Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition–parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.


■ INTRODUCTION
Caspases are an evolutionary conserved family of cysteine proteases with well-defined functions in the regulation of cell death and inflammation. 1,2More recently, physiological and disease-related functions of various caspases unrelated to cell death execution and immune response have also been described.These include activities in the developing nervous system that affect synaptic plasticity, 3 axon/dendrite pruning, 4−6 their outgrowth 7 and branching, 8 stem cell activity (self-renewal, differentiation), and thus the regeneration of various tissues. 9−19 We and others have described nonapoptotic functions of caspases in osteogenesis and bone homeostasis as well.Treatment with the inhibitor of caspase-3 (CASP-3) accelerated bone loss in ovariectomized mice, 20 Bmp4-induced osteoblastic differentiation of MC3T3-E1 is associated with increased activity of caspases-2, -3, and -8, 21 and pharmacological and genetic inhibition of caspase-8 inhibited differentiation of these cells by reducing osteocalcin expression. 22,23−27 Caspase-9 (CASP-9) is classically considered the initiator of the intrinsic apoptotic cascade.Its activation occurs in the apoptosome, a protein complex formed in response to the permeabilization of outer mitochondrial membrane. 28,29Alternative CASP-9 activation pathways have also been described. 30esides its well-described role in apoptosis, other physiological functions of CASP-9 have been identified, including regulation of myocyte cell differentiation and proliferation, 31 development of olfactory sensory neurons (axonal projections, synapse formation, neuronal maturation), 32

Sample Preparation for Proteomics Analysis
Wt, GFP-control (further labeled as mock), and Casp9 KO MC3T3-E1 cells were seeded (7 × 10 5 ) in the growth medium in four biological replicates.The cells were collected after 48 h using a 1 mM EDTA/PBS solution and then lysed in a buffer containing 8 M urea and 0.5 M TEAB (triethylammonium bicarbonate) pH 8.5, sonicated (50 W, 30 × 0.1 s, 30 s pause, 30 × 0.1 s), and incubated on ice for 75 min.Lysates were further centrifuged at 14,000g and 4 °C for 20 min.Protein concentrations in sample supernatants were determined using a RC-DC protein assay kit (Bio-Rad, USA).

Protein Digestion
Protein digestion was performed using the Filter-Aided Sample Preparation (FASP) method.50 μg of protein per sample was transferred to the Microcon filter device, 30 kDa cutoff (Millipore, Germany) containing 200 μL of 8 M urea dissolved in 0.5 M TEAB, pH 8.5.Samples were centrifuged at 14,000g and 20 °C for 15 min.100 μL of 8 M urea and 10 μL of 50 mM tris (2-carboxyethyl) phosphine were added to the filter, and samples were reduced on a thermomixer at 600 rpm and 37 °C for 60 min and centrifuged at 14,000g and 20 °C for 15 min.In the next step, 100 μL of 8 M urea and 5 μL of 200 mM methylmethanethiosulfonate were added to the samples.The samples were alkylated on a thermomixer at 600 rpm and 25 °C for 1 min, stored without stirring in the dark for 20 min, and centrifuged at 14,000g and 20 °C for 15 min.Subsequently, 100 μL of 0.5 M TEAB was added to the filter, and samples were centrifuged at 14,000g and 20 °C for 20 min.The previous step was repeated once.Enzymatic digestion of proteins was initiated by addition of 100 μL of 0.5 M TEAB and 1.67 μL of 1 μg/μL trypsin solution (Promega, USA) dissolved in 50 mM acetic acid (trypsin:cleaved protein ratio was 1:30).The samples were mixed on a thermomixer at 600 rpm and 37 °C for 1 min and digested overnight at 37 °C without shaking.The next day, peptides were eluted by centrifugation at 14,000g and 20 °C for 15 min.

Peptide Desalting
C18 Silica MicroSpin columns (NestGroup Inc., USA) were used to desalt the peptides prior to mass spectrometry (MS) analysis.The columns were washed twice with 200 μL of 0.1% trifluoracetic acid (TFA) in acetonitrile and centrifuged at 100g and RT for 3 min, which was followed by two washes with 200 μL of 0.1% TFA in water and centrifuged at 300g and RT for 3 min.Columns were left to hydrate for 15 min at RT and centrifuged at 300g and RT for 3 min.Peptide samples were added to the columns and centrifuged at 500g and RT for 3 min.Then, the columns were washed three times with 200 μL of 0.1% TFA in water and centrifuged at 500g and RT for 3 min.The elution was performed by the addition of 200 μL of 0.1% TFA in 50% acetonitrile and centrifugation at 500g and RT for 3 min, which was followed by 200 μL of 0.1% TFA in 80% acetonitrile and centrifugation under the same conditions and the addition of 200 μL of 0.1% TFA in 100% acetonitrile and centrifugation at 500g and RT for 3 min.Eluates were lyophilized in a SpeedVac and stored at −20 °C.

