Mmp13 deletion in mesenchymal cells increases bone mass and may attenuate the cortical bone loss caused by estrogen deficiency

The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal cells, but functional evidence for the mediators of these effects has only recently begun to emerge. We report that the matrix metalloproteinase 13 (MMP13) is the highest up-regulated gene in mesenchymal cells from mice lacking the estrogen receptor alpha (ERα). In sham-operated female mice with conditional Mmp13 deletion in Prrx1 expressing cells (Mmp13ΔPrrx1), the femur and tibia length was lower as compared to control littermates (Mmp13f./f). Additionally, in the sham-operated female Mmp13ΔPrrx1 mice cortical thickness and trabecular bone volume in the femur and tibia were higher and osteoclast number at the endocortical surfaces was lower, whereas bone formation rate was unaffected. Notably, the decrease of cortical thickness caused by ovariectomy (OVX) in the femur and tibia of Mmp13f./f mice was attenuated in the Mmp13ΔPrrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell–derived MMP13 may regulate osteoclast number and/or activity, bone resorption, and bone mass. And increased production of mesenchymal cell-derived factors may be important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone.


Results
Mmp13 expression is downregulated by ERα. To identify estrogen target genes that regulate osteoclastogenesis indirectly, i.e., via actions on cells of the mesenchymal lineage, we performed microarray analysis of GFP sorted Osx1 + cells without or with ERα, derived from the calvaria cells of ERα f/f ;GFP:Osx1-Cre mice or GFP:Osx1-Cre controls. The highest up-regulated gene in ERα deleted mesenchymal/stromal cells encodes the matrix metalloproteinase 13 (Mmp13), as shown in the heat map (Fig. 1a) and in the volcano plot (Fig. 1b). The microarray findings from the GFP sorted ERα deleted Osx1 + calvaria cells were confirmed by qPCR and reproduced in cultures of ERα deleted Prrx1 + bone marrow stromal cells derived from ERα f/f ;Prrx1-Cre mice (Fig. 1c). Moreover, the mRNA levels of MMP13 were fourfold higher in bone marrow stromal cell cultures from OVX C57BL/6 mice, as compared to sham controls (Fig. 1d). In line with our findings, ERα regulates the Mmp13 promoter activity in synoviocytes 14 . Mmp13 deletion in mesenchymal progenitors decrease the length of the femur and tibia. To elucidate the role of Mmp13 in bone homeostasis in vivo, we next generated mice with conditional deletion of Mmp13 in mesenchymal progenitors expressing Prrx1 (Mmp13 ΔPrrx1 ) and used floxed mice (Mmp13f. /f ) as control. The Prrx1-cre transgene targets early limb bud and a subset of craniofacial mesenchymal stem cells. We did not detect a skull phenotype. All measurements were made in the femur and/or tibia. Please note that in the following description of the results, the p values from two-way ANOVA analysis are provided below each graph. The expression of the Mmp13 mRNA in femur and tibia shafts was dramatically decreased in the Mmp13 ΔPrrx1 mice (Fig. 2a), establishing the effectiveness of the deletion. Body weight and uterine weight were not affected by the Mmp13 deletion (Fig. 2b,c). However, femur and tibia length was decreased Fig. 2d,e).
In contrast to the microarray data of Fig. 1, we did not detect a change of the mRNA levels of Mmp13 in the OVX Mmp13f. /f or Mmp13 ΔPrrx1 mice in the osteocyte-enriched bone marrow-devoid preparations we used for this measurement (Fig. 2a). As expected, OVX increased body weight in Mmp13f. /f mice (Fig. 2b) and decreased uterine weight in Mmp13f. /f and Mmp13 ΔPrrx1 mice (Fig. 2c). Femur and tibia length was not affected by OVX in either genotype (Fig. 2d,e).

Mmp13 deletion increases cortical bone and attenuates the cortical bone loss caused by OVX.
