Heparan sulfate (HS) regulates diverse cell signalling events in intervertebral disc development and homeostasis. The aim of the present study was to investigate the effect of ablation of perlecan HS/CS on murine intervertebral disc development. Genetic models carrying mutations in genes encoding HS biosynthetic enzymes have identified multiple roles for HS in tissue homeostasis. In the present study, we utilised an Hspg2 exon 3 null HS/CS-deficient mouse to assess the role of perlecan HS in disc cell regulation. HS makes many important contributions to growth factor sequestration, stabilisation/delivery, and activation of receptors directing cellular proliferation, differentiation, and assembly of extracellular matrix. Perlecan HS/CS-mediated interactions promote extracellular matrix assembly/stabilisation and tissue functional properties, and thus, removal of perlecan HS/CS should affect extracellular matrix function and homeostasis. Hspg2 exon 3 null intervertebral discs accumulated significantly greater glycosaminoglycan in the nucleus pulposus, annulus fibrosus, and vertebral growth plates than C57BL/6 wild-type (WT) I intervertebral discs. Proliferation of intervertebral disc progenitor cells was significantly higher in Hspg2 exon 3 null intervertebral discs, and these cells became hypertrophic by 12 weeks of age and were prominent in the vertebral growth plates but had a disorganised organisation. C57BL/6 WT vertebral growth plates contained regular columnar growth plate chondrocytes. Exostosis-like, ectopic bone formation occurred in Hspg2 exon 3 null intervertebral discs, and differences were evident in disc cell maturation and in matrix deposition in this genotype, indicating that perlecan HS/CS chains had cell and matrix interactive properties which repressively maintained tissue homeostasis in the adult intervertebral disc.
Heparan sulfate (HS) is an ancient glycosaminoglycan and a highly conserved molecule which has persisted throughout vertebrate and invertebrate evolution over the last 500 million years . Cells invest a significant amount of genetic information which encodes the biosynthetic enzymatic machinery required for the production of HS. The phylogenetic conservation of HS through vertebrate evolution points to vital properties this glycosaminoglycan conveys which ensures cellular survival.
Nearly all vertebrate cells express HS proteoglycans at the cell surface . The persistence of HS through millions of years of vertebrate evolution illustrates the importance of HS in physiological processes essential to cellular survival [3–5]. Over 20 biosynthetic enzymes are required for the synthesis of HS, and thus, this is a significant investment in genetic information for a cell to make [6–8]. HS proteoglycans are indispensable during embryonic development and also have essential regulatory functions in adulthood in varied pathophysiological processes [6–8]. We have previously shown the essential roles HS/CS perlecan plays in foetal human spinal development . HS proteoglycans bind to many secreted signalling proteins, including growth factors, cytokines, and morphogens, which affect their distributions in tissues and regulate cellular behaviour [7,8]. The HS chains are heterogeneous and have variable chain lengths, and may differ with regard to both degree and pattern of sulfation in specific domains which are both highly (S domain) and minimally sulfated (NS domain) in the one glycosaminoglycan chain. Glycosaminoglycans are components of the glycocalyx of all cells and the first point of contact between that cell and neighbouring cells, with the extracellular matrix or with any invading organism . Thus, there were heightened evolutionary pressures on these front-line glycosaminoglycans to develop recognition and effector functions, and a major positive selection stimulus for their diversification [11,12]. Thus, many structural permutations were explored, and the complexity of HS structures, which have persisted to the present day, is all the more remarkable when the intricately organised, precise, sequential stages in HS assembly are considered. The development of molecular recognition, information and storage, and information transfer properties in glycosaminoglycans such as HS is a remarkable feature and one which was perfected over a very significant evolutionary period [1,11,12].
Perlecan (HSPG2) has three Asp-Gly-Arg glycosaminoglycan attachment points in domain I. In perlecan synthesised by endothelial cells, all three glycosaminoglycan attachment sites are occupied by HS; however, most other cell types of synthesise perlecan where at least one of these HS chains is replaced by chondroitin sulfate (CS). Additional CS attachment sites have been reported in perlecan domain V; however, in the present study, we could find no evidence that this site was occupied by glycosaminoglycan.
The aim of the present study was to specifically ascertain the contribution of perlecan HS/CS to developmental processes in the murine intervertebral disc and to determine whether the HS chains of other HS proteoglycans can compensate for a lack of perlecan HS. Perlecan exon 3 encodes perlecan domain 1, and its deletion in Hspg2 exon 3 null mice results in a 22 kDa size reduction in the mutant perlecan core protein. Mouse perlecan also contains 14 immunoglobulin repeats in domain IV compared with 21 repeats present in human perlecan further reducing the size of the murine perlecan core protein . Previous studies with HS-deficient mice have demonstrated a reduction in the deposition of transforming growth factor (TGF)-β1 in skin, defects in fibroblast growth factor (FGF)-2 signalling, delayed vascular wound repair, altered chondrocyte responses, and reduced osteophyte formation in a murine model of post-traumatic osteoarthritis, indicating that lack of HS was chondroprotective [14–17]. Given the importance of HS in extracellular matrix organisation and tissue function [3,18], it was rather surprising that a study in tail and Achilles tendons showed that HS deficiency did not detrimentally affect tendon function but resulted in smaller more compact collagen fibrils in the collagen fibre bundles of tail and Achilles tendons in mutant mice resulting in a modest increase in their material properties .
