PDGF-AA promotes osteogenic differentiation and migration of mesenchymal stem cell by down-regulating PDGFRα and derepressing BMP-Smad1/5/8 signaling

Abstract

Platelet-derived growth factors (PDGFs) play important roles in skeletal development and bone fracture healing, yet how PDGFs execute their functions remains incompletely understood. Here we show that PDGF-AA, but not -AB or -BB, could activate the BMP-Smad1/5/8 pathway in mesenchymal stem cells (MSCs), which requires BMPRIA as well as PDGFRα. PDGF-AA promotes MSC osteogenic differentiation through the BMP-Smad1/5/8-Runx2/Osx axis and MSC migration via the BMP-Smad1/5/8-Twist1/Atf4 axis. Mechanistic studies show that PDGF-AA activates BMP-Smad1/5/8 signaling by feedback down-regulating PDGFRα, which frees BMPRI and allows for BMPRI-BMPRII complex formation to activate smad1/5/8, using BMP molecules in the microenvironment. This study unravels a physical and functional interaction between PDGFRα and BMPRI, which plays an important role in MSC differentiation and migration, and establishes a link between PDGF-AA and BMPs pathways, two essential regulators of embryonic development and tissue homeostasis.

Figure 1. PDGF-AA activates BMP-Smad1/5/8 pathway via BMPRIA in MSC cells.

A. PDGF-AA activates Smad1/5/8 but not Smad2/3 in MSC cultures. Primary MSC mouse cells were starved from serum for 4 h and then 25 ng/ml of PDGF-AA was added to the cultures. Cells were harvested at different time points and were lysed to analyze the activation of Smad1/5/8 and Smad2/3 by western blot. Right panel: quantitation data. B. BMPRIA deficiency diminished Smad1/5/8 activation at the basal level or in response to PDGF-AA. Primary mouse MSC cells were infected with control virus or virus expressing Cre and selected for 5 days, to knock out BMPRIA. These cells were serum starved and treated with PDGF-AA for 2 hrs. Cells were harvested to analyze the activation of Smad1/5/8 by western blot. Right panel: quantitation data. C. BMPRI inhibitor LDN-193189 diminished Smad1/5/8 activation at the basal level or in response to PDGF-AA. Primary mouse MSC cells were serum starved for 4 hrs, pretreated with LDN-193189 for 1 hr and then treated with PDGF-AA for different periods of time. Cells were harvested to analyze the activation of Smad1/5/8 by western blot. Data are means ±s.e.m. (n = 3 for all panels). * p<0.05, when compared to empty vectors or untreated cells.

Figure 2. PDGF-AA promotes MSC osteogenic differentiation.

A. PDGF-AA promotes ALP expression in MSC, which requires BMP-Smad1/5/8 signaling. Primary mouse MSC cells were cultured with or without PDGF-AA for 7 or 14 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. ALP was stained 7 or 14 days after culture. B. Quantitation data for ALP activities that were normalized to the total protein levels of each culture. C. PDGF-AA promotes bone mineralization in MSC, which requires BMP-Smad1/5/8 signaling. Primary mouse MSC cells were cultured in differentiation medium with or without PDGF-AA (changed every 3 days) for 28 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Mineralization was stained with Von Kossa and Alizarin red method. D. PDGF-AA up-regulated the mRNA levels of Runx2, Atf4, and Osx via the BMP-Smad1/5/8 signaling, without affecting the mRNA levels of Sox9, C/EBPα, or PPARγ. Primary MSC cells were cultured with or without PDGF-AA for 7 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Total RNA was isolated from the cells and realtime PCR was carried out to determine the mRNA levels of the six transcription factors. Data are means ±s.e.m. (n = 3 for all panels). * p<0.05, when compared to empty vectors or untreated cells. E. PDGF-AA up-regulated the protein levels of Runx2, Atf4, and Osx via the BMP-Smad1/5/8 signaling. The experiments were carried out as described in Fig. 2D and the protein levels of these proteins were determined by western blot.

Figure 3. PDGF-AA promotes MSC migration via BMP-Smad1/5/8 signaling.

