TISSUE-SPECIFIC STEM CELLS 1. Department of Endocrinology and Metabolism, Endocrine Research Laboratory (KMEB), Odense University Hospital & University of Southern Denmark, Odense, Denmark; 2. Danish Stem Cell Center (DanStem), Institute of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark. * To whom correspondence should be addressed: Moustapha Kassem, Professor, Laboratory of molecular endocrinology (KMEB), Department of Endocrinology, University of Southern Denmark and University Hospital of Odense, J.B. Winsloesvej 25, 1st floor, DK5000, Odense C, Denmark. Tel: 4565504084, Fax: +66503920; E-mail: [email protected]; This work was supported by grants from University of Southern Denmark, local government of Southern Denmark, NovoNordisk foundation, Lundbeck foundation, Danish Arthritis association, and Simon Fougner Hartmanns family foundation Received August 11, 2014; accepted for publication February 13, 2015; available online without subscription through the open access option.
Pharmacological Inhibition of PRKG1 Enhances Bone Formation by Human Skeletal Stem Cells through Activation of RhoA-Akt Signaling ABBAS JAFARI1,2, MAJKEN S. SIERSBAEK1,2, LI CHEN1, DIYAKO QANIE1, WALID ZAHER1, BASEM M. ABDALLAH1, MOUSTAPHA KASSEM1,2* Key words. human skeletal (mesenchymal) stem cells • osteoblast differentiation • bone formation • Kinase inhibitor • Akt signalling
ABSTRACT Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways and pharmacological targeting of protein kinases has led to development of novel treatments for several malignant and non-malignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo osteoblast (OB) differentiation of hMSC, in a stage- and cell type-specific manner, without affecting adipogenesis or osteoclastogenesis. Furthermore, we showed that systemic administration of H-8 enhances in vivo bone formation by hMSC, using a preclinical ectopic bone formation model in mice. Using functional screening of known H-8 targets, we demonstrated that inhibition of Protein Kinase G1 (PRKG1) and consequent activation of RhoA-Akt signaling is the main mechanism through which H-8 enhances osteogenesis. Our studies revealed PRKG1 as a novel negative regulator of OB differentiation and suggest that pharmacological inhibition of PRKG1 in hMSC implanted at the site of bone defect can enhance bone regeneration. STEM CELLS 2015; 00:000–000
©AlphaMed Press 1066-5099/2015/$30.00/0 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/stem.2013
STEM CELLS 2015;00:00-00 www.StemCells.com
©AlphaMed Press 2015
2
INTRODUCTION Developing novel approaches for enhancing bone tissue regeneration is required for optimal treatment of a number of common clinical conditions such as repair of critical size bone defects following trauma, infection, or tumor resection [1]. Stem cell-based therapy is a promising new approach where local implantation of skeletal (also known as marrow stromal or mesenchymal) stem cells (MSC) together with functionalized scaffolds containing agents enhancing osteoblast (OB) differentiation; are carried out at sites of bone defects [2]. However, identification of agents that enhance OB differentiation of MSC and in vivo bone regeneration remains a challenge [1, 3]. Protein phosphorylation is known as the most common type of post-translational modification of proteins and it is estimated that around 30% of cellular proteins are phosphorylated on at least one residue [4, 5]. Around 518 protein kinases with a wide range of structures, functions, and subcellular localizations have been identified [6-8], making protein kinases one of the largest gene families comprising ~ 2% of the human genome [4]. Many kinases have been identified to regulate osteoblastic cell functions. Several growth factors with known significant effects on osteoblast differentiation and bone formation have cognate receptors with intrinsic kinase activity e.g. Bone morphogenetic proteins (BMPs) and insulin-like growth factor (IGF)-1. In addition, several kinases are known to regulate osteoblast functions through direct activation or inactivation of key osteoblastic transcription factors e.g. Runx2 [9], Osterix [10], and ATF4 [11]. A number of kinases have also been identified as activators or inhibitors of intracellular proteins that regulate important signaling pathways in osteoblast biology e.g. P300. Akt phosphorylates P300 at Ser-1834 and promotes its function as a coactivator of Runx2; the master regulator of osteogenesis [12-14], whereas phosphorylation of P300 at Ser-89 by protein kinase C inhibits its function [15]. Small molecule protein kinase inhibitors have been developed as novel drugs for treatment of malignant and non-malignant diseases [16-18]. The USA food and drug administration (FDA) has approved several kinase inhibitor drugs alleviating concerns about their safe use as therapeutic agents [17, 19, 20]. Employing small molecule protein kinase inhibitors for targeting human MSC and enhancing bone formation has not been previously explored. The aim of the current study was to identify small molecule kinase inhibitors that enhance differentiation of hMSC to osteoblastic cells and test their ability for enhancing in vivo bone formation. Thus, we screened a small molecule kinase inhibitor library containing 80 known small molecule protein kinase inhibitors that cover a wide spectrum of signaling pathways. Our data identified H-8 as a potent stimulator of in vitro osteo-
www.StemCells.com
Inhibition of PRKG1 enhances bone formation
blast differentiation and in vivo bone formation of hMSC.