Henriksson et al. [20,21] have identified a stem cell niche at the juncture of the vertebral growth plate and outer annulus fibrosus in rabbit, minipig, rat, and human intervertebral discs. Shu et al.  also identified a discrete progenitor cell population in the same location in the newborn ovine and foetal human intervertebral disc. Perlecan was prominently produced by these progenitor/stem cells and is a component of many stem cell niches [23–27], including the perichondrium in the developmental human foetal knee, hip, and elbow [26,28]. The mouse also contains cells typical of a progenitor/stem cell niche in the outer annulus fibrosus at its juncture with the vertebral growth plate . Activated stem cells released from the niche environment express many CS sulpfation motifs including 4-C-3, 7-D-4, 3-B-3(−)  and the cell-fate receptor Notch-1 . Activated progenitor cell populations are actively involved in diarthrodial joint development [31–35], and the CS sulfation motifs they express are considered markers of tissue morphogenesis [30,35]. Particular CS sulfation motifs on stem cell surface proteoglycans in developmental studies on the human foetal intervertebral disc and knee [33,34,36,37] are useful as markers of tissue morphogenesis. Chondrocytes from superficial cartilage have also been sorted by flow cytometry using antibodies to these CS sulfation motifs to isolate progenitor cells . The isolated 4-C-3, 7-D-4, and 3-B-3(−)-positive chondrocytes were capable of synthesising a full depth neocartilage which displayed a stratified structure with appropriate proteoglycan and collagenous organisation in the superficial, mid, and deep cartilage zones identical with that seen in articular cartilage . Chondrocyte morphologies were also typical of each cartilage zone (superficial, mid, and deep zones) in this de novo neocartilage produced in vitro .
The role of the HS chains on the large modular proteoglycan perlecan in growth factor signalling and how these regulate disc cell populations in developmental processes is a topical subject . In the present study, we examined glycosaminoglycans from the Hspg2 exon 3 null (HS-deficient) mouse to determine the roles of the perlecan HS/CS chains on the regulation of cellular behaviour, matrix organisation, and tissue homeostasis.
Materials and methods
Pre-poured, 12-well, 1 mm thick NuPAGE 3–8% (w/v) SDS–Tris–acetate polyacrylamide gradient gels, electrophoresis application, and transfer buffers and HiMark® pre-stained protein standards were Novex products (Invitrogen, Mount Waverley, Victoria, Australia). Spectra® pre-stained protein standards were obtained from ThermoFisher, Pierce Biotechnology, Rocklands, IL, U.S.A. Resource Q anion exchange fast protein chromatography (FPLC) columns were purchased from GE Healthcare, Amersham Biosciences, Uppsala, Sweden. High-affinity Nunc Immuno Maxisorp flat bottom 96-well polystyrene microtitre plates, Chondroitinase ABC, GuHCl, Tris-free base, Tricine, and bovine serum albumin were purchased from Sigma–Aldrich, Sydney, Australia. Antibodies to perlecan domain III (MAb 7B5), IV (MAb A7L6, monoclonal rat anti-mouse perlecan domain IV), or to perlecan domain V (MAb H300, polyclonal rabbit anti-mouse perlecan domain V) were purchased from Zymed, Abcam (Melbourne, Victoria, Australia) or Santa Cruz Biotechnology, CA, U.S.A., respectively. Menzel and Glaser SuperFrost UltraPlus, positively charged microscope sides were obtained from Fisher Scientific, Braunschweig, GmbH. NovaRED substrate was obtained from Vector Laboratories (Burlingame, CA, U.S.A.). Rabbit or goat anti-mouse, rat, and rabbit IgG secondary antibodies conjugated with alkaline phosphatase, nitrocellulose membranes, and NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3'-indolyphosphate) development kits were purchased from Bio-Rad, North Ryde, Australia, and Heparitinase III antibodies to native HS and enzymatically generated stub epitopes (10-E-4, 3-G-10 MAbs) were purchased from Seikagaku Corp, Tokyo, Japan. Centrifugal diafiltration devices were purchased from Millipore, Sydney, Australia. 2-B-6 and 7-D-4 hybridoma-conditioned media were gifts from Prof. Bruce Caterson, University of Cardiff, U.K.
Hspg2 exon 3 null homozygous mouse breeding pairs backcrossed into a C57BL/6 background for 12 generations were kindly supplied by Dr R. Soinninen, University of Oulu BioCentre (Oulu, Finland). WT C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, Maine, U.S.A.). The WT and Hspg2 exon 3 null mouse colonies were maintained in the Kearns Facility of the Kolling Institute (Royal North Shore Hospital, St Leonards, Australia). Ethical approval for the present study was obtained from The Animal Care and Ethics Review Board of The Royal North Shore Hospital; RNSH Protocol 0709-035A Dr J. Melrose, Associate Prof. C.B. Little, Dr R.C. Appleyard Evaluation of Δ3-/Δ3- HSPG2-deficient mice.
