Mechanisms of mitotic inhibition in corneal endothelium: contact inhibition and TGF-beta2.

Contact inhibition has been implicated as an important antiproliferative mechanism in developing and mature corneal endothelium. Although exogenous TGF-beta2 and TGF-beta2 in aqueous humor suppress S-phase entry in cultured rat corneal endothelial cells, it is not known whether TGF-beta2 contributes to the mitotic inhibition that occurs during in vivo endothelial development. TGF-beta receptors I, II, and III must be coexpressed for a TGF-beta2-induced intracellular signal to be transmitted. The current study was conducted to determine whether TGF-beta2 contributes to mitotic inhibition during endothelial development, by investigating when these receptors become coexpressed in the endothelium of neonatal rats. Cyclin-dependent kinase inhibitors (CKIs), such as p27kip1 and p15INK4b, help mediate mitotic inhibition in other cell types. The role of CKIs in inhibiting proliferation in corneal endothelium was examined by first determining the kinetics of p27kip1 expression in neonatal rat corneal endothelium. Studies were then extended to cultured cells to more directly compare the effects of TGF-beta2 and cell-cell contact on the relative protein and mRNA expression of the CKIs, p27kip1, and p15INK4b.Immunocytochemistry (ICC) detected TGF-beta receptors I, II, and III (RI, RII, RIII, respectively) in the endothelium of rat corneas on postnatal days 1, 10, and 21, and in adult (3-month-old) rats. ICC for p27kip1 was conducted on postnatal days 1, 7, 14, and 21. Samples were taken for p27kip1 RT-PCR on postnatal days 7, 14, and 21 and from adult rats. The effect of TGF-beta2 on p27kip1 and p15INK4b expression was determined in G(0)-phase synchronized subconfluent rat corneal endothelial cells incubated for 24 hours in 10% serum +/- 5 ng/mL TGF-beta2. CKI expression was also examined in fully confluent cultures. RT-PCR and Western blot analysis detected p27kip1 and p15INK4b mRNA and protein expression, respectively. The effect of releasing cells from cell-cell contact on proliferation and p27kip1 protein expression was studied in confluent cultures treated for 1 hour with and without 2.0 mg/mL di-sodium EDTA and then maintained for 24 hours in 10% serum. Cultures were then either fixed for ICC of Ki67, a marker of actively cycling cells, or extracted for Western blot determination of p27kip1 protein.Positive staining for TGF-beta RIII was detected on postnatal day 10, and staining for RI and RII was detected on postnatal day 21. The endothelium stained positively for p27kip1 on postnatal day 1 and thereafter, and p27kip1 PCR product was detectable at the earliest time point tested (postnatal day 7). In cultured cells, TGF-beta2 and cell-cell contact had relatively little effect on expression of p27kip1 or p15INK4b mRNA. TGF-beta2 lowered the levels of both proteins, but p27kip1 remained at a higher level than p15INK4b. In confluent cultures, p15INK4b protein was reduced; however, p27kip1 protein levels increased 20-fold. Positive staining for Ki67 was detected, and p27kip1 protein levels substantially decreased in EDTA-treated confluent cells compared with the untreated control.Previous studies from this laboratory showed that corneal endothelial cell proliferation ceases in neonatal rat by postnatal day 13. This timing correlated with the formation of stable cell-cell contacts, implicating contact inhibition as an important mechanism of growth arrest during endothelial development. The current studies showed that coexpression of TGF-beta RI, RII, and RIII occurred too late for TGF-beta2 to have a significant role in inhibiting proliferation during endothelial development. Studies in cultured cells suggest that p27kip1 mediates inhibition of proliferation induced by TGF-beta2, although the response to this cytokine was relatively weak. ICC and RT-PCR of p27kip1 in neonatal endothelium and RT-PCR and Western blot studies in cultured cells indicate that contact inhibition is mediated, in large part, through the activity of p27kip1. These results, together with previous data from this laboratory, strongly suggest that contact inhibition is an important mechanism responsible for inducing cell cycle arrest during corneal endothelial development and for maintaining the mature monolayer in a nonproliferative state. In both cases, contact-induced inhibition is mediated, at least in part, by p27kip1. TGF-beta2 appears not to induce mitotic arrest in the developing endothelium, but may function to maintain the mature endothelium in a nonreplicative state should cell-cell contact be lost in the monolayer.