Mmp13 deletion in Prrx1 cells caused greater cortical thickness and cortical area in the femoral diaphysis as indicated by pairwise comparison between sham-operated Mmp13f. /f and sham-operated Mmp13 ΔPrrx1 mice ( Fig. 3a,b). This effect was due to a smaller medullary area (Fig. 3c) while total area did not change (Fig. 3d). The greater cortical thickness with Mmp13 deletion was less marked in the distal metaphysis of the femur (Fig. 3e) and in the diaphysis of the tibia (Fig. 3f).
OVX of the Mmp13f. /f control mice decreased cortical thickness at the femoral diaphysis and distal metaphysis as well as the tibia diaphysis (Fig. 3a,e,f). Consistent with our working hypothesis, the effects of OVX on cortical bone at the diaphysis and distal metaphysis of the femur and tibia diaphysis were attenuated in the Mmp13 ΔPrrx1 mice (Fig. 3a,e,f). Together, these data suggest that Mmp13 deletion increases cortical bone mass in femur and tibia and prevents or attenuates the loss of cortical bone caused by estrogen deficiency.
Mmp13 deletion increases trabecular bone but does not affect the loss of bone caused by OVX in this compartment. Trabecular bone volume was higher in both the distal femur and the proximal tibia by approximately 3.7-and threefold respectively, in sham-operated Mmp13 ΔPrrx1 mice as compared to shamoperated Mmp13f. /f controls (Fig. 4a-e). This difference was due to higher trabecular number (Fig. 4b) and thickness (Fig. 4c); and was mirrored by lower trabecular separation (Fig. 4d). In several mice, trabecular bone extended to the midshaft (see lower left micro-CT image in Fig. 3a).
As seen before 15,16 , at 6-months of age estrogen sufficient female mice have very little trabecular bone mass remaining at the distal femur (Fig. 4a). There was no discernible effect of the OVX at this site in Mmp13f. /f mice ( Fig. 4a-d). However, we observed a loss of trabecular bone mass in both the femur and tibia of the OVX Mmp13 ΔPrrx1 mice (Fig. 4a,e). Collectively, these data indicate that Mmp13 deletion increases trabecular bone mass but does not prevent the loss of trabecular bone caused by estrogen deficiency.
Mmp13 deletion decreases osteoclast number in cortical bone. To elucidate the cellular mechanism by which the Mmp13 deletion increased cortical bone volume, we performed histomorphometric analysis of the endocortical surface of the femur. Mmp13 deletion caused an approximately 50% reduction in osteoclast number and surface (Fig. 5a-c), as indicated by pairwise comparison between sham-operated Mmp13f. /f and sham-operated Mmp13 ΔPrrx1 mice. The MMP13 deletion had no effect on mineral apposition rate (MAR),  (Fig. 5d-g). These findings suggest that a decrease of osteoclast number and thereby resorption are responsible for the increase of cortical bone. OVX of both Mmp13f. /f and Mmp13 ΔPrrx1 mice resulted in the expected increase in osteoclast number and surface (Fig. 5a,b), while MAR, MS and BFR were not affected ( Fig. 5d-f). Surprisingly, in the OVX Mmp13 ΔPrrx1 mice the increase in osteoclast number and surface was greater (5.5-fold) as compared to the OVX-induced increase in the Mmp13f. /f mice (2-fold).

Mmp13 deletion increases whole-bone strength of the femur. It has been previously reported that
Mmp13−/− mice have increased cortical bone fragility 17 . To examine bone strength in female Mmp13 ΔPrrx1 mice we performed three-point bending of the femur (Fig. 6a). Despite the thicker cortices in female Mmp13 ΔPrrx1 mice, the moment of inertia was not different from the controls (Fig. 6b). Nonetheless, the yield load, peak load,    (Fig. 6c). With respect to the estimated material properties, female Mmp13 ΔPrrx1 mice had increased yield stress and ultimate stress but the same modulus as compared to control mice (Fig. 6d). Material density determined by micro-CT was also not different (Fig. 6e). Therefore, and in contrast to a previous report, deletion of Mmp13 led to an increase in bone structural and material properties.
