Odds ratios (OR) and 95% confidence intervals (CI) of predictors of the number of lifestyle risk factors.

<p>Odds ratios (OR) and 95% confidence intervals (CI) of predictors of the number of lifestyle risk factors.</p

Prevalence and Prevalence Odds Ratio (POR) of combinations of two lifestyle risk factors.

<p>Prevalence and Prevalence Odds Ratio (POR) of combinations of two lifestyle risk factors.</p

Characteristics of the study population, n (%).

<p>Characteristics of the study population, n (%).</p

Odds ratio (95% confidence interval)^*of socioeconomic factors (t year) for predicting number of chronic multimorbidity (t+1 year) from multinomial multilevel models fitted to Korea Health Panel (2009–2011), stratified by each socioeconomic measures and age groups.

<p>Odds ratio (95% confidence interval)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173770#t003fn002" target="_blank">*</a>}of socioeconomic factors (t year) for predicting number of chronic multimorbidity (t+1 year) from multinomial multilevel models fitted to Korea Health Panel (2009–2011), stratified by each socioeconomic measures and age groups.</p

Odds ratio (95% confidence interval)^*of socioeconomic factors for each disease groups and pairs between six disease groups from multinomial multilevel models fitted to Korea Health Panel (2009–2011).

<p>Odds ratio (95% confidence interval)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173770#t004fn002" target="_blank">*</a>}of socioeconomic factors for each disease groups and pairs between six disease groups from multinomial multilevel models fitted to Korea Health Panel (2009–2011).</p

Bivariate association between socio-demographic factors(t year) and number of chronic disease (t+1 year)in the Korea Health Panel members (2009–2011).

<p>Bivariate association between socio-demographic factors(t year) and number of chronic disease (t+1 year)in the Korea Health Panel members (2009–2011).</p

Socio-demographic and health behavioural characteristics of study participants from the Korea Health Panel members (2009–2011) by age groups.

<p>Socio-demographic and health behavioural characteristics of study participants from the Korea Health Panel members (2009–2011) by age groups.</p

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

Inhibition of the ERK or JNK signaling pathway induces TAZ cytoplasmic localization on stiff hydrogels.

<p>(A) hMSCs on a 4.47 kPa hydrogel were treated with 10 μM U0126 or 10 μM SP600125. After 12 h of treatment, cells were subjected to immunostaining with an anti-TAZ antibody. The red fluorescence signal shows the location of TAZ, and DAPI was used to stain the nuclei. The results show that even in cells on a stiff matrix, TAZ is localized evenly to the cytoplasm and nucleus following ERK or JNK inhibition. (B) Approximately 100 cells in panel (A) were counted, and TAZ localization was analyzed in these cells. The counting procedure was done using the Image J program. The number of cells that showed an even cytoplasmic-nuclear or cytoplasm-dominant TAZ localization was higher in the presence of an ERK or JNK inhibitor than in the absence of either inhibitor. (C) Cell lysates in panel (A) were prepared, and the activity of the Hippo signaling pathway components LATS and MST kinase was analyzed by immunoblotting. The phosphorylation status of LATS and MST kinase was analyzed with p-LATS1 and p-MST1/2 antibodies, respectively. Total LATS1 and MST2 levels were detected as a loading control.</p