The G protein-coupled receptor ADGRG6 maintains mouse growth plate homeostasis through IHH Signaling.

The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded (FFPE) tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of PTHrP but increased IHH signaling throughout the growth plate. Attenuation of Smoothened (SMO)-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates.

The cartilage growth plate is an essential structure for skeletal growth, however, the mechanisms that govern growth plate homeostasis are still poorly understood. In this study, we showed that an adhesion G protein-coupled receptor (GPCR) named ADGRG6 plays an essential role in maintaining the slow-cycling cells in the resting zone of the growth plate and directing appropriate proliferation and differentiation of the growth plate chondrocytes. Using a technique called spatial transcriptomics, we compared the gene expression profiles in control and Adgrg6 mutant growth plates and found that ADGRG6 prevents premature hypertrophic differentiation of the growth plate chondrocytes by negatively regulating Indian Hedgehog (IHH) signaling. In summary, our findings highlighted the essential role of a cartilage-enriched GPCR in maintaining growth plate homeostasis through IHH signaling.

Evolutionary insights into sequence modifications governing chitin recognition and chitinase inactivity in YKL-40 (HC-gp39, CHI3L1).

YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatDs) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity.

Revealing a Natural Model of Pre-Osteoarthritis of the Hip Through Study of Femoroacetabular Impingement.

Femoroacetabular impingement (FAI) is considered the mechanical cause of hip osteoarthritis (OA). Surgical intervention involves labrum repair and osteochondroplasty to remove the impingement, alleviating symptoms. Nevertheless, some patients progress to hip OA after surgery, indicating that factors other than mechanical abnormality are contributing to hip OA progression. This review article discusses our laboratory's studies on hip FAI and OA, undertaken to identify key molecular players in the progression of hip OA. Transcriptome analysis identified peroxisome proliferator activated receptor gamma (PPARγ) as a crucial molecule in early hip OA. PPARγ, widely expressed in chondrocytes, has a protective role in preventing OA, but its true mechanism remains unknown. We observed a dysregulation of DNA methyltransferase (DNMT) in the progression of hip OA, with high expression of DNMT1 and 3A and downregulation of DNMT3B. Moreover, we established that DNMT3A is the main molecule that is binding to PPARγ promoter CpG area, and hypermethylation of this area occurs during disease progression. This suggests that epigenetic changes are a main mechanism that regulates PPARγ expression. Finally, we developed a novel rabbit model of hip FAI and OA and are currently performing studies to validate our small-animal model to human FAI.

Biomechanical Impact of Phosphate Wasting on Articular Cartilage Using the Murine Hyp Model of X-linked hypophosphatemia.

Degenerative osteoarthritis (OA) is recognized as an early-onset comorbidity of X-linked hypophosphatemia (XLH), contributing to pain and stiffness and limiting range of motion and activities of daily living. Here, we extend prior findings describing biochemical and cellular changes of articular cartilage (AC) in the phosphate-wasting environment of XLH to determine the impact of these changes on the biomechanical properties of AC in compression and potential role in the etiology of OA. We hypothesize that despite increased proteoglycan biosynthesis, disruption of the mineralized zone of AC impacts the mechanical properties of cartilage that function to accommodate loads and that therapeutic restoration of this zone will improve the mechanical properties of AC. Data were compared between three groups: wild type (WT), Hyp, and Hyp mice treated with calcitriol and oral phosphate. EPIC microCT confirmed AC mineral deficits and responsiveness to therapy. MicroCT of the Hyp subchondral bone plate revealed that treatment improved trabecular bone volume (BV/TV) but remained significantly lower than WT mice in other trabecular microstructures (p < 0.05). Microindentation AC studies revealed that, compared with WT mice, the mean stiffness of tibial AC was significantly lower in untreated Hyp mice (2.65 ± 0.95 versus 0.87 ± 0.33 N/mm, p < 0.001) and improved with therapy (2.15 + 0.38 N/mm) to within WT values. Stress relaxation of AC under compressive loading displayed similar biphasic relaxation time constants (Taufast and Tauslow) between controls and Hyp mice, although Tauslow trended toward slowed relaxation times. In addition, Taufast and Tauslow times correlated with peak load in WT mice (r = 0.80; r = 0.78, respectively), whereas correlation coefficient values for Hyp mice (r = 0.46; r = 0.21) improved with treatment (r = 0.71; r = 0.56). These data provide rationale for therapies that both preserve AC stiffness and recovery from compression. The Hyp mouse also provides unique insight into determinants of structural stiffness and the viscoelastic properties of AC in the progression of OA. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Substantially Delayed Maturation of Growth Plate Chondrocytes in "Humanized" PTH1R Mice with the H223R Mutation of Jansen's Disease.

