Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation

RESEARCH ARTICLE Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation Jun-Ha Hwang1, Mi Ran Byun1, A. Rum Kim1, Kyung Min Kim1, Hang Jun Cho1, Yo Han Lee1, Juwon Kim1, Mi Gyeong Jeong2, Eun Sook Hwang2*, Jeong-Ho Hong1* a11111 1 Department of Life Sciences, School of Life Sciences and Biotechnology, Korea University, Seoul, Korea, 2 College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea * (J-H Hong); (ESH) Abstract OPEN ACCESS Citation: Hwang J-H, Byun MR, Kim AR, Kim KM, Cho HJ, Lee YH, et al. (2015) Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation. PLoS ONE 10(8): e0135519. doi:10.1371/journal.pone.0135519 Editor: Adam J. Engler, University of California, San Diego, UNITED STATES Received: May 3, 2015 Accepted: July 22, 2015 Published: August 11, 2015 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant nos. 2014R1A2A2A01006547 and 2014R1A1A2057408) and by a grant from the Korea Health Technology R&D project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry for Health & Welfare (grant no. HI14C3266), Republic of Korea. This work was also supported by a grant from Korea University. 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. Introduction The extracellular matrix (ECM) is a dynamic structure that provides structural support for organs and tissues. It closely contacts cells, activates several cellular components, and regulates cell proliferation, differentiation, and migration [1]. The chemical composition and structure of the ECM, which are unique to each tissue, are important for cell-ECM interaction and cellular function. Dysregulation of ECM composition, structure, and stiffness contribute to diverse pathological conditions [2]. In particular, ECM stiffness regulates multipotent mesenchymal stem cell (MSC) differentiation; a soft matrix has neurogenic potential, and a stiff matrix that mimics collagenous bone has osteogenic potential [3–6]. It has also been shown that stiff matrix-driven osteogenesis was induced by integrin-mediated mechanotransduction [7]. PLOS ONE | DOI:10.1371/journal.pone.0135519 August 11, 2015 1 / 16 MAPK Activation and Induced Osteogenic Differentiation on Stiff ECM Competing Interests: The authors have declared that no competing interests exist. Transcriptional coactivator with PDZ-binding motif (TAZ) and its paralog Yes associated protein (YAP) were characterized as signaling mediators of mechanotransduction [8–10]. The activity of TAZ and YAP are regulated by ECM stiffness [8]. A stiff ECM stimulates nuclear localization of TAZ/YAP and facilitates osteogenic differentiation, whereas a soft ECM inhibits their nuclear localization and induces adipogenic differentiation [8,11]. A stiff ECM activates Rho GTPase, which stimulates F-actin polymerization and activates TAZ and YAP [8]. Mechanical forces are an important regulators of TAZ/YAP activity [10,12]. TAZ regulates MSC differentiation by activating osteoblast and myoblast differentiation and inhibiting adipocyte differentiation [13–15]. TAZ stimulates Runx2 target genes, but inhibits PPARγ-mediated gene transcription [13]. TAZ and YAP are also known as effector proteins in the Hippo signaling pathway, which plays an important role in cell proliferation, tumorigenesis, and stem cell self-renewal [16,17]. In the present study, we show that a stiff ECM induces ERK and JNK activation, facilitates the nuclear localization of TAZ, and stimulates osteogenic differentiation. Results A stiff surface stimulates the nuclear localization of TAZ, a mechanotransduction effector Tissues have diverse elasticity values; normal liver and brain have values of several hundred Pascals (Pa), whereas muscle has a value of more than 12 kPa, and tendon and cartilage have values in the megapascal range [7]. To study the function of TAZ on a stiff ECM and identify the minimum stiffness required for TAZ activation in tissues, TAZ localization was analyzed in tissues grown on hydrogels with various degrees of stiffness by immunocytochemistry. Previously, increased TAZ nuclear localization was observed in cells on a 40 kPa gel matrix, but not on a 0.7 kPa matrix [8]. However, the difference was too extreme to define the minimal stiffness required for TAZ activation. Thus, we attempted to determine the minimal stiffness of gel matrix required for the nuclear localization of TAZ. We analyzed the nuclear localization of TAZ in cells on 0.7, 4.47, 8.73, and 40 kPa hydrogels by immunocytochemistry, which was evidenced by the detection of a green fluorescence signal for TAZ in the nucleus. We observed that TAZ was localized to the nucleus on hydrogels with stiffnesses greater than 4.47 kPa (S1 Fig). Next, we narrowed down the stiffness range by assessing nuclear localization on 4.47, 2.83, 1.37, and 0.7 kPa hydrogels. Eventually, we observed that a hydrogel matrix with a stiffness of 4.47 kPa is required for TAZ activation, as no activation was observed on hydrogel matrices with stiffnesses less than 2.8 kPa (Fig 1A and 1B). In addition, cells with normal spread shape and focal adhesions were observed on the 4.47 kPa hydrogel matrix, as shown by (...truncated)