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Frank J. Lovicu - One of the best experts on this subject based on the ideXlab platform.

  • negative regulation of Lens Fiber cell differentiation by rtk antagonists spry and spred
    Experimental Eye Research, 2018
    Co-Authors: Guannan Zhao, Charles G Bailey, Yue Feng, John E J Rasko, Frank J. Lovicu
    Abstract:

    Abstract Sprouty (Spry) and Spred proteins have been identified as closely related negative regulators of the receptor tyrosine kinase (RTK)-mediated MAPK pathway, inhibiting cellular proliferation, migration and differentiation in many systems. As the different members of this antagonist family are strongly expressed in the Lens epithelium in overlapping patterns, in this study we used Lens epithelial explants to examine the impact of these different antagonists on the morphologic and molecular changes associated with fibroblast growth factor (FGF)-induced Lens Fiber differentiation. Cells in Lens epithelial explants were transfected using different approaches to overexpress the different Spry (Spry1, Spry2) and Spred (Spred1, Spred2, Spred3) members, and we compared their ability to undergo FGF-induced Fiber differentiation. In cells overexpressing any of the antagonists, the propensity for FGF-induced cell elongation was significantly reduced, indicative of a block to Lens Fiber differentiation. Of these antagonists, Spry1 and Spred2 appeared to be the most potent among their respective family members, demonstrating the greatest block in FGF-induced Fiber differentiation based on the percentage of cells that failed to elongate. Consistent with the reported activity of Spry and Spred, we show that overexpression of Spry2 was able to suppress FGF-induced ERK1/2 phosphorylation in Lens cells, as well as the ERK1/2-dependent Fiber-specific marker Prox1, but not the accumulation of β-crystallins. Taken together, Spry and Spred proteins that are predominantly expressed in the Lens epithelium in situ, appear to have overlapping effects on negatively regulating ERK1/2-signaling associated with FGF-induced Lens epithelial cell elongation leading to Fiber differentiation. This highlights the important regulatory role for these RTK antagonists in establishing and maintaining the distinct architecture and polarity of the Lens.

  • wnt frizzled signaling is part of an fgf induced cascade that promotes Lens Fiber differentiation
    Investigative Ophthalmology & Visual Science, 2013
    Co-Authors: L J Dawes, Frank J. Lovicu, Yuki Sugiyama, Ana S Tanedo, John W. Mcavoy
    Abstract:

    Purpose. It is well established that Lens Fiber differentiation depends on an FGF-initiated growth factor signaling cascade. Given that recent studies indicate Wnt-Frizzled/Planar Cell Polarity (Wnt-Fz/PCP) signaling has a role in coordinating the orientation and alignment of Fibers, this study set out to investigate the relationship between this pathway and FGF-induced Fiber differentiation.

  • frs2α enhances fibroblast growth factor mediated survival and differentiation in Lens development
    Development, 2012
    Co-Authors: Bhavani P Madakashira, Frank J. Lovicu, Lixing W Reneker, Brad D Wagner, Daniel A Kobrinski, Andrew D Hancher, Elizabeth C Arneman, Fen Wang, Hailey Shin, Michael L. Robinson
    Abstract:

    Most growth factor receptor tyrosine kinases (RTKs) signal through similar intracellular pathways, but they often have divergent biological effects. Therefore, elucidating the mechanism of channeling the intracellular effect of RTK stimulation to facilitate specific biological responses represents a fundamental biological challenge. Lens epithelial cells express numerous RTKs with the ability to initiate the phosphorylation (activation) of Erk1/2 and PI3-K/Akt signaling. However, only Fgfr stimulation leads to Lens Fiber cell differentiation in the developing mammalian embryo. Additionally, within the Lens, only Fgfrs activate the signal transduction molecule Frs2α. Loss of Frs2α in the Lens significantly increases apoptosis and decreases phosphorylation of both Erk1/2 and Akt. Also, Frs2α deficiency decreases the expression of several proteins characteristic of Lens Fiber cell differentiation, including Prox1, p57KIP2, aquaporin 0 and β-crystallins. Although not normally expressed in the Lens, the RTK TrkC phosphorylates Frs2α in response to binding the ligand NT3. Transgenic Lens epithelial cells expressing both TrkC and NT3 exhibit several features characteristic of Lens Fiber cells. These include elongation, increased Erk1/2 and Akt phosphorylation, and the expression of β-crystallins. All these characteristics of NT3-TrkC transgenic Lens epithelial cells depend on Frs2α. Therefore, tyrosine phosphorylation of Frs2α mediates Fgfr-dependent Lens cell survival and provides a mechanistic basis for the unique Fiber-differentiating capacity of Fgfs on mammalian Lens epithelial cells.