Activating parathyroid hormone (PTH)/PTH-related Peptide (PTHrP) receptor (PTH1R) mutations causes Jansen's metaphyseal chondrodysplasia (JMC), a rare disease characterized by growth plate abnormalities, short stature, and PTH-independent hypercalcemia. Previously generated transgenic JMC mouse models, in which the human PTH1R allele with the H223R mutation (H223R-PTH1R) is expressed in osteoblasts via type Ia1 collagen or DMP1 promoters cause excess bone mass, while expression of the mutant allele via the type IIa1 collagen promoter results in only minor growth plate changes. Thus, neither transgenic JMC model adequately recapitulates the human disease. We therefore generated "humanized" JMC mice in which the H223R-PTH1R allele was expressed via the endogenous mouse Pth1r promoter and, thus, in all relevant target tissues. Founders with the H223R allele typically died within 2 months without reproducing; several mosaic male founders, however, lived longer and produced F1 H223R-PTH1R offspring, which were small and exhibited marked growth plate abnormalities. Serum calcium and phosphate levels of the mutant mice were not different from wild-type littermates, but serum PTH and P1NP were reduced significantly, while CTX-1 and CTX-2 were slightly increased. Histological and RNAscope analyses of the mutant tibial growth plates revealed markedly expanded zones of type II collagen-positive, proliferating/prehypertrophic chondrocytes, abundant apoptotic cells in the growth plate center and a progressive reduction of type X collagen-positive hypertrophic chondrocytes and primary spongiosa. The "humanized" H223R-PTH1R mice are likely to provide a more suitable model for defining the JMC phenotype and for assessing potential treatment options for this debilitating disease of skeletal development and mineral ion homeostasis. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Antibiotic Treatment Prior to Injury Abrogates the Detrimental Effects of LPS in STR/ort Mice Susceptible to Osteoarthritis Development.

Post traumatic osteoarthritis (PTOA) is a form of secondary osteoarthritis (OA) that develops in ~50% of cases of severe articular joint injuries and leads to chronic and progressive degradation of articular cartilage and other joint tissues. PTOA progression can be exacerbated by repeated injury and systemic inflammation. Few studies have examined approaches for blunting or slowing down PTOA progression with emphasis on systemic inflammation; most arthritis studies focused on the immune system have been in the context of rheumatoid arthritis. To examine how the gut microbiome affects systemic inflammation during PTOA development, we used a chronic antibiotic treatment regimen starting at weaning for 6 weeks before anterior cruciate ligament (ACL) rupture in STR/ort mice combined with lipopolysaccharide (LPS)-induced systemic inflammation. STR/ort mice develop spontaneous OA as well as a more severe PTOA phenotype than C57Bl/6J mice. By 6 weeks post injury, histological examination showed a more robust cartilage staining in the antibiotic-treated (AB) STR/ort mice than in the untreated STR/ort controls. Furthermore, we also examined the effects of AB treatment on systemic inflammation and found that the effects of LPS administration before injury are also blunted by AB treatment in STR/ort mice. The AB- or AB+LPS-treated STR/ort injured joints more closely resembled the C57Bl/6J VEH OA phenotypes than the vehicle- or LPS-treated STR/ort, suggesting that antibiotic treatment has the potential to slow disease progression and should be further explored therapeutically as prophylactic post injury. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Collagen X Is Dispensable for Hypertrophic Differentiation and Endochondral Ossification of Human iPSC-Derived Chondrocytes.

