YAP and TAZ Mediate Osteocyte Perilacunar/Canalicular Remodeling

Animals

Mice harboring loxP-flanked exon 3 alleles in both YAP and TAZ on a mixed C57BL/6J genetic background were kindly provided by Dr Eric Olson (University of Texas Southwestern Medical Center). Osteocytes were targeted using Cre-recombination under the control of an 8 kb fragment of the dentin matrix protein-1 promoter (8 kb-DMP1-Cre).35 All mice were fed regular chow (PicoLab Rodent Diet, cat #: 0007688, LabDiet, St. Louis, MO, USA) ad libitum and housed in cages containing 2 to 5 animals each. Mice were maintained at constant 25°C on a 12-hour light/dark cycle. Mice with homozygous floxed alleles for both YAP and TAZ (YAP^fl/fl;TAZ^fl/fl) were mated with double heterozygous conditional knockout mice (YAP^fl/+;TAZ^fl/+;DMP1-Cre) to produce eight possible genotypes in each litter, but only the genotypes in Table 1 were compared. Mice were tail or ear clipped after weaning or before euthanasia and genotyped by an external service (Transnetyx Inc, Cordova, TN, USA). Both male and female mice were evaluated with YAP^fl/fl;TAZ^fl/fl mice serving as littermate wild-type (WT) controls. The different analyses were performed in both male and female young mice at either postnatal day 28 (P28) or postnatal day 84 (P84) as indicated. All protocols were approved by the Institutional Animal Care and Use Committees at the University of Notre Dame and the University of Pennsylvania. All animal procedures were performed in adherence to federal guidelines for animal care and conform to the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines.

Microcomputed tomography

Micro-computed tomography (micro-CT) was performed according to published guidelines.36 Harvested femurs from P84 mice were stored at −20°C until evaluation. Frozen specimens were thawed and imaged using a vivaCT 80 scanner (Scanco Medical, Bruttisellen, Switzerland) to determine trabecular and cortical femoral bone architecture before mechanical testing. The mid-diaphysis and distal femur were imaged with an X-ray intensity of 114 μA, energy of 70 kVp, integration time of 300 ms, and resolution of 10 μm. Mid-diaphyseal and distal femoral 2D tomograms were manually contoured, stacked, and binarized by applying a Gaussian filter (sigma = 1, support = 1) at a threshold value of 250 mg HA/cm³. Eight mice were analyzed per group. Investigators were blinded to animal genotype during scan quantification.

Mechanical testing

After micro-CT scanning, femurs from P84 mice were tested in three-point bending to failure. Femurs were loaded with the condyles facing down onto the bending fixtures with a support span length of 4.4 mm, which attenuated the effect of femur length on the measured mechanical properties. The upper fixture was aligned with the mid-diaphysis. The femurs were loaded to failure at a rate of 0.5 mm/s using the ElectroForce 3220 Series testing system (TA Instruments, New Castle, DE, USA). Stiffness, maximum load to failure, work to maximum load, and work to failure were quantified using a custom MATLAB script.37 Eight mice were analyzed per group. Investigators were blinded to animal genotype during data quantification.

Osteocyte lacunae visualization and quantification

Nano-computed tomography (nano-CT) was used to evaluate the morphological characteristics of osteocyte lacunae in the proximal tibia (Xradia Versa XRM-520, Zeiss, Dublin, CA, USA). Tibias from P84 mice were harvested and removed of all non-osseous tissue. Samples were scanned in 70% EtOH, in custom-made sample holders that oriented the samples vertically on the stage. Tibias were originally scanned with a 4× objective to determine regions of interest. A 650 μm³ region of bone was located on the anterior medial aspect of the tibia, 8 mm from the tibiofibular junction (TFJ). Nano-computed tomography images image were collected in the region of interest using a 20× objective with energy settings of 40 V, 3.0 W, using the Air Filter and 3201 projections. To obtain a constant resolution of 0.6 μm voxel for all the samples, the source and detector distance were varied between 5.8 and 8.5 mm from the sample, and excitation ranged from 6 to 8 seconds contingent on resulting intensity values.

