ABSTRACT
Idiopathic scoliosis (IS) is the most common type of musculoskeletal defect effecting children and is classified by age of onset, location, and degree of spine curvature. Although rare, the onset of IS during infancy is the more severe and rapidly progressive form of the disease, leading to increased mortality due to significant respiratory compromise. The pathophysiology of IS, in particular for infantile IS, remain elusive. Here, we show that PRMT5 is critical for the regulation of terminal hypertrophic chondrocyte differentiation in the spine and models infantile IS in mouse. Conditional ablation of PRMT5 in osteochondral progenitors led to impaired terminal hypertrophic chondrocyte differentiation and asymmetric defects of endochondral bone formation in the perinatal spine. Analysis of several markers of endochondral ossification revealed increased COLX and Ihh expression and a dramatic reduction of Mmp13 and RUNX2 expression in the intervertebral disc and vertebral growth plate. Furthermore, we demonstrate that PRMT5 function in committed chondrogenic lineages is required for regulation of COLX expression in the adult spine. Together, our results establish PRMT5 as a critical regulator of hypertrophic chondrocyte differentiation and endochondral bone formation in spine development and maintenance.
INTRODUCTION
Idiopathic scoliosis (IS) is the most common pediatric spinal deformity characterized by a lateral curvature of the spine >10°, affecting ∼3% of children worldwide (Wise et al., 2008). Clinical sub-classifications of IS are based on age of presentation at infantile (birth to 3 years), juvenile (age 3 to 11 years), and adolescent (11 years and older) ages (Giampietro et al., 2003). Infantile IS develops rapidly and can lead to significant respiratory compromise, increased morbidity, and mortality (Cahill and Samdani, 2012; Davies and Reid, 1970; Pehrsson et al., 1992; Roye, 2018). Therefore, there is a strong need for early diagnosis and rational therapies that might halt or ameliorate the pathogenesis of infantile IS.
The etiology of IS remains poorly understood, however a genetic causality and candidate loci associated with the disease are beginning to be established. For instance, sibling risk studies report that 11.5-19% of siblings share risk for spine curvatures >10° (Riseborough and Wynne-Davies, 1973; Rogala et al., 1978). Large-scale genome wide association studies also implicated several candidate loci associated with IS, including associations with GPR126 (ADGRG6), LBX1, CHL1 and SOX9 genes (Ikegawa, 2016; Kou et al., 2013; Sharma et al., 2011; Takahashi et al., 2011). Some of these candidate genes associated with IS and their effectors, including Adgrg6/Gpr126 (Karner et al., 2015), Sox9 (Henry et al., 2012), Shp2 (Kim et al., 2013), Gdf5/6 (Settle et al., 2003), and Fgf3 (Gao et al., 2015), are known to develop spine defects and scoliosis when mutated in mouse. Interestingly, a majority of these genes are involved with the development and homeostasis of connective tissues and cartilages, indicating a potential link between cartilaginous tissues and the pathogenesis of IS (Liu and Gray, 2018).
The intervertebral disc (IVD) is a cartilaginous tissue that connects each of the vertebral bodies in the spine, functioning to both disperse mechanical loading and convey flexibility to the spine. The IVD is composed of the nucleus pulposus, the inner most gel-like center, which is surrounded by numerous fibrocartilage lamellar layers collectively known as the annulus fibrosus. The IVD is connected to the growth plate and bony vertebrae by the cartilaginous endplate. The vertebrae form via endochondral ossification of the cartilaginous anlage flanking the notochord during embryonic development and elongate through proliferation and differentiation of the growth plate during postnatal development (Smith et al., 2011).
Endochondral ossification is the process by which the majority of the mammalian skeleton is formed. It is a complex process in the vertebral bodies, which is comparable to homologous mechanisms of that in long bone (Karsenty et al., 2009; Long and Ornitz, 2013). This process is characterized by the succession of proliferative, prehypertrophic, hypertrophic, and terminal hypertrophic chondrocytes (Long and Ornitz, 2013). The ossification of the vertebra begins within the center of the cartilaginous vertebral body template. Cells within this center of ossification exit the cell cycle and initiate hypertrophy associated with the secretion of type X collagen (COLX; encoded by Col10a1). The terminal hypertrophic chondrocytes express the matrix metalloproteinase 13 (Mmp13), which is critical for terminal differentiation of hypertrophic chondrocytes and endochondral bone formation (Inada et al., 2004). Mechanistically, MMP-13 aids in the degradation of the COLX rich extracellular matrix surrounding the hypertrophic chondrocytes, while invading blood vessels attracted by vascular endothelial growth factors (VEGFs) to promote the infiltration of bone forming osteoblasts (Karsenty et al., 2009; Long and Ornitz, 2013). While many hypertrophic chondrocytes undergo apoptosis clearing a way for the elaboration of the bony matrix, recent studies demonstrate that a subset of these hypertrophic chondrocytes can transdifferentiate into osteoblasts, directly contributing to bone formation (Jing et al., 2015; Park et al., 2015; Zhou et al., 2014).
