The terminal differentiation of osteoblasts into osteocytes, the most abundant cell type in cortical bone, is critical for skeletal homeostasis. Osteocyte loss is a hallmark of bone aging and fragility, yet the mechanisms regulating osteocyte formation and survival are poorly understood. We show that inactivation of fibroblast growth factor receptor 1 (Fgfr1) in the mature osteoblast lineage results in extensive osteocyte death, identifying FGFR1 signaling as essential for osteocyte viability and bone integrity. Lineage tracing and analysis of endogenous and induced appositional bone formation revealed that newly embedded osteocytes fail to survive without FGFR1. These osteocytes exhibited ectopic expression of osteocalcin and podoplanin within sclerostin-positive, TUNEL-reactive lacunae, along with defective dendrite formation and disruption of the local lacunocanalicular network. RNA sequencing of cortical bone demonstrated reduced expression of extracellular matrix (ECM) genes and neuronal regulatory genes, while histological and ultrastructural analyses showed disorganized collagen fibrils, diminished osteoid, and abnormal mineralization. In vitro, FGF signaling in Ocy454 cells regulated gene programs involved in development, axon guidance, and bone ECM organization, highlighting a dual function for FGF signaling in which it controls both matrix-dependent and intrinsic cell differentiation mechanisms during the osteoblast-to-osteocyte transition. We propose that FGFR1 deficiency causes ECM disorganization and impaired dendrite formation, disrupting osteocyte communication with neighboring bone and vascular cells, ultimately leading to cell death. These findings establish FGFR signaling as a critical regulator of osteocyte differentiation, viability of bone-embedded osteocytes, and bone homeostasis.

After birth, tissues grow until they reach adult size, with each organ exhibiting unique cellular dynamics, growth patterns, and stem or non-stem cell sources. Using multiscale experimental and computational approaches, we found that aortic enlargement follows distinct growth principles, scaling with the vertebral column. Expansion proceeds via two temporally coordinated, spatially stochastic waves of proliferation aligned with blood flow, each with unique cell-cycle kinetics, with the first wave featuring cycles as short as 6 h. Single-cell RNA sequencing revealed increased fatty acid metabolism accompanying cell enlargement. Mathematical modeling and experiments showed that endothelial cell extrusion is essential for maintaining homeostatic aortic size as it adjusts for proliferation excess. Using a genetic model of achondroplasia, we mechanistically demonstrated that the aorta preserves proper scaling by increasing cell extrusion while keeping proliferation rates intact. These findings provide a blueprint of the principles orchestrating aortic growth, which relies entirely on the proliferation of resident differentiated cells. A record of this paper's transparent peer review process is included in the supplemental information.

Pseudoachondroplasia (PSACH), a severe dwarfing condition characterized by impaired skeletal growth and early joint degeneration, results from mutations in cartilage oligomeric matrix protein (COMP). These mutations disrupt normal protein folding, leading to the accumulation of misfolded COMP in chondrocytes. The MT-COMP mouse is a murine model of PSACH that expresses D469del human COMP in response to doxycycline and replicates the PSACH chondrocyte and clinical pathology. The basis for the mutant-COMP pathology involves endoplasmic reticulum (ER) stress signaling through the PERK/eIF2α/CHOP pathway. C/EBP homologous protein (CHOP), in conjunction with a TNFα inflammatory process, upregulates mTORC1, hindering autophagy clearance of mutant COMP protein. Life-long joint pain/degeneration diminishes quality of life, and treatments other than joint replacements are urgently needed. To assess whether molecules that reduce CHOP activity should be considered as a potential treatment for PSACH, we evaluated MT-COMP mice with 50% CHOP (MT-COMP/CHOP+/−), antisense oligonucleotide (ASO)-mediated CHOP knockdown, and complete CHOP ablation (MT-COMP/CHOP−/−). While earlier studies demonstrated that loss of CHOP in MT-COMP mice reduced intracellular retention, inflammation, and growth plate chondrocyte death, we now show that it did not normalize limb growth. ASO treatment reduced CHOP mRNA by approximately 60%, as measured by RT-qPCR, but did not improve limb length similar to MT-COMP/CHOP+/−. Interestingly, both 50% genetic reduction and complete loss of CHOP alleviated pain, while total ablation of CHOP in MT-COMP mice was necessary to preserve joint health. These results indicate that (1) CHOP reduction therapy is not an effective strategy for improving limb length and (2) pain and chondrocyte pathology are more responsive to intervention than the prevention of joint damage.