Dragonfly 3.6 (Object Research Systems [ORS] Inc, Montreal, Canada) was used for segmentation of bone types and osteocyte lacunae regions. Cortical and trabecular bone were manually segmented. Osteocyte lacunae were identified and segmented in three dimensions using a global Otsu threshold, defined for each sample to most closely match grayscale images.38 Threshold values were not statistically different between genotypes (31,382 ± 1080 for YAP^WT;TAZ^WT and 31,032 ± 1168 for YAP^cKO;TAZ^cKO; p = 0.83). Voids that were too small or too large (<50 μm³ and >1500 μm³)39-41 to be an osteocyte lacuna were removed. Osteocyte volume, surface area, and aspect ratio were measured for each osteocyte. Additionally, custom python code based on previous work39, 42, 43 was implemented to analyze osteocyte lacunar shape measures, oblateness, and stretch. Oblateness and stretch are calculated from the eigenvalues of the shape tensor for each individual osteocyte lacuna. Lacunar oblateness is defined as the “plateness” of a lacuna and ranges from [−1, 1], where −1 corresponds to a perfect rod (strongly prolate) and 1 to a perfect plate (strongly oblate).39

(1)

Similarly, lacunar stretch measures the sphericity of the osteocyte lacunae. Lacunar stretch is evaluated between [0, 1], where 0 corresponds to a perfectly spherical and 1 to an infinitely stretched object.39

(2)

Histology, immunohistochemistry, and immunofluorescence

Femurs and tibias from P28 and P84 mice were fixed with 10% neutral-buffered formalin for 48 hours at 4°C and decalcified for 4 weeks with 0.25 M EDTA (pH 7.4) at 4°C. Paraffin sections (5 μm thickness) were processed for either immunohistochemistry or histology. Primary antibodies were compared with both normal rabbit sera IgG control sections. For immunostaining, anti-CTSK (1:75, ab19027; Abcam, Cambridge, MA, USA), anti-MMP13 (1:100, ab39012; Abcam), anti-MMP14 (1:100, ab38971; Abcam), anti-YAP (1:200, 14074; Cell Signaling, Danvers, MA, USA), and anti-TAZ (1:200 NB110-58359; Novus Biologicals, Centennial, CO, USA) primary antibodies were applied overnight. Next, sections were incubated with corresponding biotinylated secondary antibody, avidin-conjugated peroxidase, and diaminobenzidine substrate chromogen system (329ANK-60; Innovex Biosciences, Richmond, CA, USA), which allowed for immunohistochemical detection of positively stained cells. Hematoxylin and eosin stains (H&E), tartrate-resistant acid phosphatase (TRAP), picrosirius red, and silver nitrate stains were used to stain for osteoblasts, osteoclasts, collagen, and the osteocyte lacunar/canalicular network as previously shown.44, 45

Femurs from P28 mice were fixed with 10% neutral-buffered formalin for 48 hours at 4°C, transferred to 30% sucrose in PBS overnight at 4°C, and then embedded in OCT compound (Tissue-Tek, Sakura Finetek USA, Torrance, CA, USA). Thin sections (7 μm thickness) were made from undecalcified femurs using cryofilm IIC tape (Section Lab Co. Ltd., Hiroshima, Japan) as previously described46 and processed for immunofluorescence. Taped sections were glued to microscope slides using chitosan adhesive glue, rehydrated, and then decalcified with 0.25 M EDTA (pH 7.4) for 5 minutes before immunofluorescence staining. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were then performed using the Click-iT Plus TUNEL Assay kit (C10618; Invitrogen, Carlsbad, CA, USA) according to the manufacturerʼs instructions. Thick sections (20 μm thickness) were processed similarly and stained for F-actin with Alexa Fluor 488-conjugated phalloidin (1:50, Life Technologies, Carlsbad, CA, USA) for confocal imaging.

Femurs from P28 mice injected with Calcein (C0875-25G; Sigma-Aldrich, St. Louis, MO, USA) at P21 and Alizarin Complexone (A3882-25G; Sigma-Aldrich) at P26 were embedded in methyl-methacrylate and processed for dynamic bone histomorphometry. Using a diamond-embedded wire saw (Delaware Diamond Knives, Wilmington, DE, USA), transverse sections (40 μm) were cut from the midshaft and ground to a final thickness of 20 μm. The slice sections were mounted on slides, and three sections per limb were analyzed.

