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, 21 (7), 1176-86

Bone Morphogenetic protein-2-induced Signaling and Osteogenesis Is Regulated by Cell Shape, RhoA/ROCK, and Cytoskeletal Tension

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Bone Morphogenetic protein-2-induced Signaling and Osteogenesis Is Regulated by Cell Shape, RhoA/ROCK, and Cytoskeletal Tension

Yang-Kao Wang et al. Stem Cells Dev.

Abstract

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.

Figures

FIG. 1.
FIG. 1.
BMP-2-induced osteogenic differentiation is regulated by cell shape. (A) Bright-field images of hMSC plated at 3,000 cells/cm2 (upper 2 panels) or 20,000 cells/cm2 (lower 2 panels), cultured for 2 weeks in the absence (left panels, Control) or presence (right panels, BMP) of BMP-2 (100 ng/mL), and stained with ALP activity. Scale bar: 20 μm. (B) Quantification plot of ratio of ALP positively stained cells versus DAPI-stained cells in 5 random selected fields of cultured cells at 1,000, 3,000, 10,000, and 20,000 cells/cm2 in the absence or presence of BMP-2 (100 ng/mL). (C) Quantification of ratio of ALP positively stained hMSCs versus DAPI-stained cells in 5 random selected fields of cells cultured at 3,000 or 20,000 cells/cm2 in the absence or presence of BMP-2 at different concentration (from 0 to 500 ng/mL). (D) Quantitative PCR results of osteogenic differentiation markers of hMSCs plated at indicated density and cultured in the absence or presence of BMP-2 (100 ng/mL) for 2 weeks. (E) Bright-field ALP images of hMSCs plated on large (10,000 μm2, upper panels) or small (625 μm2, lower panels) fibronectin islands for 2 weeks in the absence or presence of BMP-2 (100 ng/mL). Scale bar: 20 μm. (F) Quantification plot of ratio of ALP positively stained cells versus DAPI-stained cells plated on different sizes of fibronectin stamped areas (from 625 to 10,000 μm2) in the absence or presence of BMP-2. (G) Luciferase activity of SBE-luciferase transfected hMSCs plated at 3,000 cells/cm2 or 20,000 cells/cm2 in the presence or absence of BMP-2 (100 ng/mL) for 2 days. (H) Luciferase activity of SBE-luciferase transfected hMSCs cultured on fibronectin micropattern stamped spread (10,000 μm2) or unspread (625 μm2) followed by treatment with BMP-2 (100 ng/mL) for 2 days. All quantification data were presented as mean±SEM of at lease 3 independent experiments. *P<0.05 versus paired control; #P<0.05 versus low density/spread cells treated with BMP-2. ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein; hMSC, human mesenchymal stem cells; SBE, SMAD binding element; SMAD, SMA/mothers against decapentaplegic. Color images available online at www.liebertonline.com/scd
FIG. 2.
FIG. 2.
BMP stimulates RhoA signaling. (A) Western blot (upper panels) and quantification plot (lower panel) showed active RhoA in serum-starved hMSCs at 3,000 cells/cm2 with BMP-2 (100 ng/mL) treatment at different time points. (B) Western blot and quantification plot showed active RhoA in serum-starved hMSCs plated at 3,000 or 20,000 cells/cm2 with or without BMP-2 (100 ng/mL) treatment. (*P<0.05 vs. paired control; #P<0.05 vs. BMP treatment at 3,000 cells/cm2). (C) Western blot and quantification plot showed levels of recombined p-mypt after in vitro ROCK kinase assay in serum-starved hMSCs plated at 3,000 or 20,000 cells/cm2 with or without BMP-2 (100 ng/mL) treatment. (*P<0.05 vs. paired control; #P<0.05 vs. BMP treatment at 3,000 cells/cm2). (D) Western blot ppMLC/MLC and quantification plot showed activation of ppMLC in serum-starved hMSCs plated at 3,000 or 20,000 cells/cm2 with or without BMP-2 (100 ng/mL) treatment (*P<0.05 vs. paired control; #P<0.05 vs. BMP treatment at 3,000 cells/cm2). All western blot results were presented as a representative experiment of at least 3 independent experiments. ppMLC, phosphorylated myosin light chain. ROCK, Rho-associated protein kinase.
FIG. 3.
FIG. 3.