Collagen X is a non-fibril collagen produced by hypertrophic chondrocytes and was believed to associate with the calcification process of growth plate cartilage. The homozygous loss of Col10a1 gene in mice, however, demonstrated no remarkable effects on growth plate formation or skeletal development. To investigate the role of collagen X in human chondrocytes, we established human induced pluripotent stem cells (hiPSCs) with heterozygous (COL10A1 +/-) or homozygous (COL10A1 -/-) deletions of COL10A1 gene using the dual sgRNA CRISPR/Cas9 system. Several mutant clones were established and differentiated into hypertrophic chondrocytes by a previously reported 3D induction method. No remarkable differences were observed during the differentiation process between parental and mutant cell lines, which differentiated into cells with features of hypertrophic chondrocytes, indicating that collagen X is dispensable for the hypertrophic differentiation of human chondrocytes in vitro. To investigate the effects of collagen X deficiency in vivo, chondrocyte pellets at the proliferating or prehypertrophic stage were transplanted into immunodeficient mice. Proliferating pellet-derived tissues demonstrated the zonal distribution of chondrocytes with the transition to bone tissues mimicking growth plates, and the proportion of bone tended to be larger in COL10A1 -/- tissues. Prehypertrophic pellet-derived tissues produced trabecular bone structures with features of endochondral ossification, and there was no clear difference between parental- and mutant-derived tissues. A transcriptome analysis of chondrocyte pellets at the hypertrophic phase showed a lower expression of proliferating-phase genes and a higher expression of calcification-phase genes in COL10A1 -/- pellets compared with parental cell pellets. These in vitro and in vivo data suggested that collagen X is dispensable for the hypertrophic differentiation and endochondral ossification of human iPSC-derived chondrocytes, though it may facilitate the differentiation process. Thus, COL10A1 -/- iPSC lines are useful for investigating the physiological role of collagen X in chondrocyte differentiation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Role of cartilage and bone matrix regulation in early equine osteochondrosis.

The objective of this study is to better understand the pathogenesis of early equine osteochondrosis (OC) by identifying differences in gene and protein expression of extracellular matrix components and regulators in normal and diseased cartilage and bone, focusing on the osteochondral junction and cells surrounding the cartilage canals. We expected to find an upregulation of matrix metalloproteinases and a decrease in extracellular matrix constituent expression along the osteochondral junction and cells surrounding the cartilage canals in OC samples. Paraffin-embedded osteochondral samples (6 OC-affected, 8 normal controls) and cDNA from chondrocytes captured with laser capture microdissection from frozen sections (4 OC-affected, 5 normal controls) were used in this study. Quantitative real-time polymerase chain reaction was performed on 16 target genes. Immunohistochemistry was performed on osteochondral samples for Sox-9, lubricin, osteocalcin, and collagen type IIB. In OC-affected samples, there was significantly (P ≤ 0.05) decreased gene expression of collagen type IIB, aggrecan, and SOX-9 in chondrocytes surrounding the cartilage canals and decreased gene expression of PRG4 (Lubricin) and collagen type IIB in chondrocytes along the osteochondral junction. We found significantly lower collagen type IIB total matrix percentages in the middle and deep cartilage layers, lower lubricin total cellular percentage in the superficial layer, and higher Sox-9 total cellular percentage in bone of OC samples. No significant differences were found in matrix degradation molecules or HSCORE protein expression at any locations between normal and OC-affected samples in our study.

Conditional Deletion of HIF-2α in Mouse Nucleus Pulposus Reduces Fibrosis and Provides Mild and Transient Protection From Age-Dependent Structural Changes in Intervertebral Disc.

