Recombinant Human Proteoglycan 4 Research Articles

Proteoglycan 4 (PRG4) treatment improves skin wound healing in a porcine model.

Proteoglycan 4 (PRG4) is a boundary lubricant originally identified in articular cartilage and has been since shown to have immunomodulation and antifibrotic properties. Previously, we have demonstrated that recombinant human (rh)PRG4 treatment accelerates auricular cartilage injury closure through an inhibition of the fibrotic response, and promotion of tissue regeneration in mice. The purpose of the current study was to examine the effects of rhPRG4 treatment (vs. a DMSO carried control) on full-thickness skin wound healing in a preclinical porcine model. Our findings suggest that while rhPRG4 did not significantly accelerate nor impede full-thickness skin wound closure, it did improve repair quality by decreasing molecular markers of fibrosis and increasing re-vascularization. We also demonstrated that rhPRG4 treatment increased dermal adipose tissue during the healing process specifically by retaining adipocytes in the wound area but did not inhibit lipolysis. Overall, the results of the current study have demonstrated that rhPRG4 acts as antifibrotic agent and regulates dermal adipose tissue during the healing processes resulting in a tissue with a trajectory that more resembles the native skin vs. a fibrotic patch. This study provides strong rationale to examine if rhPRG4 can improve regeneration in human wounds.

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.

Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration

The wound healing response is one of most primitive and conserved physiological responses in the animal kingdom, as restoring tissue integrity/homeostasis can be the difference between life and death. Wound healing in mammals is mediated by immune cells and inflammatory signaling molecules that regulate tissue resident cells, including local progenitor cells, to mediate closure of the wound through formation of a scar. Proteoglycan 4 (PRG4), a protein found throughout the animal kingdom from fish to elephants, is best known as a glycoprotein that reduces friction between articulating surfaces (e.g. cartilage). Previously, PRG4 was also shown to regulate the inflammatory and fibrotic response. Based on this, we asked whether PRG4 plays a role in the wound healing response. Using an ear wound model, topical application of exogenous recombinant human (rh)PRG4 hastened wound closure and enhanced tissue regeneration. Our results also suggest that rhPRG4 may impact the fibrotic response, angiogenesis/blood flow to the injury site, macrophage inflammatory dynamics, recruitment of immune and increased proliferation of adult mesenchymal progenitor cells (MPCs) and promoting chondrogenic differentiation of MPCs to form the auricular cartilage scaffold of the injured ear. These results suggest that PRG4 has the potential to suppress scar formation while enhancing connective tissue regeneration post-injury by modulating aspects of each wound healing stage (blood clotting, inflammation, tissue generation and tissue remodeling). Therefore, we propose that rhPRG4 may represent a potential therapy to mitigate scar and improve wound healing.

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-1β), 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-1β. 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-1β. 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 Modulates Osteogenic Smooth Muscle Cell Differentiation during Vascular Remodeling and Intimal Calcification.

Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. PRG4 expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, Prg4 was upregulated in SMCs from partially ligated ApoE−/− mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and TGFb1. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in ApoE−/− mice on warfarin. In vitro, PRG4 was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that PRG4 expression was driven by transcription factors SMAD3 and SOX9. Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous PRG4, SMAD3 and SOX9, and restored SMC markers’ expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.

Proteoglycan 4 Reduces Neuroinflammation and Protects the Blood-Brain Barrier after Traumatic Brain Injury.