MATERIALS AND METHODS Cell culture We employed a well-characterized immortalized hMSC cell line at low passage (hMSC-TERT4) that is generated by overexpressing human telomerase reverse transcriptase gene [21, 22]. For simplicity, hMSC-TERT4 cells will hereafter be referred to as hMSC. Primary hMSC cultures were established from bone marrow aspirates and adipose tissue of different healthy donors as described [23]. The cells were cultured in a standard growth medium containing minimal essential medium (MEM) (Gibco, USA) supplemented with 10% batch-tested FBS (South America origin) (Gibco, UK) and 1% penicillin/streptomycin (Gibco, USA). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Osteoblast differentiation of Hmsc For osteogenic differentiation, cells were plated at a density of 18,000 cells/cm2 in 6-well plates in standard growth medium. At 70% confluence, the medium was replaced with osteoblastic induction medium (OIM) consisting of standard growth medium supplemented with 5 mM β-glycerophosphate (Calbiochem, Germany), 50 µg/mL L-ascorbic acid (Sigma, Denmark), 10 nM Dexamethasone (Sigma, Denmark), and 10 nM 1,25dihydroxy vitamin D3 (LEO pharma, Denmark).
Alkaline phosphatase (ALP) activity assay ALP activity was measured by using p-nitrophenyl phosphate (Fluka, UK) as substrate and normalization to cell viability was used to correct for differences in cell number, as described before [24]. Briefly, CellTiter-Blue reagent (Promega, USA) was added to culture medium, incubated at 37 °C for 1 h, and fluorescent intensity (560EX/590EM) was measured using FLUOstar Omega multimode microplate reader (BMG Labtech, Germany). Cells were then washed with Tris-buffered saline (TBS), fixed in 3.7% formaldehyde, 90% ethanol for 30 s at room temperature, incubated with substrate (1 mg/ml of p-nitrophenyl phosphate in 50 mM NaHCO3, pH 9.6, and 1mM MgCl2) at 37 °C for 20 min, and the absorbance measured at 405 nm, using FLUOstar Omega multimode microplate reader.
Alizarin red (AR-S) staining In vitro mineralization was assessed by performing alizarin red S (AR-S) staining, as described previously [25]. Briefly, cells were induced into osteoblast differentiation as described earlier for 14 days. Cells were then washed in PBS, fixed in 70% ethanol at -20 °C for 1 hour, rinsed in dH2O, and stained with 40 mM AR-S (Sigma©AlphaMed Press 2015
3 Aldrich, St. Louis, MO, USA), pH 4.2, for 10 minutes with rotation. Stained cultures were then rinsed twice with dH2O, followed by washing three times with PBS to reduce nonspecific staining. The amount of mineralized matrix (Bound stain) was quantified by elution of the Alizarin red stain, using 20 minutes incubation of the cultures in 10% (wt/vol) cetylpyridinium chloride solution on a shaker (100 RPM) at room temperature. The absorbance of the eluted dye was measured at 570 nM, using FLUOstar Omega multimode microplate reader.
Small molecule kinase inhibitors screening We screened a commercially available small molecule kinase inhibitor library (Screen-Well™ Kinase Inhibi® tor Library from Biomol international ). This library contains 80 known inhibitors and covers a wide variety of signaling pathways. hMSC cells were plated in 96 well plates (18,000 cells/cm2) in culture media. The day after, culture media was replaced with OIM. In order to determine the kinase inhibitors that have the potential to enhance the ALP activity, each kinase inhibitor was added individually to the OIM at 1 µM and 10 µM concentration. Media was renewed every 3rd day and 6 days after induction of differentiation, ALP activity was quantified as described before. In each 96 well plate, a non-induced sample, as well as the samples that had only the OIM or OIM plus the vehicle were included.
Adipocyte differentiation of hMSC For adipogenic differentiation of hMSC, cells were 3 2 plated at high densities (40 x 10 cells/cm ) in 6-well plates in standard growth medium. To induce adipocyte differentiation, one day after seeding the cells, the medium was changed to adipogenic inducing media (AIM) consisting of standard growth medium supplemented with 10% horse serum (Sigma, Denmark), 100 nM Dexamethasone (Sigma, Denmark), 450 μM 1-methyl-3isobutylxanthine( IBMX) (Sigma, Denmark), 1 μM Rosiglitazone (BRL49653) (Cayman Chemical, USA), and 3 μg/ml Insulin (Sigma, Denmark) [26-28]. The medium was changed every other day, and on day 15, cells were visualized for adipocytes by oil red O staining.