Chondrocyte maturation and growth plate development are tightly regulated by a series of growth factors and transcriptional regulators. For example, the transcriptional regulator SOX9 (SRY-related high mobility group-box 9) is required for chondrocyte proliferation and to prevent precocious hypertrophic differentiation (Akiyama et al., 2002; Karsenty et al., 2009). Whereas the transcription factor RUNX2 (Runt related transcription factor 2), which has been shown to directly regulate Col10a1, Mmp13 and Vegfa expression (Hess et al., 2001; Li et al., 2011; Takahashi et al., 2017; Zelzer et al., 2001), is an important driver of hypertrophic chondrocyte differentiation and endochondral ossification (Komori, 2010a; Komori, 2010b; Komori, 2018). Indian hedgehog (Ihh) signaling plays a critical role in chondrocyte proliferation and hypertrophic differentiation via PTHrP dependent and independent processes (Amano et al., 2014; Long and Ornitz, 2013; Mak et al., 2008).
The protein arginine methyltransferase 5 (PRMT5) is required for the maintenance of chondrocyte progenitors in embryonic limb buds (Norrie et al., 2016), where its loss results in precocious Sox9 expression followed by widespread apoptosis (Norrie et al., 2016). Mechanistically, PRMT5 mediates symmetric demethylation of arginine residues on histones H3R8, H3R2, and H4R3, which regulates a diverse set of target proteins (Bedford and Clarke, 2009; Pal et al., 2004; Stopa et al., 2015; Zhao et al., 2009). PRMT5 dependent arginine methylation can also regulate the assembly of the small nuclear ribonucleoprotein (snRNP) complex (Chari et al., 2008; Meister and Fischer, 2002; Stopa et al., 2015), effecting splicing of mRNAs and gene expression. Recent work also demonstrated that PRMT5 dependent posttranslational di-methlyation of SOX9 protein can increase its half-life (Sun et al., 2019). Given the range of possible molecular functions for PRMT5, it is not surprising that this enzyme plays a role in a number of tissue-specific differentiation pathways, including keratinocytes, muscle, nerve cells and lung differentiation (Calabretta et al., 2018; Chittka et al., 2012; Dacwag et al., 2009; Kanade and Eckert, 2012; Li et al., 2018). In this study, we used conditional mouse genetics coupled with histological and gene expression analyses to establish a novel role for PRMT5 during chondrocyte differentiation for perinatal spine development, modeling infantile IS. We additionally demonstrate a continuous role for PRMT5 in growth plate of the IVD in adult mice. Overall, our work establishes PRMT5 as a critical factor for the terminal differentiation of hypertrophic chondrocytes and endochondral ossification of the spine.
MATERIALS AND METHODS
Mice
Animal studies were approved by the Institutional Animal Care and Use Committee at the University of Texas at Austin (AUP-2018-00276 and AUP-2016-00255). All mouse strains, including Prmt5f/f (Prmt5tm2c(EUCOMM)Wtsi) (Norrie et al., 2016), Col2Cre (Long et al., 2001), ATC (Dy et al., 2012), OcCre (Zhang et al., 2002), and Rosa-LacZ (Soriano, 1999), were described previously. Doxycycline (Dox) was administered to ATC; Prmt5f/f and littermate controls or ATC; Rosa-LacZf/+ mice with intraperitoneal (IP) injections by two strategies: (i) starting at 2-weeks of age, once/week (10mg Dox/kg body weight) for 4 continuous weeks, or (ii) starting at 4-weeks of age, once/week (10mg Dox/kg body weight) for 4 continuous weeks. Mice were harvested at P1, P10, 6-weeks, 2.5-months and 4-months of age for tissue analysis.
