Journal: Biology Direct

Article Title: USP24-dependent stabilization of Runx2 recruits a p300/NCOA3 complex to transactivate ADAMTS genes and promote degeneration of intervertebral disc in chronic inflammation mice

doi: 10.1186/s13062-023-00395-5

Figure Lengend Snippet: Runx2 recruited p300 and NCOA3 to assemble a complex in vivo and in vitro (A) NCOA3 pulled down both Runx2 and p300 in vivo. Equal weights (0.05 g) of three independent IVDs from LPS-challenged mice were mixed to make a homogenate, followed by immunoprecipitation with anti-NCOA3- and IgG-coated protein A agarose. The purified complexes were used for western blotting assays with anti-NCOA3, anti-Runx2, and anti-p300 antibodies. (B) p300 pulled down both Runx2 and NCOA3 in vivo. The same homogenate used in (A) was immunoprecipitated with anti-p300- and IgG-coated protein A agarose. The purified complexes were used for western blotting assays with anti-P300, anti-Runx2, and anti-NCOA3 antibodies. (C and D) In vitro Co-IP results. Different combinations of Myc-tagged and Flag-tagged plasmids, as shown in the figure, were co-transfected into NP-1 cells. After incubation at 37 °C for 48 h, cells were used for immunoprecipitation using anti-Flag-agarose. The input and output proteins were detected with anti-Flag and anti-Myc antibodies. (C) Determination of the direct interaction of Flag Runx2- Myc p300 and Flag Runx2- Myc NCOA3. (D) Determination of the direct interaction of Flag p300- Myc Runx2 and Flag p300- Myc NCOA3.

Article Snippet: The LPS-challenged mice were then randomly divided into four subgroups and injected with either 100 µL PBS (LPS-sham group), the p300 inhibitor EML425 (15 mg/kg; MedChemExpress; Monmouth Junction, NJ, USA; # HY-110,263) (EML425 group), the p38 inhibitor doramapimod (4 mg/kg; Selleck Chemicals; Houston, TX, USA; #S1574) (doramipimod group), or the NCOA3 inhibitor bufalin (1 mg/kg; Selleck Chemicals; #S7821) (bufalin group).

Techniques: In Vivo, In Vitro, Immunoprecipitation, Purification, Western Blot, Co-Immunoprecipitation Assay, Transfection, Incubation

Journal: Biology Direct

Article Title: USP24-dependent stabilization of Runx2 recruits a p300/NCOA3 complex to transactivate ADAMTS genes and promote degeneration of intervertebral disc in chronic inflammation mice

doi: 10.1186/s13062-023-00395-5

Figure Lengend Snippet: Runx2 was phosphorylated in LPS-IVDs and LPS-treated NP-1/AF-1 cells (A) Protein levels of NCOA3-p300-Runx2 members in IVDs from sham- and LPS-treated mice. Homogenates of three lumbar discs (L1/L2) from three sham- and LPS-treated mice were used for western blotting to detect the protein levels of NCOA3, p300, Runx2, and GAPDH (loading control). (B and C) Protein levels of NCOA3-p300-Runx2 members in LPS-treated NP and AF cells. Three NP/AF cell lines (1, 2, and 3) were incubated with or without 20 ng/mL LPS for 6 h. Cell lysates were used for western blotting to determine the protein levels of NCOA3, p300, Runx2, and GAPDH (loading control). (B) NP cells; (C) AF cells. (D and E) Protein levels of NCOA3-p300-Runx2 members in NP and AF cells co-treated with LPS and phosphatase. Three NP/AF cell lines (1, 2, and 3) were incubated with or without 20 ng/mL LPS and 200 units of phosphatase for 6 h. Cell lysates were used for western blotting to determine the protein levels of NCOA3, p300, Runx2, and GAPDH (loading control). (D) NP cells; (E) AF cells. (F-H) Protein levels of different kinases in LPS-treated IVDs and LPS-treated NP-1/AF-1 cells. The same protein samples as in (A-C) were used for western blotting to determine the protein levels of p38, ERK1, ERK2, JNK1, and GAPDH (loading control). (F) IVDs; (G) NP cells; (H) AF cells

Article Snippet: The LPS-challenged mice were then randomly divided into four subgroups and injected with either 100 µL PBS (LPS-sham group), the p300 inhibitor EML425 (15 mg/kg; MedChemExpress; Monmouth Junction, NJ, USA; # HY-110,263) (EML425 group), the p38 inhibitor doramapimod (4 mg/kg; Selleck Chemicals; Houston, TX, USA; #S1574) (doramipimod group), or the NCOA3 inhibitor bufalin (1 mg/kg; Selleck Chemicals; #S7821) (bufalin group).

