Evolutionary Selection and Constraint on Human Knee Chondrocyte Regulation Impacts Osteoarthritis Risk.

During human evolution, the knee adapted to the biomechanical demands of bipedalism by altering chondrocyte developmental programs. This adaptive process was likely not without deleterious consequences to health. Today, osteoarthritis occurs in 250 million people, with risk variants enriched in non-coding sequences near chondrocyte genes, loci that likely became optimized during knee evolution. We explore this relationship by epigenetically profiling joint chondrocytes, revealing ancient selection and recent constraint and drift on knee regulatory elements, which also overlap osteoarthritis variants that contribute to disease heritability by tending to modify constrained functional sequence. We propose a model whereby genetic violations to regulatory constraint, tolerated during knee development, lead to adult pathology. In support, we discover a causal enhancer variant (rs6060369) present in billions of people at a risk locus (GDF5-UQCC1), showing how it impacts mouse knee-shape and osteoarthritis. Overall, our methods link an evolutionarily novel aspect of human anatomy to its pathogenesis.

Decellularized nucleus pulposus matrix/chitosan hybrid hydrogel combined with nucleus pulposus stem cells and GDF5-loaded microspheres for intervertebral disc degeneration prevention.

BACKGROUND: Intervertebral disc degeneration (IDD) is considered an important pathological basis for spinal degenerative diseases. Tissue engineering is a powerful therapeutic strategy that can effectively restore the normal biological properties of disc units. In this study, hydrogels loaded with growth/differentiation factor 5 (GDF5) and stem cells were combined to provide an effective strategy for nucleus pulposus regeneration. METHODS: Nucleus pulposus stem cells (NPSCs) were obtained by low-density inoculation and culture, and their stem cell characteristics were verified by flow cytometry and a tri-lineage-induced differentiation experiment. A decellularized nucleus pulposus matrix (DNPM) and chitosan hybrid hydrogel was prepared, and GDF5-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres were incorporated into the hydrogels to obtain a composite hydrogels with GDF5-loaded microspheres. Taking bone marrow mesenchymal stem cells (BMSCs) as a reference, the effect of composite hydrogels with GDF5-loaded microspheres on the chondrogenic differentiation of NPSCs was evaluated. A model of intervertebral disc degeneration induced by acupuncture on the tail of rats was constructed, and the repair effect of composite hydrogels with GDF5-loaded microspheres combined with NPSCs on IDD was observed. RESULTS: Stem cell phenotype identification, stemness gene expression and tri-lineage-induced differentiation confirmed that NPSCs had characteristics similar to those of BMSCs. The rat DNPM and chitosan hybrid hydrogels had good mechanical properties, and the GDF5-loaded microspheres sustainably released GDF5. NPSCs grew normally in the composite hydrogels and gradually expressed a chondrocyte phenotype. Animal experiments showed that the composite hydrogels with GDF5-loaded microspheres combined with NPSCs effectively promoted nucleus pulposus regeneration and that the effect of the hydrogels on the repair of IDD was significantly better than that of BMSCs. CONCLUSION: GDF5-loaded microspheres combined with DNPM/chitosan composite hydrogels can effectively promote the differentiation of NPSCs into nucleus pulposus-like cells and effectively preventIDD.

Relaxed selection in evolution of genes regulating limb development gives clue to variation in forelimb morphology of cetaceans and other mammals.

Cetaceans have evolved unique limb structures, such as flippers, due to genetic changes during their transition to aquatic life. However, the full understanding of the genetic and evolutionary mechanisms behind these changes is still developing. By examining 25 limb-related protein-coding genes across various mammalian species, we compared genetic changes between aquatic mammals, like whales, and other mammals with unique limb structures such as bats, rodents and elephants. Our findings revealed significant modifications in limb-related genes, including variations in the Hox, GDF5 and Evx genes. Notably, a relaxed selection in several key genes was observed, suggesting a lifting of developmental constraints, which might have facilitated the emergence of morphological innovations in cetacean limb morphology. We also uncovered non-synonymous changes, insertions and deletions in these genes, particularly in the polyalanine tract of HOXD13, which are distinctive to cetaceans or convergent with other aquatic mammals. These genetic variations correlated with the diverse and specialized limb structures observed in cetaceans, indicating a complex interplay of relaxed selection and specific mutations in mammalian limb evolution.