  • growth factor signaling in vitreous humor induced Lens Fiber differentiation
    Investigative Ophthalmology & Visual Science, 2010
    Co-Authors: Qian Wang, John W. Mcavoy, Frank J. Lovicu
    Abstract:

    The Lens has a distinctive architecture, with an anterior monolayer of Lens epithelial cells overlying a mass of elongated Fiber cells. The Lens grows as Lens epithelial cells proliferate at the Lens equator and differentiate into secondary Fiber cells. The surrounding ocular media, the aqueous humor that bathes the Lens epithelial cells and the vitreous humor that bathes the Lens Fiber cells, have been shown to be important in the maintenance of this distinctive polarity and architecture of the Lens.1 The ocular media contain members of several growth factor families (see Ref. 2) including, insulin-like growth factor (IGF),3 fibroblast growth factor (FGF),4 platelet-derived growth factor (PDGF),5 epidermal growth factor (EGF),6 hepatocyte growth factor (HGF),7 and vascular endothelial growth factor (VEGF).8,9 Studies over the past two decades have linked these growth factors in various ways to the process of Lens Fiber differentiation. Extensive experiments with Lens epithelial explants have shown that FGF induces Lens Fiber cell differentiation.10 In vitro, FGF has been shown to induce many of the morphologic and molecular changes associated with Fiber differentiation, including cell elongation, the loss of cytoplasmic organelles, denucleation, and the accumulation of Fiber-specific β- and γ-crystallins.11–13 Of interest, these experiments showed that FGF could also induce Lens epithelial cell proliferation, with a low dose of FGF inducing cell proliferation and a much higher concentration required to induce Fiber differentiation.14 Based on this, and together with studies examining the distribution of FGF in the eye, it was proposed that an anteroposterior gradient of FGF in the eye may determine Lens polarity,10,15 with a low concentration of FGF in aqueous stimulating proliferation and a higher concentration of FGF in the vitreous inducing Lens Fiber differentiation. In situ experiments using transgenic mice to misexpress FGFs and/or FGF receptors, were also shown to impair the normal development and growth of the Lens,16–22 further underpinning the important and essential role for FGF in Lens differentiation. To date, FGF has been demonstrated to be the only growth factor able to induce Lens Fiber differentiation; however, other growth factors such as IGF and/or PDGF and Wnts have been shown to potentiate FGF-induced Lens Fiber differentiation in vitro.23–26 In transgenic mice that overexpressed IGF in the Lens, the germinative and transitional zones were shown to expand, but no inappropriate differentiation of Lens epithelial cells was observed.27 Overexpression of PDGF in the Lens resulted in enlarged Lenses that developed cataracts.28 The Lens epithelium of these mice became multilayered with some evidence of Fiber differentiation changes28; however, subsequent in vitro experiments confirmed that PDGF cannot induce this Fiber differentiation process without FGF.25 Taken together, these in vitro and in vivo experiments suggest that IGF or PDGF do not directly induce Lens Fiber differentiation, but may play some role in this process. High-affinity receptors for FGF, IGF, PDGF, and EGF belong to different subclasses of RTKs29; however, they all can activate similar intercellular signaling pathways, including the ERK1/2 and PI3-K pathways through their association with common adaptor proteins.30–32 It has been established that ERK1/2 signaling is essential for both Lens cell proliferation and differentiation.33–35 More recent studies have shown that the duration of ERK1/2 phosphorylation is associated with a specific cell fate in the Lens.36 Although both FGF and vitreous can induce Lens Fiber differentiation, their ability to stimulate phosphorylation of ERK1/2 and Akt differs to some degree.36,37 As other vitreous-derived growth factors can also stimulate the phosphorylation of ERK1/2 and Akt signaling pathways, we propose that a combination of factors participate in vitreous-induced Lens Fiber differentiation. To better define the role of these growth factors in the vitreous, we compared their ability to phosphorylate ERK1/2 and Akt in Lens epithelial cells, either independently or in combination with FGF. Our findings demonstrate a correlation between the different ERK/Akt phosphorylation profiles and the degree of growth factor-induced Fiber differentiation. The results clearly show that IGF, PDGF, and EGF can all prolong and potentiate FGF-induced ERK1/2 activation, leading to Lens Fiber differentiation. Our blocking studies using a range of selective RTK inhibitors to block vitreous-induced ERK/Akt signaling and Fiber differentiation show that FGF is an essential growth factor involved in vitreous-induced Lens Fiber differentiation and that IGF, PDGF, and EGF may also be involved in this process by regulating ERK1/2 and Akt phosphorylation. Finally, these studies indicate that prolonged phosphorylation of ERK1/2 is necessary but not sufficient for Fiber differentiation to proceed.

