Regulation of ciliary function by fibroblast growth factor signaling identifies FGFR3-related disorders achondroplasia and thanatophoric dysplasia as ciliopathies.

Cilia project from almost every cell integrating extracellular cues with signaling pathways. Constitutive activation of FGFR3 signaling produces the skeletal disorders achondroplasia (ACH) and thanatophoric dysplasia (TD), but many of the molecular mechanisms underlying these phenotypes remain unresolved. Here, we report in vivo evidence for significantly shortened primary cilia in ACH and TD cartilage growth plates. Using in vivo and in vitro methodologies, our data demonstrate that transient versus sustained activation of FGF signaling correlated with different cilia consequences. Transient FGF pathway activation elongated cilia, while sustained activity shortened cilia. FGF signaling extended primary cilia via ERK MAP kinase and mTORC2 signaling, but not through mTORC1. Employing a GFP-tagged IFT20 construct to measure intraflagellar (IFT) speed in cilia, we showed that FGF signaling affected IFT velocities, as well as modulating cilia-based Hedgehog signaling. Our data integrate primary cilia into canonical FGF signal transduction and uncover a FGF-cilia pathway that needs consideration when elucidating the mechanisms of physiological and pathological FGFR function, or in the development of FGFR therapeutics.

Dominant-negative SOX9 mutations in campomelic dysplasia.

Campomelic dysplasia (CD) is an autosomal dominant, perinatal lethal skeletal dysplasia characterized by a small chest and short long bones with bowing of the lower extremities. CD is the result of heterozygosity for mutations in the gene encoding the chondrogenesis master regulator, SOX9. Loss-of-function mutations have been identified in most CD cases so it has been assumed that the disease results from haploinsufficiency for SOX9. Here, we identified distal truncating SOX9 mutations in four unrelated CD cases. The mutations all leave the dimerization and DNA-binding domains intact and cultured chondrocytes from three of the four cases synthesized truncated SOX9. Relative to CD resulting from haploinsufficiency, there was decreased transactivation activity toward a major transcriptional target, COL2A1, consistent with the mutations exerting a dominant-negative effect. For one of the cases, the phenotypic consequence was a very severe form of CD, with a pronounced effect on vertebral and limb development. The data identify a novel molecular mechanism of disease in CD in which the truncated protein leads to a distinct and more significant effect on SOX9 function.

An inactivating mutation in intestinal cell kinase, ICK, impairs hedgehog signalling and causes short rib-polydactyly syndrome.

The short rib polydactyly syndromes (SRPS) are a group of recessively inherited, perinatal-lethal skeletal disorders primarily characterized by short ribs, shortened long bones, varying types of polydactyly and concomitant visceral abnormalities. Mutations in several genes affecting cilia function cause SRPS, revealing a role for cilia function in skeletal development. To identify additional SRPS genes and discover novel ciliary molecules required for normal skeletogenesis, we performed exome sequencing in a cohort of patients and identified homozygosity for a missense mutation, p.E80K, in Intestinal Cell Kinase, ICK, in one SRPS family. The p.E80K mutation abolished serine/threonine kinase activity, resulting in altered ICK subcellular and ciliary localization, increased cilia length, aberrant cartilage growth plate structure, defective Hedgehog and altered ERK signalling. These data identify ICK as an SRPS-associated gene and reveal that abnormalities in signalling pathways contribute to defective skeletogenesis.

Altered Sox9 and FGF signaling gene expression in Aga2 OI mice negatively affects linear growth.

Osteogenesis imperfecta (OI), or brittle bone disease, is a disorder characterized by bone fragility and increased fracture incidence. All forms of OI also feature short stature, implying an effect on endochondral ossification. Using the Aga2+/- mouse, which has a mutation in type I collagen, we show an affected growth plate primarily due to a shortened proliferative zone. We used single-cell RNA-Seq analysis of tibial and femoral growth plate tissues to understand transcriptional consequences on growth plate cell types. We show that perichondrial cells, which express abundant type I procollagen, and growth plate chondrocytes, which were found to express low amounts of type I procollagen, had ER stress and dysregulation of the same unfolded protein response pathway as previously demonstrated in osteoblasts. Aga2+/- proliferating chondrocytes showed increased FGF and MAPK signaling, findings consistent with accelerated differentiation. There was also increased Sox9 expression throughout the growth plate, which is expected to accelerate early chondrocyte differentiation but reduce late hypertrophic differentiation. These data reveal that mutant type I collagen expression in OI has an impact on the cartilage growth plate. These effects on endochondral ossification indicate that OI is a biologically complex phenotype going beyond its known impacts on bone to negatively affect linear growth.

