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Hepatocyte Growth Factor, Keratinocyte Growth Factor, and Other Growth Factor-Receptor Systems in the Lens

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Hepatocyte Growth Factor, Keratinocyte Growth Factor, and Other Growth Factor-Receptor Systems in the Lens Hepatocyte Growth Factor, Keratinocyte Growth Factor, and Other Growth Factor-Receptor Systems in the Lens Jian Weng,* Qianwa Liang,~\ Rajiv R. Mohan,~f Qian Li* and Steven E. Wilsonf Purpose. To examine the expression and function of hepatocyte growth factor (HGF), kera- tinocyte growth factor (KGF), epidermal growth factor (EGF) and other growth factor- cytokine-receptor systems in lens epithelial cells. Methods. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot anal- ysis were used to examine the expression of messenger RNAs in primary cultured rabbit and human lens cells and in ex vivo rabbit lens tissue. Protein expression and the effect of HGF and KGF on crystallin expression in lens epithelial cells were evaluated by immunoprecipita- tion and Western blot analysis. The effect of exogenous HGF, KGF, and EGF and of the coculture of lens epithelial cells with corneal endothelial cells on the proliferation of rabbit lens cells in a Transwell system was determined by cell counting. Results. Messenger RNAs and proteins of HGF and KGF were expressed in primary rabbit lens epithelial cells and in ex vivo rabbit lens epithelial tissue. Human lens cells also expressed the mRNAs. Other growth factors and receptor messenger RNAs were also expressed. Hepato- cyte and keratinocyte growth factors, and coculture with corneal endothelial cells stimulated proliferation of rabbit lens epithelial cells. In first-passage rabbit lens cells, HGF, KGF, and EGF increased the expression of alpha and beta crystallins. Conclusions. Hepatocyte and keratinocyte growth factor-receptor systems are expressed in lens cells. HGF and KGF are not expressed in epithelial cells in such tissues as skin, cornea, and lacrimal gland in which fibroblastic and epithelial cells interact in the formation of an organ. Expression of these growth factors in the lens may have evolved because the lens cells are relatively isolated within the anterior chamber of the eye. Our results suggest, however, that growth factors released by the corneal endothelium also could modulate lens functions (aquecrine interactions). Invest Ophthalmol Vis Sci. 1997;38:1543-1554. VFrowth factors modulate such functions as prolifera- factor (PDGF), and insulin-like growth factor (IGF) tion, motility, differentiation, and apoptosis in lens have been implicated in the regulation of lens cell epithelial cells.1"3 Acidic fibroblast growth factor functions.1"3 (FGF-1), basic fibroblast growth factor (FGF-2), epi- The anatomic position of the lens within the ante- dermal growth factor (EGF), platelet-derived growth rior segment presents a regulatory milieu that is unique to the eye. Because the adult lens is avascular From the "Department of Ophthalmology, University of Texas Southwestern Medical and lacks innervation, all of the regulatory signals Center at Dallas, Texas, and the -fEye Institute and the Department of Cell Biology, must be derived from the lens cells themselves or from The Cleveland Clinic foundation, Ohio. Presented in pan at the annual meeting of the Association for Research in Vision signals delivered through the aqueous humor. All of and Ophthalmology, Fort Lauderdale, Florida, May, 1995. the nutritional requirements of the lens are provided Supported in part by U. S. Public Health Service grant EY10056from the National Eye Institute, National Institutes of Health, Bethesda, Maryland and The Cleveland by the aqueous humor, and it therefore seems plausi- Clinic Foundation, Cleveland, OH. ble that important regulatory signals could also be Submitted for publication September 23, 1996; revised February 20, 1997; accepted February 21, 1997. delivered through this medium. Proprietary interest category: p(SEW); N (JW, QL, RRM, QL) has financial interest in a patent related to applications of HGF and KGF to the eye. None of the other In results of recent studies on the expression and authors has any commercial or proprietary interest. function of corneal hepatocyte growth factor (HGF) Reprint requests: Steven. E. Wilson, Eye Institute and Department of Cell Biology/ A31, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH and of keratinocyte growth factor (KGF), we were sur- 44195. prised to discover that corneal endothelial cells pro- investigative Ophthalmology & Visual Science, July 1997, Vol. 38, No. 8 Copyright © Association for Research in Vision and Ophthalmology 1543 Downloaded from iovs.arvojournals.org on 09/26/2021 1544 Investigative Ophthalmology & Visual Science, July 1997, Vol. 38, No. 8 duce HGF and KGF.4 These two growth factors have Board of the University of Texas Southwestern Medi- been identified