All mice were caged in groups (n = 2–5 mice per 500 cm2 cage floor space) and received acidified water and complete pelleted food ad libitum. All cages were individually ventilated with filter lids, sterilised Aspen chip bedding, environmental enrichment (tissues, house), and maintained at 20–22°C, 50–60% (v/v) humidity, with a 12 h light–dark cycle regimen. Only male mice were used, in the present study, to avoid complicating and variable contributions from female sex hormones which are known to affect cartilage and bone metabolism. Tissue sampling methods are outlined below. Mice from the two genotypes were weighed throughout the full experimental period to assess any changes in body mass.
Genotyping of Hspg2 exon 3 null mice
Perlecan exon 3 null mice had a pGK-neo cassette inserted to replace exon 3 in the Hspg2Δ3−/Δ3− mice resulting in a 22 kDa reduction in size of the mutant perlecan core protein. Genomic DNA from WT and Hspg2 exon 3 null murine tail tips was isolated using commercial kits (Qiagen). Regions of the murine Hspg2 gene were amplified by PCR using primers for intron 2 of mouse Hspg2 (GTA GGG ACA CTT GTC ATC CT), exon 3 (CTG CCA AGG CCA TCT GCA AG), and exon 3 null (AGG AGT AGA AGG TGG CGC GAA GG), and the PCR products were identified following separation by 2% (w/v) agarose gel electrophoresis. This identified 310 and 445 bp bands amplified from PCR of the genomic DNA of the WT and Hspg2Δ3−/Δ3− mice.
Histological processing of spinal segments
Six thoracolumbar spines from each of the mouse genotypes of 6- and 12-week-old animals were examined in the present study, and eight intervertebral discs were examined per spine giving 48 intervertebral discs from each sample group for each time point examined. Prior to fixing, the spinal segments were trimmed of all surrounding soft connective tissue and fixed in 10% (w/v) neutral-buffered formalin for 48 h, then decalcified in 10% (v/v) formic acid and 5% (v/v) neutral-buffered formalin with constant agitation and frequent changes of decalcification solution for up to 5 days. The specimens were then dehydrated in serial graded ethanols and xylene, and embedded in paraffin wax. Serial longitudinal 4 μm vertical microtome sections were cut from the embedded tissue blocks. Approximately fifty sections were taken until mid-sagittal sections were available, and these were used in comparisons between spines in each genotype. Spine sections were attached to SuperFrost Plus glass microscope slides (Menzel-Glaser, Germany), de-paraffinised in xylene (two changes × 5 min), and re-hydrated through graded ethanol washes (100–70% (v/v)) to water.
Toluidine blue staining: Mid-line sagittal longitudinal sections of the mouse spinal segments (4 μm) were stained for 10 min in 0.04% (w/v) toluidine blue in 0.1 M sodium acetate buffer, pH 4.0, to visualise glycosaminoglycan followed by a 2-min counterstain in 0.1% (w/v) fast green FCF. A standardised protocol was used to minimise variation in toluidine blue staining intensity, and the slides were stained as a single batch and equivalent regions of the intervertebral disc, and the spinal level was examined in each genotype.
Quantitation of toluidine blue staining using Adobe Photoshop CS-4 software
Tiff files (8-bit, RGB colour space) of images of the 12-week toluidine blue-stained tissue sections were opened in Photoshop and the wand tool was used to delineate specific areas of toluidine blue staining. Toluidine blue staining of the vertebral growth plate, nucleus pulposus, and the glycosaminoglycan-rich matrix surrounding the anterior progenitor cell population was quantified separately for each of the mouse genotypes. Quantitation of the inner and mid-annulus fibrosus was only possible in the Hspg2 exon 3 null slides due to the extremely low annulus fibrosus staining in the other samples. Measurement of the area of the toluidine blue-stained regions of interest was taken, and the intensity and number of stained pixels in the blue channel were measured, and the integrated toluidine blue staining for each of the areas was calculated. This was used as an index of the toluidine blue proteoglycan staining in each region of interest and the data were used to confirm the glycosaminoglycan quantitation obtained by ELISA.
Extraction of perlecan from tissues
Hip cartilage was harvested from twelve 3-week-old mice and finely diced and extracted in 4MGuCl 50 mM Tris–HCl (pH 7.2) containing broad-spectrum protease inhibitors (Roche Australia, North Ryde, NSW, Australia) for 72 h at 4°C with constant end-over-end mixing. Pooled mouse longissimus or multifidus muscle was harvested from the dorsal spine of six animals, and plantaris muscle was also harvested from the hind legs of two animals and finely diced. Muscle tissues were extracted with 2 M NaCl + protease inhibitors for 48 h at 4°C. Tissue extracts were then subjected to centrifugal diafiltration to remove the extractants and equilibrate them in appropriate buffers for SDS–PAGE or Resource Q anion exchange FPLC.
