Ubiquitin E3 ligase Wwp1 negatively regulates osteoblast function by inhibiting osteoblast differentiation and migration

Abstract

Ubiquitin E3 ligase-mediated protein degradation promotes proteasomal degradation of key positive regulators of osteoblast functions. For example, the E3 ligases--SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1), Itch, and WW domain-containing E3 ubiquitin protein ligase 1 (Wwp1)--promote degradation of Runt-related transcription factor 2 (Runx2), transcription factor jun-B (JunB), and chemokine (C-X-C) receptor type 4 (CXCR-4) proteins to inhibit their functions. However, the role of E3 ligases in age-associated bone loss is unknown. We found that the expression level of Wwp1, but not Smurf1 or Itch, was significantly increased in CD45-negative (CD45(-)) bone marrow-derived mesenchymal stem cells from 6-month-old and 12-month-old wild-type (WT) mice. Wwp1 knockout (Wwp1(-/-)) mice developed increased bone mass as they aged, associated with increased bone formation rates and normal bone resorption parameters. Bone marrow stromal cells (BMSCs) from Wwp1(-/-) mice formed increased numbers and areas of alkaline phosphatase(+) and Alizarin red(+) nodules and had increased migration potential toward chemokine (C-X-C motif) ligand 12 (CXCL12) gradients. Runx2, JunB, and CXCR-4 protein levels were significantly increased in Wwp1(-/-) BMSCs. Wwp1(-/-) BMSCs had increased amount of ubiquitinated JunB protein, but Runx2 ubiquitination was no change. Knocking down JunB in Wwp1(-/-) BMSCs returned Runx2 protein levels to that in WT cells. Thus, Wwp1 negatively regulates osteoblast functions by affecting both their migration and differentiation. Mechanisms designed to decrease Wwp1 levels in BMSCs may represent a new approach to prevent the decrease in osteoblastic bone formation associated with aging.

Keywords: BONE FORMATION; MESENCHYMAL STEM CELLS; OSTEOBLASTS; WWP1.

Figure 1. MSC-enriched CD45⁻ cells from old mice express decreased osteoblast marker genes and increased E3 ligase Wwp1

Different ages of WT mice were used. CD45⁻ cells were isolated from bone marrow cells via negative selection using micro-beads conjugated with anti-mouse CD45 antibody. The gene expression levels of osteoblast markers (ALP and Runx2, A) and E3 ligases (Wwp1, Smurf1 and itch, B) were determined by qPCR. Values are the means ± SD of 3 culture dishes. (C) 3rd passage of bone marrow stromal cells (BMSCs) were stained with MSC and non-MSC surface markers and subjected to FACS analysis. (D) Protein expression of Wwp1 and Smurf1 in BMSCs from young (1-month-old) and old (12-month-old) mice was determined by Western blot analysis. The fold changes were calculated from the intensity of bands on Western blot image using Scion Image Beta 4.02 (Scion Corporation, NIH). Values are the means ± SD of 3 separate experiments. *, p<0.05 vs data from 1-month-old mice.

Figure 2. Increased trabecular bone volume in Wwp1^−/− mice

Femurs isolated from 1-month-, 6-month- and 12-month-old Wwp1^−/− mice and WT littermates were subjected to micro-CT analysis. (A) Representative 3D reconstructed images show significantly increased trabecular structure parameters in bones of Wwp1^−/− mice. (B) Trabecular bone parameters from microCT analysis. Values are the means ± SD of 5 mice. *, p<0.05 vs data from WT mice.

Figure 3. Increased bone formation in Wwp1^−/− mice

Six-month-old Wwp1^−/− mice and WT littermates were labeled with calcein, and femurs were subjected to histologic examination. (A) Representative images of calcein-labeled trabecular and cortical bone. (B) Histomorphometric analyses of calcein-labeled sections. (C) Right panels: representative TRAP-stained sections. Left panels: histomorphometric analyses of TRAP-stained sections. Values are the means ± SD of 5 mice. **, p<0.01 vs data from WT mice.

