Expression of integrin-linked kinase in the murine lens is consistent with its role in epithelial-mesenchymal transition of lens epithelial cells in vitro

Abstract

Purpose: To evaluate the expression and location of integrin-linked kinase (ILK) within the mouse lens and to characterize the role of this protein during mouse lens epithelial cells (LEC) differentiation in vitro.

Methods: Transcription levels of ILK mRNA were determined by RT-PCR in cultured cells and lens tissue. ILK protein was detected by immunoblotting, immunocytochemistry, immunohistochemistry, and immunoprecipitation. A role for ILK in the outgrowth of LEC from dissected mouse lens explants was determined by the use of ILK short interfering RNA (siRNA). Affinity-purified polyclonal anti-recombinant human ILK IgG was prepared and characterized for these experiments. A comparison of several anti-ILK antibodies was performed by immunoblotting, immunoprecipitation, and ELISA.

Results: ILK was transcribed in LEC and lens fiber cells in vivo. ILK protein was expressed in the differentiating LEC at the equatorial region of the lens and, to a lesser extent, within the cortical and nuclear fiber cells. LEC in vitro produced copious ILK, which exhibited a filamentous pattern throughout the cytoplasm. The expression of ILK was increased during epithelial-mesenchymal-transition (EMT) of LEC from lens explants, whereas inhibition of ILK by siRNA delayed expression of the EMT markers smooth muscle alpha-actin and fibronectin.

Conclusions: Analysis of ILK expression, localization, and activity in the mouse lens and cultured LEC is substantially facilitated by the generation of a multi-functional, polyclonal, affinity-purified anti-ILK antibody. Expressed in most tissues and cells lines, ILK is unexpectedly restricted to the equatorial LEC and differentiated fiber cells of the mouse lens. The occurrence of ILK expression with LEC differentiation is consistent with the positive regulatory function of ILK, which is revealed in a model of EMT in vitro. This is the first study to show the expression of ILK in the lens and its unique distribution pattern within cultured lens epithelia.

Figure 1

Characterization of polyclonal ILK antibody. A: Purified rhILK used for antigen in rabbits. Lane 1, molecular weight standards; lane 2, 5 μg rhILK; lane 3, 10 μg rhILK. B: Immunoblot of immortalized mouse LEC lysates probed with affinity-purified polyclonal ILK IgG (R3B1; column 1), in comparison with commercially-available anti-ILK monoclonal (column 2) and polyclonal antibodies (column 3), before (-) and after (+) reduction with dithiothreitol (DTT). C: ELISA showing binding of affinity-purified anti-ILK IgG (R3B1) to rhILK (triangle) in comparison with commercially-available anti-ILK polyclonal antibody (circle) and monoclonal antibodies (square). Each point represents three independent experiments, ±SD. D: Comparison of ILK immunoprecipitated from mouse LEC lysates with commercial and R3B1 IgG and subsequently probed with commercial monoclonal antibody. Lane 1, rhILK; lane 2, lysate only (no antibody); lane 3, non-specific rabbit IgG; lane 4, commercial polyclonal antibody; lane 5, R3B1. In A, B, and D, molecular weight markers are shown on the left of each panel.

Figure 2

ILK in the mouse lens and in immortalized lens cells. A: Western blot of lysates from cortical and nuclear fiber cells dissected from mouse lenses. Additional probe for GAPDH is shown as a protein loading control. Molecular weight markers are indicated on the left. B: RT-PCR for ILK performed on RNA isolated from immortalized mouse (lane 1) and human LEC (lane 2), as well as mouse lens tissue dissected into capsule/epithelial (lane 3) and lens fiber fractions (lane 4). Probe for GAPDH mRNA is shown as a control for cellular RNA.

Figure 3

Localization of ILK in the mouse lens. A: Hematoxylin and eosin-stained mouse lens. The red arrow denotes the lens epithelial cell layer. Boxes indicate anterior (B,C), equatorial (D,E), and posterior (F,G) areas imaged for immunohistochemistry. B: Immunohistochemistry of mouse lens anterior LEC and fiber cells with affinity-purified anti-ILK IgG (R3B1). FITC-conjugated donkey anti-rabbit IgG was used as a secondary antibody. C: Immunohistochemistry of anterior LEC with commercial polyclonal anti-ILK antibody. D: Immunohistochemistry of lens equatorial region with R3B1. E: Immunohistochemistry of lens equatorial region with R3B1 pre-absorbed with 10 μg/ml rhILK. F: Immunohistochemistry of the posterior lens with R3B1. G: Immunohistochemistry of the posterior lens with rabbit IgG control. The scale bar is equal to 10 μm.