Intervertebral disc degeneration is rescued by TGFß/BMP signaling modulation in an ex vivo filamin B mouse model.

Spondylocarpotarsal syndrome (SCT) is a rare musculoskeletal disorder characterized by short stature and vertebral, carpal, and tarsal fusions resulting from biallelic nonsense mutations in the gene encoding filamin B (FLNB). Utilizing a FLNB knockout mouse, we showed that the vertebral fusions in SCT evolved from intervertebral disc (IVD) degeneration and ossification of the annulus fibrosus (AF), eventually leading to full trabecular bone formation. This resulted from alterations in the TGFß/BMP signaling pathway that included increased canonical TGFß and noncanonical BMP signaling. In this study, the role of FLNB in the TGFß/BMP pathway was elucidated using in vitro, in vivo, and ex vivo treatment methodologies. The data demonstrated that FLNB interacts with inhibitory Smads 6 and 7 (i-Smads) to regulate TGFß/BMP signaling and that loss of FLNB produces increased TGFß receptor activity and decreased Smad 1 ubiquitination. Through the use of small molecule inhibitors in an ex vivo spine model, TGFß/BMP signaling was modulated to design a targeted treatment for SCT and disc degeneration. Inhibition of canonical and noncanonical TGFß/BMP pathway activity restored Flnb-/- IVD morphology. These most effective improvements resulted from specific inhibition of TGFß and p38 signaling activation. FLNB acts as a bridge for TGFß/BMP signaling crosstalk through i-Smads and is key for the critical balance in TGFß/BMP signaling that maintains the IVD. These findings further our understanding of IVD biology and reveal new molecular targets for disc degeneration as well as congenital vertebral fusion disorders.

4-PBA Treatment Improves Bone Phenotypes in the Aga2 Mouse Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetically heterogenous disorder most often due to heterozygosity for mutations in the type I procollagen genes, COL1A1 or COL1A2. The disorder is characterized by bone fragility leading to increased fracture incidence and long-bone deformities. Although multiple mechanisms underlie OI, endoplasmic reticulum (ER) stress as a cellular response to defective collagen trafficking is emerging as a contributor to OI pathogenesis. Herein, we used 4-phenylbutiric acid (4-PBA), an established chemical chaperone, to determine if treatment of Aga2+/- mice, a model for moderately severe OI due to a Col1a1 structural mutation, could attenuate the phenotype. In vitro, Aga2+/- osteoblasts show increased protein kinase RNA-like endoplasmic reticulum kinase (PERK) activation protein levels, which improved upon treatment with 4-PBA. The in vivo data demonstrate that a postweaning 5-week 4-PBA treatment increased total body length and weight, decreased fracture incidence, increased femoral bone volume fraction (BV/TV), and increased cortical thickness. These findings were associated with in vivo evidence of decreased bone-derived protein levels of the ER stress markers binding immunoglobulin protein (BiP), CCAAT/-enhancer-binding protein homologous protein (CHOP), and activating transcription factor 4 (ATF4) as well as increased levels of the autophagosome marker light chain 3A/B (LC3A/B). Genetic ablation of CHOP in Aga2+/- mice resulted in increased severity of the Aga2+/- phenotype, suggesting that the reduction in CHOP observed in vitro after treatment is a consequence rather than a cause of reduced ER stress. These findings suggest the potential use of chemical chaperones as an adjunct treatment for forms of OI associated with ER stress. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Skeletal diseases caused by mutations in PTH1R show aberrant differentiation of skeletal progenitors due to dysregulation of DEPTOR.