Imaging and histomorphometric analysis

Histological and immunohistochemical sections were imaged on either a 90i Upright/Widefield Research Microscope (Nikon Instruments, Melville, NY, USA) at the 20× and 40× objectives or an Axio Observer Z1 (Zeiss) at the 20×, 40×, and 63× objectives. Quantification of paraffin immunohistochemistry and histology was performed using ImageJ (NIH) and Osteomeasure (OsteoMetrics, Decatur, GA, USA). To determine the number of positively immunostained cells, three fields of view per mouse per antibody were manually scored as either positive or negative and reported as percentage positively stained lacunae per total number of lacunae using ImageJ (NIH). To determine canalicular length, primary canaliculi emanating from each lacuna and extending as a single, unbranched process30 were traced with ImageJ (NIH). The mean length was taken from at least 5 osteocytes per field within the mid-cortex, with 3 fields per mouse and 6 mice per group.30 Osteoblast number per bone surface, osteocyte number per bone area, and osteoclast surface per bone surface were quantified using Osteomeasure (OsteoMetrics) on H&E- and TRAP-stained sections in the metaphyseal cancellous bone with 8 mice per group.

The samples tested in three-point bending were stained with picrosirius red to construct a multivariate regression model that included collagen organization and content as a predictor of mechanical behavior. These samples were imaged both under polarized light using an Eclipse ME600 Microscope (Nikon Instruments) at the 20× objective and using second harmonic generated (SHG) microscopy. SHG images were taken on a multiphoton-enabled Fluoview Research Microscope (Olympus) at a fundamental wavelength of 875 nm with the 25× objective on sections oriented in the same direction for all groups. All SHG images were quantified using ImageJ (NIH) and reported as mean pixel intensity within the mid-cortical region relative to WT bone. Mean pixel intensities across four separate regions of interest within each image of the cortex were averaged as technical replicates for a given sample.

Confocal images were acquired using an LSM 710 confocal (Zeiss) with the 63× objective on 20-micron-thick sections. The 15-micron-thick z-stack images were acquired using a step size of 0.5 microns. Three separate regions of interest within the mid-cortex were averaged as technical replicates for a given animal sample. All confocal image stacks were quantified using ImageJ (NIH). Stacks were smoothed by applying a Gaussian filter (sigma = 0.3), then skeletonized using the Skeletonize3D function. The AnalyzeSkeleton2D/3D function was then used to quantify mean branch length, number of branches, and number of junctions.47 Six YAP^WT;TAZ^WT and four YAP^cKO;TAZ^cKO mice were analyzed.

Immunofluorescence sections of P28 femurs were imaged on an Axio Observer Z1 (Zeiss) at the 20×, 40×, and 63× objectives. To determine the number of positively TUNEL-stained lacunae, 3 fields of view per mouse in the metaphyseal cancellous bone were manually scored as either positive or negative and reported as percent positively stained per total number of lacunae scored in ImageJ (NIH). Dynamic bone histomorphometry parameters were quantified using Osteomeasure (OsteoMetrics). The following primary data were collected: total bone surface length (BS); single label perimeter (sL.Pm); double label perimeter (dL.Pm); and double label width (dL.Ith). From primary data, we derived mineralizing surface (MS/BS = [1/2sL.Pm + dL.Pm]/B.Pm × 100; %); mineral apposition rate (MAR = dL.Ith/5 days; μm/d) and bone formation rate (BFR/BS = MAR × MS/BS; μm³/μm² per day). Six mice were analyzed per group. Investigators were blinded to animal genotype during image acquisition and quantification of all histology, immunohistochemistry, and immunofluorescent images.

Cell culture

IDG-SW3 cells were cultured in α-MEM supplemented with 10% fetal bovine serum, 1% antibiotic/antimycotic (15240062; Gibco) and 1 U/mL INF-γ (PMC4031; Invitrogen) at 33°C. OCY454 cells were cultured in α-minimum essential media (α-MEM) supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic (Gibco) at 33°C. Before treatment, OCY454 cells were differentiated for 12 days at 37°C, whereas IDG-SW3 cells were differentiated for 21 days at 37°C in α-MEM supplemented with 10% fetal bovine serum (100 to 106; Gemini Bio-Products, West Sacramento, CA, USA), 1% antibiotic/antimycotic (15240062; Gibco), 50 μg/mL ascorbic acid (A-4544; Sigma-Aldrich), and 4 mM β-glycerophosphate (G-9422; Sigma-Aldrich) without INF-γ. Before treatment on the last day of differentiation, both OCY454 and IDG-SW3 cells were treated with 3 μM verteporfin (SML0534; Sigma-Aldrich) overnight in 1% fetal bovine serum. The next morning, the media was changed and supplemented with 3 μM verteporfin and 5 ng/mL TGF-β1 (HZ1011; Humanzyme, Rosemont, IL, USA) for 6 hours before mRNA isolation.

