Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation
Fig 6
Inhibition of ROCK/F-actin represses the transcriptional activity of TAZ in hMSCs.
(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 CTGF and CYR61 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 DLX5, MSX2, osteocalcin, and RUNX2, were analyzed by qRT-PCR. Target gene expression was normalized to GAPDH. (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 TAZ. Gene expression was normalized to GAPDH.