Alterations in the balance between skeletogenesis and adipogenesis is a pathogenic feature in multiple skeletal disorders. Clinically, enhanced bone marrow adiposity in bones impairs mobility and increases fracture risk, reducing the quality of life of patients. The molecular mechanism that underlies the balance between skeletogenesis and adipogenesis is not completely understood but alterations in skeletal progenitor cells' differentiation pathway plays a key role. We recently demonstrated that parathyroid hormone (PTH)/PTH-related peptide (PTHrP) control the levels of DEPTOR, an inhibitor of the mechanistic target of rapamycin (mTOR), and that DEPTOR levels are altered in different skeletal diseases. Here, we show that mutations in the PTH receptor-1 (PTH1R) alter the differentiation of skeletal progenitors in two different skeletal genetic disorders and lead to accumulation of fat or cartilage in bones. Mechanistically, DEPTOR controls the subcellular localization of TAZ (transcriptional co-activator with a PDZ-binding domain), a transcriptional regulator that governs skeletal stem cells differentiation into either bone and fat. We show that DEPTOR regulation of TAZ localization is achieved through the control of Dishevelled2 (DVL2) phosphorylation. Depending on nutrient availability, DEPTOR directly interacts with PTH1R to regulate PTH/PTHrP signaling or it forms a complex with TAZ, to prevent its translocation to the nucleus and therefore inhibit its transcriptional activity. Our data point DEPTOR as a key molecule in skeletal progenitor differentiation; its dysregulation under pathologic conditions results in aberrant bone/fat balance.

Biallelic mutations in LAMA5 disrupts a skeletal noncanonical focal adhesion pathway and produces a distinct bent bone dysplasia.

BACKGROUND: Beyond its structural role in the skeleton, the extracellular matrix (ECM), particularly basement membrane proteins, facilitates communication with intracellular signaling pathways and cell to cell interactions to control differentiation, proliferation, migration and survival. Alterations in extracellular proteins cause a number of skeletal disorders, yet the consequences of an abnormal ECM on cellular communication remains less well understood METHODS: Clinical and radiographic examinations defined the phenotype in this unappreciated bent bone skeletal disorder. Exome analysis identified the genetic alteration, confirmed by Sanger sequencing. Quantitative PCR, western blot analyses, immunohistochemistry, luciferase assay for WNT signaling were employed to determine RNA, proteins levels and localization, and dissect out the underlying cell signaling abnormalities.  Migration and wound healing assays examined cell migration properties. FINDINGS: This bent bone dysplasia resulted from biallelic mutations in LAMA5, the gene encoding the alpha-5 laminin basement membrane protein. This finding uncovered a mechanism of disease driven by ECM-cell interactions between alpha-5-containing laminins, and integrin-mediated focal adhesion signaling, particularly in cartilage. Loss of LAMA5 altered ß1 integrin signaling through the non-canonical kinase PYK2 and the skeletal enriched SRC kinase, FYN. Loss of LAMA5 negatively impacted the actin cytoskeleton, vinculin localization, and WNT signaling. INTERPRETATION: This newly described mechanism revealed a LAMA5-ß1 Integrin-PYK2-FYN focal adhesion complex that regulates skeletogenesis, impacted WNT signaling and, when dysregulated, produced a distinct skeletal disorder. FUNDING: Supported by NIH awards R01 AR066124, R01 DE019567, R01 HD070394, and U54HG006493, and Czech Republic grants INTER-ACTION LTAUSA19030, V18-08-00567 and GA19-20123S.

The PTH/PTHrP-SIK3 pathway affects skeletogenesis through altered mTOR signaling.

Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)-salt-inducible kinase 3 (SIK3)-mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.

A Chaperone Complex Formed by HSP47, FKBP65, and BiP Modulates Telopeptide Lysyl Hydroxylation of Type I Procollagen.