Neuroinflammation and dysfunction of the blood–brain barrier (BBB) are two prominent mechanisms of secondary injury in neurotrauma. It has been suggested that Toll-like receptors (TLRs) play important roles in initiating and propagating neuroinflammation resulting from traumatic brain injury (TBI), but potential beneficial effects of targeting these receptors in TBI have not been broadly studied. Here, we investigated the effect of targeting TLRs with proteoglycan 4 (PRG4) on post-traumatic neuroinflammation and BBB function. PRG4 is a mucinous glycoprotein with strong anti-inflammatory properties, exerting its biological effects by interfering with TLR2/4 signaling. In addition, PRG4 has the ability to inhibit activation of cluster of differentiation 44 (CD44), a cell-surface glycoprotein playing an important role in inflammation. Using the controlled cortical impact model of TBI in rats, we showed a rapid and prolonged upregulation of message for TLR2/4 and CD44 in the injured cortex. In the in vitro model of the BBB, recombinant human PRG4 (rhPRG4) crossed the endothelial monolayers through a high-capacity, saturable transport system. In rats sustaining TBI, PRG4 delivery to the brain was enhanced by post-traumatic increase in BBB permeability. rhPRG4 injected intravenously at 1 h post-TBI potently inhibited post-traumatic activation of nuclear factor kappa B and extracellular signal-regulated kinases 1/2, the two major signal transduction pathways associated with TLR2/4 and CD44, and curtailed the post-traumatic influx of monocytes. In addition, PRG4 restored normal BBB function after TBI by preventing the post-traumatic loss of tight junction protein claudin 5 and reduced neuronal death. Our observations provide support for therapeutic strategies targeting TLRs in TBI.

Investigating the Synergistic Interactions of Surface Immobilized and Free Natural Ocular Lubricants for Contact Lens Applications: A Comparative Study between Hyaluronic Acid and Proteoglycan 4 (Lubricin).

The main reasons for the discontinuation of contact lens wear are ocular dryness and discomfort. Proteoglycan 4 (PRG4), a mucinous glycoprotein, and hyaluronic acid (HA), a nonsulfated linear glycosaminoglycan, are naturally present in the eye and contribute to ocular hydration and lubrication. This study aimed to investigate the impact of the structure of the recombinant human PRG4 (rhPRG4)/HA complex on contact lens properties, when one agent is grafted and the counterpart is physisorbed on the surface of model conventional or silicone contact lens materials. Investigation of the wettability, water retention, antifouling, and boundary lubricant properties of the prepared hydrogels showed that the rhPRG4/HA interactions varied with the rhPRG/HA configuration on the hydrogel surface as well as the composition of the underlying substrate used. The rhPRG4-physisorbed/HA-grafted sample was characterized by better antifouling and boundary lubricant properties on the model conventional hydrogels, while the HA-physisorbed/rhPRG4-grafted sample exhibited improved surface wettability, antifouling, and water-retentive properties on the model silicone hydrogels. The results of this study contribute to the design of biomimetic contact lens surfaces that work synergistically with ocular fluid-phase biological agents to enhance compatibility between the contact lens and the ocular environment, alleviating dry eye symptoms and improving comfort.

The Effect of Intense Exercise on Equine Serum Proteoglycan-4/Lubricin.

Objective: Local biological and biomechanical-stimuli modulate proteoglycan-4 secretion within synovial joints. For the horse, changes to proteoglycan-4 concentration and function are notable in acute joint injury and osteoarthritis. Proteoglycan-4 (also known as Lubricin) is present in the blood, however the effect of exercise on equine serum levels is unknown. The overall objective of this study was, therefore, to investigate the effect of intense exercise on serum proteoglycan-4 in thoroughbred horses.Methods: Samples of blood were taken from thoroughbreds (n = 12) during a chuckwagon racing event (Alberta, Canada). The chuckwagon race is a sprint racing event where teams of horses pull a combined 1,325 lbs (601 kg) of wagon and driver around a 5/8th mile (1 km) of dirt track, racing at full gallop to the finish. Blood samples were collected 30-min before the race start, and several timepoints post-race: 5-min, 90-min, 3-h, 12-h, and 23-h. Proteoglycan-4 concentrations in serum were quantified by enzyme-linked-immunosorbent-assay using recombinant-human proteoglycan-4 standards and anti-proteoglycan-4 mAb 9G3. The molecular weight of immunoreactive proteoglycan-4 in serum was assessed by western blot.Results: Proteoglyan-4 in serum demonstrated the expected high MW immunoreactivity to mAb 9G3, consistent with that of full length PRG4. Serum proteoglycan-4 decreased five-minutes post-race from baseline concentration (0.815 ± 0.175 to 0.466 ± 0.090 μg/mL, μ ± SEM, p < 0.01).Conclusions: The concentration of serum proteoglycan-4 in horses decreased significantly five min post-exercise. A potential explanation for this finding could be increased proteoglycan-4 clearance from the circulation. Further investigations could extend to complete the detailed characterization of proteoglycan-4 structure and its potential function within the blood as it relates to joint health and exercise.

