Affymetrix gene chip characterization of IL-1β regulated genes

Many of IL-1β effects on chondrocyte gene expression were postulated to be due to up regulation of iNOS and enhanced NO production, however many of them were based just on the correlation between increased NO production and observed effects.

Therefore we performed a detailed analysis of IL-1 regulated genes also in the cells, which were stimulated in the presence of iNOS inhibitors to elucidate direct effects of IL-1 on gene expression and these which could be NO mediated.

In our first experimental setting we investigated IL-1 regulated genes in chondrogenic pellets from 4 bone marrow donors. Pellets were stimulated for 24h with 1nM IL-1β at different time points of differentiation (0, 7, 11, 14, 18, 20, 33, 52 days). Afterwards results obtained for cells stimulated with IL-1 were compared with results obtained for control pellets at each differentiation time point using following calculations:

• T test, median fold change cutoff 0.7/1.3x; p value cutoff 0.001

(comparisons at days 0 and 11) and 0.01 (comparisons at days 7 and 14)

• log log plots of all vs all within a certain day group, fold change cutoff 1.5x (comparisons at days 18 and 53), 2x (comparison at day 20) and 3x (comparison at day 33); all days where further reduced by median fold change cutoff 0.7/1.3x

• total probe sets (ps) from above methods:

– 139 upregulated ps – 35 downregulated ps

• absent/present analysis, p value cutoff 0.05

combinations days: 0-7, 0-11, 0-14, 7-11, 7-14, 11-14 – 70 upregulated ps

– 8 downregulated ps

Lists of IL-1 regulated genes are presented in the supplement: Tables: VII - X.

Among up-regulated genes after IL-1 treatment are e.g. NFκB, COX-2 (PTGS2), PTGES, PTGER4 (prostaglandin E receptor subtype EP4), interleukins (1β, 6, 8 and 11), chemokines, metalloproteases (MMP1, MMP3, MMP12, ADAMTS5), GTP cyclohydrolase 1 (GCH1), enzyme which synthesizes tetrahydrobiopterin, a cofactor for iNOS, genes related to TNFα but also adhesion molecules as ICAM1, VCAM1 and NRCAM.

Some anabolic genes like BMP2 and FGF2 were also up-regulated.

Interestingly significantly elevated levels of metallothioneins (1F, 1G, 1H, 1K, 1X, 2A) were expressed after IL-1β stimulation and also SOD2 was heavily induced.

Interestingly, among up-regulated genes there are several genes like proteases which have been shown as related to the development of OA but also genes, which haven’t been previously associated with this disease and could serve as potential targets for further investigations.

4.5.2. Affymetrix gene chip characterization of IL-1β regulated genes in human chondrocytes, effect of iNOS inhibition

The predominant role of IL-1β in the development of OA is well established, however several detrimental effects of this cytokine have been implicated to be dependent on up-regulation of iNOS and NO synthesis.

To elucidate which effects are mediated by NO and which are directly due to the action of IL-1 human chondrocytes in alginate beads were stimulated for 24h with IL-1β in presence or absence of iNOS inhibitors. We used two inhibitors: 1400W (10µM) and BYK191023 (30µM) to exclude substance-specific effects. Additionally substances were also tested in control conditions.

After 24h stimulation was controlled with Griess assay and cells were harvested for RNA isolation. Experiments were performed in 5 replicates. RNA was assayed with Affymetrix chips HG-U133_Plus_2 containing 54675 probe sets.

Calculations of results was performed as listed below:

• comparisons performed:

– control vs IL1B

– IL1B vs IL1B+1400W Æ target effects – IL1B vs IL1B+191023 Æ target effects – control vs 1400W Æ off-target effects – control vs 191023 Æ off-target effects

None of the genes which were regulated already by iNOS inhibitors in control conditions (off-target effects) were upregulated in cells stimulated with IL-1 (target effects).

• tests applied for filtering of regulated probe sets in obtained data:

– absence / presence anlysis, p value cutoff 0.05

– T-test, p value cut-off 0.001; median fold change, cutoff 0.7/1.3x

Using the t-test method 441 probe sets were identified as increased over 1,3-fold after IL-1β treatment in comparison to control. Additionally 149 probe sets were detected using absence/presence analysis. Both analyses revealed 590 probe sets up regulated after IL-1 treatment in human chondrocytes.

All IL-1β up-regulated genes are listed in the supplement Table XI.

Between IL-1β up-regulated genes we have found: NFκB, several chemokine ligands, interleukins (IL-1α and β, 10, 11, 20, 24), iNOS, GCH1, PLA2 (phospholipase A2), COX-2, PTGER2 (prostaglandin E receptor subtype EP2), MMP10, TNFα and a number of genes related to TNFα, GCH1 and ICAM1.

Interestingly also genes related to chondrocyte differentiation were up-regulated by IL-1β as e.g. SOX 7,11,17, WNT5A, BMP6.

Afterwards a search for IL-1 up-regulated genes, which were down regulated with iNOS inhibitors, was performed (T-test, p value cutoff 0.001; median fold change IL-1 vs IL-1 + inhibitor cutoff 0.7x; additionally absence/presence analysis, p value cutoff 0.05).

Analysis revealed 5 genes down-regulated by treatment with Byk191023 and upregulated in IL-1 alone and 30 genes down-regulated by treatment with 1400W.

