Cover and contents : “Proceedings of the 11th CEReS Symposium on Enviromental Remote Sensing” February 23, 2009

2009年2月23日（月） 於 千葉大学けやき会

Proteasome inhibition partially reverts the effect of SIK inhibitor on c-Fos reduction.

<p>RAW264.7 cells were preincubated with vehicle (0.03% DMSO) or 0.5 μM of SIK inhibitor HG-9-91-01 for 30 min followed by RANKL (50 ng/ml) stimulation for 24h. MG132 (10 μM) was added during the last 4h of incubation. Total protein extracts (50 μg/lane) were separated by 10% SDS-PAGE followed by immunoblotting using specific antibodies. <i>Inset</i> show a representative example depicting c-Fos, NFATc1 and GAPDH. c-Fos expression levels in presence or absence of MG132 treatment were quantified relative to GAPDH and represented as ratio of their respective RANKL stimulated control (n = 3). Data are expressed as mean +/- SD. * P<0.05 <i>vs</i>. no MG132 treatment.</p

SIK inhibitor HG-9-91-01 reduces RANKL-induced osteoclast differentiation markers and bone-resorbing activity.

<p><b>A</b>) RAW264.7 cells were preincubated with vehicle (0.03% DMSO) or indicated concentration of SIK inhibitor HG-9-91-01 for 30 min followed by RANKL (50 ng/ml) stimulation. After 4 days mRNA expression levels of MMP9, Cathepsin K, and TRAP genes were determined by RT-qPCR using 18S rRNA as normalization control (n = 5). <b>B)</b> Cell viability was determined in the supernatants by measuring LDH release. The histograms represent the levels of mRNA or LDH (%) compared with that of the RANKL control (set = 100%). <b>C)</b> RAW264.7 cells were cultured as above in 24 well Osteo plate in presence of 0.5 μM of HG-9-91-01. After 4 days the cells were removed with 5% sodium hypochlorite solution and resorbed areas were measured. The histograms represent the relative resorbed area (%) compared with that of RANKL-stimulated control (set = 100%). A representative example is depicted. <b>D)</b> BMM cells (n = 4) were preincubated with vehicle (0.03% DMSO) or the indicated concentration of SIK inhibitor HG-9-91-01 for 30 min followed by RANKL stimulation (50ng/ml) and MCSF (30 ng/ml) for 3 days. mRNA analysis was performed as above. <b>E)</b> BMM were cultured as above in 24 well Osteo plate in presence of 0.1 or 0.5 μM of HG-9-91-01. After 3 days resorbed areas were measured as above. The histograms represent the relative resorbed area (%) compared with that of RANKL-stimulated control (set = 100%). A representative example is depicted. Data are expressed as mean +/- SD. * <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 significant <i>vs</i>. RANKL control.</p

SIK2 or -3 silencing by CRISPR/Cas9 mediated gene editing reduces osteoclast differentiation and impairs resorption activity upon RANKL stimulation.

<p><b>A)</b> RAW264.7 cells were infected with pLentiCRISPR-V2 viruses targeting SIK1, SIK2, and SIK3, respectively as detailed in <i>Material and Methods</i> and SIK expression was determined in cell lysates (25 μg/lane) form RAW 264.7 infected cells or wild type cells separated by 7.5% PAGE-SDS and probed with specific antibodies and anti-tubulin. A representative example is depicted (n = 3). <b>B</b>) SIK KO or wild type RAW cells were incubated with 50 ng/ml RANKL for 4 days and then were fixed and stained with TRAP. The TRAP positive multinucleated cells (TRAP+ MNCs) were visualized by digital microphotography and the TRAP+ MNCs containing more than 3 nuclei from four randomly selected magnification fields (scale bar 100 μm) were counted. Error bars = mean +/- SD, from one independent experiment performed in triplicate. <b>C</b>) Cells Lysates obtained from SIK KO cells or wild type RAW 264.7 cells cultured in absence or presence of RANKL (50 ng/ml) for 24 h were separated by 7.5% PAGE-SDS and SIK expression determined as above (<b>i</b>), in the same lysates separated on 10% PAGE-SDS (50 μg/lane) the expression of c-Fos and NFATC1 were determined (<b>ii</b>). GAPDH expression was used as loading control. A representative example (n = 3) is depicted. <b>D)</b> SIK KO cell or wild type RAW were cultured as above in 24 well osteo plates. After 4 days the cells were removed with 5% sodium hypochlorite solution and resorbed areas were measured. Four randomly selected magnification fields (scale bar 100 μm) for each condition were analyzed. The histograms represent the relative resorbed area (%) compared with that of wild type RAW or SIK1 control (set = 100%). Data are expressed as mean +/- SD, from one experiment performed in triplicate. Data are representative of two independent experiments. <i>Insets</i> show a representative example for each condition. * P<0.05, **P<0.01, ***P<0.001.</p

Expression of SIK1-3 in osteoclasts derived from RAW264.7 cells or BMM.