BMP stimulates cytoskeletal tension. (A) Epifluorescence images of stress fibers in hMSCs plated at 3,000 (upper panels) or 20,000 (lower panels) cells/cm2 in the absence (left panels, Control) or presence of BMP-2 (right panels, BMP, 100 ng/mL) for 1 h. (B) Quantification results of stress fiber formation in hMSCs plated at 3,000 or 20,000 cells/cm2 in the absence or presence of BMP-2 (100 ng/mL) (*P<0.05 vs. paired control.). A.U. represents arbitrary unit. (C) Representative images of control (C), BMP-2 (100 ng/mL) (BMP) and BMP-2 plus Y27632 (25 μM) (BMP+Y27) treated hMSC on mPAD (red, mPAD; green, actin cytoskeleton; blue, nuclei). Plot of average traction force exerted on each underlying post were presented as mean±SEM of 3 independent experiments (*P<0.01 vs. C; #P<0.01 between BMP-2 and BMP-2+ Y27). Scale bar: 20 μm. (D) Fluorescence images and quantification plot of stress fibers in hMSCs plated at 3,000 cells/cm2 and treated with Y27632 (10 μM) in the presence of BMP-2 (100 ng/mL) for 1 h. mPAD, microfabricated postarray detector. Color images available online at www.liebertonline.com/scd
FIG. 4.
FIG. 4.
BMP-induced osteogenic differentiation depends on ROCK and myosin activity. (A) Bright-field images of hMSC plated at 3,000 cells/cm2 for 2 weeks in the absence (Control) or presence of BMP-2 (100 ng/mL), BMP-2 plus Y27632 (BMP+Y27, 10 μM), and BMP-2 plus blebbistatin (BMP+Bleb, 25 μM) and stained with ALP activity. Scale bar: 20 μm. (B) Quantification plot of ratio of ALP positively stained hMSCs versus DAPI-stained cells in 5 random selected fields as indicated. All results were presented as mean±SEM (*P<0.05 vs. paired control; #P<0.05 vs. paired BMP only). (C) Bright-field images of hMSC with knock down of specific gene using small-interfering RNA (as indicated) and plated at 3,000 cells/cm2 for 2 weeks in the absence (Control) or presence of BMP-2 (BMP, 100 ng/mL) and stained with ALP activity. (D) Quantification plot of ratio of ALP positively stained hMSCs versus DAPI-stained cells in 5 random selected fields as indicated. Color images available online at www.liebertonline.com/scd
FIG. 5.
FIG. 5.
SMAD-dependent gene expression is regulated by ROCK and myosin activity. (A) Luciferase activity of SBE-luciferase-transfected hMSCs plated at 3,000 cells/cm2 and in the absence or presence of BMP-2 (100 ng/mL) or Y27632 (10 μM) for 2 days. (B) Luciferase activity of SBE-luciferase transfected hMSCs plated at 3,000 cells/cm2 and in the absence or presence of BMP-2 (100 ng/mL) or blebbistatin (25 μM) for 2 days. Cotransfection of renilla-luciferase served as an internal control. All data were presented as mean±SEM. *P<0.05 versus paired control; #P<0.05 versus treated with BMP-2.
FIG. 6.
FIG. 6.
BMP-induced phosphorylation of SMAD and SMAD complex formation is ROCK and tension dependent. (A) A representative western blot result of levels of p-SMAD1/5/8 in hMSC plated at 3,000 cells/cm2 in the absence or presence of BMP (100 ng/mL), BMP+Y27632 (BMP+Y27, 10 μM), BMP+blebbistatin (BMP+Bleb, 25 μM), Y27632 (Y27, 10 μM), and blebbistatin (Bleb, 25 μM). GAPDH served as an internal control. (B) Quantification plot of ratio of pSMAD1/5/8 levels of (A). (C, E) Cell lysates from different treatments were immunoprecipitated with anti-SMAD4 followed by immunoblotting with either anti-p-SMAD1 or SMAD4 antibodies. (D, F) Quantification results of immunoprecipitated pSMAD1 with SMAD4 in MSC treated with either Y27632 (10 μM) (D) or blebbistatin (25 μM) (F).
FIG. 7.
FIG. 7.
p-SMAD1/5/8 nuclear translocation requires cell spreading and ROCK signaling. (A) Immunofluorescence images of serum-starved hMSCs plated on fibronectin stamped islands and treated with or without BMP-2 (100 ng/mL) for 1 h. Green, stress fiber; red, p-SMAD1/5/8; blue, DAPI. (B) Quantification of ratio of nuclear translocated p-SMAD1/5/8 in hMSCs plated on different sizes of fibronectin-stamped area. (C) Immunofluorescence images of Y27632 (10 μM) or blebbstatin (25 μM) pretreated hMSCs followed by BMP treatment for 1 h. Green, stress fiber; red, p-SMAD1/5/8; blue, DAPI. (D) Quantification of BMP-2-induced nuclear translocated p-SMAD1/5/8 in the presence of Y27632 or blebbstatin. *P<0.05 versus paired control; #P<0.05 versus BMP-treatment at 10,000 μm2 and BMP treat alone. Scale bar, 20 μm. (E) Model of cell shape regulates BMP-2-induced osteogenic differentiation. Cell shape acts as a mechanical cue that cooperates with BMP to induced osteogenesis. Rho/ROCK-mediated cell tension is not only required but also induced in response to BMP-2 treatment. The RhoA/ROCK signaling regulates nuclear translocation of p-SMAD, which is responsible for BMP-2 induced osteogenesis.

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