Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a β-catenin-dependent mechanism

Abstract

Wnt10b is an established regulator of mesenchymal stem cell (MSC) fate that inhibits adipogenesis and stimulates osteoblastogenesis, thereby impacting bone mass in vivo. However, downstream mechanisms through which Wnt10b exerts these effects are poorly understood. Moreover, whether other endogenous Wnt ligands also modulate MSC fate remains to be fully addressed. In this study, we identify Wnt6 and Wnt10a as additional Wnt family members that, like Wnt10b, are downregulated during development of white adipocytes in vivo and in vitro, suggesting that Wnt6 and/or Wnt10a may also inhibit adipogenesis. To assess the relative activities of Wnt6, Wnt10a and Wnt10b to regulate mesenchymal cell fate, we used gain- and loss-of function approaches in bipotential ST2 cells and in 3T3-L1 preadipocytes. Enforced expression of Wnt10a stabilizes β-catenin, suppresses adipogenesis and stimulates osteoblastogenesis to a similar extent as Wnt10b, whereas stable expression of Wnt6 has a weaker effect on these processes than Wnt10a or Wnt10b. In contrast, knockdown of endogenous Wnt6 is associated with greater preadipocyte differentiation and impaired osteoblastogenesis than knockdown of Wnt10a or Wnt10b, suggesting that, among these Wnt ligands, Wnt6 is the most potent endogenous regulator of MSC fate. Finally, we show that knockdown of β-catenin completely prevents the inhibition of adipogenesis and stimulation of osteoblast differentiation by Wnt6, Wnt10a or Wnt10b. Potential mechanisms whereby Wnts regulate fate of MSCs downstream of β-catenin are also investigated. In conclusion, this study identifies Wnt10a and Wnt6 as additional regulators of MSC fate and demonstrates that mechanisms downstream of β-catenin are required for Wnt6, Wnt10a and Wnt10b to influence differentiation of mesenchymal precursors.

Figure 1. Wnt6, Wnt10a and Wnt10b mRNA expression decreases during adipogenesis in vivo and in vitro

(A) Epididymal WAT from C57BL/6J mice was separated into stromovascular (SVF) and Adipocyte fractions. Total RNA was isolated from each fraction, and the expression of FABP4, PPARγ, Wnt10b, Wnt10a and Wnt6 was analyzed by qPCR and normalized to 18S rRNA. Data are presented relative to the maximum expression for each transcript as mean ± SD (n = 5 or 6). (B-E) ST2 bipotential cells (B, D) or 3T3-L1 preadipocytes (C, E) were induced to differentiate into adipocytes. (B, C) Adipogenesis was assessed by staining with Oil Red-O at day 0 or days 6-8 post-induction. Micrographs of stained cells, representative of at least three independent experiments, are shown. (D, E) Total RNA was isolated at the indicated time points and the transcript expression of Wnt6, Wnt10a and Wnt10b was analyzed by qPCR and normalized to 18S rRNA. Data are presented relative to expression at day 0 as mean ± SD of three independent experiments. Statistically significant differences between SVF and Adipocytes (A) or compared to transcript levels at day 0 (D, E) are indicated as follows: * = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Figure 2. Wnt6, Wnt10a and Wnt10b inhibit adipogenesis

ST2 cells or 3T3-L1 preadipocytes were infected with retroviruses for stable expression of Wnt6, Wnt10a, Wnt10b or an EV control. (A, B) Cells were grown to confluence, total RNA was isolated and the expression of Wnt6, Wnt10a, Wnt10b, PPARγ, FABP4 and CEBPα was analyzed by qPCR. (C, D) β-catenin was analyzed by immunoblotting in cytosolic lysates of confluent EV- and Wnt-expressing cells; α-tubulin was used as a loading control. (E-H) EV and Wnt-expressing ST2 cells (E, G) or 3T3-L1 preadipocytes (F, H) were induced to differentiate into adipocytes. At day 6 (ST2) or day 8 (3T3-L1) post-induction, extent of adipogenesis was assessed by Oil Red-O staining (E, F) and analysis of adipocyte gene expression (G, H). Transcript expression in (A), (B), (G) and (H) was normalized to 18S rRNA and is presented relative to the maximum expression of each transcript as mean ± SD of at least three independent experiments. Statistical significance compared to transcript expression in EV cells is indicated as follows: * = P < 0.05; ** = P < 0.01; *** = P < 0.001. Immunoblots and images of Oil Red-O-stained cells are representative of at least three independent experiments.

Figure 3. Wnt6, Wnt10a and Wnt10b stimulate osteoblastogenesis

(A) Expression of alkaline phosphatase (Alpl), osteocalcin (Ocn), Osterix (Osx), Twist1, Dlx5 and Runx2 in RNAs from ST2 cells (Fig. 2A) was analyzed by qPCR. Transcripts were normalized to 18S rRNA and are presented relative to maximum expression as mean ± SD of at least three experiments. (B, C) EV- or Wnt-expressing ST2 cells were induced to differentiate into osteoblasts. The extent of osteoblastogenesis was assessed at day nine post-induction by staining for matrix mineralization with Alizarin red (B) and by quantification of matrix calcium content (C). Micrographs and wells of stained cells (B) are representative of at least three independent experiments. Calcium content (C) was normalized to cellular protein content and is presented as mean ± SD of four biological replicates per cell type, representative of two experiments. Statistically significant differences in transcript expression or calcium content between Wnt-expressing and EV cells are indicated as follows: * = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Figure 4. Wnt knockdown enhances adipogenesis