<p>RAW264.7 cells were stimulated for 4 days with RANKL (50 ng/ml) to induce differentiation into osteoclasts. <b>A)</b> mRNA expression levels of Cathepsin K, MMP9 and TRAP genes were determined by RT-qPCR using 18S rRNA as normalization control and calculated by the 2-^ΔΔCt method (n = 5). Data are expressed as fold changes versus untreated RAW264.7 cells (set as one). A representative example of formation of giant multinucleated cells detected by TRAP staining is depicted. <b>B)</b> Relative SIK1, SIK2 and SIK3 mRNA expression (<b>i</b>) in RAW 264.7 cells (n = 6) following RANKL stimulation (50ng/ml, 4days). mRNA was measured by RT-qPCR using 18S rRNA as normalization control and calculated by the 2-^ΔΔCt method. Protein expression levels (<b>ii</b>) of SIK1, SIK2 and SIK3 in cell lysates separated on a 7.5% SDS-PAGE (50 μg protein/lane) were determined by Western blot and normalized for tubulin expression (n = 4) <b>C)</b> Relative SIK1, SIK2 and SIK3 mRNA expression (<b>i</b>) in BMM cells (n = 7) following RANKL stimulation (50ng/ml) and MCSF (30 ng/ml), for 3 days. mRNA was measured by RT-qPCR using 18S rRNA as normalization control and calculated by the 2-^ΔΔCt method. Protein expression levels (<b>ii</b>) of SIK1, SIK2 and SIK3 in cell lysates separated on a 7.5% SDS-PAGE (50 μg protein/lane) were determined by Western blot and normalized for tubulin expression (n = 5). Protein data are expressed as fold changes versus control cells (set as one). Data are expressed as mean +/- SD. Statistical significance is reported as * <i>P</i><0.05.</p

Effect of SIK inhibitor HG-9-91-01 on RANKL-induced signalling pathways.

<p>Effect of HG-9-91-01 pretreatment on NF-κB, MAPKs and JNK activation in RANKL stimulated RAW264.7 cells. Cells were cultured in 1% serum for 16h, preincubated with vehicle (0.03% DMSO) or 0.5 μM of SIK inhibitor HG-9-91-01 for 30 min followed by RANKL (50 ng/ml) stimulation for the indicated time points. Total protein extracts (25 μg/lane) were separated by 10% SDS-PAGE followed by immunoblotting using specific antibodies against <b>A)</b> phospho-ERK 1/2, phospho-p38 and phospho-JNK and <b>B)</b> phospho-p65, phospho IκB or GAPDH. Membranes were stripped and reprobed with anti ERK1/2, anti-p38, anti-JNK, anti-p65 or anti-IκB antibodies, respectively. <i>Insets</i> show a representative example of four independent experiments. Effect of HG-9-91-01 pretreatment on <b>(C)</b> c-Fos and <b>(D)</b> NFATc1 expression. RAW264.7 cells were preincubated with vehicle (0.03% DMSO) or 0.5 μM of SIK inhibitor HG-9-91-01 for 30 min followed by RANKL (50 ng/ml) stimulation for 24h. mRNA expression levels of c-Fos and NFATc1 genes were determined by RT-qPCR using 18S rRNA as normalization control (n = 9). Total protein extracts (50 μg/lane) were separated by 10% SDS-PAGE followed by immunoblotting using specific antibodies (n = 6). <i>Insets</i> show a representative example depicting c-Fos, NFATc1 and GAPDH expression. c-Fos and NFATc1 were quantified relative to GAPDH. Data are presented as fold changes versus untreated cells (set as one) and expressed as mean +/- SD. * P<0.05, **P<0.01, significant vs. RANKL-stimulated control.</p

Oligonucleotides used for CISPR/Cas9 gene editing in murine RAW 264.7cells, in italics recognition sequence of BsmBI.

