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Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis


Authors: David Mellis aff001;  Katherine A. Staines aff002;  Silvia Peluso aff003;  Ioanna Ch. Georgiou aff004;  Natalie Dora aff003;  Malgorzata Kubiak aff001;  Rob van’t Hof aff005;  Michela Grillo aff001;  Colin Farquharson aff006;  Elaine Kinsella aff001;  Anna Thornburn aff003;  Stuart H. Ralston aff005;  Donald M. Salter aff005;  Natalia A. Riobo-Del Galdo aff004;  Robert E. Hill aff003;  Mark Ditzel aff001
Authors place of work: Edinburgh CRUK Cancer Research Centre, MRC Institute for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom aff001;  School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom aff002;  MRC Human Genetics Unit, MRC Institute for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom aff003;  Leeds Institute of Medical Research and School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom aff004;  Centre for Genomic and Experimental Medicine, MRC Institute for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom aff005;  Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh, United Kingdom aff006
Published in the journal: Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis. PLoS Genet 17(4): e1009275. doi:10.1371/journal.pgen.1009275
Category: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009275

Summary

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

Keywords:

Ankle joints – Hedgehog signaling – Chondrocytes – Knee joints – Signal processing – Tendons – PKA signaling cascade

Introduction

Ubiquitin ligases target proteins for ubiquitination which can modulate protein function by regulating protein degradaton, protein–protein interactions, and protein localization [14], and thus, provide important post-translational mechanisms essential for a variety of cellular processes. The Drosophila homologue of the mammalian Ubiquitin Protein Ligase E3 Component N-Recognin 5 (UBR5), designated as hyperplastic discs (Hyd), was originally identified as a Drosophila tumor suppressor protein [57] and regulator of Hedgehog (HH) signalling [6]. Physical and genetic interactions with established components of the HH signalling pathway [7,8] strengthened Hyd’s role as a regulator of HH signalling. We previously addressed a possible conserved role for UBR5 in HH-mediated processes in mice [9]. Although no overt effects were seen in patterning of the developing limb bud in mouse embryogenesis; here, we show that the coordinated action of Ubr5 with HH signalling is crucial to maintain skeletal tissue homeostasis associated with the appendicular skeleton postnatally and in adult mice.

HH signalling regulates cell processes that are critical for skeletal tissue development, growth and homeostasis [10]. Two HH ligands, Sonic- and Indian-Hedgehog (SHH and IHH, respectively) are widely expressed and function as extracellular signalling molecules that bind to cells expressing HH receptors such as patched-1 (PTCH1). Binding to PTCH1 results in de-repression of the G protein-coupled receptor, smoothened (SMO), and activation of SMO-associated canonical and non-canonical signalling pathways [1113]. Activation of the SMO-associated canonical pathway results in stimulation of GLI-mediated transcription and expression of crucial target genes [7]. Activation of the recently identified SMO-associated non-canonical pathway relies on SMO’s GPCR activity [14,15] and results in inhibitory heterotrimeric G protein-mediated inhibition of adenylate cyclase and a concomitant reduction in cyclic AMP (cAMP) levels [14,16,17]. Although not yet experimentally addressed, non-canonical signalling may also contribute to many of the well-described roles for canonical HH signalling in normal skeletal formation, maturation and maintenance [10,18].

At birth, IHH is the ligand that drives HH signalling within the growing limbs. Expression of Ihh is localized to a zone of postmitotic, prehypertrophic chondrocytes immediately adjacent to the zone of proliferating chondrocytes [1820] and is essential for endochondral ossification but also induces osteoblast differentiation in the perichondrium [21]. Dysregulation of this signalling pathway is detrimental to musculoskeletal tissue homeostasis [22,23]. Notably, studies have shown that increased HH signalling can drive pathological ectopic cartilage and bone formation in soft tissues [10] through the process of heterotopic chondrogenesis and heterotopic ossification (HO) [24]. Upregulation of HH signalling is believed to contribute to the rare disorder, progressive osseous heteroplasia (POH), which includes in its phenotypic spectrum soft tissue ossification. POH is caused by loss-of-function of GNAS, a G protein alpha subunit and activator of adenylate cyclase. A murine model of POH demonstrated that increased HH signalling as a consequence of GNAS loss-of-function in mesenchymal limb progenitor cells drove heterotopic ossification [25]. Similarly, synovial chondromatosis, a disease resulting in ossification of synovial tissue is associated with increased canonical HH signalling [26]. However, in contrast with cartilage and bone gain, elevated HH signalling is also associated with the cartilage degradation and loss [27,28]. Hence, appropriate HH signalling is normally involved in the suppression of ectopic, and genesis and maintenance of normtopic, cartilage and bone.

Here, we show that the loss of Ubr5 function in Ubr5mt mice resulted in diverse musculoskeletal defects including spontaneous, progressive and tissue-specific patterns of ectopic chondrogenesis and ossification as well as articular cartilage degeneration and shedding. Surprisingly, reducing SMO function in UBR5-deficient mice led to a dramatic reduction in the age of onset and increased severity of the Ubr5mt phenotype. These observations challenge the existing dogma by highlighting an important role for Smo, in the absence of UBR5, in suppressing, rather than promoting, ectopic chondrogenesis, tissue calcification/ossification and articular cartilage damage. We, therefore, reveal a previously unknown physiological role for Ubr5 and highlight its genetic interaction with Smo in regulating cellular and tissue-homeostasis. These findings may influence current therapeutic approaches modulating HH signalling for the treatment of degenerative musculoskeletal conditions such as osteoarthritis and heterotopic ossification.

Results

Loss of Ubr5 function causes skeletal heterotopias at 6 months

To overcome the embryonic lethality associated with germline mutant animals [29], we combined a Ubr5 conditional loss-of-function gene trap (Ubr5gt) [9] with Prx1-Cre [30] (Prx1-Cre;Ubr5gt/gt animals henceforth, referred to as Ubr5mt) to ensure that adult tissues derived from early limb bud mesenchyme, predominantly bone and connective tissue, were Ubr5 deficient. Since the HH pathway affects embryonic limb patterning and bone growth, the Ubr5 deficient fetuses (at E15.5) were initially examined and bones and joints appeared to develop normally [9]. However, the HH pathway continues to function in postnatal bone growth and homeostasis [10] and thus, at approximately 6 months of age, we noticed that mice began to display defects in locomotion. Control animals normally remained supported by their hindlimbs (‘sprung’), whereas, Ubr5mt animals rested their posteriors directly upon the floor (‘squat’) (S1A–S1C Fig). Considering the tissue targeted by the conditional mutation, the observed phenotype indicated a potential musculoskeletal system defect which prompted the examination of hindleg bone and joint structures.

At 6 months of age, X-ray imaging revealed that Ubr5mt animals exhibited abnormally shaped and/or ectopic signals around knee and ankle joints (S1D–S1G Fig). 3D micro-computed tomography (μCT) revealed that, whereas Prx1-Cre control joints appeared normal with no evidence of ectopic structures (Fig 1A), the knees of all Ubr5mt mice (n = 10) exhibited isolated ectopic signals clearly separated from the adjacent femoral condyles and tibia (Fig 1B). Surface rendering of the μCT scans demonstrated that the array of knee-associated sesamoid bones (patella and fabella) and calcified menisci (Fig 1C and 1D) were abnormal. Ubr5mt knees presented with large ectopic structures on all four faces of the knee joint, as well as enlarged and irregularly shaped fabella and patella sesamoid bones (Fig 1E and 1F). In addition, the Ubr5mt animals exhibited multiple ectopic signals around the ankle joint (Fig 1G and 1H), with the most striking one appearing consistently on the dorsal side running parallel to the long axis of the tibia (Fig 1H, open arrows) associated with the Achilles tendon (AT). This ectopic signal remained isolated from the calcaneus and tibia. Other ectopic structures included two ectopic U-shaped signals on the ventral and lateral sides of the tibia (Fig 1H).

<i>Ubr5</i><sup><i>mt</i></sup> animals exhibit multiple ectopic structures around the knee and ankle joints in <i>Ubr5</i> expressing tissue.
Fig. 1. Ubr5mt animals exhibit multiple ectopic structures around the knee and ankle joints in Ubr5 expressing tissue.
6-month old control and Ubr5mt animals were analysed by μCT. (A, B) Color-coded density maps revealed presence of ectopic, non-uniform density structures (open arrowheads) around the tibia (closed arrowheads) and femoral condyles (arrows). (C, D) Knee joints in ventral and (E, F) in medial (lower panels) aspect. Closed arrowheads indicate normal structures: the patella (Pa), menisci (Mn) and fabella (Fb). Open arrowheads indicate ectopic signals (Es) present in the Ubr5mt knee joint. (G) Control ankle joints exhibit a signal extending from the ventral face of the tibia (closed arrowhead) and a small structure presumed to be a sesamoid bone (arrow). (H) Multiple ectopic signals were present around the Ubr5mt ankle joint (arrowheads), including a large dorsally located and well-isolated structure in the location of the AT (open arrowhead). (I-R) 20-week-old Prx1-Cre control and Ubr5mt ankle and knee joints were stained for β-gal activity. Whole mount knee (I, K) and ankle (J, L) are shown with subsequent sagittal sections for the knee (M) and the boxed area magnified in (N) shows the outer layer of the menisci (open arrowhead), and the adjacent synovium (closed arrowhead) stained positive for β-gal expression. Sagittal sections for the ankle are shown in (O) and magnified in (P) showing staining of the AT and superficial digital flexor tendon (open and closed arrowheads, respectively). (Q, R) Expression of β-gal in the articular cartilage of the knee and the red dashed box in Q shown at higher magnification in R.