LC-MS/MS Identification of Peptides in DIA Mode
LC-MS/MS analyses of all peptides were done using nanoElute system (Bruker, USA) connected to a timsTOF Pro spectrometer (Bruker, USA).One column (no trapping column; separation column: Aurora C18, 75 μm ID, 250 mm long, 1.6 μm particles; Ion Opticks, Australia) mode was used on a nanoElute system with default equilibration and sample loading conditions (separation column equilibration: 4 column volumes at 800 bar; sample loading at 800 bar using 2× pick up volume + 2 μL).Concentrated peptides were eluted by a 120 min linear gradient program (flow rate 300 nL/min, 3−30% of mobile phase B; mobile phase A, 0.1% FA in water; mobile phase B, 0.1% FA in acetonitrile) followed by a system wash step at 80% mobile phase B. The analytical column was placed inside the Column Toaster (40 °C; Bruker, USA) and its emitter side was installed into CaptiveSpray ion source (Bruker, USA).
MSn data were acquired using the data-independent acquisition−parallel accumulation serial fragmentation (diaPA-SEF) approach with a base method m/z range of 100−1700 and 1/k0 range of 0.6−1.6V × s × cm −2 .The Supplementary Data 1 file defines the m/z 400−1100 precursor range with equal windows size of 26 Th (including 1 Th overlaps) using two steps each PASEF scan and a cycle time of 100 ms locked to 100% duty cycle.

Processing of LC-MS/MS Data
Quantitative analysis of the LC-MS/MS DIA data was performed in Spectronaut 15.1 (Biognosys, Switzerland) software using the directDIA approach against the Mus musculus UniProt/SwissProt database (2021_03, 17,519 sequences, downloaded on 7/29/2021).Precursor q-value cutoff and experiment protein q-value cutoff were set to 0.01.Peptides identified with q-value < 0.01 in at least 4 of 16 analyses were Journal of Proteome Research included (q-value percentile 0.25 setting).Fixed modifications were set to Methylthio (C), and variable modifications were set to Acetyl (Protein N-term) and Oxidation (M).Other parameters were set as default.Differential abundance testing was performed using Student's t test in Spectronaut 15.1; proteins with absolute log2 fold change (|log2FC|) > 0.58 and with q-value < 0.05 were considered differentially abundant between the sample groups.An ANOVA test and the visualization of ANOVA significant proteins in a heatmap were performed using Perseus software 38 version 2.0.11.0.

Gene Set Enrichment Analysis
GSEA analysis was performed using the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt). 39,40This analysis included all identified proteins sorted by ranking metrics computed as negative log2 of the q-value with the sign of the log2-fold change for each comparison.The organism of interest was set to Mus musculus, and the method of interest to GSEA.Analysis was performed against the Gene Ontology Biological Process (GO BP) database with minimum number of genes for a category set to 3 and with FDR significance level 0.05.The results were visualized in R Statistical Software version 4.3.1 using the ggplot2 package 41 version 3.4.4.The Venn diagram was created using the Venny 2.1 tool. 42

Enrichment Analysis of Molecular Pathways
Sets of genes encoding proteins that were either statistically (qvalue < 0.05) significantly upregulated (log2FC > 0.58) or downregulated (log2FC < −0.58) in both clones against mock were separately submitted to pathway enrichment analysis using g:Profiler tool 43 that implements Fisher exact test and multipletest correction to evaluate pathway enrichment.Lists were added as an unordered query.A list of gene names of all proteins identified in our proteomics experiment was used as a landscape for statistical testing.The organism of interest was set to Mus musculus.Pathways from Gene Ontology Biological Process (GOBP), Gene Ontology Molecular Function (GOMF), and Gene Ontology Cellular Compartment (GOCC) databases were included.Electronic GO annotations were excluded.The minimal pathway size was set to 15, and the maximum was set to 1500.The results were visualized using the Cytoscape software (version 3.10.) 44with the use of EnrichmentMap application (version 3.3.6) 45with the FDR q-value cutoff 0.05 and Edge cutoff (Similarity) 0.375.