Figure 4

Coincidence of ILK with β₁-integrin but not EEA1 in the mouse lens. A: Immunohistochemistry with R3B1 (red) and anti-β₁ integrin antibody (green). Yellow indicates coincidence of staining (arrows). The scale bar is equal to 1 μm. B: Immunohistochemistry with R3B1 (green; arrows) and anti-EEA1 antibody (red; arrowheads). No coincidental staining is evident. The scale bar is equal to 10 μm. Morphological alterations and non-specific staining for ILK in the capsule (A, extreme right) occurred with the staining protocol required for integrin immunohistochemistry.

Figure 5

ILK is induced during LEC differentiation and inhibition of ILK retards expression of EMT markers α-SMA and fibronectin. A: Immunoblot of lysates (20 μg/lane) from immortalized and primary mouse LEC that grew out from dissected lens capsules (0-14 days). Lane 1, rhILK (500 ng); lane 2, immortalized LEC; lane 3, primary LEC, 0 days; lane 4, primary LEC, 7 days; lane 5, primary LEC, 14 days. Lysates were probed for ILK and α-SMA as an indicator of EMT. rhILK (lane 1) was used for protein size comparison, and a probe for GAPDH was used to normalize protein loading. B: Densitometry of protein bands from the blot shown in A. Relative expression values are normalized to those of GAPDH. C: Immunoblot of lysates (2 capsules/lane) from primary mouse LEC that grew out from dissected lens capsules (0-14 days). Lane 1, primary LEC + ILK-targeting siRNA, 7 days; lane 2, primary LEC + ILK-targeting siRNA, 14 days; lane 3, primary LEC + GFP-targeting siRNA (control), 7 days; lane 4, primary LEC + GFP-targeting siRNA. Lysates were probed for ILK to verify ILK-targeting siRNA efficiency. Fibronectin and α-SMA were monitored as indicators of EMT. An additional probe for GAPDH was used to normalize protein loading. D: Densitometry of protein bands from the blot shown in C. ILK- and GFP-siRNA-treated explants show a significant difference in protein expression after 14 days of culture (the asterisk indicates a p<0.02). Relative values are normalized to that of GAPDH.

Figure 6

Production and localization of ILK in cultured mouse LEC. A: Immunoblot of lysates (20 μg/lane) from resting (lane 1) and serum-deprived cultured LEC (lane 2). Additional probe for GAPDH is shown as a protein loading control. Molecular weight standards are indicated at left. B: Immunoblot of phosphorylated MBP (MBP-P) following immunoprecipitation of ILK with commercial (#06-592) or R3B1 polyclonal antibodies, followed by in vitro kinase assay. C: Immunocytochemistry with affinity-purified anti-ILK IgG (R3B1) of LEC plated on glass coverslips; arrows indicate ILK. FITC-conjugated donkey anti-rabbit IgG was used as a secondary antibody. D: Staining performed with rabbit IgG control. E: Staining with R3B1 antibody preincubated with 10 mu g/ml rhILK. F: Staining with R3B1 (red) and anti-β₁ integrin antibody (green). Yellow indicates coincidence of staining (arrows). G: Staining of cells with R3B1 (red) and anti-α5β₁ integrin antibody (green). H: Staining with R3B1 (red) and anti-α5β₁ integrin antibody (green) of cells plated on glass coverslips coated with 10 μg/ml fibronectin. Arrows in G and H indicate coincidence of staining. I-J: Staining with R3B1 (red) and anti-EEA1 antibody (green). Arrows indicate ILK; arrowheads indicate EEA1. The scale bar is equal to 1 μm.

Figure 7

Localization and increased production of ILK in cultured human RPE. Immunocytochemistry with affinity-purified anti-ILK IgG (R3B1) of hRPE plated on glass coverslips. FITC-conjugated donkey anti-rabbit IgG was used as a secondary antibody. A: Non-stressed hRPE stained with R3B1 (green). B: hRPE deprived of serum for 24 h and subsequently stained with R3B1 (green). Increased ILK expression was apparent after stress. Arrows indicate localized ILK staining in hRPE. The scale bar is equal to 1 μm.