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Endothelial mesenchymal transformation mediated by IL-1β-induced FGF-2 in corneal endothelial cells
Experimental Eye Research 95 (2012) 35e39
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Endothelial mesenchymal transformation mediated by IL-1b-induced FGF-2 in corneal endothelial cells Jeong Goo Lee a, MinHee K. Ko a, EunDuck P. Kay a, b, * a b
Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2011 Accepted in revised form 3 August 2011 Available online 17 August 2011
This review describes the molecular mechanism of endothelial mesenchymal transformation (EMT) mediated by fibroblast growth factor-2 (FGF-2) in corneal endothelial cells (CECs). Corneal fibrosis is not frequently observed in corneal endothelium/Descemet’s membrane complex; but when this pathologic tissue is produced, it causes a loss of vision by physically blocking light transmittance. Herein, we will address the cellular activities of FGF-2 and its signaling pathways during the EMT process. Furthermore, we will discuss the role of inflammation on FGF-2-mediated EMT. Interleukin-1b (IL-1b) greatly upregulates FGF-2 production in CECs, thus leading to FGF-2-mediated EMT; the whole spectrum of the injury-mediated inflammation (IL-1b pathway) and the subsequent EMT process (FGF-2 pathway) will be briefly discussed. Intervention in the two pathways will provide the means to block EMT before inflammation causes an irreversible change, such as the production of retrocorneal fibrous membrane observed in human eyes. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: corneal endothelial cells endothelial mesenchymal transformation retrocorneal fibrous membrane FGF-2 PI 3-kinase IL-1b Rho kinases type I collagen p27
1. Introduction Corneal endothelium plays a critical role in maintaining corneal hydration and corneal transparency. Adult human corneal endothelial cells (CECs) are unique in that they are mitotically inactive and are arrested at the G1 phase of the cell cycle (Joyce, 2003). Despite the presence of fibroblast growth factor-2 (FGF-2), a potent mitogenic factor that is stored in Descemet’s membrane (Kay et al., 1993), corneal endothelium in vivo remains anti-proliferative throughout its life span. When cornea is injured, the wound repair process of corneal endothelium appears to have two distinct pathways: 1) the regenerative pathway, by which CECs do not replicate but are replaced by migration and spreading of existing endothelial cells; and 2) the nonregenerative pathway (or fibrosis), by which transformed endothelial cells not only resume proliferation but alter their cell morphology and collagen phenotypes, leading to the production of an abnormal fibrillar extracellular matrix (ECM). One such clinical example is the formation of a retrocorneal fibrous
* Corresponding author. Doheny Eye Institute, 1355 San Pablo St., DVRC203, Los Angeles, CA 90033, USA. Tel.: þ1 323 442 6625; fax: þ1 323 442 6688. E-mail address: [email protected] (E.P. Kay). 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.08.003
membrane (RCFM) between Descemet’s membrane and the corneal endothelium, the physical presence of which causes loss of vision (Waring, 1982). A RCFM can be developed by epithelial, keratocytic, and endothelial origins (Jakobiec and Bhat, 2010). This review, however, will only discuss endothelial mesenchymal transformation (EMT) observed in RCFM of endothelial origin. Fig. 1A shows such endothelial origin-RCFM produced in a rabbit cornea using a transcorneal freezing procedure (Kay et al., 1982). An in vitro model to elucidate the molecular mechanism of RCFM led us to the finding that FGF-2 exerts a key role in such endothelial to mesenchymal transformation (Fig. 1B): first, FGF-2 signaling directly regulates cell cycle progression by degrading p27Kip1 (p27), a negative regulator of the G1 phase of the cell cycle, leading to a marked stimulation of cell proliferation (Lee and Kay, 2007, 2008); second, FGF-2 signaling upregulates the steady-state levels of a1(I) collagen RNA by stabilizing the message and subsequently facilitates synthesis and secretion of type I collagen into the extracellular space (Ko and Kay, 2005); and third, FGF-2 signaling induces a change in cell shape from a polygonal to a fibroblastic morphology and causes the loss of the contact-inhibited monolayer (Fig. 1B) (Lee and Kay, 2006a). We also discovered that interleukin1b (IL-1b) exerts a critical role as a switch of the FGF-2-mediated EMT; IL-1b induces FGF-2 through the phosphatidylinositol (PI) 3-kinase and p38 pathways in CECs (Lee and Kay, 2009). We also
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including Sprouty, Casitas B-lineage lymphoma (Cbl), and phosphatases (Turner and Grose, 2010). Thus, FGF signaling is modulated by both positive and negative mechanisms, and subtle changes in the signal may be of great importance for determining the biological responses during development, homeostasis, or wound healing. We will only discuss the signal transduction triggered by the 18 kDa isoform, which causes EMT in CECs. 3. FGF-2: Its role in mitogenic pathway
Fig. 1. Phase-contrast microscopy of normal cornea and of RCFM-containing cornea (A), and primary CECs and transformed CECs (B). A. RCFM was produced in rabbit eyes using transcorneal freezing procedures that were performed at evenly spaced biweekly intervals for four sessions. Rabbits were sacrificed 1 week after the final cryoapplication (Kay et al., 1982). Freshly removed rabbit corneas and corneas containing RCFM were embedded in OCT compound and frozen in liquid nitrogen. Bar, 50 mm. B. Primary cultures of rabbit CECs were treated with FGF-2 (10 ng/ml) and passaged three times in the presence of FGF-2. The resultant multilayers of fibroblast-like cells were designated transformed CECs. Bar, 200 mm.
