Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation

<div><p>Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.</p></div

Inhibition of ROCK/F-actin represses the transcriptional activity of TAZ in hMSCs.

<p>(A) hMSCs cultured on a 4.47kPa hydrogel were treated with a ROCK inhibitor (Y27632, 50 μM) or an F-actin inhibitor (latrunculin A, 0.5 μM). After 12 h of treatment, total RNA was prepared, and <i>CTGF</i> and <i>CYR61</i> expression was assessed by qRT-PCR. (B) The CTGF-luc reporter gene construct or the pGL3-basic control vector was transfected into hMSCs, and after 16 h, the transfected cells were plated on 4.47 kPa hydrogels. After 24 h, luciferase reporter gene activity was analyzed. To inhibit ROCK or F-actin, cells were pretreated with 50 μM Y27632 or 0.5 μM latrunculin A 12 h before reporter gene analysis. A Renilla luciferase-expressing vector was used as a transfection control. Luciferase activity was normalized to Renilla luciferase activity. (C) hMSCs on 4.47 kPa hydrogels were differentiated into osteoblasts for 6 days in the presence of 50 μM Y27632 or 0.5 μM latrunculin A. DMSO was used as the vehicle control. The expression of osteoblastic marker genes, including <i>DLX5</i>, <i>MSX2</i>, osteocalcin, and <i>RUNX2</i>, were analyzed by qRT-PCR. Target gene expression was normalized to <i>GAPDH</i>. (D) hMSCs were transfected with 6OSE2-luc or pGL3-basic (control) along with a Renilla luciferase-expressing construct. Then, the cells were plated on a 4.47 kPa hydrogel. After 24 h, luciferase reporter gene activity was analyzed. To inhibit ROCK or F-actin, the cells were pretreated with 50 μM Y27632 or 0.5 μM latrunculin A 12 h before the reporter gene assay. Luciferase activity was normalized to Renilla luciferase activity. (***p < 0.005, t-test). (E) Total RNAs of hMSCs in panel (A) were prepared and qRT-PCR was assessed to analyze the transcription of <i>TAZ</i>. Gene expression was normalized to <i>GAPDH</i>.</p

ECM stiffness regulates the cellular phenotype of human mesenchymal stem cells (hMSCs) and the localization and transcriptional activity of TAZ.

<p>(A) In indicated hydrogels, cell adhesion and morphology were visualized by light microscopy. Bright field images were taken 24hr after seeding. (B) ECM stiffness controls focal adhesion complex formation and TAZ localization. hMSCs were immunostained with an anti-vinculin antibody to detect focal adhesions (green fluorescence signal) 24 h after seeding. TAZ localization was visualized as a red fluorescence signal. DAPI was used to stain the cell nucleus. (C) The expression of TAZ target genes, including <i>CTGF</i> and <i>CYR61</i>, were analyzed by qRT-PCR using the cells in panel (A). Target gene expression was normalized to the <i>GAPDH</i> expression. Data is shown as fold induction. Asterisks indicate statistical significance (***p < 0.005, t-test). (D) The luciferase reporter gene CTGF-luc was introduced into hMSCs. After 16 h, the transfected cells were plated on 1.37 or 4.47 kPa hydrogels. After 24 h, luciferase reporter gene activity was analyzed. The pGL3-basic luciferase reporter gene, which has no promoter for transcription, was used as a negative control. A Renilla luciferase-expressing vector was used as a transfection control. Luciferase activity was normalized to Renilla luciferase activity and is expressed as relative fold induction. (***p < 0.005, t-test). (E) hMSCs were seeded on a 1.37 or 4.47 kPa hydrogel, and twenty four hours after seeding, total RNAs were isolated and qRT-PCR analysis was assessed to see the expression of <i>TAZ</i> gene. Gene expression was normalized to <i>GAPDH</i>.</p