All Byk191023 down-regulated genes in IL-1 stimulated cells are listed in the Table11.

Table 11. Genes detected as down-regulated by Byk191023 [30µM] and upregulated in IL-1 alone. In the table are given: gene symbol; the mean of normalized expression values for control cells, IL-1 stimulated cells, cells stimulated in the presence of Byk191023 and 1400W (for comparison); description of the gene and Affymetrix identifier.

Symbol control IL1B IL1+191023 IL1+1400W Description Probe set 1557826_at 0 164 0 0 Hypothetical protein LOC338817,

mRNA 1557826_at

1568799_at 0 162 0 132 Clone IMAGE:4798168, mRNA 1568799_at 225033_at 2449 8606 6530 6627 Clone IMAGE:4401795, mRNA 225033_at ENO2 3068 10580 3681 2704 enolase 2 (gamma, neuronal) 201313_at GBE1 28370 38100 27130 25160 glucan (1,4-alpha-), branching

enzyme 1 203282_at

All 1400W down-regulated genes in IL-1 stimulated cells are listed in the Table 12.

Both iNOS inhibitors down regulated only two genes: enolase 2 gamma, neuronal (ENO2) and glucan (1,4-alpha-), branching enzyme1 (glycogen branching enzyme) (GBE1).

Table 12. Genes detected as down-regulated by 1400W [10µM] and upregulated in IL-1 alone. In the table are given: gene symbol; the mean of normalized expression values for control cells, IL-1 stimulated cells, cells stimulated in the presence of 1400W and Byk191023 (for comparison); description of the gene and Affymetrix identifier.

Symbol control IL1B IL1+1400W IL1+191023 Description Probe set AGMAT 0 168 0 176 agmatine ureohydrolase

(agmatinase) 219792_at

C20orf139 2774 4650 3010 4273 chromosome 20 open reading frame

139 225252_at

CDKN1A 3554 7398 5686 6006 cyclin-dependent kinase inhibitor 1A

(p21, Cip1) 202284_s_at

EGLN3 622 1164 517 407 egl nine homolog 3 (C. elegans) 219232_s_at ENO2 3068 10580 2704 3681 enolase 2 (gamma, neuronal) 201313_at ERO1L 1528 3093 1446 1758 ERO1-like (S. cerevisiae) 225750_at EVC 0 312 0 279 Ellis van Creveld syndrome 219432_at GBE1 28370 38100 25160 27130 glucan (1,4-alpha-), branching

enzyme 1 203282_at

HK2 3710 12730 6680 6872 hexokinase 2 202934_at HNRPA1 9823 15010 9661 9382 heterogeneous nuclear

ribonucleoprotein A1 214280_x_at IGFBP3 20060 76840 28900 37170 insulin-like growth factor binding

protein 3 212143_s_at

JMJD1A 2042 4632 2483 2696 jumonji domain containing 1A 212689_s_at LIMK2 511 1116 399 554 LIM domain kinase 2 221756_at LOC284207 10650 20400 14440 15114 hypothetical protein LOC284207 225955_at LOC56270 3596 6001 4406 3933 hypothetical protein 628 209076_s_at Lrp2bp 2190 4353 2244 2702 low density lipoprotein

receptor-related protein binding protein 227337_at MXI1 5584 10050 4185 4356 MAX interactor 1 202364_at PFKFB3 6984 17230 8865 9544

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 202464_s_at

PFKP 9251 17647 11360 13190 phosphofructokinase, platelet 201037_at RAI3 846 1776 974 1040 retinoic acid induced 3 212444_at RIS1 2201 6993 2713 4274 Ras-induced senescence 1 213338_at SLC16A1 1176 3426 1685 2041 solute carrier family 16 member 1 202234_s_at SLC2A1 1889 5156 1652 2421 solute carrier family 2 member 1 201250_s_at SUI1 26771 47233 32953 37010 putative translation initiation factor 202021_x_at SUI1 28650 46808 35450 38792 putative translation initiation factor 212130_x_at TFRC 17347 26073 17873 21340 transferrin receptor (p90, CD71) 207332_s_at TNFRSF10

D 7299 12320 9072 9522 tumor necrosis factor receptor

superfamily, member 10d 227345_at TRIM16 537 2125 868 1111 tripartite motif-containing 16 204341_at TXNIP 8775 14190 6764 8779 thioredoxin interacting protein 201010_s_at 232484_at 706 1135 823 874 LOC388443 (LOC388443), mRNA 232484_at

The same criteria was used for selection of IL-1 down-regulated genes. 1343 probe sets were found as down-regulated after IL-1 treatment in human chondrocytes. As expected expression of several genes related to cartilage matrix was down-regulated (collagens, integrins, hyaluronan and proteoglycan link protein 1).

All IL-1β down-regulated genes are listed in the supplement Table XII.

Analysis revealed 175 genes regulated by treatment with 1400W and 91 genes up-regulated by treatment with Byk191023. These genes are listed in the supplement Table XIII and XIV.

However, iNOS inhibitors used had very weak effect on the gene expression in human chondrocytes. This was confirmed by clustering analysis, were experiments formed two main clusters: control and IL-1 (Figure 54).

Figure 54. Clustering analysis of Affymetrix experiments obtained for human chondrocytes in alginate beads (description in text).