confirmed this to be the case in vivo, in which polymorphonuclear leukocytes (PMNs) infiltrating the anterior chamber are a major source of IL-1b, which subsequently induces production of FGF-2 in corneal endothelium (Song et al., 2010). These in vivo findings provide a mechanism to link between the injury-mediated inflammation and FGF-2-mediated EMT. 2. FGF-2 and its signaling pathways FGF-2 belongs to the 23-member FGF family. This ubiquitous and multifunctional regulator is involved in the proliferation, angiogenesis, and differentiation of a broad spectrum of mesodermal and neuro-ectodermal cells (Bikfalvi et al., 1997; Ornitz and Itoh, 2001). Five isoforms of FGF-2, with molecular weights ranging from 18 to 34 kDa, have been identified; all five are derived from a single messenger RNA: four high molecular weight (HMW) isoforms that arise from upstream CUG codons and one 18-kDa ECM isoform that arises from the downstream AUG codon (Arnaud et al., 1999; Prats et al., 1989). The HMW isoforms contain nuclearlocalization signals; thus these proteins are found in the nucleus. The 18-kDa FGF-2, on the other hand, is primarily a cytosolic protein without a signal peptide sequence; but it is generally sequestered to the extracellular matrix by heparan sulphate proteoglycans (HSPGs). Recent studies described unconventional secretion of FGF-2, which is independent of the endoplasmic reticulum-Golgi pathway; FGF-2 is initially recruited to the inner leaflet of plasma membrane, followed by membrane translocation in an HSPGs-dependent manner (Nickel, 2007). In mammals, there are four transmembrane FGF receptors with tyrosine kinase activity (Johnson and Williams, 1993; Hynes and Dey, 2010). Following ligand binding and receptor dimerization, the kinase domains transphosphorylate each other, leading to the docking of adaptor proteins and the activation of four key downstream pathways: Ras-Raf-MAPK, PI 3-kinase-Akt, signal transducer and activator of transcription, and PKC activated via phospholipase Cg (Dailey et al., 2005). Signaling is also negatively regulated at several levels by the induction of negative regulators,
In the FGF-2-mediated mitotic pathway, cell proliferation of CECs progresses by removal of p27 from the G1 phase of the cell cycle (Lee and Kay, 2007). The important regulatory role of p27 in CECs during development was reported in a study that showed p27 was involved in regulating proliferation in the corneal endothelium of the developing mouse cornea (Yoshida et al., 2004). The activity of p27 is greatly controlled by its concentration; the level of p27 is mainly regulated by ubiquitin-proteasome-mediated proteolysis (Pagano et al., 1995; Lu et al., 2009). The cell cycle-dependent degradation of p27 requires phosphorylation of p27 at threonine (Thr) or serine residues (Ser); there are at least four known phosphorylation sites: Thr187, Ser10, Thr157, and Thr198. The cyclindependent kinase 2 (Cdk2)-Cyclin E complex is responsible for phosphorylation of p27 at Thr187 (Sherr and Roberts, 1999; Lee and Kay, 2007), while Akt phosphorylates p27 on Ser10, Thr157, and Thr198 (Fujita et al., 2002; Liang et al., 2002). The Ser10 residue is also phosphorylated by kinase-interacting stathmin (KIS), a nuclear serine-threonine kinase (Boehm et al., 2002; Lee and Kay, 2011). Our kinetic studies (Lee and Kay, 2007, 2008) using the two sites of Ser10 and Thr187 led us to conclude that phosphorylated p27 at Thr187 and at Ser10 represents two distinct populations of p27 in the G1 phase of the cell cycle. We reported that phosphorylation of p27 at Ser10 is an early G1 event, as opposed to the late G1 event observed with phosphorylation of p27 at Thr187, and that phosphorylation of p27 at Ser10 is the major mechanism for G1/S transition in response to FGF-2 stimulation. We also found that rabbit CECs (rCECs) employ both PI 3-kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in parallel for G1/S transition following FGF-2 stimulation (Lee and Kay, 2011). Both PI 3-kinase and ERK1/2 pathways are involved in phosphorylation of p27 at Ser10 by KIS and at Thr187 by Cdk2 activated by Cdc25A (cell division cycle 25A), which dephosphorylates the inactive phospho-Cdk2 at Tyr15 (Chen and Gardner, 2004). We further demonstrated that FGF-2 induces expression of KIS and Cdc25A through PI 3-kinase and ERK1/2 pathways. Thus, CECs under proliferative control employ multiple pathways to remove the strong G1 inhibitor (p27) from the scene. 4. FGF-2: its role in actin cytoskeleton and morphogenic pathway The cytoskeletal elements with their polymerization dynamics are central to many cellular activities, including morphogenesis and wound healing. Assembly and organization of the actin cytoskeleton is regulated by Rho small GTPases, Rho, Rac, and Cdc42 (Etienne-Manneville and Hall, 2002). The conclusion that Rho, Rac, and Cdc42 regulate three separate signal transduction pathways (stress fiber, lamellipodia, and filopodia) linking the plasma membrane receptor to the assembly of distinct F-actin structures has been confirmed in a wide variety of cell systems. Unlike in these other cell systems, the formation of lamellipodia and filopodia is not readily observed in CECs in culture (Lee and Kay, 2006a). We showed that FGF-2 disrupts stress fibers and reorganizes actin cytoskeleton at the cortex by inhibiting Rho activity and activating Rac. Such inactivation of Rho is mediated by
J.G. Lee et al. / Experimental Eye Research 95 (2012) 35e39