Hypoxia-inducible factors (HIFs) are critical to the development and homeostasis of hypoxic tissues. Although HIF-2α, one of the main HIF-α isoforms, is expressed in nucleus pulposus (NP) cells, its functions remain unknown. We deleted HIF-2α in the NP tissue using a notochord-specific FoxA2Cre allele to study HIF-2α function in the adult intervertebral disc. Unlike observations in HIF-1αcKO mice, fate mapping studies using Rosa26-mTmG reporter showed that HIF-2α loss in NP did not negatively impact cell survival or affect compartment development. Rather, loss of HIF-2α resulted in slightly better attributes of NP morphology in 14-month-old HIF-2αcKO mice as evident from lower scores of degeneration. These 14-month-old HIF-2αcKO mice also exhibited significant reduction in NP tissue fibrosis and lower collagen turnover in the annulus fibrosis (AF) compartment. Imaging-Fourier transform-infrared (FTIR) analyses showed decreased collagen and protein content in the NP and maintained chondroitin sulfate levels in 14-month-old HIF-2αcKO . Mechanistically, global transcriptomic analysis showed enrichment of differentially expressed genes with Gene Ontology (GO) terms related to metabolic processes and cell development, molecular functions concerned with histone and protein binding, and associated pathways, including oxidative stress. Noteworthy, these morphological differences were not apparent in 24-month-old HIF-2αcKO , indicating that aging is the dominant factor in governing disc health. Together these data suggest that loss of HIF-2α in the NP compartment is not detrimental to the intervertebral disc development but rather mitigates NP tissue fibrosis and offers mild but transient protection from age-dependent early degenerative changes. © 2022 American Society for Bone and Mineral Research (ASBMR).

Type VI Collagen Regulates Endochondral Ossification in the Temporomandibular Joint.

For many years there has been a keen interest in developing regenerative treatment for temporomandibular joint-osteoarthritis (TMJ-OA). Currently, there is no consensus treatment due to the limited self-healing ability of articular cartilage and lack of understanding of the complex mechanisms regulating cartilage development in the TMJ. Endochondral ossification, the process of subchondral bone formation through chondrocyte differentiation, is critical for TMJ growth and development, and is tightly regulated by the composition of the extracellular matrix (ECM). Type VI collagen is a highly expressed ECM component in the TMJ cartilage, yet its specific functions are largely unknown. In this study, we investigated α2(VI)-deficient (Col6a2-knockout [KO]) mice, which are unable to secret or incorporate type VI collagen into their ECM. Compared with wild-type (WT) mice, the TMJ condyles of Col6a2-KO mice exhibit decreased bone volume/tissue volume (BV/TV) and a larger bone marrow space, suggesting the α2(VI)-deficient condyles have a failure in endochondral ossification. Differentiating chondrocytes are the main source of bone cells during endochondral ossification. Our study shows there is an increased number of chondrocytes in the proliferative zone and decreased Col10-expressing chondrocytes in Col6a2-KO cartilage, all pointing to abnormal chondrocyte differentiation and maturation. In addition, RNA sequencing (RNAseq) analysis identified distinct gene expression profiles related to cell cycle and ECM organization that were altered in the mutant condyles. These data also suggest that bone morphogenetic protein 2 (BMP2) activity was deregulated during chondrocyte differentiation. Immunohistochemical analysis indicated an upregulation of Col2 and Acan expression in Col6a2-KO cartilage. Moreover, the expression of pSmad1/5/8 and Runx2 was decreased in the Col6a2-KO cartilage compared with WT controls. Taken together, our data indicate that type VI collagen expressed in the TMJ cartilage is important for endochondral ossification, possibly by modulating the ECM and altering/disrupting signaling pathways important for TMJ chondrocyte differentiation. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Overexpression of transcription factor FoxA2 in the developing skeleton causes an enlargement of the cartilage hypertrophic zone, but it does not trigger ectopic differentiation in immature chondrocytes.