Following Prx1-Cre mediated recombination of the Ubr5gt gene-trap construct, the reporter gene lacZ (encodes β-galactosidase [β-gal] activity) is activated and expressed under the influence of Ubr5 gene regulators [9] acting as proxy for Ubr5 expression. β-gal activity is initially detected in limb mesenchyme at early embryonic stages [9] and at the postnatal stages examined here, activity is restricted to limb tissue derived from this embryonic mesenchyme. Analysis of lacZ expression in 20 week-old control and Ubr5mt mice indicated that Ubr5 is expressed around the knee (Fig 1I and 1K) and ankle (Fig 1J and 1L) joints. Further analysis revealed strong β-gal activity around the periphery of the menisci and synovium (Fig 1 M and 1N). The ankle also revealed β-gal activity within the AT and superficial digital flexor tendon and in a large ectopic structure within the AT midbody (Fig 1O and 1P). Thus, the tissues that exhibit Cre-mediated expression of the lacZ gene are affected in the mutant phenotype.

Ubr5mt-associated ectopic structures exhibit chondrogenesis and calcification

The morphology of these ectopic structures around the knee (see Fig 1D and 1F) and ankle (see Fig 1H) joints were further investigated to determine the cellular composition and possible derivation of these ectopias. As shown by μCT, both knee (Fig 2A and 2B) and ankle (Fig 2D and 2E) ectopic structures harbored X-ray dense internal structures indicative of bone. Accordingly, in these joints (Fig 2C and 2F) von Kossa staining, which is widely used to detect abnormal calcium deposits, revealed staining in these ectopic structures. Von Kossa staining in the Ubr5mt knee joints revealed positive stained structures within the synovium deep in the patellar tendon (Fig 2C) and near the ankle joint staining within the AT (Fig 2F). In addition, in the control AT, the expected ordered stacking of tenocytes along the anterior-posterior axis (Fig 2G) and an absence of toluidine blue staining associated with proteoglycans (Fig 2H) was observed. In contrast, these regions of the Ubr5mt AT were devoid of tenocytes, which were replaced by long columns of proteoglycan-expressing hypertrophic chondrocytes (Fig 2I and 2J). Histological analysis of control knee joints revealed a synoviocyte-rich intimal layer of the synovium (Fig 2K), whereas Ubr5mt knee joints exhibited bone- (Fig 2L) and cartilage-like (Fig 2M) ectopic structures. The observed mutant morphology was consistent with the formation of ectopic bone around the knee and in the AT, which was further associated with the presence of ectopic chondrocytes and the production of extracellular matrix.Thus, an abnormal phenotype consisting of ectopic chondrogenesis, calcification and ossification (hereafter, referred as ECCO) of the synovium and tendons in Ubr5mt tissues was observed. We concluded that Ubr5 normally functions to prevent spontaneous ectopic formation of chondrocytes in tissues and calcification and/or ossification in cartilage.

<i>Ubr5</i><sup><i>mt</i></sup> limbs exhibit ectopic chondrogenesis, cartilage formation and calcification and ossification.
Fig. 2. Ubr5mt limbs exhibit ectopic chondrogenesis, cartilage formation and calcification and ossification.
(A, B, D, E) Colour-coded X-ray density maps of volume rendered Ubr5mt knee (A B) and ankle (D, E) joints. (B, E) show cross-sections through the joint to reveal the internal structure and density. Arrowheads indicate ectopic structures indicated in Fig 1F and 1H. Low density = blue and High density = red. Sagittal sections (C, F) from 20-week-old or 24-week-old animals are shown. (C, F) Von Kossa staining of Ubr5mt knee (C) and ankle joints (F). (C) Ubr5mt knee joints revealed ectopic signals in the synovium (dashed box and arrowhead) lying within the synovium and under the patellar tendon. (F) Ubr5mt ankle revealed ectopic signals in AT (dashed box and arrowhead) on the deep face of the AT. (G, I) H&E and (H, J) toluidine blue staining of the midbody of Achilles tendons. The left panel of each pair shows a low magnification image of the tendon. A higher magnification of the boxed region is shown in the right panel. (G, H) Control tendons showed the expected columns of tenocytes and very little toluidine blue staining. (I, J) Ubr5mt tendons harbour chondrocytes that coincide with regions of toluidine blue staining. (K) Image of control synovium (arrowhead) located underneath the patella (Pa) and patellar tendon (PT) and adjacent to the tibial articular cartilage (AC). (L, M) Ubr5mt synovium harbours ectopic tissue. (L) The synovium harbours a bone-like structure (arrowhead). (M) In other regions, the synovium abutting the patella appeared thickened but not ossified showing cartilage harbouring chondrocytes. Pt = patellar tendon; Pa = patella; Mn = meniscus.

Loss of Ubr5 function causes articular cartilage degradation

μCT analysis of 6-month old control (Fig 3A) and Ubr5mt (Fig 3D) knee revealed the previously described overt morphological differences (see above), but further analysis at the surface of the joints, showed a significantly increased volume of high subchondral bone density (designated by red in Fig 3B, 3C, 3E and 3F) in the mutant. The increase in volume as a percentage of total subchondral bone (depicted in S1H–S1J Fig) is quantified in Fig 3G. In addition, histological assessment showed a dramatic loss of articular cartilage (AC) from the lateral tibial and femoral surfaces of all Ubr5mt knee joints assessed (Fig 3H and 3I); a condition not detected in any control mice at this stage. Further examination of the exposed subchondral bone in these Ubr5mt mice revealed abnormal intermixed bone and cartilage within this region (Fig 3J and 3K). Analysis for lacZ expression revealed β-gal activity within the upper layer chondrocytes of the femoral and tibial AC (Fig 1Q and 1R) which corresponds with a role for Ubr5 in this tissue. Thus an overview of the hindlegs at 24 weeks reveals a diverse range of cartilaginous defects which include firstly, metaplastic conversion of connective tissue associated with the knee and ankle and secondly, severe degradation of AC causing exposure of the subchondral bone at the joint surface.

<i>Ubr5</i><sup><i>mt</i></sup> animals show subchondral bone defects and AC cellular and extracellular abnormalities.
Fig. 3. Ubr5mt animals show subchondral bone defects and AC cellular and extracellular abnormalities.
(A, D) Surface rendered μCT-based 3D models images of knee joints of Prx1-Cre (Control) (A) and Ubr5mt (D). Volume rendered 3D models of 26-week-old tibial subchondral bone (outlined in A, D) showing (B, E) ventral, and (C, F) anterior views. High-density volume shown in red and low-density in blue. (G) Graph of percentage of high-density signal volume as a percentage of total subchondral bone volume. s.e.m indicated. n = three biological replicates per genotype. t-test. p = 0.0103. H&E-based histological analysis of (H-K) 26-week-old Prx1-Cre (Control) and Ubr5mt tissues. (H, I) Low magnification view of the interface at the knee, showing control (dashed lines underscore the AC) and Ubr5mt which exhibited extended regions that lacked AC and exposed subchondral bone (dashed lines). Peripheral regions retained some AC (white arrowheads in I). (J) 26-week-old control tibial AC was normal and (K) Ubr5mt AC revealed exposed subchondral bone (black arrowheads) and intercalated cartilage (white arrowheads)).

Ubr5 deficiency results in a postnatal, progressive phenotype

To establish the approximate age at which this striking ECCO phenotype was initially detectable, a timed series of in vivo μCT scans on ageing, live animals was followed. Ubr5mt animals at 3-weeks of age revealed no marked abnormal phenotype in knee or ankle joints (S2A–S2D Fig), suggesting that the ectopic structures did not form during fetal development but rather formed postnatally. Between 6 and 12 weeks of age, the ectopic structures began to emerge (Fig 4A and 4B), initially on the ventral side of the tibia. Dorsally located ectopic signals associated with the AT emerged by 16 weeks of age (Fig 4C) and all ectopic structures were enlarged by 24 weeks of age (Fig 4D). Ubr5 deficiency, therefore, led to enhanced, progressive chondrogenesis and osteogenesis in the connective tissue of the knee and ankle of the aging Ubr5mt mouse.