Recombinant Human Proteoglycan-4 Mediates Interleukin-6 Response in Both Human and Mouse Endothelial Cells Induced Into a Sepsis Phenotype.

Sepsis is a leading cause of death in the United States. Putative targets to prevent systemic inflammatory response syndrome include antagonism of toll-like receptors 2 and 4 and CD44 receptors in vascular endothelial cells. Proteoglycan-4 is a mucinous glycoprotein that interacts with CD44 and toll-like receptor 4 resulting in a blockade of the NOD-like receptor pyrin domain-containing-3 pathway. We hypothesized that endothelial cells induced into a sepsis phenotype would have less interleukin-6 expression after recombinant human proteoglycan 4 treatment in vitro. Enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction to measure interleukin-6 protein and gene expression. Research laboratory. Human umbilical vascular endothelial cells, human lung microvascular endothelial cells, and transgenic mouse (wild type) (Cd44 +/+/Prg4 +/+), Cd44 -/- (Cd44 tm1Hbg Prg4 +/+), Prg4 GT/GT (Cd44 +/+ Prg4 tm2Mawa/J), and double knockout (Cd44 tm1Hbg Prg4 tm2Mawa/J) lung microvascular endothelial cells. Cells were treated with 100 or 250 ng/mL lipopolysaccharide-Escherichia coli K12 and subsequently treated with recombinant human proteoglycan 4 after 30 minutes. Interleukin-6 levels in conditioned media were measured via enzyme-linked immunosorbent assay and gene expression was measured via reverse transcriptase-quantitative polymerase chain reaction with ΔΔ-Ct analysis. Additionally, human umbilical vascular endothelial cells and human lung microvascular endothelial cells were treated with 1:10 diluted plasma from 15 patients with sepsis in culture media. After 30 minutes, either 50 or 100 µg/mL recombinant human proteoglycan 4 was administered. Interleukin-6 protein and gene expression were assayed. Proteoglycan 4 levels were also compared between control and sepsis patient plasma. Human umbilical vascular endothelial cell, human lung microvascular endothelial cell, and mouse lung microvascular endothelial cell treated with lipopolysaccharide had significantly increased interleukin-6 protein compared with controls. Recombinant human proteoglycan-4 significantly reduced interleukin-6 in human and mouse endothelial cells. Interleukin-6 gene expression was significantly increased after lipopolysaccharide treatment compared with controls. This response was reversed by 50 or 100 µg/mL recombinant human proteoglycan-4 in 80% of sepsis samples in human umbilical vascular endothelial cells and in 60-73% in human lung microvascular endothelial cells. In Cd44 -/- genotypes of the mouse lung microvascular endothelial cells, recombinant human proteoglycan-4 significantly reduced interleukin-6 protein levels after lipopolysaccharide treatment, indicating that Cd44 is not needed for recombinant human proteoglycan-4 to have an effect in a toll-like receptor 4 agonist inflammation model. Patient sepsis samples had higher plasma levels of native proteoglycan-4 than controls. Recombinant human proteoglycan-4 is a potential adjunct therapy for sepsis patients and warrants future in vivo model studies.

Proteoglycan 4 is Increased in Human Calcified Aortic Valves and Enhances Valvular Interstitial Cell Calcification.

