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Laboratory Sciences |

Transcription, Translation, and Function of Lubricin, a Boundary Lubricant, at the Ocular Surface

Tannin A. Schmidt, PhD; David A. Sullivan, PhD; Erich Knop, MD, PhD; Stephen M. Richards, ALM; Nadja Knop, MD, PhD; Shaohui Liu, MD, PhD; Afsun Sahin, MD; Raheleh Rahimi Darabad, MD; Sheila Morrison, BSc; Wendy R. Kam, MSc; Benjamin D. Sullivan, PhD
JAMA Ophthalmol. 2013;131(6):766-776. doi:10.1001/jamaophthalmol.2013.2385.
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Importance Lubricin may be an important barrier to the development of corneal and conjunctival epitheliopathies that may occur in dry eye disease and contact lens wear.

Objective To test the hypotheses that lubricin (ie, proteoglycan 4 [PRG4 ]), a boundary lubricant, is produced by ocular surface epithelia and acts to protect the cornea and conjunctiva against significant shear forces generated during an eyelid blink and that lubricin deficiency increases shear stress on the ocular surface and promotes corneal damage.

Design, Setting, and Participants Human, porcine, and mouse tissues and cells were processed for molecular biological, immunohistochemical, and tribological studies, and wild-type and PRG4 knockout mice were evaluated for corneal damage.

Results Our findings demonstrate that lubricin is transcribed and translated by corneal and conjunctival epithelial cells. Lubricin messenger RNA is also present in lacrimal and meibomian glands, as well as in a number of other tissues. Absence of lubricin in PRG4 knockout mice is associated with a significant increase in corneal fluorescein staining. Our studies also show that lubricin functions as an effective friction-lowering boundary lubricant at the human cornea-eyelid interface. This effect is specific and cannot be duplicated by the use of hyaluronate or bovine serum albumin solutions.

Conclusions and Relevance Our results show that lubricin is transcribed, translated, and expressed by ocular surface epithelia. Moreover, our findings demonstrate that lubricin presence significantly reduces friction between the cornea and conjunctiva and that lubricin deficiency may play a role in promoting corneal damage.

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Figure 1. Identification of lubricin messenger RNA (mRNA) in human and mouse ocular tissues and cells by reverse transcriptase–polymerase chain reaction. A, RNA samples in lanes 1 through 4 are a human liver standard, a corneoscleral rim of a 24-year-old woman, a corneoscleral rim of a 51-year-old woman, and immortalized human conjunctival epithelial cells. The anticipated amplicon size of the human lubricin transcript was 526 base pairs (bp), and all bands fell within the 10% error of the Bioanalyzer (2100; Agilent Technologies) when using a 1000-bp sizing kit (internal controls = 15 and 1500 bp). Samples were sequenced and shown to be lubricin. Analogous bands were also found in RNAs prepared from the corneoscleral rim of a 64-year old man, pools of 3 male and 3 female conjunctival tissues, primary cultures of human male and female corneal epithelial cells, and immortalized human corneal and meibomian gland epithelial cells (data not shown). L indicates ladder. B, Presence of lubricin mRNA in impression cytology samples from the human ocular surface. Samples in lanes 1 through 9 are from a 29-year-old man, 31-year-old man, 59-year-old woman, 36-year-old woman, 24-year-old woman, 29-year-old woman, 37-year-old woman, 27-year-old man, and 35-year-old woman, respectively. The RNA in lane 10 was from the conjunctival epithelial cells shown in part A and was used here as a positive control. C, Identification of lubricin mRNA in mouse lacrimal and meibomian glands. Samples in lanes 1 through 5 are a no template control, female NOD/LtJ mouse lacrimal glands, male NOD/LtJ mouse lacrimal glands, female BALB/c mouse meibomian glands, and male BALB/c mouse meibomian glands. The bands in these lanes are equivalent to the anticipated 367-bp size. The NOD/LtJ mouse lacrimal glands were obtained from 5 adult mice of each sex (ie, 10 glands per sample), and the BALB/c mouse meibomian glands were isolated from the eyelids of 7 adult mice of each sex (ie, 28 eyelids/sample).

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Figure 2. Lubricin protein expression in human ocular surface epithelia and cartilage. Immunohistochemical evaluation for lubricin (A-C) shows a strong staining of the ocular surface epithelia of the cornea and the palpebral (eyelid) and bulbar (eyeball) conjunctivae. In the conjunctiva, the goblet cells showed less intense or absent staining and hence appeared as bright spots (arrowhead in C, inset). Negative controls with omission of antibody (diluent control) (D-F) are completely negative and normal rabbit IgG is almost completely negative (G-I). An irrelevant rabbit antibody (anti-CD8) (J-L) shows no specific lubricin staining. A, D, G, and J, Overview of the ocular surface with the cornea and palpebral and bulbar conjunctiva (original magnification ×2.5). B, E, H, and K, Cornea (original magnification ×20). C, F, I, and L, Conjunctiva (original magnification ×10). Strong staining similar to that at the ocular surface is seen in the positive control tissue (cartilage) with a known presence of lubricin protein (M-O) (same enlargements). Lubricin staining is seen at the surface of the cartilage and at the bone-cartilage junction. Chondrocytes are marked by arrows and the lubricin depositions in the cartilage matrix are marked by arrowheads (N and O). The size markers indicate either 1000 μm (A, D, G, J, and M) or 100 μm.