Ubr5mt mice exhibit degenerative, age related defects.
Fig. 4. Ubr5mt mice exhibit degenerative, age related defects.
(A-D) Consecutive uCT scans of live Ubr5mt animals at the indicated ages. Ubr5mt ankles form (i) ventral ectopic signals (arrowheads) around (B) 12 weeks of age and (ii) dorsal ectopic signals around (C) 16 weeks of age that (D) increased in size over time. Three-week-old knee joints, stained with H&E, of Prx1-Cre (control) (E) and Ubr5mt (F). Black dashed lines in (F) demarcate the Ubr5mt apical acellular region. (G-M) Six-week-old (G,H) Prx1-Cre (control) and (I,J,L) Ubr5mt proximal tibial AC were analysed. (G) Control AC and at higher magnification (H) revealed the expected chondrocyte profile along the apical to basal axis, namely (I) superficial chondrocytes lining the apical surface; (II) non-hypertrophic rounded/oblong nuclei chondrocytes; (III) larger pre-hypertrophic-like chondrocytes within the central zone; and (IV) large hypertrophic chondrocytes located near the border with the underlying subchondral bone. SB = subchondral bone. (I) Ubr5mt tibial AC and at higher magnification (J) revealed abnormal chondrocytes and an acellular apical layer lacking superficial chondrocytes. (K) Graph of the percentage of superficial chondrocytes in six-week-old tibial AC. N = three biological replicates of each genotype. Mean and s.e.m indicated. Chi square test on pooled cell counts. p = <0.0001. (L) Ubr5mt tibial AC exhibited clusters of eosin positive chondrocytes (dashed lines) and multiple tidemarks (arrows). (M) Graph of percentage of eosinophilic chondrocytes in six-week-old AC. Chi square test on pooled cell counts. N = three biological replicates of each genotype. p = <0.001. (N-Q) Postnatal pCAGG-Cre-mediated recombination of the Ubr5gt construct (O), but not pCAGG-Cre expression alone (N), resulted in X-ray dense ectopic signals forming in the AT region. (P) Counts of animals exhibiting ankle-associated ectopic signals, scored for the absence (Normal) or presence of ectopic signals (Ectopic), n = >4 for each genotype. Fisher’s exact test, p value = 0.0048. (Q) Volumetric measurement of ectopic signals designated HO (heterotopic ossification) volume from each animal n = 4. Unpaired t test, p = 0.0079. Standard error indicated. Control AC (R) exhibited superficial chondrocytes (arrows). (S) pCAGG-Ubr5mt AC exhibited an acellular apical layer (arrowheads), multiple tidemarks (arrows), and surface damage (black arrowhead). (T) Graph of percentage of sections with acellular regions and AC damage. Mean and s.e.m indicated. n = three biological replicates. Three slides analysed from each animal. Average plotted. Fishers exact on pooled section counts. p = 0.0434. (U) pCAGG-Ubr5mt AC also exhibited a reduction in superficial chondrocytes and an increase in empty apically-located lacunae (arrowheads). Graph of (V) superficial chondrocytes and (W) empty lacunae expressed as number per mm of AC. n = three biological replicates. Analysis of two sections per animal. Individual slide values plotted. Mean and s.d indicated. Fishers exact test on pooled counts p = <0.0001.

These metaplastic conversions within the connective tissue supporting the knee and ankle, however, contrasted with the changes demonstrated in the AC which manifested as a degenerative phenotype. To investigate the timing of AC degradation, we examined mice at 3 and 6 weeks. No gross structural disruption of the AC in the Ubr5mt animals at 3-weeks of age was detected (Fig 4E and 4F). By 6-weeks of age, Ubr5mt articular cartilage exhibited an irregular osteochondral interface (Fig 4G and 4I), clusters of large, hypertrophic-like chondrocytes (Fig 4H and 4J) and a reduction in the number of superficial chondrocytes (Fig 4K). Ubr5mt articular cartilage also exhibited multiple tidemarks and regions of strongly eosin positive nuclei indicative of necrosis (Fig 4L and 4M) that were absent in controls. Thus, progressive damage was found in the AC due to the loss of Ubr5 but in this case, early cellular and extracellular abnormalities occurred prior to AC degradation, which included increased subchondral bone density and exposure of subchondral bone

Despite loss of UBR5 in early limb mesenchyme, these data indicated that the ectopic structures arose postnatally and subsequently progressed with age. To directly address if postnatal UBR5 function was required to suppress ECCO and the degradation of the AC, we utilised a mouse line carrying a tamoxifen-inducible, conditional Cre, pCAGG-CreERT2 [30]. Control pCAGG-CreERT2 (pCAGG-Con) or pCAGG-CreERT2;Ubr5gt/gt (pCAGG-Ubr5mt) animals were treated with tamoxifen (administered on two consecutive days) at six weeks of age. Staining for β-gal activity, although more broadly distributed, confirmed tamoxifen-mediated recombination of the Ubr5mt gene trap and its associated β-gal expression in tissues that included muscles and tendons (S2E and S2F Fig), and within the midbody ectopia at the AT (S2G Fig). μCT analysis at 8 weeks revealed that tamoxifen-treated control animals exhibited no ectopic signals (Fig 4N), whereas pCAGG-Ubr5mt animals exhibited AT -associated ectopic signals (Fig 4O). Scoring (Fig 4P) and heterotopic ossification (HO) volumetric analysis (Fig 4Q) confirmed that only tamoxifen-treated pCAGG-Ubr5mt animals exhibited ectopic signals. Comparison of 12 week control to treated pCAGG-Ubr5mt (Fig 4R and 4S) knees revealed Ubr5mt-associated apical acellular layer (Fig 4S and 4T), damage to the apical surface, multiple tidemarks, reduced superficial zone chondrocytes (Fig 4V) and increased numbers of empty lacunae (Fig 4U and 4W). We concluded that postnatal Ubr5 function was both necessary and sufficient to maintain AC homeostasis and prevent ECCO.

Inhibition of Smo promotes Ubr5mt-associated ECCO and enhances Ubr5mt-mediated AC degradation

As UBR5/HYD regulates HH signalling in Drosophila [7,8], we next used a genetic approach to address whether aberrant HH signaling contributed to the Ubr5mt ECCO and AC phenotypes. The Smo gene encodes a core membrane component, regulated by the HH receptor PTCH1, that initiates the downstream signalling cascade leading to GLI-dependent transcription (canonical signalling) or Gi protein-dependent events that are tissue specific (non-canonical signalling). We reasoned that reduction in Smo expression levels would sensitize the HH pathway; thus, heterozygosity for a Smo loss of function allele (SmoLoF) [31] was used in a cross to Ubr5mt to create Prx1-Cre;Ubr5gt/gt;SmoLoF/+ animals (Ubr5mt+SmoLoF).

In contrast to our expectations, μCT analysis of 12-week Ubr5mt+SmoLoF mice exhibited significantly more severe defects than those of age-matched Ubr5mt (Fig 5A–5C) and SmoLoF/+ mice (which were indistinguishable from wildtype), with multiple, large ectopic signals apparent around the knee (Fig 5A–5F) and ankle joints (Fig 5H–5N). Volumetric analysis revealed a significant increase in the volume of Ubr5mt+SmoLoF femoral-associated ectopic bodies compared to Ubr5mt alone (Fig 5G) and the ankles harboured a 20-fold increase in the volume of ectopic signals (Fig 5O). In agreement, histological analysis of the Ubr5mt+SmoLoF joints revealed an enhanced phenotype to that described in Ubr5mt (Figs 1 and 2). Ubr5mt+SmoLoF synovium harboured large ectopic tissue masses (Fig 6A) with extensive vascularisation (Fig 6B) and chondrocytes lining the surface (Fig 6C) with deeper calcified cartilage and vascularization (Fig 6D). Sagittal sectioning through the ankle revealed large ectopic structures within the superficial digital flexor tendon (Fig 6E), consisting of bone and cartilaginous tissue (Fig 6F and 6H), and at the tendon interface (Fig 6G). Large swathes of chondrocytes were present within the superficial digital flexor and AT that coincided with an absence of tenocytes (Fig 6I and 6J), as previously reported in the Ubr5mt (Fig 2). In addition, the AC in Ubr5mt+SmoLoF knee joints exhibited extensive loss over both tibial and femoral surfaces at this young age (Fig 6M and 6N), while Ubr5mt knee joints exhibited only tears within the AC (Fig 6K, 6L and quantification in 6O). Importantly, the loss of a single copy of Smo alone (Prx1-Cre;SmoLoF/+) resulted in no structural or AC damage (Fig 6P).

