Cataract- and lens-specific upregulation of ARK receptor tyrosine kinase in Emory mouse cataract

Abstract

Purpose: The Emory mouse is a well-characterized model for age-onset cataract. The purpose of the present study was to identify differentially expressed genes between pre- and postcataract Emory mouse lenses.

Methods: Eyes were extracted from Emory mice at 3 weeks (precataract) and 7.5 months (postcataract) of age, and lenses were dissected. Lens RNA was compared for gene expression differences by RT-PCR differential display, and transcripts exhibiting altered levels of gene expression were cloned and identified by sequencing. The levels of two transcripts were further evaluated by RT-PCR in 3-week- and 7.5-month-old lenses and the remainder of the eye. The same transcripts were also measured in lenses from three non-Emory mouse strains (FVB/N, 129Sv, and CD1) ages 4 weeks to 11.5 months.

Results: Three transcripts were identified as exhibiting altered levels of gene expression between 3-week- and 7.5-month-old Emory mouse lenses. These encoded alphaA-crystallin (decreased), betaA3/A1-crystallin (decreased), and adhesion-related kinase (ARK) receptor tyrosine kinase (increased). Decreased alphaA-crystallin and increased ARK expression were not detected in lenses isolated from three non-Emory mouse strains of similar age. Increased expression of ARK was not detected between 3-week- and 7.5-month-old Emory mouse eye nonlens tissues.

Conclusions: The present data confirm that expression of the alphaA-crystallin gene is decreased in cataract in the Emory mouse lens relative to age-matched control lenses and they provide evidence for cataract- and lens-specific upregulation of the ARK receptor tyrosine kinase in the Emory mouse.

Figures

Figure 1

Photographs of 3-week-and 7.5-month-old Emory mouse lenses. (A) An Emory mouse lens at 3 weeks, showing no evidence of cataract. (B) An Emory mouse lens at 7.5 months, showing obvious signs of opacity.

Figure 2

RT-PCR differential display profile of 7.5-month-old (7.5 M) versus 3-week-old (3 Wk) Emory mouse lenses. The autoradiogram was obtained by differential display of 7.5-month-old (24 pooled) versus 3-week-old (20 pooled) Emory mouse lenses. Lanes 1 to 3: 400, 200, and 150 ng of 7.5-month-old Emory mouse RNA, respectively; lanes 4 to 6: 100 ng each of 3-week-old Emory mouse RNA. Arrows: bands chosen for further analysis. Cloning and sequencing of these bands identified them as βA3/A1-crystallin (bands 1, 2), αA-crystallin (band 3), and ARK (band 4). Approximate sizes are indicated.

Figure 3

Confirmation of differential ARK and αA-crystallin transcript levels in 3-week-old (3 Wk) and 7.5-month-old (7.5 M) Emory mouse lenses. Ethidium bromide–stained gels show the transcript levels detected by RT-PCR, using 43 ng of Emory mouse lens RNA at the indicated number of PCR cycles. (A) Ethidium bromide–stained gels show levels of ARK transcript obtained using primer set 1 (Table 1). (B) Levels of αA-crystallin transcript obtained with the same RNAs.

Figure 4

Reconfirmation of differential ARK and αA-crystallin transcript levels in 3-week-old (3 Wk) and 7.5-month-old (7.5 M) Emory mouse lenses. Ethidium bromide–stained gels show the transcript levels detected by RT-PCR, using 100 ng of Emory mouse lens RNA at the indicated number of PCR cycles. (A) Levels of ARK transcript obtained using primer set 2 (Table 1). (B) Levels of αA-crystallin transcript obtained with the same RNAs.

Figure 5

ARK levels in 3-week-old (3 Wk) and 7.5-month-old (7.5 M) Emory mouse lenses and the remainder of the eye minus the lens. Ethidium bromide–stained gels show the transcript levels detected by RT-PCR, using the indicated amounts of RNA obtained from Emory mouse lens (A) or the remainder of the eye minus the lens (B).

Figure 6

Expression levels of ARK and αA-crystallin in Emory mouse versus non–Emory mouse lenses. Ethidium bromide-stained gels show transcript levels detected by RT-PCR, using 100 ng RNA isolated from the lenses of Emory and non-Emory mouse strains at indicated ages. W, week; M, month.