Similar to females, Mmp13 deletion in males increases trabecular bone mass and whole-bone strength of the femur, but has no effect on cortical bone. The bone phenotype of sex steroid sufficient male Mmp13 ΔPrrx1 and Mmp13f. /f mice was analyzed at 4 and 6 month of age. Body weight was not affected by the Mmp13 deletion at either age ( Fig. 7a), but femur length decreased at 4 and 6 months of age (Fig. 7b), as it did in females. In difference to females, cortical thickness in male mice was not affected by the Mmp13 deletion (Fig. 7c). Trabecular bone volume increased 1.4-and 1.6-fold at 4 and 6 months, respectively (Fig. 7d), but this increase was lower compared to the one we observed in Mmp13 ΔPrrx1 female mice. The increased trabecular bone volume was associated with an increase in the number of trabeculae and a decrease in trabecular separation ( Fig. 7e,g), but no change in trabecular thickness (Fig. 7f).  www.nature.com/scientificreports/ Finally, similar to females, three-point bending of the femur in male Mmp13 ΔPrrx1 mice revealed higher bone strength including an increase in stiffness and modulus (Fig. 7h-j), with no change in material density (Fig. 7k); but unlike females the moment of inertia was decreased in males (Fig. 7h).

Discussion
In this paper we show that the highest upregulated mRNA in mesenchymal lineage cells lacking ERα encoded MMP13; and loss of ovarian function in mice increased the expression of the mmp13 mRNA in ex vivo bone marrow stromal cell cultures. In estrogen sufficient (sham-operated) Mmp13 ΔPrrx1 mice cortical thickness and trabecular bone volume in the femur and tibia were increased as compared to littermate controls, while femur and tibia length was decreased. These bone phenotypic changes were associated with a decrease in osteoclast number, but no changes in bone formation. Moreover, the loss of cortical bone caused by OVX in the femur and tibia was attenuated in the Mmp13 ΔPrrx1 mice. The effect of OVX on trabecular bone, on the other hand, was not affected. These results elucidate an important role of mesenchymal cell-derived MMP13 on osteoclast number or activity, bone resorption, and bone mass. We had previously shown that the OVX-induced loss of cortical, but not trabecular bone, was attenuated in mice with conditional Cxcl12 deletion in Prrx1 expressing cells. Taken together, the functional genetic evidence obtained by these two studies suggests that increased production of  The expression of MMP13 was increased in calvaria or bone marrow derived cells from ERα conditional KO mice and in ex vivo bone marrow derived stromal cell cultures from OVX mice. In contrast, loss of estrogen with OVX did not alter the expression of MMP13 in osteocyte-enriched cortical bone shafts from the femur. These findings suggest that estrogens attenuate the expression of MMP13 in stromal cells or osteoblasts, but not osteocytes. Others have shown before that the MMP13 content of osteoblastic cells in the proximal tibia increases with OVX in the rat 12 . Furthermore, in human articular chondrocytes, estradiol suppresses the expression of MMP13 18 ; and an increase in MMP13 has been associated with the deleterious effects of estrogen deficiency in osteoarthritis and intravertebral disc degeneration 19 . In contrast, estrogens may promote temporomandibular joint disorders via induction of MMP-9 and MMP13 in fibrochondrocytes 20 . In synoviocytes, ERα may regulate the expression of MMP13 through the AP-1 transcriptional regulatory site 21 . However, other transcription factor binding sites such as Runx, PEA-3 and p53 in conjunction with AP-1 appear to be critical for the transcriptional regulation of Mmp13 in other cells 22,23 , perhaps explaining the different responses of this gene to estrogens in different bone cell types.