  • a role for wnt planar cell polarity signaling during Lens Fiber cell differentiation
    Seminars in Cell & Developmental Biology, 2006
    Co-Authors: Yongjuan Chen, John W. Mcavoy, Frank J. Lovicu, R J W Stump
    Abstract:

    Wnt signaling through frizzled (Fz) receptors plays key roles in just about every developmental system that has been studied. Several Wnt-Fz signaling pathways have been identified including the Wnt/planar cell polarity (PCP) pathway. PCP signaling is crucial for many developmental processes that require major cytoskeletal rearrangements. Downstream of Fz, PCP signaling is thought to involve the GTPases, Rho, Rac and Cdc42 and regulation of the JNK cascade. Here we report on the localization of these GTPases and JNK in the Lens and assess their involvement in the cytoskeletal reorganisation that is a key element of FGF-induced Lens Fiber cell differentiation.

John W. Mcavoy - One of the best experts on this subject based on the ideXlab platform.

  • wnt frizzled signaling is part of an fgf induced cascade that promotes Lens Fiber differentiation
    Investigative Ophthalmology & Visual Science, 2013
    Co-Authors: L J Dawes, Frank J. Lovicu, Yuki Sugiyama, Ana S Tanedo, John W. Mcavoy
    Abstract:

    Purpose. It is well established that Lens Fiber differentiation depends on an FGF-initiated growth factor signaling cascade. Given that recent studies indicate Wnt-Frizzled/Planar Cell Polarity (Wnt-Fz/PCP) signaling has a role in coordinating the orientation and alignment of Fibers, this study set out to investigate the relationship between this pathway and FGF-induced Fiber differentiation.

  • growth factor signaling in vitreous humor induced Lens Fiber differentiation
    Investigative Ophthalmology & Visual Science, 2010
    Co-Authors: Qian Wang, John W. Mcavoy, Frank J. Lovicu
    Abstract:

