Generation of a mouse line harboring a bi‐transgene expressing luciferase and tamoxifen‐activatable creER<sup>T2</sup> recombinase in cartilage

Cascio, Leandro Lo; Liu, Ke; Nakamura, Hiroyuki; Chu, Grace; Lim, Ngee Han; Chanalaris, Anastasios; Saklatvala, Jeremy; Nagase, Hideaki; Bou‐Gharios, George

doi:10.1002/dvg.22734

Cited by 17 publications

(16 citation statements)

References 27 publications

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“…MyD88‐null and TAK‐1 (MAP3K7) loxP‐flanked (floxed) mice were originally acquired from The Jackson Laboratory. The CreER aggrecan promoter line was generated at the Kennedy Institute by George Bou‐Gharios. Animal experiments were performed after ethics and statutory approval was obtained in accordance with local policy.…”

Section: Methodsmentioning

confidence: 99%

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K⁶³‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Ismail

Didangelos

Vincent

et al. 2017

Arthritis & Rheumatology

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ObjectiveMechanical injury to cartilage predisposes to osteoarthritis (OA). Wounding of the articular cartilage surface causes rapid activation of MAP kinases and NF‐κB, mimicking the response to inflammatory cytokines. This study was undertaken to identify the upstream signaling mechanisms involved.MethodsCartilage was injured by dissecting it from the articular surface of porcine metacarpophalangeal (MCP) joints or by avulsing murine proximal femoral epiphyses. Protein phosphorylation was assayed by Western blotting of cartilage lysates. Immunolocalization of phosphorylated activating transcription factor 2 (ATF‐2) and NF‐κB/p65 was detected by confocal microscopy. Messenger RNA (mRNA) was measured by quantitative reverse transcriptase–polymerase chain reaction (qRT‐PCR). Receptor associated protein 80 (RAP‐80) ubiquitin interacting motif agarose was used in a pull‐down assay to obtain K63‐polyubiquitinated proteins. Ubiquitin linkages on immunoprecipitated transforming growth factor β–activated kinase 1 (TAK‐1) were analyzed with deubiquitinases.ResultsSharp injury to porcine cartilage caused rapid activation of JNK and NF‐κB pathways and the upstream kinases MKK‐4, IKK, and TAK‐1. Pharmacologic inhibition of TAK‐1 in porcine cartilage abolished JNK and NF‐κB activation and reduced the injury‐dependent inflammatory gene response. High molecular weight species of phosphorylated TAK‐1 were induced by injury, indicating its ubiquitination. An overall increase in K63‐linked polyubiquitination was detected upon injury, and TAK‐1 was specifically linked to K63‐ but not K48‐polyubiquitin chains. In mice, avulsion of wild‐type femoral epiphyses caused similar intracellular signaling that was reduced in cartilage‐specific TAK‐1–null mice. Epiphyseal cartilage of MyD88‐null and TRAF‐6–null mice responded to injury, suggesting the involvement of a ubiquitin E3 ligase other than TRAF‐6.ConclusionActivation of TAK‐1 by phosphorylation and K63‐linked polyubiquitination by injury indicates its role in driving cell activation. Further studies are needed to identify the upstream ubiquitination mechanisms, including the E3 ligase involved.

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Section: Methodsmentioning

confidence: 99%

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K⁶³‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Ismail

Didangelos

Vincent

et al. 2017

Arthritis & Rheumatology

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“…Mice used in this study were in third generation backcross to C57Bl/ 6J. Dot1l fl/fl were also bred to B6.Cg-Acan tm1(cre/ERT2)Crm /J mice (Jax019148) 19 (from Dr. G. Bou-Gharios and the Kennedy Institute, Oxford, UK) to generate inducible cartilage-specific Dot1l knockout mice (Dot1l fl/fl ;Acan-Cre-ER þ/or Dot1l CondCART-KO ). Mice used in this study were in second generation backcross to C57Bl/6J.…”

Section: Micementioning

confidence: 99%

“…Mice used in this study were in second generation backcross to C57Bl/6J. Tamoxifen was injected at the age of 8 weeks (2 mg, 3 injections, every other day) 19 , to generate conditional cartilage-specific knockout mice. Genotypes of animals were confirmed by PCR upon weaning and after sacrifice 12 .…”