Lysine hydroxylation of type I collagen telopeptides varies from tissue to tissue, and these distinct hydroxylation patterns modulate collagen cross-linking to generate a unique extracellular matrix. Abnormalities in these patterns contribute to pathologies that include osteogenesis imperfecta (OI), fibrosis, and cancer. Telopeptide procollagen modifications are carried out by lysyl hydroxylase 2 (LH2); however, little is known regarding how this enzyme regulates hydroxylation patterns. We identified an ER complex of resident chaperones that includes HSP47, FKBP65, and BiP regulating the activity of LH2. Our findings show that FKBP65 and HSP47 modulate the activity of LH2 to either favor or repress its activity. BiP was also identified as a member of the complex, playing a role in enhancing the formation of the complex. This newly identified ER chaperone complex contributes to our understanding of how LH2 regulates lysyl hydroxylation of type I collagen C-telopeptides to affect the quality of connective tissues. © 2017 American Society for Bone and Mineral Research.

A postnatal role for embryonic myosin revealed by MYH3 mutations that alter TGFß signaling and cause autosomal dominant spondylocarpotarsal synostosis.

Spondylocarpotarsal synostosis (SCT) is a skeletal disorder characterized by progressive vertebral, carpal and tarsal fusions, and mild short stature. The majority of affected individuals have an autosomal recessive form of SCT and are homozygous or compound heterozygous for nonsense mutations in the gene that encodes the cytoskeletal protein filamin B (FLNB), but a subset do not have FLNB mutations. Exome sequence analysis of three SCT patients negative for FLNB mutations identified an autosomal dominant form of the disease due to heterozygosity for missense or nonsense mutations in MYH3, which encodes embryonic myosin. Cells transfected with the MYH3 missense mutations had reduced TGFß signaling, revealing a regulatory role for embryonic myosin in the TGFß signaling pathway. In wild-type mice, there was persistent postnatal expression of embryonic myosin in the small muscles joining the neural arches of the spine suggesting that loss of myosin function in these muscles contribute to the disease. Our findings demonstrate that dominant mutations in MYH3 underlie autosomal dominant SCT, identify a postnatal role for embryonic myosin and suggest that altered regulation of signal transduction in the muscles within the spine may lead to the development of vertebral fusions.

Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress.

Facioscapulohumeral muscular dystrophy is an autosomal dominant disorder resulting from an unusual genetic mechanism. The mutation, a deletion of 3.3 kb subtelomeric repeats, appears to disrupt the regional regulation of 4q35 g ene expression. The specific gene(s)responsible for facioscapulohumeral muscular dystrophy have not been identified. However, the 'vacuolar/necrotic' phenotype exhibited by facioscapulohumeral muscular dystrophy myoblasts suggests that aberrant gene expression occurs early in facioscapulohumeral muscular dystrophy muscle development. In order to test this hypothesis, global gene expression profiling and in vitro characterization of facioscapulohumeral muscular dystrophy and control myoblasts were carried out. Genes involved in several cellular processes such as oxidative stress were found to be dysregulated. In vitro studies confirmed this susceptibility to oxidative stress, as proliferative stage facioscapulohumeral muscular dystrophy myoblasts exhibit greatly reduced viability when exposed to the oxidative stressor paraquat. This effect was not seen in either normal or disease control myoblasts, or in any of the cell lines upon differentiation to multinucleated myotubes. Immunocytochemical studies of the cyclin dependent kinase inhibitor p21 demonstrated increased expression in facioscapulohumeral muscular dystrophy myoblasts, suggesting an early cell cycle arrest. Another process distinguishing facioscapulohumeral muscular dystrophy from controls involves the transcription of extracellular matrix components. Expression of elastin, decorin, lumican and the extracellular matrix remodeling factor TIMP3 were reduced in facioscapulohumeral muscular dystrophy myoblasts. These studies suggest that facioscapulohumeral muscular dystrophy muscular dystrophy results from a defect in early myogenesis, manifested as increased susceptibility to oxidative stress, morphological aberrations and early cell cycle arrest.

Analysis of allele-specific RNA transcription in FSHD by RNA-DNA FISH in single myonuclei.

