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Post-transcriptional gene regulation following exposure of osteoarthritic human articular chondrocytes to hyperosmotic conditions  S.R. Tew, O. Vasieva,

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Presentation on theme: "Post-transcriptional gene regulation following exposure of osteoarthritic human articular chondrocytes to hyperosmotic conditions  S.R. Tew, O. Vasieva,"— Presentation transcript:

1 Post-transcriptional gene regulation following exposure of osteoarthritic human articular chondrocytes to hyperosmotic conditions  S.R. Tew, O. Vasieva, M.J. Peffers, P.D. Clegg  Osteoarthritis and Cartilage  Volume 19, Issue 8, Pages (August 2011) DOI: /j.joca Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

2 Fig. 1 Exposure to hyperosmotic conditions leads to increases in mRNAs enriched with ARE-containing genes. (A) Pie charts illustrating the occurrence of ARE’s in genes significantly upregulated (P<0.01, uncorrected paired t test) following 5h exposure to hyperosmotic conditions. Different classes of AREs are categorised according to the ARED cluster system. (B) Hierarchical clustering analysis of genes upregulated 1.4-fold after a 5h exposure to hyperosmolarity. Columns show all combinations of individual treatments (C – control, H – hyperosmotic) and samples (samples are M – male or F – female, with age denoted afterward). Green in the rightmost column indicates the presence of an ARE in the genes 3′UTR. (C) Real time PCR analysis of expression of 15 candidate genes chosen for further examination. Bars represent means with 95% confidence intervals. All genes except SLC5A3 demonstrated upregulation that was significant (*P values: COBLL1<0.001, RGS16=0.007, PGM2II<0.001, GLS=0.006, PFKFB3=0.017, GEM=0.006, IL6=0.009, HSPH1<0.001, ADAMTS1=0.009, ARNTL<0.001, BAMBI=0.007, COX2=0.001, SGK1<0.001, SLC5A3=0.227) using paired t tests (data is derived from experiments carried out with four separate donors). Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

3 Fig. 2 IPA of hyperosmotically regulated genes identified enrichment ion those associated with TGFβ signalling. Schematic demonstrating enrichment of hyperosmotically regulated genes within the TGFβ signalling pathway. Red indicates upregulation (>1.4-fold) and green downregulation (>1.4-fold). Grey indicates significantly regulated genes controlled between 1.1- and 1.4-fold. Stronger colour intensity indicates stronger regulation of each gene. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

4 Fig. 3 Hyperosmolarity regulates a diverse set of chondrocyte genes at the post-transcriptional level. RNA decay curves generated from actinomycin D chase experiments. Freshly isolated HAC were treated under control (380mOsm, diamonds) or hyperosmotic (550mOsm, squares) conditions for 2h and then actinomycin D was added to the media. mRNA levels were then determined at 0, 1, 3 and 5h by qRT-PCR. Data points represent the average of decay curves obtained from two different donors – bars represent 95% confidence interval. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

5 Fig. 4 Increased expression of hyperosmotically regulated chondrocyte genes requires MAPK signalling. Western blot analysis of (A) phosphorylated and total ERK1/2 and (B) phosphorylated p38 MAPK and GAPDH in freshly isolated HAC cultured for 5h under 380 or 550mOsm conditions. Real time PCR analysis of freshly isolated HAC grown for 5h in hyperosmotic conditions in the presence or absence of (C) the p38 MAPK inhibitor SB or (D) the MEK1/2 inhibitor U0126. Vertical axis represents fold change vs HAC cultured in 380mOsm conditions. Bars indicate mean and 95% confidence intervals of data from four (SB202190) or five (U0126) independent experiments. *P<0.05 vs without inhibitor, paired t test. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

6 Fig. 5 miRNAs are not strongly regulated following 5h exposure to hyperosmolarity. Heat map illustrating the expression of those miRNAs that demonstrated a change in expression within a 95% confidence interval following hyperosmotic stimulation. Donor (M=male, F=female+age) is denoted at top of each column as well as treatment (380mOsm or 550mOsm). The data has been subjected to hierarchical clustering. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

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