After birth, tissues grow continuously until reaching adult size, with each organ exhibiting unique cellular dynamics, growth patterns, and (stem or non-stem) cell sources. Using a suite of experimental and computational multiscale approaches, we found that aortic expansion is guided by specific biological principles and scales with the vertebral column rather than animal body weight. Expansion proceeds via two distinct waves of arterial cell proliferation along blood flow that are spatially stochastic, yet temporally coordinated. Each wave exhibits unique cell cycle kinetics and properties, with the first wave exhibiting cell cycle durations as fast as 6 hours. Single-cell RNA sequencing showed changes in fatty acid metabolism concomitant with an increase in cell size. Mathematical modeling and experiments indicated endothelial cell extrusion is essential for homeostatic aortic growth and balancing excess proliferation. In a genetic model of achondroplasia, the aorta achieves proper scaling through enhanced cell extrusion while maintaining normal proliferation dynamics. Collectively, these results provide a blueprint of the principles that orchestrate aortic growth which depends entirely on differentiated cell proliferation rather than resident stem cells.

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFBs) and a stable but poorly described population of lipid-rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFBs). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single-cell RNA sequencing and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a MyoFB differentiation program that is distinct from other mesenchymal cell types and increases the known repertoire of mesenchymal cell types in the neonatal lung.

The enthesis is a transitional tissue between tendon and bone that matures postnatally. The development and maturation of the enthesis involve cellular processes likened to an arrested growth plate. In this study, we explored the role of fibroblast growth factor 9 ( Fgf9 ), a known regulator of chondrogenesis and vascularization during bone development, on the structure and function of the postnatal enthesis. First, we confirmed spatial expression of Fgf9 in wildtype tendon and enthesis using in situ hybridization. We then used Cre recombinase driven by the scleraxis promoter (ScxCre) to conditionally inactivate Fgf9 in mouse tendon and enthesis. Characterization of enthesis morphology and mechanical properties in Fgf9 ScxCre and wildtype (WT) entheses showed a smaller calcaneal and humeral apophyses, thinner cortical bone at the attachment, increased cellularity, and reduced failure load in mature entheses in Fgf9 ScxCre compared to WT littermates. During postnatal development, we found reduced chondrocyte hypertrophy and disrupted type X collagen (Col X) in Fgf9 ScxCre entheses. These findings support a model in which tendon-derived Fgf9 regulates the functional development of the enthesis, including its postnatal mineralization.

Pseudoachondroplasia (PSACH), a severe dwarfing condition associated with early-onset joint degeneration and lifelong joint pain, is caused by mutations in cartilage oligomeric matrix protein (COMP). The mechanisms underlying the mutant-COMP pathology have been defined using the MT-COMP mouse model of PSACH that has the common D469del mutation. Mutant-COMP protein does not fold properly, and it is retained in the rough endoplasmic reticulum (rER) of chondrocytes rather than being exported to the extracellular matrix (ECM), driving ER stress that stimulates oxidative stress and inflammation, driving a self-perpetuating cycle. CHOP (ER stress signaling protein) and TNFα inflammation drive high levels of mTORC1 signaling, shutting down autophagy and blocking ER clearance, resulting in premature loss of chondrocytes that negatively impacts linear growth and causes early joint degeneration in MT-COMP mice and PSACH. Previously, we have shown that resveratrol treatment from birth to 20 weeks prevents joint degeneration and decreases the pathological processes in articular chondrocytes. Resveratrol's therapeutic mechanism of action in the mutant-COMP pathology was shown to act by primarily stimulating autophagy and reducing inflammation. Importantly, we demonstrated that MT-COMP mice experience pain consistent with PSACH joint pain. Here, we show, in the MT-COMP mouse, that resveratrol treatment must begin within 4 weeks to preserve joint health and reduce pain. Resveratrol treatment started at 6 or 8 weeks (to 20 weeks) was not effective in preventing joint degeneration. Collectively, our findings in MT-COMP mice show that there is a postnatal resveratrol treatment window wherein the inevitable mutant-COMP joint degeneration and pain can be prevented.

