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S. Chakravarti, L. Roberts, N. Vij, S. Joyce; Lumican–deficiency leads to elevated cyclins and increased proliferation in cultured cells and corneal stroma. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2222.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The corneal stromal cells proliferate during development and on injury. We investigated the role of lumican, a major corneal proteoglycan in regulating stromal cell proliferation. Methods: Primary cultures of mouse embryonic fibroblasts (MEF) and corneal fibroblasts (CF) were derived from E14 embryos and corneas, respectively. Cell growth was measured by Aqueous One Cell Proliferation assay and DNA synthesis by immunostaining for incorporated BrdU. Postnatal 10–day (P–10) and 6–week old (adult) mouse cornea were immunostained for Ki67 positive (proliferation marker) cells. Proliferation in cell culture was detected by immunostaining for PCNA and Ki67. MEF proliferation was assayed after transfection with a recombinant lumican construct or in medium with recombinant lumican. MDM2, p53, p21, cyclins A, D1 and E levels were estimated by semi–quantitative immunoblotting. Results: Lum–/– MEF and CF grow faster than Lum+/+ cells. Increased DNA synthesis (BrdU assay) and immunostaining of PCNA in Lum –/– cells indicate this higher growth to be in part due to increased proliferation. Ectopic expression of lumican in Lum–/– MEF or recombinant lumican–supplemented culture medium restored proliferation to lower wild type rates. Compared to Lum+/+, Lum–/– P–10 corneas showed an increase in proliferating stromal cells (p=0.01). To elucidate the underlying mechanism in lumican–dependent regulation of cell proliferation, we assessed levels of cell cycle regulators, p53, p21 and the cyclins. Cyclins A, D1 and E were elevated, while the cdk inhibitor p21 was decreased in MEF and the cornea of Lum–/– mice. In addition a decrease in p53 and an increase in Mdm2 (p53 ubiquitination/degradation) suggest higher turnover of p53 in Lum–/–. Conclusion:Lumican suppresses proliferation and helps to maintain quiescent differentiated cells in the adult healthy cornea. While lumican deficiency clearly down regulates p21, elevating G1–S cyclins and cell proliferation, the link between extracellular lumican and the intracellular cell cycle regulators remains elusive.
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