

Runx2 (runt-related transcription factor 2) is a master regulator of skeletogenesis. Distinct promoters in the Runx2 gene transcribe the “bone-related”Runx2-II and non-osseousRunx2-I isoforms that differ only in their respective N termini. Existing mutant mouse models with both isoforms deleted exhibit an arrest of osteoblast and chondrocyte maturation and the complete absence of mineralized bone, but they do not distinguish the separate functions of the two N-terminal isoforms. To elucidate the function of the bone-related isoform, we generated selectiveRunx2-II-deficient mice by the targeted deletion of the distal promoter and exon 1. HomozygousRunx2-II-deficient (Runx2-II-/-) mice unexpectedly formed axial, appendicular, and craniofacial bones derived from either intramembranous ossification or mesenchymal cells of the bone collar, but they failed to form the posterior cranium and other bones derived from endochondral ossification. HeterozygousRunx2-II-deficient mice had grossly normal skeletons, but were osteopenic. The commitment of mesenchymal cellsex vivoto the osteoblast lineage occurred inRunx2-II-/-mice, but osteoblastic gene expression was impaired. Chondrocyte maturation appeared normal, but the zone of hypertrophic chondrocytes was not transformed into metaphyseal bone, leading to widened growth plates inRunx2-II-/-mice. Compensatory increments inRunx2-I expression occurred inRunx2-II-/-mice but were not sufficient to normalize osteoblastic maturation or transcriptional activity. Our findings support distinct functions ofRunx2-II and -I in the control of skeletogenesis.Runx2-I is sufficient for early osteoblastogenesis and intramembranous bone formation, whereasRunx2-II is necessary for complete osteoblastic maturation and endochondral bone formation.

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Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

Stem Cells International ◽

10.1155/2019/7281912 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

Thu T. Duong ◽

James Lim ◽

Vidyullatha Vasireddy ◽

Tyler Papp ◽

Hung Nguyen ◽

...

Keyword(s):

Cortical Neurons ◽

Transgene Expression ◽

Fluorescent Protein ◽

Ex Vivo ◽

Cell Types ◽

Egfp Expression ◽

Cell Type ◽

Egfp Gene ◽

Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

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Conditional inactivation of VEGF-A in areas of collagen2a1 expression results in embryonic lethality in the heterozygous state

Development ◽

10.1242/dev.127.7.1445 ◽

2000 ◽

Vol 127 (7) ◽

pp. 1445-1453 ◽

Cited By ~ 5

Author(s):

J.J. Haigh ◽

H.P. Gerber ◽

N. Ferrara ◽

E.F. Wagner

Keyword(s):

Bone Formation ◽

Ischemic Cardiomyopathy ◽

Embryonic Lethality ◽

Limb Bud ◽

Endochondral Bone Formation ◽

Functional Importance ◽

Mouse Development ◽

Endochondral Bone ◽

Myocardial Layers ◽

Transgenic Lines

VEGF-A has been implicated in regulating the initial angiogenic invasion events that are essential for endochondral bone formation. VEGF-A mRNA expression was indeed found in the sclerotome of the developing somite and in the limb-bud mesenchyme at E10.5 in mouse development but declined during chondrogenesis and became upregulated in hypertrophic chondrocytes prior to angiogenic invasion. To determine the functional importance of VEGF-A expression in the developing chondrogenic tissues, VEGF-A was conditionally inactivated during early embryonic development using Collagen2a1-Cre transgenic lines. Deletion of a single VEGF-A allele in Collagen2a1-Cre-expressing cells results in embryonic lethality around E10.5. This lethality is characterized by aberrant development of the dorsal aorta and intersomitic blood vessels, along with defects in the developing endocardial and myocardial layers of the heart. A small percentage of VEGF(Flox)/+, Collagen2a1-Cre fetuses survive until E17.5, show aberrant endochondral bone formation and develop a heart phenotype resembling a dilated form of ischemic cardiomyopathy. These results provide insights into the function of VEGF-A in heart and endochondral bone formation and underscore the importance of tightly controlled levels of VEGF-A during development.

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Prolactin inhibits the apoptosis of chondrocytes induced by serum starvation

Journal of Endocrinology ◽

10.1677/joe.1.06766 ◽

2006 ◽

Vol 189 (2) ◽

pp. R1-R8 ◽

Cited By ~ 18

Author(s):

C Zermeño ◽

J Guzmán-Morales ◽

Y Macotela ◽

G Nava ◽

F López-Barrera ◽

...

Keyword(s):

Articular Chondrocytes ◽

Cell Types ◽

Cartilage Degradation ◽

Chondrocyte Apoptosis ◽

Heat Inactivation ◽

Serum Starvation ◽

Endochondral Bone Formation ◽

Endochondral Bone ◽

Therapeutic Value ◽

Apoptotic Effect

The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.