<i>Smo</i><sup><i>LoF</i></sup> enhances the <i>Ubr5</i><sup><i>mt</i></sup> ECCO phenotype.
Fig. 5. SmoLoF enhances the Ubr5mt ECCO phenotype.
Analysis of 12-week-old knee (A-G) and ankle (H-O) joints by μCT-based 3D models. (A-C) Ubr5mt and (D-F) Ubr5mt+SmoLoF knee joints revealed ectopic structures marked by red dashed lines and open arrowheads. Sesamoid bones indicated by closed arrowhead. Asterisk marks an ectopic structure displacing the patella. Images (A, B, D, E) are surface rendered 3D models, while (C, F) are optical cross sections through the volume-rendered model revealing the internal structure and X-ray densities of the (open arrowhead) ectopic sesamoid and (closed arrowhead) abnormal patella structures. (G) Volumetric analysis of ectopic structures revealed Ubr5mt+SmoLoF exhibited a dramatic increase in total ectopic volume over Ubr5mt alone. Mean and s.e.m indicated. n = six knees from three animals for each genotype. t-test. p = 0.0002. (H-J) Ubr5mt ankle joints exhibited a few small ectopic signals. (K-N) Ubr5mt+SmoLoF ankles joints exhibited large (closed arrowhead) and small (arrow) ectopic signals in addition to an abnormal and enlarged calcaneus (open arrowhead). (N) Optical cross sections through volume-rendered model revealed the internal structure and X-ray densities of the (open arrowhead) calcaneus and (closed arrowhead) ectopic structure. (O) Volumetric quantification of ectopic structures in the indicated genotypes. n = five animals per genotype. t test, p value = 0.0293. Mean and s.e.m. indicated.
<i>Smo</i><sup><i>LoF</i></sup> enhances the <i>Ubr5</i><sup><i>mt</i></sup> AC phenotype.
Fig. 6. SmoLoF enhances the Ubr5mt AC phenotype.
(A) Ubr5mt+ SmoLoF synovium exhibited large ectopic tissue deep to the patella and adjacent to the femur (open arrowhead). Three black dashed boxes, from left to right, are enlarged in (B), (C) and (D), respectively. (B) Sub-intimal synovial layer abutting the ectopic tissue was highly vascularized (arrowheads). (C) The region interfacing with the ectopic tissue harboured plump spindle-like cells and chondroid-like cells (arrowheads). (D) The core of the ectopic tissue resembled calcified cartilage undergoing endochondral ossification and harboured vascularized cavities (arrowheads). (E) Ubr5mt+SmoLoF ankle exhibited a large ectopic structure (black arrowhead) and abnormal superficial digital flexor tendon (open arrowhead) and calcaneus (asterisk). The upper and lower dashed boxed region are enlarged in (F) and (G), respectively. (F) The ectopic mass contained bone-like (black dashed lines) and cartilaginous tissues (white dashed lines) surrounded by extensively vascularised synovium (open arrowheads). (G) The presumed superficial digital flexor tendon attached to the ectopic bone (black arrowhead), harboured chondrocytes and resembled cartilage (encircled by black dash line). The adjacent periosteum of the calcaneus was highly vascularised (open arrowheads). (H) Cartilaginous thickening of the outer calcaneus. (I) The AT was thickened. The dashed box enlarged in (J) shows columns of chondrocytes (open arrowheads). (K-N) Analysis of 12-week-old knee joints of Ubr5mt and Ubr5mt+SmoLof by H&E stained histological sections of the lateral condyles. (K, L) Ubr5mt exhibited tears in the AC (open arrowhead) and (M, N) Ubr5mt+SmoLoF exhibited extensive loss of AC (dashed lines) and damaged apical surfaces (arrowhead). Dashed boxes in (K) and (M) indicate the enlarged regions in (L) and (N), respectively. (O) Percentage of sections bearing no damage (‘None’), tears (‘Tears’) or exposed calcified cartilage (Exposed), revealed a significance difference between the genotypes. Mean and s.e.m indicated. Chi-square test on pooled slide counts. p = 0.0027. (P’) SmoLoF AC showed no signs of AC damage.

Ubr5 suppresses canonical HH signalling and PKA activity

A functional link between UBR5 activity and HH signalling was further examined in 6-week old Ubr5mt mice. At this age ectopic structures were not detectable (Fig 4), thereby increasing the likelihood of detecting potential causative changes in expression patterns. Immunohistochemistry on Ubr5mt knee intimal (Fig 7A–7F) and subintimal synovium (Fig 7G–7L) revealed increased Gli1 expression in comparison to Prx1-Cre control animals (Fig 7B, 7E, 7H and 7K; respectively), indicative of increased canonical HH signalling. qRT-PCR analysis also confirmed increased expression of markers of canonical HH signalling in RNA from isolated synovium (Gli1 and Ptc1) (Fig 7M). Additionally, intimal and sub-intimal Ubr5mt synovium exhibited increased phosphorylated PKA substrate (PPS) staining suggesting decreased Gi proteins activation, characteristic of non-canonical HH signalling (Fig 7C, 7F, 7I and 7L). Consistent with the observations in the synovium, Ubr5mt AC exhibited markers of increased canonical (Fig 8A–8D) and decreased non-canonical HH signalling (Fig 8E and 8F). Although little change for PTCH1 was detected (Fig 8G) there was significant differences for Gli1 expression and PKA substrate staining (Fig 8H and 8I).

<i>Ubr5</i><sup><i>mt</i></sup> synovium exhibits markers of increased canonical and decreased non-canonical HH signalling.
Fig. 7. Ubr5mt synovium exhibits markers of increased canonical and decreased non-canonical HH signalling.
Immunohistochemical localization of markers of canonical and non-canonical HH signalling (A-L) six-week-old sagittal sections of Prx1-Cre control (Con) and Ubr5mt animals. In general, control (A-C) intimal and (G-I) subintimal layers exhibited weaker GLI1 and PPS staining than in comparable Ubr5mt sections (D-F and J-L, respectively). (B) GLI1 staining in the control intimal layer was located to the vasculature (closed arrowheads) and some adipocytes (open arrowheads). (C) PPS staining was in the vasculature (closed arrowheads) and within adipocytes (open arrowheads). (E) GLI1 and (F) PPS staining were throughout the subintimal layer. (H) GLI1 staining of control synoviocytes within the subintimal layer (arrowhead). (I) PPS staining in sub-intimal layer synoviocytes (arrowhead). (K) GLI1 and (L) PPS staining were strongly expressed within hyperplastic and thickened synovial sub-intimal layer. Synoviocytes exhibited robust nuclear and cytoplasmic staining for GLI1 (K) and PPS (L). (M) qRT-PCR on synovium RNA for expression of canonical HH pathway expression markers Ptch1 and Gli1. Graph indicates mean and s.e.m. n = three animals. t-test. Ptch1 p = 0.0273 and Gli1 p = 0.0477.
Impaired Ubr5 function results in increased canonical and decreased non-canonical HH signalling.
Fig. 8. Impaired Ubr5 function results in increased canonical and decreased non-canonical HH signalling.
Immunohistochemical analysis of six-week-old control and Ubr5mt tibial AC examined for markers of canonical HH pathway activity. Relative to (A, C) control, (B, D) Ubr5mt AC displayed increased staining intensities for PTCH1 (A, B) and GLI1 (C, D) with GLI1 exhibiting expanded expression domains (D, double-headed arrows). (E, F) Staining for PKA phosphorylated substrates (PPS) revealed (Q) Ubr5mt AC exhibited increased numbers of robust staining cells. The number of expressing cells is quantified in (G-I). Quantification confirmed Ubr5mt AC to harbour increased numbers of positive cells for all antigens except PTCH1. Graphs represent the percentage of positive cells, regardless of staining intensity, with the mean and s.e.m indicated. n = 3 biological replicates. Chi-square test on pooled cells count data. p = <0.0008, except PTCH1 which was not significant.

UBR5mt AC and damaged human AC exhibits both aberrant expression of markers of chondrogenesis and HH signalling

As seen in murine Ubr5mt AC, osteoarthritic AC from patients also exhibits markers of increased canonical HH signalling [32]. We next addressed (i) UBR5 expression and (ii) markers of decreased non-canonical HH signalling (PPS) in human AC. Graded samples from (OA) patients (S3A–S3C Fig) undergoing total joint replacement were assessed for UBR5 expression (S3E, S3G and S3I Fig) and PKA activity (PPS in S3D, S3F and S3H Fig). As in the murine model, PPS IHC staining increased (S3J Fig), and hUBR5 staining decreased (S3K Fig) with decreasing AC health. Observations of changes in markers consistent with increased canonical and decreased non-canonical HH signalling in Ubr5mt synovium and AC were echoed in human OA samples.

To further delineate whether mammalian Ubr5 could influence markers of canonical and non-canonical HH signalling, murine NIH3T3 cells were engineered to either exhibit increased (cDNA overexpression) or decreased (shRNA knock-down) Ubr5 expression. Cells were then transfected with constructs encoding (i) Shh, (ii) constitutively active Smo mutant (Smo-M2) or (iii) Gli1. Canonical pathway activity was measured using a Gli-responsive luciferase reporter assay. While perturbation of Ubr5 expression had no effect on Shh- or Smo-M2-mediated signalling (Fig 9A and 9B), Ubr5 overexpression caused a significant reduction (Fig 9A, P<0.001), and Ubr5 shRNA-mediated knockdown caused a significant increase (Fig 9B, P<0.05), in Gli1-mediated luciferase activity. However, Ubr5-overexpression did not perturb the expression level of endogenous or exogenous GLI1 protein (Fig 9C), excluding a role for UBR5-mediated degradation. Therefore, UBR5 appeared to only suppress canonical HH signalling associated with overexpression of GLI1.