    The Lens has a distinctive architecture, with an anterior monolayer of Lens epithelial cells overlying a mass of elongated Fiber cells. The Lens grows as Lens epithelial cells proliferate at the Lens equator and differentiate into secondary Fiber cells. The surrounding ocular media, the aqueous humor that bathes the Lens epithelial cells and the vitreous humor that bathes the Lens Fiber cells, have been shown to be important in the maintenance of this distinctive polarity and architecture of the Lens.1 The ocular media contain members of several growth factor families (see Ref. 2) including, insulin-like growth factor (IGF),3 fibroblast growth factor (FGF),4 platelet-derived growth factor (PDGF),5 epidermal growth factor (EGF),6 hepatocyte growth factor (HGF),7 and vascular endothelial growth factor (VEGF).8,9 Studies over the past two decades have linked these growth factors in various ways to the process of Lens Fiber differentiation. Extensive experiments with Lens epithelial explants have shown that FGF induces Lens Fiber cell differentiation.10 In vitro, FGF has been shown to induce many of the morphologic and molecular changes associated with Fiber differentiation, including cell elongation, the loss of cytoplasmic organelles, denucleation, and the accumulation of Fiber-specific β- and γ-crystallins.11–13 Of interest, these experiments showed that FGF could also induce Lens epithelial cell proliferation, with a low dose of FGF inducing cell proliferation and a much higher concentration required to induce Fiber differentiation.14 Based on this, and together with studies examining the distribution of FGF in the eye, it was proposed that an anteroposterior gradient of FGF in the eye may determine Lens polarity,10,15 with a low concentration of FGF in aqueous stimulating proliferation and a higher concentration of FGF in the vitreous inducing Lens Fiber differentiation. In situ experiments using transgenic mice to misexpress FGFs and/or FGF receptors, were also shown to impair the normal development and growth of the Lens,16–22 further underpinning the important and essential role for FGF in Lens differentiation. To date, FGF has been demonstrated to be the only growth factor able to induce Lens Fiber differentiation; however, other growth factors such as IGF and/or PDGF and Wnts have been shown to potentiate FGF-induced Lens Fiber differentiation in vitro.23–26 In transgenic mice that overexpressed IGF in the Lens, the germinative and transitional zones were shown to expand, but no inappropriate differentiation of Lens epithelial cells was observed.27 Overexpression of PDGF in the Lens resulted in enlarged Lenses that developed cataracts.28 The Lens epithelium of these mice became multilayered with some evidence of Fiber differentiation changes28; however, subsequent in vitro experiments confirmed that PDGF cannot induce this Fiber differentiation process without FGF.25 Taken together, these in vitro and in vivo experiments suggest that IGF or PDGF do not directly induce Lens Fiber differentiation, but may play some role in this process. High-affinity receptors for FGF, IGF, PDGF, and EGF belong to different subclasses of RTKs29; however, they all can activate similar intercellular signaling pathways, including the ERK1/2 and PI3-K pathways through their association with common adaptor proteins.30–32 It has been established that ERK1/2 signaling is essential for both Lens cell proliferation and differentiation.33–35 More recent studies have shown that the duration of ERK1/2 phosphorylation is associated with a specific cell fate in the Lens.36 Although both FGF and vitreous can induce Lens Fiber differentiation, their ability to stimulate phosphorylation of ERK1/2 and Akt differs to some degree.36,37 As other vitreous-derived growth factors can also stimulate the phosphorylation of ERK1/2 and Akt signaling pathways, we propose that a combination of factors participate in vitreous-induced Lens Fiber differentiation. To better define the role of these growth factors in the vitreous, we compared their ability to phosphorylate ERK1/2 and Akt in Lens epithelial cells, either independently or in combination with FGF. Our findings demonstrate a correlation between the different ERK/Akt phosphorylation profiles and the degree of growth factor-induced Fiber differentiation. The results clearly show that IGF, PDGF, and EGF can all prolong and potentiate FGF-induced ERK1/2 activation, leading to Lens Fiber differentiation. Our blocking studies using a range of selective RTK inhibitors to block vitreous-induced ERK/Akt signaling and Fiber differentiation show that FGF is an essential growth factor involved in vitreous-induced Lens Fiber differentiation and that IGF, PDGF, and EGF may also be involved in this process by regulating ERK1/2 and Akt phosphorylation. Finally, these studies indicate that prolonged phosphorylation of ERK1/2 is necessary but not sufficient for Fiber differentiation to proceed.