Genetics of hip dysplasia - a systematic literature review.

BACKGROUND: Developmental dysplasia of the hip (DDH) is a congenital condition affecting 2-3% of all newborns. DDH increases the risk of osteoarthritis and is the cause of 30% of all total hip arthroplasties in adults < 40 years of age. We aim to explore the genetic background of DDH in order to improve diagnosis and personalize treatment. METHODS: We conducted a structured literature review using PRISMA guidelines searching the Medline, Embase and Cochrane databases. We included 31 case control studies examining single nucleotide polymorphisms (SNPs) in non-syndromic DDH. RESULTS: A total of 73 papers were included for full text review, of which 31 were single nucleotide polymorphism (SNP) case/control association studies. The literature review revealed that the majority of published papers on the genetics of DDH were mostly underpowered for detection of any significant association. One large genome wide association study has been published (N = 9,915), establishing GDF5 as a plausible risk factor. CONCLUSIONS: DDH is known to be congenital and heritable, with family occurrence of DDH already included as a risk factor in most screening programs. Despite this, high quality genetic research is scarce and no genetic risk factors have been soundly established, prompting the need for more research.

A single cell transcriptional atlas of early synovial joint development.

Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

Growth differentiation factor 5 inhibits lipopolysaccharide-mediated pyroptosis of nucleus pulposus mesenchymal stem cells via RhoA signaling pathway.

BACKGROUND: Degenerative disc disease(DDD)is one of the most important causes of low back pain (LBP). Programmed death of human nucleus pulposus mesenchymal stem cells (NPMSCs) plays an important role in the progression of DDD. Growth differentiation factor-5 (GDF-5) is a protein that promotes chondrogenic differentiation, and has been reported to slow the expression of inflammatory factors in nucleus pulposus cells. Compared with those in normal rats, MRI T2-weighted images show hypointense in the central nucleus pulposus region of the intervertebral disc in GDF-5 knockout rats. METHODS AND RESULTS: We aimed to evaluate the role of GDF-5 and Ras homolog family member A (RhoA) in NPMSCs. We used lipopolysaccharide (LPS) to simulate the inflammatory environment in degenerative disc disease, and performed related experiments on the effects of GDF-5 on NPMSCs, including the effects of pyroptosis, RhoA protein, and the expression of extracellular matrix components, and the effects of GDF-5, on NPMSCs. In addition, the effect of GDF-5 on chondroid differentiation of NPMSCs was included. The results showed that the addition of GDF-5 inhibited the LPS-induced pyroptosis of NPMSCs, and further analysis of its mechanism showed that this was achieved by activating the RhoA signaling pathway. CONCLUSION: These findings suggest that GDF-5 plays an important role in inhibiting the pyroptosis of NPMSCs and GDF-5 may have potential for degenerative disc disease gene-targeted therapy in the future.

Are commercial genetic injury tests premature?

INTRODUCTION: Several direct-to-consumer (DTC) genetic testing companies have emerged that claim to be able to test for susceptibility for musculoskeletal injuries. Although there are several publications on the emergence of this industry, none have critically evaluated the evidence for the use of genetic polymorphisms in commercial tests. The aim of this review was to identify, where possible, the polymorphisms and to evaluate the current scientific evidence for their inclusion. RESULTS: The most common polymorphisms included COL1A1 rs1800012, COL5A1 rs12722, and GDF5 rs143383. The current evidence suggests that it is premature or even not viable to include these three polymorphisms as markers of injury risk. A unique set of injury-specific polymorphisms, which do not include COL1A1, COL5A1, or GDF5, identified from genome-wide association studies (GWAS) is used by one company in their tests for 13 sports injuries. However, of the 39 reviewed polymorphisms, 22 effective alleles are rare and absent in African, American, and/or Asian populations. Even when informative in all populations, the sensitivity of many of the genetic markers was low and/or has not been independently validated in follow-up studies. CONCLUSIONS: The current evidence suggests it is premature to include any of the reviewed polymorphisms identified by GWAS or candidate gene approaches in commercial genetic tests. The association of MMP7 rs1937810 with Achilles tendon injuries, and SAP30BP rs820218 and GLCCI1 rs4725069 with rotator cuff injuries does warrant further investigation. Based on current evidence, it remains premature to market any commercial genetic test to determine susceptibility to musculoskeletal injuries.