Ubr5 functions as a negative regulator of HH signalling ex vivo.
Fig. 9. Ubr5 functions as a negative regulator of HH signalling ex vivo.
Analysis of HH pathway activity in murine NIH3T3 cells in response to modulation of Ubr5 expression. (A) Cells were transfected with empty pN21 vector (grey bars) or pN21-Ubr5 (black bars) together with plasmids encoding Shh, Smo-M2 or Gli1 and 8xGLI-Firefly and pTK-Renilla luciferase reporters in growth medium (DMEM with 10% FBS). After 24 h, serum was reduced to 0.5% and the Firefly/Renilla luciferase activity was measured 48 h later. Bars represent mean +/- s.e.m. of n = 3 independent experiments. (B) A similar GLI-luciferase assay was carried out in NIH3T3 cells stably expressing Ubr5 shRNA (black bars) or scrambled shRNA (grey bars). Bars represent mean +/- s.e.m. of n = 3 independent experiments. (C) NIH3T3 cells were co-transfected with pN21-Ubr5 (Ubr5) or empty vector (Control) and Gli1-myc or empty pcDNA3.1, followed by Western blot analysis of Gli1 expression (arrowhead) using β-actin as loading control. (D) Stable knockdown of Ubr5 impaired readouts of non-canonical HH signalling. Production of cAMP by control scrambled shRNA (black bars) or Ubr5 shRNA stable cells (grey bars) following acute treatment with the adenylate cyclase activator forskolin (For) or forskolin plus the SMO agonist purmorphamine (For/Pur), compared to DMSO vehicle as control (-). Forskolin-stimulated cAMP production in Ubr5 shRNA cells was significantly elevated compared to control cells (p = 0.0368; t-test). Purmorphamine suppressed forskolin-mediated cAMP production in both scramble control (p = 0.0318; t-test) and Ubr5 (p = 0.0160; t-test) shRNA cell lines. Graphs indicate mean and s.e.m.; n = 4 independent experiments. (E) Proposed model of UBR5 function in HH signalling: UBR5 negatively regulates canonical HH signalling downstream of SMO, hypothetically through facilitating the function of the HH negative regulator Sufu, despite simultaneously inhibiting adenylate cyclase (AC). In this context, loss of Ubr5 could increase Gli1 expression by two means: 1) impairment of Sufu negative regulation and 2) stimulation of Gli1 transcriptional activity by increasing PKA-dependent phosphorylation of BRD4. The convergence of Ubr5 and SMO to suppress adenylate cyclase activity could explain the phenotypic enhancement observed in compound mice with loss of function of Ubr5 and Smo. Green and red arrows indicate established modes of activation and repression, respectively.

We then addressed whether loss of Ubr5 function would also affect cAMP production as a readout of Gi protein activity, an indirect marker of non-canonical HH signalling. Ubr5 shRNA cells showed an ~2-fold increase in maximal cAMP production in response to forskolin, an adenylate cyclase agonist (Fig 9D) [33]. Moreover, simultaneous addition of forskolin and purmorphamine, a SMO agonist, lowered maximal cAMP generation, but its effect was suppressed by Ubr5 shRNA (Fig 9D). Together, the in vitro findings suggest that Ubr5 loss results in reduced stimulation of Gi proteins by Smo, leading to increased cAMP/PKA activity levels. Overall, these data supported our in vivo observations that Ubr5 normally acts to suppress GLI1 activity while promoting PKA activity.

Discussion

Ubr5 mutation causes musculoskeletal tissue defects

We report a role for mammalian Ubr5 in adult skeletal homeostasis that impacts upon and genetically interacts with, components of the HH signalling pathway. These findings add to the emerging importance of the N-end rule ligases in regulating important signaling and cellular processes in human, and animal health and disease [34,35]. Loss of the Ubr5 gene in early limb mesenchyme resulted in postnatal defects in and around joints within the fore and hind-limb. The mutant phenotype displayed a degree of variability between individual mice; however, the abnormalities were pervasive in the mutants and were never observed in wildtype or control mice. The defects that were consistently observed included ectopic bone and cartilage formation, and articular cartilage degradation. The spectrum of defects observed are summarized in S4 Fig.

Our data indicates metaplastic production of chondrocytes and/or ectopic endochondral ossification as a major component of Ubr5mt-associated ECCO. Comparison of the Ubr5mt-associated ECCO phenotype with that of human inherited HO diseases reveals some similarities and differences. Within the ECCO-prone tissues there were distinct tissue-specific responses; for example, the knee-associated synovium underwent ectopic chondrogenesis, calcification and ossification to produce bone, whereas the Achilles tendon only underwent ectopic chondrogenesis and calcification. The abnormalities of the knee-associated synovium which display heterotopic chondrogenesis are reminiscent of human benign bone tumours called osteochondromas [36], whereas the heterotopic tissue calcification without ossification seen in the AT resembles a form of calcific tendinopathy [37]. The mouse Ubr5 mutation, thus, provides a genetic model for the generation of these bone abnormalities and suggests that the processes of chondrogenesis, tissue calcification and ossification represent discrete, albeit interrelated, steps that when deregulated can individually, or collectively, contribute to distinct tissue pathologies.

Our findings also demonstrated an important role for Ubr5 in regulating AC homeostasis, where its loss led to dramatic cellular, extracellular and structural defects. The observed defects in HH signalling could have been causative in nature as HH signalling is intimately linked to both stem cell [22] and chondrocyte biology [10]. One of the more distinctive Ubr5mt AC defects was the tearing along the tidemark between non-calcified and calcified cartilage. This focal failure suggested the interface was prone to transverse shear forces and ‘slipping’ of one layer (i.e., non-calcified cartilage) relative to the other (i.e., calcified cartilage). Interestingly, this mode of AC shedding and the associated regions of necrosis mirrored defects observed in mammalian osteochondrosis [38,39].

Recently, increased Ubr5 expression was correlated with muscle growth, hypertrophy [40] and recovery from injury [41] suggesting a role in muscle maintenance. These reports suggest that Ubr5 may have a broad role in the musculoskeletal system of the limbs affecting homeostasis not only in the cartilaginous connective tissue but also in the associated musculature.

UBR5 influences markers of canonical and non-canonical HH signalling

Based on the current dogma, we hypothesized that the Ubr5mt-associated ECCO was caused by increased HH signalling. In contrast, the introduction of SmoLoF heterozygosity into a Ubr5mt background both (i) exacerbated Ubr5mt-associated defects as well as elicited novel defects not observed by loss of Ubr5 function alone (e.g., ECCO of the calcaneal periosteum and the superficial digital flexor tendons and increased volume and altered shape of normotopic sesamoid bones). This combined ability to influence both normotopic and heterotopic bones (S4 Fig), highlights the importance of UBR5 in normal and pathological skeletal tissue homeostasis. Furthermore, our genetic analysis exposed a pro-homeostatic function for SMO–and by extension HH signaling–in suppressing Ubr5mt ECCO.

In vivo and in vitro observations identified a loss of Ubr5 associated with predictors of increased (GLI1 activity) and decreased (PKA activity) canonical HH signalling. Based on the current dogma, it is difficult to reconcile increased GLI activity in the context of increased PKA activity, given that PKA phosphorylates other GLI family members, GLI2 and GLI3, targeting them for processing into transcriptional repressors [14,42]. However, the evolving breadth of the HH pathway (Fig 9E) provides potential mechanistic explanations for this apparently paradoxical observation.

Recent evidence expanded the role of PKA to promote canonical HH signalling by promoting BRD4-mediated stimulation of GLIs transcriptional activity (Fig 9E) [4345]. Interestingly, HO-associated with increased HH signalling was suppressed by the BRD4 inhibitor JQ1 [46], which clearly demonstrated a role for a cAMP-PKA-BRD4-GLI1 axis in skeletal tissue homeostasis. A non-canonical role of SMO as a G protein-coupled receptor (14, 15) provides a mechanism to control PKA activity. Upon stimulation, SMO activates heterotrimeric Gi proteins, which, upon dissociation, inhibit adenylate cyclase through the Gα subunit to reduce cAMP production and PKA activation [15,47,48]. Therefore, SMO inhibition can lead to increased cAMP-mediated PKA activity accounting for SMO modification of the Ubr5mt phenotype, as impairment of either UBR5 or SMO leads to increased cAMP-mediated PKA activity–with their combined impairment leading to either additive or synergistic effects. Interestingly, our preliminary research (personal communication NDGR) supports a role for UBR5 in regulating readouts of non-canonical HH signalling other than PKA (i.e.; RhoA) [16]. Although our data reveal a genetic interaction between UBR5 and an essential component of the HH signalling pathway, we cannot fully establish the underlying mechanism(s) driving Ubr5mt-associated ECCO. Future work will require developing the tools to differentiate between causative individual, or combined, contributions of aberrant canonical or non-canonical HH signalling. The addition of SmoLoF into a Ubr5mt background would have exacerbated a pre-existing imbalance between the pathway outputs to drive ECCO.