Cell Proliferation
3 × 10 4 of MC3T3-E1 wt, mock, and Casp9 KO cells were cultured in 6-well plates for 4 days.The cells were counted daily using a CASY cell counter (Roche).

Cell Migration
Two different methods were used to analyze the cell migration.First, the migration of control and Casp9 KO MC3T3-E1 cells was monitored using an xCELLigence instrument (Roche, Switzerland) as described previously. 36Briefly, CIM-plates 16 with complete growth medium (10% FBS) in the bottom chambers were assembled.Cells were serum starved for 2 h, detached with 1 mM EDTA/PBS, washed with PBS, counted, and plated in serum-free medium in the upper chambers in duplicates at a density of 7.5 × 10 4 per well.Impedance (displayed as dimensionless parameter cell index) was monitored every 15 min for 8 h.Second, a scratch (wound healing) assay was used to monitor the migration of control and Casp9 KO MC3T3-E1 cells.The cells were seeded in a 24-well plate at a density of 3 × 10 4 per well.The cell monolayer was wounded with a sterile pipet tip 72 h after seeding.Subsequently, the fresh medium or medium supplemented with inhibitor/ DMSO as a control was added.The cells were photographed every 3 h for 9 or 12 h postwounding using an Olympus IX53 microscope (×40), and cell migration was analyzed by Fiji (NIH, USA) as changes in wound area (%).A wound healing assay was performed subsequently also with wt MC3T3-E1 cells treated with 100 μM CASP-9 inhibitor (218776, Sigma-Aldrich), 100 μM CASP-3/-7 inhibitor (218832, Sigma-Aldrich) or vehicle.

qRT-PCR
Total RNA was isolated using the GenElute Total RNA Purification Kit (Sigma-Aldrich, USA) and cDNA was isolated using the QuantiTect RT Kit (Qiagen, Germany).qPCR was performed with the KAPA SYBR Fast Master mix (KAPA Biosystems, USA) with primers spanning exon−exon junctions (Supplementary Data 2) using the LightCycler 480 (Roche, Switzerland).Mouse Gapdh was used as the internal control.The qRT-PCR data were analyzed by the ΔΔCt method.

Immunohistochemistry
Mouse front limbs and heads (CD1 mouse strain) were collected fresh post-mortem, and prenatal (E) stage E15 was examined.The samples were obtained in agreement with the recent legislation in the Czech Republic, law 359/2012 Sb., in which there is no specific requirement for post-mortem sampling.Histological sections were deparaffinized in xylene and rehydrated in a gradient series of ethanol.Consecutive sections were pretreated in citrate buffer (10 min/98 °C) for antigen retrieval and then incubated with ABHD2 antibody (14039-1-AP, Proteintech, Germany) or antibody specific to cleaved CASP-9 (9509, Cell Signaling Technology, USA) overnight.After treatment with primary antibodies, the samples were exposed to the secondary anti-rabbit antibody Alexa Fluor 488 (Thermo Fisher Scientific) for 40 min at RT. Nuclei were detected by a ProLong Gold Antifade reagent with DAPI (Thermo Fisher Scientific).

Statistics
Statistical analysis was performed with Prism v8.0.1 (GraphPad Software, La Jolla, CA).All experimental data are presented as mean ± SD and were analyzed with an unpaired t test unless stated otherwise.

Casp9 KO Affects the Proteotype of Osteoblastic Cells
To investigate the function of CASP-9 in osteoblastic cells, two independent MC3T3-E1 Casp9 KO clones (A6 and B1) were generated using the CRISPR/Cas9 approach.The absence of the CASP-9 protein was confirmed by immunoblotting (Figure 1A) and the presence of a short Ins/Del within the Casp9 gene was validated by DNA sequencing.Depletion of CASP-9 did not alter cell morphology, as shown in Figure 1B.Next, to identify proteins associated with Casp9 deficiency in MC3T3-E1 cells, proteome changes in wt, mock, and both Casp9 KO clones were evaluated in four biological replicates.Proteins were identified and quantified using LC-MS/MS analysis in the diaPASEF mode.