activated Rac through the action of PI 3-kinase, suggesting that Rac is upstream to Rho. Our study demonstrates the antagonizing activities between Rho and Rac and between Rho and Cdc42. Simultaneously, the polygonal cell morphology of CECs is altered to the elongated shape as actin cytoskeleton is reorganized from the stress fiber phenotype to the cortical actin phenotype. Of great interest, PI 3-kinase regulates all of these cellular activities related to actin cytoskeleton and cellular morphology. We also reported that activation of Cdc42 and inactivation of RhoA are required to acquire the elongated cell shape with protrusive processes (Lee and Kay, 2006b); CECs transiently transfected with constitutive active Cdc42G12V formed protrusive processes, whereas CECs expressing dominant negative Cdc42T17N demonstrated stress fiber formation in polygonal cells. We further demonstrated the elongated cells with protrusive process are actively involved in cell migration. We conclude that the organization of actin cytoskeleton in response to FGF-2 stimulation is an orderly event: disruption of stress fibers is prerequisite to cortical actin formation, which is followed by the formation of protrusive processes. It is likely that these elongated cells with prominent protrusive processes may represent the wound phenotypes that are actively involved in migration into the injury sites. Thus, Cdc42 appears to be involved in the final stage of actin cytoskeleton reorganization necessary for the endothelial to mesenchymal transformation of CECs. On the other hand, corneal endothelium in vivo organizes actin at the cortex (Lee and Kay, 2003); the FGF-2 present in Descemet’s membrane probably facilitates the organization of actin cytoskeleton at the cortex. Under physiological conditions, CECs in vivo maintain their polygonal cell shape with cortical actin under the minimal influence of FGF-2. When injury-caused inflammation modulates the local concentration of FGF-2, the FGF-2 is able to further exert its activities on Cdc42, causing the polygonal cells to convert to spindle-shaped cells with prominent protrusive processes that may be responsible for the non-regenerative wound healing.
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fibrillar collagen and that this action of FGF-2 is mediated by PI 3kinase. 6. IL-1b: its role in production of FGF-2 Corneal fibrosis represents a significant pathophysiological problem that causes blindness by physically blocking light transmittance. RCFM is believed to represent an end-stage disease process of the corneal endothelium that can lead to corneal opacity and blindness. We reported that activated PMNs transform the type IV collagen-synthesizing polygonal endothelial cells (Kay et al., 1984a) to type I collagen-synthesizing fibroblastic cells and that PMNs release a 17-kDa protein (Kay et al., 1984b). Using ProteinChip Array technology, we identified the protein obtained from the activated PMNs as IL-1b (Lee et al., 2004). IL-1b is a major proinflammatory cytokine that plays an important role in acute and chronic inflammatory diseases (Dinarello, 2009), including inflammation and wound healing on the ocular surface (Kimura et al., 2009). One well-known role of IL-1b is its stimulation of the expression of a variety of genes necessary for the wound repair process. Both IL-1a and IL-1b markedly stimulate synthesis and release of FGF-2 in various cells (Cronauer et al., 1999). Likewise, CECs produce all isoforms of FGF-2 in response to IL-1b stimulation through the PI 3-kinase pathway with p38 as a downstream effector (Lee et al., 2004; Lee and Kay, 2009). We further showed that such is the case in vivo using a transcorneal freeze injury model (Song et al., 2010). Transcorneal freezing on rabbit eyes facilitates infiltration of PMNs, which subsequently release IL-1b into the aqueous humor. Production of all isoforms of FGF-2 is observed in corneal endothelium immediately following transcorneal freezing, and such production is maintained for 48 h after the injury. Simultaneously, transcorneal
5. FGF-2: its role in synthesis and secretion of type I collagen The physiologic collagen phenotypes of CECs are types IV and VIII collagen (Kay et al., 1984a); but CECs also synthesize type I collagen, which is intracellularly degraded immediately after synthesis (Ko and Kay, 2001). Such intracellular degradation of type I collagen in CECs is essential to maintain a healthy cornea because secretion of type I collagen into Descemet’s membrane would adversely affect corneal function. On the other hand, type I collagen is a major constituent in RCFM (Kay et al., 1982). Our attempt to investigate how type I collagen becomes the major ECM component in RCFM tissue led us to the finding that FGF-2 regulates expression of type I collagen through the action of PI 3-kinase (Ko and Kay, 2005). When CECs were continuously maintained in FGF-2 for 3 passages, cells completely lost their contact-inhibited phenotypes and formed multi-layers of elongated cells; these cells were designated transformed CECs (Fig. 1B). The steady-state level of a1(I) collagen RNA was greatly up-regulated through stabilization of the message in transformed CECs. Of interest, transformed CECs predominantly secreted homotrimeric type I collagen, [a1(I)]3, with heterotrimeric type I collagen, [a1(I)2a2(I)], as a minor species. We further demonstrated that type I collagen in the transformed CECs was preferentially associated and colocalized with Hsp47 at Golgi, suggesting that the molecule is correctly targeted for the secretory pathway. PI 3-kinase inhibitor reduced the steady-state levels and stability of a1(I) and a2(I) collagen RNAs and the secretion of type I collagen. These findings indicate that FGF-2 is able to completely switch the collagen phenotypes from basement membrane to
Fig. 2. Putative events leading to endothelial mesenchymal transformation following injury to cornea. When injury is inflicted to the cornea, inflammatory cells (PMNs) infiltrate into the anterior chamber and corneal tissue. IL-1b released by PMNs rapidly activates PI 3-kinase, which in turn, greatly facilitates synthesis of FGF-2, as a direct mediator of EMT. FGF-2 alters three major phenotypes through PI 3-kinase pathways and leads to endothelial mesenchymal transformation and the non-physiological wound healing.