Pseudoachondroplasia (PSACH), a short limb skeletal dysplasia associated with premature joint degeneration, is caused by misfolding mutations in cartilage oligomeric matrix protein (COMP). Here, we define mutant-COMP-induced stress mechanisms that occur in articular chondrocytes of MT-COMP mice, a murine model of PSACH. The accumulation of mutant-COMP in the ER occurred early in MT-COMP articular chondrocytes and stimulated inflammation (TNFα) at 4 weeks, and articular chondrocyte death increased at 8 weeks while ER stress through CHOP was elevated by 12 weeks. Importantly, blockage of autophagy (pS6), the major mechanism that clears the ER, sustained cellular stress in MT-COMP articular chondrocytes. Degeneration of MT-COMP articular cartilage was similar to that observed in PSACH and was associated with increased MMPs, a family of degradative enzymes. Moreover, chronic cellular stresses stimulated senescence. Senescence-associated secretory phenotype (SASP) may play a role in generating and propagating a pro-degradative environment in the MT-COMP murine joint. The loss of CHOP or resveratrol treatment from birth preserved joint health in MT-COMP mice. Taken together, these results indicate that ER stress/CHOP signaling and autophagy blockage are central to mutant-COMP joint degeneration, and MT-COMP mice joint health can be preserved by decreasing articular chondrocyte stress. Future joint sparing therapeutics for PSACH may include resveratrol.

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells, via single-cell RNA-Seq and protein mass spectrometry, demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro, recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells, with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells, S1P ablation results in an increased production of granulocyte-macrophage progenitors, the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche.