We previously found that FoxA factors are necessary for chondrocyte differentiation. To investigate whether FoxA factors alone are sufficient to drive chondrocyte hypertrophy, we build a FoxA2 transgenic mouse in which FoxA2 cDNA is driven by a reiterated Tetracycline Response Element (TRE) and a minimal CMV promoter. This transgenic line was crossed with a col2CRE;Rosa26rtTA/+ mouse line to generate col2CRE;Rosa26rtTA/+;TgFoxA2+/- mice for inducible expression of FoxA2 in cartilage using doxycycline treatment. Ectopic expression of FoxA2 in the developing skeleton reveals skeletal defects and shorter skeletal elements in E17.5 mice. The chondro-osseous border was frequently mis-shaped in mutant mice, with small islands of col.10+ hypertrophic cells extending in the metaphyseal bone. Even though overexpression of FoxA2 causes an accumulation of hypertrophic chondrocytes, it did not trigger ectopic hypertrophy in the immature chondrocytes. This suggests that FoxA2 may need transcriptional co-factors (such as Runx2), whose expression is restricted to the hypertrophic zone, and absent in the immature chondrocytes. To investigate a potential FoxA2/Runx2 interaction in immature chondrocytes versus hypertrophic cells, we separated these two subpopulations by FACS to obtain CD24+CD200+ hypertrophic chondrocytes and CD24+CD200- immature chondrocytes and we ectopically expressed FoxA2 alone or in combination with Runx2 via lentiviral gene delivery. In CD24+CD200+ hypertrophic chondrocytes, FoxA2 enhanced the expression of chondrocyte hypertrophic markers collagen 10, MMP13, and alkaline phosphatase. In contrast, in the CD24+CD200- immature chondrocytes, neither FoxA2 nor Runx2 overexpression could induce ectopic expression of hypertrophic markers MMP13, alkaline phosphatase, or PTH/PTHrP receptor. Overall these findings mirror our in vivo data, and suggest that induction of chondrocyte hypertrophy by FoxA2 may require other factors in addition to Runx2 (i.e., Hif2α, MEF2C, or perhaps unknown factors), whose expression/activity is rate-limiting in immature chondrocytes.

Suppression of Sost/Sclerostin and Dickkopf-1 Augment Intervertebral Disc Structure in Mice.

Intervertebral disc (IVD) degeneration is a leading cause of low back pain, characterized by accelerated extracellular matrix breakdown and IVD height loss, but there is no approved pharmacological therapeutic. Deletion of Wnt ligand competitor Lrp5 induces IVD degeneration, suggesting that Wnt signaling is essential for IVD homeostasis. Therefore, the IVD may respond to neutralization of Wnt ligand competitors sost(gene)/sclerostin(protein) and/or dickkopf-1 (dkk1). Anti-sclerostin antibody (scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We aimed to (i) determine if pharmacological neutralization of sclerostin, dkk1, or their combination would stimulate Wnt signaling and augment IVD structure and (ii) determine the prolonged adaptation of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n = 6-7/group) were subcutaneously injected 2×/week for 5.5 weeks with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg), or vehicle (veh). Separately, IVD of sost KO and wild-type (WT) mice (n = 8/group) were harvested at 16 weeks of age. First, compared with vehicle, injection of scl-Ab, dkk1-Ab, and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and ß-catenin gene expression. Despite these similarities, only injection of scl-Ab alone strengthened IVD mechanical properties and decreased heat shock protein gene expressions. Genetically and compared with WT, sost KO enlarged IVD height, increased proteoglycan staining, and imbibed IVD hydration. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling co-transcription factor ß-catenin in the IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and revealed a reduction of cellular stress-related pathways. Together, suppression of sost/sclerostin or dkk1 each augmented IVD structure by stimulating Wnt signaling, but scl-Ab outperformed dkk1-Ab in strengthening the IVD. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health. © 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).

The Posterior Part Influences the Anterior Part of the Mouse Cranial Base Development.