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freezing disrupts the actin cytoskeleton at the cortex, and cell shapes are altered from cobblestone to irregular shape. Blockade of PI 3-kinase/p38 pathways reverses the altered actin cytoskeleton and cell shape. Taken together, it is likely that IL-1b initiates the endothelial to mesenchymal transformation of CECs through its inductive activity on FGF-2, a direct mediator of EMT. 7. Central role of PI 3-kinase We have shown the evidence that CECs predominantly utilize PI 3-kinase pathways during endothelial to mesenchymal transformation (Fig. 2). We also showed that IL-1b released by PMNs induces FGF-2 production through the PI 3-kinase pathway (Fig. 2). A marked increase of PI 3-kinase activity was observed in CECs stimulated with IL-1b for 10 min (Lee et al., 2004). Such a rapid activation of PI 3-kinase leads to up-regulation of FGF-2 synthesis: a threefold increase in FGF-2 production was observed in CECs treated with IL-1b for 1 h, after which the levels of FGF-2 were further increased in a time-dependent manner (12- to 16-fold during 24- to 72-h stimulation). Then the IL-1b-induced FGF-2 employs totally different kinetics of PI 3-kinase activation; delayed and sustained activation of PI 3-kinase is observed in all three phenotypes modulated by FGF-2 during EMT: cell proliferation, cell shape change, and induction of type I collagen synthesis. Thus intervention of both IL-1b-mediated and FGF-2-mediated pathways will provide the means to block EMT before inflammation causes an irreversible change, such as endothelial origin RCFM. In both pathways, PI 3-kinase can be used as a potential therapeutic target. 8. Clinical relevance of EMT and conclusion Descemet’s stripping endothelial keratoplasty (DSEK) has become widely accepted as the preferred method for treating endothelial dysfunction (Banitt and Chopra, 2010; Price and Price, 2007). Compared with standard penetrating keratoplasty, DSEK provides quicker visual rehabilitation and an improved safety profile with less immune rejection. In spite of widespread adoption of DSEK, graft failure has been recognized, and one of the DSEK failures is RCFM production, albeit at a low frequency (Young et al., 2009). Thus, studying the mechanism of EMT is becoming even more important with the emergence of endothelial keratoplasty and the current main surgical approach to endothelial disease. References Arnaud, E., Touriol, C., Boutonnet, C., Gensac, M.C., Vagner, S., Prats, H., Prats, A.C., 1999. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol. Cell Biol. 19, 505e514. Banitt, M.R., Chopra, V., 2010. Descemet’s stripping with automated endothelial keratoplasty and glaucoma. Curr. Opin. Ophthalmol. 21, 144e149. Bikfalvi, A., Klein, S., Pintucci, G., Rifkin, D.B., 1997. Biological roles of fibroblast growth factor-2. Endocr. Rev. 18, 26e45. Boehm, M., Yoshimoto, T., Crook, M.F., Nallamshetty, S., True, A., Nabel, G.J., Nabel, E.G., 2002. A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression. EMBO J. 21, 3390e3401. Chen, S., Gardner, D.G., 2004. Suppression of WEE1 and stimulation of CDC25A correlates with endothelin-dependent proliferation of rat aortic smooth muscle cells. J. Biol. Chem. 279, 13755e13763. Cronauer, M.V., Stadlmann, S., Klocker, H., Abendstein, B., Eder, I.E., Rogatsch, H., Zeimet, A.G., Marth, C., Offner, F.A., 1999. Basic fibroblast growth factor synthesis by human peritoneal mesothelial cells: induction by interleukin-1. Am. J. Pathol. 155, 1977e1984. Dailey, L., Ambrosetti, D., Mansukhani, A., Basilico, C., 2005. Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev. 16, 233e247. Dinarello, C.A., 2009. Immunological and inflammatory functions of the interleukin1 family. Annu. Rev. Immunol. 27, 519e550.