Section: Micementioning

confidence: 99%

“…To study the impact of the postnatal deletion of Dot1l in mouse cartilage, we bred homozygous floxed Dot1l mice (Dot1l fl/ fl ) with an Acan-Cre-ER mouse strain (Acan-Cre-ER þ/-) 19 to obtain postnatal tamoxifen-inducible specific recombination in chondrocytes (Dot1l fl/fl ;Acan-Cre-ER þ/-) (Dot1l CondCART-KO ). To induce recombination, mice were injected with tamoxifen at the age of 8 weeks 19 [ Fig. 4(A)].…”

Section: Postnatal Tamoxifen-induced Conditional Dot1l Cart-ko Mice Dmentioning

confidence: 99%

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Increased susceptibility to develop spontaneous and post-traumatic osteoarthritis in Dot1l-deficient mice

Cornelis

Storms

et al. 2019

Osteoarthritis and Cartilage

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Objective: We earlier identified that the histone methyltransferase Disruptor of telomeric silencing 1-like (DOT1L) is as a master protector of cartilage health via limiting excessive activation of the Wnt pathway. However, cartilage-specific homozygous Dot1l knockout mice exhibited a severe growth phenotype and perinatal death, which hampered their use in induced or ageing models of osteoarthritis (OA). The aim of this study was to generate and examine haploinsufficient and inducible conditional Dot1l-deficient mouse models to evaluate the importance of DOT1L during post-traumatic or ageing-associated OA onset and progression. Method: We used cartilage-specific heterozygous and postnatal tamoxifen-inducible Dot1l knockout mice and performed destabilization of the medial meniscus (DMM) and ageing as OA models. Mice were examined histologically using X-rays and micro-computed tomography (mCT), and cartilage damage and osteophyte formation were assessed based on OARSI guidelines. Immunohistochemistry of DOT1L, H3K79me2, TCF1 and COLX was performed. Results: Both Dot1l-deficient strains exhibit a phenotype characterized by joint remodeling with extensive osteophyte formation and ectopic ossification upon ageing, indicating accelerated development of spontaneous osteoarthritis. In the DMM-induced OA mouse model, absence of Dot1l resulted in increased cartilage damage. Wnt signalling hyper-activation and ectopic chondrocyte hypertrophy were observed in the articular cartilage of both Dot1l-deficient mice. Conclusions: This study demonstrated the functional relevance of DOT1L in vivo during the development of OA using genetically modified mice. Thus, maintaining or enhancing DOT1L activity during ageing or after trauma might prevent OA onset and progression.

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“…Tamoxifen was administered to 2-month-old TSC1CKO ER mice (100 mg/g body weight, intraperitoneally, daily for 5 days) 16 .…”

Section: Tamoxifen Administration and Mouse Oa Modelmentioning

confidence: 99%

mTORC1 activation downregulates FGFR3 and PTH/PTHrP receptor in articular chondrocytes to initiate osteoarthritis

Zhang

Wang

Zeng

et al. 2017

Osteoarthritis and Cartilage

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Generation of a mouse line harboring a bi‐transgene expressing luciferase and tamoxifen‐activatable creER^T2 recombinase in cartilage

Cited by 17 publications

References 27 publications

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K⁶³‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K⁶³‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Increased susceptibility to develop spontaneous and post-traumatic osteoarthritis in Dot1l-deficient mice

mTORC1 activation downregulates FGFR3 and PTH/PTHrP receptor in articular chondrocytes to initiate osteoarthritis

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Generation of a mouse line harboring a bi‐transgene expressing luciferase and tamoxifen‐activatable creERT2 recombinase in cartilage

Cited by 17 publications

References 27 publications

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K63‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K63‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Increased susceptibility to develop spontaneous and post-traumatic osteoarthritis in Dot1l-deficient mice

mTORC1 activation downregulates FGFR3 and PTH/PTHrP receptor in articular chondrocytes to initiate osteoarthritis

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Generation of a mouse line harboring a bi‐transgene expressing luciferase and tamoxifen‐activatable creER^T2 recombinase in cartilage

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K⁶³‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K⁶³‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage