Inhibition of Phlpp1 preserves the mechanical integrity of articular cartilage in a murine model of post-traumatic osteoarthritis.

OBJECTIVE: Phlpp1 inhibition is a potential therapeutic strategy for cartilage regeneration and prevention of post-traumatic osteoarthritis (PTOA). To understand how Phlpp1 loss affects cartilage structure, cartilage elastic modulus was measured with atomic force microscopy (AFM) in male and female mice after injury. METHODS: Osteoarthritis was induced in male and female Wildtype (WT) and Phlpp1-/- mice by destabilization of the medial meniscus (DMM). At various timepoints post-injury, activity was measured, and knee joints examined with AFM and histology. In another cohort of WT mice, the PHLPP inhibitor NSC117079 was intra-articularly injected 4 weeks after injury. RESULTS: Male WT mice showed decreased activity and histological signs of cartilage damage at 12 but not 6-weeks post-DMM. Female mice showed a less severe response to DMM by comparison, with no histological changes seen at any time point. In both sexes the elastic modulus of medial condylar cartilage was decreased in WT mice but not Phlpp1-/- mice after DMM as measured by AFM. By 6-weeks, cartilage modulus had decreased from 2 MPa to 1 MPa in WT mice. Phlpp1-/- mice showed no change in modulus at 6-weeks and only a 25% decrease at 12-weeks. The PHLPP inhibitor NSC117079 protected cartilage structure and prevented signs of OA 6-weeks post-injury. CONCLUSIONS: AFM is a sensitive method for detecting early changes in articular cartilage post-injury. Phlpp1 suppression, either through genetic deletion or pharmacological inhibition, protects cartilage degradation in a model of PTOA, validating Phlpp1 as a therapeutic target for PTOA.

Prg4-Expressing Chondroprogenitor Cells in the Superficial Zone of Articular Cartilage.

Joint-resident chondrogenic precursor cells have become a significant therapeutic option due to the lack of regenerative capacity in articular cartilage. Progenitor cells are located in the superficial zone of the articular cartilage, producing lubricin/Prg4 to decrease friction of cartilage surfaces during joint movement. Prg4-positive progenitors are crucial in maintaining the joint's structure and functionality. The disappearance of progenitor cells leads to changes in articular hyaline cartilage over time, subchondral bone abnormalities, and the formation of ectopic ossification. Genetic labeling cell technology has been the main tool used to characterize Prg4-expressing progenitor cells of articular cartilage in vivo through drug injection at different time points. This technology allows for the determination of the origin of progenitor cells and the tracking of their progeny during joint development and cartilage damage. We endeavored to highlight the currently known information about the Prg4-producing cell population in the joint to underline the significance of the role of these cells in the development of articular cartilage and its homeostasis. This review focuses on superficial progenitors in the joint, how they contribute to postnatal articular cartilage formation, their capacity for regeneration, and the consequences of Prg4 deficiency in these cells. We have accumulated information about the Prg4+ cell population of articular cartilage obtained through various elegantly designed experiments using transgenic technologies to identify potential opportunities for further research.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843.

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally. Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed. ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion. Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

Mechanical motion promotes expression of Prg4 in articular cartilage via multiple CREB-dependent, fluid flow shear stress-induced signaling pathways.

Lubricin is a secreted proteoglycan encoded by the Prg4 locus that is abundantly expressed by superficial zone articular chondrocytes and has been noted to both be sensitive to mechanical loading and protect against the development of osteoarthritis. In this study, we document that running induces maximal expression of Prg4 in the superficial zone of knee joint articular cartilage in a COX-2-dependent fashion, which correlates with augmented levels of phospho-S133 CREB and increased nuclear localization of CREB-regulated transcriptional coactivators (CRTCs) in this tissue. Furthermore, we found that fluid flow shear stress (FFSS) increases secretion of extracellular PGE2, PTHrP, and ATP (by epiphyseal chondrocytes), which together engage both PKA- and Ca(++)-regulated signaling pathways that work in combination to promote CREB-dependent induction of Prg4, specifically in superficial zone articular chondrocytes. Because running and FFSS both boost Prg4 expression in a COX-2-dependent fashion, our results suggest that mechanical motion may induce Prg4 expression in the superficial zone of articular cartilage by engaging the same signaling pathways activated in vitro by FFSS that promote CREB-dependent gene expression in this tissue.

Quadruped Gait and Regulation of Apoptotic Factors in Tibiofemoral Joints following Intra-Articular rhPRG4 Injection in Prg4 Null Mice.

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome leads to diarthrodial joint arthropathy and is caused by the absence of lubricin (proteoglycan 4-PRG4), a surface-active mucinous glycoprotein responsible for lubricating articular cartilage. In this study, mice lacking the orthologous gene Prg4 served as a model that recapitulates the destructive arthrosis that involves biofouling of cartilage by serum proteins in lieu of Prg4. This study hypothesized that Prg4-deficient mice would demonstrate a quadruped gait change and decreased markers of mitochondrial dyscrasia, following intra-articular injection of both hindlimbs with recombinant human PRG4 (rhPRG4). Prg4-/- (N = 44) mice of both sexes were injected with rhPRG4 and gait alterations were studied at post-injection day 3 and 6, before joints were harvested for immunohistochemistry for caspase-3 activation. Increased stance and propulsion was shown at 3 days post-injection in male mice. There were significantly fewer caspase-3-positive chondrocytes in tibiofemoral cartilage from rhPRG4-injected mice. The mitochondrial gene Mt-tn, and myosin heavy (Myh7) and light chains (Myl2 and Myl3), known to play a cytoskeletal stabilizing role, were significantly upregulated in both sexes (RNA-Seq) following IA rhPRG4. Chondrocyte mitochondrial dyscrasias attributable to the arthrosis in CACP may be mitigated by IA rhPRG4. In a supporting in vitro crystal microbalance experiment, molecular fouling by albumin did not block the surface activity of rhPRG4.

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells.

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

Regulatory Mechanisms of Prg4 and Gdf5 Expression in Articular Cartilage and Functions in Osteoarthritis.

Owing to the rapid aging of society, the numbers of patients with joint disease continue to increase. Accordingly, a large number of patients require appropriate treatment for osteoarthritis (OA), the most frequent bone and joint disease. Thought to be caused by the degeneration and destruction of articular cartilage following persistent and excessive mechanical stimulation of the joints, OA can significantly impair patient quality of life with symptoms such as knee pain, lower limb muscle weakness, or difficulty walking. Because articular cartilage has a low self-repair ability and an extremely low proliferative capacity, healing of damaged articular cartilage has not been achieved to date. The current pharmaceutical treatment of OA is limited to the slight alleviation of symptoms (e.g., local injection of hyaluronic acid or non-steroidal anti-inflammatory drugs); hence, the development of effective drugs and regenerative therapies for OA is highly desirable. This review article summarizes findings indicating that proteoglycan 4 (Prg4)/lubricin, which is specifically expressed in the superficial zone of articular cartilage and synovium, functions in a protective manner against OA, and covers the transcriptional regulation of Prg4 in articular chondrocytes. We also focused on growth differentiation factor 5 (Gdf5), which is specifically expressed on the surface layer of articular cartilage, particularly in the developmental stage, describing its regulatory mechanisms and functions in joint formation and OA pathogenesis. Because several genetic studies in humans and mice indicate the involvement of these genes in the maintenance of articular cartilage homeostasis and the presentation of OA, molecular targeting of Prg4 and Gdf5 is expected to provide new insights into the aetiology, pathogenesis, and potential treatment of OA.

PRG3 and PRG5 C-Termini: Important Players in Early Neuronal Differentiation.

The functional importance of neuronal differentiation of the transmembrane proteins' plasticity-related genes 3 (PRG3) and 5 (PRG5) has been shown. Although their sequence is closely related, they promote different morphological changes in neurons. PRG3 was shown to promote neuritogenesis in primary neurons; PRG5 contributes to spine induction in immature neurons and the regulation of spine density and morphology in mature neurons. Both exhibit intracellularly located C-termini of less than 50 amino acids. Varying C-termini suggested that these domains shape neuronal morphology differently. We generated mutant EGFP-fusion proteins in which the C-termini were either swapped between PRG3 and PRG5, deleted, or fused to another family member, plasticity-related gene 4 (PRG4), that was recently shown to be expressed in different brain regions. We subsequently analyzed the influence of overexpression in immature neurons. Our results point to a critical role of the PRG3 and PRG5 C-termini in shaping early neuronal morphology. However, the results suggest that the C-terminus alone might not be sufficient for promoting the morphological effects induced by PRG3 and PRG5.

Proteoglycan 4 (PRG4) expression and function in dry eye associated inflammation.

Dry eye disease (DED) affects hundreds of millions of people worldwide. It is characterized by the production of inflammatory cytokines and chemokines as well as damaging matrix metalloproteinases (MMPs) at the ocular surface. While proteoglycan 4 (PRG4), a mucin-like glycoprotein present at the ocular surface, is most well known as a boundary lubricant that contributes to ocular surface integrity, it has been shown to blunt inflammation in various cell types, suggesting a dual mechanism of action. Recently, full-length recombinant human PRG4 (rhPRG4) has been shown to improve signs and symptoms of DED in humans. However, there remains a significant need for basic science research on rhPRG4's biological properties and its potential therapeutic mechanisms of action in treating DED. Therefore, the objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine whether exogenous rhPRG4 modulates cytokine and chemokine secretion in response to dry eye associated inflammation (TNFα and IL-1ß), explore interactions between rhPRG4 and MMP-9, and understand how experimental dry eye (EDE) in mice affects PRG4 expression. PRG4 secretion from hTCEpi cells was quantified by Western blot and expression visualized by immunocytochemistry. Cytokine/chemokine production was measured by ELISA and Luminex, while rhPRG4's effect on MMP-9 activity, binding, and expression was quantified using an MMP-9 inhibitor kit, surface plasmon resonance, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Finally, EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and by Western blot in lacrimal gland lysate. In vitro results demonstrate that hTCEpi cells synthesize and secrete PRG4, and PRG4 secretion is inhibited by TNFα and IL-1ß. In response to these pro-inflammatory stresses, exogenous rhPRG4 significantly reduced the stimulated production of IP-10, RANTES, ENA-78, GROα, MIP-3α, and MIG, and trended towards a reduction of MIP-1α and MIP-1ß. The hTCEpi cells were also able to internalize fluorescently-labelled rhPRG4, consistent with a mechanism of action that includes downstream biological signaling pathways. rhPRG4 was not digested by MMP-9, and it did not modulate MMP-9 gene expression in hTCEpi cells, but it was able to bind to MMP-9 and inhibited in vitro activity of exogenous MMP-9 in the presence of human tears. Finally, in vivo results demonstrate that EDE significantly decreased immunolocalization of PRG4 on the corneal epithelium and trended towards a reduction of PRG4 in lacrimal gland lysate. Collectively these results demonstrate rhPRG4 has anti-inflammatory properties on corneal epithelial cells, particularly as it relates to mitigating chemokine production, and is an inhibitor of MMP-9 activity, as well as that in vivo expression of PRG4 can be altered in preclinical models of DED. In conclusion, these findings contribute to our understanding of PRG4's immunomodulatory properties in the context of DED inflammation and provide the foundation and motivation for further mechanistic research of PRG4's properties on the ocular surface as well as expanding clinical evaluation of its ability as a multifunctional therapeutic agent to effectively provide relief to those who suffer from DED.

Proteoglycan 4: From Mere Lubricant to Regulator of Tissue Homeostasis and Inflammation: Does proteoglycan 4 have the ability to buffer the inflammatory response?

Proteoglycan 4 (PRG4), first identified in synovial fluid, is an extracellular matrix structural protein in the joint implicated in reducing shear at the cartilage surface as well as controlling adhesion-dependent synovial growth and regulating bulk protein deposition onto the cartilage. However, recent evidence suggests that it can bind to and effect downstream signaling of a number of cell surface receptors implicated in regulating the inflammatory response. Therefore, we pose the hypothesis: Does PRG4 regulate the inflammatory response and maintain tissue homeostasis? Based on these novel findings implicating PRG4 as an inflammatory signaling molecule, we will present and discuss several hypotheses regarding potential mechanisms by which PRG4 may be able to regulate inflammation. If future studies can demonstrate that PRG4 is a potent inflammatory mediator, this will change current paradigms in the musculoskeletal and ophthalmological fields regarding the how the inflammatory microenvironment is regulated in these tissues and potentially others.

Characteristics of MSCs in Synovial Fluid and Mode of Action of Intra-Articular Injections of Synovial MSCs in Knee Osteoarthritis.

We have been studying mesenchymal stem cells (MSCs) in synovial fluid and the intra-articular injection of synovial MSCs in osteoarthritis (OA) knees. Here, mainly based on our own findings, we overview the characteristics of endogenous MSCs in the synovial fluid of OA knees and their mode of action when injected exogenously into OA knees. Many MSCs similar to synovial MSCs were detected in the synovial fluid of human OA knees, and their number correlated with the radiological OA grade. Our suspended synovium culture model demonstrated the release of MSCs from the synovium through a medium into a non-contacting culture dish. In OA knees, endogenous MSCs possibly mobilize in a similar manner from the synovium through the synovial fluid and act protectively. However, the number of mobilized MSCs is limited; therefore, OA progresses in its natural course. Synovial MSC injections inhibited the progression of cartilage degeneration in a rat OA model. Injected synovial MSCs migrated into the synovium, maintained their MSC properties, and increased the gene expressions of TSG-6, PRG-4, and BMP-2. Exogenous synovial MSCs can promote anti-inflammation, lubrication, and cartilage matrix synthesis in OA knees. Based on our findings, we have initiated a human clinical study of synovial MSC injections in OA knees.

Heparan sulfate deficiency leads to hypertrophic chondrocytes by increasing bone morphogenetic protein signaling.

OBJECTIVE: Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext-/- mouse model. METHOD: We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice. RESULTS: Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007). CONCLUSIONS: HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

Inhibitory Effects of PRG4 on Migration and Proliferation of Human Venous Cells.

BACKGROUND: Proteoglycan 4 (PRG4; lubricin) is a member of two gene co-expression network modules associated with human vein graft failure. However, little is known about PRG4 and the vascular system. Therefore, we have investigated the effects of recombinant human PRG4 (rhPRG4) on cell migration and proliferation in human veins. METHODS: Effects of rhPRG4 on cell migration, proliferation, and neointima formation were determined in human venous tissue and cultured venous smooth muscle cells (SMCs), adventitial cells, and endothelial cells. Expression of PRG4 by cultured human saphenous veins, failed vein grafts, and varicose veins was determined by immunostaining or Western blotting. RESULTS: Limited expression of PRG4 in fresh saphenous veins was dramatically increased around medial SMCs after culture ex vivo. rhPRG4 inhibited the migration of cultured SMCs, adventitial cells, and endothelial cells, as well as the proliferation of endothelial cells. rhPRG4 also inhibited the migration of SMCs and adventitial cells from tissue explants, but there was no effect on cell proliferation or neointima formation in ex vivo whole veins. Finally, PRG4 was largely absent in two examples of venous pathology, that is, failed human vein grafts and varicose veins. CONCLUSIONS: Although rhPRG4 can inhibit the migration of venous SMCs, endothelial cells, and adventitial cells, and the proliferation of endothelial cells, PRG4 was only increased around medial SMCs in veins after ex vivo culture. PRG4 was not observed around medial SMCs in failed human vein grafts and varicose veins, suggesting the possibility that a failure of PRG4 upregulation may promote these pathologies.

Ablation of the miRNA Cluster 24 Has Profound Effects on Extracellular Matrix Protein Abundance in Cartilage.