SDS–PAGE and immunoblotting of perlecan
Aliquots of longissimus/multifidus muscle extracts (20 μg protein) from each of the mouse genotypes were electrophoresed on pre-cast 3–8% (w/v) polyacrylamide–SDS–Tris–acetate gradient mini-gels for 1 h at 200 V in Tris–Tricine buffer (50 mM Tricine, 50 mM Tris base, 0.1% (w/v) SDS, pH 8.3). Molecular mass standards (HiMark® and Spectra®) were also electrophoresed on each gel. The gel was stained with Coomassie R250. Segments of gel corresponding to the elution position of perlecan were excised from unstained gel segments and the proteins were electroeluted using a little blue tank electroelutor/electroconcentrator cell (Isco through Nova Biotech, CA, U.S.A.) for further examination by immunoblotting and by ELISA to identify the glycosaminoglycans present on the perlecan core protein. Samples were then transferred onto nitrocellulose membranes using 5 mM Bicine, 5 mM Bis–Tris, 0.2 mM EDTA, 0.005% (w/v) SDS, 10% (v/v) methanol, pH 7.2, in a semi-dry blotter at 300 mA and 20 V for 1 h. The membrane was blocked with 1% (w/v) bovine serum albumin in Tris-buffered saline for 2 h at room temperature followed by incubation with rabbit anti-perlecan domain V (1/1000 dilution) at 4°C overnight. After washing, anti-rabbit IgG conjugated with alkaline phosphatase 1/1000 dilution was added for 1 h at room temperature. The blot was visualised with NBT/BCIP using 20 min colour development.
Resource Q anion exchange FPLC of selected tissue extracts
Extracts of hip cartilage and plantaris leg muscle from 12-week-old C57BL/6 and perlecan exon 3 null mice equilibrated in 20 mM Tris–HCl (pH 7.2) (Resource Q starting buffer) were applied to a 1 ml column of Resource Q and the column was eluted with starting buffer (4 ml) followed by a linear gradient of NaCl (0–1.0 M) in starting buffer (17.5 ml). Perlecan was identified in eluting fractions 14–18 under these conditions, while perlecan from WT tissue samples was identified in fractions 22–24 using dot blotting with antibody A7L6 for detection. These fractions were pooled for ELISA analysis of the perlecan glycosaminoglycan chains.
Immunolocalisation of perlecan in mouse tissues
Mouse longissimus/multifidus muscle samples from C57BL/6 and perlecan exon 3 null mice were fixed in 10% (v/v) neutral-buffered formalin, dehydrated, and embedded in paraffin. Longitudinal tissue sections were used for the perlecan immunolocalisations with MAb A7L6 and HRP-conjugated anti-rat IgG secondary antibody using Nova RED as a substrate for colour development. Human foetal elbow tissues containing myotubes in longitudinal and in cross-section and a prominent nerve plexus plus human foetal cardiac muscle were used as positive control tissues. Negative controls were also examined by substituting an irrelevant antibody for the authentic rat anti-mouse perlecan domain IV (MAb A7L6).
ELISA plate assay of perlecan glycosaminoglycan side chains
Replicate (n = 6) 100 μl perlecan samples (10 μg/ml) were attached to 96-well flat bottom polystyrene high-binding ELISA microtitre plates for 2 h at 25°C. Wells were rinsed twice with phosphate-buffered saline (PBS), pH 7.4, and blocked with 5% (w/v) BSA in PBS for 1 h at 25°C. Selected wells were digested with Chondroitinase ABC (0.05 U/ml) and/or Heparitinase III (0.01 U/ml) in PBS, pH 7.2, for 16 h at 37°C and rinsed in PBS 1% (w/v) Tween-20 (PBS–Tween). Primary antibodies diluted in 2% (w/v) bovine serum albumin in PBS were applied to the wells for 2 h at 25°C. These included anti-perlecan domain IV or V antibodies A7L6 (5 μg/ml) and H300 (5 μg/ml), 2-B-6 mouse hybridoma-conditioned media to chondroitin-4-sulfate stubs (1/50 dilution), 7-D-4 hybridoma-conditioned medium (1/20 dilution), mouse 10-E-4 anti-native HS (2 μg/ml), and mouse 3-G-10 anti-HS stub antibody (2 μg/ml dilution). Bound primary antibody was detected by incubation with biotinylated mouse or rat IgG secondary antibodies (1 : 1000) diluted in 2% (w/v) BSA in PBS for 1 h at room temperature. Bound biotinylated secondary antibody was detected using avidin-HRP conjugate (1 : 500) with 30 min colour development at 25°C using 2 mM 2,2′-azino-di-3-ethylbenzthiazoline sulfonic acid and H2O2 substrates in 50 mM sodium citrate, pH 4.6. Absorbance values were measured at 405 nm using a plate reader.
All statistical analyses were performed using Stata 14. Mixed ordinal logistic models grouped by mouse genotype and tissue zones as variables for comparisons and, if significant, differences between groups were evident, and they were assessed using Mann–Whitney U-ranked tests. The Benjamin–Hochberg false-positive correction for multiple tests was performed and gave a corrected P-value of 0.045 for significance at an α-value of 5%. Data were presented as box plots with median, 25 and 75% percentiles and range shown.
The schema in Figure 1a,b depicts the replacement of perlecan exon 3 with a pGK-neo cassette in the Hspg2Δ3−/Δ3− mice. Hspg2Δ3−/Δ3− mice were fertile and litters were of expected size. There were no gross abnormalities or differences in appearance between WT and Hspg2Δ3−/Δ3− animals at birth. By 3 weeks of age, the previously reported micropthalia in Hspg2Δ3−/Δ3− animals was evident. Age-matched Hspg2Δ3−/Δ3− mouse body weights were less than corresponding WT mice of 10–18 weeks of age (Figure 1c). Although smaller, Hspg2Δ3−/Δ3− mice had similar skeletal proportions to WT mice and no apparent musculoskeletal abnormalities. Mutant mice were more docile when handled, but no other behavioural abnormalities were noted. Initial examination of toluidine blue-stained mid-saggittal sections of thoracolumbar glycosaminoglycans from each mouse genotype identified the superior and inferior vertebral growth plate cartilages adjacent to the glycosaminoglycans (Figure 1d). The nucleus pulposus had a flattened appearance in the CB57L/6 mice and had a prominent glycosaminoglycan poor notochondral remnant centrally. Some glycosaminoglycan staining was evident on the periphery of the nucleus pulposus. The structural organisation in the Hspg2 exon 3 null intervertebral discs was quite different from the CB57L/6 mouse intervertebral discs. The perlecan exon 3 null mice had an oval nucleus pulposus with greater glycosaminoglycan staining within and around its periphery, and the notochordal remnant again was a flattened structure within the nucleus pulposus but contained more prominent peripheral glycosaminoglycan staining. The inner annulus fibrosus and outer annulus fibrosus, to a lesser extent in perlecan exon 3 null intervertebral discs, had prominent toluidine blue glycosaminoglycan staining at 6 weeks and this increased at 12 weeks. This was not the case for the WT mice. Toluidine blue staining was also more intense in the vertebral growth plates of the perlecan exon 3 null mice compared with the CB57L/6 mice. A prominent population of hypertrophic cells at the juncture of the outer annulus fibrosus and vertebral growth plate in the perlecan exon 3 null mice were surrounded by a glycosaminoglycan-rich matrix. A similar population of cells was also observed in the WT mouse intervertebral discs, but these were less numerous and fewer had a hypertrophic morphology.
Genomic organisation of Wild type C57BL/6 and perlecan exon 3 null mice, body weights of the two genotypes over 10–20 weeks of age and glycosaminoglycan deposition in macroscopic views of lumbar intervertebral discs at 6 and 12 weeks of age.
Comparison of the morphology of intervertebral discs from both mouse genotypes at higher magnification showed clear differences between them (Figure 2a,b). Major differences were evident in the accumulation of glycosaminoglycan in and around the nucleus pulposus, inner-mid-annulus fibrosus, and in the vertebral growth plate (Figure 2). Marginal progenitor cells at the juncture of the outer annulus fibrosus–vertebral growth plate were also more prominent in the Hspg2 exon 3 null mouse (Figure 2b) than in WT intervertebral disc, and at 12 weeks, these extended across the whole breadth of the growth plate in 12 of the 32 intervertebral discs examined (Figure 3c,d). This was not observed in the WT intervertebral discs where the vertebral growth plate chondrocytes were aligned in small columns (Figure 3e–g). Another abnormality evident in the Hspg2 exon 3 null mouse, but absent from the WT intervertebral disc, was the presence of exostose's formations in 14 of the 32 intervertebral discs examined (Figure 3a,b,h). Glycosaminoglycan accumulation was also more prominent in the nucleus pulposus and annulus fibrosus of the perlecan exon 3 null intervertebral discs (Figure 3h,i).
Glycosaminoglycan deposition in representative C57BL/6 and perlecan exon 3 null intervertebral discs stained with toluidine blue-fast green.
Glycosaminoglycan deposition in intervertebral discs and vertebral growth plates of wild type C57BL/6 and perlecan exon 3 null intervertebral discs in specific disc regions illustrating differences in glycosaminoglycan distribution, cell hypertrophy, cellular proliferation and exostosis formations.
