T1-Weighted Magnetic Resonance Imaging (MRI).

<p>Representative MRI images from the (a) sham group, (b) I/R group (12 h after I/R), (c) I/R group (24 h after I/R), (d) I/R group (48 h after I/R), (e) I/R group (72 h after I/R), (f) I/R+bexarotene group (12 h after I/R), (g) I/R+bexarotene group (24 h after I/R), (h) I/R+bexarotene group (48 h after I/R), and (i) I/R+bexarotene group (72 h after I/R). The red arrows point to the signal intensity-enhanced regions with larger regions and more pronounced signal intensities indicating more serious brain damage. (B) MRI signal values at each time point are expressed as means±SDs (n = 5 per group; *<i>P</i><0.05 versus the sham group, **<i>P</i><0.01 versus the sham group; #<i>P</i><0.05 versus the I/R group, and ##<i>P</i><0.01 versus the I/R group).</p

MMP-9 mRNA Expression.

<p>MMP-9 mRNA expression level in the ischemic zone of the rat brain was determined by RT-PCR with β-actin used as an internal control. (B) Densitometric analysis. Values are expressed as means±SDs (n = 5 per group; *<i>P</i><0.05 versus the sham group, **<i>P</i><0.01 versus the sham group; #<i>P</i><0.05 versus the I/R group, and ##<i>P</i><0.01 versus the I/R group).</p

MMP-9 Activity.

<p>(A) MMP-9 activity in the ischemic zone of the rat brain was determined by gelatin zymography. (B) Densitometric analysis. Values are expressed as means±SDs (n = 5 per group; *<i>P</i><0.05 versus the sham group, **<i>P</i><0.01 versus the sham group; #<i>P</i><0.05 versus the I/R group, and ##<i>P</i><0.01 versus the I/R group).</p

Evans Blue (EB) Method.

<p>Increased EB content in brain tissue confirms increased BBB permeability post-I/R. Values are expressed in μg/g as means±SDs (n = 5 per group; *<i>P</i><0.05 versus the sham group, **<i>P</i><0.01 versus the sham group; #<i>P</i><0.05 versus the I/R group, and ##<i>P</i><0.01 versus the I/R group).</p

ApoE, MMP-9, Claudin-5, and Occludin Protein Expression.

<p>(A) ApoE (B) MMP-9, (C) claudin-5, and (D) occludin protein expression levels in the ischemic zone of the rat brain were determined by Western blotting and densitometric analysis with β-actin used as an internal control. Values are expressed as means±SDs (n = 5 per group; *<i>P</i><0.05 versus the sham group, **<i>P</i><0.01 versus the sham group; #<i>P</i><0.05 versus the I/R group, and ##<i>P</i><0.01 versus the I/R group).</p

Brain Water Content.

<p>Brain water content was measured by the dry wet weight method. Values are expressed in % as means±SDs (n = 5 per group; *<i>P</i><0.05 versus the sham group, **<i>P</i><0.01 versus the sham group; #<i>P</i><0.05 versus the I/R group, and ##<i>P</i><0.01 versus the I/R group).</p

Schematic of Experimental Protocol.

<p>Schematic of Experimental Protocol.</p

P38 and ERK1/2 MAPKs Act in Opposition to Regulate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells

<div><p>Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation and bone formation, the precise molecular mechanism involved remains to be fully elucidated. In this current study, we explore the possible involvement and detail effects of p38 and ERK1/2 MAPKs on BMP9-indcued osteogenic differentiation of mesenchymal progenitor cell (MPCs). We find that BMP9 simultaneously stimulates the activation of p38 and ERK1/2 in MPCs. BMP9-induced early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as matrix mineralization and osteocalcin (OC) are inhibited by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. BMP9-induced activation of Runx2 and Smads signaling are reduced by SB203580, and yet increased by PD98059 in MPCs. The <em>in vitro</em> effects of inhibitors are reproduced with adenoviruses expressing siRNA targeted p38 and ERK1/2, respectively. Using mouse calvarial organ culture and subcutaneous MPCs implantation, we find that inhibition of p38 activity leads to significant decrease in BMP9-induced osteogenic differentiation and bone formation, however, blockage of ERK1/2 results in effective increase in BMP9-indcued osteogenic differentiation <em>in vivo</em>. Together, our results reveal that p38 and ERK1/2 MAPKs are activated in BMP9-induced osteogenic differentiation of MPCs. What is most noteworthy, however, is that p38 and ERK1/2 act in opposition to regulate BMP9-induced osteogenic differentiation of MPCs.</p> </div

BMP9 induces phosphorylation/activation of p38 and ERK1/2 in MPCs.

<p>(<b>A</b>). Western blotting analysis of BMP9-induced phosphorylation of Samd1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of Smad1/5/8, p38 and ERK1/2 was analyzed by western blotting. β-actin was used to demonstrate equal loading of all samples. (<b>B</b>) Western blotting analysis of BMP9-induced phosphorylation of p38 and ERK1/2 in MEFs. MEFs were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed by western blotting. (<b>C</b>) Western blotting analysis of BMP9-induced phosphorylation of p38 and ERK1/2 in C2C12 cells. C2C12 cell were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed by western blotting. (<b>D</b>) Western blotting analysis of BMP9 conditioned medium (BMP9-CM)-induced phosphorylation of p38 and ERK1/2 in C3H10T1/2 cells. C3H10T1/2 cells were treated with BMP9-CM, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed at indicated time points by western blotting.</p

Opposing effects of p38 and ERK1/2 on BMP9-induced ALP activity of MPCs.

<p>(<b>A</b>) Inhibition of BMP9-induced ALP activity by p38 inhibitor SB203580 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with varying concentrations (0, 2, 5 and 10 µM) of SB203580. ALP activity was quantitative measured at day 7. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (<i>vs</i>. control groups); “*”, p<0.05 (<i>vs</i>. control groups). (<b>B</b>) Enhancement of BMP9-induced ALP activity by ERK1/2 inhibitor PD98059 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with varying concentrations (0, 10, 25 and 50 µM) of PD98059. ALP activity was quantitative measured at 7 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01; “*”, p<0.05 (<i>vs</i>. control groups). (<b>C</b>) Opposing effects of SB203580 and PD98059 on BMP9-induced ALP activity in MEFs. MEFs were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with fixed concentrations of SB203580 (25 µM) or PD98059 (10 µM). ALP activity was quantitative measured at 5 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (<i>vs</i>. control groups). (<b>D</b>) Opposing effects of SB203580 and PD98059 on BMP9-induced ALP activity in C2C12 cells. C2C12 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with fixed concentrations of SB203580 (25 µM) or PD98059 (10 µM). ALP activity was quantitative measured at 3 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (<i>vs</i>. control groups).</p