It has been suggested before, that stimulation of collagenase activity, particularly by MMP13, acts as a coupling factor for the activation of osteoclasts 9 . However, other reports suggest that MMP13 can stimulate osteoclast activity independent of its enzymatic activity 11 . Mmp13 null mice are resistant to the loss of bone mass caused by multiple myeloma, though the number of osteoclasts on bone was unaffected by the MMP13 deletion 24 . In In line with previous evidence from Mmp13 null mice 6 , we found that Mmp13 deletion in the Prrx1 targeted mesenchymal progenitors decreased the length of the femur and tibia in both male and female mice. This effect most likely results from the expansion of hypertrophic cartilage in the growth plate that occurs during development and growth and it is caused by the deletion of Mmp13 in growth plate chondrocytes 7 , which are targeted by Prrx1-Cre. We also confirmed herein that Mmp13 deletion increases trabecular bone volume in the femur and tibia. This effect was seen in both sexes. However, Mmp13 ΔPrrx1 females exhibited a bigger increase than males. Deletion of Mmp13 in Col1-Cre or Dmp1-Cre targeted cells also increases trabecular bone mass, indicating that osteoblasts and/or osteocytes are the major source of MMP13 responsible for this effect 7,8 . Nonetheless, we cannot exclude the possibility that MMP13 in cells of the mesenchymal lineage other than osteoblasts and osteocytes contributes to the changes in bone mass.
In the present report we show for the first time that Mmp13 deletion in females increases cortical thickness; and this effect may be associated with a decrease in osteoclast number and/or activity in the endocortical  www.nature.com/scientificreports/ surface with no changes in bone formation. Interestingly, and similar to previous reports in male Mmp13 KO mice 17 , cortical thickness was unaffected in our male Mmp13 ΔPrrx1 mice. Despite the increase in trabecular bone mass, we found no changes in osteoclast number or bone formation in trabecular bone in 6-month-old females. Nonetheless, Nakatani et al. 25 have shown that the number of osteoclasts is severely decreased in the trabecular bone of 8-day-old Mmp13 KO mice. Thus, it is possible that osteoclast number was decreased at an earlier age in our mice. Collectively, this evidence indicates that MMP13 is a potent stimulant of bone resorption, particularly in female mice. It has been reported previously that male mice with global Mmp13 deletion or osteocyte-specific Mmp13 ablation have defective osteocyte perilacunar remodeling and decreased bone toughness 17 . Albeit, the mice with the osteocyte-specific Mmp13 ablation exhibited incongruent changes in cortical bone biomechanical properties: decreased ultimate load but increased yield load and yield stress. The Prrx1-Cre transgene in our Mmp13 ΔPrrx1 mice has inexorably deleted Mmp13 in osteocytes. Yet, in contrast to these previous findings, both male and female Mmp13 ΔPrrx1 mice exhibited increased femoral bone strength. We have not performed an examination of the osteocyte canalicular network in our mice, but the increase in femoral strength we found in Mmp13 ΔPrrx1 mice argues against a biomechanically consequential change of the lacuna-canalicular network.
Cellular senescence is a hallmark of aging [26][27][28][29] and a state in which cells secrete an array of pro-inflammatory cytokines, chemokines and proteases, known collectively as the Senescence Associated Secretory Phenotype (SASP) 30,31 . Work by us and others has shown that osteoprogenitors and osteocytes from old mice exhibit markers of senescence and SASP, including higher levels of MMP13 and CXCL12 and that the adverse effects of aging on murine cortical bone are due, at least in part, to cellular senescence [32][33][34] . As shown herein and in our previously published studies with Cxcl12 ΔPrrx1 mice, both MMP13 and CXCL12 contribute to the loss of cortical bone caused by estrogen deficiency. We find it intriguing that some of the same cytokines may be responsible for the increase in osteoclast number and loss of cortical bone caused by both estrogen deficiency and old age.
In conclusion, the work described herein, adds to and fully supports a long line of evidence that MMP13 plays an important role, not only in bone development, but also during bone remodeling in postnatal life. These effects are evidently mediated by changes in osteoclast number or perhaps activity during the resorption phase of remodeling. Furthermore, Mmp13 is a target gene of ERα signaling in mesenchymal progenitors and their descendants, such as bone marrow stromal cells, osteoblast progenitors, and matrix synthesizing mature osteoblasts. Loss of the restraining effect of estrogens on MMP13 in estrogen deficient states, such as OVX in mice and menopause in women, may therefore be an important culprit of the increased resorption associated with these states. Importantly, even though mesenchymal cell-derived MMP13 influences trabecular bone mass, it plays no role in the loss of trabecular bone caused by estrogen deficiency, highlighting the striking divergence of the cellular targets and downstream mediators of the effects of ERα activation by estrogens in cortical versus trabecular bone. Whether MMP13 plays also a role in the SASP-mediated loss of cortical bone in old age needs to be functionally investigated in future studies.