Aortic valve stenosis (AVS), a consequence of increased fibrosis and calcification of the aortic valve leaflets, causes progressive narrowing of the aortic valve. Proteoglycans, structural components of the aortic valve, accumulate in regions with fibrosis and moderate calcification. Particularly, proteoglycan 4 (PRG4) has been identified in fibrotic parts of aortic valves. However, the role of PRG4 in the context of AVS and aortic valve calcification has not yet been determined. Here, transcriptomics, histology, and immunohistochemistry were performed in human aortic valves from patients undergoing aortic valve replacement. Human valve interstitial cells (VICs) were used for calcification experiments and RNA expression analysis. PRG4 was significantly upregulated in thickened and calcified regions of aortic valves compared with healthy regions. In addition, mRNA levels of PRG4 positively associated with mRNA for proteins involved in cardiovascular calcification. Treatment of VICs with recombinant human PRG4 enhanced phosphate-induced calcification and increased the mRNA expression of bone morphogenetic protein 2 and the runt-related transcription factor 2. In summary, PRG4 was upregulated in the development of AVS and promoted VIC osteogenic differentiation and calcification. These results suggest that an altered valve leaflet proteoglycan composition may play a role in the progression of AVS.

Localization of full-length recombinant human proteoglycan-4 in commercial contact lenses using confocal microscopy

The aim of this study was to determine the sorption location of full-length recombinant human proteoglycan 4 (rhPRG4) tagged with fluorescein isothiocyanate (FITC) to four silicone hydrogel contact lenses [balafilcon A (PureVision, Bausch + Lomb), senofilcon A (Acuvue Oasys, Johnson & Johnson), comfilcon A (Biofinity, CooperVision), lotrafilcon B (Air Optix, Alcon)] and one conventional hydrogel lens [etafilcon A (Acuvue 2, Johnson & Johnson)], using confocal laser scanning microscopy (CLSM). Lenses (n = 3 each) were incubated under two conditions: (1) FITC-rhPRG4 solution at 300 μg/mL and (2) phosphate-buffered saline, for 1 h at 37 °C in darkness with gentle shaking. The central 4 mm of each lens was removed and viewed with the Zeiss 510 CLSM using an argon laser at 488 nm (FITC excitation 495 nm, emission 521 nm). Depth scans were taken at 1 μm intervals to a maximum depth of 100 μm. All lens materials demonstrated sorption of rhPRG4. Both senofilcon A and balafilcon A revealed FITC-rhPRG4 penetration into the bulk of the lens, generally favoring the surface. rhPRG4 was observed exclusively on the surface of lotrafilcon B, with no presence within the bulk of the lens. rhPRG4 was evenly distributed throughout the bulk of the lens, as well as on the surface, for comfilcon A and etafilcon A. The sorption profile of FITC-rhPRG4 was successfully visualized using CLSM in various contact lens materials. The polymer composition, surface treatment and pore size of the material can influence the sorption of rhPRG4.

Recombinant human PRG4 (rhPRG4) suppresses breast cancer cell invasion by inhibiting TGFβ-Hyaluronan-CD44 signalling pathway.

Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an anti-invasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFβ) to induce phenotypic disruption of three-dimensional human breast cancer cell-derived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFβ-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFβ-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFβ has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFβ has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFβ actions. Finally, these findings point to rhPRG4’s potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer.

Absence of Proteoglycan 4 (Prg4) Leads to Increased Subchondral Bone Porosity Which Can Be Mitigated Through Intra-Articular Injection of PRG4.

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein important for joint health. Mice lacking Prg4 demonstrate degeneration of the cartilage and altered skeletal morphology. The purpose of this study was to examine if Prg4 deficiency leads to subchondral bone defects and if these defects could be mitigated through intra-articular injection of recombinant human PRG4 (rhPRG4). Mice deficient in Prg4 expression demonstrated increased cartilage thickness and increased subchondral bone porosity compared with C57BL/6 controls. While the porosity of the subchondral bone of Prg4-/- mice decreased over time with maturation, intra-articular injection of rhPRG4 was able to forestall the increase in porosity. In contrast, neither hyaluronan (HA) nor methylprednisolone injections had beneficial effects on the subchondral bone porosity in the Prg4 knockout mice. Bone marrow progenitor cells from Prg4-/- mice demonstrated reduced osteogenic differentiation capacity at 4 weeks of age, but not at 16 weeks of age. While most studies on PRG4/lubricin focus on the health of the cartilage, this study demonstrates that PRG4 plays a role in the maturation of the subchondral bone. Furthermore, increasing joint lubrication/viscosupplementation through injection of HA or controlling joint inflammation through injection of methylprednisolone may help maintain the cartilage surface, but had no positive effect on the subchondral bone in animals lacking Prg4. Therefore, alterations in the subchondral bone in models with absent or diminished Prg4 expression should not be overlooked when investigating changes within the articular cartilage regarding the pathogenesis of osteoarthritis/arthrosis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2077-2088, 2019.