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Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Endothelial mesenchymal transformation mediated by IL-1b-induced FGF-2 in corneal endothelial cells Jeong Goo Lee a, MinHee K. Ko a, EunDuck P. Kay a, b, * a b
Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2011 Accepted in revised form 3 August 2011 Available online 17 August 2011
This review describes the molecular mechanism of endothelial mesenchymal transformation (EMT) mediated by fibroblast growth factor-2 (FGF-2) in corneal endothelial cells (CECs). Corneal fibrosis is not frequently observed in corneal endothelium/Descemet’s membrane complex; but when this pathologic tissue is produced, it causes a loss of vision by physically blocking light transmittance. Herein, we will address the cellular activities of FGF-2 and its signaling pathways during the EMT process. Furthermore, we will discuss the role of inflammation on FGF-2-mediated EMT. Interleukin-1b (IL-1b) greatly upregulates FGF-2 production in CECs, thus leading to FGF-2-mediated EMT; the whole spectrum of the injury-mediated inflammation (IL-1b pathway) and the subsequent EMT process (FGF-2 pathway) will be briefly discussed. Intervention in the two pathways will provide the means to block EMT before inflammation causes an irreversible change, such as the production of retrocorneal fibrous membrane observed in human eyes. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: corneal endothelial cells endothelial mesenchymal transformation retrocorneal fibrous membrane FGF-2 PI 3-kinase IL-1b Rho kinases type I collagen p27
1. Introduction Corneal endothelium plays a critical role in maintaining corneal hydration and corneal transparency. Adult human corneal endothelial cells (CECs) are unique in that they are mitotically inactive and are arrested at the G1 phase of the cell cycle (Joyce, 2003). Despite the presence of fibroblast growth factor-2 (FGF-2), a potent mitogenic factor that is stored in Descemet’s membrane (Kay et al., 1993), corneal endothelium in vivo remains anti-proliferative throughout its life span. When cornea is injured, the wound repair process of corneal endothelium appears to have two distinct pathways: 1) the regenerative pathway, by which CECs do not replicate but are replaced by migration and spreading of existing endothelial cells; and 2) the nonregenerative pathway (or fibrosis), by which transformed endothelial cells not only resume proliferation but alter their cell morphology and collagen phenotypes, leading to the production of an abnormal fibrillar extracellular matrix (ECM). One such clinical example is the formation of a retrocorneal fibrous
* Corresponding author. Doheny Eye Institute, 1355 San Pablo St., DVRC203, Los Angeles, CA 90033, USA. Tel.: þ1 323 442 6625; fax: þ1 323 442 6688. E-mail address: [email protected] (E.P. Kay). 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.08.003
membrane (RCFM) between Descemet’s membrane and the corneal endothelium, the physical presence of which causes loss of vision (Waring, 1982). A RCFM can be developed by epithelial, keratocytic, and endothelial origins (Jakobiec and Bhat, 2010). This review, however, will only discuss endothelial mesenchymal transformation (EMT) observed in RCFM of endothelial origin. Fig. 1A shows such endothelial origin-RCFM produced in a rabbit cornea using a transcorneal freezing procedure (Kay et al., 1982). An in vitro model to elucidate the molecular mechanism of RCFM led us to the finding that FGF-2 exerts a key role in such endothelial to mesenchymal transformation (Fig. 1B): first, FGF-2 signaling directly regulates cell cycle progression by degrading p27Kip1 (p27), a negative regulator of the G1 phase of the cell cycle, leading to a marked stimulation of cell proliferation (Lee and Kay, 2007, 2008); second, FGF-2 signaling upregulates the steady-state levels of a1(I) collagen RNA by stabilizing the message and subsequently facilitates synthesis and secretion of type I collagen into the extracellular space (Ko and Kay, 2005); and third, FGF-2 signaling induces a change in cell shape from a polygonal to a fibroblastic morphology and causes the loss of the contact-inhibited monolayer (Fig. 1B) (Lee and Kay, 2006a). We also discovered that interleukin1b (IL-1b) exerts a critical role as a switch of the FGF-2-mediated EMT; IL-1b induces FGF-2 through the phosphatidylinositol (PI) 3-kinase and p38 pathways in CECs (Lee and Kay, 2009). We also
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including Sprouty, Casitas B-lineage lymphoma (Cbl), and phosphatases (Turner and Grose, 2010). Thus, FGF signaling is modulated by both positive and negative mechanisms, and subtle changes in the signal may be of great importance for determining the biological responses during development, homeostasis, or wound healing. We will only discuss the signal transduction triggered by the 18 kDa isoform, which causes EMT in CECs. 3. FGF-2: Its role in mitogenic pathway
Fig. 1. Phase-contrast microscopy of normal cornea and of RCFM-containing cornea (A), and primary CECs and transformed CECs (B). A. RCFM was produced in rabbit eyes using transcorneal freezing procedures that were performed at evenly spaced biweekly intervals for four sessions. Rabbits were sacrificed 1 week after the final cryoapplication (Kay et al., 1982). Freshly removed rabbit corneas and corneas containing RCFM were embedded in OCT compound and frozen in liquid nitrogen. Bar, 50 mm. B. Primary cultures of rabbit CECs were treated with FGF-2 (10 ng/ml) and passaged three times in the presence of FGF-2. The resultant multilayers of fibroblast-like cells were designated transformed CECs. Bar, 200 mm.