Analyses of mice
Skeletal preparations were performed as previously described (Allen et al., 2011). Radiographs of mouse skeleton were generated using a Kubtec DIGIMUS X-ray system (Kubtec T0081B) with auto exposure under 25 kV. Cobb angle was measured on high resolution X-ray images with ImageJ as previously described (Cobb, 1948). Histological analysis was performed on thoracic spines fixed in 10% neutral-buffered formalin for 3 days at room temperature followed by 1-week decalcification in Formic Acid Bone Decalcifier (Immunocal, StatLab). After decalcification, bones were embedded in paraffin and sectioned at 5μm thickness. Alcian Blue Hematoxylin/Orange G (ABH/OG) and Safranin O/Fast Green (SO/FG) staining were performed following standard protocols. Quantification of IVD clefts and cell layers were performed on ABH/OG stained sections of 3 control and 3 mutant mice. 3-6 IVDs were analyzed per mouse. Immunohistochemical analyses were performed on paraffin sections after antigen retrieval using 10mM Tris and 1mM EDTA (with 0.05% Triton-X-100, pH 9.0) (PRMT5 and RUNX2), 4mg/ml pepsin (COLII and COLX), 100μg/ml hyaluronidase (PRG4), or 10μg/ml Proteinase K (SOX9), and colorimetric development methodologies with the following primary antibodies: anti-PRMT5 (Abcam, ab109451, 1:100), anti-Collagen Type II (Thermo Scientific, MS235B, 1:100), anti-Collagen Type X (Quartett, 1-CO097-05, 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095, 1:50), anti-Lubricin (PRG4) (Abcam, ab28484, 1:400), and anti-RUNX2 (Medical & Biological Laboratories, D130-3, 1:100). The Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) cell death assay was performed on paraffin sections with the In Situ Cell Death Detection Kit, Fluorescein (Roche) according to the manufacturer’s instructions. Quantification of TUNEL positive cells were performed on sections of 3 control and 3 mutant mice. 2-4 IVDs were analyzed per mouse. The beta-galactosidase staining was performed on frozen sections as previously described (Liu et al., 2015). Briefly, spines were harvested and fixed in 4% paraformaldehyde for 2 hours at 4 °C and decalcified with 14% EDTA at 4 °C for 1 week. Tissues were washed in sucrose gradient, embedded with Tissue-Tek OCT medium, snap-frozen in liquid nitrogen, and sectioned at 10μm with a Thermo Scientific HM 550 cryostat. In situ hybridization using a Digoxygenin-labeled antisense riboprobes for Mmp13, Ihh, and Bmp4 were performed on 5μm paraffin sections as described previously with modifications (Karner et al., 2015), and detected with a tyramine-amplified fluorescent antibody (Perkin Elmer, NEL753001KT).
RNA isolation and Real-time RT-PCR
Intervertebral discs from the thoracic spine of the 2.5-month ATC; Prmt5f/f and control mice (Dox induced from 4-weeks) were isolated in cold PBS, pooled together, snap frozen and pulverized in liquid nitrogen. Three control and three mutant mice were used in each group. Total RNA was isolated using the TRlzol Reagent (Invitrogen, 15596026), and cleaned up with the Direct-zol RNA miniprep kit (Zymo Research, Z2070). Reverse transcription was performed using 500ng total RNA with the iScript cDNA synthesis kit (BioRad). Real-time RT-PCR analyses were performed as previously described (Liu et al., 2015). Gene expression was normalized to b-actin mRNA and relative expression was calculated using the 2-(ΔΔCt) method. Primers sequences are listed in Table S1.
RESULTS
Conditional loss of Prmt5 in the spine models infantile IS in mouse
In order to test the role of PRMT5 in the spine we crossed mice harboring a Prmt5f/f conditional allele (Norrie et al., 2016) to Collagen2al-Cre (Col2Cre) mice (Long et al., 2001). This Col2Cre allele demonstrates robust Cre activity in osteochondral progenitors (OCPs) and effectively labeling most structural elements of the spine including the entire IVD, the periosteum, and trabecular bone (Supplemental Fig. 1). Col2Cre;Prmt5f/f mutant pups were produced at mendelian ratios when assessed at E18.5 (22.2%, n=18). However, all mutant animals died of unknown causes prior to weaning (11.9% survival assessed at Postnatal (P) day 1, n=42; and 5.4% survival assessed at P10, n=62) (Fig. 1A). We did not recover any Col2Cre;Prmt5f/f mutants after P14 (0%, n=23). Two of the mutant mice that survived at P10 (n=4) were much smaller than the control mice (Fig. 1F), while the other two were comparable to the littermate controls, suggesting variable penetrance. Interestingly at E18.5, analysis by whole-mount skeletal prep revealed no obvious defects in the size, patterning, and maturation of the skeleton in the Col2Cre;Prmt5f/f mutants (Fig. 1C; n=4). However, at P10 we observed obvious IS-like thoracic scoliosis (red arrowhead; Fig. 1F) with an average cobb angle of 30±4° in Col2Cre;Prmt5f/f mutants (Fig. 1D; n=4/4). Lateral X-rays had loss of signal attenuation in the distal ribs indicative of reduced ossification in mutant mice (Fig. 1F”) relative to littermate controls (yellow arrowheads; Fig. 1E”). Immunohistochemistry (IHC) against PRMT5 revealed robust protein expression throughout the IVD and growth plate in wild-type mice at P1 and P10, which was consistently reduced in Col2Cre;Prmt5f/f mutant mice (Fig. 1G-J’). We did not observe PRMT5 expression in the trabecular bone or cortical bone (Supplemental Fig. 2), suggesting that PRMT5 has a limited role in committed osteoblast lineages.
