IGF-II is present in bovine corneal stroma and activates keratocytes to proliferate in vitro

Abstract

Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate. We fractionated stromal extract on a column of Sephacryl S-300 and tested the fractions for mitogenic activity using cell culture and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. We found that the mitogenic activity in the extract separated into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. We also compared the effects of 10 ng IGF-II/ml on keratocytes in culture to that of 2 ng TGF-beta/ml over a 7-day culture period. We found that IGF-II and TGF-beta, alone or combined, increased both (3)H-thymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to alpha-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no alpha-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-beta expressed alpha-SM actin, fibronectin, SPARC and lumican, and expressed no or low levels of keratocan, indicating a myofibroblast phenotype. Keratocytes cultured in IGF-II plus TGF-beta, however, expressed alpha-SM actin, fibronectin, SPARC, lumican, and keratocan by day 7 of culture. The results of this study show that IGF-II to be present in the corneal stroma, to stimulate keratocyte proliferation while maintaining native phenotype and to override the TGF-beta mediated down regulation of keratocan production. The IGF-II in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scarring effects of TGF-beta.

Fig. 1

SDS/PAGE of concentrated stromal extract on 4–12% gels. Lane 1: Coomassie blue stain of 20 micro liters of extract. Lane 2: Western blot of 20 micro liters of extract using antibodies to IGF-II. Lane 3: Western blot of 2 micrograms of IGF-II using antibodies to IGF-II. Lane 4: Western blot of 20 micro liters of extract using antibodies to IGFBP. Lane 5: Western blot of 2 micrograms of IGFBP using antibodies to IGFBP.

Fig. 2

Chromatography of stromal extract on a column of Sephacryl S-300. Upper panel: fractions 11–30 were monitored for absorbance at 280 nm and for the ability to stimulate 3H thymidine incorporation in keratocytes in culture. Lower panel: fractions 11–30 were analyzed by Western blot using antibodies to IGF-II and to IGFBP. The pixel density of the IGF-II and IGFPB bands was measured and graphed.

Fig. 3

DNA content of keratocyte cultures. Keratocytes were cultured in media without (diamonds) or with IGF-II (squares), TGF-β (triangles) or IGF-II plus TGF-β (X). Compared to the DNA content of control day 1 cultures, the day 7 IGF-I, the day 7 TGF-β, and the day 7 IGF-II plus TGF-β cultures were significantly higher. P values are listed on figure.

Fig. 4

Incorporation of ³H thymidine by keratocyte cultures. Keratocytes were cultured in media without (control) or with IGF-II, TGF-β or IGF-II plus TGF-β from days 1 to 7. The media contained ³H thymidine from days 4 to 7. Compared to the control, incorporation of ³H thymidine by cells cultured in IGF-II, TGF-β, and IGF-II plus TGF-β were significantly higher. Cultures in TGF-β were also significantly higher than cultures in IGF-II and significantly lower than cultures in IGF-II plus TGF-β. P values are listed on figure.

Fig. 5

Western blots of extracts of cells after four (Day 4) and seven (Day 7) days of culture. Keratocytes were cultured in media without (C) or with IGF-II (I), TGF-β (T), or IGF-I plus TGF-β (I/T). Blots were probed with antibodies to α-SM actin (alpha-SMA) or to aldehyde dehydrogenase (ALDH).

Fig. 6

Western blot of culture media collected days 1–4 (Day 4) and days 4–7 (Day 7). Keratocytes were cultured in media without (C) or with IGF-II (I), TGF-β (T), or IGF-I plus TGF-β (I/T). Blots were probed with antibodies to fibronectin (FBN), to SPARC (SPARC), to keratocan (KER), or to lumican (LUM).