Activation of metallothioneins and alpha-crystallin/sHSPs in human lens epithelial cells by specific metals and the metal content of aging clear human lenses

Abstract

Purpose: To identify those metallothionein and alpha-crystallin/small heat-shock genes induced by toxic metals in human lens cells and to evaluate the levels of these metals between young and aged human lenses.

Methods: Human SRA01/04 and primary human lens epithelial cells were cultured and exposed to Cd(2+), Cu(2+), and Zn(2+). The levels of lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock (alphaA-crystallin, alphaB-crystallin, and HSP27) genes were analyzed by semiquantitative and quantitative competitive RT-PCR. The content of aluminum, cadmium, calcium, chromium, copper, iron, lead, magnesium, manganese, nickel, potassium, sodium, and zinc in young (mean, 32.8 years), middle-aged (mean, 52.3 years), and old (mean, 70.5 years) human lenses was analyzed by inductively coupled plasma-emission spectroscopy.

Results: Lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock genes (alphaA-crystallin, alphaB-crystallin, and HSP27) were differentially induced by specific metals in SRA01/04 human lens epithelial cells. Cd(2+) and Zn(2+), but not Cu(2+), induced the metallothioneins, whereas Cd(2+) and Cu(2+), but not Zn(2+), induced alphaB-crystallin and HSP27. alphaA-crystallin was induced by Cu(2+) only. Similar responses of the metallothionein IIa gene were detected in identically treated primary human lens epithelial cells. Cd(2+) and Zn(2+) induced metallothionein IIa to five times higher levels than metallothionein Ig. Of 13 different metals, only iron was altered, exhibiting an 81% decrease in old versus young lenses.

Conclusions: Induction of metallothioneins and alpha-crystallin/small heat shock proteins by different metals indicates the presence of metal-specific lens regulatory pathways that are likely to be involved in protection against metal-associated stresses.

Figure 1

Schematic representation of the MTIIa mimic cDNA (A). A 138-bp internal sequence (+18 to +155 bp from the start of translation) was deleted from the MTIIa cDNA to create the 99-bp MTIIa mimic cDNA. Shown for comparison is the full-length 237-bp MTIIa cDNA. Schematic representation of the MTIg mimic cDNA (B). A 118-bp internal sequence (+18 to +135 base pairs, from the start of translation) was deleted from the MTIg cDNA to create the 99-bp MTIg mimic cDNA. Shown for comparison is the full-length 217-bp MTIg cDNA. Indicated are the primer binding sites.

Figure 2

Cell toxicity resulting from Cd²⁺, Cu²⁺, and Zn²⁺ treatment of HLE cells at the metal concentrations shown below the metals (in micromolar). HLE cells were treated with indicated metals for 8 hours, and cell death was examined by trypan blue exclusion. Data are the mean ± SD of results in three separate experiments.

Figure 3

Ethidium bromide–stained gels showing quantitative competitive RT-PCR analysis of MTIIa induced after 8 hours of Cd²⁺, Cu²⁺, or Zn²⁺ treatment. RNA (300 ng) was amplified in the presence of increasing amounts (0–500 pg) of competing MTIIa mimic DNA. Indicated are the metal concentrations, the 237-bp MTIIa cDNA, the 99-bp MTIIa mimic DNA PCR products, and the calculated radioactivity incorporated in each PCR product.

Figure 4

Ethidium bromide–stained gels showing quantitative competitive RT-PCR analysis of MTIg induced after 8 hours of Cd²⁺, Cu²⁺, or Zn²⁺ treatment, in conditions identical with those described in Figure 3. Indicated are the metal concentrations, the 217-bp MTIg cDNA, and the 99-bp MTIg mimic PCR products.

Figure 5

Ethidium bromide–stained gel showing the levels of MTIIa detected by RT-PCR in 50 ng RNA isolated from primary HLE cells induced by Cd²⁺, Cu²⁺, and Zn²⁺ for 8 hours at the indicated concentrations for a total of 28 PCR cycles. Shown as the control are the corresponding GAPDH levels.

Figure 6

Ethidium bromide–stained gels showing the levels of metallothionein (left) and small heat-shock (right) genes detected by RT-PCR in 100 ng of RNA isolated from HLE cells induced by Cd²⁺, Cu²⁺, and Zn²⁺ for 8 hours at the indicated concentrations.