Probing the Molecular Interactions and Lubrication Mechanisms of Purified Full-Length Recombinant Human Proteoglycan 4 (rhPRG4) and Hyaluronic Acid (HA).

Probing the adsorption and lubrication behavior of lubricin, also known as proteoglycan 4 (PRG4), is important for understanding the ultralow friction of cartilage lubrication. Most previous research has focused on native lubricin either purified from synovial fluid or articular cartilage explant culture media. In this work, the adsorption behavior and lubrication mechanism of full-length recombinant human PRG4 (rhPRG4) on mica as well as the effect of adding hyaluronic acid (HA, a polysaccharide) were systematically investigated using a surface forces apparatus (SFA) technique. A low friction coefficient (μ ∼ 0.04) was measured when multilayer rhPRG4 (∼31 nm) was confined in between mica surfaces, even when the load increased to ∼1.2 MPa. Intriguingly, a previously unreported ultralow friction coefficient (μ < 0.005) was observed at a low sliding velocity ( v = 0.14 μm/s) with the applied load P reaching ∼3.6 MPa when a diluted rhPRG4 solution (∼90 μg/mL) was used. The distinct friction behavior is likely due to the smooth and more close-packed lubricin coating, as made evident by the atomic force microscope imaging. Adding HA onto multilayer rhPRG4-coated mica increased the friction coefficient μ to ∼0.1; however, the load bearing property increased, indicating potential synergistic effect between rhPRG4 and HA, which was further demonstrated by the weak adhesion observed when separating rhPRG4-coated mica and HA-coated aminopropyltriethoxysilane-mica (APTES-mica). Alternatively, adding premixed rhPRG4-HA on mica had a friction coefficient (μ ∼ 0.1) close to that of injecting concentrated rhPRG4 (∼450 μg/mL) with lower load sustainability. Our results provide fundamental insights into the adsorption and lubrication behavior of lubricin and its interaction with HA, with useful implications for the underlying mechanism of ultralow friction provided by synovial fluid.

Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

BackgroundGout is an inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystals’ joint deposition. MSU phagocytosis by resident macrophages is a key step in gout pathogenesis. MSU phagocytosis triggers nuclear factor kappa B (NFκB) activation and production of cytokines and chemokines. Proteoglycan-4 (PRG4) is a glycoprotein produced by synovial fibroblasts and exerts an anti-inflammatory effect in the joint mediated by its interaction with cell surface receptor CD44. PRG4 also binds and antagonizes TLR2 and TLR4. The objective of this study is to evaluate the efficacy of recombinant human PRG4 (rhPRG4) in suppressing MSU-induced inflammation and mechanical allodynia in vitro and in vivo.MethodsTHP-1 macrophages were incubated with MSU crystals ± rhPRG4 or bovine submaxillary mucin (BSM), and crystal phagocytosis, cytokines and chemokines expression and production were determined. NFκB p65 subunit nuclear translocation, NLRP3 induction, caspase-1 activation and conversion of proIL-1β to mature IL-1β were studied. MSU phagocytosis by Prg4+/+ and Prg4−/− peritoneal macrophages was determined in the absence or presence of rhPRG4, BSM, anti-CD44, anti-TLR2, anti-TLR4 and isotype control antibodies. Rhodamine-labeled rhPRG4 was incubated with murine macrophages and receptor colocalization studies were performed. Lewis rats underwent intra-articular injection of MSU crystals followed by intra-articular treatment with PBS or rhPRG4. Weight bearing and SF myeloperoxidase activities were determined.ResultsrhPRG4 reduced MSU crystal phagocytosis at 4 h (p < 0.01) and IL-1β, TNF-α, IL-8 and MCP-1 expression and production at 6 h (p < 0.05). BSM did not alter MSU phagocytosis or IL-1β production in human and murine macrophages. rhPRG4 treatment reduced NFκB nuclear translocation, NLRP3 expression, caspase-1 activation and generation of mature IL-1β (p < 0.05). MSU-stimulated IL-1β production was higher in Prg4−/− macrophages compared to Prg4+/+ macrophages (p < 0.001). rhPRG4, anti-CD44, anti-TLR2 and anti-TLR4 antibody treatments reduced MSU phagocytosis and IL-1β production in murine macrophages (p < 0.05). rhPRG4 preferentially colocalized with CD44 on Prg4−/− peritoneal macrophages compared to TLR2 or TLR4 (p < 0.01). rhPRG4 normalized weight bearing and reduced SF myeloperoxidase activity compared to PBS in vivo.ConclusionrhPRG4 inhibits MSU crystal phagocytosis and exhibits an anti-inflammatory and anti-nociceptive activity in vitro and in vivo. rhPRG4’s anti-inflammatory mechanism may be due to targeting CD44 on macrophages.