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Development of a multiomics and functional drug sensitivity platform to investigate cell type specific drug effects in AML.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e19013 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e19013-e19013

Author(s):

Marianne T. Santaguida ◽

Ryosuke Kita ◽

Steven A. Schaffert ◽

Erica K. Anderson ◽

Kamran A Ali ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Response ◽

Drug Sensitivity ◽

Ex Vivo ◽

Cell Types ◽

Specific Cell ◽

Rna Seq ◽

Cell Type ◽

Drug Sensitivity Assay ◽

Cell Type Specific

e19013 Background: Understanding the heterogeneity of AML is necessary for developing targeted drugs and diagnostics. A key measure of heterogeneity is the variance in response to treatments. Previously, we developed an ex vivo flow cytometry drug sensitivity assay (DSA) that predicted response to treatments in myelodysplastic syndrome. Unlike bulk cell viability measures of other drug sensitivity assays, our flow cytometry assay provides single cell resolution. The assay measures a drug’s effect on the viability or functional state of specific cell types. Here we present the development of this technology for AML, with additional measurements of DNA-Seq and RNA-Seq. Using the data from this assay, we aim to characterize the heterogeneity in AML drug sensitivity and the molecular mechanisms that drive it. Methods: As an initial feasibility analysis, we assayed 1 bone marrow and 3 peripheral blood AML patient samples. For the DSA, the samples were cultured with six AML standard of care (SOC) compounds across seven doses, in addition to two combinations. The cells were stained to detect multiple cell types including tumor blasts, and drug response was measured by flow cytometry. For the multi-omics, the cells were magnetically sorted to enrich for blasts and then assayed using a targeted 400 gene DNA-Seq panel and whole bulk transcriptome RNA-Seq. For comparison with BeatAML, Pearson correlations between gene expression and venetoclax sensitivity were investigated. Results: In our drug sensitivity assay, we measured dose response curves for the six SOC compounds, for each different cell type across each sample. The dose responses had cell type specific effects, including differences in drug response between CD11b+ blasts, CD11b- blasts, and other non-blast populations. Integrating with the DNA-Seq and RNA-Seq data, known associations between ex vivo drug response and gene expression were identified with additional cell type specificity. For example, BCL2A1 expression was negatively correlated with venetoclax sensitivity in CD11b- blasts but not in CD11b+ blasts. To further corroborate, among the top 1000 genes associated with venetoclax sensitivity in BeatAML, 93.7% had concordant directionality in effect. Conclusions: Here we describe the development of an integrated ex vivo drug sensitivity assay and multi-omics dataset. The data demonstrated that ex vivo responses to compounds differ between cell types, highlighting the importance of measuring drug response in specific cell types. In addition, we demonstrated that integrating these data will provide unique insights on molecular mechanisms that affect cell type specific drug response. As we continue to expand the number of patient samples evaluated with our multi-dimensional platform, this dataset will provide insights for novel drug target discovery, biomarker development, and, in the future, informing treatment decisions.

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

Endocrinology ◽

10.1210/en.2014-1109 ◽

2014 ◽

Vol 155 (8) ◽

pp. 3123-3135 ◽

Cited By ~ 25

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.

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A Transgenic Rat for Noninvasive Assessment of Chondrogenesis in Vivo

Cartilage ◽

10.1177/19476035211057243 ◽

2021 ◽

pp. 194760352110572

Author(s):

Elisabeth Ferreira ◽

Landon B. Gatrell ◽

Luke Childress ◽

Hong Wu ◽

Ryan M. Porter

Keyword(s):

Bone Formation ◽

Heterotopic Ossification ◽

Ex Vivo ◽

Firefly Luciferase ◽

Bone Morphogenetic Protein 2 ◽

Endochondral Bone Formation ◽

Transgenic Rat ◽

Regulatory Sequences ◽

Endochondral Bone

Objective To support the preclinical evaluation of therapeutics that target chondrogenesis, our goal was to generate a rat strain that can noninvasively report endogenous chondrogenic activity. Design A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. Candidate lines were established on a Lewis background and characterized by serial bioluminescence imaging as well as ex vivo measurement of molecular reporter levels in several tissues. The sensitivity and specificity of the reporter strain were assessed in models of orthotopic and ectopic chondrogenesis. Results Substantial bioluminescence signal was detected from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance was intense at 1 month of age, rapidly declined with continued development, yet remained detectable in 2-year-old animals. Explant imaging and immunohistochemistry confirmed that both molecular reporters were localized to cartilage. Implantation of wild-type bone marrow stromal cells into osteochondral defects made in both young adult and aged reporter rats led to a time-dependent elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair. To stimulate ectopic, endochondral bone formation, bone morphogenetic protein 2 was overexpressed in the gastrocnemius muscle, which led to bioluminescent signal that closely preceded heterotopic ossification. Conclusions This strain can help develop strategies to stimulate cartilage repair and endochondral bone formation or to inhibit chondrogenesis associated with heterotopic ossification.

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Ex vivo gene therapy-induced endochondral bone formation: comparison of muscle-derived stem cells and different subpopulations of primary muscle-derived cells

Bone ◽

10.1016/j.bone.2004.01.028 ◽

2004 ◽

Vol 34 (6) ◽

pp. 982-992 ◽

Cited By ~ 48

Author(s):

Hsain-Chung Shen ◽

Hairong Peng ◽

Arvydas Usas ◽

Brian Gearhart ◽

James Cummins ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Formation ◽

Ex Vivo ◽

Endochondral Bone Formation ◽

Endochondral Bone ◽

Ex Vivo Gene Therapy

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Constitutive E2F1 Overexpression Delays Endochondral Bone Formation by Inhibiting Chondrocyte Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.23.10.3656-3668.2003 ◽

2003 ◽

Vol 23 (10) ◽

pp. 3656-3668 ◽

Cited By ~ 28

Author(s):

Blanca Scheijen ◽

Marieke Bronk ◽

Tiffany van der Meer ◽

René Bernards

Keyword(s):

Bone Formation ◽

Endochondral Ossification ◽

Bone Growth ◽

Late Phase ◽

Type Ii Collagen ◽

Endochondral Bone Formation ◽

Nodule Formation ◽

Chondrocyte Differentiation ◽

Endochondral Bone ◽

Chondrocyte Maturation

ABSTRACT Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation.

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