Oil Red O staining Oil Red O is a fat-soluble dye which is used for staining of neutral triglycerides and lipids. Adipogenic cultures of day 15 were fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with 3% isopropanol solution, and stained with Oil Red O (Sigma, USA) solution (25 mg Oil Red O dye, 5 ml of 100% isopropanol, and 3.35 ml of H2O) for 1 h at room temperature. The bound dye wad eluted by 100% isopropanol and its absorbance was measured at 490 nM, using FLUOstar Omega multimode microplate reader.
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Inhibition of PRKG1 enhances bone formation
Isolation and osteoclast differentiation of CD14+ mononuclear cells Osteoclast differentiation of human peripheral blood mononuclear cells (PBMC) was performed as described previously [29]. Briefly, human osteoclast precursors were isolated from the blood of healthy donors provided anonymously by the blood bank of Odense University Hospital. PBMC were first separated by centrifugation on Ficoll-Paque PLUS; then, CD14+ cells (monocytes) were isolated by magnetic cell sorting according to the manufacturer's instruction. Briefly, PBMCs were resuspended in PBS containing 2% FBS, incubated (15 min at 4°C) with biotinylated anti-human CD14 goat antibody (R&D Systems, UK), and then incubated with Magcellect streptavidin ferrofluid (R&D Systems, UK) (15 min at 4°C). A magnetic device was used to retain the tagged cells and negative cells were discarded by extensive washes in PBS containing 2% FBS. Sorted cells were cultured in culture medium; containing α-MEM supplemented with 10% FBS and 30 ng/ml rhM-CSF for 3 days at 37°C. For osteoclast differentiation, adherent monocytes were trypsinized and reseeded in 96 well plates (250,000 cells/well), in culture medium supplemented with 30 ng/ml rhM-CSF. The day after, media was replaced by osteoclast differentiation medium containing both 30 ng/ml rhM-CSF and 30 ng/ml rhRANKL with replacement of medium every second day. To monitor osteoclast differentiation, tartrate resistant acid phosphatase (TRAP) staining was performed 5 days after induction of osteoclast differentiation [29]. Briefly, cells were fixed with 4% formaldehyde and stained for TRAP using the Leukocyte Acid Phosphatase kit (Sigma, USA) according to the manufacturer’s protocol. TRAP-positive multinucleated cells (MNCs) with more than four nuclei were scored as osteoclasts (OC).
Total RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated using TRIzol according to the manufacturer’s instructions. First-strand complementary cDNA was synthesized using a revertAid H minus first-strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. qPCR was performed using the StepOnePlus qPCR system and Fast SYBR® Green master mix as a double strand DNA–specific binding dye. The comparative threshold cycle (CT) between target genes and the reference genes was used to measure the expression level of each target gene using the formula (1/(2ΔCT)) in which ΔCT is the difference between the CT value of the target gene and the CT value of the reference genes. Following MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [30], two reference genes; β2m and TBP were used for normalization of RT-qPCR data. Supplementary table 1 shows the primers used for RT-qPCR. ©AlphaMed Press 2015
4
Inhibition of PRKG1 enhances bone formation
Western blot analysis of proteins
KINOMEscanTM kinase assay
Cells were lysed using RIPA buffer (Sigma, USA) containing phosphatase inhibitor cocktail (Sigma, USA) and protease inhibitor cocktail (Sigma, USA). Cell lysates were centrifuged at 12000 g for 10 minutes at 4 °C. Total protein concentrations were measured using Bradford assay (Thermo Fisher Scientific, USA) and equal amount of protein was loaded on a 10% polyacrylamide gel (Invitrogen, USA). Blotted PVDF membranes were incubated overnight at 4°C with antibodies against PAkt (Ser473, cell signaling, USA), P-RhoA (Ser188, Abcam, USA), p-mTOR (Ser2448, cell signaling, USA), and Actin (Sigma, USA). Membranes were incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, USA) for 45 min at room temperature, and protein bands were visualized using Amersham ECL chemiluminescence detection system (GE Healthcare Bio-Sciences Corp, USA). Western blot band intensities were measured by image J and presented as relative to the control. All antibodies were used at a 1:1000 dilution in 5% blotting grade milk solution prepared in PBST (PBS supplemented with 0.1% Tween® 20).
Inhibition of PRKG1 by H-8 was determined using KINOMEscanTM kinase assay, performed at the LeadHunter™ Discovery Services (DiscoveRx Corporation, USA). KINOMEscanTM is a novel and proprietary active site-directed competition binding assay that directly and quantitatively measures the interactions between test compounds and kinases, by determining binding of the small molecule kinase inhibitors to the kinase ATP binding site [36]. KINOMEscan™ assay do not require ATP and thereby report true thermodynamic interaction affinities, as opposed to IC50 values, which can depend on the ATP concentration. In addition, the assay has a wide dynamic range and can measure bindings at concentrations as low as 1–10 pM. TM More information about KINOMEscan assay can be found at www.discoverx.com.