Figure 4. MSC-enriched bone marrow stromal cells from Wwp1^−/− mice have increased osteoblast differentiation

BMSCs from 6-month-old Wwp1^−/− mice and WT littermates were used. (A) Cells were cultured in the basal (CFU-F) or in the osteoblast-inducing medium (CFU-ALP) for 14 or 21 days respectively. Cells were stained with H&E or ALP. The number of colonies was counted (upper panels). The bone marrow cells from Wwp1^−/− mice and WT littermates were stained with a panel of antibodies for MSC marker and subjected to FACS analysis (lower panels). (B) Primary bone marrow cells were stained with MSC and non-MSC surface markers and subjected to FACS analysis. CD45⁻ cells were examined the frequency of cells expressing Sca1, CD105 and Ter119. (C) Cells were cultured in osteoblast-inducing medium for 21 days. Cells were stained with Alizarin Red and the area of calcified nodules was measured. (D) The expression levels of osteoblast markers (ALP and Runx2) were determined by qPCR. (E) Cells were cultured in adipocyte-inducing medium for 6 days and subjected to Oil red O staining. (F) Expression levels of Runx2 and JunB proteins by Western blot analysis. The fold changes were calculated as Fig. 1D. Values are the means ± SD of 3 separate dishes. *, p<0.05 or **, p<0.01 vs WT cells.

Figure 5. MSC-enriched bone marrow stromal cells from Wwp1^−/− mice migrate faster to a CXCL12 gradient

(A) BMSCs from WT mice were labeled with calcein AM and seeded in the upper chamber of a transwell dish. CXCL12 (10 ng/ml) was added to the lower chamber and cells were incubated for different times (upper panel) or different doses of CXCL12 were added to the lower chamber and cells were incubated for 24 hrs (lower panel). The % of cells that migrated to the lower chamber was determined by measuring calcein intensity. (B) BMSCs from Wwp1^−/− mice and WT littermates were labeled and seeded as above. CXCL12 was added to the lower chamber and cells were incubated for 24hrs. The % of cells that migrated to the lower chamber was determined. Values are the means ± SD of 3 well/group. *, p<0.05, **, p<0.01, and ***, p<0.001 vs PBS or WT group.

Figure 6. MSC-enriched bone marrow stromal cells from Wwp1^−/− mice have decreased CXCL12-induced CXCR4 degradation

(A–C) C3H10T1/2 cells were used. (A) Cells were labeled and seeded as Fig. 5A. CXCL12 was added to the lower chamber and cells were incubated for different times. The % of cells that migrated to the lower chamber was determined. Values are the means ± SD of 3 wells/group. **, p<0.01 vs PBS group. (B) Expression of CXCR4 protein in CXCL12-treated cells was determined by Western blot analysis. (C) Cells were pre-treated with the proteasome inhibitor, MG132, or the lysosome inhibitor, chloroquine (CQ), and then treated with CXCL12 for 12 hours. Expression of CXCR4 protein was determined. (D) BMSCs from Wwp1^−/− mice and WT littermates were pre-treated with MG132 or CQ and then with CXCL12 as above. Expression of CXCR4 protein was determined by Western blot analysis and the fold changes were calculated as Fig. 1D.

Figure 7. Decreased bone volume and osteoblast function in old mice is rescued in Wwp1^−/−mice

(A–C) BMSCs from young (1-month-old) and old (12-month-old) WT mice were used. (A) The area of alizarin-red-stained calcified nodules. (B) The % of CXCL12-mediated migration. Values are the means ± SD of 3 wells/group. *, p<0.05, **, p<0.01 vs data from young mice. (C) Expression of Runx2, JunB and CXCR4 proteins was determined by Western blot analysis and the fold changes were calculated as Fig. 1D. (D–F) BMSCs from old Wwp1^−/− mice and WT littermates were used. (D) The area of alizarin-red-stained calcified nodules. (E) The % of CXCL12-mediated migration. Values are the means ± SD of 3 wells/group. *, p<0.05, **, p<0.01 vs data from WT littermates. (F) Expression of Runx2, JunB and CXCR4 proteins was determined by Western blot analysis and the fold changes were calculated as Fig. 1D. (G) BMSCs derived from Wwp1^−/− mice and WT littermates were treated with MG 132 (10 μM) for 4 hours and then lysed for ubiquitination assays to detect endogenous ubiquitinated JunB and Runx2 proteins. (H) BMSCs from Wwp1^−/− mice and WT littermates were transfected with JunB siRNA or control siRNA. Protein expressions of Runx2 and JunB were determined by Western blot analysis.