Purchase this article with an account.
K. Runager, I. B. Thøgersen, H. Karring, G. K. Klintworth, T. Møller-Pedersen, J. J. Enghild; Biosynthesis of Transforming Growth Factor Beta Induced Protein in Keratocytes and Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5866.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor beta induced protein (TGFBIp) is found in large quantities in the human cornea. It has been shown to be linked to several corneal dystrophies, but the function of the protein is still largely unknown. In a previous study we have shown that mature corneal TGFBIp from human and porcine is proteolytically processed immediately after the RGD sequence. To determine the processing event of TGFBIp in the cornea, we investigated the biosynthesis of TGFBIp in keratocytes and corneal fibroblasts from porcine
Keratocytes were isolated from porcine corneas and allowed to grow overnight in D-MEM medium (+ platelet poor horse serum). Serum-cultured corneal fibroblasts were obtained using explant technique and grown in 10% FBS. A pulse-chase experiment was employed starting with a starvation period (no Met/Cys), after which the ‘pulse’ was initiated with the addition of 35S Met/Cys (100 µCi) for 15 minutes followed by a ‘chase’ period. Cells and media were stopped at times 0, 5, 15, 30, 60, 120 and 180 minutes and TGFBIp was isolated by immunoprecipitation using a highly specific polyclonal antibodies directed against recombinant full-length TGFBIp. The immunoprecipitates were analyzed by SDS-PAGE and autoradiography.
The data revealed that both cell phenotypes produced TGFBIp. For the corneal fibroblasts, TGFBIp appeared in the media 30 minutes after the onset of biosynthesis. For keratocytes, however, no TGFBIp was detected in the media. In addition, the pulse-chase experiments revealed differences in the processing of TGFBIp between the keratocyte and corneal fibroblast phenotypes.
The pulse-chase experiments reveal that both keratocytes and corneal fibroblasts produce TGFBIp. Since we observe a production of the protein in keratocytes but do not detect it in the media, we hypothesize that TGFBIp binds to receptors, such as integrins, on the surface of the cells. The results suggest that the C-terminal processing predominantly occurs extracellularly.
This PDF is available to Subscribers Only