1. Hypoxia potentiates the BMP-2 driven COL2A1 stimulation in human articular chondrocytes via p38 MAPK 
J.E. Lafont, F.-A. Poujade, M. Pasdeloup, P. Neyret, F. Mallein-Gerin 
Osteoarthritis and Cartilage 
Volume 24, Issue 5, Pages (May 2016)
DOI: /j.joca
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Hypoxic environment dramatically improves the anabolic response of HACs to the chondrogenic growth factor BMP-2. HACs were placed in normoxic (21%O2), or hypoxic (1%O2) environment and treated with BMP-2 for 5 days. Cell lysates (A) or culture medium (B) were analysed by western immunoblot using anti-type II collagen, anti-type I collagen, anti-Sox9 or anti-Actin antibody. In order to be compared with each other, equivalent volumes of conditioned medium (or adjusted volumes to a similar amount of cells) were loaded on polyacrylamide gels. Arrows indicate the forms of collagen partially matured. Corresponding quantification of the bands of interest is stated as arbitrary unit below each blot. Gene expression analysis of COL2A1, COL9A1, SOX9, AGGRECAN, RUNX2 or COL1A1 (C). All values are the means ± 95% CI of relative mRNA level, normalised to RPL0 from n = 8 donors. Significance of oxygen tension on the effect of BMP-2 is indicated in the graph with the corresponding P-values.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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3. Fig. 2
Hypoxic environment strongly potentiates the BMP-2 effect on the type II collagen deposition by HACs. HACs were placed in normoxic (21%O2), or hypoxic (1%O2) environment and treated with BMP-2 for 5 days. Images from one representative donor out of three donors are shown. Cells were then fixed and stained for type II collagen (with Cy2-conjugated secondary antibody, red) and for SOX9 (with Cy3-conjugated secondary antibody, green). Nuclei were stained with Hoechst dye (blue) (×20 magnification). The scale bar represents 100 μm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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4. Fig. 3
Increased deposition of type II collagen by articular chondrocytes is associated with nuclear localisation of SOX9. HACs were placed in normoxic (21%O2), or hypoxic (1%O2) environment and treated with BMP-2 for 5 days. Cells were then fixed and stained for type II collagen (with Cy2-conjugated secondary antibody, red) and for SOX9 (with Cy3-conjugated secondary antibody, green). Nuclei were stained with Hoechst dye (blue). Images from one representative donor out of three donors are shown. (A) Strong staining for collagen is observed around the plasma membrane and inside the cytoplasm (white arrow), while SOX9 is mainly restricted to the nucleus when HACs are stimulated under hypoxic environment (1%O2) as assessed with Hoechst dye (empty arrow). Panel (B) shows the stimulation of HACs with BMP-2 under hypoxic environment leads to the absence of SOX9 patches in the cytoplasm (asterisk) compared to the other culture conditions (white arrow) (×40 magnification). The scale bar represents 20 μm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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5. Fig. 4
mRNA levels of BMP receptors are not modified by hypoxia, but in the BMP-stimulated HACs, the canonical Smad pathway is inhibited under hypoxic environment whereas p38 MAP kinase is activated. HACs were placed in normoxic (21%O2), or hypoxic (1%O2) environment and treated with BMP-2 for 5 days. Gene expression analysis of BMPR-IA, BMPR-II, ID-1, ID-3 (A). All values are the means ± 95% CI of relative mRNA level, normalised to RPL0 from n = 5 donors. Significance of oxygen tension on the effect of BMP-2 is indicated in the graph with the corresponding P-values. Cell lysates were analysed by western immunoblot to detect phospho-Smad 1/5/8, Smad 5, phospho-p38 MAPK and p38 MAPK (B). Experiments are representative of at least three experiments (three donors).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
The over-stimulation of type II collagen deposition by stimulated HACs under hypoxia is prevented by p38 MAP kinase inhibition. HACs were placed in normoxic (21%O2), or hypoxic (1%O2) environment for 5 days in the presence of the p38 MAP kinase inhibitor SB (1 μM). Sox9 aggregates can be detected in the cytoplasm when p38 is inhibited (arrows), or absent in a standard BMP-2 stimulation (star). Images from one representative donor out of three donors are shown. Cells were then fixed and stained for type II collagen (with Cy2-conjugated secondary antibody, red), and for SOX9 (with Cy3-conjugated secondary antibody, green). Nuclei were stained with Hoechst dye (blue) (×40 magnification). Blocking the p38 MAP kinase prevents the enhanced deposition of type II collagen. The scale bar represents 20 μm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
The hypoxia-induced anabolism of HACs is prevented by p38 MAP kinase inhibition. HACs were placed in normoxic (21%O2), or hypoxic (1%O2) environment for 5 days with the p38 MAP kinase inhibitor SB (1 μM). Blots from one representative donor out of three donors are shown. Cell lysates or conditioned medium were analysed by western immunoblot using anti-SOX9 (A), anti-type II collagen (B), or anti-actin antibodies. In order to be compared with each other, equivalent volumes of conditioned medium (or adjusted volumes to a similar amount of cells) were loaded on polyacrylamide gels. (C) Quantification of band intensities are presented for the procollagen α1(II), mature collagen α1(II) and cross-linked interchains of type II collagen.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

8. Fig. 7
The enhanced responsiveness toward anabolism of HACs under hypoxia is prevented by p38 MAP kinase inhibition. BMP-2 stimulated chondrocytes were placed in normoxic (21%O2), or hypoxic (1%O2) environment for 5 days with the p38 MAP kinase inhibitor SB (1 μM). Blots from one representative donor out of three donors are shown. Cell lysates or conditioned medium (A) were analysed by western immunoblot using anti-type II collagen, anti-SOX9, or anti-actin antibodies. (B) Quantification of band intensities are presented for the procollagen α1(II), mature collagen α1(II), and cross-linked interchains of type II collagen. EMSA was performed with nuclear extracts from HACs stimulated with BMP-2 under hypoxic environment and treated or not with p38 MAPK inhibitor SB (C). Probes containing regulatory sequences of the COL2A1 gene located in the intron 1 (probe#1) were used to assess the binding activity of SOX9.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

9. Fig. 8
The deposition of type II collagen induced by BMP-2 in a normoxic environment (21% oxygen) is greatly enhanced when HACs are cultured under hypoxia. Lowering the level of oxygen leads to SOX9 accumulation in the nucleus of stimulated chondrocytes. As a result, hypoxia enables more COL2A1 transcription and more type II collagen-containing matrix deposition. Such a synergy between BMP-2 and hypoxic stimulations involves a cross-talk through p38 MAP kinase pathway, whereas the Smad signalling is repressed. Thus hypoxic environment strongly increases the articular chondrocytes response toward anabolism.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions