Dataset Information

Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation.

ABSTRACT: A previously identified enhancer 10?kb upstream of the Aggrecan (Acan) gene (UE) can drive cartilage specific reporter expression in vivo. Here, we report that the paralogous transcription factors PAX1 and PAX9 differentially drive UE, depending on the presence or absence of SOX9-driven transactivation. In the developing vertebral column, PAX1/9 expression was inversely correlated with Acan expression. Moreover, PAX1/9 was co-expressed with SOX9/5/6 in the intervertebral mesenchyme and the inner annulus fibrosus (AF), and with SOX9 in the outer AF. Significant Acan upregulation was observed during chondrification of Pax1-silenced AF cells, while, Acan was significantly downregulated by persistent expression of Pax1 in cartilage. Deletion of UE using CRISPR/Cas9 resulted in ~30% and ~40% reduction of Acan expression in cartilage and the AF, respectively. In the UE, PAX1/9 acts as weak transactivators through a PAX1/9-binding site partially overlapped with a SOX9-binding site. In the presence of SOX9, which otherwise drives robust Acan expression along with SOX5/6, PAX1/9 competes with SOX9 for occupancy of the binding site, resulting in reduced transactivation of Acan. Coimmunoprecipitation revealed the physical interaction of Pax1 with SOX9. Thus, transactivation of the UE is differentially regulated by concerted action of PAX1/9, SOX9, and SOX5/6 in a context-dependent manner.

SUBMITTER: Takimoto A

PROVIDER: S-EPMC6418084 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation.

Takimoto Aki A Kokubu Chikara C Watanabe Hitomi H Sakuma Tetsushi T Yamamoto Takashi T Kondoh Gen G Hiraki Yuji Y Shukunami Chisa C

Scientific reports 20190314 1

A previously identified enhancer 10 kb upstream of the Aggrecan (Acan) gene (UE) can drive cartilage specific reporter expression in vivo. Here, we report that the paralogous transcription factors PAX1 and PAX9 differentially drive UE, depending on the presence or absence of SOX9-driven transactivation. In the developing vertebral column, PAX1/9 expression was inversely correlated with Acan expression. Moreover, PAX1/9 was co-expressed with SOX9/5/6 in the intervertebral mesenchyme and the inner ...[more]

PMID: 30872687

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Project description:OBJECTIVE:Osteoarthritis (OA) chondrocytes exhibit impairment of autophagy, one arm of the proteostasis network that coordinates proteome and organelle quality control and degradation. Deficient proteostasis impacts differentiation and viability, and inflammatory processes in aging and disease. The present study was undertaken to assess ubiquitin proteasome system proteasomal function in OA chondrocytes. METHODS:We evaluated human knee cartilage by immunohistochemistry, and assessed proteasomal function, levels of proteasomal core subunits and chaperones, and autophagy in cultured chondrocytes. Assays included Western blotting, quantitative reverse transcription-polymerase chain reaction, proteasomal protease activity assessment, and cell immunofluorescence analysis. RESULTS:Human knee OA cartilage exhibited polyubiquitin accumulation, with increased ubiquitin K48-linked polyubiquitinated proteins in situ, suggesting proteasomal impairment. Cultured OA chondrocytes demonstrated accumulation of K48 polyubiquitinated proteins, significantly reduced 20S proteasome core protease activity, and decreased levels of phosphorylated FOXO4 and proteasome 26S subunit, non-ATPase 11 (PSMD11), a FOXO4-inducible promoter of proteasomal activation. Levels of proteasome subunit β type 3 (PSMB3), PSMB5, PSMB6, and proteasome assembly chaperone 1 were not decreased in OA chondrocytes. In normal chondrocytes, PSMD11 small interfering RNA knockdown stimulated certain autophagy machinery elements, increased extracellular nitric oxide (NO) levels, and reduced chondrocytic master transcription factor SOX9 protein and messenger RNA (mRNA) and aggrecan (AGC1) mRNA. PSMD11 gain-of- function by transfection increased proteasomal function, increased levels of SOX9-induced AGC1 mRNA, stimulated elements of the autophagic machinery, and inhibited extracellular levels of interleukin-1-induced NO and matrix metalloproteinase 13 in OA chondrocytes. CONCLUSION:Deficient PSMD11, associated with reduced phosphorylated FOXO4, promotes impaired proteasomal function in OA chondrocytes, dysregulation of chondrocytic homeostasis, and decreased levels of SOX9 mRNA, SOX9 protein, and AGC1 mRNA. Chondrocyte proteasomal impairment may be a therapeutic target for OA.

Dataset Information

Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation.

Publications

Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation.

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