<p>Oligonucleotides used for CISPR/Cas9 gene editing in murine RAW 264.7cells, in italics recognition sequence of BsmBI.</p

Oligonucleotides used for real-time PCR analysis.

<p>Oligonucleotides used for real-time PCR analysis.</p

RANKL stimulation modulates the LKB1-SIK signaling axis in RAW cells.

<p>RAW264.7 cells were cultured in 1% serum for 16h, preincubated with vehicle (0.03% DMSO) or 0.5 μM of SIK inhibitor HG-9-91-01 for 30 min followed by RANKL (50 ng/ml) stimulation for the indicated time points. Total protein extracts (25 μg/lane) were separated by 10% SDS-PAGE followed by immunoblotting using specific antibodies against <b>A)</b> phospho-Ser428 LKB1 and <b>B)</b> phospho-Ser259 HDAC5. Membranes were stripped and reprobed with anti-LKB1 and GAPDH, respectively. <i>Insets</i> show a representative example of four independent experiments. <b>C)</b> Effect of HG-9-91-01 on RANKL-induced phosphorylation of HDAC5 and CRTC3. RAW264.7 cells were preincubated with vehicle (0.03% DMSO) or 0.5 μM inhibitor for 30 min followed by RANKL (50 ng/ml) stimulation for 90 min. Total protein extracts (25 μg/lane) were separated by 10% SDS-PAGE followed by immunoblotting using specific antibodies against phospho-Ser259 HDAC5, pSer370-CRTC3 and GAPDH. Membranes were stripped and reprobed with anti-HDAC5, anti-CRTC3, respectively. <i>Inset</i> shows a representative example of three independent experiments. <b>D)</b> Effect of HG-9-91-01 on cytosolic and nuclear redistribution of HDAC5 and CRTC3 upon RANKL stimulation. RAW264.7 cells were preincubated with vehicle (0.03% DMSO, control) or 0.5 μM HG-9-91-01 for 30 min followed by RANKL (50 ng/ml) stimulation for 90 min. Cytosolic and nuclear proteins extracts were prepared as detailed in <i>Material and Methods</i>. Fifty μg/lane were separated by 10% SDS-PAGE followed by immunoblotting using specific antibodies against phospho-Ser259 HDAC5 and phosphoSer370-CRTC3. Membranes were stripped and reprobed with anti-HDAC5 and CRTC3. Probing with LSD1 and GAPDH antibodies was used as qualitative control of the nuclear and cytosolic preparations, respectively. Data are presented as fold changes versus control cells (set as one) and represent the mean +/- SD. <i>Inset</i> shows a representative example of three independent experiments. * P<0.05, **P<0.01.</p

A77 1726 increases IL-1 receptor antagonist (IL-1Ra) secretion in human synovial fibroblasts and articular chondrocytes in the presence of IL-1β

<p><b>Copyright information:</b></p><p>Taken from "The active metabolite of leflunomide, A77 1726, increases the production of IL-1 receptor antagonist in human synovial fibroblasts and articular chondrocytes"</p><p>Arthritis Research & Therapy 2004;6(3):R181-R189.</p><p>Published online 19 Feb 2004</p><p>PMCID:PMC416438.</p><p>Copyright © 2004 Palmer et al., licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Human osteoarthritis (OA) synovial fibroblasts (passage 2) and de-differentiated human OA articular chondrocytes (passage 7) were stimulated (closed symbols) or not (open symbols) with 1 ng/ml IL-1β alone or in combination with 50 μmol/l or 100 μmol/l A77 1726 for 48 hours. Freshly isolated normal human articular chondrocytes were stimulated (black columns) or not (white columns) with 100 μmol/l A77 1726 alone or in combination with 1 ng/ml IL-1β for 48 hours. A77 1726 was added 2 hours before stimulation with IL-1β. IL-1Ra concentrations in culture supernatants were measured using ELISA. Results are expressed as means ± SEM for three determinations in a representative experiment. Similar results were obtained with cells from three different donors (three OA samples) for synovial fibroblasts and three different donors (three OA samples) for de-differentiated chondrocytes. On freshly isolated chondrocytes, a stimulatory effect of A77 1726 was observed in cells from 12 (five normal adult, six OA and one normal paediatric sample) out of 18 different donors. *< 0.05 versus control; < 0.05 versus IL-1β alone