Involvement of C-Kit Receptor Tyrosine Kinase in the Maintenance of Ciliary Epithelial Neural Stem Cells: Interaction with Notch Signaling

Abstract

Abstract: : Purpose: The adult mammalian ciliary epithelium (CE) harbors mitotically quiescent population of neural stem cells. We used microarray analysis of global gene expression to identify factors that are critical regulators of these cells (Das et al., 2002 ARVO Abstract). One of the candidate genes that emerged from this analysis is the c-Kit receptor tyrosine kinase (c-Kit RTK) that has been shown to regulate stem cells during spermatogenesis, hematopoiesis and melanogenesis. Here we demonstrate that c-Kit RTK is likely a key factor in the maintenance of CE neural stem cells and a positive regulator of Notch signaling. Methods: CE neural stem cells were isolated and cultured as previously described (Ahmad et al., Biochem. Biophys. Res. Commun. 2000,270; 515-521). The relative levels of c-Kit RTK in CE neural stem cells in proliferation and differentiation condition were ascertained by immunocytochemical and RT-PCR analyses. The functional analysis of c-Kit RTK in the maintenance of CE neural stem cells included the following approaches: 1) Activation of c-Kit RTK using its ligand, stem cell factor (SCF). 2) Inhibition of c-Kit RTK by c-kit antibody. 3) Inhibition of intracellular aspect of c-Kit RTK signaling by Rapamycin. In order to understand the relationship between signaling through c-Kit RTK and Notch receptor, another key regulator of stem cells, levels of Notch1 mRNA were analyzed in response to perturbation of c-Kit signaling. Results: Immunocytochemical analysis showed that ~70% of the proliferating cells expressed c-Kit RTK. Exposure of neurospheres to SCF alone and in the presence of c-Kit antibody resulted in an increase and decrease in the incorporation of Brdu, respectively, suggesting the involvement of c-Kit RTK in the self-renewal of CE neural stem cells. This observation was further supported by using Rapamycin, which compromised the self-renewal and suggested that the intracellular effect of c-Kit RTK involves ATK kinase. In addition, we observed that the c-Kit RTK signaling positively regulates the expression of Notch 1 receptor. Conclusions: Preliminary observations suggest that c-Kit signaling plays an important role in the maintenance and proliferation of CE neural stem cells. The c-Kit RTK may also influence the maintenance indirectly by positively regulating Notch signaling.

Keywords: retinal development • ciliary body • cell-cell communication