Vav1 Regulates Mesenchymal Stem Cell Differentiation Decision Between Adipocyte and Chondrocyte via Sirt1

Abstract

Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in the bone marrow. MSCs have the potential to differentiate to adipocytes, chondrocytes, and other types of cells. In this study, we investigated the molecular mechanism that controls MSC cell fate decisions for differentiation. We found that Vav1, a guanine nucleotide exchange factor for Rho GTPase, was highly expressed in MSCs. Interestingly, loss of Vav1 in MSCs led to spontaneous adipogenic but impaired chondrogenic differentiation, and accordingly Vav1 null mice displayed an increase in fat content and a decrease in cartilage. Conversely, ectopic expression of Vav1 in MSCs reversed this phenotype, and led to enhanced MSC differentiation into chondrocyte but retarded adipogenesis. Mechanistically, loss of Vav1 reduced the level of Sirt1, which was responsible for an increase of acetylated PPARγ. As acetylation activates PPARγ, it increased C/EBPα expression and promoted adipogenesis. On the other hand, loss of Vav1 resulted in an increase of acetylated Sox9, a target of Sirt1. As acetylation represses Sox9 activity, it led to a dramatic reduction of collagen 2α1, a key regulator in chondrocyte differentiation. Finally, we found that Vav1 regulates Sirt1 in MSCs through Creb. Together this study reveals a novel function of Vav1 in regulating MSC cell fate decisions for differentiation through Sirt1. Sirt1 deacetylates PPARγ and Sox9, two key mediators that control adipocyte and chondrocyte differentiation. The acetylation status of PPARγ and Sox9 has opposite effects on its activity, thereby controlling cell fate decision. Stem Cells 2016;34:1934-1946.

Keywords: Adipogenesis; Chondrogenesis; MSC; Sirt1; Vav1.

Figure 1.

Vav1 is expressed in murine MSCs and that negatively regulates MSC differentiation to adipocytes. (A): Cultured MSCs at the second passage from WT and Vav1 null bone marrow were analyzed for Vav1 expression by Western blot. (B): Oil red O staining was performed on the fourth passage MSCs cultured in Mesencult basal medium. FABP4 expression in the cells above was analyzed by immunofluorescent staining. Representative images were shown (bar = 50 μm). (C): FABP4+ cells were enumerated in 10 randomly selected microscopic fields from cultures in each group. (D): MSCs at the fourth passage were harvested and the mRNA levels of Hp, leptin, and adiponectin were analyzed by qPCR. MSCs were pooled from three mice in each experiment, and each experiment was repeated twice. **, p < .01. Abbreviations: Hp, haptoglobin; MSCs, mesenchymal stem cells; WT, wild type.

Figure 2.

Ectopic expression of Vav1 in MSCs inhibited adipogenic differentiation. (A): WT MSCs were infected with a lentiviral vector for Vav1 (Vav1) or a vector control (vector), respectively. The levels of Vav1 were determined by qPCR. (B): The cells were cultured in an adipogenic differentiation medium for 14 days, followed by immuno-fluorescent staining for FABP4. Representative images were shown. 100x mag. (C): The numbers of FABP4+ cells were counted in 10 randomly selected fields under microscopy. (D): The mRNA levels of Hp, leptin, and adiponectin in MSCs was analyzed by qPCR. MSCs were pooled from three mice in each experiment, and each experiment was repeated twice. **, p < .01. Abbreviations: Hp, haptoglobin; MSCs, mesenchymal stem cells; WT, wild type.

Figure 3.

Genetic deletion of Vav1 in mice resulted in increased body fat content. (A): The body weight of 12 weeks old female mice was measured. (B): Total body fat was determined by NMR using the Burker Minispec. (C): Serum leptin levels were measured by ELISA. (D): Subcutaneous and abdominal visceral fat was harvested and processed for HE staining and oil red staining. Representative images were shown. 100x mag. (E): The numbers of adipocytes were determined by measuring the DNA content in epididymal fat pads. (F): The distribution of diameter of adipocytes in the epididymal fat pads of 12 weeks old female WT and Vav1 KO mice. (G): qPCR analysis for UCP1 and PGC-1α in BAT; and chemerin and Resistin in epididymal WAT between WT and Vav1 null mice. Values represent mean ± SD. *, p < .05; **, p < .01. Abbreviations: BAT, brown adipocytic tissue; HE, hematoxylin and eosin; WAT, white adipocytic tissue; WT, wild type.

Figure 4.

