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-β/ml over a 7-day culture period. We found that IGF-II and TGF-β, alone or combined, increased both 3 Hthymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to α-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no α-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-β expressed α-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-β, however, expressed α-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-β 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-β.

Head and neck squamous cancer stromal fibroblasts produce growth factors influencing phenotype of normal human keratinocytes

“…High expression levels of IGF-2 were observed also immunohistochemically and at mRNA level in tumor and in stromal cells (Nosho et al 2005;Sayer et al 2005;Diehl et al 2006;Kornprat et al 2006;Pavelic et al 2007;Weber et al 2007). While IGF-2 is important for normal epithelial cells, it is also able to stimulate proliferation of tumor cells (Brady et al 2007;Musselmann et al 2008). We observed the positive effect of SCCF on epithelial-mesenchymal transition (Lacina et al 2007a), and this phenomenon seems to be positively controlled by IGF-2 (Arciniegas et al 2006).…”

Differential gene expression patterns of the developing and adult mouse cornea compared to the lens and tendon

“…Gpc functions may be mediated via IGF-II, and Igf2 was elevated in P10 corneas (Supplemental Table 1). A recent study also reported IGF-II in the bovine cornea and its role in regulating stromal keratocyte proliferation (Musselmann et al 2008). Igfbp2, encoding an IGF binding protein that modulates IGF levels and its autocrine and paracrine functions was also found to be over expressed in the cornea of both ages by microarray and RT-PCR.…”

Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose

“…High levels of insulin would not normally be present in the corneal stroma, however IGF-II, another ligand for IGF-IR, is in the bovine corneal stroma and it causes bovine keratocytes to proliferate and maintain their normal phenotype in vitro (Musselmann et al 2008). IGF-I has previously been shown to stimulate the proliferation of rabbit keratocytes in vitro while maintaining their dendritic morphology (Jester and Ho-Chang 2003).…”