The 19S proteasomal lid subunit POH1 enhances the transcriptional activation by Mitf in osteoclasts

Abstract

The microphthalmia-associated transcription factor (Mitf) regulates gene expression required for osteoclast differentiation. Genes regulated by Mitf have been previously identified. However, proteins that interact and regulate Mitf's activity in osteoclasts are not well known. Here, we report that POH1, a subunit of the 19S proteasome lid is a regulator of Mitf. We show that POH1 and Mitf interact in osteoclasts and that this interaction is dependent on RANKL signaling. Overexpression of POH1 increased Mitf's activation of 5XGal4-TK and Acp5 promoters. The amino terminus of POH1 mediates the binding to Mitf and is sufficient to increase Mitf's transcriptional activity. Finally, we show that mutations in the JAMM motif of POH1 reduced Mitf activation of promoters. In summary, our results identify a novel mechanism of Mitf regulation in osteoclasts by POH1.

Figure 1. Mitf interacts with POH1 in osteoclasts

Representative Western blot of Mitf immunoprecipitates from bone marrow derived osteoclasts stimulated with M-CSF (10 ng/mL) or M-CSF and RANKL (60 ng/mL) for 5 days and immunoblotted against POH1 (Zymed) and Mitf.

Figure 2. POH1 reduces ubiquitination of Mitf

(A) Representative Western blot of Mitf immunoprecipitates from RAW 264.7 c4 cells stimulated with M-CSF (10 ng/mL) or M-CSF (10 ng/mL) and RANKL (60 ng/mL) for 2 days and immunoblotted against ubiquitin (Cell Signaling). (B) Representative Western blot of 293T cells transiently transfected with FLAG-Mitf, HA-ubiquitin and wild type or mutant POH1. Lysates were collected 24 hours following transfection and analyzed by Western blotting with a HA-tag antibody. The lower panel was immunoblotted with a FLAG antibody.

Figure 3. POH1 increases Mitf’s ability to activate transcription

(A) NIH 3T3 cells were transiently transfected with TRAP luciferase reporter construct and/or Mitf and POH1. *p≤0.05 vs. Acp5-luc+Mitf. (B) NIH 3T3 cells were transiently transfected with 5X Gal4-TK-luciferase reporter construct and/or pMI-Mitf and full length POH1. pMI-MITF encodes amino terminus (amino acids 1-185) of Mitf fused in frame to the Gal4 DNA binding domain. Luciferase reporter activity is presented as relative luciferase units (RLU) and the results of three experiments each performed in duplicate are presented. *p≤0.05 vs.gal4-TK-luc+Mitf. (C) Real time RT-PCR analysis of osteoclasts treated with or without 25 µM MG132 6 hours before harvest for expression of Acp5 (encoding TRACP) or cathepsin K RNA. ***p≤0.0005 vs. M-CSF treatment and *p≤0.05 vs. RANKL treatment for Ctsk and *p≤0.05 vs. M-CSF treatment and NS, not significant vs. RANKL treatment for Acp5.

Figure 4. Amino terminus of POH1 is sufficient to interact with Mitf

(A) Myc-tagged POH1 constructs and FLAG-tagged Mitf were expressed in 293T cells and complexes from whole cell lysates were immunoprecipitated with FLAG antibody and analyzed by Western blot with the indicated antibodies. (B) NIH 3T3 cells were transiently transfected with 5X Gal4-TK-luciferase reporter construct and/or pMI-Mitf and amino terminus of POH1 (amino acids 1-180). Luciferase reporter activity is presented as relative luciferase units (RLU) and the results of three experiments each performed in duplicate are presented. **p≤0.005 vs. Gal4-TK-luc+pMI-Mitf.

Figure 5. MPN+ domain of POH1 is required for regulation of Mitf’s activity

(A) NIH 3T3 cells were transiently transfected with 5X Gal4-TK-luciferase reporter construct and/or pMI-Mitf POH1 point mutants in the MPN+/JAMM motif. Luciferase reporter activity is presented as relative luciferase units (RLU) and the results of three experiments each performed in duplicate are presented. *p≤ 0.05 vs. Gal4-TK-luc+pMI-Mitf. (B) Real time RT-PCR from RAW 264.7 c4 cells that were either mock transfected, transiently transfected with full length POH1 or full length POH1 containing the point mutant C120S. Cells were stimulated with M-CSF (10 ng/mL) or M-CSF and RANKL (60 ng/mL) for 5 days. **p≤0.005 vs. M-CSF treatment.

Figure 6. POH1 enhances RAW 264.7c4 differentiation

RAW 264.7c4 cells were either mock transfected or transfected with wild type full length POH1, amino terminal portion of POH (amino acids 1-180) or full length C120S POH1. Cells were cultured in M-CSF and RANKL for 7 days. The number of multinuclear cells (greater than 2 nuclei/cell) was counted and the results of two experiments performed in triplicate are presented. *p≤0.05 and ***p≤0.0005 vs. mock transfected

Figure 7. Model of Mitf and POH1 interaction

(A) M-CSF stimulation of osteoclast activates ERK1/2, which phosphorylates Mitf on serine residue 73 [Weilbaecher et al., 2001]. The serine at residue 73 has been shown to be necessary for Mitf to be ubiquinated [Xu et al., 2000]. (B) RANKL stimulation activates the p38MAPK pathway resulting in Mitf phosphorylated on serine residue 307 [Mansky et al., 2002]. We hypothesize that POH1 interacts with Mitf when it is phosphorylated on serine residue 307. Mitf’s interaction with POH1 prevents Mitf’s ubiquitination allowing for persistent activation of genes necessary for osteoclast differentiation.