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is likely caused by epigenetic alterations in chromatin involving contraction of the D4Z4 repeat array near the telomere of chromosome 4q. The precise mechanism by which deletions of D4Z4 influence gene expression in FSHD is not yet resolved. Regulatory models include a cis effect on proximal gene transcription (position effect), DNA looping, non-coding RNA, nuclear localization and trans-effects. To directly test whether deletions of D4Z4 affect gene expression in cis, nascent RNA was examined in single myonuclei so that transcription from each allele could be measured independently. FSHD and control myotubes (differentiated myoblasts) were subjected to sequential RNA-DNA FISH. A total of 16 genes in the FSHD region (FRG2, TUBB4Q, FRG1, FAT1, F11, KLKB1, CYP4V2, TLR3, SORBS2, PDLIM3 (ALP), LRP2BP, ING2, SNX25, SLC25A4 (ANT1), HELT and IRF2) were examined for interallelic variation in RNA expression within individual myonuclei. Sequential DNA hybridization with a unique 4q35 chromosome probe was then applied to confirm the localization of nascent RNA to 4q. A D4Z4 probe, labeled with a third fluorochrome, distinguished between the deleted and normal allele in FSHD nuclei. Our data do not support an FSHD model in which contracted D4Z4 arrays induce altered transcription in cis from 4q35 genes, even for those genes (FRG1, FRG2 and SLC25A4 (ANT1)) for which such an effect has been proposed.

Localization of 4q35.2 to the nuclear periphery: is FSHD a nuclear envelope disease?

Facioscapulohumeral muscular dystrophy (FSHD) may be a new member of the class of neuromuscular diseases (NMD) due to defects in the nuclear envelope. Unlike other NMDs with primary defects in nuclear envelope proteins, however, FSHD may result from inappropriate chromatin interactions at the envelope. 3D Immuno-FISH and a novel method of 3D by 2D analysis using NucProfile were developed to examine nuclear organization of the FSHD genomic region. In contrast to most other telomeres, the FSHD region at 4q35.2 localizes to the nuclear periphery. This localization is consistent in normal myoblasts, myotubes, fibroblasts and lymphoblasts, does not vary significantly throughout the cell cycle, and is independent of chromosome territory effects. The nuclear lamina protein lamin A/C is required for FSHD region chromatin localization to the nuclear envelope, as the association is lost in lamin A/C null fibroblasts. As both normal and affected alleles (deleted for the subtelomeric repeat D4Z4) localize to the nuclear periphery, FSHD likely arises instead from improper interactions with transcription factors or chromatin modifiers at the nuclear envelope. Interestingly, it is not D4Z4 itself that mediates interaction with the envelope, as sequences proximal to D4Z4 (i.e. D4S139) localize closer to the nuclear periphery, perhaps accounting for the chromosome 4 specificity of the disease.

Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation.

The neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) results from integral deletions of the subtelomeric repeat D4Z4 on chromosome 4q. A disruption of chromatin structure affecting gene expression is thought to underlie the pathophysiology. The global gene expression profiling of mature muscle tissue presented here provides the first insight into an FSHD-specific defect in myogenic differentiation. FSHD expression profiles generated by oligonucleotide microarrays were compared with those from normal muscle as well as other types of muscular dystrophies (DMD, aSGD) in order to determine FSHD-specific changes. In addition, matched biopsies (affected and unaffected muscle) from individuals with FSHD served to monitor expression changes during the progression of the disease as well as to diminish non-specific changes resulting from individual variability. Among genes altered in an FSHD-specific and highly significant manner, many are involved in myogenic differentiation and suggest a partial block in the normal differentiation program. Indeed, many of the transcripts affected in FSHD represent direct targets of the transcription factor MyoD. Additional mis-expressed genes confirm a diminished capacity to buffer oxidative stress, as demonstrated in FSHD myoblasts. This enhanced vulnerability of proliferative stage myoblasts to reactive oxygen species is also disease-specific, further implicating a defect in FSHD muscle satellite cells. Importantly, none of the genes localizing to the FSHD region at 4q35 were found to exhibit a significantly altered pattern of expression in FSHD muscle. This finding was corroborated by expression analysis of FSHD muscle using a custom cDNA microarray containing 51 genes and ESTs from the 4q35 region. Disruptions in FSHD myogenesis and oxidative capacity may therefore not arise from a position effect mechanism as has been previously suggested, but rather from a global effect on gene regulation. Improper nuclear localization of 4qter is discussed as an alternative model for FSHD gene regulation and pathogenesis.