L-Sox5 and Sox6 Drive Expression of the Aggrecan Gene in Cartilage by Securing Binding of Sox9 to a Far-Upstream Enhancer

Han, Yu; Lefebvre, Véronique

doi:10.1128/mcb.00695-08

Cited by 257 publications

(294 citation statements)

References 55 publications

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“…To this end, we examined SOX9 acetylation level and binding to a previously described ACAN enhancer,(Han & Lefebvre, 2008), in various experimental settings, including OA articular cartilage. To further understand the role of SOX9 acetylation, we aimed to investigate the impact of this protein modification on the stability and nuclear trafficking of SOX9.…”

Section: Introductionmentioning

confidence: 99%

Acetylation reduces SOX 9 nuclear entry and ACAN gene transactivation in human chondrocytes

Kumar

Elayyan

et al. 2016

Aging Cell

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SummaryChanges in the content of aggrecan, an essential proteoglycan of articular cartilage, have been implicated in the pathophysiology of osteoarthritis (OA), a prevalent age‐related, degenerative joint disease. Here, we examined the effect of SOX9 acetylation on ACAN transactivation in the context of osteoarthritis. Primary chondrocytes freshly isolated from degenerated OA cartilage displayed lower levels of ACAN mRNA and higher levels of acetylated SOX9 compared with cells from intact regions of OA cartilage. Degenerated OA cartilage presented chondrocyte clusters bearing diffused immunostaining for SOX9 compared with intact cartilage regions. Primary human chondrocytes freshly isolated from OA knee joints were cultured in monolayer or in three‐dimensional alginate microbeads (3D). SOX9 was hypo‐acetylated in 3D cultures and displayed enhanced binding to a −10 kb ACAN enhancer, a result consistent with higher ACAN mRNA levels than in monolayer cultures. It also co‐immunoprecipitated with SIRT1, a major deacetylase responsible for SOX9 deacetylation. Finally, immunofluorescence assays revealed increased nuclear localization of SOX9 in primary chondrocytes treated with the NAD SIRT1 cofactor, than in cells treated with a SIRT1 inhibitor. Inhibition of importin β by importazole maintained SOX9 in the cytoplasm, even in the presence of NAD. Based on these data, we conclude that deacetylation promotes SOX9 nuclear translocation and hence its ability to activate ACAN.

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Section: Introductionmentioning

confidence: 99%

Acetylation reduces SOX 9 nuclear entry and ACAN gene transactivation in human chondrocytes

Kumar

Elayyan

et al. 2016

Aging Cell

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“…AF-MSCs only expressed FABP4 and LEP that are associated with the intermediate and late phases in developing adipocytes (Bernlohr et al 1985), which may be led to a distinct developmental program characteristic of stem cells (Zuk et al 2002). The potential of bovine MSCs to undergo chondrogenesis has been assessed by staining the cells with Alcian blue and confirmed using markers commonly associated with the chondrocyte phenotype: the early marker SOX9, collagen type 2 and ACAN, the most essential cartilage proteoglycan, and key markers of chondrocyte differentiation (Han & Lefebvre 2008). In AECs and AF-MSCs, the expression of SOX9, which has been reported to regulate the rate of chondrocyte differentiation by controlling the expression of a series of chondrocyte-specific genes including ACAN and COL2A1 (Tchetina et al 2003), was detected.…”

Section: Discussionmentioning

confidence: 99%

Mesenchymal stem cells from amnion and amniotic fluid in the bovine

Corradetti¹,

Meucci²,

Bizzaro³

et al. 2013

Reproduction

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Amnion and amniotic fluid (AF) are noncontroversial and inexhaustible sources of mesenchymal stem cells (MSCs) that can be harvested noninvasively at low cost. As in humans, also in veterinary field, presumptive stem cells derived from these tissues reveal as promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. The aim of this work is to obtain and characterize, for the first time in bovine species, presumptive MSCs from the epithelial portion of the amnion (AECs) and from the AF (AF-MSCs) to be used for clinical applications. AECs display a polygonal morphology, whereas AF-MSCs exhibit a fibroblastic-like morphology only starting from the second passage, being heterogeneous during the primary culture. For both lines, the proliferative ability has been found constant over the ten passages studied and AECs show a statistically lower (P!0.05) doubling time with respect to AF-MSCs. AECs express MSC-specific markers (ITGB1 (CD29), CD44, ALCAM (CD166), ENG (CD105), and NT5E (CD73)) from P1 to P3; in AF-MSCs, only ITGB1, CD44, and ALCAM mRNAs are detected; NT5E is expressed from P2 and ENG has not been found at any passage. AF-MSCs and AECs are positive for the pluripotent markers (POU5F1 (OCT4) and MYC (c-Myc)) and lack of the hematopoietic markers. When appropriately induced, both cell lines are capable of differentiating into ectodermal and mesodermal lineages. This study contributes to reinforce the emerging importance of these cells as ideal tools in veterinary medicine. A deeper evaluation of the immunological properties needs to be performed in order to better understand their role in cellular therapy.

