A RHO-ASSOCIATED PROTEIN-KINASE - DIFFERENTIALLY DISTRIBUTED IN LIMBAL AND CORNEAL EPITHELIA

PURPOSE. The authors have developed monoclonal antibodies (mAbs) to ch aracterize the sequential biochemical changes in corneal epithelial ce lls after they differentiate from stem cells, located in the limbus, a nd migrate centripetally to follow the pathway of terminal differentia tion. The purpose of this study was to identify a protein (recognized by mAb HE1/11F) with increased expression associated with the transiti on of the limbal epithelium to corneal epithelium. METHODS. The distri bution and identification of the protein(s) were performed using an in direct immunohistochemical staining technique and a western blot analy sis, respectively. A rabbit corneal epithelial cDNA library, construct ed in the Uni-Zap XR vector, was screened with mAb HE1/11F to select c DNA clones expressing polypeptide(s) recognized by this mAb. Additiona l overlapping cDNA clones were oh tained from a primer extension cDNA library to determine the sequence of the complete open reading frame e ncoding the protein recognized by mAb HE1/11F. RESULTS. Rabbit corneal epithelium exhibited strong immunostaining with mAb HE1/11F, however, the limbal epithelial cells stained weakly. HE1/11F recognized 160-kD a (HEBM1) and 100-kDa (HEBM2) polypeptides in the corneal epithelial e xtracts. The amino acid sequence of the protein deduced from the nucle otide sequence of the cDNA exhibited a close homology to that of a Rho A (Ras-related small GTPase)-associated serine-threonine kinase (ROCK- I or Rho-associated coiled-coil kinase). A 160-kDa RhoA-binding polype ptide with a molecular mass similar to that of HEBM1 and ROCK-I was de tected in the corneal epithelial extracts. These findings strongly sug gested that HEBM1 was rabbit ROCK-I. The identity of HEBM1 was further confirmed from the reactivity of mAb HE1/11F with ROCK-I immunoprecip itated from rabbit corneal epithelial extracts using anti-ROCK-I antib odies. CONCLUSIONS. The increased expression of a protein identified a s ROCK-I from cDNA analyses is associated with rabbit corneal epitheli al differentiation and transition from the limbal to corneal surface. Therefore, a RhoA signaling pathway is likely to be associated with co rneal epithelial differentiation (maturation). A close homology among the cDNA sequences of rabbit, mouse, rat, and human ROCK-I indicates t hat this RhoA-associated kinase is a well-conserved protein.