In vivo bone regeneration assays in immunodeficient mice
For ectopic bone formation assay, hMSC (5 x 105) were mixed with hydroxyapatite-tricalcium phosphate ceramic powder (HA-TCP, 40 mg; Zimmer Scandinavia, Denmark) and transplanted subcutaneously into the dorsal surface of 2-month-old female NOD/SCID mice (NOD/LtSz- Prkdcscid), as described previously [25, 31]. The implants were removed after 8 weeks and transferred to 4% neutral buffered formalin for 24 hours; afterwards, formic acid was added for 3 days. Using standard histopathologic methods, the HA-TCP implants were embedded in paraffin, and tissue sections (4 mm thick) were cut and stained with hematoxylin and eosin. The total bone volume per total volume was quantified as described previously [25, 31]. Healos® is an osteoconductive carrier that is composed of cross-linked type I collagen fibers fully coated with hydroxyapatite and has been employed in clinical trials before [32, 33]. For critical-size calvarial defect model, hMSC (15000/cm2) were seeded on Healos® scaffolds (DePuy Spine, Inc.) and treated with vehicle or H-8 (25 µM) for 5 days, before implantation into mouse calvarial defects. Calvarial defects (3-mm) were created in the right and left parietal of 2-month-old female NOD/SCID mice, using a biopsy punch, as described previously [34]. In each mouse, one defect was implanted with vehicle-treated and the other with H-8 treated cells. Survival and localization of the implanted hMSC were evaluated using bioluminescent imaging of the animals as described before [35]. To evaluate bone formation, microcomputed tomographical (μCT) scanning images were obtained from mice at 1, 4, and 6 weeks after the surgery, using a VivaCT40 scanner (Scanco Medical AG, Bassersdorf, Switzerland).
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G-LISA RhoA activation assay RhoA activity was determined using the active RhoA colorimetric ELISA assay (Cytoskeleton Inc., Sweden) according to the manufacturer’s instructions. Briefly, cells were serum starved for 24 hours at 70% confluence and then stimulated with either vehicle or H-8 (10 µM) for 15 minutes. Cells were then lysed using the provided lysis buffer (Cytoskeleton Inc., Sweden) supplemented with 0.001% protease inhibitor cocktail (Cytoskeleton Inc., Sweden). Protein concentrations were determined using Precision Red Advanced Protein Assay Reagent (Cytoskeleton Inc., Sweden). Lysate concentrations were equilibrated to 2 mg/ml. The amount of active RhoA was determined using the absorbance based G-LISA RhoA activation assay kit (Cytoskeleton Inc., Sweden). In this assay, the plate wells are coated with a Rho-GTP binding protein that binds active (GTP-bound) RhoA. 50 µL of cell lysate and blank were pipetted, in triplicate, into wells coated with a Rho-GTP binding protein and active RhoA levels were determined using incubation with the provided anti-RhoA primary antibody and the horseradish peroxidase (HRP) conjugated secondary antibody, and measurement of absorbance (490 nm) using FLUOstar Omega multimode microplate reader.
siRNA transfections For siRNA transfections, we used the non-targeting control siRNA#1 and #2 (Ambion, USA) as negative control. All siRNAs were Silencer Select® siRNA (Ambion, USA) that are chemically modified with locked nucleic acid (LNA) residues that results in higher stability, less off-target effects, and less immune-stimulatory effects. Supplementary table 2 shows the sequence of the targeting siRNAs. We employed a reverse transfection protocol using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, USA). Briefly, 70% confluent hMSC cultures were trypsinyzed and 18000 cells/cm2 were reverse-transfected with siRNAs (25 nM) using ©AlphaMed Press 2015
5 MEM supplemented with 10% FBS, and after 8 hours, the transfection media was replaced with normal culture media (CM). Two days after transfection, osteogenic induction media (OIM) was added to hMSC cultures. For the screening study, each of the 3 independent siRNAs against each target were reverse-transfected separately, and changes in OB differentiation were determined using ALP activity quantitation on day 6 of OB differentiation. The screening experiment was performed twice and the average ALP activity of the 3 independent siRNAs from two biological replicates was used for the analysis. To avoid selection of a false positive hit and to ensure the specificity of the effects observed by PRKG1 siRNAs, we performed follow up studies using a new siRNA targeting a different region of PRKG1 mRNA, compared to the siRNAs used in the screening study. RT-qPCR analysis of PRKG1 expression on days2, 6, and 12 after siRNA transfection was used to determine the knockdown efficiency. Changes in OB differentiation and mineralization were determined using ALP activity quantitation (day 6 of OB differentiation), alizarin red staining (day 12 of OB differentiation), and RT-qPCR analysis of OB marker gene expression (day 4 of OB differentiation).