The importance of balanced canonical and non-canonical HH signalling was recently demonstrated in osteogenesis [49]. Loss of the cilia regulatory protein IFT80 resulted in impaired osteoblast differentiation and coincided with (i) decreased expression of canonical target genes and (ii) increased non-canonical activity. The authors proposed that the non-canonical HH pathway prevented, and the canonical pathway promoted, formation of osteoblasts. Due to the emerging importance of non-canonical HH signalling [12], we also propose that the combined effects on canonical and non-canonical HH signalling contributed to the observed loss of tissue homeostasis in Ubr5mt animals. Overall, our detection of Ubr5mt-associated increased canonical (GLI1 activity) and indications of decreased non-canonical HH signalling (cAMP-PKA) are in general agreement with a reported pro-osteogenic environment conducive to HO [49]. UBR5 may therefore join IFT80 [49] and DYRK1B [50] as differential regulators of canonical and non-canonical HH signalling. Our future work will involve establishing which of the various non-canonical, SMO’s GPCR-associated downstream effectors (e.g., PKA, RHOA, RAC1, PI3K etc.) [51,52] drive ECCO.

In summary, we reveal a previously unknown role for Ubr5 in influencing HH signalling, tissue homeostasis and preventing spontaneous ECCO. A role for UBR5 in regulating HH signalling and tissue homeostasis supports the classification of human UBR5 as a Tier 1 human cancer susceptibility gene (Sanger Cancer Gene Consensus). We believe the Ubr5mt mouse model could assist in uncovering mechanisms that lead to disorders including characterisation of early pathological events and elucidation of pro-homeostatic mechanisms capable of promoting general bone health. In the future, manipulation of human UBR5 and SMO function could potentially provide a means of preventing pathological, and promoting beneficial, chondrogenesis and ossification in both the clinic and in biomedical engineering applications.

Materials and methods

Ethics statement

Human material

Human AC was obtained from knee joint arthroplasty specimens with ethical approval from the Lothian Research Ethics Committee. Written formal consent was obtained.

Murine studies

All animal experiments were reviewed and approved by the University of Edinburgh Animal Welfare and Ethics Committee and were conducted with appropriate licensing under Animals (Scientific Procedures) Act 1986 (license number PPL 60/4424).

Prx1-Cre;Ubr5gt/gt experimental animals (referred to as Ubr5mt) and their respective littermate controls were generated and all experiments were conducted in accordance with the ARRIVE guidelines. Animals were routinely weighed and there were no significant differences between experimental and control animals. Males animals were used for analysis unless otherwise stated. Tamoxifen (0.1mg/kg body weight) in corn oil, or vehicle only, were administered i.p to six-week-old animals on two consecutive days. For X-gal staining, embryos and postnatal hind limbs were dissected, fixed in 4% formaldehyde (from paraformaldehyde [PFA]) at 4°C, washed and stained in X-Gal stain solution (XRB supplemented with 1mg/ml X-Gal) overnight [20].

Histology

Hindlimbs were fixed in 4% formaldehyde (fromPFA)) for 72hrs at 4°C before being decalcified 0.5M ethylenediaminetetraacetic acid (EDTA) pH7.4 at 4°C. Samples were embedded in paraffin wax blocks and 5μm sagittal sections cut. For cryotome sectioning, samples were equilibrated in a 30% sucrose/phosphate buffered saline (PBS) solution at 4°C and then embedded in OCT compound (Fisher Scientific, Loughborough, UK) before 10μm sagittal sections were cut. For human material, 8x3mm blocks of AC were cut from femoral tibial condyles and fixed in neutral buffered formalin and then paraffin wax embedded. Histological staining with Von Kossa (Abcam, Cambridge, UK), toluidine blue (Sigma) and haematoxylin and eosin (Sigma) were carried out according to standard procedures. All histological scoring was carried out on the lateral tibial condyle with AC damage determined by a binary scoring system, of ‘normal’ or ‘damaged’. At least three slides separated by 25μm were analysed for each limb. For cell and immunohistochemical scoring, cell-types or positive staining cells were expressed as a percentage of the total chondrocyte count. The number of empty lacunae were expressed per mm of AC analysed.

Immunohistochemistry

Primary antibodies: rabbit anti-IHH (1:200, Millipore, Billerica, US); goat anti-PTCH1 (1:50, Santa Cruz, Dallas, US); rabbit anti-GLI1 (1:50, Cell Signalling); rabbit anti-SOX9 (1:50 Santa Cruz); rabbit anti-RUNX2 (1:250, Sigma); PKA phosphorylated substrates (1:150, Cell Signalling); rabbit anti-EDD1 (HsUBR5) (1:100, Bethyl Labs, Montgomery, US). Biotinylated secondary antibodies: goat anti-rabbit and horse anti-goat (1:200, Vector Labs).

Paraffin sections were de-waxed, blocked for endogenous peroxidase and underwent antigen retrieval in 10mM sodium citrate pH6 at 80°C for 30–60 minutes. Slides were blocked with serum-free pan-species block (DAKO, Glostrup, Denmark), incubated with primary antibodies overnight at 4°C, and incubated with biotinylated secondary antibodies for 45mins at room temperature. Sections underwent streptavidin-mediated signal amplification (ELITE ABC, Vectorlabs, Burlingame, US) prior to incubation with peroxidase substrate kit DAB (Vectorlabs).

μCT monitoring

For longitudinal studies, eight-week-old mice were anesthetized with isoflurane prior to in-vivo μCT scanning using a Skyscan 1076 (Bruker, USA, MA) at a resolution of 18μm isotropic voxel size. Procedures were repeated every four weeks and carried out under local ethical approval (ERF WGH-14-74). Fixed limbs were imaged at 18μm resolution using a Skyscan 1076 (Brucker, USA, MA) at 65kV, 110uA, 0.5mm Al filter, 700ms exposure, 0.6-degree rotation step, 180-degree rotation, 2 frame averaging.

μCT image processing

Raw μCT image stacks was reconstructed using GPU-based NRecon (Bruker, USA, MA) using identical parameters within each study type (10% beam hardening and image conversion range of 0.003–0.1125. Reconstructed image stacks were imported into CTAn (Bruker, USA, MA) for selecting regions of interest and acquiring 2D density maps, volumetric quantification of ectopic structures and generation of surface rendered 3D models. Surface rendered 3D were visualized in CTVol (Bruker, USA, MA). 3D volume rendered models were generated in CTVox using a colour-coded transfer functions to identify low, medium and high densities. Longitudinal studies used IMARIS (Bitplane, UK) to create, view and perform volumetric quantification of 3D models.

RNA extraction and q-RT-PCR analysis

Individual joint components were micro-dissected and stored in liquid nitrogen. RNA was extracted using Trizol reagent (Life Technologies), according to manufacturer’s instructions. RNA was reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen). The qRT-PCR was performed using LightCycler 480 SYBR Green I Master (Roche, Germany) and target gene expression normalized to Rpl5 and analysed using the ΔΔCT method [53].

Plasmid constructs

The Shh and SmoM2 (W593L) expression vectors were provided by P. Beachy (Stanford University, USA, CA). mGli1 expression and the reporter vectors 8xGBS-luc were a gift from H. Sasaki (Osaka University, Japan). pCMV-dR8.2 dvpr (8455) and pCMV-VSV-G (8454) were generated in the Weiner lab and obtained from Addgene (USA). pRL-TK was obtained from Promega (USA) and pcDNA3.1+ was purchased from Invitrogen (USA). Recombinant SHH ligand was synthesized and purified as described previously [54].

The complete Ubr5 cDNA was synthesised from murine embryonic stem cells total RNA [9] and cloned into a modified pcDNA5/FRT vector (Life Technologies) containing an amino-terminal 2×HA/2×Strep. NIH3T3 cells (American Type Culture Collection, USA) were seeded at a density of 100,000/ml and transfected after 24hr with pcDNA3.1 alone, UBR5 and pcDNA3.1, Gli1 and pcDNA3.1, or Gli1 and Ubr5 using FuGENE6 (Roche). After 48hrs, the medium was replaced by DMEM/0.5% FCS, and cells were lysed 24 hrs later in Laemmli buffer. Whole cell lysate was separated on a 6% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in 5% non-fat milk, incubated with primary antibodies overnight at 4°C at 1:1,000 dilution for GLI1 (Cell Signalling) or 1:10,000 dilution for β-actin (Sigma). Secondary HRP-conjugated-anti-mouse antibody was applied at a 1:2,000 dilution for 1hr at room temperature. The membranes were developed using the Clarity western ECL substrate (BioRad, USA, CA).

Retrovirus production and stable Ubr5 silencing

Previously validated shRNA-encoding oligos targeting murine Ubr5 and or a scrambled sequence were cloned into pLKO.1-puro (Sigma). shUBR5 and shScrambled-pLKO.1-puro were co-transfected with pCMV-VSV-G and pCMV-dR8.2 dvpr plasmids into HEK 293T cells using TransIT293 reagent (Mirus Bio LLC, USA). To generate stable silenced shUBR5 cells, NIH3T3 cells were seeded at 120,000 cells/ml and infected with 0.5 ml shScramble or shUBR5 retroviral supernatant in the presence of 8 mg/ml polybrene (Sigma). The media was changed after 24 hrs and cells were selected with 2 mg/ml puromycin 48 hrs post-infection.