RNA isolation and qPCR

P84 femur samples were dissected and removed of all non-osseous tissue. The ends were cut at the growth plate and marrow flushed before being snap-frozen in liquid nitrogen-cooled isopentane for 1 minute before storage at −80°C until processing. Tissues were then homogenized via mortar and pestle and RNA from the sample was collected using Trizol Reagant (15596026; Life Technologies) followed by centrifugation in chloroform. RNA from femur tissue and cell culture experiments were purified using the RNA Easy Kit (74106; Qiagen, Valencia, CA, USA) and quantified by spectrophotometry using a NanoDrop 2000 (Thermo-Fisher Scientific, Waltham, MA, USA). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed on 0.5 μg/μL concentration of RNA using the High-Capacity cDNA Reverse Transcription Kit (4368814; Thermo-Fisher Scientific). Quantitative polymerase chain reaction (qPCR) assessed RNA amount using a StepOnePlus Real-Time PCR System (Thermo-Fisher Scientific) relative to the internal control of 18S ribosomal RNA (18S rRNA). Data are presented using the ΔΔCt method. Six mice per group were used. Specific mouse primer sequences are listed (Supplemental Table S1).

Statistics and regression

Sample sizes were selected a priori by power analyses based on effect sizes and population standard deviations taken from published data on YAP^fl/fl;TAZ^fl/fl mice in other tissues,48 assuming a power of 80% and α = 0.05. All statistics and regression analyses were performed in GraphPad Prism (GraphPad, La Jolla, CA, USA) or using R (Version 3.5.1). Repeated-measures ANOVA with post hoc Tukey's HSD comparison test was performed using R (Version 3.5.1) for osteocyte lacunae quantification. All other comparisons between two groups were made using the two-tailed Student's t test, provided the data were normally distributed according to D'Agostino-Pearson omnibus normality test and homoscedastic according to Bartlett's test in GraphPad Prism. When parametric test assumptions were not met, data were log-transformed, and residuals were evaluated. If necessary, the nonparametric Mann–Whitney test was used. A p value < 0.05 was considered significant. Post hoc power analyses were performed using R (Version 3.5.1) for phenotypic results as indicated (Supplemental Table S2). Data are presented as bars and individual samples with lines corresponding to the mean and standard error of the mean (SEM).

Multivariate regression analyses were performed as described previously,49 using R (Version 3.5.1) with some modifications.37 Briefly, we used an exhaustive best subsets algorithm to determine the best predictors of maximum load and stiffness from a subset of morphological parameters measured, which included moment of inertia (I) or section modulus (I/c), micro-CT-measured tissue mineral density (TMD), SHG intensity, and femur length. The best subsets algorithm selects the optimal model using the Akaike's information criterion (AIC), which gives preference to less complex models with fewer explanatory parameters to avoid overfitting the data.50 The overall “best” multivariate model for each predicted mechanical property was selected with the lowest relative AIC value, indicative of having the least variables with the greatest predictive power.

DMP1-Cre conditionally ablates YAP and TAZ primarily in osteocytes

To determine the roles of YAP and TAZ in osteocyte-mediated bone remodeling, we used Cre-lox to selectively delete YAP and TAZ from 8 kb-DMP1-Cre expressing cells.35, 51 We used a breeding strategy that generated YAP/TAZ allele dosage-dependent DMP1-conditional knockouts.37 All genotypes (Table 1) appeared at expected Mendelian ratios. By early skeletal maturity (P84), YAP/TAZ allele dosage-dependent DMP1-conditional deletion did not significantly alter body mass in either males or females (Supplemental Fig. S1A). YAP/TAZ deletion reduced femoral length at P84 only in double homozygous knockouts, for both sexes (Fig. 1 A, B; Supplemental Fig. S1B). A single copy of either gene was sufficient to rescue this defect. Therefore, for further analyses, we selected littermate YAP^fl/fl;TAZ^fl/fl WT (YAP^WT;TAZ^WT) and YAP^fl/fl;TAZ^fl/fl;8 kb-DMP1-Cre conditional double knockout (YAP^cKO;TAZ^cKO) mice for comparison.