confirmed this to be the case in vivo, in which polymorphonuclear leukocytes (PMNs) infiltrating the anterior chamber are a major source of IL-1b, which subsequently induces production of FGF-2 in corneal endothelium (Song et al., 2010). These in vivo findings provide a mechanism to link between the injury-mediated inflammation and FGF-2-mediated EMT. 2. FGF-2 and its signaling pathways FGF-2 belongs to the 23-member FGF family. This ubiquitous and multifunctional regulator is involved in the proliferation, angiogenesis, and differentiation of a broad spectrum of mesodermal and neuro-ectodermal cells (Bikfalvi et al., 1997; Ornitz and Itoh, 2001). Five isoforms of FGF-2, with molecular weights ranging from 18 to 34 kDa, have been identified; all five are derived from a single messenger RNA: four high molecular weight (HMW) isoforms that arise from upstream CUG codons and one 18-kDa ECM isoform that arises from the downstream AUG codon (Arnaud et al., 1999; Prats et al., 1989). The HMW isoforms contain nuclearlocalization signals; thus these proteins are found in the nucleus. The 18-kDa FGF-2, on the other hand, is primarily a cytosolic protein without a signal peptide sequence; but it is generally sequestered to the extracellular matrix by heparan sulphate proteoglycans (HSPGs). Recent studies described unconventional secretion of FGF-2, which is independent of the endoplasmic reticulum-Golgi pathway; FGF-2 is initially recruited to the inner leaflet of plasma membrane, followed by membrane translocation in an HSPGs-dependent manner (Nickel, 2007). In mammals, there are four transmembrane FGF receptors with tyrosine kinase activity (Johnson and Williams, 1993; Hynes and Dey, 2010). Following ligand binding and receptor dimerization, the kinase domains transphosphorylate each other, leading to the docking of adaptor proteins and the activation of four key downstream pathways: Ras-Raf-MAPK, PI 3-kinase-Akt, signal transducer and activator of transcription, and PKC activated via phospholipase Cg (Dailey et al., 2005). Signaling is also negatively regulated at several levels by the induction of negative regulators,
In the FGF-2-mediated mitotic pathway, cell proliferation of CECs progresses by removal of p27 from the G1 phase of the cell cycle (Lee and Kay, 2007). The important regulatory role of p27 in CECs during development was reported in a study that showed p27 was involved in regulating proliferation in the corneal endothelium of the developing mouse cornea (Yoshida et al., 2004). The activity of p27 is greatly controlled by its concentration; the level of p27 is mainly regulated by ubiquitin-proteasome-mediated proteolysis (Pagano et al., 1995; Lu et al., 2009). The cell cycle-dependent degradation of p27 requires phosphorylation of p27 at threonine (Thr) or serine residues (Ser); there are at least four known phosphorylation sites: Thr187, Ser10, Thr157, and Thr198. The cyclindependent kinase 2 (Cdk2)-Cyclin E complex is responsible for phosphorylation of p27 at Thr187 (Sherr and Roberts, 1999; Lee and Kay, 2007), while Akt phosphorylates p27 on Ser10, Thr157, and Thr198 (Fujita et al., 2002; Liang et al., 2002). The Ser10 residue is also phosphorylated by kinase-interacting stathmin (KIS), a nuclear serine-threonine kinase (Boehm et al., 2002; Lee and Kay, 2011). Our kinetic studies (Lee and Kay, 2007, 2008) using the two sites of Ser10 and Thr187 led us to conclude that phosphorylated p27 at Thr187 and at Ser10 represents two distinct populations of p27 in the G1 phase of the cell cycle. We reported that phosphorylation of p27 at Ser10 is an early G1 event, as opposed to the late G1 event observed with phosphorylation of p27 at Thr187, and that phosphorylation of p27 at Ser10 is the major mechanism for G1/S transition in response to FGF-2 stimulation. We also found that rabbit CECs (rCECs) employ both PI 3-kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in parallel for G1/S transition following FGF-2 stimulation (Lee and Kay, 2011). Both PI 3-kinase and ERK1/2 pathways are involved in phosphorylation of p27 at Ser10 by KIS and at Thr187 by Cdk2 activated by Cdc25A (cell division cycle 25A), which dephosphorylates the inactive phospho-Cdk2 at Tyr15 (Chen and Gardner, 2004). We further demonstrated that FGF-2 induces expression of KIS and Cdc25A through PI 3-kinase and ERK1/2 pathways. Thus, CECs under proliferative control employ multiple pathways to remove the strong G1 inhibitor (p27) from the scene. 4. FGF-2: its role in actin cytoskeleton and morphogenic pathway The cytoskeletal elements with their polymerization dynamics are central to many cellular activities, including morphogenesis and wound healing. Assembly and organization of the actin cytoskeleton is regulated by Rho small GTPases, Rho, Rac, and Cdc42 (Etienne-Manneville and Hall, 2002). The conclusion that Rho, Rac, and Cdc42 regulate three separate signal transduction pathways (stress fiber, lamellipodia, and filopodia) linking the plasma membrane receptor to the assembly of distinct F-actin structures has been confirmed in a wide variety of cell systems. Unlike in these other cell systems, the formation of lamellipodia and filopodia is not readily observed in CECs in culture (Lee and Kay, 2006a). We showed that FGF-2 disrupts stress fibers and reorganizes actin cytoskeleton at the cortex by inhibiting Rho activity and activating Rac. Such inactivation of Rho is mediated by