MicroRNAs (miRNAs) regulate cartilage differentiation and contribute to the onset and progression of joint degeneration. These small RNA molecules may affect extracellular matrix organization (ECM) in cartilage, but for only a few miRNAs has this role been defined in vivo. Previously, we showed that cartilage-specific genetic ablation of the Mirc24 cluster in mice leads to impaired cartilage development due to increased RAF/MEK/ERK pathway activation. Here, we studied the expression of the cluster in cartilage by LacZ reporter gene assays and determined its role for extracellular matrix homeostasis by proteome and immunoblot analysis. The cluster is expressed in prehypertrophic/hypertrophic chondrocytes of the growth plate and we now show that the cluster is also highly expressed in articular cartilage. Cartilage-specific loss of the cluster leads to increased proteoglycan 4 and matrix metallopeptidase 13 levels and decreased aggrecan and collagen X levels in epiphyseal cartilage. Interestingly, these changes are linked to a decrease in SRY-related HMG box-containing (SOX) transcription factors 6 and 9, which regulate ECM production in chondrocytes. Our data suggests that the Mirc24 cluster is important for ECM homoeostasis and the expression of transcriptional regulators of matrix production in cartilage.

Stable recombinant production of codon-scrambled lubricin and mucin in human cells.

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition. The codon-scrambling strategy was applied to generate synonymous genes, or "synDNAs," for two mucins of commercial interest: lubricin and mucin 1. Stable, long-term recombinant production in suspension-adapted human 293-F cells was demonstrated for the synonymous lubricin complementary DNA (cDNA), which we refer to as SynLubricin. Under optimal conditions, a 293-F subpopulation produced recombinant SynLubricin at more than 200 mg/L of media and was stable throughout 2 months of continuous culture. Functionality tests confirmed that the recombinant lubricin could effectively inhibit cell adhesion and lubricate cartilage explants. Together, our work provides a viable workflow for cDNA design and stable mucin production in mammalian host production systems.

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

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

PRG4 expression in myxoid liposarcoma maintains tumor cell growth through suppression of an antitumor cytokine IL-24.

PRG4 is one of the downstream molecules of the myxoid liposarcoma (MLS)-specific fusion oncoproteins TLS-CHOP and EWS-CHOP. Exogenous PRG4 expression increases the tumorigenicity of cells injected in nude mice. The molecular functions of PRG4 in tumorigenesis and/or tumor progression of MLS cells, however, still remain unclear. In this report, we demonstrated that siRNA-mediated knockdown of PRG4 suppressed the growth of the MLS-derived cell lines 1955/91 and 2645/94. In addition, PRG4 knockdown promoted adipocytic differentiation in 1955/91 cells. Thus, PRG4 may play essential roles in MLS cell growth and have potential as a therapeutic target. On the other hand, our previous study has revealed that TLS-CHOP suppresses expression of an anti-tumor cytokine IL-24, contributing to tumor cell survival. In this study, we found that double knockdown of PRG4 and IL-24 did not inhibit MLS cell growth, and single knockdown of PRG4 remarkably increased IL-24 expression. These results suggest that the growth inhibitory effect of PRG4 knockdown is caused by induction of IL-24 expression, and PRG4 may contribute to maintain MLS cell growth through repression of IL-24 expression.

Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice.

OBJECTIVE: Previous studies have shown that Transforming growth factor-ß (TGF-ß)/TGFßRII-Smad3 signaling is involved in articular cartilage homeostasis. However, the role of TGF-ß/ALK5 signaling in articular cartilage homeostasis has not been fully defined. In this study, a combination of in vitro and in vivo approaches was used to elucidate the role of ALK5 signaling in articular cartilage homeostasis and the development of osteoarthritis (OA). DESIGN: Mice with inducible cartilage-specific deletion of Alk5 were generated to assess the role of ALK5 in OA development. Alterations in cartilage structure were evaluated histologically. The expressions of genes associated with articular cartilage homeostasis and TGF-ß signaling were analyzed by qRT-PCR, western blotting and immunohistochemistry. The chondrocyte apoptosis was detected by TUNEL staining and immunohistochemistry. In addition, the molecular mechanism underlying the effects of TGF-ß/ALK5 signaling on articular cartilage homeostasis was explored by analyzing the TGF-ß/ALK5 signaling-induced expression of proteoglycan 4 (PRG4) using specific inhibitors. RESULTS: Postnatal cartilage-specific deletion of Alk5 induced an OA-like phenotype with degradation of articular cartilage, synovial hyperplasia, osteophyte formation, subchondral sclerosis, as well as enhanced chondrocyte apoptosis, overproduction of catabolic factors, and decreased expressions of anabolic factors in chondrocytes. In addition, the expressions of PRG4 mRNA and protein were decreased in Alk5 conditional knockout mice. Furthermore, our results showed, for the first time, that TGF-ß/ALK5 signaling regulated PRG4 expression partially through the protein kinase A (PKA)-CREB signaling pathway. CONCLUSIONS: TGF-ß/ALK5 signaling maintains articular cartilage homeostasis, in part, by upregulating PRG4 expression through the PKA-CREB signaling pathway in articular chondrocytes.