Loss of Prmt5 in osteochondral progenitors results in asymmetric defects of endochondral ossification in the spine
Using histological approaches, we observed impaired endochondral ossification of the vertebral body in Col2Cre;Prmt5f/f mutant mice at P1 (Fig. 2B). In addition, mutant IVDs show an increase of midline clefts in the endplate and growth plate (Fig. 2B’, B”, quantified in E), possibly resulting from the failure of midline fusion during the development of the vertebral bodies and the IVDs (Smith et al., 2011). At P10, there is impaired endochondral ossification and reduced trabecular and cortical bone formation in the vertebral body in a highly heterogeneous manner (Fig. 2D, D”); in some instances, with stark asymmetries of endochondral ossification even in adjacent vertebrae (yellow asterisks; Fig. 2D). These regions of persistent cartilage tissues in the vertebrae displayed a general disorganization of the chondrocytes, some of which form into columns or clusters (red, yellow dashed outlines; Fig. 2 D”). These asymmetrical defects in endochondral ossification are likely to translate to anisotropic mechanical properties of the spine, which in turn may drive the onset of scoliosis observed in Col2Cre;Prmt5f/f mutant mice.
We also observed morphological alterations in the mutant IVD at P10. In controls, the IVD endplate contains several layers of flat cells that lightly stain with Alcian blue (green bracket; Fig. 2C’). The proliferative/prehypertrophic zone of the control growth plate is made up of several layers of flat chondrocytes that organize into columns (orange bracket; Fig. 2C’), while the hypertrophic zone of the growth plate is composed of 2-4 layers of enlarged hypertrophic chondrocytes (red bracket, Fig 2C’). In Col2Cre;Prmt5f/f mutant mice we observed alterations of these regions of the IVD. For instance, the cells of the endplate appeared larger, and the surrounding matrix was more heavily stained by Alcian blue (green bracket; Fig. 2D’), suggesting inappropriate differentiation of the endplate tissue. Quantitative analysis in these IVD tissues demonstrated increased number of cell layers in the hypertrophic zone of the growth plate, but not in the endplate of the Col2Cre;Prmt5f/f mutant mice (Fig. 2F). We also observed a mild increased of cell layers in proliferative/prehypertrophic zone (Fig. 2F). Collectively, these data suggest a model in which PRMT5 is critical for development of vertebra formation due to defects in hypertrophic chondrocyte differentiation.
Deletion of Prmt5 in osteochondral progenitors results in altered extracellular matrix components and accumulation of type X collagen in the cartilaginous tissues of the spine
We next assayed several established regulators of hypertrophic chondrocyte differentiation(Long and Ornitz, 2013) in order to assess potential mechanisms for PRMT5-dependent regulation of IVD development. IHC analysis of the hypertrophic chondrocyte marker type X collagen (COLX) revealed ectopic, expanded expression throughout the entire IVD in Col2Cre;Prmt5f/f mutant mice, and highlighted the expansion of the hypertrophic zone of the growth plate (Fig. 3B, B’), compared to controls (Fig. 3C, C’). On the other hand, the expression of proteoglycan 4 (PRG4/ Lubricin), a common marker of healthy IVD (Jay, 1992; Jay and Waller, 2014; Teeple et al., 2015), was remarkably absent in the IVDs of Col2Cre;Prmt5f/f mutant mice at P10 (Fig. 3D, D’). The expression of SOX9, a key transcriptional regulator of chondrogenesis, was reduced in the cartilaginous endplate of mutant mice (Supplemental Fig. 3C-D’), but was not obviously affected in the proliferative growth plate. Hypertrophic chondrocytes of the vertebral growth plate downregulate Sox9 expression during the process of terminal hypertrophic chondrocyte differentiation (Supplemental Fig. 3C’). However, Col2Cre;Prmt5f/f mutant mice displayed ectopic SOX9 expression in these phenotypically hypertrophic cells of the growth plate (Supplemental Fig. 3D’). Despite these transcriptional alterations of Sox9, we found that type II Collagen (COLII) expression was not obviously changed in Col2Cre;Prmt5f/f mutant IVDs at P10 (Supplemental Fig. 3A-B’). Next, we performed the same IHC analysis in the vertebral body of the Col2Cre;Prmt5f/f mutant mice at P10 as shown in (Fig. 2D, D”). Within these areas of persistent cartilage in the vertebral bodies, we observed expression of SOX9 and COLX but not PRG4 expression (Supplemental Fig. 4), suggesting these persistent areas of cartilage in the vertebral bone were the result of similar alterations of hypertrophic chondrocyte differentiation as was observed in the vertebral growth plate. Together our results suggest that PRMT5 regulates the normal development of hypertrophic chondrocytes of the growth plate and vertebral bodies.