Techniques: Western Blot, Control, Incubation

Journal: Biology Direct

Article Title: USP24-dependent stabilization of Runx2 recruits a p300/NCOA3 complex to transactivate ADAMTS genes and promote degeneration of intervertebral disc in chronic inflammation mice

doi: 10.1186/s13062-023-00395-5

Figure Lengend Snippet: The binding affinity on the promoters of ADAMTS genes was much higher for pRunx2 S28 than for Runx2 (A) Greater amounts of p300 and NCOA3 protein were pulled down by pRunx2 than by Runx2. Equal weights (0.05 g) of three independent IVDs from LPS-challenged mice were mixed to make a homogenate, followed by immunoprecipitation with anti-Runx2-, anti-pRunx2 S28 , and IgG-coated protein A agarose. The purified complexes were used to determine the protein levels of Runx2, pRunx2 S28 , p300, and NCOA3. (B-F) Occupancies of Runx2 and pRunx2 S28 on the promoters of ADAMTS1/2/3/4/5 . The NP-1 cells were incubated with or without 20 ng/mL LPS for 6 h, followed by ChIP assays with anti-Runx2-, anti-pRunx2 S28 , and IgG-coated protein A agarose. The input and output DNA samples were used for RT-qPCR analyses to examine the occupancies of Runx2 and pRunx2 S28 on the promoters of ADAMTS1 (B) , ADAMTS4 (C) , ADAMTS5 (D) , ADAMTS2 (E) , and ADAMTS3 (F) . * P < 0.05; *** P < 0.001; ns: no significant difference

Article Snippet: The LPS-challenged mice were then randomly divided into four subgroups and injected with either 100 µL PBS (LPS-sham group), the p300 inhibitor EML425 (15 mg/kg; MedChemExpress; Monmouth Junction, NJ, USA; # HY-110,263) (EML425 group), the p38 inhibitor doramapimod (4 mg/kg; Selleck Chemicals; Houston, TX, USA; #S1574) (doramipimod group), or the NCOA3 inhibitor bufalin (1 mg/kg; Selleck Chemicals; #S7821) (bufalin group).

Techniques: Binding Assay, Immunoprecipitation, Purification, Incubation, Quantitative RT-PCR

Journal: Biology Direct

Article Title: USP24-dependent stabilization of Runx2 recruits a p300/NCOA3 complex to transactivate ADAMTS genes and promote degeneration of intervertebral disc in chronic inflammation mice

doi: 10.1186/s13062-023-00395-5

Figure Lengend Snippet: Inhibitors of p38/NCOA3/p300 decreased the expression of ADAMTS genes and slowed the degeneration of IVDs in LPS-challenged mice (A) The injection time points for LPS and p38/NCOA3/p300 inhibitors. (B) Representative MRI images of lumbar IVDs from different groups of mice. (C) Representative picrosirius red staining images of IVDs from different groups of mice. Bars = 100 μm. (D-G) Serum concentrations of proinflammatory cytokines: IL-1β (D) , IL-6 (E) , IL-15 (F) , and TNFα (G). (H-M) The mRNA expression levels of ADAMTS genes in different groups of mice (n = 8 for each group). (H) ADAMTS1 ; (I) ADAMTS2 ; (J) ADAMTS3 ; (K) ADAMTS4 ; (L) ADAMTS5 ; (M) ADAMTS6 ; * P < 0.05; ** P < 0.01; *** P < 0.001; ns: no significant difference

Article Snippet: The LPS-challenged mice were then randomly divided into four subgroups and injected with either 100 µL PBS (LPS-sham group), the p300 inhibitor EML425 (15 mg/kg; MedChemExpress; Monmouth Junction, NJ, USA; # HY-110,263) (EML425 group), the p38 inhibitor doramapimod (4 mg/kg; Selleck Chemicals; Houston, TX, USA; #S1574) (doramipimod group), or the NCOA3 inhibitor bufalin (1 mg/kg; Selleck Chemicals; #S7821) (bufalin group).

Techniques: Expressing, Injection, Staining

Journal: Biology Direct

Article Title: USP24-dependent stabilization of Runx2 recruits a p300/NCOA3 complex to transactivate ADAMTS genes and promote degeneration of intervertebral disc in chronic inflammation mice

doi: 10.1186/s13062-023-00395-5

Figure Lengend Snippet: Schematic model for the degeneration of IVDs by the transactivation of ADAMTS genes by the NCOA3-p300-pRunx2 complex (A) Schematic model of the transactivation of ADAMTS genes by the NCOA3-p300-pRunx2 complex in the degeneration of IVDs. Chronic inflammation activates p38 kinase, which phosphorylates Runx2 at the Ser28 site. pRunx2 S28 then recruits p300 and NCOA3 to assemble a complex that binds to the promoters of ADAMTS1/4/5/6/7/8/9/10/12/13/14/15/16/17/18/20 to transactivate their expression. The induction of these ADAMTS genes promotes ECM degradation and leads to IDD. (B) Schematic model depicting how inhibition of the NCOA3-p300-pRunx2 complex decreases the degeneration of IVDs. Administration of p38/NCOA3/p300 inhibitors in LPS-challenged mice decreases the expression of ADAMTS1/4/5/6/7/8/9/10/12/13/14/15/16/17/18/20 , thereby retarding ECM degradation and slowing the IDD process

Article Snippet: The LPS-challenged mice were then randomly divided into four subgroups and injected with either 100 µL PBS (LPS-sham group), the p300 inhibitor EML425 (15 mg/kg; MedChemExpress; Monmouth Junction, NJ, USA; # HY-110,263) (EML425 group), the p38 inhibitor doramapimod (4 mg/kg; Selleck Chemicals; Houston, TX, USA; #S1574) (doramipimod group), or the NCOA3 inhibitor bufalin (1 mg/kg; Selleck Chemicals; #S7821) (bufalin group).

Techniques: Expressing, Inhibition