Purpose: Site-1 protease (S1P) is a proprotein convertase known primarily for its roles in lipid homeostasis and the unfolded protein response. In previous studies, we demonstrated in mice the importance of S1P to overall skeletal development. A recent study in a human with defective S1P functions coupled to skeletal abnormalities validated the importance of S1P to skeletal development. S1P ablation in chondroprogenitors results in mice with no endochondral bone. S1P ablation in postnatal chondrocytes completely eliminates the primary postnatal growth plate by preventing chondrocyte maturation with a profound effect on trabecular bone growth. Our studies to analyze a direct role for S1P in bone development by ablating S1P in the Osterix (Osx)-lineage demonstrated a vital role in bone development. These mice have osteopenia with a significant reduction in bone formation and mineral apposition rates. Besides having delicate bones, these mice also develop scoliosis very early postnatally. These mice also demonstrated drastically reduced skeletal stem cells in the bone marrow. Furthermore, bone marrow stromal cells were unable to differentiate into osteoblasts in vitro. To investigate the mechanistic link between S1P-ablation, osteopenia and bone marrow anomalies in these mice, we investigated bone marrow-derived cells by single cell sequencing and proteomic analysis. Methods: To generate mice with S1P ablation in the Osx-lineage (Cko), S1Pf/f mice (mice homozygous for the floxed exon 2 of S1P) were bred with S1P+/f;Osx-Cre (Osx1-GFP::Cre) mice. Male and female mice showed identical phenotypes. For proteomic analysis of the bone marrow, non-RBC bone marrow cells harvested from postnatal 21-day old (P21) WT and Cko mice were lysed and analyzed by tryptic digestion and nano-LC/MS/MS technology after tandem mass tag labeling. For single cell RNA sequencing of GFP+ cells from the bone marrow of P21 Cko mice, single GFP+ cells were FACS-sorted and sequenced by RNA-Seq. For a global understanding of the bone marrow, non-RBC, single bone marrow cells were also analyzed by the 10X Genomics platform. Bone marrow cells were also analyzed by FACS using fluorescent-conjugated antibodies to neutrophil and hematopoietic stem cell (HSC) markers. Immunofluorescence for stefin proteins were performed on formalin-fixed, paraffin-embedded mouse hind limbs using a rabbit anti-human Stefin A antibody. Results: LC/MS/MS proteomic analysis of lysates from bone marrow cells harvested from WT and Cko mice showed that the stefin A (Stfa) family of proteins (Stfa1/Stfa2/Stfa3/Stfa2l1) was significantly upregulated in the Cko, when compared to WT. This data suggested a disruption of the bone marrow niche in the Cko. The bone marrow in the Cko is also marked by the presence of GFP+ cells, not seen in WT, heterozygote or control Osx-Cre mice. FACS-sorting of these GFP+ cells from the Cko followed by sequencing revealed an expression profile similar to neutrophils, a hematopoietic cell; furthermore, these GFP+ cells also showed expression of the stefin protein family. As the GFP is expressed as a Cre-GFP fusion protein from the Osx1 promoter, this data indicated that Osx-Cre is also active in hematopoietic cells, rather than only in osteoblast progenitors in the bone marrow. Sequencing of several thousands of WT and Cko bone marrow cells by the 10X Genomics platform revealed that the Cko bone marrow, while similar to WT, was also much altered. tSNE plots identified ten clusters for both WT and Cko with seven clusters exhibiting almost identical expression profiles, and three clusters deviating significantly from each other. One of these three clusters in the Cko showed an expression profile that suggested duplication of the neutrophil population that also expressed additional members of the Stfa gene family, not seen in WT neutrophils. The Cko also had an additional Ly6C-expressing cluster indicative of an increase in other hematopoietic cells such as monocytes or dendritic cells in the Cko bone marrow. The doubling of neutrophils in the Cko bone marrow was confirmed by FACS analysis for CD45 CD11b Ly6G triple-positive markers for neutrophils and was statistically significant. FACS analysis for HSCs showed that the increase in neutrophils is due to an increase in granulocyte-macrophage progenitors (GMP) in the Cko bone marrow. Immunofluorescence analysis for stefin showed very high expression for stefin in the Cko bone marrow in mice as young as P3. Conclusions: Our study shows that S1P ablation in the Osterix lineage results in the disruption of the bone marrow niche. This disruption is manifested as an increase in neutrophils indicating an inflammatory disposition in the Cko bone marrow. The Cko bone marrow also demonstrates an increase in Stfa proteins, which are known cathepsin inhibitors. While stefins are part of the normal expression profile in neutrophils, more members of this family are expressed in Cko neutrophils, with the doubling of the neutrophils contributing to stefin overexpression in the Cko bone marrow. The increase in inflammatory components such as neutrophils via a skewed increase in GMPs may induce an undesirable cross talk between the bone marrow niche and stem cell development pathways, which may be responsible for the decrease in skeletal progenitors in the Cko. How exactly stefin proteins contribute to this scenario needs to be investigated further, but our study indicates that S1P regulates both stefin production and the stem cell pool in the bone marrow and may be required to balance the production of skeletal and hematopoietic stem cell populations. Our study thus suggests that S1P is involved in the mechanistic crosstalk between the osteogenic and hematopoietic developmental programs.