Skeletal Muscle-Derived Exosomal miR-146a-5p Inhibits Adipogenesis by Mediating Muscle-Fat Axis and Targeting GDF5-PPARγ Signaling.

Skeletal muscle-fat interaction is essential for maintaining organismal energy homeostasis and managing obesity by secreting cytokines and exosomes, but the role of the latter as a new mediator in inter-tissue communication remains unclear. Recently, we discovered that miR-146a-5p was mainly enriched in skeletal muscle-derived exosomes (SKM-Exos), 50-fold higher than in fat exosomes. Here, we investigated the role of skeletal muscle-derived exosomes regulating lipid metabolism in adipose tissue by delivering miR-146a-5p. The results showed that skeletal muscle cell-derived exosomes significantly inhibited the differentiation of preadipocytes and their adipogenesis. When the skeletal muscle-derived exosomes co-treated adipocytes with miR-146a-5p inhibitor, this inhibition was reversed. Additionally, skeletal muscle-specific knockout miR-146a-5p (mKO) mice significantly increased body weight gain and decreased oxidative metabolism. On the other hand, the internalization of this miRNA into the mKO mice by injecting skeletal muscle-derived exosomes from the Flox mice (Flox-Exos) resulted in significant phenotypic reversion, including down-regulation of genes and proteins involved in adipogenesis. Mechanistically, miR-146a-5p has also been demonstrated to function as a negative regulator of peroxisome proliferator-activated receptor γ (PPARγ) signaling by directly targeting growth and differentiation factor 5 (GDF5) gene to mediate adipogenesis and fatty acid absorption. Taken together, these data provide new insights into the role of miR-146a-5p as a novel myokine involved in the regulation of adipogenesis and obesity via mediating the skeletal muscle-fat signaling axis, which may serve as a target for the development of therapies against metabolic diseases, such as obesity.

The role of Gdf5 in the development of the zebrafish fin endoskeleton.

BACKGROUND: The development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate the correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected in fin mesenchyme condensations and segmentation zones as well as the jaw joint, however, little is known about the functional role of Gdf5 outside of Amniota. RESULTS: We generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analyzed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, as a consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed disorganization and reduced length of endoskeletal elements in the median fins, while joints and mineralization seemed unaffected. CONCLUSIONS: Our study demonstrates the importance of Gdf5 in the development of the zebrafish pectoral and median fin endoskeleton and reveals that the severity of the effect increases from anterior to posterior elements. Our findings are consistent with phenotypes observed in the human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.

Hox11 expression characterizes developing zeugopod synovial joints and is coupled to postnatal articular cartilage morphogenesis into functional zones in mice.