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Figure 3. Specificity of lubricin staining in human ocular surface epithelia. The strong lubricin staining (A-C) in the cornea and conjunctiva was distinctly reduced after preincubation of the lubricin antibody with a sense peptide (D-F). In contrast, preadsorption to a random peptide (nonsense) did not affect the staining (G-I). The apparent staining of artificial folds in the cornea (A, D, and G) is a technical artifact, due to the entrapment of reagents. A, D, and G, Overview of the ocular surface with the cornea and palpebral and bulbar conjunctivae (original magnification ×2.5). B, E, and H, Cornea (original magnification ×20). C, F, and I, Conjunctiva (original magnification ×10). The size markers indicate either 1000 μm (A, D, and G) or 100 μm.

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Figure 4. Specificity of lubricin staining in human cartilage. A section of articular cartilage is seen in low magnification (A, D, and G) as well as in higher magnification at the surface (B, E, and H) and bone-cartilage junction (C, F, and I). Lubricin staining (A-C) was blocked by antibody preincubation with the sense (D-F), but not a random (G-I), peptide. Lubricin staining at the cartilage surface forms a thin surface layer (arrowheads), and narrow flattened chrondrocytes may be seen (arrows). At the bone-cartilage junction, chondrocyte lacunae (arrows) occur as bright spaces that are encircled by lubricin (arrowheads). The overview image for the sense peptide preincubation (D) has superimposed areas of bone marrow that spilled over the section during the preparation process. Magnification of the area indicated by a rectangle shows that this material is equally present over and outside the surface and the staining is not specific. Size markers indicate 1000 μm (A, D, and G) or 100 μm.

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Figure 5. Identification of lubricin protein in porcine corneal epithelium and bovine synovial fluid (SF) by Western blot. Lanes contain molecular weight markers; blots shown with (LPN) and without (−) exposure to antilubricin antibody (Ab). Arrows indicate lubricin protein.

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Figure 6. Effect of lubricin deficiency on the pattern of corneal fluorescein staining. A, Columns and vertical bars equal the mean (standard error) of the degree of staining on the left, right, or both corneas. Staining was significantly (* P < .05 and † P < .05, 1-tailed) greater in the knockout than the wild-type mice. B, Example of fluorescein staining of the wild-type mouse ocular surface. Relatively little damage is evident, marked by superficial stippling and occasional micropunctate staining. C, Example of fluorescein staining of the lubricin PRG4 −/− knockout mouse ocular surface. Numerous macropunctate coalescent areas and patches of contiguous staining are evident. Photographs (original magnification ×1.6) were taken after instillation of fluorescein drops into the inferior conjunctival sac. The degree of staining in these images of the superior, medial, inferior, lateral, and central regions of the cornea were all 1 in the wild-type mice (total score = 5) and 2, 3, 1, 2, and 1, respectively, in the knockout mice (total score = 9).

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Figure 7. Effects of lubricin and sliding velocity on static and kinetic friction at a human cornea-eyelid interface. A, Tissue preparation. An annulus (outer radius = 3.2 mm, inner radius = 1.5 mm) was punched from the eyelid, then attached to a nonpermeable, rigid plastic annular cylinder. B, Test setup. The corneal ocular surface was fastened to the spherical end of an inert nonpermeable semirigid rubber plug cylinder (radius = 6 mm) by applying superglue to the sclera. This plug was attached to the rotational actuator of the BOSE ELF 3200 mechanical testing machine, thus forming the bottom articular surface. The annulus was attached to the linear actuator coupled with an axial load (N) and torsion (τ) load cell, thus forming the upper articulating surface. The lubricant bath was formed by securing an inert tube around the cylinder. C, Static friction of saline, AQuify Long-Lasting Comfort Drops (which contain hyaluronic acid, 0.1%; CIBA Vision), and lubricin. D, Static friction of saline and bovine serum albumin (BSA). E, Kinetic friction of saline, AQuify, and lubricin. F, Kinetic friction of saline and BSA. Lubricin and BSA were tested at 300 μg/mL. Values are the mean (standard error) of 6 (C and E) and 3 (D and F) tests. Values of μStatic, Neq were greatest in saline and similar in AQuify, whereas values in lubricin were statistically lower than those in both saline and AQuify at all effective sliding velocities (veff) (P < .001- P < .05). Values of < μKinetic, Neq > in lubricin were lowest and statistically different from saline at all veff (P < .001- P < .01) and from AQuify at the lower veff of 1 and 0.3 mm/s (both P < .01), where a boundary mode of lubrication is more operative, yet not at the higher veff of 30 and 10 mm/s (P = .33 and .06, respectively). The friction-lowering effect of lubricin appeared to be specific, because BSA did not reduce friction compared with saline.

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