Site-1 protease (S1P) is a proprotein convertase with essential functions in the conversion of precursor proteins to their active form. In earlier studies, we demonstrated that S1P ablation in the chondrocyte lineage results in a drastic reduction in endochondral bone formation. To investigate the mechanistic contribution of S1P to bone development we ablated S1P in the osterix lineage in mice. S1P ablation in this lineage results in osteochondrodysplasia and variable degrees of early postnatal scoliosis. Embryonically, even though Runx2 and osterix expression are normal, S1P ablation results in a delay in vascular invasion and endochondral bone development. Mice appear normal when born, but by day 7 display pronounced dwarfism with fragile bones that exhibit significantly reduced mineral density, mineral apposition rate, bone formation rate and reduced osteoblasts indicating severe osteopenia. Mice suffer from a drastic reduction in bone marrow mesenchymal progenitors as analyzed by colony-forming unit-fibroblast assay. Fluorescence-activated cell sorting analysis of the skeletal mesenchyme harvested from bone marrow and collagenase-digested bone show a drastic reduction in hematopoietic lineage-negative, endothelial-negative, CD105(+) skeletal stem cells. Bone marrow mesenchymal progenitors are unable to differentiate into osteoblasts in vitro, with no effect on adipogenic differentiation. Postnatal mice have smaller growth plates with reduced hypertrophic zone. Thus, S1P controls bone development directly by regulating the skeletal progenitor population and their differentiation into osteoblasts. This article has an associated First Person interview with the first author of the paper.

Site-1 protease (S1P), encoded by MBTPS1, is a serine protease in the Golgi. S1P regulates lipogenesis, endoplasmic reticulum (ER) function, and lysosome biogenesis in mice and in cultured cells. However, how S1P differentially regulates these diverse functions in humans has been unclear. In addition, no human disease with S1P deficiency has been identified. Here, we report a pediatric patient with an amorphic and a severely hypomorphic mutation in MBTPS1. The unique combination of these mutations results in a frequency of functional MBTPS1 transcripts of approximately 1%, a finding that is associated with skeletal dysplasia and elevated blood lysosomal enzymes. We found that the residually expressed S1P is sufficient for lipid homeostasis but not for ER and lysosomal functions, especially in chondrocytes. The defective S1P function specifically impairs activation of the ER stress transducer BBF2H7, leading to ER retention of collagen in chondrocytes. S1P deficiency also causes abnormal secretion of lysosomal enzymes due to partial impairment of mannose-6-phosphate-dependent delivery to lysosomes. Collectively, these abnormalities lead to apoptosis of chondrocytes and lysosomal enzyme-mediated degradation of the bone matrix. Correction of an MBTPS1 variant or reduction of ER stress mitigated collagen-trafficking defects. These results define a new congenital human skeletal disorder and, more importantly, reveal that S1P is particularly required for skeletal development in humans. Our findings may also lead to new therapies for other genetic skeletal diseases, as ER dysfunction is common in these disorders.