Previous studies on mouse embryo limbs have established that interzone mesenchymal progenitor cells emerging at each prescribed joint site give rise to joint tissues over fetal time. These incipient tissues undergo structural maturation and morphogenesis postnatally, but underlying mechanisms of regulation remain unknown. Hox11 genes dictate overall zeugopod musculoskeletal patterning and skeletal element identities during development. Here we asked where these master regulators are expressed in developing limb joints and whether they are maintained during postnatal zeugopod joint morphogenesis. We found that Hoxa11 was predominantly expressed and restricted to incipient wrist and ankle joints in E13.5 mouse embryos, and became apparent in medial and central regions of knees by E14.5, though remaining continuously dormant in elbow joints. Closer examination revealed that Hoxa11 initially characterized interzone and neighboring cells and was then restricted to nascent articular cartilage, intra joint ligaments and structures such as meniscal horns over prenatal time. Postnatally, articular cartilage progresses from a nondescript cell-rich, matrix-poor tissue to a highly structured, thick, zonal and mechanically competent tissue with chondrocyte columns over time, most evident at sites such as the tibial plateau. Indeed, Hox11 expression (primarily Hoxa11) was intimately coupled to such morphogenetic processes and, in particular, to the topographical rearrangement of chondrocytes into columns within the intermediate and deep zones of tibial plateau that normally endures maximal mechanical loads. Revealingly, these expression patterns were maintained even at 6 months of age. In sum, our data indicate that Hox11 genes remain engaged well beyond embryonic synovial joint patterning and are specifically tied to postnatal articular cartilage morphogenesis into a zonal and resilient tissue. The data demonstrate that Hox11 genes characterize adult, terminally differentiated, articular chondrocytes and maintain region-specificity established in the embryo.

BMPR1A is necessary for chondrogenesis and osteogenesis, whereas BMPR1B prevents hypertrophic differentiation.

BMP2 stimulates bone formation and signals preferably through BMP receptor (BMPR) 1A, whereas GDF5 is a cartilage inducer and signals preferably through BMPR1B. Consequently, BMPR1A and BMPR1B are believed to be involved in bone and cartilage formation, respectively. However, their function is not yet fully clarified. In this study, GDF5 mutants with a decreased affinity for BMPR1A were generated. These mutants, and wild-type GDF5 and BMP2, were tested for their ability to induce dimerization of BMPR1A or BMPR1B with BMPR2, and for their chondrogenic, hypertrophic and osteogenic properties in chondrocytes, in the multipotent mesenchymal precursor cell line C3H10T1/2 and the human osteosarcoma cell line Saos-2. Mutants with the lowest potency for inducing BMPR1A-BMPR2 dimerization exhibited minimal chondrogenic and osteogenic activities, indicating that BMPR1A is necessary for chondrogenic and osteogenic differentiation. BMP2, GDF5 and the GDF5 R399E mutant stimulated expression of chondrogenic and hypertrophy markers in C3H10T1/2 cells and chondrocytes. However, GDF5 R399E, which induces the dimerization of BMPR1B and BMPR2 more potently than GDF5 or BMP2, displayed reduced hypertrophic activity. Therefore, we postulate that stronger BMPR1B signaling, compared to BMPR1A signaling, prevents chondrocyte hypertrophy and acts as a cartilage stabilizer during joint morphogenesis.This article has an associated First Person interview with the first author of the paper.

Novel NFκB Inhibitor SC75741 Mitigates Chondrocyte Degradation and Prevents Activated Fibroblast Transformation by Modulating miR-21/GDF-5/SOX5 Signaling.

Osteoarthritis (OA) is a common articular disease manifested by the destruction of cartilage and compromised chondrogenesis in the aging population, with chronic inflammation of synovium, which drives OA progression. Importantly, the activated synovial fibroblast (AF) within the synovium facilitates OA through modulating key molecules, including regulatory microRNAs (miR's). To understand OA associated pathways, in vitro co-culture system, and in vivo papain-induced OA model were applied for this study. The expression of key inflammatory markers both in tissue and blood plasma were examined by qRT-PCR, western blot, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays. Herein, our result demonstrated, AF-activated human chondrocytes (AC) exhibit elevated NFκB, TNF-α, IL-6, and miR-21 expression as compared to healthy chondrocytes (HC). Importantly, AC induced the apoptosis of HC and inhibited the expression of chondrogenesis inducers, SOX5, TGF-ß1, and GDF-5. NFκB is a key inflammatory transcription factor elevated in OA. Therefore, SC75741 (an NFκB inhibitor) therapeutic effect was explored. SC75741 inhibits inflammatory profile, protects AC-educated HC from apoptosis, and inhibits miR-21 expression, which results in the induced expression of GDF-5, SOX5, TGF-ß1, BMPR2, and COL4A1. Moreover, ectopic miR-21 expression in fibroblast-like activated chondrocytes promoted osteoblast-mediated differentiation of osteoclasts in RW264.7 cells. Interestingly, in vivo study demonstrated SC75741 protective role, in controlling the destruction of the articular joint, through NFκB, TNF-α, IL-6, and miR-21 inhibition, and inducing GDF-5, SOX5, TGF-ß1, BMPR2, and COL4A1 expression. Our study demonstrated the role of NFκB/miR-21 axis in OA progression, and SC75741's therapeutic potential as a small-molecule inhibitor of miR-21/NFκB-driven OA progression.