CASP-9 is Associated with Pathways of Cellular Migration and Adhesion
To describe changes in protein abundances after Casp9 KO on a proteotype-wide level, GSEA analysis was performed against the Gene Ontology Biological Process database using the WebGestalt tool. 39Casp9 KO in A6 and B1 clones compared  to mock cells was associated with statistically significant (FDR qvalue < 0.05) positive enrichment (normalized enrichment score (NES) > 0) of 71 and 91 GO BP pathways, respectively, and negative enrichment (FDR q-value < 0.05, NES < 0) of 20 and 12 GO BP pathways, respectively (Figure 2A,B, Supplementary Data 9).In total, 30 and 6 GO BP pathways were positively and negatively enriched, respectively, in both clones compared to mock cells (Figure 2C, Supplementary Data 10).The positively enriched pathways in both clones were frequently associated with cytoskeletal organization, morphogenesis, adhesion, and locomotion.On the other hand, DNA replication, recombination and repair were found in negatively enriched pathways.As a negative control for the Casp9 KO clones, we compared the proteotypes of wt cells to the mock cells and performed GSEA to define GO BP pathways associated with transfection using the control plasmid.In this comparison, no GO BP pathways were positively enriched, and a total of 35 pathways were negatively enriched (Supplementary Data 9).None of the pathways were negatively enriched in A6 and B1 clones compared with mock cells.These results suggest that the deregulated mechanisms observed in A6 and B1 clones are specific to cells with silenced Casp9 gene and depend on CASP-9 function.
Next, an enrichment analysis of Gene Ontology pathways, including Biological Processes (GOBP), Molecular Function (GOMF), and Cellular Compartment (GOCC) terms, was performed using the g:Profiler tool 43 to define biological pathways consisting of proteins strictly up-or downregulated by Casp9 KO.These analyses included lists of 283 significantly upregulated or 141 significantly downregulated proteins in both clones simultaneously compared to the mock cell line.Enriched pathways among the upregulated proteins included 8 GOBP and 1 GOCC terms (Figure 3, Supplementary Data 11).These include regulation of cell migration and motility, cell adhesion, and proteins located on the plasma membrane.On the other hand, downregulated proteins are involved in 3 GOBP pathways that participate in DNA replication (Figure 3, Supplementary Data 11).
The pathways associated with cellular migration enriched in GSEA and g:Profiler analyses included 9 negative regulators of cellular migration that were upregulated in both clones compared to mock and wt cell lines (Table 1).

CASP-9 Regulates Migration but Not Proliferation of MC3T3-E1 Cells
Proteomic data analysis suggested that CASP-9 may target proteins involved in regulating cell proliferation and migration of MC3T3-E1 cells.Proliferation analysis revealed no difference in the growth rate of Casp9 KO cells compared to parental and mock cells (Figure 4A).However, the ability of Casp9 KO cells to migrate was reduced compared to parental and mock cells in both wound healing and transwell/xCELLigence assays (Figure 4B,C).To further confirm the involvement of CASP-9 in regulating the migration of MC3T3-E1 cells, cells were treated with a CASP-9 inhibitor or vehicle, and their migration was analyzed using a wound healing assay.Again, inhibition of CASP-9 enzymatic activity resulted in the reduced migration of MC3T3-E1 cells (Figure 4D).Interestingly, treatment with CASP-3/-7 inhibitor did not affect the migration of MC3T3-E1 cells, suggesting that the migration-promoting role of CASP-9 is not dependent on the activity of downstream caspases (Figure 4E).

ABHD2 Protein: Possible Substrate of CASP-9
Proteomic analysis revealed 9 possible substrates of CASP-9 that are upregulated in Casp9 KO cells and were considered negative regulators of cell motility by g:Profiler Gene Ontology pathway analysis (Table 1).After a literature search and screening of available databases, 48,49 these proteins have not been identified as CASP-9 substrates.Interestingly, four of these proteins (ADAM15, fibulin-1, BST-2, and ABHD2) were found previously to be upregulated in micromass cultures treated with CASP-9 inhibitor by proteomic screen. 47Therefore, we further focused our attention on these four proteins.BST-2 was not detected by immunoblotting (Supplementary Data 12) and fibulin-1 has been recently identified as a substrate of CASP-3, 50 a downstream molecule of CASP-9 in proteolytic cascade, so these two proteins were excluded from further analyses.Increased levels of ABHD2 and ADAM15 proteins were confirmed in both Casp9 KO clones (Figure 5A).Subsequent qRT-PCR analysis revealed no significant differences in Abhd2 and Adam15 expression between the control and Casp9 KO cells, suggesting that their deregulation occurs at the protein level (Figure 5B).
To confirm the role of CASP-9 in the regulation of ABHD2 and ADAM15, MC3T3-E1 cells were treated with a CASP-9 inhibitor.Subsequent immunoblotting analysis revealed that the ABHD2 protein level increased after CASP-9 inhibition but the ADAM15 protein level remained unchanged (Figure 6).Interestingly, neither ABHD2 nor ADAM15 protein levels were altered by treatment with CASP-3/-7 inhibitor (Figure 6), suggesting that downstream caspases are not involved in the regulation/cleavage of these proteins.
ABHD2 is a widely expressed protein known primarily for its function in sperm activation via progesterone signaling. 51,52owever, its function and expression in osteoblasts during bone development have never been demonstrated.Therefore, to investigate the presence of ABHD2 in osteoblasts in vivo and to analyze the colocalization of ABHD2 with cleaved CASP-9, consecutive sections of mouse frontal limbs at prenatal stage E15 were examined by immunofluorescence.In all tested samples, a positive signal of ABHD2 was detected in osteoblasts, and the signal overlapped with that of active CASP-9 (Figure 7).These data confirm the in vivo relevance of the results obtained from the cell cultures.