Statistical analysis Data are represented as mean ± standard deviation of at least 3 independent experiments with at least 3 replicates for each biological replicate, unless otherwise stated. Differences between variables were calculated using standard two-tailed unpaired student t-tests. p
Pharmacological Inhibition of PRKG1 Enhances Bone Formation by Human Skeletal Stem Cells through Activation of RhoA-Akt Signaling ABBAS JAFARI1,2, MAJKEN S. SIERSBAEK1,2, LI CHEN1, DIYAKO QANIE1, WALID ZAHER1, BASEM M. ABDALLAH1, MOUSTAPHA KASSEM1,2* Key words. human skeletal (mesenchymal) stem cells • osteoblast differentiation • bone formation • Kinase inhibitor • Akt signalling
ABSTRACT Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways and pharmacological targeting of protein kinases has led to development of novel treatments for several malignant and non-malignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo osteoblast (OB) differentiation of hMSC, in a stage- and cell type-specific manner, without affecting adipogenesis or osteoclastogenesis. Furthermore, we showed that systemic administration of H-8 enhances in vivo bone formation by hMSC, using a preclinical ectopic bone formation model in mice. Using functional screening of known H-8 targets, we demonstrated that inhibition of Protein Kinase G1 (PRKG1) and consequent activation of RhoA-Akt signaling is the main mechanism through which H-8 enhances osteogenesis. Our studies revealed PRKG1 as a novel negative regulator of OB differentiation and suggest that pharmacological inhibition of PRKG1 in hMSC implanted at the site of bone defect can enhance bone regeneration. STEM CELLS 2015; 00:000–000
©AlphaMed Press 1066-5099/2015/$30.00/0 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/stem.2013
STEM CELLS 2015;00:00-00 www.StemCells.com
©AlphaMed Press 2015
2
INTRODUCTION Developing novel approaches for enhancing bone tissue regeneration is required for optimal treatment of a number of common clinical conditions such as repair of critical size bone defects following trauma, infection, or tumor resection [1]. Stem cell-based therapy is a promising new approach where local implantation of skeletal (also known as marrow stromal or mesenchymal) stem cells (MSC) together with functionalized scaffolds containing agents enhancing osteoblast (OB) differentiation; are carried out at sites of bone defects [2]. However, identification of agents that enhance OB differentiation of MSC and in vivo bone regeneration remains a challenge [1, 3]. Protein phosphorylation is known as the most common type of post-translational modification of proteins and it is estimated that around 30% of cellular proteins are phosphorylated on at least one residue [4, 5]. Around 518 protein kinases with a wide range of structures, functions, and subcellular localizations have been identified [6-8], making protein kinases one of the largest gene families comprising ~ 2% of the human genome [4]. Many kinases have been identified to regulate osteoblastic cell functions. Several growth factors with known significant effects on osteoblast differentiation and bone formation have cognate receptors with intrinsic kinase activity e.g. Bone morphogenetic proteins (BMPs) and insulin-like growth factor (IGF)-1. In addition, several kinases are known to regulate osteoblast functions through direct activation or inactivation of key osteoblastic transcription factors e.g. Runx2 [9], Osterix [10], and ATF4 [11]. A number of kinases have also been identified as activators or inhibitors of intracellular proteins that regulate important signaling pathways in osteoblast biology e.g. P300. Akt phosphorylates P300 at Ser-1834 and promotes its function as a coactivator of Runx2; the master regulator of osteogenesis [12-14], whereas phosphorylation of P300 at Ser-89 by protein kinase C inhibits its function [15]. Small molecule protein kinase inhibitors have been developed as novel drugs for treatment of malignant and non-malignant diseases [16-18]. The USA food and drug administration (FDA) has approved several kinase inhibitor drugs alleviating concerns about their safe use as therapeutic agents [17, 19, 20]. Employing small molecule protein kinase inhibitors for targeting human MSC and enhancing bone formation has not been previously explored. The aim of the current study was to identify small molecule kinase inhibitors that enhance differentiation of hMSC to osteoblastic cells and test their ability for enhancing in vivo bone formation. Thus, we screened a small molecule kinase inhibitor library containing 80 known small molecule protein kinase inhibitors that cover a wide spectrum of signaling pathways. Our data identified H-8 as a potent stimulator of in vitro osteo-
www.StemCells.com
Inhibition of PRKG1 enhances bone formation
blast differentiation and in vivo bone formation of hMSC.