Surface-Functionalized Model Contact Lenses with a Bioinspired Proteoglycan 4 (PRG4)-Grafted Layer.

Ocular dryness and discomfort are the primary reasons for the discontinuation of contact lens wear. This is mainly due to poorly hydrated contact lens surfaces and increased friction, particularly at the end of the day and can potentially cause reduced vision or even inflammation. Proteoglycan 4 (PRG4) is a mucinous glycoprotein with boundary lubricating properties, naturally found in the eye, able to prevent tear film evaporation and protect the ocular surface during blinking. Aiming to improve the interfacial interactions between the ocular surface and the contact lens, the synthesis and characterization of surface-modified model contact lenses with PRG4 is described. Full-length recombinant human PRG4 (rhPRG4) was successfully grafted onto the surface of model conventional and silicone hydrogel (SiHy) contact lenses via its somatomedin B-like end-domain using N, N'-carbonyldiimidazole linking chemistry. Grafting was assessed by Fourier transform infrared spectroscopy-attenuated total reflectance, X-ray photoelectron spectroscopy, and radioactive (I131) labeling. Surface immobilization of rhPRG4 led to model conventional and SiHy materials with improved antifouling properties, without impacting optical transparency or causing any toxic effects to human corneal epithelial cells in vitro. The surface wettability and the boundary friction against human corneal tissue were found to be substrate-dependent, with only the rhPRG4-grafted model SiHy exhibiting a reduced contact angle and kinetic friction coefficient compared to the unmodified surfaces. Hence, clinical grade rhPRG4 can be an attractive candidate for the development of novel bioinspired SiHy contact lenses, providing improved comfort and overall lens performance.

Degradation of proteoglycan 4/lubricin by cathepsin S: Potential mechanism for diminished ocular surface lubrication in Sjögren's syndrome

