Transforming growth factor-β enables NFATc1 expression during osteoclastogenesis

Osteoclastogenesis is dependent on distinct stimuli that prime and activate osteoclast differentiation. One cytokine needed to prime monocytes for osteoclastogenesis is TGF-β, which enables and augments RANKL and TNF-α-induced osteoclast differentiation. However, the precise time-period during which this occurs and the molecular mechanism mediating this action are unknown. We report here TGF-β prime monocytes for osteoclast formation within 24 h by regulating expression of NFATc1, a key osteoclastic transcription factor. TGF-β directly induces cytoplasmic NFATc1 expression within 24 h, but is unable to stimulate NFATc1 nuclear translocation. Furthermore, RANKL-induced NFATc1 expression is dependent on the presence of TGF-β during the early stages of osteoclastogenesis. Similarly, TNF-α activates osteoclastogenesis by stimulating translocation of TGF-β-induced NFATc1. In light of these findings, it is apparent that osteoclast formation is dependent on coordinated interactions between TGF-β and RANKL/TNF-α that regulate the expression and intracellular distribution of NFATc1 during early stages of osteoclast differentiation

Transcriptomic Analysis of Induced Pluripotent Stem Cells Derived from Patients with Bipolar Disorder from an Old Order Amish Pedigree

<div><p>Fibroblasts from patients with Type I bipolar disorder (BPD) and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs). Established iPSCs were subsequently differentiated into neuroprogenitors (NPs) and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs, NPs and neurons matured in culture for either 2 weeks (termed early neurons, E) or 4 weeks (termed late neurons, L). Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate, enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically, 328 genes were differentially expressed between BPD and control L neurons including GAD1, glutamate decarboxylase 1 (2.5 fold) and SCN4B, the voltage gated type IV sodium channel beta subunit (-14.6 fold). Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology, GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism, protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD.</p></div

Quantitative RT-PCR of RNA samples from NPs and neurons.

<p>RNA samples were collected from NPs, E and L neurons and markers of neuronal gene expression were evaluated. Pan-neuronal markers, (A,B), cortical neuronal marker (C), and synaptic markers (D,E) were evaluated. Each bar represents mean ± standard error (n = 8, combining 4 BPD and 4 controls). One-way ANOVA was performed and Bonferroni’s Multiple Comparison post-hoc test was done. * p < 0.01, ** p < 0.001, *** p < 0.0001 compared to NP.</p

Microarray data analysis.

<p>A. Principal Component Analysis (PCA) plot shows three distinct clusters: iPSCs, NPs and E plus L neurons as outlined with blue, green and red lines, respectively. The symbols represent undifferentiated iPSCs (blue x), control NP (green cross; +), control E (black circle; ○), control L (red triangle; Δ) and BPD NP (grey asterisk; *), BPD E (turquoise diamond; ♢) and BPD L (pink inverted triangle; ▽). B. Clustered heat map of differentially expressed genes in both control and BPD neurons demonstrates that the genes are clustered closely for E and L neurons regardless of disease status and separate from NPs and iPSCs. Horizontal axis shows genes used for clustering in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142693#pone.0142693.s005" target="_blank">S2 Table</a> and vertical axis shows sample clustering. C. Changes in expression of genes associated with axonal guidance (Ingenuity pathway analysis) shown as a clustered heat map show that the expression of genes associated with axonal guidance was higher in E and L neurons than in NPs. Horizontal axis shows genes used for clustering as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142693#pone.0142693.s006" target="_blank">S3 Table</a>.</p

Confirmational quantitative RT-PCR.

<p>RNA samples collected during differentiation at NP, E and L neuron stages for microarray studies were analyzed by quantitative RT-PCR. Genes previously implicated to be involved with BPD from genomic studies were evaluated: ANK3, ODZ4 and CACNA1C were analyzed (A-C) and did not show differences in expression at any stage. GSK3B did not show significant differences in BPD and control at any stage (D). SCN4B showed trends of down regulation both in E and L neurons (E), but did not meet the statistical significance. Expression of GAD1 was upregulated in L of BPD compare to control. The box on the plots represents the 25 and 75 percentiles. The horizontal bar within the box represents the median. Individual data points are represented by triangles (BPD) or squares (Controls). n = 4 except for SCN4B NP and L controls and E BPD where n = 3. Two-way ANOVA was performed and Bonferroni’s Multiple Comparison post-hoc test was done. ** p < 0.01 when compared to control.</p