MATERIALS AND METHODS Cell culture We employed a well-characterized immortalized hMSC cell line at low passage (hMSC-TERT4) that is generated by overexpressing human telomerase reverse transcriptase gene [21, 22]. For simplicity, hMSC-TERT4 cells will hereafter be referred to as hMSC. Primary hMSC cultures were established from bone marrow aspirates and adipose tissue of different healthy donors as described [23]. The cells were cultured in a standard growth medium containing minimal essential medium (MEM) (Gibco, USA) supplemented with 10% batch-tested FBS (South America origin) (Gibco, UK) and 1% penicillin/streptomycin (Gibco, USA). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Osteoblast differentiation of Hmsc For osteogenic differentiation, cells were plated at a density of 18,000 cells/cm2 in 6-well plates in standard growth medium. At 70% confluence, the medium was replaced with osteoblastic induction medium (OIM) consisting of standard growth medium supplemented with 5 mM β-glycerophosphate (Calbiochem, Germany), 50 µg/mL L-ascorbic acid (Sigma, Denmark), 10 nM Dexamethasone (Sigma, Denmark), and 10 nM 1,25dihydroxy vitamin D3 (LEO pharma, Denmark).
Alkaline phosphatase (ALP) activity assay ALP activity was measured by using p-nitrophenyl phosphate (Fluka, UK) as substrate and normalization to cell viability was used to correct for differences in cell number, as described before [24]. Briefly, CellTiter-Blue reagent (Promega, USA) was added to culture medium, incubated at 37 °C for 1 h, and fluorescent intensity (560EX/590EM) was measured using FLUOstar Omega multimode microplate reader (BMG Labtech, Germany). Cells were then washed with Tris-buffered saline (TBS), fixed in 3.7% formaldehyde, 90% ethanol for 30 s at room temperature, incubated with substrate (1 mg/ml of p-nitrophenyl phosphate in 50 mM NaHCO3, pH 9.6, and 1mM MgCl2) at 37 °C for 20 min, and the absorbance measured at 405 nm, using FLUOstar Omega multimode microplate reader.
Alizarin red (AR-S) staining In vitro mineralization was assessed by performing alizarin red S (AR-S) staining, as described previously [25]. Briefly, cells were induced into osteoblast differentiation as described earlier for 14 days. Cells were then washed in PBS, fixed in 70% ethanol at -20 °C for 1 hour, rinsed in dH2O, and stained with 40 mM AR-S (Sigma©AlphaMed Press 2015
3 Aldrich, St. Louis, MO, USA), pH 4.2, for 10 minutes with rotation. Stained cultures were then rinsed twice with dH2O, followed by washing three times with PBS to reduce nonspecific staining. The amount of mineralized matrix (Bound stain) was quantified by elution of the Alizarin red stain, using 20 minutes incubation of the cultures in 10% (wt/vol) cetylpyridinium chloride solution on a shaker (100 RPM) at room temperature. The absorbance of the eluted dye was measured at 570 nM, using FLUOstar Omega multimode microplate reader.
Small molecule kinase inhibitors screening We screened a commercially available small molecule kinase inhibitor library (Screen-Well™ Kinase Inhibi® tor Library from Biomol international ). This library contains 80 known inhibitors and covers a wide variety of signaling pathways. hMSC cells were plated in 96 well plates (18,000 cells/cm2) in culture media. The day after, culture media was replaced with OIM. In order to determine the kinase inhibitors that have the potential to enhance the ALP activity, each kinase inhibitor was added individually to the OIM at 1 µM and 10 µM concentration. Media was renewed every 3rd day and 6 days after induction of differentiation, ALP activity was quantified as described before. In each 96 well plate, a non-induced sample, as well as the samples that had only the OIM or OIM plus the vehicle were included.
Adipocyte differentiation of hMSC For adipogenic differentiation of hMSC, cells were 3 2 plated at high densities (40 x 10 cells/cm ) in 6-well plates in standard growth medium. To induce adipocyte differentiation, one day after seeding the cells, the medium was changed to adipogenic inducing media (AIM) consisting of standard growth medium supplemented with 10% horse serum (Sigma, Denmark), 100 nM Dexamethasone (Sigma, Denmark), 450 μM 1-methyl-3isobutylxanthine( IBMX) (Sigma, Denmark), 1 μM Rosiglitazone (BRL49653) (Cayman Chemical, USA), and 3 μg/ml Insulin (Sigma, Denmark) [26-28]. The medium was changed every other day, and on day 15, cells were visualized for adipocytes by oil red O staining.
Oil Red O staining Oil Red O is a fat-soluble dye which is used for staining of neutral triglycerides and lipids. Adipogenic cultures of day 15 were fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with 3% isopropanol solution, and stained with Oil Red O (Sigma, USA) solution (25 mg Oil Red O dye, 5 ml of 100% isopropanol, and 3.35 ml of H2O) for 1 h at room temperature. The bound dye wad eluted by 100% isopropanol and its absorbance was measured at 490 nM, using FLUOstar Omega multimode microplate reader.