Sjögren's syndrome (SS) is an autoimmune disease affecting the lacrimal and salivary glands with hallmark clinical symptoms of dry eye and dry mouth. Recently, markedly increased cathepsin S (CTSS) activity has been observed in the tears of SS patients. Proteoglycan 4 (PRG4), also known as lubricin, is an effective boundary lubricant that is naturally present on the ocular surface. While PRG4 is susceptible to proteolytic digestion, the potential effect of CTSS on PRG4 remains unknown. The objective of this study was to assess the ability of CTSS to enzymatically degrade purified PRG4, and PRG4 naturally present in human tears, and alter ocular surface boundary lubricating properties. To assess the potential time course and dose-dependency of PRG4 digestion by CTSS, full-length recombinant human PRG4 (rhPRG4) was incubated at 37 °C with or without CTSS in an enzymatic digestion buffer. Digestion of PRG4 by CTSS was also examined within normal human tear samples, both with and without supplementation by rhPRG4. Finally, digestion of endogenous PRG4 by CTSS, and the effect of a CTSS inhibitor, was examined in SS tears on Schirmer strips. Digestion products were separated on 3–8% SDS-PAGE and visualized by protein staining and western blotting. The boundary lubricating ability of rhPRG4 samples was assessed using an in vitro human eyelid-cornea friction test. Finally, SDS-PAGE protein stain bands resulting from rhPRG4 digestion were submitted for tandem mass spectrometry analysis to confirm their identity as PRG4 and identify non-tryptic cleavage sites. CTSS digested rhPRG4 in a time and dose dependent manner. CTSS digestion of rhPRG4 at 1% (where % is the mass ratio of CTSS to rhPRG4) resulted in a time dependent decrease in the full-length, ∼460 kDa, monomeric rhPRG4 band, and an appearance of lower MW fragments. After 20 h, no full-length rhPRG4 was observed. Furthermore, with an increased relative enzyme concentration of 3%, no protein bands were observed after 2 h, indicating complete digestion of rhPRG4. Western blotting demonstrated PRG4 is present in normal human tears, and that rhPRG4, tears, and tears supplemented with rhPRG4 incubated with 3–9% CTSS demonstrated decreased intensity of high MW PRG4 bands, indicative of partial degradation by CTSS. Similarly, western blotting of PRG4 in SS tears incubated with CTSS demonstrated decreased intensity of high MW PRG4 bands, which was reversed in the presence of the CTSS inhibitor. CTSS treatment of rhPRG4 resulted in an increased friction coefficient, compared to untreated controls. Lastly, the lower MW bands were confirmed to be PRG4 fragments by tandem mass spectrometry, and 6 non-tryptic cleavage sites were identified. rhPRG4 is susceptible to proteolytic digestion by CTSS, both alone and in human tears, which results in diminished ocular surface boundary lubricating ability. Moreover, endogenous PRG4 is susceptible to proteolytic digestion by CTSS, both in normal and SS tears. Given the elevated activity of CTSS in SS tears, and the role intact PRG4 plays in ocular surface health and lubrication, degradation of PRG4 by CTSS is a potential mechanism for diminished ocular surface lubrication in SS. Collectively these results suggest that tear supplementation of PRG4 may be beneficial for SS patients.

The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes

BackgroundLubricin/proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. Recently, we showed that recombinant human PRG4 (rhPRG4) is a putative ligand for CD44 receptor. rhPRG4-CD44 interaction inhibits cytokine-induced rheumatoid arthritis synoviocyte proliferation. The objective of this study is to decipher the autocrine function of PRG4 in regulating osteoarthritic synoviocyte proliferation and expression of catabolic and pro-inflammatory mediators under basal and interleukin-1 beta (IL-1β)-stimulated conditions.MethodsCytosolic and nuclear levels of nuclear factor kappa B (NFκB) p50 and p65 subunits in Prg4+/+ and Prg4-/- synoviocytes were studied using western blot. Nuclear translocation of p50 and p65 proteins in osteoarthritis (OA) fibroblast-like synoviocytes (FLS) in response to IL-1β stimulation in the absence or presence of rhPRG4 was studied using DNA binding assays. OA synoviocyte (5000 cells per well) proliferation following IL-1β (20 ng/ml) treatment in the absence or presence of rhPRG4 (50–200 μg/ml) over 48 hours was determined using a colorimetric assay. Gene expression of matrix metalloproteinases (MMPs), tissue inhibitor of metallproteinases-1 (TIMP-1), TIMP-2, IL-1β, IL-6, IL-8, TNF-α, cycloxygenae-2 (COX2) and PRG4 in unstimulated and IL-1β (1 ng/ml)-stimulated OA synoviocytes, in the presence or absence of rhPRG4 (100 and 200 μg/ml), was studied following incubation for 24 hours.ResultsPrg4-/- synoviocytes contained higher nuclear p50 and p65 levels compared to Prg4+/+ synoviocytes (p < 0.05). rhPRG4 (100 μg/ml) reduced p50 and p65 nuclear levels in Prg4+/+ and Prg4-/- synoviocytes (p < 0.001). Similarly, rhPRG4 (200 μg/ml) inhibited NFκB translocation and cell proliferation in OA synoviocytes in a CD44-dependent manner (p < 0.001) via inhibition of IκBα phosphorylation. IL-1β reduced PRG4 expression in OA synoviocytes and rhPRG4 (100 μg/ml) treatment reversed this effect (p < 0.001). rhPRG4 (200 μg/ml) reduced basal gene expression of MMP-1, MMP-3, MMP-13, IL-6, IL-8, and PRG4 in OA synoviocytes, while increasing TIMP-2 and cycloxygenase-2 (COX2) expression (p < 0.001). rhPRG4 (200 μg/ml) reduced IL-1β induction of MMP-1, MMP-3, MMP-9, MMP-13, IL-6, IL-8, and COX2 expression in a CD44-dependent manner (p < 0.001).ConclusionPRG4 plays an important anti-inflammatory role in regulating OA synoviocyte proliferation and reduces basal and IL-1β-stimulated expression of catabolic mediators. Exogenous rhPRG4 autoregulates native PRG4 expression in OA synoviocytes.