SOX9 protein is stabilized by TGF-ß and regulates PAPSS2 mRNA expression in chondrocytes.

OBJECTIVE: We previously identified 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) as a transcriptional target of transforming growth factor ß (TGF-ß) in chondrocytes. PAPSS2 is required for proper sulfation of proteoglycans in cartilage. Defective sulfation in the matrix results in alterations in mechanical properties of the cartilage that would be expected to result in degeneration. The objective of this study was to identify factors that regulate PAPSS2 expression and compare to a known TGF-ß responsive gene, proteoglycan 4/lubricin (PRG4). In this study, TGF-ß-mediated regulation of SOX9 was characterized, and the involvement of SOX9 in regulation of PAPSS2 mRNA was investigated. DESIGN: Primary bovine articular chondrocytes grown in micromass culture and ATDC5 cells were used as the model system. Adenoviruses were used to express SOX9 and SMAD3. siRNA was used to knock-down Sox9 and Smad3. Western blot and real-time quantitative RT-PCR (qPCR) were used to measure changes in protein and mRNA levels in response to treatment. RESULTS: Over-expression of SOX9 was sufficient to up-regulate PAPSS2 mRNA. TGF-ß treatment of SOX9-expressing cells resulted in enhanced up-regulation of PAPSS2 mRNA, suggesting that SOX9 cooperates with TGF-ß signaling. Furthermore, Sox9 was required for full TGF-ß-mediated induction of Papss2. In contrast, PRG4 was regulated by SMAD3 but not SOX9. SOX9 protein levels were increased after treatment with TGF-ß, although SOX9 mRNA was not. SOX9 protein was post-translationally stabilized after treatment with TGF-ß. CONCLUSIONS: TGF-ß stabilizes SOX9 protein, and SOX9 is sufficient and necessary for TGF-ß-mediated regulation of PAPSS2 mRNA, providing a novel mechanism for TGF-ß-mediated gene regulation in chondrocytes.

Friction-Induced Mitochondrial Dysregulation Contributes to Joint Deterioration in Prg4 Knockout Mice.

Deficiency of PRG4 (lubricin), the boundary lubricant in mammalian joints, contributes to increased joint friction accompanied by superficial and upper intermediate zone chondrocyte caspase-3 activation, as shown in lubricin-null (Prg4-/-) mice. Caspase-3 activity appears to be reversible upon the restitution of Prg4 either endogenously in vivo, in a gene trap mouse, or as an applied lubricant in vitro. In this study we show that intra-articular injection of human PRG4 in vivo in Prg4-/- mice prevented caspase-3 activation in superficial zone chondrocytes and was associated with a modest decrease in whole joint friction measured ex vivo using a joint pendulum method. Non-lubricated Prg4-/- mouse cartilage shows caspase cascade activation caused by mitochondrial dysregulation, and significantly higher levels of peroxynitrite (ONOO- and -OH) and superoxide (O-2) compared to Prg4+/+ and Prg4+/- cartilage. Enzymatic activity levels of caspase 8 across Prg4 mutant mice were not significantly different, indicating no extrinsic apoptosis pathway activation. Western blots showed caspase-3 and 9 activation in Prg4-/- tissue extracts, and the appearance of nitrosylated Cys163 in the active cleft of caspase-3 which inhibits its enzymatic activity. These findings are relevant to patients at risk for arthrosis, from camptodactyl-arthropathy-coxa vara-pericarditis (CACP) syndrome and transient lubricin insufficiency due to trauma and inflammation.