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Inhibition of PRKG1 enhances bone formation
Isolation and osteoclast differentiation of CD14+ mononuclear cells Osteoclast differentiation of human peripheral blood mononuclear cells (PBMC) was performed as described previously [29]. Briefly, human osteoclast precursors were isolated from the blood of healthy donors provided anonymously by the blood bank of Odense University Hospital. PBMC were first separated by centrifugation on Ficoll-Paque PLUS; then, CD14+ cells (monocytes) were isolated by magnetic cell sorting according to the manufacturer's instruction. Briefly, PBMCs were resuspended in PBS containing 2% FBS, incubated (15 min at 4°C) with biotinylated anti-human CD14 goat antibody (R&D Systems, UK), and then incubated with Magcellect streptavidin ferrofluid (R&D Systems, UK) (15 min at 4°C). A magnetic device was used to retain the tagged cells and negative cells were discarded by extensive washes in PBS containing 2% FBS. Sorted cells were cultured in culture medium; containing α-MEM supplemented with 10% FBS and 30 ng/ml rhM-CSF for 3 days at 37°C. For osteoclast differentiation, adherent monocytes were trypsinized and reseeded in 96 well plates (250,000 cells/well), in culture medium supplemented with 30 ng/ml rhM-CSF. The day after, media was replaced by osteoclast differentiation medium containing both 30 ng/ml rhM-CSF and 30 ng/ml rhRANKL with replacement of medium every second day. To monitor osteoclast differentiation, tartrate resistant acid phosphatase (TRAP) staining was performed 5 days after induction of osteoclast differentiation [29]. Briefly, cells were fixed with 4% formaldehyde and stained for TRAP using the Leukocyte Acid Phosphatase kit (Sigma, USA) according to the manufacturer’s protocol. TRAP-positive multinucleated cells (MNCs) with more than four nuclei were scored as osteoclasts (OC).
Total RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated using TRIzol according to the manufacturer’s instructions. First-strand complementary cDNA was synthesized using a revertAid H minus first-strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. qPCR was performed using the StepOnePlus qPCR system and Fast SYBR® Green master mix as a double strand DNA–specific binding dye. The comparative threshold cycle (CT) between target genes and the reference genes was used to measure the expression level of each target gene using the formula (1/(2ΔCT)) in which ΔCT is the difference between the CT value of the target gene and the CT value of the reference genes. Following MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [30], two reference genes; β2m and TBP were used for normalization of RT-qPCR data. Supplementary table 1 shows the primers used for RT-qPCR. ©AlphaMed Press 2015
4
Inhibition of PRKG1 enhances bone formation
Western blot analysis of proteins
KINOMEscanTM kinase assay
Cells were lysed using RIPA buffer (Sigma, USA) containing phosphatase inhibitor cocktail (Sigma, USA) and protease inhibitor cocktail (Sigma, USA). Cell lysates were centrifuged at 12000 g for 10 minutes at 4 °C. Total protein concentrations were measured using Bradford assay (Thermo Fisher Scientific, USA) and equal amount of protein was loaded on a 10% polyacrylamide gel (Invitrogen, USA). Blotted PVDF membranes were incubated overnight at 4°C with antibodies against PAkt (Ser473, cell signaling, USA), P-RhoA (Ser188, Abcam, USA), p-mTOR (Ser2448, cell signaling, USA), and Actin (Sigma, USA). Membranes were incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, USA) for 45 min at room temperature, and protein bands were visualized using Amersham ECL chemiluminescence detection system (GE Healthcare Bio-Sciences Corp, USA). Western blot band intensities were measured by image J and presented as relative to the control. All antibodies were used at a 1:1000 dilution in 5% blotting grade milk solution prepared in PBST (PBS supplemented with 0.1% Tween® 20).
Inhibition of PRKG1 by H-8 was determined using KINOMEscanTM kinase assay, performed at the LeadHunter™ Discovery Services (DiscoveRx Corporation, USA). KINOMEscanTM is a novel and proprietary active site-directed competition binding assay that directly and quantitatively measures the interactions between test compounds and kinases, by determining binding of the small molecule kinase inhibitors to the kinase ATP binding site [36]. KINOMEscan™ assay do not require ATP and thereby report true thermodynamic interaction affinities, as opposed to IC50 values, which can depend on the ATP concentration. In addition, the assay has a wide dynamic range and can measure bindings at concentrations as low as 1–10 pM. TM More information about KINOMEscan assay can be found at www.discoverx.com.