Cell numbers were manually counted in tissue sections of the anterior and posterior progenitor cell populations of the C57BL/6 and Hspg2 exon 3 null mice (Figure 4a). This showed that the anterior cell populations from the Hspg2 exon 3 null mice were higher than in the other samples. Morphometric image analysis of toluidine blue staining in tissue sections of 12-week-old mice was also done for the vertebral growth plate cartilage associated with the anterior progenitor cell population, nucleus pulposus, and inner and outer annulus fibrosus (Figure 4b). This showed quantitatively higher glycosaminoglycan staining levels in the vertebral growth plate, progenitor cell cartilage, nucleus pulposus, and annulus fibrosus in the Hspg2 exon 3 null mice. ELISA of perlecan glycosaminoglycan fractions from each of the mouse genotypes showed that the perlecan exon 3 null samples contained no detectable HS and minor amounts of CS, whereas abundant HS and moderate CS levels were detected in the perlecan samples from the WT intervertebral disc (Figure 5a,b). ELISA analyses were conducted on perlecan samples using antibodies to a terminal native CS epitope 2-B-6(−), to native HS (10-E-4), to a native CS neoepitope (7-D-4), to a heparitinase III-generated Δ-HS stub epitope (3-G-10), and to a chondroitinase ABC-generated Δ-chondroitin-4-sulfate stub epitope [2-B-6(+)] (Figure 5a,b). This confirmed the absence of HS in the perlecan exon 3 null mice samples (Figure 5a,b). Antibodies to perlecan domain III (MAb 7B5), domain IV (MAb A7L6), and domain V (antibody H300) confirmed the efficient binding of perlecan to the ELISA plates (Figure 5c).
Morphometric determination of cell numbers and quantitation of toluidine blue stained glycosaminoglycan deposition at 6 and 12 weeks of age in specific regions of C57BL/6 wild type and perlecan exon 3 null intervertebral discs and vertebral growth plates.
Quantitation of specific CS and HS epitopes by ELISA in wild type and perlecan exon 3 null perlecan samples electroeluted from specific regions of 3–8% polyacrylamide gradient electrophoresis gels or isolated by Resource Q anion exchange FPLC and demonstration of their reactivities with antibodies to perlecan domains III, IV and V.
Attempts to micro-dissect murine intervertebral discs to obtain specific areas of the vertebral growth plate, annulus fibrosus, and nucleus pulposus yielded insufficient material for large scale extraction of perlecan or RNA. However, a survey of other mouse tissues identified abundant levels of perlecan in the paraspinal and leg muscles and hip articular cartilage, and these tissues could be easily sampled. Immunolocalisations of mouse muscle samples also confirmed the presence of perlecan in these tissues (Figure 6a–e).
Immunolocalisation of perlecan associated with myotubes and cardiomyocytes and a paraspinal nerve plexus in positive control human foetal and murine muscle from wild type and perlecan exon 3 null mice and Resource Q anion exchange FPLC of perlecan samples from the two mouse genotypes.
Extraction and isolation of perlecan from hip cartilage and muscle using Resource Q anion exchange chromatography was undertaken, and dot blotting using antibody H300 was used to identify perlecan-containing fractions (Figure 6g–j). These fractions were further examined by SDS–PAGE and Western blotting (Figure 7). Gel segments from non-stained gel areas corresponding to high molecular mass proteins of similar size to 460 kDa pre-stained protein standards were electroeluted and further examined by Western blotting. This confirmed the presence of perlecan in these electroeluted samples; however, the core protein of the Hspg2 exon 3 null perlecan was smaller, consistent with the deletion of perlecan domain I and its attached glycosaminoglycan (Figure 7). Immunoblotting of perlecan samples also identified the presence of HS and CS in the cartilage and muscle perlecan samples from the WT mice (Figure 8, lanes 2, 3, 7, and 8) but not in the perlecan exon 3 null mice (Figure 8, lanes 1, 5, and 6). This also showed that CS or HS did not occupy domain V in perlecan samples from the exon 3 null mice.
Coomassie R250 stained polyacrylamide gel segments of perlecan samples.
Immunoblotting of perlecan from C57BL/6 wild type and perlecan exon 3 null tissue samples.
Immunolocalisation studies on the foetal human IVD have identified aggrecan, the major IVD proteoglycan, versican, biglycan, decorin, fibromodulin, lumican, and keratocan as components of the IVD ECM. Surprisingly, no definitive study has documented all of these proteoglycans in the murine intervertebral disc [9,40–43]. Perlecan is a major functional component of the foetal human intervertebral disc; however, its tissue levels steadily decline with ageing. Immunolocalisation of perlecan and aggrecan in knee and hip articular cartilage and meniscus also follows a similar decline with ageing [40–45]. Toluidine blue is a non-specific proteoglycan stain, and all of the aforementioned proteoglycans containing sulfated glycosaminoglycan would elicit a metachromatic response with this stain. Thus, no information on individual proteoglycans was obtained in the present study using toluidine blue staining of intervertebral disc tissue sections, and this must be considered a limitation of our study. However, numerous studies consider that aggrecan is the most abundant disc proteoglycan, and thus, the toluidine blue staining of murine IVD tissues we observed in the present study would be expected to largely reflect changes in aggrecan levels in response to ablation of HS/CS from perlecan.
The chondrogenic activity of perlecan has been mapped to domain I ; however, perlecan domain IV contains binding sites for nidogens, laminin–nidogen complex, fibronectin, and fibulin-2, and thus, has matrix-stabilising properties. Furthermore, perlecan domain IV also has cell signalling properties [47–49].