Methods
Animal experimentation. Mice with conditional deletion of ERα using Osx1-and Prrx1-Cre and respective littermates were generated as previously described 35 . Mice with conditional deletion of Mmp13 in the mesenchymal lineage were generated by a two-step breeding strategy. Hemizygous Prrx1-cre transgenic male mice (B6.Cg-Tg(Prrx1-cre)1Cjt/J; Jackson Laboratories, stock #5584) were crossed with female Mmp13 floxed ( f/f ) mice (FVB.129S-Mmp13 tm1Werb /J, Jackson Laboratories, stock # 005710) to generate mice heterozygous for the Mmp13 floxed allele with and without the Cre allele. These mice were intercrossed to generate Mmp13f. /f (used as control) and Mmp13 ΔPrrx1 mice. Offspring were genotyped by PCR using the following primer sequences: TGA TGA CGT TCA AGG AAT TCA GTT T, wild-type, product size 572 bp, CCA CAC TGC TCG ACA TTG, mutant, product size 372 bp and GGT GGT ATG AAC AAG TTT TCT GAG C, heterozygote, product size 372 bp and 572 bp. Offspring from all genotypes were tail-clipped for DNA extraction at the time of weaning (21 days) and then group-housed with same sex littermates. Mice were maintained at a constant temperature of 23 °C, a 12-h light/dark cycle, and had access to food and water ad libitum. All mice used in these experiments were obtained from the same group of breeders in 2 consecutive breeding cycles separated by 30 days.
Mmp13f. /f and Mmp13 ΔPrrx1 littermate male mice were harvested and analyzed at 16 weeks (13 Mmp13f. /f and 12 Mmp13 ΔPrrx1 ) and 24 weeks of age (14 Mmp13f. /f and 10 Mmp13 ΔPrrx1 ). Twenty-week-old female Mmp13 ∆Prrx1 mice and Mmp13f. /f littermates were either OVX or sham-operated after being stratified by body weight (fifteen Mmp13f. /f and 15 Mmp13 ΔPrrx1 were sham operated; 12 Mmp13f. /f and 12 Mmp13 ΔPrrx1 were OVX). Specifically, within each genotype, mice were sorted from low to high weight values. Mice were then assigned the numbers 1 and 2, successively. All animals with the same number were assigned to the same group. Weight means and standard deviation for each group were calculated and compared by t-test to assure that means were similar. Surgeries were performed in the morning under sedation with 2% isoflurane, as previously described 36 . Mice were injected with calcein (Sigma-Aldrich, C0876; 35 mg/kg body weight) 7 and 3 days before euthanasia for quantification of bone formation. Animals were euthanized 8 weeks after surgery and tissues dissected for further analyses. Whole body weight was measured 2 days before surgery, before calcein injections and before euthanasia. Uterine weights were obtained to confirm depletion of sex steroids. Investigators were blinded during animal handling and endpoint measurements. No adverse events occurred during surgeries, calcein injections and harvest procedures. For bone strength measurements 13 Mmp13f. /f and 14 Mmp13 ΔPrrx1 females with 26 weeks old were euthanized and femurs were harvested. All animal experiments followed ARRIVE guidelines and approved by the animal care guidelines for the Care and Use of Institutional Animal Care and Use Committees of UAMS and the Central Arkansas Veterans Healthcare System. www.nature.com/scientificreports/ Bone imaging. Right femurs and tibias from male and female Mmp13f. /f and Mmp13 ΔPrrx1 mice were dissected, cleaned from adherent muscle and fixed in 10% Millonig's formalin, dehydrated, and kept in 100% ethanol at 4 °C. Two female tibiae were damaged during the harvest procedure and discarded. Femur and tibia lengths were measured with a micrometer followed by micro-CT analysis using a µCT40 (Scanco Medical, Bruttisellen, Switzerland). Bones were scanned at 12 µm nominal isotropic voxel size, 500 projection (medium resolution, E = 55 kVp, I = 72 µA, 4 W, integration time 150 ms and threshold 200 mg/cm 3 ), integrated into 3-D voxel images (1024 × 1024 pixel matrices for each individual planar stack) from the distal epiphysis of the femur and the proximal epiphysis of the tibia towards the mid-diaphysis to obtain a number of slices variable between 