To analyze the specificity of 8 kb-DMP1-Cre-mediated YAP/TAZ deletion, we evaluated YAP/TAZ expression in osteocytes, osteoblasts, and growth plate chondrocytes. YAP mRNA expression was significantly reduced by 67% in YAP^cKO;TAZ^cKO femoral bone preparations (Fig. 1 C). YAP protein expression was significantly reduced in osteocytes (58% reduction) but not osteoblasts (0% reduction; p = 0.98) (Fig. 1 D, E). Similarly, TAZ mRNA expression was significantly reduced by 72% in YAP^cKO;TAZ^cKO femoral bone preparations (Fig. 1 F), and TAZ protein expression was significantly reduced in osteocytes (79% reduction) but not osteoblasts (6% reduction; p = 0.12) (Fig. 1 G, H). The reduction in YAP^cKO;TAZ^cKO growth plate thickness did not reach statistical significance within the hypertrophic zone (16% reduction; p = 0.26), the proliferating zone (12% reduction; p = 0.39), or total growth plate thickness (13% reduction; p = 0.27; Supplemental Fig. S1C). Further, YAP^cKO;TAZ^cKO growth plate chondrocytes did not show differential expression of YAP or TAZ in either proliferating or hypertrophic zones (Supplemental Fig. S1D–G).

DMP1-conditional YAP/TAZ deletion impaired bone accrual

Dual homozygous YAP/TAZ deletion from DMP1-expressing cells reduced bone accrual and microarchitectural parameters in both the cancellous and cortical compartments of P84 femurs. In metaphyseal cancellous bone, conditional YAP/TAZ deletion significantly reduced bone volume fraction, trabecular number, and thickness and increased trabecular spacing and structural model index (Fig. 2 A–F). In mid-diaphyseal femoral cortical bone, conditional YAP/TAZ deletion reduced cortical thickness and bone area without changing medullary area (Fig. 2 G–I; Supplemental Fig. S2). Differences in TMD (p = 0.24), periosteal perimeter (p = 0.15), and moment of inertia (p = 0.08) were not statistically significant (Fig. 2 J–L).

To determine if the low bone mass phenotype resulted from decreased bone formation and/or increased bone resorption, we evaluated osteoblast and osteoclast activity. Conditional YAP/TAZ deletion increased osteoclast surface per bone surface (Fig. 3 A, B) and decreased osteoblast number per bone surface (Fig. 3 C, D) in P84 distal femur metaphyseal cancellous bone. YAP/TAZ deletion decreased bone formation rate, reducing both mineralizing surface percentage and mineral apposition rate at P28 in distal femur metaphyseal cancellous bone (Fig. 3 E–H). In the cortical compartment, YAP/TAZ deletion similarly reduced osteoblast number per endosteal bone surface (Supplemental Fig. S3A, B) and decreased bone formation rate by reducing mineral apposition rate without altering mineralizing surface percentage (Supplemental Fig. S3C–F).

Further, YAP/TAZ deletion significantly reduced mRNA expression of YAP/TAZ-TEAD target genes, Cyr61 and Ctgf in femoral bone at P84 (Fig. 3 I, J). However, YAP/TAZ deletion did not alter sclerostin (SOST), receptor activator of nuclear factor kappa-B ligand (Rankl), or osteoprotegerin (Opg) transcript expression (Supplemental Fig. S4).

DMP1-conditional YAP/TAZ deletion impaired bone mechanical properties and matrix collagen composition

To determine whether YAP/TAZ deletion impaired functional mechanical properties, we tested P84 femurs in three-point bending to failure (Fig. 4 A). Conditional YAP/TAZ deletion did not significantly reduce maximum load to failure (Fig. 4 B; p = 0.07) but significantly reduced bending stiffness, work to maximum load, and work to failure (Fig. 4 C–E).