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“…DNA fragments from the mouse Col2a1 gene that were used to construct plasmids were obtained by PCR amplification using primers and the RP23-345G Bac clone (BacPac Resources) as the template as described elsewhere (9). The plasmids carrying human SOX9, SOX6, SOX5, or SOX5 mutants have been previously described (9,17,32). The constructs used to verify the effect of the deletion in intron 6 on splicing were constructed using the N1-pEGFP vector (Clontech).…”

Section: Methodsmentioning

confidence: 99%

A Novel Regulatory Mechanism of Type II Collagen Expression via a SOX9-dependent Enhancer in Intron 6

Yasuda

Chen

et al. 2017

Journal of Biological Chemistry

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Edited by Eric R. FearonType II collagen ␣1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases. Its expression has been shown to be dependent on SOX9, a master transcription factor required for chondrogenesis that binds to an enhancer region in intron 1. However, ChIP sequencing revealed that SOX9 does not strongly bind to intron 1, but rather it binds to intron 6 and a site 30 kb upstream of the transcription start site. Here, we aimed to determine the role of the novel SOX9-binding site in intron 6. We prepared reporter constructs that contain a Col2a1 promoter, intron 1 with or without intron 6, and the luciferase gene. Although the reporter constructs were not activated by SOX9 alone, the construct that contained both introns 1 and 6 was activated 5-10-fold by the SOX9/SOX5 or the SOX9/SOX6 combination in transient-transfection assays in 293T cells. This enhancement was also observed in rat chondrosarcoma cells that stably expressed the construct. CRISPR/Cas9-induced deletion of intron 6 in RCS cells revealed that a 10-bp region of intron 6 is necessary both for Col2a1 expression and SOX9 binding. Furthermore, SOX9, but not SOX5, binds to this region as demonstrated in an electrophoretic mobility shift assay, although both SOX9 and SOX5 bind to a larger 325-bp fragment of intron 6 containing this small sequence. These findings suggest a novel mechanism of action of SOX5/6; namely, the SOX9/5/6 combination enhances Col2a1 transcription through a novel enhancer in intron 6 together with the enhancer in intron 1.SOX9 is a major transcription factor that regulates chondrocyte differentiation (1-3). The role of SOX9 in chondrogenesis was suggested first by the identification of heterozygous mutations in and around the SOX9 gene in the human genetic disease campomelic dysplasia, which is a cartilage disease that causes severe skeletal malformations and is often associated with XY female sex reversal (4, 5). Furthermore, site-and stage-specific down-regulation of SOX9 mRNA by mutations in a noncoding region near the SOX9 gene have been shown to cause Pier-Robinson syndrome, which is characterized by the abnormal positioning of the jaw, cleft palate, and airway obstruction (1).During chondrocyte differentiation, SOX9 regulates each step, from mesenchymal condensation and chondrocyte differentiation to the formation of hypertrophic chondrocytes (6). Many genes have been shown to be direct targets of this transcription factor, including genes encoding extracellular matrix (ECM) 4 proteins, membrane proteins, other transcription factors, and signaling molecules (7-9). Some of the ECM proteins that are regulated by SOX9 include COL2A1, COL9A2, ACAN, PRELP, and MATN 3.The expression of one of these ECM proteins, type II collagen ␣1 (COL2A1) is necessary for chondrocyte differentiation (10). Type II collagen ␣1 is specific for cartilaginous tissues and necessary for the normal embryonic development of the skeleton. Mutations in this gene cause achondrogenesis, chondrodys...

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L-Sox5 and Sox6 Drive Expression of the Aggrecan Gene in Cartilage by Securing Binding of Sox9 to a Far-Upstream Enhancer

Cited by 257 publications

References 55 publications

Acetylation reduces SOX 9 nuclear entry and ACAN gene transactivation in human chondrocytes

Acetylation reduces SOX 9 nuclear entry and ACAN gene transactivation in human chondrocytes

Mesenchymal stem cells from amnion and amniotic fluid in the bovine

A Novel Regulatory Mechanism of Type II Collagen Expression via a SOX9-dependent Enhancer in Intron 6

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