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activated Rac through the action of PI 3-kinase, suggesting that Rac is upstream to Rho. Our study demonstrates the antagonizing activities between Rho and Rac and between Rho and Cdc42. Simultaneously, the polygonal cell morphology of CECs is altered to the elongated shape as actin cytoskeleton is reorganized from the stress fiber phenotype to the cortical actin phenotype. Of great interest, PI 3-kinase regulates all of these cellular activities related to actin cytoskeleton and cellular morphology. We also reported that activation of Cdc42 and inactivation of RhoA are required to acquire the elongated cell shape with protrusive processes (Lee and Kay, 2006b); CECs transiently transfected with constitutive active Cdc42G12V formed protrusive processes, whereas CECs expressing dominant negative Cdc42T17N demonstrated stress fiber formation in polygonal cells. We further demonstrated the elongated cells with protrusive process are actively involved in cell migration. We conclude that the organization of actin cytoskeleton in response to FGF-2 stimulation is an orderly event: disruption of stress fibers is prerequisite to cortical actin formation, which is followed by the formation of protrusive processes. It is likely that these elongated cells with prominent protrusive processes may represent the wound phenotypes that are actively involved in migration into the injury sites. Thus, Cdc42 appears to be involved in the final stage of actin cytoskeleton reorganization necessary for the endothelial to mesenchymal transformation of CECs. On the other hand, corneal endothelium in vivo organizes actin at the cortex (Lee and Kay, 2003); the FGF-2 present in Descemet’s membrane probably facilitates the organization of actin cytoskeleton at the cortex. Under physiological conditions, CECs in vivo maintain their polygonal cell shape with cortical actin under the minimal influence of FGF-2. When injury-caused inflammation modulates the local concentration of FGF-2, the FGF-2 is able to further exert its activities on Cdc42, causing the polygonal cells to convert to spindle-shaped cells with prominent protrusive processes that may be responsible for the non-regenerative wound healing.
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fibrillar collagen and that this action of FGF-2 is mediated by PI 3kinase. 6. IL-1b: its role in production of FGF-2 Corneal fibrosis represents a significant pathophysiological problem that causes blindness by physically blocking light transmittance. RCFM is believed to represent an end-stage disease process of the corneal endothelium that can lead to corneal opacity and blindness. We reported that activated PMNs transform the type IV collagen-synthesizing polygonal endothelial cells (Kay et al., 1984a) to type I collagen-synthesizing fibroblastic cells and that PMNs release a 17-kDa protein (Kay et al., 1984b). Using ProteinChip Array technology, we identified the protein obtained from the activated PMNs as IL-1b (Lee et al., 2004). IL-1b is a major proinflammatory cytokine that plays an important role in acute and chronic inflammatory diseases (Dinarello, 2009), including inflammation and wound healing on the ocular surface (Kimura et al., 2009). One well-known role of IL-1b is its stimulation of the expression of a variety of genes necessary for the wound repair process. Both IL-1a and IL-1b markedly stimulate synthesis and release of FGF-2 in various cells (Cronauer et al., 1999). Likewise, CECs produce all isoforms of FGF-2 in response to IL-1b stimulation through the PI 3-kinase pathway with p38 as a downstream effector (Lee et al., 2004; Lee and Kay, 2009). We further showed that such is the case in vivo using a transcorneal freeze injury model (Song et al., 2010). Transcorneal freezing on rabbit eyes facilitates infiltration of PMNs, which subsequently release IL-1b into the aqueous humor. Production of all isoforms of FGF-2 is observed in corneal endothelium immediately following transcorneal freezing, and such production is maintained for 48 h after the injury. Simultaneously, transcorneal
5. FGF-2: its role in synthesis and secretion of type I collagen The physiologic collagen phenotypes of CECs are types IV and VIII collagen (Kay et al., 1984a); but CECs also synthesize type I collagen, which is intracellularly degraded immediately after synthesis (Ko and Kay, 2001). Such intracellular degradation of type I collagen in CECs is essential to maintain a healthy cornea because secretion of type I collagen into Descemet’s membrane would adversely affect corneal function. On the other hand, type I collagen is a major constituent in RCFM (Kay et al., 1982). Our attempt to investigate how type I collagen becomes the major ECM component in RCFM tissue led us to the finding that FGF-2 regulates expression of type I collagen through the action of PI 3-kinase (Ko and Kay, 2005). When CECs were continuously maintained in FGF-2 for 3 passages, cells completely lost their contact-inhibited phenotypes and formed multi-layers of elongated cells; these cells were designated transformed CECs (Fig. 1B). The steady-state level of a1(I) collagen RNA was greatly up-regulated through stabilization of the message in transformed CECs. Of interest, transformed CECs predominantly secreted homotrimeric type I collagen, [a1(I)]3, with heterotrimeric type I collagen, [a1(I)2a2(I)], as a minor species. We further demonstrated that type I collagen in the transformed CECs was preferentially associated and colocalized with Hsp47 at Golgi, suggesting that the molecule is correctly targeted for the secretory pathway. PI 3-kinase inhibitor reduced the steady-state levels and stability of a1(I) and a2(I) collagen RNAs and the secretion of type I collagen. These findings indicate that FGF-2 is able to completely switch the collagen phenotypes from basement membrane to
Fig. 2. Putative events leading to endothelial mesenchymal transformation following injury to cornea. When injury is inflicted to the cornea, inflammatory cells (PMNs) infiltrate into the anterior chamber and corneal tissue. IL-1b released by PMNs rapidly activates PI 3-kinase, which in turn, greatly facilitates synthesis of FGF-2, as a direct mediator of EMT. FGF-2 alters three major phenotypes through PI 3-kinase pathways and leads to endothelial mesenchymal transformation and the non-physiological wound healing.