The cranial base is a critical structure in the head, which is composed of endoskeletal and dermal skeletal. The braincase floor, part of the cranial base, is a midline structure of the head. Because it is a midline structure connecting the posterior skull with the facial region, braincase floor is critical for the orientation of the facial structure. Shortened braincase floor leads to mid-facial hypoplasia and malocclusions. During embryonic development, elongation of the braincase floor occurs through endochondral ossification in the parachordal cartilage, hypophyseal cartilage, and trabecular cartilage, which leads to formation of basioccipital (BO), basisphenoid (BS), and presphenoid (PS) bones, respectively. Currently, little is known about whether maturation of parachordal cartilage, hypophyseal cartilage, and trabecular cartilage occurs in a simultaneous or sequential manner and if the formation of one impacts the others. Our previous studies demonstrated that loss of function of ciliary protein Evc2 leads to premature fusion in the intersphenoid synchondrosis (ISS). In this study, we take advantage of Evc2 mutant mice to delineate the mechanism governing synchondrosis formation. Our analysis supports a cascade mechanism on the spatiotemporal regulation of the braincase floor development that the hypertrophy of parachordal cartilage (posterior side) impacts the hypertrophy of hypophyseal cartilage (middle) and trabecular cartilage (anterior side) in a sequential manner. The cascade mechanism well explains the premature fusion of the ISS in Evc2 mutant mice and is instructive to understand the specifically shortened anterior end of the braincase floor in various types of genetic syndromes. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Differentiation of Induced Pluripotent Stem Cells Into Chondrocytes: Methods and Applications for Disease Modeling and Drug Discovery.

Induced pluripotent stem cell (iPSC) technology allows pathomechanistic and therapeutic investigation of human heritable disorders affecting tissue types whose collection from patients is difficult or even impossible. Among them are cartilage diseases. Over the past decade, iPSC-chondrocyte disease models have been shown to exhibit several key aspects of known disease mechanisms. Concurrently, an increasing number of protocols to differentiate iPSCs into chondrocytes have been published, each with its respective (dis)advantages. In this review we provide a comprehensive overview of the different differentiation approaches, the hitherto described iPSC-chondrocyte disease models and mechanistic and/or therapeutic insights that have been derived from their investigation, and the current model limitations. Key lessons are that the most appropriate differentiation approach is dependent upon the cartilage disease under investigation and that further optimization is still required to recapitulate the in vivo cartilage. © 2022 American Society for Bone and Mineral Research (ASBMR).

Integrated View on the Role of Vitamin D Actions on Bone and Growth Plate Homeostasis.

1,25(OH)2D3, the biologically active form of vitamin D3, is a major regulator of mineral and bone homeostasis and exerts its actions through binding to the vitamin D receptor (VDR), a ligand-activated transcription factor that can directly modulate gene expression in vitamin D-target tissues such as the intestine, kidney, and bone. Inactivating VDR mutations or vitamin D deficiency during development results in rickets, hypocalcemia, secondary hyperparathyroidism, and hypophosphatemia, pointing to the critical role of 1,25(OH)2D3-induced signaling in the maintenance of mineral homeostasis and skeletal health. 1,25(OH)2D3 is a potent stimulator of VDR-mediated intestinal calcium absorption, thus increasing the availability of calcium required for proper bone mineralization. However, when intestinal calcium absorption is impaired, renal calcium reabsorption is increased and calcium is mobilized from the bone to preserve normocalcemia. Multiple cell types within bone express the VDR, thereby allowing 1,25(OH)2D3 to directly affect bone homeostasis. In this review, we will discuss different transgenic mouse models with either Vdr deletion or overexpression in chondrocytes, osteoblasts, osteocytes, or osteoclasts to delineate the direct effects of 1,25(OH)2D3 on bone homeostasis. We will address the bone cell type-specific effects of 1,25(OH)2D3 in conditions of a positive calcium balance, where the amount of (re)absorbed calcium equals or exceeds fecal and renal calcium losses, as well as during a negative calcium balance, due to selective Vdr knockdown in the intestine or triggered by a low calcium diet. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Epigenetic Mechanisms Mediating Cell State Transitions in Chondrocytes.