Purpose: Site-1 protease (S1P) is a proprotein convertase that processes latent, endoplasmic reticulum (ER) membrane-bound transcription factors SREBPs and ATF6 to their active form. Cartilage-specific ablation of S1P in mice (S1Pcko) using Col2-Cre results in poor cartilage due to the entrapment of type IIB procollagen (pro-Col IIB) in chondrocytes and a complete lack of endochondral bone formation. Ablation of S1P in postnatal chondrocytes using Col2-CreERT mice, gradually eliminates the primary growth plate with a gradual abolition of Ihh and type X collagen expression in the growth plate. In this study we used Osx-Cre to ablate S1P to study the contribution of specific cells types to growth plate and bone development. Methods: S1Pf/f mice (homozygous for the S1Pflox allele in which exon 2 of S1P is floxed) were crossed with Osx-Cre mice (in which the Cre recombinase is under the influence of the Osterix (Osx) promoter) to obtain mice with Osx-Cre-directed S1P ablation (S1POsx mice). S1POsx mice and wild type (WT) littermates were studied by histology, in situ hybridization analysis (ISH), immunofluorescence (IF), real-time (RT) PCR and micro-computed tomography (μCT) techniques. Osteogenic differentiation assays analyzing for osteoblast differentiation in vitro were conducted with bone marrow harvested from 21 day old WT and mutant mice. Results: Mice with a homozygous deletion for S1P (S1Pf/f;Osx-Cre mice or S1Pcko-Osx) suffer from severe chondrodysplasia; mice heterozygous for S1P deletion (S1P+/f-Osx) have a size intermediate to S1Pcko-Osx mice and WT littermates. Additionally, these mice are also kyphotic that gets more pronounced with age and was confirmed by μCT analysis. In situ hybridization analysis (ISH) showed S1P ablation specifically in the prehypertrophic/hypertrophic chondrocyte zone and GFP lineage tracing also demonstrated Osx-Cre activity (the Cre recombinase from Osx-Cre is made as a fusion protein with GFP) in the bone collar of both mutant mice. Both S1P+/f-Osx and S1Pcko-Osx mice exhibited a delay in the onset for endochondral bone formation. In IF studies for pro-Col IIB distribution at E16.5, S1Pcko-Osx mice (but not S1P+/f-Osx) showed pro-Col IIB entrapment but only in the prehypertrophic/hypertrophic zone, the zones where S1P is ablated. However these mice were able to clear the trapped Col II with no evidence of apoptosis in the growth plate. ISH studies demonstrated normal Col1a1 gene expression at E16.5 in both S1P+/f-Osx and S1Pcko-Osx mice. In IF analysis at E15.5, both S1P+/f-Osx and S1Pcko-Osx mice showed normal Runx2 expression in the prehypertrophic/hypertrophic zone and in the perichondrium. While there is a delay in onset of endochondral ossification, mutant mice are ultimately able to make endochondral bone and appear no different from WT mice on birth. However, by seven days postnatally both mutant mice show stunted growth with fragile bones and a drop in expression of mature osteoblast markers Col1a1, Bglap (osteocalcin), and ALP (alkaline phosphatase) by RT-PCR. μCT analysis also showed decreased bone mineralization in both mutant mice with the S1Pcko-Osx mice showing a drastic decrease than the heterozygote S1P+/f-Osx mice. In osteogenic differentiation assays with harvested bone marrow, both S1P+/f-Osx and S1Pcko-Osx mice showed an inability to generate mature osteoblasts in vitro. Conclusions: Our previous study had demonstrated an indispensable need for S1P in cartilage development where it was required for the secretion of pro-Col IIB from the chondrocytes in to the cartilage matrix. Our current study demonstrates the indispensable nature of S1P for bone development and osteoblast maintenance. The lack of Col II entrapment in the heterozygous mice but a delay in onset for endochondral bone development suggest that this defect is not due to faulty chondrocyte activity, but due to a defect in bone progenitors that lack S1P in the periosteum where Osx-Cre is also active. The resulting chondrodysplasia seen by postnatal day 7 in both S1P+/f-Osx and S1Pcko-Osx and the dramatically compromised osteogenic differentiation ability in vitro indicates an indispensable role for S1P in maintaining postnatal osteoblast precursor pool and/or differentiation for postnatal bone development. This suggests novel S1P substrates in bone development pathways and endorses the importance of S1P to overall skeletal development.

ABSTRACTIn this study, we examined early time‐dependent changes in articular cartilage and synovium in response to tibial compression and sought the plausible origin of cells that respond to compression in the healer (LGXSM‐6) and non‐healer (LGXSM‐33) recombinant inbred mouse strains. The right knee of 13‐week old male mice was subjected to tibial compression using 9N axial loading. The contralateral left knee served as a control. Knees were harvested at 5, 9, and 14 days post‐injury. Histological changes in cartilage and synovium, immunofluorescence pattern of CD44, aggrecan, type‐II collagen, cartilage oligomeric matrix protein and the aggrecan neo‐epitope NITEGE, and cell apoptosis (by TUNEL) were examined. We used a double nucleoside analog cell‐labeling strategy to trace cells responsive to injury. We showed that tibial compression resulted in rupture of anterior cruciate ligament, cartilage matrix loss and chondrocyte apoptosis at the injury site. LGXSM‐33 showed higher synovitis and ectopic synovial chondrogenesis than LGXSM‐6 with no differences for articular cartilage lesions. With loading, an altered pattern of CD44 and NITEGE was observed: cells in the impacted area underwent apoptosis, cells closely surrounding the injured area expressed CD44, and cells in the intact area expressed NITEGE. Cells responding to injury were found in the synovium, subchondral bone marrow and the Groove of Ranvier. Taken together, we found no strain differences in chondrocytes in the early response to injury. However, the synovial response was greater in LGXSM‐33 indicating that, at early time points, there is a genetic difference in synovial cell reaction to injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:524–536, 2017.