■ DISCUSSION
Although caspases are known primarily for their role in various forms of cell death and inflammation, 1,2 recent studies have identified other physiological and pathophysiological functions of these proteases. 2,53,54This also applies for CASP-9 as well.We have previously observed the expression of active CASP-9 in nonapoptotic osteoblasts within the ossification zone of developing long bones. 55To the best of our knowledge, the functions of CASP-9 in osteoblasts, beyond the execution of apoptosis, have not been studied yet.We thus performed a proteomic screen to identify possible CASP-9 targets in MC3T3-E1 cells, an osteoblastic cell line derived from mouse calvaria, the common in vitro model for osteoblastic lineage.MC3T3-E1 cells with depleted CASP-9 were generated using the CRISPR/Cas9 approach, and their proteome was compared to the proteome of parental/mock-transfected cells.To map the changes in protein abundances associated with CASP-9 depletion, we used the diaPASEF approach that combines peptide separation using trapped ion mobility spectrometry and  precursor m/z window cycling. 56diaPASEF provides sensitive peptide detection and data completeness 56 and we have previously shown this strategy to achieve superior proteome coverage in chondrogenic micromass cultures compared to other commonly used LC-MS/MS-based proteomics workflows. 47In the current study, Casp9 KO significantly affected, namely, proteins associated with cell migration/motility and DNA replication.
Using two different assays, we confirmed that Casp9 KO results in impaired migration of the MC3T3-E1 cells.Cell migration was also inhibited by an inhibitor of CASP-9 enzymatic activity, suggesting that the proteolytic activity of CASP-9 plays a role.−64 Both enzymatic and nonenzymatic functions were involved.However, studies investigating the role of CASP-9 in the regulation of cell migration are rather limited.Inhibition of DRONC, the fly ortholog of CASP-9, affected border cell migration in Drosophila ovary. 65−70 Our proteomic screen identified proteins, described as negative regulators of cell migration, that were upregulated in Casp9 KO MC3T3-E1 cells and thus represent possible CASP-9 substrates.This study focused on two of them: ABHD2 and ADAM15.ABHD2 is a member of a family of α/β hydrolase fold domain proteins that mediate lipid metabolism and signal transduction.It is ubiquitously expressed protein known for its role in progesterone-mediated activation of sperm, regulation of calcium signaling, lung development and function, monocyte/ macrophage recruitment/differentiation/activity, regulation of viral replication, etc. 52,71 ABHD2 deficiency enhances migration of vascular smooth muscle cells, resulting in intimal hyperplasia in mice. 34However, the expression and function of ABHD2 in osteoblasts have never been determined.We found that the ABHD2 protein is expressed in osteoblasts of the developing mouse limb bones, its protein expression colocalizes with that of active CASP-9, and its level is regulated by CASP-9 in MC3T3-E1 osteoblasts.While genetic depletion or pharmacological inhibition of CASP-9 in MC3T3-E1 cells resulted in an increase of ABHD2 protein, activation of CASP-9 has an opposite effect.Moreover, inhibition of downstream CASP-3 has no effect on the ABHD2 protein, suggesting that this effector caspase is not involved.Thus, our results suggest that ABHD2 is a direct target of CASP-9, although we cannot exclude the possibility that other downstream caspases and/or proteases activated by CASP-9 may play a role.
ADAM15 is another protein identified as upregulated in the proteomic screen of Casp9 KO cells.Using immunoblotting, we detected a higher level of its 75 kDa form that has been reported to correspond to the mature form of this enzyme. 72,73However, treatment with CASP-9 inhibitor did not confirm the deregulation of ADAM15.We hypothesize that the alterations in the mature form of ADAM15 are caused by the rather longterm deregulation of CASP-9 in CRISPR clones.Consistent with this hypothesis, a proteomic screen of chondrogenic micromass cultures revealed higher ADAM15 levels after 7 days of treatment with CASP-9 inhibitor. 47In MC3T3-E1 cells, however, prolonged treatment with CASP-9 inhibitor significantly reduces their viability, making longer exposure time intervals difficult to achieve.We thus hypothesize that ADAM15 may not be a direct target of CASP-9 but may rather be deregulated or cleaved by other proteases in response to longterm CASP-9 inhibition.
Another upregulated protein in the proteomic screen of Casp9 KO cells is Bst-2.Bst-2 is a transmembrane protein with putative immunomodulatory functions. 74−77 To the best of our knowledge, no function of Bst-2 in bone homeostasis has been described.To confirm the data obtained by mass spectrometry, we analyzed the expression of Bst-2 in MC3T3-E1 control and Casp9 KO cells by immunoblotting using three different antibodies.However, in neither case were we able to obtain any reliable signal at a MW corresponding to the mouse Bst-2 protein (30−35 kDa).We are aware that the Bst-2 protein was detected using mass spectrometry, but the success of immunoblotting detection is highly dependent on the quality of the available antibodies.Although antibody detection may be more sensitive than mass spectrometry, the quality and specificity of antibodies are crucial.The question of Bst-2 as a possible substrate of CASP-9 and the physiological relevance of Bst-2 for bone homeostasis remain thus open.
Bone formation, especially during bone remodeling and fracture repair, requires mature osteoblasts to migrate to specific sites in the three-dimensional environment.Understanding this process is necessary as alterations in osteoblast migration and navigation might significantly affect bone development and metabolic bone diseases such as osteoporosis. 78Identification of mechanisms that regulate these processes is thus an important prerequisite for the design of targeted therapies.