PRMT5 regulates the expression of critical factors of chondrocyte differentiation during endochondral ossification in the spine
We next set out to determine the mechanisms by which PRMT5 regulates terminal hypertrophic differentiation. RUNX2 is a critical driver of terminal differentiation of hypertrophic chondrocytes and osteoblast differentiation in mouse (Long and Ornitz, 2013; Takarada et al., 2013), and acts in part through the activation of Mmp13 (Inada et al., 1999; Komori, 2010a; Komori, 2018). The expression of RUNX2 was markedly diminished in Col2Cre;Prmt5f/f mutant IVD at P10 (Fig. 4B, B’), while RUNX2 expression in control mice remained robust throughout the IVD (Fig. 4A, A’). In contrast, we did not observe alterations of RUNX2 expression earlier in development in Col2Cre;Prmt5f/f mutant IVDs at P1 (Supplemental Fig. 5A, B). Next, we performed fluorescent in situ hybridization (FISH) using an Mmp13 specific riboprobe on medial sectioned IVDs. At P10, there was an obvious reduction of Mmp13 expression in the growth plate and trabecular bone of the Col2Cre;Prmt5f/f mutants (Fig. 4D-D”) compared with typical robust expression pattern in the hypertrophic zone in control IVDs (Fig. 4C-C”). Col2Cre;Prmt5f/f mutant mice also had an obvious decrease in Mmp13 expression in the presumptive center of ossification and vertebral growth plate at P1 (Supplementary Fig. 5D-D”), compared with a robust Mmp13 signal in ossification center and hypertrophic growth plate in control mice (Supplementary Fig. 5C-C”). Interestingly, we observed ectopic Mmp13 signal within the nucleus pulposus and the endplate in mutant IVDs at P1 (Supplemental Fig. 5D-D”), which was not observed in control mice. The consistent reduction of RUNX2 and - its downstream effector - Mmp13 in the IVD and vertebral growth plate of Col2Cre;Prmt5f/f mutant mice supports a mechanistic role for PRMT5 in the regulation of hypertrophic chondrocytes differentiation and endochondral ossification during perinatal development of the spine.
To better understand the regulatory mechanisms of PRMT5 during endochondral bone formation, we next assayed Indian hedgehog (Ihh) expression in both control and Col2Cre;Prmt5f/f mutant mice. Ihh signaling maintains chondrocyte proliferation and negatively regulates chondrocyte hypertrophy by regulation of PTHrP expression (Long and Ornitz, 2013). On the other hand, Ihh acts independently of PTHrP to promote chondrocyte hypertrophy, and induces COLX expression through RUNX2/Smad interactions (Amano et al., 2014; Mak et al., 2008). FISH analysis with an Ihh specific riboprobe shows comparable expression pattern in both control and Col2Cre;Prmt5f/f mutant mice at P1, confirming that loss of PRMT5 does not affect early hypertrophic differentiation (Supplemental Fig. 5E, F). However, at P10 we observed a dramatic increase of Ihh expression in the mutant IVD and growth plate (Fig. 4F-F”), including abnormal expression of Ihh in the proliferative growth plate, as well as in the majority of cells of the endplate and annulus fibrosus (Fig. 4F-F”). Our results demonstrate that PRMT5 regulates terminal hypertrophic differentiation through positive regulation of RUNX2 and Mmp13 expression and by negative regulation of Ihh expression during perinatal development, resulting in an obvious delay of terminal hypertrophic chondrocyte differentiation. We suggest this is in part due to the loss of RUNX2 and Mmp13 expression which promote terminal hypertrophic chondrocyte differentiation as well as, due to increased, ectopic Ihh expression may act to counter hypertrophic differentiation. Taken together, our results suggest that misregulation of several critical regulators of hypertrophic chondrocyte differentiation leads to attenuated terminal hypertrophic differentiation in vertebral growth plate, changes in gene expression in the IVD, and ultimately leads to impaired endochondral ossification during perinatal spine development (see discussion).
PRMT5 regulates Bmp4 expression in the spine
Loss of Prmt5 in limb bud and lung tissue induces ectopic, elevated Bmp4 expression which is accompanied by increased apoptosis in these tissues (Li et al., 2018; Norrie et al., 2016). Consistent with previous observations in Prmt5-deficient limb (Norrie et al., 2016) and lung epithelium (Li et al., 2018) in mouse, we also observed increased ectopic Bmp4 signal in the growth plate of Col2Cre;Prmt5f/f mutant IVDs at P10 that was not present in controls (Supplemental Fig. 6B, B’). We did not observe upregulation of Bmp4 in the IVD at P1 mice in either genotype (Supplemental Fig. 6C-D’), suggesting that this increased expression accumulates during perinatal development. We also detected a mild increase in cell death in the IVDs of Col2Cre;Prmt5f/f mutant mice in the annulus fibrosus, endplate, and growth plate regions at both P1 and P10 (Supplemental Fig. 7B, D, E, F). Taken together, these data demonstrate that as in other tissues and organs PRMT5 is required for regulation of Bmp4 and apoptosis in the cartilaginous tissues of the IVD and growth plate.