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freezing disrupts the actin cytoskeleton at the cortex, and cell shapes are altered from cobblestone to irregular shape. Blockade of PI 3-kinase/p38 pathways reverses the altered actin cytoskeleton and cell shape. Taken together, it is likely that IL-1b initiates the endothelial to mesenchymal transformation of CECs through its inductive activity on FGF-2, a direct mediator of EMT. 7. Central role of PI 3-kinase We have shown the evidence that CECs predominantly utilize PI 3-kinase pathways during endothelial to mesenchymal transformation (Fig. 2). We also showed that IL-1b released by PMNs induces FGF-2 production through the PI 3-kinase pathway (Fig. 2). A marked increase of PI 3-kinase activity was observed in CECs stimulated with IL-1b for 10 min (Lee et al., 2004). Such a rapid activation of PI 3-kinase leads to up-regulation of FGF-2 synthesis: a threefold increase in FGF-2 production was observed in CECs treated with IL-1b for 1 h, after which the levels of FGF-2 were further increased in a time-dependent manner (12- to 16-fold during 24- to 72-h stimulation). Then the IL-1b-induced FGF-2 employs totally different kinetics of PI 3-kinase activation; delayed and sustained activation of PI 3-kinase is observed in all three phenotypes modulated by FGF-2 during EMT: cell proliferation, cell shape change, and induction of type I collagen synthesis. Thus intervention of both IL-1b-mediated and FGF-2-mediated pathways will provide the means to block EMT before inflammation causes an irreversible change, such as endothelial origin RCFM. In both pathways, PI 3-kinase can be used as a potential therapeutic target. 8. Clinical relevance of EMT and conclusion Descemet’s stripping endothelial keratoplasty (DSEK) has become widely accepted as the preferred method for treating endothelial dysfunction (Banitt and Chopra, 2010; Price and Price, 2007). Compared with standard penetrating keratoplasty, DSEK provides quicker visual rehabilitation and an improved safety profile with less immune rejection. In spite of widespread adoption of DSEK, graft failure has been recognized, and one of the DSEK failures is RCFM production, albeit at a low frequency (Young et al., 2009). Thus, studying the mechanism of EMT is becoming even more important with the emergence of endothelial keratoplasty and the current main surgical approach to endothelial disease. References Arnaud, E., Touriol, C., Boutonnet, C., Gensac, M.C., Vagner, S., Prats, H., Prats, A.C., 1999. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol. Cell Biol. 19, 505e514. Banitt, M.R., Chopra, V., 2010. Descemet’s stripping with automated endothelial keratoplasty and glaucoma. Curr. Opin. Ophthalmol. 21, 144e149. Bikfalvi, A., Klein, S., Pintucci, G., Rifkin, D.B., 1997. Biological roles of fibroblast growth factor-2. Endocr. Rev. 18, 26e45. Boehm, M., Yoshimoto, T., Crook, M.F., Nallamshetty, S., True, A., Nabel, G.J., Nabel, E.G., 2002. A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression. EMBO J. 21, 3390e3401. Chen, S., Gardner, D.G., 2004. Suppression of WEE1 and stimulation of CDC25A correlates with endothelin-dependent proliferation of rat aortic smooth muscle cells. J. Biol. Chem. 279, 13755e13763. Cronauer, M.V., Stadlmann, S., Klocker, H., Abendstein, B., Eder, I.E., Rogatsch, H., Zeimet, A.G., Marth, C., Offner, F.A., 1999. Basic fibroblast growth factor synthesis by human peritoneal mesothelial cells: induction by interleukin-1. Am. J. Pathol. 155, 1977e1984. Dailey, L., Ambrosetti, D., Mansukhani, A., Basilico, C., 2005. Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev. 16, 233e247. Dinarello, C.A., 2009. Immunological and inflammatory functions of the interleukin1 family. Annu. Rev. Immunol. 27, 519e550.
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