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.