A. Transwell experiments and quantitation data. Migration of MSCs was evaluated using a transwell chamber equipped with an 8-µm-pore filter membrane. Cells were plated at 5×10⁴ cells/well in serum-free media onto the upper compartment of the chamber and cultured in the absence or presence of PDGF-AA for 24 h. The filter membrane was removed, fixed with methanol, and stained with 0.1% crystal violet. Lower panel: the number of cells that had migrated to the lower surface of the filter membrane was counted in 5 random fields under a light scope (×200). B. PDGF-AA promotes expression of BMP-Smad1/5/8-controlled genes that regulate cell migration. Primary mouse MSC cells were cultured with or without PDGF-AA for 7 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Total RNA was isolated from the cells and realtime-PCR was carried out to determine the mRNA levels of genes that are involved in regulating cell migration. Data are means±s.e.m. (n = 3). * p<0.05, **, P<0.01 when compared to empty vectors or untreated cells.

Figure 4. PDGFRα shows an inhibitory effect on Smad1/5/8 activation.

A. PDGFRα knockdown enhanced Smad1/5/8 activation at the basal level but diminished PDGF-AA-induced Smad1/5/8 activation. Primary mouse MSC cells were transfected with control or siRNA against PDGFRα for 2 days. The cells were then treated with PDGF-AA for 2 hrs. Cells were harvested to analyze the activation of Smad1/5/8 by western blot. Right panel: quantitation data. B. PDGFRα knockdown enhanced Smad1/5/8 activation in response to BMP2. Primary mouse MSC cells were transfected with control or siRNA against PDGFRα for 2 days. The cells were then treated with 50 ng/ml of BMP2 for 0.5 or 1 hr. Cells were harvested to analyze the activation of Smad1/5/8 by western blot. Right panel: quantitation data. C. Inhibition of lysosome-mediated protein degradation diminished PDGF-AA-induced Smad1/5/8 activation. Primary mouse MSC cells were pretreated with chloroquine or NH₄Cl for 6 h and then treated with PDGF-AA for 1 or 2 hrs. Cells were harvested to analyze the activation of Smad1/5/8 by western blot. Lower panel: quantitation data. Data are means ±s.e.m. (n = 3 for all panels). *, p<0.05, when compared to empty vectors or untreated cells.

Figure 5. PDGFRα interacts with BMPRIA.

A. PDGFRα (His tagged) was co-expressed with BMPRIA(HA tagged) in 293T cells. The cells were treated with 25 ng/ml PDGF-AA for 0.5 or 2 h or untreated. Cell lysates were divided into two parts for analysis of PDGFRα and BMPRIA expression by western blot, or immunoprecipitation of PDGFRα to test whether BMPRIA was brought down with PDGFRα. B. PDGFRα (His tagged) was co-expressed with BMPRIA(HA tagged) in 293T cells. The cells were treated with 25 ng/ml PDGF-AA for 0.5 or 2 h or untreated. Cell lysates were divided into two parts for analysis of PDGFRα and BMPRIA expression by western blot, or immunoprecipitation of BMPRIA to test whether PDGFRα was brought down with BMPRIA. C. Co-immunoprecipitation of endogenous PDGFRα and BMPRIA. Primary MSC cells were lyzed and cleaned lysates were incubated with IgG, anti-BMPRIA antibodies, or anti-PDGFRα antibodies overnight at 4°C. The immunoprecipitated proteins and their interacting proteins were detected by western blot.

Figure 6. PDGFRα interferes with BMPRI-BMPRII interaction.

A. PDGFRα (His tagged) was co-expressed with BMPRIA (HA tagged) and BMPRII (Flag tagged) in 293T cells. The cells were treated with 25 ng/ml PDGF-AA for 0.5 or 2 h or untreated. Cell lysates were divided into two parts for analysis of BMPRIA and BMPRII expression by western blot, or immunoprecipitation of BMPRIA to test whether the level of BMPRII brought down with BMPRIA was affected by expression of PDGFRα. Right panel: quantitation data. *, p<0.05, when compared to empty vectors transfected cells. B. PDGFRα (His tagged) was co-expressed with BMPRIA (HA tagged) and BMPRII (Flag tagged) in 293T cells. The cells were treated with 25 ng/ml PDGF-AA for 0.5 or 2 h or untreated. Cell lysates were divided into two parts for analysis of BMPRIA and BMPRII expression by western blot, or immunoprecipitation of BMPRII to test whether the level of BMPRIA brought down with BMPRII was affected by expression of PDGFRα. Right panel: quantitation data. *, p<0.05, when compared to empty vectors transfected cells.