PRMT5 is not required in the mature osteoblast lineages for spine development and stability
Giving the clear alterations in endochondral bone formation in Col2Cre;Prmt5f/f mutant mice, we next sought to determine if PRMT5 also functions in committed bone-forming lineages. To address this, we utilized an Osteocalcin (Oc) Cre transgenic mouse to specifically remove PRMT5 function in mature osteoblasts (Zhang et al., 2002). These conditional mutant mice were born at Mendelian ratios and were all adult viable, displaying no obvious phenotypes (n=7). X-ray analysis showed no scoliosis in the OcCre;Prmt5f/f mutant mice at P10 (Supplemental Fig. 8A, B, n=4) or at 2 months of age (data not shown, n=3). Histological analysis at P10 showed no obvious alterations of spine tissues in OcCre;Prmt5f/f mutant mice (Supplemental Fig. 8C, D). As described previously we also did not detect PRMT5 expression in the cortical or trabecular bone of the spine (Supplemental Fig. 2). Taken together, we conclude that PRMT5 functions in chondrocyte lineages to promote endochondral ossification, and does not have an obvious role in mature osteoblast lineages for this process.
PRMT5 has a role in homeostasis of the adult intervertebral disc
To gain insight into whether PRMT5 has a role in homeostasis of the spine in adult mice, we assayed the expression of PRMT5 in wild-type IVD by IHC at 6-weeks and 4-months-of-age. We observed low level of PRMT5 expression in a group of cells bordering the ligament insertion and perichondrium region of the IVD at both time points (Supplemental Fig. 9).
Given indication of continued PRMT5 in adult IVD, we decided to assay the role of PRMT5 in spinal homeostasis. To specifically remove PRMT5 function in cartilaginous tissues of the spine in adult mouse, we cross Prmt5f/f mice with an Acan enhancer-driven, Tetracycline-inducible Cre (ATC) transgenic mouse which targets committed chondrocyte lineages (Dy et al., 2012), and induced recombination from 4 to 8 weeks of age (Fig. 5A). Beta-galactosidase staining in ATC;Rosa-LacZ f/+ mice revealed near complete recombination in nucleus pulposus, endplate, and annulus fibrosus of the IVD, as wells as over 50% recombination in growth plate of the at one week post injection (5 weeks-of-age) using this protocol for recombination (Fig. 5C). We first assayed several markers of IVD using qPCR analysis of whole IVD cDNA libraries in experimental groups at 6 weeks post injection (2.5 months-of-age) (Fig. 5A). We found that consistent with the observations in Col2Cre;Prmt5f/f mutant mice, there was a ∼2-fold reduction in Prmt5 expression, as well as ∼2-fold reduction in the expression of Mmp13 and Prg4, respectively. In contrast, the expression of Sox9, Col2a1, Acan, and Col10a1 were not strongly affected at this time point (Fig. 5D). By the age of 4 months (3 months post induction), there were no overt signs of degenerative histopathology in ATC;Prmt5f/f mutant IVD (Fig. 5F), with the exception of a minor increase of acellular clefts at the midline of the endplate (yellow arrow; Fig. 5F), which we occasionally observe in wild type IVD as well (p=0.047; two-tailed student t test; n=3 mice for each group). However, we observed increased, ectopic COLX expression in the endplate and growth plate of ATC;Prmt5f/f mutant IVDs (Fig. 5H, H’). Taken together, our data shows that loss of PRMT5 in adult IVD results in reduced expression of normal extracellular matrix component Prg4, coupled with increased expression of hypertrophic marker COLX and decreased expression of Mmp13, consistent with a continuous role for PRMT5 in regulating hypertrophic chondrocyte turnover and homeostasis of the IVD in adulthood. We did not observe scoliosis in ATC;Prmt5f/f mutant mice when induced from either 2-weeks-of-age or 4-weeks-of-age by dorsal X-ray imaging (Supplemental Fig. 10 and data not shown). Taken together, our study demonstrates a novel role for PRMT5 in cartilaginous lineages for the regulation of adult IVD homeostasis, but has a limited temporal role during perinatal development for the regulation of spine stability.
