STAT 3 Regulates the Proliferation and Differentiation of Rabbit Limbal Epithelial Cells via a Np 63-Dependent Mechanism

The ocular epithelium consists of corneal, limbal, and conjunctival epithelial cells that are derived from two distinct phenotypic cell lineages, namely, the cornea and conjunctiva. An overwhelming body of evidence has firmly suggested that limbus is the location of corneal epithelial stem cells. The fact that limbal epithelial stem cell bear several inherent properties of adult stem cells is well established in that they (1) constitute a small population of relatively undifferentiated cells; (2) have a high proliferative potential and unlimited self-renewal ability; (3) are slow-cycling or labelretaining cells; (4) undergo asymmetric cell division; and (5) reside in a specialized niche. In severe ocular surface disorders, limbal epithelial stem cells are extensively damaged with a consequent failure in the supply of corneal keratinocytes, leading eventually to corneal inflammation (erosion, ulceration, or even perforation) and conjunctivalization (opacification and vascularization), and hence visual impairment. The nuclear transcription factor p63, a member of the p53 family, was found to be specifically expressed in the basal cells of many human epithelia and was reckoned to be a specific marker for human corneal epithelial stem cell. Six p63 isoforms have been reported, and Np63 has been shown to be the major isoform expressed in the basal layer of limbal epithelium. Recently we showed that the suppression of Np63 expression inhibits proliferation of rabbit limbal keratinocytes cultured on human amniotic membrane. Moreover, Np63 has been shown to be a marker of both resting and activated limbal epithelial stem cells. Collectively these results implied that p63 may be crucial in regulating the proliferation and differentiation of limbal epithelial stem cell. The proliferation and differentiation of stem cell are regulated by several signaling pathways. Among them, transcription factor signal transducer and activator of transcription 3 (STAT3) has been proved to play a pivotal role in modulating self-renewal of the embryonic and hematopoietic stem cells. Recently we showed that Np63 autoregulates its own expression via the activation of STAT3 pathway in human nasopharyngeal carcinoma cells. In a more recent report using human liver carcinoma cells, we found that expression of Np63 exerts a positive regulatory effect (autoregulation) on the activity of Np63 promoter through activation, phosphorylation, dimerization, and nuclear translocation of STAT3. Taken together, these observations clearly suggested that STAT3 plays important roles in the expression of Np63 in tumor cells. However, whether similar regulatory mechanisms exist in limbal epithelial stem cell has yet to be elucidated. In the present study, we aimed to delineate the role of STAT3 in the regulation of p63 expression, limbal epithelial cell proliferation, and differentiation by using limbal explant cultures with or without STAT3 silencing. Our results indicate that in limbal epithelial outgrowth, STAT3 is localized mostly in the nucleus and is correlated with a higher Np63 expression. Knockdown of Np63 expression by siRNA suppresses the expansion of limbal epithelial outgrowth. Moreover, knockdown of STAT3 downregulates Np63 expression and suppresses limbal epithelial outgrowth. Interestingly, such growth suppression is rescued by ectopically expressed Np63.