Fibroblast Growth Factor-2 Promotes Keratan Sulfate Proteoglycan Expression by Keratocytes in Vitro

Abstract

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [35 S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor ␤, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.The corneal stroma is a disc of connective tissue that constitutes about 90% of the mammalian cornea. This tissue consists of a unique transparent extracellular matrix populated by keratocytes, flattened mesenchymal cells responsible for production and maintenance of this matrix. In healing corneal wounds keratocytes become activated, begin mitosis, and migrate to the wound location, where they secrete nontransparent scar components (1, 2). Cells in the healing wound are characterized by secretion of pro-inflammatory cytokines such as interleukin-1␣ and proteolytic enzymes involved in tissue remodeling, collagenase, gelatinase, and stromelysin (3, 4). This remodeling (fibroblastic) phenotype is simulated in vitro when keratocytes are cultured in medium containing fetal bovine serum and subcultured by trypsinization (5).The extracellular matrix of the normal corneal stroma is characterized by a unique class of molecules known as the corneal keratan sulfate proteoglycans (KSPG). 1 These consist of three structurally related proteins modified with N-linked keratan sulfate chains (6). The three proteins (lumican, keratocan, and mimecan) are found in a number of connective tissues but are expressed at much higher levels in the cornea compared with noncorneal tissues (7-9). In noncorneal tissues, these proteins are not modified with keratan sulfate (10). The high level of expression of these three proteins combined with a specialized glycosylation is a property unique to keratocytes and constitutes an essential feature of the role of the keratocyte in maintenance of corneal transparency. This conclusion is supported by the recent demonstration that mice bearing null mutations in the lumican gene lose corneal transparency, whereas mice with a similar...

A Rapid Transient Increase in Hyaluronan Synthase-2 mRNA Initiates Secretion of Hyaluronan by Corneal Keratocytes in Response to Transforming Growth Factor β

“…In vitro, keratocytes under quiescent serum-free conditions secrete matrix components similar or identical to those they produce in vivo (7)(8)(9). On exposure to serum and TGF␤, keratocytes alter their morphology and matrix secretion in a manner similar to cells in healing stromal wounds (8,10).…”

Gene expression profiling of advanced ovarian cancer: characterization of a molecular signature involving fibroblast growth factor 2

“…The gene list defined herein also included several members of the FGF2 signaling pathway, which are known to be expressed in ovarian and EOC cells (Crickard et al, 1994;Di Blasio et al, 1995;Valve et al, 2000), and several other genes regulated by FGF2 in various contexts: CDH11 is specifically regulated by FGF2 in models of kidney fibrosis (Strutz et al, 2002), LUM expression is induced by FGF2 in keratocytes of the corneal stroma (Long et al, 2000), while BGN is similarly controlled in aortic endothelial cells (Kinsella et al, 1997). Coexpression of the latter molecules could also be related to the evidence that FGF family members bind to heparan sulfate proteoglycans of the ECM, with such interaction comprising part of the regulation of the FGF signaling unit (Plotnikov et al, 1999;Plotnikov et al, 2000;Schlessinger et al, 2000).…”

“…The largest functional category clearly contained numerous genes encoding structural proteins of the ECM (such as collagens and proteoglycans) and genes involved in cell adhesion and signaling. Notably, this list of genes also contained fibroblast growth factor 2 (FGF2), fibroblast growth factor receptor 4 (FGFR4), as well as other ECM-related genes reported to be regulated by FGF2 in various cellular contexts: OB cadherin (CDH11) (Strutz et al, 2002), lumican (LUM) (Long et al, 2000), biglycan (BGN) (Kinsella et al, 1997). At least two other functional categories were identified within the retrieved gene list, that is, several genes associated with the immune/inflammatory response of the host and various genes involved in transcription regulation (Supplementary section c), although they were not significant in terms of GO-terms distribution.…”

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

“…Keratocytes isolated from the stroma by collagenase digestion and cultured in serum free media retain their dendritic morphology and their quiescence (Jester et al 1996;Beales et al 1999) but can be activated to proliferate by a number of growth factors including, FGF(fibroblast growth factor)-2, PDGF(platelet derived growth factor), TGF(transforming growth factor)-β, IL(interleukin)-1-α, IGF(insulin like growth factor)-I and insulin (Long et al 2000;Jester et al 2002;Jester and Ho-Chang 2003;Musselmann et al 2005). Antibodies to TGF-β have been shown to reduce keratocyte activation in corneal wounds (Jester et al 1997) and this suggests that TGF-β is present in wounds and activates keratocytes in vivo as well.…”