DISCUSSION
The formation of the vertebral bodies, like the long bones, ossify through a process of endochondral ossification where hypertrophic chondrocytes are replaced by and in some cases contribute to bone-forming osteoblast lineages (Aghajanian and Mohan, 2018). Our findings indicate that PRMT5 has an important role in the process of terminal differentiation of hypertrophic chondrocytes and endochondral bone formation, in part by positive regulation Mmp13 and RUNX2 expression and negative regulation of Ihh expression. We observed that loss of PRMT5 function in osteochondral progenitors results in the expansion of hypertrophic chondrocytes in the vertebral growth plate. However, ossification of the vertebrae is severely impaired during perinatal development, mimicking an Mmp13 loss-of-function phenotype (Inada et al., 2004). Finally, we show that PRMT5 regulation of perinatal endochondral bone formation is a potential mechanism underlying infantile IS. Taken together, these results establish PRMT5 as a fundamental regulator of terminal hypertrophic chondrocyte differentiation and endochondral bone formation, as well as a critical regulator for perinatal spine integrity.
Regulation of Terminal Chondrocyte Differentiation and Endochondral Bone Formation by PRMT5
One of the most important regulators of hypertrophic chondrocyte differentiation and endochondral bone formation is RUNX2 (Komori, 2010b; Komori, 2018). Runx2-deficient mice exhibit a complete loss of mature osteoblasts and endochondral bone formation (Chen et al., 2014; Ducy et al., 1997; Takarada et al., 2013). RUNX2 has been shown to directly regulate Col10a1, Mmp13 and Vegfa expression (Hess et al., 2001; Li et al., 2011; Takahashi et al., 2017; Zelzer et al., 2001), and therefore plays a crucial role in driving chondrocyte differentiation and endochondral ossification (Fig. 6A). Mmp13, a direct target of RUNX2 (Fig. 6A), is also required for these processes as mice lacking Mmp13 display expanded hypertrophic zone and delay in endochondral ossification in the long bone (Inada et al., 2004; Stickens et al., 2004). Here, we found that Col2Cre;Prmt5f/f mutant mice displayed an obvious reduction in RUNX2 and Mmp13 expression in proliferative and hypertrophic chondrocytes of the perinatal growth plate (Fig. 4 and Supplemental Fig. 5). Meanwhile, the Col2Cre;Prmt5f/f mutant mice showed an expansion of the hypertrophic growth plate with ectopic, expanded COLX expression. These results indicate that loss of PRMT5 in osteochondral progenitors allows hypertrophic differentiation to commence; however, the process of removing COLX positive hypertrophic cells is severely impaired due to loss of RUNX2/Mmp13 expression (Fig. 6B). How hypertrophic chondrocyte differentiation is able to proceed, albeit in a severely delayed manner, without these factors is still under investigation. It is possible that undetectable levels of these proteins are being made or that other RUNX-family members or co-factors that are important for driving terminal differentiation, such as RUNX3, MEF2C, or FOXA2 as indicated in long bone (Arnold et al., 2007; Tan et al., 2018; Yoshida et al., 2004), are able to act in a semi-redundant manner for this process.
Another important regulator of hypertrophic chondrocyte differentiation is IHH. Ihh signaling plays critical role in both chondrocyte proliferation and hypertrophic differentiation. It can directly promote proliferation and inhibit premature hypertrophy of the proliferative chondrocyte via IHH/PTHrP negative feedback loop (Long and Ornitz, 2013). On the other hand, it can promote hypertrophy and induce COLX expression in a PTHrP independent manner (Amano et al., 2014; Mak et al., 2008). We observed that Col2Cre;Prmt5f/f mutant mice displayed ectopic expansion of Ihh expression throughout the hypertrophic growth plate, proliferative growth plate, and some cells within the endplate of IVD at perinatal (P10) stage but not neonatal stage (P1) (Fig. 4F and Supplemental Fig. 5F), which overlaps with the regions of ectopic, expanded COLX expression (Fig. 2B and Fig. 6B). These results indicate that loss of PRMT5 in osteochondral progenitors allows chondrocyte proliferation and hypertrophic differentiation, however it fails to shut down/restrict Ihh expression during perinatal development, which may contribute to increased production and accumulation of COLX positive cells in the growth plate. We speculate that the misregulation of Mmp13 and Ihh in Col2Cre;Prmt5f/f mutant mouse spines act synergistically to promote the accumulation of hypertrophic chondrocytes in the growth plate and inhibit their terminal differentiation, which leads to reduced endochondral bone formation during perinatal skeletogenesis.
In addition, PRMT5 has been shown to regulate Bmp4 expression in several contexts. For example, loss of Prmt5 in mouse mesenchymal progenitor cells led to upregulated Bmp4 in mouse limb (Norrie et al., 2016) and PRMT5 directly associated with chromatin of Bmp4 to suppress its transcription during lung branching morphogenesis (Li et al., 2018). In agreement, Col2Cre;Prmt5f/f mutant mice also display ectopic elevation of Bmp4 expression in the hypertrophic growth plate (Supplemental Fig. 6). Interestingly, in vitro studies have shown that Bmp4 can stimulate chondrocyte hypertrophy and induce COLX expression (Clark et al., 2009; Hatakeyama et al., 2004; Minina et al., 2001; Steinert et al., 2009). However, whether PRMT5-dependent regulation of Bmp4 expression is related to defective hypertrophic chondrocyte differentiation remains to be determined.