Full-Length Recombinant Human Proteoglycan 4 Interacts with Hyaluronan to Provide Cartilage Boundary Lubrication.

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid and at the surface of articular cartilage. The objectives of this study were to (1) assess the articular cartilage surface adsorption and in vitro cartilage boundary lubricating ability of full-length recombinant human PRG4 (rhPRG4), and (2) cartilage boundary lubricating ability of purified rhPRG4, both alone and in combination with hyaluronan (HA). rhPRG4 adsorption onto articular cartilage explants was assessed by immunohistochemistry and dot blot. An in vitro cartilage-cartilage friction test was used to assess rhPRG4's cartilage boundary lubricating ability compared to bovine PRG4, and that of purified rhPRG4 both alone and in combination with HA. rhPRG4 was able to adsorb to the articular surface, as well as the cut surface, of cartilage explants. The kinetic coefficient of friction of rhPRG4 was similar to that of PRG4 (p = 0.16) and lower than phosphate-buffered saline (p < 0.05), while that of purified rhPRG4 + HA was significantly lower than rhPRG4 alone (p < 0.05). This study demonstrates that rhPRG4 can adsorb to an intact articular cartilage surface and functions as an effective boundary lubricant, both alone and with HA, and provides the foundation for in vivo evaluation of this clinically relevant full-length rhPRG4 for treatment of osteoarthritis.

Effects of concentration and structure on proteoglycan 4 rheology and interaction with hyaluronan.

The contribution of proteoglycan 4 (PRG4) to synovial fluid and hyaluronan (HA) solution rheology are poorly understood. The effects of PRG4 disulfide-bonded structure on viscosity and viscosity of newly available full-length recombinant human PRG4 (rhPRG4) have not previously been reported. This study determined the viscosity of PRG4 and rhPRG4, R/A (reduced and alkylated) PRG4 and rhPRG4, and PRG4 and rhPRG4+HA solutions. Steady shear viscosities of 1.5 MDa HA, PRG4 from bovine cartilage explant culture, rhPRG4 and (rh)PRG4+HA solutions were measured with 40 mm parallel plate fixtures. PRG4 demonstrated shear-dependent viscosity at high concentrations, but Newtonian behaviour at low concentrations and when disulfide-bonded/multimeric structure was disrupted by R/A. rhPRG4 demonstrated Newtonian behaviour over all concentrations tested and upon R/A. At high HA concentrations, rhPRG4 reduced solution viscosity, suggesting a binding interaction. At low HA concentrations, solution viscosity was increased relative to HA alone, possibly due to self-association of rhPRG4. Effects of PRG4 on HA solution viscosity were dependent on PRG4's disulfide-bonded structure. The finding that rhPRG4 can increase the viscosity of low concentration HA solutions suggests that supplementation with rhPRG4 may help mitigate the loss in synovial fluid viscosity experienced with decreased HA concentration in osteoarthritis.