Epigenetic modifications play critical roles in regulating cell lineage differentiation, but the epigenetic mechanisms guiding specific differentiation steps within a cell lineage have rarely been investigated. To decipher such mechanisms, we used the defined transition from proliferating (PC) into hypertrophic chondrocytes (HC) during endochondral ossification as a model. We established a map of activating and repressive histone modifications for each cell type. ChromHMM state transition analysis and Pareto-based integration of differential levels of mRNA and epigenetic marks revealed that differentiation-associated gene repression is initiated by the addition of H3K27me3 to promoters still carrying substantial levels of activating marks. Moreover, the integrative analysis identified genes specifically expressed in cells undergoing the transition into hypertrophy. Investigation of enhancer profiles detected surprising differences in enhancer number, location, and transcription factor binding sites between the two closely related cell types. Furthermore, cell type-specific upregulation of gene expression was associated with increased numbers of H3K27ac peaks. Pathway analysis identified PC-specific enhancers associated with chondrogenic genes, whereas HC-specific enhancers mainly control metabolic pathways linking epigenetic signature to biological functions. Since HC-specific enhancers show a higher conservation in postnatal tissues, the switch to metabolic pathways seems to be a hallmark of differentiated tissues. Surprisingly, the analysis of H3K27ac levels at super-enhancers revealed a rapid adaption of H3K27ac occupancy to changes in gene expression, supporting the importance of enhancer modulation for acute alterations in gene expression. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Pleckstrin homology (PH) domain and Leucine Rich Repeat Phosphatase 1 (Phlpp1) Suppresses Parathyroid Hormone Receptor 1 (Pth1r) Expression and Signaling During Bone Growth.

Endochondral ossification is tightly controlled by a coordinated network of signaling cascades including parathyroid hormone (PTH). Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 (Phlpp1) affects endochondral ossification by suppressing chondrocyte proliferation in the growth plate, longitudinal bone growth, and bone mineralization. As such, Phlpp1-/- mice have shorter long bones, thicker growth plates, and proportionally larger growth plate proliferative zones. The goal of this study was to determine how Phlpp1 deficiency affects PTH signaling during bone growth. Transcriptomic analysis revealed greater PTH receptor 1 (Pth1r) expression and enrichment of histone 3 lysine 27 acetylation (H3K27ac) at the Pth1r promoter in Phlpp1-deficient chondrocytes. PTH (1-34) enhanced and PTH (7-34) attenuated cell proliferation, cAMP signaling, cAMP response element-binding protein (CREB) phosphorylation, and cell metabolic activity in Phlpp1-inhibited chondrocytes. To understand the role of Pth1r action in the endochondral phenotypes of Phlpp1-deficient mice, Phlpp1-/- mice were injected with Pth1r ligand PTH (7-34) daily for the first 4 weeks of life. PTH (7-34) reversed the abnormal growth plate and long-bone growth phenotypes of Phlpp1-/- mice but did not rescue deficits in bone mineral density or trabecular number. These results show that elevated Pth1r expression and signaling contributes to increased proliferation in Phlpp1-/- chondrocytes and shorter bones in Phlpp1-deficient mice. Our data reveal a novel molecular relationship between Phlpp1 and Pth1r in chondrocytes during growth plate development and longitudinal bone growth. © 2021 American Society for Bone and Mineral Research (ASBMR).

IRX3 and IRX5 Inhibit Adipogenic Differentiation of Hypertrophic Chondrocytes and Promote Osteogenesis.

Maintaining the correct proportions of different cell types in the bone marrow is critical for bone function. Hypertrophic chondrocytes (HCs) and osteoblasts are a lineage continuum with a minor contribution to adipocytes, but the regulatory network is unclear. Mutations in transcription factors, IRX3 and IRX5, result in skeletal patterning defects in humans and mice. We found coexpression of Irx3 and Irx5 in late-stage HCs and osteoblasts in cortical and trabecular bone. Irx3 and Irx5 null mutants display severe bone deficiency in newborn and adult stages. Quantitative analyses of bone with different combinations of functional alleles of Irx3 and Irx5 suggest these two factors function in a dosage-dependent manner. In Irx3 and Irx5 nulls, the amount of bone marrow adipocytes was increased. In Irx5 nulls, lineage tracing revealed that removal of Irx3 specifically in HCs exacerbated reduction of HC-derived osteoblasts and increased the frequency of HC-derived marrow adipocytes. ß-catenin loss of function and gain of function specifically in HCs affects the expression of Irx3 and Irx5, suggesting IRX3 and IRX5 function downstream of WNT signaling. Our study shows that IRX3 and IRX5 regulate fate decisions in the transition of HCs to osteoblasts and to marrow adipocytes, implicating their potential roles in human skeletal homeostasis and disorders.