Loss of Vav1 in MSCs retarded chondrocyte differentiation. (A): MSCs at passage 2 were cultured in chondrogenic differentiation medium for 21 days, followed by immuno fluorescent staining for Collagen II (bar= 100 μm). Representative images were shown. (B): The numbers of Collagen II positive cells were counted in 10 randomly selected microscopic fields. (C): The levels of mRNA for Col2α1, aggrecan, Sox5, Sox6, and Coll10 were analyzed by qPCR. (D): The sections of distal femoral from 4 months old WT and Vav1 null mice (n = 5) were stained with 0.1% safranin O. Representative images were shown. All other experiments were repeated at least twice. Values represent mean ± SD, *, p < .05; **, p < .01. Abbreviations: MSCs, mesenchymal stem cells; WT, wild type.

Figure 5.

Ectopic expression of Vav1 promoted MSC chondrogenic differentiation. (A): Wild type MSCs were infected with a lentiviral vector for Vav1 (Vav1) or vector control (Vector), followed by culturing the cells in chondrogenic induction medium for 10 days. The cells were immuno-stained for Collagen II (bar = 100 μm). Representative images were shown. (B): The numbers of Collagen II + cells were counted in 10 randomly selected microscopic fields. (C): The levels of mRNA for Col2α1 and Aggrecan, Sox5, Sox6, and Coll10 were analyzed by qPCR. Each experiment was done in triplicate and repeated twice. *, p < .05; **, p < .01. Abbreviations: MSCs, mesenchymal stem cells.

Figure 6.

Vav1 regulates MSC differentiation through Sirt1. (A): MSCs at the second to third passage were harvested and evaluated for Sirt1 and C/EBPα expression by Western blot. (B): WT MSCs were cultured in adipocyte differentiation induction medium. Cells were harvested at indicated time points and subjected to Western blot for Vav1, Sirt1, and C/EBPα (C): Cell lysates of MSCs at the second to third passage were immunoprecipitated with a PPARγ antibody, followed by Western blot for total PPARγ and acetylated PPARγ. (D): Second passage MSCs from WT or Vav1 null mice were cultured in chondrocyte culture medium for 3 weeks. Cell lysates were analyzed for Col2α1 expression by Western blot. (E): Cell lysates of MSCs after chondrocyte differentiation induction were immunoprecipitated with a Sox9 antibody, followed by Western blot for total Sox9 and acetylated Sox9. (F): WT MSCs were cultured in chondrocyte differentiation induction medium. Cells were harvested at indicated time points and subjected to Western blot for Vav1. (G): WT MSCs were cultured in the presence of Ex527 at 10 μM for 48 hours, followed by the evaluation for total PPARγ and Sox9 proteins as well as acetylated PPARγ and Sox9 proteins. (H): The levels of Sirt1, C/EBPα, and Col2α1 were evaluated after incubation with Ex527. (I, J, and K): Vav1 null MSCs were infected with lentiviral vectors for Sirt1 or vector control for 48 hours. The cell lysates were subjected to Western blot analysis for Sirt1, acetylated PPARγ, and total PPARγ. Oil red O staining was performed on cultured cells (bar = 50 μm). Abbreviations: MSCs, mesenchymal stem cells; WT, wild type.

Figure 7.

Vav1 regulates MSC differentiation through Creb-mediated Sirt1 expression. (A and B): WT MSCs were infected with lentiviral vectors for Vav1 or vector controls for 48 hours. The cell lysates were subjected to Western blot analysis for Vav1, Sirt1, C/EBPα, and Col2α1, as well as IP-Western for total PPARγ and acetylated PPARμ, and total Sox9 and acetylated Sox9. (C): WT MSCs were infected with lentiviral vectors for Vav1 or vector controls for 48 hours in the absence or presence of Ex527 at l0 μM. The cell lysates were subjected to Western blot analysis for Vav1, Sirt1, C/EBPα, and Col2α1. The protein levels were quantitated by measuring the intensity of Western blot. Each experiment was repeated at least twice. *, p < .05; **, p < .01. (D): Cell lysates from second passage MSCs of WT or Vav1 null mice were subjected to Western blot analysis for phosphor-Creb and total Creb protein. (E): WT MSCs were infected with lentiviral vectors for Vav1 or vector controls for 48 hours. The cell lysates were subjected to Western blot analysis for Vav1, phosphor-Creb, and total Creb protein. (F): WT MSCs were infected with shRNA for Creb or vector controls for 48 hours. The cell lysates were subjected to Western blot analysis for Sirt1 and total Creb protein. (G): The protein levels of Vav1 and Sirt1 were evaluated from both human bone marrow and peripheral blood cell derived MSCs, which were infected with either shRNA for Vav1 or vector controls and cultured in MSCGM Mesenchymal Stem Cell Growth Medium for 48 hours. (H): A diagram of putative signaling pathway, by which Vav1 acts as a central regulator via Creb/Sirt1 to control the MSC commitment towards adipocytes versus chondrocytes. Abbreviations: BM, bone marrow; MSCs, mesenchymal stem cells; PBC, peripheral blood cell; WT, wild type.