Gli-luciferase assay

NIH3T3 cells were seeded, and after reaching 70% confluence transfected with pcDNA3.1, Shh, SmoM2, or Gli1 together with Gli-luciferase and Renilla luciferase reporter plasmids with or without pcDNA5-HA-Strep-Ubr5, using FuGENE 6 transfection reagent (Roche) according to the manufacturer’s protocol. For Ubr5 knockdown studies, stable shScramble and shUbr5 NIH3T3 cells were transfected with pcDNA3.1, Shh, SmoM2, or Gli1, together with Gli-luciferase and Renilla luciferase reporter plasmids. In both cases, after the cells reached 100% confluency, the medium was replaced with DMEM/0.5% FCS. After 24 hrs, Firefly and Renilla luciferase activities were determined with the Dual Luciferase Reporter Assay System (Promega).

cAMP assay

Control (scrambled) or knock down (Ubr5 shRNA) NIH3T3 cells were seeded at 130,000 cells/ml, serum starved overnight, and stimulated with 10μM forskolin (FORSK) for 5min. Cells were pre-incubated with 5μM purmorphamine for 10min before addition of FORSK. Cells were processed according to Parameter cAMP Enzyme Immune Assay (R&D Systems) instructions.

Statistical analysis

Data analysis and statistics was performed using PRISM software (GraphPad, La Jolla, US). Count data was analysed using a contingency table and either two-sided Chi square or Fisher’s exact tests according to count size. Continuous data was analysed using unpaired, two-tailed Students t-tests. The level of significance for all tests was set at p = <0.05.

Supporting information

S1 Fig [c]
mice exhibit gait abnormalities and ectopic X-ray-dense signals and analysis of subchondral bone.

S2 Fig [e]
Ectopic structures are not detected in three-week-old control or ankle or knee joints and require postnatal expression of .

S3 Fig [a]
UBR5 and PPS levels correlate with human AC damage.

S4 Fig [tif]
Spectrum of and +Smo associated tissue-specific metaplastic responses.


Zdroje

1. Hershko A, Ciechanover A, Rose IA. Identification of the active amino acid residue of the polypeptide of ATP-dependent protein breakdown. J Biol Chem. 1981;256(4):1525–8. Epub 1981/02/25. 6257674.

2. Mitch WE, Goldberg AL. Mechanisms of muscle wasting. The role of the ubiquitin-proteasome pathway. N Engl J Med. 1996;335(25):1897–905. Epub 1996/12/19. doi: 10.1056/NEJM199612193352507 8948566.

3. Mukhopadhyay D, Riezman H. Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science. 2007;315(5809):201–5. Epub 2007/01/16. doi: 10.1126/science.1127085 17218518.

4. Schnell JD, Hicke L. Non-traditional functions of ubiquitin and ubiquitin-binding proteins. J Biol Chem. 2003;278(38):35857–60. Epub 2003/07/16. doi: 10.1074/jbc.R300018200 12860974.

5. Mansfield E, Hersperger E, Biggs J, Shearn A. Genetic and molecular analysis of hyperplastic discs, a gene whose product is required for regulation of cell proliferation in Drosophila melanogaster imaginal discs and germ cells. Dev Biol. 1994;165(2):507–26. Epub 1994/10/01. doi: 10.1006/dbio.1994.1271 7958417.

6. Lee JD, Amanai K, Shearn A, Treisman JE. The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms. Development. 2002;129(24):5697–706. Epub 2002/11/08. doi: 10.1242/dev.00159 12421709.

7. Moncrieff S, Moncan M, Scialpi F, Ditzel M. Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3beta Homologue, Shaggy. PLoS One. 2015;10(9):e0136760. Epub 2015/09/04. doi: 10.1371/journal.pone.0136760 26334301; PubMed Central PMCID: PMC4559392.

8. Wang G, Tang X, Chen Y, Cao J, Huang Q, Ling X, et al. Hyperplastic discs differentially regulates the transcriptional outputs of hedgehog signaling. Mech Dev. 2014;133:117–25. Epub 2014/05/24. doi: 10.1016/j.mod.2014.05.002 24854243; PubMed Central PMCID: PMC4351657.

9. Kinsella E, Dora N, Mellis D, Lettice L, Deveney P, Hill R, et al. Use of a Conditional Ubr5 Mutant Allele to Investigate the Role of an N-End Rule Ubiquitin-Protein Ligase in Hedgehog Signalling and Embryonic Limb Development. PLoS One. 2016;11(6):e0157079. Epub 2016/06/15. doi: 10.1371/journal.pone.0157079 27299863; PubMed Central PMCID: PMC4907512.

10. Alman BA. The role of hedgehog signalling in skeletal health and disease. Nat Rev Rheumatol. 2015;11(9):552–60. Epub 2015/06/17. doi: 10.1038/nrrheum.2015.84 26077918.

11. Briscoe J, Therond PP. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol. 2013;14(7):416–29. Epub 2013/05/31. doi: 10.1038/nrm3598 23719536.

12. Teperino R, Aberger F, Esterbauer H, Riobo N, Pospisilik JA. Canonical and non-canonical Hedgehog signalling and the control of metabolism. Semin Cell Dev Biol. 2014;33:81–92. Epub 2014/05/28. doi: 10.1016/j.semcdb.2014.05.007 24862854; PubMed Central PMCID: PMC4130743.

13. Pandit T, Ogden SK. Contributions of Noncanonical Smoothened Signaling During Embryonic Development. J Dev Biol. 2017;5(4). Epub 2018/02/06. doi: 10.3390/jdb5040011 29399514; PubMed Central PMCID: PMC5794034.

14. Riobo NA, Saucy B, Dilizio C, Manning DR. Activation of heterotrimeric G proteins by Smoothened. Proc Natl Acad Sci U S A. 2006;103(33):12607–12. Epub 2006/08/04. doi: 10.1073/pnas.0600880103 16885213; PubMed Central PMCID: PMC1567926.

15. Shen F, Cheng L, Douglas AE, Riobo NA, Manning DR. Smoothened is a fully competent activator of the heterotrimeric G protein G(i). Mol Pharmacol. 2013;83(3):691–7. Epub 2013/01/08. doi: 10.1124/mol.112.082511 23292797; PubMed Central PMCID: PMC3583497.

16. Polizio AH, Chinchilla P, Chen X, Kim S, Manning DR, Riobo NA. Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration. J Biol Chem. 2011;286(22):19589–96. Epub 2011/04/09. doi: 10.1074/jbc.M110.197111 21474452; PubMed Central PMCID: PMC3103338.

17. Polizio AH, Chinchilla P, Chen X, Manning DR, Riobo NA. Sonic Hedgehog activates the GTPases Rac1 and RhoA in a Gli-independent manner through coupling of smoothened to Gi proteins. Sci Signal. 2011;4(200):pt7. Epub 2011/11/25. doi: 10.1126/scisignal.2002396 22114142; PubMed Central PMCID: PMC5811764.

18. Long F, Ornitz DM. Development of the endochondral skeleton. Cold Spring Harb Perspect Biol. 2013;5(1):a008334. Epub 2013/01/04. doi: 10.1101/cshperspect.a008334 23284041; PubMed Central PMCID: PMC3579395.

19. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 1999;13(16):2072–86. Epub 1999/08/31. doi: 10.1101/gad.13.16.2072 10465785; PubMed Central PMCID: PMC316949.

20. Karp SJ, Schipani E, St-Jacques B, Hunzelman J, Kronenberg H, McMahon AP. Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone related-protein-dependent and -independent pathways. Development. 2000;127(3):543–8. Epub 2000/01/13. 10631175.

21. Yang J, Andre P, Ye L, Yang YZ. The Hedgehog signalling pathway in bone formation. Int J Oral Sci. 2015;7(2):73–9. Epub 2015/05/30. doi: 10.1038/ijos.2015.14 26023726; PubMed Central PMCID: PMC4817553.

22. Petrova R, Joyner AL. Roles for Hedgehog signaling in adult organ homeostasis and repair. Development. 2014;141(18):3445–57. Epub 2014/09/04. doi: 10.1242/dev.083691 25183867; PubMed Central PMCID: PMC4197719.

23. Peng T, Frank DB, Kadzik RS, Morley MP, Rathi KS, Wang T, et al. Hedgehog actively maintains adult lung quiescence and regulates repair and regeneration. Nature. 2015;526(7574):578–82. Epub 2015/10/06. doi: 10.1038/nature14984 26436454; PubMed Central PMCID: PMC4713039.

24. Cholok D, Chung MT, Ranganathan K, Ucer S, Day D, Davis TA, et al. Heterotopic ossification and the elucidation of pathologic differentiation. Bone. 2018;109:12–21. Epub 2017/10/11. doi: 10.1016/j.bone.2017.09.019 28987285; PubMed Central PMCID: PMC6585944.

25. Regard JB, Malhotra D, Gvozdenovic-Jeremic J, Josey M, Chen M, Weinstein LS, et al. Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification. Nat Med. 2013;19(11):1505–12. Epub 2013/10/01. doi: 10.1038/nm.3314 24076664; PubMed Central PMCID: PMC3917515.