The HS chains of perlecan interact with signalling proteins, growth factors, and extracellular matrix-stabilising structural glycoproteins regulating protein distributions and growth factor bio-availability by acting as cell surface co-receptors . Ternary complex formation between perlecan HS, fibroblast growth factors (FGFs), and their receptors (FGFRs) results in FGF–HS–FGFR signalling complexes; however, perlecan also signals with vascular endothelial cell growth factor, platelet-derived growth factor, Wnt family proteins, and the bone morphogenetic protein family, and thus, perlecan acts as a cell signalling hub [50–58]. Perlecan's modular design also facilitates interactions with a diverse range of extracellular matrix molecules such as PRELP (proline/arginine-rich and leucine-rich repeat protein), a basement membrane anchorage protein, and von Willebrand A domain-related protein (WARP); Type IV, V, VI, XI, XIII, and XVIII collagen; nidogen, laminin, fibronectin, fibulin 2, fibrillin 1, latent TGF-β1-binding protein-1, 2; elastin through which it stabilised and organised the extracellular and pericellular matrix [59–61]. The essential role of perlecan in skeletogenesis is amply demonstrated in perlecan knockout mice . However, this is a lethal condition and few mouse pups survive to birth, and thus, this model cannot be used to examine the role of perlecan in postnatal skeletal development. A milder model was therefore developed and used in the present study by ablation of Hspg2 exon 3 producing a mutant perlecan devoid of ∼20 kDa of domain I of its core protein and the glycosaminoglycan chains normally attached to this region; glycosaminoglycan is also reported to be present in perlecan domain V [14,16,17]. There was no detectable glycosaminoglycan substitution in domain V in the present study.
It has been reported that antagonism of HS with the chemical agent Surfen [63,64] or its degradation by heparanase also promotes chondrogenesis . In vitro studies with chondrocytes cultured in the presence of heparanase displayed greater proliferative rates and glycosaminoglycan synthesis than control cultures where heparanase was not added . Moreover, cellular studies have shown that steep decreases in local HS levels which occur in EXT1 mutations disrupt the normal signalling pathways which maintain the perichondrium as a mesenchymal tissue and cause excessive BMP signalling, ectopic chondrogenesis, and osteochondroma formation . In the present study, ablation of perlecan HS resulted in a markedly greater accumulation of glycosaminoglycan in the vertebral growth plate, nucleus pulposus, and annulus fibrosus that was not observed to the same extent in the C57BL/6 mice. Chondrogenesis in the Hspg2 exon 3 null intervertebral disc was also greater and was particularly evident in a discrete group of cells at the periphery of the annulus fibrosus with the vertebral growth plate. These cells laid down greater glycosaminoglycan levels than equivalent cell populations in the C57BL/6 mice, and they also became hypertrophic earlier and were associated with ectopic bone development at the juncture of the outer annulus fibrosus and vertebral growth plate by 12 weeks. Progenitor cells have been shown in the same region of human foetal and newborn ovine intervertebral disc . In human, an exostosis or osteochondroma is an aberrant bony growth occurring adjacent to growth plates/perichondrium. Mutations in the exostosin 1 (EXT1) or exostosin 2 (EXT2) gene cause hereditary multiple exostosis syndrome and some isolated osteochondromas mainly associated with the long bones or ribs [67–69]. EXT1 and EXT2 are glycosyltransferases in the Golgi apparatus which add repeating N-acetyl glucosamine and d-glucuronic acid to the repeat disaccharide region of the emerging HS chains. Perichondrial stem cells give rise to chondrocytes that clonally expand and differentiate to develop into an exostosis, and HS is markedly diminished in exostosis cartilage and perlecan displays an abnormal distribution . Perlecan has critical roles to play in cartilage development and skeletogenesis, and the maintenance of an adult chondrocyte phenotype [3,71]. The perlecan domain 1 HS chains interact with bone morphogenetic protein 2 to stimulate chondrogenic differentiation and promote bone formation [72,73]. Ablation of HS chains from perlecan in the Hspg2 exon 3 null mice therefore produced a similar phenotype in the present study to mutations or deletions in EXT1 and EXT2.