Intrinsic bone material properties are determined, in part, by collagen content and organization.37 To test whether osteocyte YAP/TAZ regulate the local collagen matrix, we performed polarized light and SHG microscopy (Fig. 4 F). YAP/TAZ deletion qualitatively reduced picrosirius red staining and significantly reduced SHG intensity per bone area, indicative of both collagen content and organization (Fig. 4 G). Both cross-sectional bone geometry (section modulus and moment of inertia) and collagen matrix content and organization significantly contributed to bone stiffness according to a multivariate best-subsets regression analysis using AIC, which selects the model with the lowest number of predictors with the greatest predictive power.37, 50 Neither micro-CT-measured TMD nor femur length contributed to the overall best model according to AIC for stiffness and maximum load to failure (Fig. 4 H, Supplemental Fig. S5).

DMP1-conditional YAP/TAZ deletion impaired the osteocyte canalicular network, but not lacunar morphology

We initially hypothesized that the primary function of YAP/TAZ in osteocytes was to regulate expression of genes that control osteocyte-osteoblast and -osteoclast communication, such as secreted factors CYR61 and CTGF. However, the reduced bone mechanical properties and disorganized collagen matrix in mice lacking YAP/TAZ from osteocytes phenocopied matrix metalloproteinase-13 (MMP13) knockout mice, which had defective collagen organization as a result of impaired perilacunar/canalicular remodeling.52 This led us to ask whether YAP/TAZ deletion from osteocytes impaired perilacunar/canalicular remodeling.

Conditional YAP/TAZ deletion did not significantly alter osteocyte density but increased empty lacunae percentage in cancellous metaphyseal bone (Fig. 5 A–C). Accordingly, conditional YAP/TAZ deletion increased the number of TUNEL-positive lacunae, suggesting an increase in osteocyte apoptosis within the cancellous bone compartment (Fig. 5 D, E). Conditional YAP/TAZ deletion significantly reduced canalicular density and mean process length, measured in silver nitrate-stained, mid-cortical bone sections (Fig. 5 F–H).

As the canalicular system is a three-dimensional network of branched cell processes that physically connect osteocytes to their neighbors,12 we next quantified the 3D canalicular network by laser scanning confocal microscopy of the osteocyte actin cytoskeleton, labeled with phalloidin (Fig. 6 A). YAP/TAZ deletion significantly reduced branch length (Fig. 6 B, C), number of branches per cell (Fig. 6 D), and number of junctions per cell (Fig. 6 E).

To determine the effect of YAP/TAZ deletion on osteocyte lacunar morphology, we performed high-resolution (0.6 um voxel) X-ray microscopy (XRM) imaging of both cortical and cancellous bone in the proximal tibia metaphysis (Fig. 6 F). YAP/TAZ deletion did not significantly alter lacuna volume (Fig. 6 G, H) or shape (Fig. 6 I, J), as significant interaction factors were observed between sex, bone compartment, and genotype for all lacunar morphology parameters. Using post hoc multiple comparisons, no differences in lacunar parameters were observed between genotypes within either a single sex or bone compartment.

DMP1-conditional YAP/TAZ deletion reduced osteocyte-mediated bone matrix remodeling

Perilacunar/canalicular remodeling involves both the deposition and degradation of the bone matrix directly surrounding the osteocytes. Prior studies used ³H-proline pulses and tetracycline labeling to identify osteocyte lacunae as sites of active collagen deposition and mineralization.15, 53, 54 Here, we measured collagen I gene expression and flourochrome incorporation in osteocyte lacunae to assess peri-osteocyte matrix deposition. Conditional YAP/TAZ deletion reduced the percentage of Calcein-labeled osteocyte lacunae (Fig. 7 A, B) and reduced transcript expression of Col1a1 (Fig. 7 C). However, YAP/TAZ deletion did not alter expression of either osteogenic genes, including alkaline phosphatase (Alp), osteocalcin (Ocn), and bone sialoprotein (Bsp), or osteocyte-marker genes, dentin matrix protein-1 (Dmp1) or phosphate-regulating neutral endopeptidase, X-linked (Phex) (Supplemental Fig. S6).