■ CONCLUSIONS
We revealed CASP-9 as a modulator of osteoblastic cell migration, and using a proteomic screen, we identified its possible relevant targets.The ABHD2 protein, a known regulator of cell migration, was subsequently validated as a possible CASP-9 substrate using experiments with the genetic and pharmacological inhibition of this protease.These data may indicate a novel nonapoptotic function of CASP-9 in bone remodeling and fracture repair, as the regulation of osteoblastic cell migration is a key component of these physiological processes.

Figure 1 .
Figure 1.(A) Protein expression of CASP-9 in parental (wt), mock, and Casp9 KO clones of MC3T3-E1 cell line; α-tubulin was used as a loading control.(B) Morphology of wt, mock, and Casp9 KO clones of MC3T3-E1 cell line; phase contrast microscopy, total magnification ×40.(C) Heatmap of biological replicates and protein groups clustering of wt, mock, and Casp9 KO MC3T3-E1 clones according to the sample protein profile.(D) Volcano plot of differential protein abundance analysis between Casp9 KO A6 clone and mock cells.(E) Volcano plot of differential protein abundance analysis between Casp9 KO B1 clone and mock cells.

Figure 2 .
Figure 2. Top 10 significantly positively and negatively enriched GO Biological Process pathways in the WebGestalt GSEA analysis: (A) of the A6 clone proteotype compared to the control mock cell line and (B) of the B1 clone proteotype compared to the control mock cell line.(C) Overlap of significantly enriched GO Biological process pathways in WebGestalt GSEA analysis between A6 vs mock comparison and B1 vs mock comparison.

Figure 3 .
Figure 3. Gene Ontology terms enriched in g:Profiler analysis among up-and downregulated proteins simultaneously in both clones with Casp9 KO.

Figure 5 .
Figure 5. (A) Protein and (B) mRNA levels of ABHD2 and ADAM15 in parental, mock, and Casp9 KO MC3T3-E1 cells; α-tubulin was used as a loading control for immunoblotting.Data represents means ± SD from at least three independent experiments.

Table 1 .
Proteins Acting as Negative Regulators of Cell Migration Upregulated after Casp9 KO in Both Clones Compared to Mock and wt Cells a aPreviously identified proteins upregulated in micromass cultures incubated with CASP-9 inhibitor 47 are in bold.