Visualization of Mineral-Targeted Alkaline Phosphatase Binding to Sites of Calcification In Vivo.

A mineral-targeted form of recombinant tissue-nonspecific alkaline phosphatase (TNAP), asfotase alfa, was approved multinationally as an enzyme replacement therapy for hypophosphatasia in 2015. Two reports to date have shown evidence of binding of this drug to mineralizing tissues using histochemistry and immunohistochemistry. Here, we sought to expand on those earlier studies by directly visualizing the in vivo binding of asfotase alfa conjugated with AnaTag HiLyte Fluor 750 or Alexa Fluor 647 fluorescent dye to sites of skeletal/dental mineralization and ectopic calcification. We utilized 40-day-old Tagln-Cre; HprtALPL/Y mice, a model of severe medial vascular calcification; Tie2-Cre; HprtALPL/Y mice, a model of severe intimal calcification; and sibling WT HprtALPL/Y mice, devoid of soft-tissue calcification. A single dose of 8 mg/kg labeled asfotase alfa was injected via the retro-orbital route. Skeletal tissues and soft organs were imaged ex vivo 2 days after the injection. Strong fluorescence signal was observed in all skeletal tissues (calvaria, vertebra, long bones, jaw, and mandibles) from mutant and WT mice. Fluorescence analysis of histological sections from bones revealed strong binding of asfotase alfa. Asfotase alfa binding to sites of ectopic calcification in the heart, aorta, and renal artery were found in both the Tagln-Cre; HprtALPL/Y and Tie2-Cre; HprtALPL/Y mice but not in WT mice. In addition, asfotase alfa binding was also found in the kidney stroma and brain of the Tie2-Cre; HprtALPL/Y mice. Our results show that fluorescence-labeled asfotase alfa administered in vivo binds not only to sites of skeletal and dental mineralization but also to sites of ectopic calcification in these animal models. © 2020 American Society for Bone and Mineral Research.

Chondrocyte-Specific RUNX2 Overexpression Accelerates Post-traumatic Osteoarthritis Progression in Adult Mice.

RUNX2 is a transcription factor critical for chondrocyte maturation and normal endochondral bone formation. It promotes the expression of factors catabolic to the cartilage extracellular matrix and is upregulated in human osteoarthritic cartilage and in murine articular cartilage following joint injury. To date, in vivo studies of RUNX2 overexpression in cartilage have been limited to forced expression in osteochondroprogenitor cells preventing investigation into the effects of chondrocyte-specific RUNX2 overexpression in postnatal articular cartilage. Here, we used the Rosa26Runx2 allele in combination with the inducible Col2a1CreERT2 transgene or the inducible AcanCreERT2 knock-in allele to achieve chondrocyte-specific RUNX2 overexpression (OE) during embryonic development or in the articular cartilage of adult mice, respectively. RUNX2 OE was induced at embryonic day 13.5 (E13.5) for all developmental studies. Histology and in situ hybridization analyses suggest an early onset of chondrocyte hypertrophy and accelerated terminal maturation in the limbs of the RUNX2 OE embryos compared to control embryos. For all postnatal studies, RUNX2 OE was induced at 2 months of age. Surprisingly, no histopathological signs of cartilage degeneration were observed even 6 months following induction of RUNX2 OE. Using the meniscal/ligamentous injury (MLI), a surgical model of knee joint destabilization and meniscal injury, however, we found that RUNX2 OE accelerates the progression of cartilage degeneration following joint trauma. One month following MLI, the numbers of MMP13-positive and TUNEL-positive chondrocytes were significantly greater in the articular cartilage of the RUNX2 OE joints compared to control joints and 2 months following MLI, histomorphometry and Osteoarthritis Research Society International (OARSI) scoring revealed decreased cartilage area in the RUNX2 OE joints. Collectively, these results suggest that although RUNX2 overexpression alone may not be sufficient to initiate the OA degenerative process, it may predetermine the rate of OA onset and/or progression following traumatic joint injury. © 2019 American Society for Bone and Mineral Research.