In addition to local matrix deposition, osteocytes also locally degrade their extracellular matrix. Many of the matrix metalloproteinases and other enzymes employed by osteoclasts to resorb bone are also expressed by osteocytes and are critical to perilacunar/canalicular remodeling.17, 52, 55 Therefore, to test whether YAP/TAZ regulate direct matrix remodeling by osteocytes, we measured mRNA and protein expression of the matrix proteases, cathepsin K (CTSK), matrix metalloproteinase-13 (MMP13), and matrix metalloproteinase-14 (MMP14). Conditional YAP/TAZ deletion reduced the percentage of osteocytes that stained positive for CTSK (Fig. 7 D, E), MMP13 (Fig. 7 G, H), and MMP14 (Fig. 7 J, K) and decreased mRNA expression of Ctsk (Fig. 7 F), Mmp13 (Fig. 7 I), and Mmp14 (Fig. 7 L) in vivo.

YAP/TAZ inhibition abrogated TG-β-induced gene expression in vitro

Though unexplored in osteocytes, YAP and TAZ are known to mediate TGF-β signaling in a variety of other cell types.31-34 Further, either pharmacologic inhibition or genetic ablation of TGF-β receptors from osteocytes caused defective perilacunar/canalicular remodeling and associated gene expression.30 Therefore, we next tested whether YAP/TAZ mediate TGF-β signaling, Cyr61/Ctgf expression, and perilacunar/canalicular remodeling-associated gene expression in osteocytes by treating two osteocyte-like cell lines with combinatorial TGF-β and/or verteporfin (VP) treatment. VP is a small molecule inhibitor that physically blocks the interaction of YAP and TAZ with their transcriptional co-effectors, particularly the TEAD transcription factors, preventing YAP/TAZ-TEAD transcriptional activity.37, 56

We first used mouse osteocyte-derived IDG-SW3 cells, which carry a DMP1 cis-regulatory system driving GFP expression, as a marker of living osteocytes.57, 58 As shown previously, IDG-SW3 cells exhibited robust DMP1-GFP transgene expression after 21 days of in vitro osteocytic differentiation (Fig. 8 A). Beginning at day 20, cells were treated with vehicle (DMSO) or 3 μM verteporfin (Fig. 8 A). At day 21, cells were treated with 5 ng/mL TGF-β or PBS vehicle for 6 hours and gene expression was evaluated by qPCR. Similar experiments were conducted in mouse osteocyte-derived OCY454 cells59 (Supplemental Fig. S7A).

First, to test whether verteporfin blocked TGF-β-induced YAP/TAZ signaling, we evaluated expression of serpin family E member 1 (SerpinE1) and expression of known osteoblast/osteoclast paracrine signaling factors, Cyr61 and Ctgf. Each of these genes are established TGF-β-inducible, YAP/TAZ-TEAD target genes.25, 26, 60, 61 As expected, TGF-β robustly induced SerpinE1, Cyr61, and Ctgf mRNA expression, which was abrogated by verteporfin treatment (Fig. 8 B–D). Next, to test whether YAP/TAZ mediate TGF-β induction of genes associated with perilacunar/canalicular remodeling, we quantified mRNA expression of Ctsk, Mmp13, and Mmp14. TGF-β significantly induced expression of Mmp13 and Mmp14, but not Ctsk, in IDG-SW3 cells, and verteporfin treatment abrogated expression of all three genes (Fig. 8 E–G). In OCY454 cells, verteporfin similarly abrogated TGF-β-induced expression of Ctgf, Cyr61, and SerpinE1 mRNA (Supplemental Fig. S7B–D) as well as Mmp14 and Ctsk but not Mmp13 (Supplemental Fig. S7E–G). Finally, we evaluated Sost, Opg, and Rankl expression in both IDG-SW3 and OCY454 cells as potential YAP/TAZ-regulated mediators of osteocyte-osteoblast/osteoclast signaling. In IDG-SW3 cells, both verteporfin and TGF-β significantly reduced Sost transcript expression, while verteporfin abrogated TGF-β-induced Rankl and Opg expression (Supplemental Fig. S8A–C). In OCY454 cells, verteporfin abrogated TGF-β-induced Opg and reduced Sost expression but did not significantly alter Rankl expression (Supplemental Fig. S8D–F). In contrast to IDG-SW3 cells, treatment of OCY454 cells with TGF-β alone significantly reduced Rankl transcript expression (Supplemental Fig. S8E).