in many tissues as paracrine modula- cal Center approved the use of human tissues in this tors of stromal-epithelial interactions secreted by fi- study. broblastic cells to regulate epithelial cell functions. Proliferation experiments were performed by plat- What functions could be served by endothelial-derived ing 2 X 104 first-passage rabbit cells per well in six- HGF and KGF? Is it possible that these growth factors well cluster plates (Falcon, Lincoln Park, NJ). Twenty- could be released from the endothelium to modulate four hours after seeding, cells were transferred into 2 the functions of epithelial-derived lens tissue that is ml of keratinocyte-defined medium (KDM: modified continuously exposed to the aqueous humor? We have MCDB 153, 5 //g/ml insulin, 0.5 /Ltg/ml hydrocorti- speculated previously that these types of growth fac- sone, and 0.15 mM Ca2+ without EGF; Clonetics, San tor- or cytokine-mediated communications might oc- Diego, CA) without growth factors, serum, or other cur between different cell types exposed to the aque- extracts. Growth factors to be tested were added to ous humor; and we have termed these hypothetical the medium. 4 regulatory mechanisms aquecrine interactions because At the time of addition, growth factors were di- this mode of communication is not defined ade- luted in sterile, phosphate-buffered saline (PBS) with quately by terms that are currently available to de- 0.2% gelatin (Sigma, St. Louis, MO) to provide 5 /il scribe growth factor-cytokine function (that is, auto- of additional volume to each well and to obtain the crine, paracrine, endocrine, and juxtacrine). This final concentration of growth factor. Each growth fac- study examined the expression of the HGF and KGF tor was tested in six wells in each experiment. Five growth factor-receptor systems in the lens and the microliters of PBS with 0.2% gelatin as a vehicle was effect of HGF, KGF, and cocultured corneal endothe- added to control flasks. Media were changed at 3-day lial cells on the proliferation of lens cells. The expres- intervals with the addition of fresh growth factors. sion of other growth factor-receptor system messen- Human recombinant double-chain HGF was ob- ger ribonucleic acids (mRNAs) in the human and rab- tained from Genentech (South San Francisco, CA), bit lens was also evaluated. human EGF from Collaborative Biomedical Products (Bedford, MA), and human KGF from Bachem (Phila- delphia, PA). Each of these human growth factors METHODS stimulates proliferation of rabbit corneal epithelial cells (Wilson SE, unpublished data, 1994), demonstra- Cell Culture, Ex Vivo Tissue, and Proliferation ting the cross-species efficacy of the growth factors. Assays Concentrations of HGF and KGF were at 5 ng/ml and Primary cultures of human5 and rabbit6 lens epithelial EGF was at 10 ng/ml, because these concentrations cells and of rabbit corneal endothelial7 cells were es- are optimal for a wide variety of cell types.8"15 Aliquots tablished by previously described methods from ex- of stock solutions of the growth factors were placed plants in Dulbecco's minimal essential medium in siliconized micro tubes and were stored at — 80°C to (DMEM; Gibco, Gaithersburg, MD) with 10% fetal minimize denaturation caused by repeated freezing bovine serum (Gibco). Human cells were derived and thawing. from fresh eyes of donors less than 2 years old in Coculture of first-passage rabbit lens epithelial compliance with the Helsinki Accord on Human cells with rabbit corneal endothelial cells was per- Rights. Rabbit cells were obtained from 7-week-old formed in six-well Transwell plates (Falcon, Lincoln New Zealand White rabbits. Rabbit lens tissue for re- Park, NJ) with a 0.45-/L/M pore size. Two X 104 first- verse transcription-polymerase chain reaction (RT- passage rabbit lens cells per well were plated in the PCR) was obtained by euthanizing New Zealand White bottom of the well with a monolayer of first-passage rabbits, immediately removing the corneoscleral rim rabbit corneal endothelial cells on the Transwell mem- with sharp scissors, expressing the intact lens, strip- brane insert. Two milliliters of KDM were added to ping the lens capsule with jeweler's forceps, transfer- the wells and changed at 3-day intervals. Lens epithe- ring the capsule with associated lens epithelial tissue lial cells alone with an empty Transwell insert or cor- into a 1.5-ml microfuge tube containing 0.75 ml of neal endothelial cells alone on the insert served as guanidinium thiocyanate solution (GTC)7 for RNA ex- control specimens. traction, and pulverizing with a Teflon disposable pes- After a 2-week exposure to growth factors or to tle (Fisher Scientific, Pittsburgh, PA). Extraction of cocultured endothelial cells, lens cells were photo- RNA from the GTC solution was achieved as previously graphed with an inverted phase-contrast microscope, described.7 These
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