ROCK inhibitor (Y27632) increases apoptosis and disrupts the actin cortical mat in embryonic avian corneal epithelium

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Abstract
The embryonic chicken corneal epithelium is a unique tissue that has been used as an in vitro epithelial sheet organ culture model for over 30 years (Hay and Revel [1969] Fine structure of the developing Avian cornea. Basel, Switzerland: S. Karger A.G.). This tissue was used to establish that epithelial cells could produce extracellular matrix (ECM) proteins such as collagen and proteoglycans (Dodson and Hay [1971] Exp Cell Res 65:215–220; Meier and Hay [1973] Dev Biol 35:318–331; Linsenmayer et al. [1977] Proc Natl Acad Sci U S A 74:39–43; Hendrix et al. [1982] Invest Ophthalmol Vis Sci 22:359–375). This historic model was also used to establish that ECM proteins could stimulate actin reorganization and increase collagen synthesis (Sugrue and Hay [1981] J Cell Biol 91:45–54; Sugrue and Hay [1982] Dev Biol 92:97–106; Sugrue and Hay [1986] J Cell Biol 102:1907–1916). Our laboratory has used the model to establish the signal transduction pathways involved in ECM‐stimulated actin reorganization (Svoboda et al. [1999] Anat Rec 254:348–359; Chu et al. [2000] Invest Ophthalmol Vis Sci 41:3374–3382; Reenstra et al. [2002] Invest Ophthalmol Vis Sci 43:3181–3189). The goal of the current study was to investigate the role of ECM in epithelial cell survival and the role of Rho‐associated kinase (p160 ROCK, ROCK‐1, ROCK‐2, referred to as ROCK), in ECM and lysophosphatidic acid (LPA) ‐mediated actin reorganization. Whole sheets of avian embryonic corneal epithelium were cultured in the presence of the ROCK inhibitor, Y27632 at 0, 0.03, 0.3, 3, or 10 μM before stimulating the cells with either collagen (COL) or LPA. Apoptosis was assessed by Caspase‐3 activity assays and visualized with annexin V binding. The ROCK inhibitor blocked actin cortical mat reformation and disrupted the basal cell lateral membranes in a dose‐dependent manner and increased the apoptosis marker annexin V. In addition, an in vitro caspase‐3 activity assay was used to determine that caspase‐3 activity was higher in epithelia treated with 10 μM Y‐27632 than in those isolated without the basal lamina or epithelia stimulated with fibronectin, COL, or LPA. In conclusion, ECM molecules decreased apoptosis markers and inhibiting the ROCK pathway blocked ECM stimulated actin cortical mat reformation and increased apoptosis in embryonic corneal epithelial cells. Developmental Dynamics 229:579–590, 2004.