650 and 690. Cortical thickness, total and medullary area of the diaphysis were determined using 18 slices at the femur and tibia mid-shafts and cortical thickness of the distal metaphysis was determined analyzing slices 300 to 350 (counting from midshaft region). Cortical analysis was performed with a threshold of 200 mg/cm 3 . Twodimensional evaluation of trabecular bone was performed on contours of the cross sectional acquired images excluding the primary spongiosa and cortex. Contours were drawn starting 8-10 slices away from the growth plate from the distal metaphysis towards the diaphysis of the femur to obtain 151 slices (12 µm/slice), or 8-10 slices away from the growth plate of the proximal metaphysis towards the diaphysis of the tibia to obtain 120 slices (12 µm/slice). For all trabecular bone measurements contours were drawn every 10 to 20 slices. Voxel counting was used for bone volume per tissue volume measurements and sphere filling distance transformation indices were used for trabecular microarchitecture with a threshold value of 220 mg/cm 3 , without pre-assumptions about the bone shape as a rod or plate. Micro-CT measurements were expressed in 3-D nomenclature as recommended by ASBMR standard guidelines 37 .
Histology and histomorphometry analysis. After µCT analysis, the right femurs from female Mmp13f. www.nature.com/scientificreports/ Next, we used FSC-H (height) vs FSC-A (area) dot plot to gate out doublets. Then, we used SSC vs GFP dot plot to gate on GFP positive cells using an unstained sample as a guide to draw our positive gate. The sorted cells were harvested for RNA isolation as described above. One µg total RNA per sample was hybridized to MouseRef-8 v1 Expression beadchips (Illumina, San Diego, CA) following protocols listed on the Gene Expression and Genomics Unit of the NIA (http:// www. grc. nia. nih. gov/ branc hes/ rrb/ dna/ index/ proto cols. htm). Microarray florescent signals were extracted using an Illumina BeadArray 500GX reader. The data analyses of microarray were performed in R software. The signals on each sample are preprocess and normalized using lumi package 40 . The microarray data of this study is deposited at GEO database under accession number GSE191214. Raw signal intensity files from the BeadStudio of all samples -GFP-sorted Osx1 + cells without or with ERα, derived from calvaria cells of ERα ΔGFP:Osx1 mice or GFP:Osx1-Cre controls -were processed together by lumi package 40 under R suite software. Quantile normalization was performed to make data comparable across samples. Differential gene expression between the two groups was evaluated by moderated Student's t test using limma package 41 . The statistical P values were further adjusted for multiple testing using the Benjamini-Hochberg method.
Statistical analysis. For statistical analysis and preparation of graph plots we used GraphPad Prism 9 software, and R (v. 4.0). Data are presented as dot plots with the central line representing the mean and error bars representing standard deviation. After determining that data were normally distributed and exhibited equivalent variances among groups, mean values were compared by two-way ANOVA with Bonferroni multiple comparison test or by unpaired student t-test. When data were not normally distributed, a non-parametric method was used such as Mann-Whitney U test instead of Student's t test (Fig. 1c,d). In cases where data violated ANOVA assumptions (e.g., normality) we used non-parametric Kruskal-Wallis tests and p values were corrected for multiple comparisons using the Benjamin-Hochberg (BH) method (Figs. 2c, 4, 5). Outliers were identified and removed by the ROUTE method with a Q = 1% or by the Grubbs test with α = 5% when data is normally distributed. Exact p values are shown for relevant comparisons. In line with the recommendations of the American Statistical Association, summarized by Amrhein et al. 42 , a threshold value of p was not used to define a statistically significant effect.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.