Henrickson et al. [20,21] have identified cell proliferation zones and progenitor cells in potential stem cell ‘niches’ in discs from the rat, minipig, rabbit, and degenerative human. A prominent niche location Henrickson proposed was at the juncture of the outer annulus fibrosus and vertebral growth plate [20,74] in the same area where hypertrophic progenitor cells were observed in the present study and where exostosis bone formations were also observed in a significant number of the Hspg2 exon 3 null intervertebral discs. Discrete populations of cells were also observed in the same locations in the C57BL/6 mouse intervertebral disc, but were less numerous than in the mutant mice and less of these cells progressed to a hypertrophic phenotype in the 12-week experimental period. A prominent feature in the present study was a markedly greater deposition of glycosaminoglycan in the HS-deficient intervertebral disc which was quantitated by morphometric image analysis, and glycosaminoglycans have diverse roles in stem cell differentiation and bone formation [75–81]. The treatment of cultured rat articular chondrocytes with heparitinase I results in an enhanced accumulation of glycosaminoglycan . Furthermore, treatment with an activity-inhibiting antibody to perlecan increased the production of type II collagen. Perlecan also has diverse effects on cells from the central nervous system. Perlecan inhibited astrocyte proliferation in vitro, but neural stem/progenitor cell proliferation was promoted . Perlecan has anti-proliferative [83–86] and anti-adhesive effects  on smooth muscle cells. In contrast, an LG3 C-terminal fragment of perlecan promotes the migration of mesenchymal–stromal stem cells to neo-intimal defects through interactions with α2β1 integrin and phosphorylation and activation of ERK-1/2 leading to thickening of the neo-intima . These results indicate that perlecan HS has diverse effects on different cell populations [50,89] and is a negative regulator of adult chondrocyte differentiation in vitro . HS-perlecan also has diverse effects on mesenchymal–stromal stem cell differentiation in vitro. Perlecan added to mesenchymal–stromal stem cell cultures suppressed adipogenic but enhanced osteogenic differentiation . Removal of the HS chains of perlecan by the inclusion of heparitinase I in these cultures did not alter this suppressive effect on adipogenesis, but decreased the stimulatory effect on osteogenesis, indicating that osteogenesis was HS dependent . Deficiency of perlecan/HSPG2 during bone development enhances osteogenesis, but decreases bone quality in adult mice and alters cartilage matrix patterning co-ordinately influencing bone formation and calcification . Perlecan may protect vascular smooth muscle cells from factors that promote vascular calcification. Perlecan expression is reduced in the calcified aortae of hyperparathyroid rats and is a risk factor for vascular calcification, and HS inhibits osteogenesis in the aorta . Bone formation by endochondral ossification is an intricately orchestrated process involving the coordination of cell–cell and cell–matrix growth factor signalling to produce mineralised bone from a cartilaginous template . Chondrogenic and osteogenic differentiation occurs in sequence during this process, and the temporo-spatial patterning requires the action of heparin-binding growth factors and their receptors. Heparanase produced by osteoblasts at the chondro-osseous junction has an important role to play in osteogenesis. Heparanase facilitates cartilage replacement by bone at the chondro-osseous junction by removing the HS side chains of perlecan/HSPG2, which otherwise prevent osteogenic cells from remodelling the hypertrophic cartilage to bone . Focal release of HS-bound growth factors by heparanase or by the action of enzymes like matrix metalloprotease-13 in the hypertrophic growth plate cartilage drives the ossification process at the chondro-osseous junction . The HS and CS, side chains of perlecan and syndecan directly regulate bone morphogenetic protein-mediated differentiation of human mesenchymal stem cells into osteoblasts and also have roles in the attainment of mesenchymal stem cell pluripotency [75,95]. Long-term culture of mesenchymal stem cells in HS- and CS-degrading enzymes markedly increases bone nodule formation, calcium accumulation, and the expression of osteoblast markers such as alkaline phosphatase, RUNX2, and osteocalcin, and alters BMP and Wnt cell signalling activity enhancing the osteogenic differentiation of human mesenchymal stem cells . The HS chains on perlecan therefore appear to negatively regulate the proliferation and maturation of articular chondrocytes which are held in a low metabolic state where they slowly turn over matrix components to replenish extracellular matrix components and maintain tissue homeostasis but do not undergo overt growth. Thus, in the present study, ablation of perlecan HS chains resulted in this regulatory property being lost, and matrix accumulation and cell hypertrophy was therefore enhanced in the Hspg2 exon 3 null mice. Perlecan is widely reported to promote anabolic processes in skeletogenesis, and findings in the present study also show that perlecan through its HS chains has repressive regulatory properties associated with cartilage maturation presumably to maintain tissue homeostasis.
Loss of the HS chains of perlecan induces changes in many murine skeletal tissues. In the G intervertebral disc cellular proliferation and matrix glycosaminoglycan deposition was enhanced with HS deficiency in Hspg2 exon 3 null mice and chondrocyte hypertrophy was more advanced, indicating that the HS chains of perlecan negatively regulated these processes. This repressive effect of HS was evident in mature tissues where isolated chondrocytes do not divide and overt biosynthetic steps for ECM production are also held in check. Basal levels of ECM synthesis still occur in mature tissues to replenish components turned over to maintain tissue homeostasis. HS deficiency promotes osteogenic effects in intervertebral disc tissues with exostosis formation observed in 43% of the Hspg2 exon 3 null intervertebral discs, but was not observed in the intervertebral discs of WT mice.
fibroblast growth factor
fibroblast growth factors and their receptors
fibroblast growth factors
fast protein chromatography
transforming growth factor
C.C.S. undertook the day-to-day running of the study, maintained the mouse colonies, collected and processed mouse tissue samples, undertook glycosaminoglycan analyses and statistical comparisons. S.M.S. undertook and interpreted the histology analyses. C.B.L. provided a scientific overview, intellectual input into experimental design and interpretation of data. J.M. supervised and participated in glycosaminoglycan ELISA, perlecan isolation, anion exchange FPLC, size exclusion chromatography, SDS–PAGE and Western blotting and co-ordinated manuscript writing contributions from authors. All authors contributed to manuscript writing and review and all approved the final version of the manuscript.
The present study was funded by The National Health and Medical Research Council Project Grant 1004032.
The Authors declare that there are no competing interests associated with the manuscript.