Excess Biglycan Interferes with TGF– Signaling Required for Eyelid Morphogenesis

Abstract

Abstract: : Purpose: We previously showed that transgenic mice (tg), Kerapr3.2–Bgn, over–expressing biglycan under the keratocan promoter exhibited exposure keratitis. These mice revealed a different premature eye opening phenotype from other failure in eyelid closure such as eye–open at birth (eob), lidgap–Gates (lgGa), open–eyes (oe), and gaping lids (gp). This study is to characterize the epithelial–mesenchymal interactions essential for eyelid morphogenesis and uncover the mechanism by which excess biglycan interrupts this important developmental process.Methods: Developing embryos of tg and non–tg littermates were evaluated at days 13.5∼ 17.0 of gestation. Light, confocoal, and scanning electron microspcopes were used to examine the pathology in tg. Cell migration assay was used to test if eyelid mesenchymal cell migration can be induced by eyelid epithelium. Neutralizing antibodies against TGF–α, TGF–ßs, FGFs, and BMPs in cell migration assays was used to determine the identity of the cytokine released by the eyelid epithelium. Ip–western analysis was employed to identify cytokine–biglycan complex. In situ hybridization and immunostaining were performed to show if autocrine/paracrine loop was altered in tg. Results: The Kerapr3.2–Bgn mice exhibited premature eye opening and exposure keratitis due to perturbation of eyelid muscle formation and the failure of Meibomian glands formation. In vitro analysis revealed that biglycan, and anti–TGF–α and EGFR antibodies, inhibit mesenchymal cell migration induced by eyelid epithelium, and that biglycan binds to TGF–α. The defects of TGF–α signaling by excess biglycan were further augmented by the interruption of the autocrine/paracrine loop of the EGFR signaling pathway of HB–EGF expression elicited by TGF–α and resulted in eyelid malformation.Conclusions: Excess biglycan sequesters TGF–α and disrupts its morphogene gradient, therefore, results in eyelid malformation. These results are consistent with the notion that biglycan serves as a regulatory molecule for the formation of a TGF–α morphogen gradient essential for eyelid morphogenesis.

Keywords: eyelid • gene/expression • growth factors/growth factor receptors