Interestingly, the formation of the cartilaginous templates and some ossification occurs normally in these conditional Col2Cre;Prmt5f/f mutant mice (Fig. 1C) despite Prmt5 deletion in osteochondral progenitor lineages. This is in contrast to the more severe loss of cartilage template in Prx1Cre;Prmt5f/f, resulting in a dramatic reduction of the mouse forelimb (Norrie et al., 2016). Taken together, these findings support a model of distinct mechanistic roles PRMT5 function (i) for initiation of early chondrocyte progenitors and (ii) for regulation of hypertrophic chondrocyte differentiation.
PRMT5 regulates homeostasis of the adult IVD
We found that postnatal loss of PRMT5 in cartilaginous tissues of the IVD resulted in alterations of the normal gene expression including reduced Prg4 expression as well as increased COLX expression, which are both markers of early-onset degenerative disc. These findings demonstrate that PRMT5 has an important role in the homeostasis of cartilaginous tissues of the IVD. It will be important to determine if the ablation of PRMT5 in the IVD of mature adult mice can generate susceptibility to the onset of pathogenic changes of the IVD and spine due to trauma or aging in mice. While the IVD has been considered an organ with little or no regenerative capacity, several studies have recently identified the presence of cells expressing stem/progenitor markers in this tissue (Blanco et al., 2010; Henriksson and Brisby, 2013; Risbud et al., 2007). Interestingly, a study using BrdU labeling identified label-retaining cells in the annulus fibrosus located in a region bordering the ligament insertion and the perichondrium region (Henriksson et al., 2009). This region overlaps with the expression of PRMT5 in the adult IVD in mouse (Supplemental Fig. 9), and suggests a possible role for PRMT5 in maintaining adult progenitor/stem cell pools which are critical for IVD homeostasis. Additional labeling studies will be necessary to test this model.
Asymmetrical properties of the spine result in scoliosis
The molecular genetics and underlying pathology of IS are largely unknown, more so for infantile onset forms of the disease. Asymmetries of the vertebral body growth and of the mechanical properties of the bony prominences, cartilaginous joints of the vertebral bodies, and musculature attachments of the spinal column have long been hypothesized to underlie the formation of idiopathic scoliosis (Liu and Gray, 2018). Our results suggest that the onset of infantile IS in Col2Cre;Prmt5f/f mutant mice is the result of obvious defects in endochondral ossification of the spine. We suggest a model where asymmetrical defects of ossification of the vertebrae result in anisotropic mechanical properties of the spinal column causing vertebral rotation and scoliosis of the spine during perinatal development. This model is supported by our observation of a complete lack of scoliosis in conditional ATC;Prmt5f/f mutant mice recombined at 2 or 4-weeks-of-age, which also display alterations in terminal chondrocyte differentiation, yet have a spinal column that has already undergone substantial ossification. This suggests that infantile IS may be the result in subtle defects and delays in endochondral ossification during perinatal development. Our current studies suggest that the cartilaginous tissues play a prominent role in the pathogenesis of infantile IS. In agreement, genetic defects in osteochondral progenitors of the spine inducing IS in mice also occurs in other genetic mouse models displaying IS (Liu and Gray, 2018), including conditional mutant mice of the Gpr126/Adgrg6 (Karner et al., 2015), Sox9 (Henry et al., 2012), and Gdf5/6 (Settle et al., 2003) genes. These findings underscore the importance of proper cartilage maturation, including endochondral ossification, for maintaining spine stability during maturation and development of the spine. Taken together, our data for the first time demonstrates that PRMT5 is a critical factor for perinatal spine stability via regulation of hypertrophic differentiation pathways. Our study further demonstrates that PRMT5 may have an important function in IVD progenitor/stem cells for maintenance of IVD homeostasis. Therefore, PRMT5 may provide a key target for the development of novel therapeutics to treat infantile IS and degenerative changes to the IVDs.
Authors’ roles
Study design: ZL and RSG. Study conduct and Data collection: ZL and JR. Data analysis: ZL. Data interpretation and approving final version of manuscript: ZL, JR, SV and RSG. ZL and RSG take responsibility for the integrity of the data analysis.
ACKNOWLEDGEMENTS
We thank Dr. Fanxin Long for sharing Col2Cre mice and Dr. Véronique Lefebvre for sharing ATC mice. We thank Dr. Matthew Hilton for providing the Mmp13 and Ihh in situ probe templates. We acknowledge Dr. Courtney Karner for helpful comments on this manuscript prior to submission. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01-AR072009 (R.S.G.), F32-AR073648 (Z.L.). It is also supported by NIH grant R01-HD073151 (S.V.), and a UT Austin Provost Graduate Excellence Fellowship (JR).