Future Cell and Gene Therapy for Osteoarthritis (OA): Potential for Using Mammalian Protein Production Platforms, Irradiated and Transfected Protein Packaging Cell Lines for Over-Production of Therapeutic Proteins and Growth Factors.

In this paper I provide a personal perspective on future prospects for cell and gene therapy for osteoarthritis (OA) and how mammalian protein production platforms, virally transfected and irradiated protein packaging cell lines may be used as "cellular factories" for over-production of therapeutic proteins and growth factors, particularly in the context of intra-articular regenerative therapies. I will also speculate on future opportunities and challenges in this area of research and how new innovations in biotechnology will impact on the field of cell and gene therapy for OA, related osteoarticular disorders and the broader discipline of regenerative medicine for musculoskeletal disorders. Mammalian protein production platforms are likely to have a significant impact on synovial joint diseases that are amenable to cell and gene therapy using therapeutic proteins and growth factors. Future cell and gene therapy for OA will need to re-consider the current strategies that employ primary, aged and senescent cells with feeble regenerative properties and seriously consider the use of mammalian protein production platforms.

Increasing the Medium Osmolarity Reduces the Inflammatory Status of Human OA Chondrocytes and Increases Their Responsiveness to GDF-5.

The environment surrounding chondrocytes changes drastically in osteoarthritis (OA). For instance, the osmolarity in cartilage (ranging from 350 to 460 mOsm in healthy tissue) decreases during the progression of OA, reaching 270 mOsm. The objective of this study was to evaluate how osmolarity influences human OA chondrocytes. For this purpose, the osmolarity of the culture medium (340 mOsm) was increased to 380, 420 or 460 mOsm and its effect on the phenotype, matrix production, protease expression, cytokine release and growth and differentiation factor-5 (GDF-5) receptor expression in human OA chondrocytes was evaluated in a monolayer. Afterwards, the same parameters, as well as the responsiveness to GDF-5, were evaluated in 3D culture at 340 and 380 mOsm. Our results revealed that increasing the medium osmolarity increased matrix production but also reduced cytokine release, type I collagen and protease expression. It was also demonstrated that at 380 mOsm, the response to GDF-5 in 3D culture was more robust than at 340 mOsm. For the first time, it was established that a decreased osmolarity plays a role in sustaining inflammation and catabolic activities in OA chondrocytes and decreases their responsiveness to GDF-5. This indicates that osmolarity is a critical aspect of OA pathobiology.

Complex Phenotypes: Mechanisms Underlying Variation in Human Stature.

PURPOSE OF REVIEW: The goal of the review is to provide a comprehensive overview of the current understanding of the mechanisms underlying variation in human stature. RECENT FINDINGS: Human height is an anthropometric trait that varies considerably within human populations as well as across the globe. Historically, much research focus was placed on understanding the biology of growth plate chondrocytes and how modifications to core chondrocyte proliferation and differentiation pathways potentially shaped height attainment in normal as well as pathological contexts. Recently, much progress has been made to improve our understanding regarding the mechanisms underlying the normal and pathological range of height variation within as well as between human populations, and today, it is understood to reflect complex interactions among a myriad of genetic, environmental, and evolutionary factors. Indeed, recent improvements in genetics (e.g., GWAS) and breakthroughs in functional genomics (e.g., whole exome sequencing, DNA methylation analysis, ATAC-sequencing, and CRISPR) have shed light on previously unknown pathways/mechanisms governing pathological and common height variation. Additionally, the use of an evolutionary perspective has also revealed important mechanisms that have shaped height variation across the planet. This review provides an overview of the current knowledge of the biological mechanisms underlying height variation by highlighting new research findings on skeletal growth control with an emphasis on previously unknown pathways/mechanisms influencing pathological and common height variation. In this context, this review also discusses how evolutionary forces likely shaped the genomic architecture of height across the globe.

Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs.

Limb synovial joints are composed of distinct tissues, but it is unclear which progenitors produce those tissues and how articular cartilage acquires its functional postnatal organization characterized by chondrocyte columns, zone-specific cell volumes and anisotropic matrix. Using novel Gdf5CreERT2 (Gdf5-CE), Prg4-CE and Dkk3-CE mice mated to R26-Confetti or single-color reporters, we found that knee joint progenitors produced small non-migratory progenies and distinct local tissues over prenatal and postnatal time. Stereological imaging and quantification indicated that the columns present in juvenile-adult tibial articular cartilage consisted of non-daughter, partially overlapping lineage cells, likely reflecting cell rearrangement and stacking. Zone-specific increases in cell volume were major drivers of tissue thickening, while cell proliferation or death played minor roles. Second harmonic generation with 2-photon microscopy showed that the collagen matrix went from being isotropic and scattered at young stages to being anisotropic and aligned along the cell stacks in adults. Progenitor tracing at prenatal or juvenile stages showed that joint injury provoked a massive and rapid increase in synovial Prg4+ and CD44+/P75+ cells some of which filling the injury site, while neighboring chondrocytes appeared unresponsive. Our data indicate that local cell populations produce distinct joint tissues and that articular cartilage growth and zonal organization are mainly brought about by cell volume expansion and topographical cell rearrangement. Synovial Prg4+ lineage progenitors are exquisitely responsive to acute injury and may represent pioneers in joint tissue repair.

microRNA-665 promotes the proliferation and matrix degradation of nucleus pulposus through targeting GDF5 in intervertebral disc degeneration.

Growing evidences suggested that microRNAs (miRNAs) played important roles in the development of intervertebral disc degeneration (IDD). However, the expression level and function of miR-665 in IDD remain unknown. In this study, we showed that the expression level of miR-665 was upregulated in degenerative human NP samples. In addition, miR-665 expression level gradually increased with the exacerbation of disc degeneration grade. Moreover, miR-665 expression level was positively associated with the Pfirrmann grade. Ectopic expression of miR-665 promoted NP cell growth. Furthermore, miR-665 overexpression decreased aggrecan and Col II expression and ectopic expression of miR-665 increased MMP-3 and MMP-13 expression in NP cell. We identified growth differentiation factor 5 (GDF5) was a direct target gene of miR-665 in NP cell and enforced expression of miR-665 decreased GDF5 expression. Elevated expression of miR-665 enhanced NP cell proliferation and decreased aggrecan and Col II expression. In addition, ectopic expression of miR-665 increased MMP-3 and MMP-13 expression through inhibiting GDF5 expression in NP cells. These results suggested that dysregulated miR-665 expression might act an important role in the development of IDD.

Dach2-Hdac9 signaling regulates reinnervation of muscle endplates.

Muscle denervation resulting from injury, disease or aging results in impaired motor function. Restoring neuromuscular communication requires axonal regrowth and endplate reinnervation. Muscle activity inhibits the reinnervation of denervated muscle. The mechanism by which muscle activity regulates muscle reinnervation is poorly understood. Dach2 and Hdac9 are activity-regulated transcriptional co-repressors that are highly expressed in innervated muscle and suppressed following muscle denervation. Dach2 and Hdac9 control the expression of endplate-associated genes such as those encoding nicotinic acetylcholine receptors (nAChRs). Here we tested the idea that Dach2 and Hdac9 mediate the effects of muscle activity on muscle reinnervation. Dach2 and Hdac9 were found to act in a collaborative fashion to inhibit reinnervation of denervated mouse skeletal muscle and appear to act, at least in part, by inhibiting denervation-dependent induction of Myog and Gdf5 gene expression. Although Dach2 and Hdac9 inhibit Myog and Gdf5 mRNA expression, Myog does not regulate Gdf5 transcription. Thus, Myog and Gdf5 appear to stimulate muscle reinnervation through parallel pathways. These studies suggest that manipulating the Dach2-Hdac9 signaling system, and Gdf5 in particular, might be a good approach for enhancing motor function in instances where neuromuscular communication has been disrupted.