(A-D) ST2 cells were infected with retroviruses for expression of shRNAs against Wnt6, Wnt10a, Wnt10b or scrambled control (shControl). (E-H) 3T3-L1 preadipocytes were infected with retroviruses for expression of shWnt6 or shControl. (A, E) Total RNA was isolated from confluent cells and the expression of the indicated transcripts was analyzed by qPCR. (B, F) β-catenin was analyzed by immunoblotting in whole-cell protein lysates of confluent cells; α-tubulin was used as a loading control. (C, D) shRNA-expressing ST2 cells were induced for adipogenesis with MDI +/− TZD (5 μM). At day 6 post-induction, cells were stained with Oil Red-O (C) or total RNA was isolated for analysis of adipocyte gene expression by qPCR (D). (G, H) shControl and shWnt6-expressing 3T3-L1 preadipocytes were induced for adipogenesis as indicated. At day 8 post-induction, cells were stained with Oil Red-O (G) or whole-cell protein lysates were immunoblotted for PPARγ and FABP4; ERK1/2 was used as a loading control (H). Data in (A), (D) and (E) were normalized to 18S rRNA or TBP mRNA and are presented relative to the maximum expression of each transcript as mean +/− SD of 3-4 biological replicates representative of one to four independent experiments. Statistical significance compared to transcript expression in shControl cells is indicated as follows: * = P < 0.05; ** = P < 0.01; *** = P < 0.001. Immunoblots (B, F, H) and images of Oil Red-O-stained cells (C, G) are representative of at least three independent experiments.

Figure 5. Wnt knockdown impairs osteoblastogenesis

(A) Alkaline phosphatase expression in RNAs from confluent ST2 cells (Fig. 4A) was analyzed by qPCR and normalized to TBP expression and is presented relative to expression in shControl cells as mean +/− SD of four biological replicates. (B, C) shControl, shWnt6, shWnt10a and shWnt10b ST2 cells were induced to differentiate into osteoblasts. At day 6 post-induction, the extent of osteoblastogenesis was assessed by staining with Alizarin red (B) and by quantification of matrix calcium content (C). Plates and micrographs of Alizarin red-stained cells (B) are representative of three independent experiments. Calcium content (C) was normalized to cellular protein content and is presented as mean +/− SD of four biological replicates per cell type, representative of two experiments. Statistically significant differences in Alp expression or calcium content between shControl and shWnt cells are indicated by *** (= P < 0.001).

Figure 6. β-catenin is required for suppression of PPARγ by Wnt6, Wnt10a or Wnt10b

Wnt-expressing ST2 cells or 3T3-L1 preadipocytes were infected with retroviruses expressing shRNAs against β-catenin (shβ-cat) or a control shRNA against firefly luciferase (shControl). (A) Total RNA was isolated from confluent cells and expression of the indicated transcripts was analyzed by qPCR and normalized to TBP mRNA (ST2s) or 18S rRNA (3T3-L1s). Data are presented relative to the maximum expression of each mRNA as mean +/− SD of three independent experiments. Statistically significant differences in transcript expression are indicated as follows: Wnt-expressing cells vs EV, * = P < 0.05; ** = P < 0.01; *** = P < 0.001. shControl vs shβ-catenin cells, # = P < 0.05; ## = P < 0.01; ### = P < 0.001. (B) Knockdown of β-catenin was confirmed by immunoblotting of whole-cell lysates prior to induction of differentiation; α-tubulin or ERK1/2 were used as loading controls. Immunoblots are representative of three independent experiments.

Figure 7. β-catenin is required for suppression of adipogenesis by Wnt6, Wnt10a or Wnt10b

Wnt-expressing shControl or shβ-catenin ST2 cells and 3T3-L1 preadipocytes were induced to differentiate into adipocytes and the extent of differentiation at day 6 (ST2) or day 8 (3T3-L1) was assessed by Oil Red-O (A) or by immunoblotting for PPARγ and FABP4 in whole-cell lysates, with ERK1/2 used as a loading control (B). These data are representative of at least three independent experiments.

Figure 8. β-catenin is required for stimulation of osteoblastogenesis by Wnt6, Wnt10a or Wnt10b

(A) Alkaline phosphatase expression in RNAs from Wnt-expressing shControl or shβ-catenin ST2 cells (Fig. 6A) was analyzed by qPCR and normalized to TBP mRNA. Data are presented as mean +/− SD of three biological replicates from a representative experiment. (B, C) Wnt-expressing shControl or shβ-catenin ST2 cells were induced to differentiate into osteoblasts in the absence or presence of CHIR99021 (3 μM). At day 8 post-induction, the extent of osteoblastogenesis was assessed by staining with Alizarin red (A) and by quantification of matrix calcium content. Plates and micrographs of Alizarin red-stained cells (A) are representative of three independent experiments. Calcium content (B) was normalized to cellular protein content and is presented as mean ± SD of four replicate samples per cell type, from one of two independent experiments. Statistically significant differences in mRNA expression or calcium content are indicated as follows: Wnt-expression or CHIR99021 treatment vs EV cells, * = P < 0.05; ** = P < 0.01; *** = P < 0.001. shControl vs shβ-catenin cells, # = P < 0.05; ## = P < 0.01; ### = P < 0.001.

Figure 9. Expression of adipogenic regulators in Wnt-expressing shControl and shβ-catenin 3T3-L1 preadipocytes and ST2 cells

Expression of the indicated mRNAs was analyzed in RNA samples from Wnt-expressing shControl or shβ-catenin 3T3-L1 preadipocytes or ST2 cells (Fig. 6A) by qPCR and normalized to 18S rRNA (A) or TBP mRNA (B). Data are presented relative to the maximum expression for each transcript as mean +/− SD of three independent experiments. Statistically significant differences in transcript expression are indicated as follows: Wnt-expressing cells vs EV, * = P < 0.05; ** = P < 0.01; *** = P < 0.001. shControl vs shβ-catenin cells, # = P < 0.05; ## = P < 0.01; ### = P < 0.001.