26. Hopyan S, Nadesan P, Yu C, Wunder J, Alman BA. Dysregulation of hedgehog signalling predisposes to synovial chondromatosis. J Pathol. 2005;206(2):143–50. Epub 2005/04/19. doi: 10.1002/path.1761 15834844.

27. Lin AC, Seeto BL, Bartoszko JM, Khoury MA, Whetstone H, Ho L, et al. Modulating hedgehog signaling can attenuate the severity of osteoarthritis. Nat Med. 2009;15(12):1421–5. Epub 2009/11/17. doi: 10.1038/nm.2055 19915594.

28. Zhou J, Chen Q, Lanske B, Fleming BC, Terek R, Wei X, et al. Disrupting the Indian hedgehog signaling pathway in vivo attenuates surgically induced osteoarthritis progression in Col2a1-CreERT2; Ihhfl/fl mice. Arthritis Res Ther. 2014;16(1):R11. Epub 2014/01/17. doi: 10.1186/ar4437 24428864; PubMed Central PMCID: PMC3978435.

29. Saunders DN, Hird SL, Withington SL, Dunwoodie SL, Henderson MJ, Biben C, et al. Edd, the murine hyperplastic disc gene, is essential for yolk sac vascularization and chorioallantoic fusion. Mol Cell Biol. 2004;24(16):7225–34. Epub 2004/07/30. doi: 10.1128/MCB.24.16.7225-7234.2004 15282321; PubMed Central PMCID: PMC479729.

30. Logan M, Martin JF, Nagy A, Lobe C, Olson EN, Tabin CJ. Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis. 2002;33(2):77–80. Epub 2002/07/12. doi: 10.1002/gene.10092 12112875.

31. Long F, Zhang XM, Karp S, Yang Y, McMahon AP. Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation. Development. 2001;128(24):5099–108. Epub 2001/12/19. 11748145.

32. Xiao WF, Li YS, Deng A, Yang YT, He M. Functional role of hedgehog pathway in osteoarthritis. Cell Biochem Funct. 2020;38(2):122–9. Epub 2019/12/14. doi: 10.1002/cbf.3448 31833076.

33. Metzger H, Lindner E. The positive inotropic-acting forskolin, a potent adenylate cyclase activator. Arzneimittelforschung. 1981;31(8):1248–50. Epub 1981/01/01. 7197529.

34. Tasaki T, Sriram SM, Park KS, Kwon YT. The N-end rule pathway. Annu Rev Biochem. 2012;81:261–89. Epub 2012/04/25. doi: 10.1146/annurev-biochem-051710-093308 22524314; PubMed Central PMCID: PMC3610525.

35. Gibbs DJ, Bacardit J, Bachmair A, Holdsworth MJ. The eukaryotic N-end rule pathway: conserved mechanisms and diverse functions. Trends Cell Biol. 2014;24(10):603–11. Epub 2014/05/31. doi: 10.1016/j.tcb.2014.05.001 24874449.

36. Hakim DN, Pelly T, Kulendran M, Caris JA. Benign tumours of the bone: A review. J Bone Oncol. 2015;4(2):37–41. Epub 2015/11/19. doi: 10.1016/j.jbo.2015.02.001 26579486; PubMed Central PMCID: PMC4620948.

37. Oliva F, Via AG, Maffulli N. Physiopathology of intratendinous calcific deposition. BMC Med. 2012;10:95. Epub 2012/08/25. doi: 10.1186/1741-7015-10-95 22917025; PubMed Central PMCID: PMC3482552.

38. McCoy AM, Toth F, Dolvik NI, Ekman S, Ellermann J, Olstad K, et al. Articular osteochondrosis: a comparison of naturally-occurring human and animal disease. Osteoarthritis Cartilage. 2013;21(11):1638–47. Epub 2013/08/21. doi: 10.1016/j.joca.2013.08.011 23954774; PubMed Central PMCID: PMC3815567.

39. Carey JL, Wall EJ, Grimm NL, Ganley TJ, Edmonds EW, Anderson AF, et al. Novel Arthroscopic Classification of Osteochondritis Dissecans of the Knee: A Multicenter Reliability Study. Am J Sports Med. 2016;44(7):1694–8. Epub 2016/05/10. doi: 10.1177/0363546516637175 27159302.

40. Seaborne RA, Hughes DC, Turner DC, Owens DJ, Baehr LM, Gorski P, et al. UBR5 is a novel E3 ubiquitin ligase involved in skeletal muscle hypertrophy and recovery from atrophy. J Physiol. 2019;597(14):3727–49. Epub 2019/05/17. doi: 10.1113/JP278073 31093990.

41. Seaborne RA, Strauss J, Cocks M, Shepherd S, O’Brien TD, van Someren KA, et al. Human Skeletal Muscle Possesses an Epigenetic Memory of Hypertrophy. Sci Rep. 2018;8(1):1898. Epub 2018/02/01. doi: 10.1038/s41598-018-20287-3 29382913; PubMed Central PMCID: PMC5789890.

42. Wang B, Fallon JF, Beachy PA. Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell. 2000;100(4):423–34. Epub 2000/02/29. doi: 10.1016/s0092-8674(00)80678-9 10693759.

43. Long J, Li B, Rodriguez-Blanco J, Pastori C, Volmar CH, Wahlestedt C, et al. The BET bromodomain inhibitor I-BET151 acts downstream of smoothened protein to abrogate the growth of hedgehog protein-driven cancers. J Biol Chem. 2014;289(51):35494–502. Epub 2014/10/31. doi: 10.1074/jbc.M114.595348 25355313; PubMed Central PMCID: PMC4271234.

44. Tang Y, Gholamin S, Schubert S, Willardson MI, Lee A, Bandopadhayay P, et al. Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition. Nat Med. 2014;20(7):732–40. Epub 2014/06/30. doi: 10.1038/nm.3613 24973920; PubMed Central PMCID: PMC4108909.

45. Martin RD, Sun Y, MacKinnon S, Cuccia L, Page V, Hebert TE, et al. Differential Activation of P-TEFb Complexes in the Development of Cardiomyocyte Hypertrophy following Activation of Distinct G Protein-Coupled Receptors. Mol Cell Biol. 2020;40(14). Epub 2020/04/29. doi: 10.1128/MCB.00048-20 32341082; PubMed Central PMCID: PMC7324848.

46. Feng H, Xing W, Han Y, Sun J, Kong M, Gao B, et al. Tendon-derived cathepsin K-expressing progenitor cells activate Hedgehog signaling to drive heterotopic ossification. J Clin Invest. 2020. Epub 2020/08/28. doi: 10.1172/JCI132518 32853181.

47. Carbe CJ, Cheng L, Addya S, Gold JI, Gao E, Koch WJ, et al. Gi proteins mediate activation of the canonical hedgehog pathway in the myocardium. Am J Physiol Heart Circ Physiol. 2014;307(1):H66–72. Epub 2014/05/13. doi: 10.1152/ajpheart.00166.2014 24816261; PubMed Central PMCID: PMC4080174.

48. Cheng L, Al-Owais M, Covarrubias ML, Koch WJ, Manning DR, Peers C, et al. Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K(+) currents in cardiomyocytes and prolongs cardiac action potential duration. J Biol Chem. 2018;293(28):11022–32. Epub 2018/05/29. doi: 10.1074/jbc.RA118.001989 29802197; PubMed Central PMCID: PMC6052211.

49. Yuan X, Cao J, He X, Serra R, Qu J, Cao X, et al. Ciliary IFT80 balances canonical versus non-canonical hedgehog signalling for osteoblast differentiation. Nat Commun. 2016;7:11024. Epub 2016/03/22. doi: 10.1038/ncomms11024 26996322; PubMed Central PMCID: PMC4802171.

50. Singh R, Dhanyamraju PK, Lauth M. DYRK1B blocks canonical and promotes non-canonical Hedgehog signaling through activation of the mTOR/AKT pathway. Oncotarget. 2017;8(1):833–45. Epub 2016/12/03. doi: 10.18632/oncotarget.13662 27903983; PubMed Central PMCID: PMC5352201.

51. Mukhopadhyay S, Rohatgi R. G-protein-coupled receptors, Hedgehog signaling and primary cilia. Semin Cell Dev Biol. 2014;33:63–72. Epub 2014/05/23. doi: 10.1016/j.semcdb.2014.05.002 24845016; PubMed Central PMCID: PMC4130902.

52. O’Hayre M, Degese MS, Gutkind JS. Novel insights into G protein and G protein-coupled receptor signaling in cancer. Curr Opin Cell Biol. 2014;27:126–35. Epub 2014/02/11. doi: 10.1016/j.ceb.2014.01.005 24508914; PubMed Central PMCID: PMC4021379.

53. Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2^(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 2013;3(3):71–85. Epub 2013/08/01. 25558171; PubMed Central PMCID: PMC4280562.

54. Martinez-Chinchilla P, Riobo NA. Purification and bioassay of hedgehog ligands for the study of cell death and survival. Methods Enzymol. 2008;446:189–204. Epub 2008/07/08. doi: 10.1016/S0076-6879(08)01611-X 18603123.


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