Purpose: Site-1 protease (S1P) is a proprotein convertase that is primarily known for processing of latent, endoplasmic reticulum (ER) membrane-bound transcription factors SREBPs and ATF6 to their free and active form. SREBPs are involved in cholesterol and fatty acid homeostasis; ATF6 is involved in ER stress response. Cartilage-specific ablation of S1P in mice (S1Pcko) using Col2-Cre results in poor cartilage with a drastic decrease of collagen type IIB (Col IIB) in the matrix and a complete lack of endochondral bone formation, even though the molecular program for chondrocyte maturation is not disrupted. In a postnatal model using Col2-CreERT mice, ablation of S1P in postnatal chondrocytes gradually eliminates the primary growth plate with a gradual abolition of Ihh and type X collagen expression in the growth plate indicating a cessation of hypertrophic chondrocyte differentiation. The goal of this study is to understand the importance of S1P in chondrocyte maturation in the growth plate. Methods: The osterix protein is expressed in prehypertrophic chondrocytes (as well as in osteoblasts). S1Pf/f mice (homozygous for the S1Pflox allele in which exon 2 of S1P is floxed) were crossed with Osx-Cre mice (in which the Cre recombinase is under the influence of the Osterix (Osx) promoter) to obtain mice with S1P ablation in prehypertrophic chondrocytes (S1POsx mice). S1POsx mice and wild type littermates were studied by histology, immunofluorescence (IF), and micro-computed tomography (μCT) techniques. Results: Mice with a homozygous deletion for S1P (S1Pf/f;Osx-Cre mice or S1Pcko-Osx) suffer from severe chondrodysplasia that is visible in their gross morphology beginning from postnatal day 7. Mice which are heterozygous for S1P deletion (S1P+/f-Osx) have a size intermediate to the S1Pcko-Osx mice and their wild type littermates; additionally, these mice are also hunchbacks (kyphosis) which gets more pronounced with age. Interestingly, μCT analysis not only confirmed the kyphosis in S1P+/f-Osx mice, but also revealed scoliosis in the S1Pcko-Osx mice. Micro-CT analysis also showed decreased bone mineralization in both mutant mice with the S1Pcko-Osx mice showing a more drastic decrease than the heterozygote resulting in fragile bones. In embryonic studies, S1Pcko-Osx mice exhibited a delay in the onset for endochondral bone formation, while the S1P+/f-Osx showed normal onset of endochondral bone formation as the wild type. In immunofluorescence studies for type II collagen (Col II) distribution, while the Col2-Cre-directed S1Pcko mice exhibited Col II entrapment in all zones of the growth plate including the resting and proliferative zone, S1Pcko-Osx mice showed Col II entrapment only in the hypertrophic zone, but not the resting and proliferative zones; negligible Col II entrapment was observed in the growth plate of S1P+/f-Osx mice. Conclusions: These results indicate the indispensable nature of S1P for cartilage and overall skeletal development. S1P is essential for normal growth plate activity and cartilage formation. The current study also endorses the importance of S1P in normal skeletal development and is important for both axial and appendicular skeletal development. For the first time, we demonstrate an important functional link between S1P and diseases of the vertebral column. These studies also suggest an indispensable role for S1P in osteoblasts. Thus understanding the contributions of S1P in cartilage, bone and vertebrae development will contribute to the understanding of overall skeletal homeostasis.