In vivo bone regeneration assays in immunodeficient mice
For ectopic bone formation assay, hMSC (5 x 105) were mixed with hydroxyapatite-tricalcium phosphate ceramic powder (HA-TCP, 40 mg; Zimmer Scandinavia, Denmark) and transplanted subcutaneously into the dorsal surface of 2-month-old female NOD/SCID mice (NOD/LtSz- Prkdcscid), as described previously [25, 31]. The implants were removed after 8 weeks and transferred to 4% neutral buffered formalin for 24 hours; afterwards, formic acid was added for 3 days. Using standard histopathologic methods, the HA-TCP implants were embedded in paraffin, and tissue sections (4 mm thick) were cut and stained with hematoxylin and eosin. The total bone volume per total volume was quantified as described previously [25, 31]. Healos® is an osteoconductive carrier that is composed of cross-linked type I collagen fibers fully coated with hydroxyapatite and has been employed in clinical trials before [32, 33]. For critical-size calvarial defect model, hMSC (15000/cm2) were seeded on Healos® scaffolds (DePuy Spine, Inc.) and treated with vehicle or H-8 (25 µM) for 5 days, before implantation into mouse calvarial defects. Calvarial defects (3-mm) were created in the right and left parietal of 2-month-old female NOD/SCID mice, using a biopsy punch, as described previously [34]. In each mouse, one defect was implanted with vehicle-treated and the other with H-8 treated cells. Survival and localization of the implanted hMSC were evaluated using bioluminescent imaging of the animals as described before [35]. To evaluate bone formation, microcomputed tomographical (μCT) scanning images were obtained from mice at 1, 4, and 6 weeks after the surgery, using a VivaCT40 scanner (Scanco Medical AG, Bassersdorf, Switzerland).
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G-LISA RhoA activation assay RhoA activity was determined using the active RhoA colorimetric ELISA assay (Cytoskeleton Inc., Sweden) according to the manufacturer’s instructions. Briefly, cells were serum starved for 24 hours at 70% confluence and then stimulated with either vehicle or H-8 (10 µM) for 15 minutes. Cells were then lysed using the provided lysis buffer (Cytoskeleton Inc., Sweden) supplemented with 0.001% protease inhibitor cocktail (Cytoskeleton Inc., Sweden). Protein concentrations were determined using Precision Red Advanced Protein Assay Reagent (Cytoskeleton Inc., Sweden). Lysate concentrations were equilibrated to 2 mg/ml. The amount of active RhoA was determined using the absorbance based G-LISA RhoA activation assay kit (Cytoskeleton Inc., Sweden). In this assay, the plate wells are coated with a Rho-GTP binding protein that binds active (GTP-bound) RhoA. 50 µL of cell lysate and blank were pipetted, in triplicate, into wells coated with a Rho-GTP binding protein and active RhoA levels were determined using incubation with the provided anti-RhoA primary antibody and the horseradish peroxidase (HRP) conjugated secondary antibody, and measurement of absorbance (490 nm) using FLUOstar Omega multimode microplate reader.
siRNA transfections For siRNA transfections, we used the non-targeting control siRNA#1 and #2 (Ambion, USA) as negative control. All siRNAs were Silencer Select® siRNA (Ambion, USA) that are chemically modified with locked nucleic acid (LNA) residues that results in higher stability, less off-target effects, and less immune-stimulatory effects. Supplementary table 2 shows the sequence of the targeting siRNAs. We employed a reverse transfection protocol using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, USA). Briefly, 70% confluent hMSC cultures were trypsinyzed and 18000 cells/cm2 were reverse-transfected with siRNAs (25 nM) using ©AlphaMed Press 2015
5 MEM supplemented with 10% FBS, and after 8 hours, the transfection media was replaced with normal culture media (CM). Two days after transfection, osteogenic induction media (OIM) was added to hMSC cultures. For the screening study, each of the 3 independent siRNAs against each target were reverse-transfected separately, and changes in OB differentiation were determined using ALP activity quantitation on day 6 of OB differentiation. The screening experiment was performed twice and the average ALP activity of the 3 independent siRNAs from two biological replicates was used for the analysis. To avoid selection of a false positive hit and to ensure the specificity of the effects observed by PRKG1 siRNAs, we performed follow up studies using a new siRNA targeting a different region of PRKG1 mRNA, compared to the siRNAs used in the screening study. RT-qPCR analysis of PRKG1 expression on days2, 6, and 12 after siRNA transfection was used to determine the knockdown efficiency. Changes in OB differentiation and mineralization were determined using ALP activity quantitation (day 6 of OB differentiation), alizarin red staining (day 12 of OB differentiation), and RT-qPCR analysis of OB marker gene expression (day 4 of OB differentiation).
Statistical analysis Data are represented as mean ± standard deviation of at least 3 independent experiments with at least 3 replicates for each biological replicate, unless otherwise stated. Differences between variables were calculated using standard two-tailed unpaired student t-tests. p
