Increased expression of osteonectin/SPARC mRNA and protein in age-related human cataracts and spatial expression in the normal human lens

Abstract

Purpose: We have previously reported increased levels of Osteonectin/SPARC transcript in age-related cataractous compared to normal human lenses. The purpose of the present study was to evaluate the corresponding levels of osteonectin/SPARC protein in age-related cataractous relative to normal lenses and to evaluate the levels of osteonectin/SPARC transcript in specific types of age-related human cataracts. The spatial expression of osteonectin/SPARC was also evaluated in normal human lenses.

Methods: Specific types of age-related cataracts were collected and graded. Normal human lenses were microdissected into epithelia and fibers. Osteonectin/SPARC protein levels were monitored by Western immunoblotting, and transcript levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Osteonectin/SPARC expression patterns were examined by RT-PCR and by immunostaining.

Results: Higher levels of osteonectin/SPARC protein were detected in age-related cataractous relative to normal human lenses. Increased levels of osteonectin/SPARC transcript were also detected in posterior-subcapsular and nuclear cataractous lenses relative to normal lenses. Osteonectin/SPARC transcripts were detected in the lens epithelium but not fibers. Osteonectin/SPARC protein levels were highest in the peripheral lens epithelium.

Conclusions: Consistent with our previous studies on osteonectin/SPARC mRNA levels, osteonectin/SPARC protein levels were also elevated in cataractous compared to normal human lenses. Increased levels of osteonectin/SPARC mRNA were also found in nuclear and posterior-subcapsular cataracts relative to normal lenses. Osteonectin/SPARC expression is confined to the lens epithelium, and osteonectin/SPARC levels are highest in the peripheral lens epithelium.

Figure 1

Western immunoblotting of osteonectin/SPARC in lens extracts. Extracts were prepared and polypeptides were separated and blotted as described in Materials and Methods. Shown is the autoradiogram of the corresponding blot. Lane 1 contains purified osteonectin/SPARC, lane 2 contains cataractous lens extract (cat), and lane 3 contains normal lens extract (norm) probed with anti-SPARC antibody. Lane 4 contains cataractous lens extract (cat) and lane 5 contains normal lens extract (norm) probed with anti-αB-crystallin antibody. Also shown are the osteonectin/SPARC and the αB-crystallin bands with their corresponding molecular weights. The protein added to each well is shown below the blot.

Figure 2

RT-PCR analysis of osteonectin/SPARC in specific cataracts. Ethidium bromide stained gel showing the levels of osteonectin/SPARC and GAPDH transcripts after 30 PCR cycles in nuclear cataracts (lane 1), posterior subcapsular cataracts (lane 2), and normal lenses (lane 3). The osteonectin/SPARC (419 bp) and GAPDH (600 bp) bands and the number of lenses used for each sample are indicated.

Figure 3

RT-PCR analysis of osteonectin/SPARC in specific cataracts. Ethidium bromide stained gel showing the levels of osteonectin/SPARC and GAPDH in microdissected normal lens epithelia (0.5 μg) and fiber cells (0.5 μg) after 35 PCR cycles. GAPDH transcript (600 bp) levels for lens epithelia (Epi) and fiber cells (Fib) are shown in lanes 1 and 2, respectively. Osteonectin/SPARC transcript (419 bp) levels for lens epithelia (Epi) and fiber cells (Fib) are shown in lanes 3 and 4, respectively.

Figure 4

Immunostaining of human lens with anti-osteonectin/SPARC. A normal lens epithelium examined with (A) anti-osteonectin/SPARC antibody, (B) anti-αB-crystallin antibody, and (C) pre-immune serum. The central lens capsule (CC), central lens epithelium (CE), peripheral lens capsule (PC), and peripheral lens epithelium (PE) are marked with arrows.