Impact of broad regulatory regions on Gdf5 expression and function in knee development and susceptibility to osteoarthritis.

OBJECTIVES: Given the role of growth and differentiation factor 5 (GDF5) in knee development and osteoarthritis risk, we sought to characterise knee defects resulting from Gdf5 loss of function and how its regulatory regions control knee formation and morphology. METHODS: The brachypodism (bp) mouse line, which harbours an inactivating mutation in Gdf5, was used to survey how Gdf5 loss of function impacts knee morphology, while two transgenic Gdf5 reporter bacterial artificial chromosome mouse lines were used to assess the spatiotemporal activity and function of Gdf5 regulatory sequences in the context of clinically relevant knee anatomical features. RESULTS: Knees from homozygous bp mice (bp/bp) exhibit underdeveloped femoral condyles and tibial plateaus, no cruciate ligaments, and poorly developed menisci. Secondary ossification is also delayed in the distal femur and proximal tibia. bp/bp mice have significantly narrower femoral condyles, femoral notches and tibial plateaus, and curvier medial femoral condyles, shallower trochlea, steeper lateral tibial slopes and smaller tibial spines. Regulatory sequences upstream from Gdf5 were weakly active in the prenatal knee, while downstream regulatory sequences were active throughout life. Importantly, downstream but not upstream Gdf5 regulatory sequences fully restored all the key morphological features disrupted in the bp/bp mice. CONCLUSIONS: Knee morphology is profoundly affected by Gdf5 absence, and downstream regulatory sequences mediate its effects by controlling Gdf5 expression in knee tissues. This downstream region contains numerous enhancers harbouring human variants that span the osteoarthritis association interval. We posit that subtle alterations to morphology driven by changes in downstream regulatory sequence underlie this locus' role in osteoarthritis risk.

Association between GDF5 rs143383 genetic polymorphism and musculoskeletal degenerative diseases susceptibility: a meta-analysis.

BACKGROUND: Several studies have assessed the association between GDF5 rs143383 polymorphism and the susceptibility of musculoskeletal degenerative diseases, such as intervertebral disc degeneration (IDD) and osteoarthritis (OA), but the results are inconsistent. The aim of our study was to evaluate the association between them comprehensively. METHODS: A systematical search was conducted on PubMed, Scopus, Web of Science (WOS), Embase, and the Cochrane Library databases updated to April 20, 2018. Eligible studies about polymorphisms in GDF5 gene and risk of IDD or OA were included. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were utilized. RESULTS: Fifteen studies with a total of 5915 cases and 12,252 controls were finally included in our study. Meta-analysis of GDF5 rs143383 polymorphism was statistically associated with increased risk of musculoskeletal degenerative diseases under each genetic model (allele model: OR = 1.32, 95% CI 1.19-1.48, P = 0.000; homozygote model: OR = 1.80, 95%CI 1.49-2.16, P = 0.000; heterozygote model: OR = 1.37, 95%CI 1.21-1.55, P = 0.000; dominant model: OR = 1.56, 95%CI 1.39-1.75, P = 0.000; recessive model: OR = 1.39, 95%CI 1.20-1.60, P = 0.000). Stratified analyses based on disease type showed a significant association between the GDF5 rs143383 polymorphism and increased risk of IDD and OA under all genetic models studied. When stratified with ethnicity, pooled outcomes revealed that this polymorphism was significantly related with increased risk of musculoskeletal degenerative diseases in both Asian and Caucasian populations under all genetic models studied. CONCLUSIONS: The present study suggested that GDF5 rs143383 polymorphism was significantly associated with susceptibility to musculoskeletal degenerative diseases.