Salt-inducible kinases (SIK) inhibition reduces RANKL-induced osteoclastogenesis

Osteoclasts are large multinucleated cells responsible for bone resorption. Excessive inflammatory activation of osteoclasts leads to bony erosions, which are the hallmark of several diseases such as rheumatoid arthritis (RA). Salt-inducible kinases (SIK) constitute a subfamily of kinases comprising three members (SIK1, -2, and -3). Inhibition of SIK kinase activity induces an anti-inflammatory phenotype in macrophages. Since osteoclasts originate from precursors of macrophage origin, we hypothesized a role of SIK in osteoclastogenesis. We analyzed SIK1, -2 and -3 expression and function in osteoclast differentiation using the mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMM). We show that all three SIK are expressed in fully differentiated osteoclasts and that in BMM-derived osteoclasts there is an increased expression of SIK1 and SIK3 proteins. Interestingly, the pan-SIK inhibitor HG-9-91-01 significantly inhibited osteoclastogenesis by dose dependently reducing osteoclast differentiation markers (i.e. CathepsinK, MMP-9 and TRAP) and bone resorbing activity. Analysis of the signaling pathways activated by RANKL in RAW cells showed that SIK inhibitors did not affect RANKL-induced ERK1/2, JNK, p38 or NF-κB activation, but induced a significant downregulation in c-Fos and NFATc1 protein levels, the two main transcription factors involved in the regulation of osteoclast-specific genes. Moreover, SIK inhibition partially increased the proteasome-mediated degradation of c-Fos. SIK2 and SIK3 knockout RAW cells were generated by the CRISPR/Cas9 approach. SIK2 KO and, to a lesser extent, SIK3 KO recapitulated the effect of SIK small molecule inhibitor, thus confirming the specificity of the effect of SIK inhibition on the reduction of osteoclastogenesis. Overall, our results support the notion that the SIK signaling pathway plays a significant role among the check-points controlling osteoclastogenesis. SIK kinase inhibitors could thus represent a potential novel therapy to prevent bone erosions

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

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

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

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

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

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