The proprotein convertase site-1 protease (S1P) converts latent ER-membrane bound transcription factors SREBPs and ATF6 to their active forms. SREBPs are involved in cholesterol and fatty acid homeostasis whereas ATF6 is involved in unfolded protein response pathways (UPR). Cartilage-specific ablation of S1P in mice (S1Pcko) results in abnormal cartilage devoid of type II collagen protein (Col II). S1Pcko mice also lack endochondral bone development. To analyze S1Pcko cartilage we performed double-labeled immunofluorescence studies for matrix proteins that demonstrated that type IIB procollagen is trapped inside the ER in S1Pcko chondrocytes. This retention is specific to type IIB procollagen; other cartilage proteins such as type IIA procollagen, cartilage oligomeric matrix protein (COMP) and aggrecan are not affected. The S1Pcko cartilage thus exhibits COMP-, aggrecan-, and type IIA procollagen-derived matrices but is characterized by the absence of a type IIB procollagen-derived matrix. To understand the molecular reason behind S1Pcko phenotypes we performed genome-wide transcriptional profiling of cartilage isolated from S1Pcko and wild type littermates. While the UPR pathways are unaffected, the SREBPs-directed cholesterol and fatty acid pathways are significantly down-regulated in S1Pcko chondrocytes, with maximal down-regulation of the stearoyl-CoA desaturase-1 (Scd1) gene. However, mouse models that lack Scd1 or exhibit reduction in lipid homeostasis do not suffer from the ER retention of Col II or lack endochondral bone. These studies indicate an indispensable role for S1P in type IIB procollagen trafficking from the ER. This role appears not to be related to lipid pathways or other current known functions of S1P and is likely dependent on additional, yet unknown, S1P substrates in chondrocytes.

This article summarizes the current understanding of the pathophysiology of knee osteoarthritis (OA). OA is a common disease of diarthrodial joints with marked prevalence in aging individuals. It is a complex, heterogeneous, whole-joint disease with multiple etiologies. Although articular cartilage degeneration is a hallmark end-stage finding of OA, not all individuals with cartilage damage develop OA. The exact trigger of OA remains to be unequivocally identified, although a number of variable secondary components including age, obesity, and inflammation determine the extent of disease progression. OA bears a strong hereditary component. However, its different genetic etiologies are not equally shared in the diverse human population. In addition, its variable clinical features have made the understanding of its pathophysiology a daunting task. Several diagnostic tools are available and many more are under development to diagnose early stage OA. The advent of biomarkers and genetic tools for OA research is very promising for stratifying individuals at higher risk for developing OA.

ObjectiveHigher body mass index (BMI) increases the risk of meniscus injury and knee osteoarthritis (OA). However, it is unknown if and how obesity affects meniscus biology. We analyzed transcriptome‐wide gene expression profiles of injured human menisci to test the hypothesis that meniscal gene expression signatures relate to patient BMI.MethodsMeniscus samples were obtained from patients undergoing arthroscopic partial meniscectomy. Transcriptome‐wide analysis of gene expression followed by validation of selected transcripts by QuantiGene Plex assay was performed. Correlations of gene expression with BMI and relative fold changes (≥1.5‐fold) in 3 BMI categories (lean [BMI 18.5–24.9 kg/m2], overweight [BMI 25.0–29.9 kg/m2], and obese [BMI >30.0 kg/m2]) were analyzed, and integrated functional classifications were probed computationally.ResultsThe obese versus overweight comparison resulted in the largest set of differences (565 transcripts) followed by obese versus lean (280 transcripts) and overweight versus lean (125 transcripts). Biologic reproducibility was confirmed by cluster analysis of expressed transcripts. Differentially regulated transcripts represented important functional classifications. Transcripts associated with oxygen transport, calcium ion binding, and cell homeostasis were elevated with BMI, while those related to extracellular matrix deposition, cell migration, and glucosamine metabolic processes were repressed. While these functional classifications may play key roles in cartilage/meniscus homeostasis, failure of extracellular matrix deposition and increase in calcium ion binding likely contribute to OA development following meniscal injury.ConclusionOur results indicate greater differences in gene expression between obese and overweight groups than between overweight and lean groups. This may indicate that there is a weight threshold at which injured meniscus responds severely to increased BMI. BMI‐related changes in gene expression present a plausible explanation for the role of meniscal injury in OA development among obese patients.