  • a role for wnt planar cell polarity signaling during Lens Fiber cell differentiation
    Seminars in Cell & Developmental Biology, 2006
    Co-Authors: Yongjuan Chen, John W. Mcavoy, Frank J. Lovicu, R J W Stump
    Abstract:

    Wnt signaling through frizzled (Fz) receptors plays key roles in just about every developmental system that has been studied. Several Wnt-Fz signaling pathways have been identified including the Wnt/planar cell polarity (PCP) pathway. PCP signaling is crucial for many developmental processes that require major cytoskeletal rearrangements. Downstream of Fz, PCP signaling is thought to involve the GTPases, Rho, Rac and Cdc42 and regulation of the JNK cascade. Here we report on the localization of these GTPases and JNK in the Lens and assess their involvement in the cytoskeletal reorganisation that is a key element of FGF-induced Lens Fiber cell differentiation.

  • aberrant Lens Fiber differentiation in anterior subcapsular cataract formation a process dependent on reduced levels of pax6
    Investigative Ophthalmology & Visual Science, 2004
    Co-Authors: Frank J. Lovicu, Shizuya Saika, Philipp Steven, John W. Mcavoy
    Abstract:

    PURPOSE. TGFβ can induce development in Lenses of opaque subcapsular fibrotic plaques that have many features of human subcapsular cataracts. To understand further the events associated with the onset and progression of TGFβ-induced cataract, several different models for anterior subcapsular cataract (ASC) were used and characterized. METHODS. Anterior subcapsular plaques were induced in rat Lenses cultured with TGFβ and in transgenic mice overexpressing TGFβ in the Lens. ASC was also examined in Lenses of mice haploinsufficient for Pax6, as well as in human biopsy specimens. Immunofluorescence and in situ hybridization labeling were used to examine changes in patterns of gene expression associated with cataract formation in these models. RESULTS. Examination of TGFβ-induced cataract in transgenic mice established that the subcapsular plaques are composed of a heterogenous cell population: a population of myofibroblastic cells as well as a population of Lens-Fiber-like cells. Further support for phenotypic change comes from the observation that the cells in these plaques no longer expressed Lens epithelial markers, such as Pax6 and Connexin43. Subsequent examination of human biopsy specimens of ASC, as well as Lenses from Pax6-deficient mice, showed that the anterior subcapsular plaques in both cases were also composed of a heterogenous population of cells. In contrast, anterior subcapsular plaques that developed in vitro in response to TGFβ did not have this same cellular heterogeneity, as no Fiber-like cells were present. CONCLUSIONS. These findings suggest that in vivo, during TGFβ-induced cataract formation, some Lens epithelial cells transform into myofibroblastic cells, whereas others differentiate into Fiber cells. As this pathologic change is accompanied by altered expression of genes characteristic of the normal Lens epithelial cell phenotype and as Lenses from Pax6-deficient mice exhibit development of anterior subcapsular plaques closely resembling those induced by TGFβ in transgenic mice, the authors propose that a reduction in Pax6 levels may be essential for this pathologic process to progress. Furthermore, it is clear from these in vitro studies that TGFβ alone cannot reproduce the same morphologic and molecular changes associated with ASC formation in vivo, indicating that additional molecule(s) in the eye are important in this process.

  • aberrant Lens Fiber differentiation in anterior subcapsular cataract formation a process dependent on reduced levels of pax6
    Investigative Ophthalmology & Visual Science, 2004
    Co-Authors: Frank J. Lovicu, Shizuya Saika, Philipp Steven, John W. Mcavoy
    Abstract:

    PURPOSE: TGFbeta can induce development in Lenses of opaque subcapsular fibrotic plaques that have many features of human subcapsular cataracts. To understand further the events associated with the onset and progression of TGFbeta-induced cataract, several different models for anterior subcapsular cataract (ASC) were used and characterized. METHODS: Anterior subcapsular plaques were induced in rat Lenses cultured with TGFbeta and in transgenic mice overexpressing TGFbeta in the Lens. ASC was also examined in Lenses of mice haploinsufficient for Pax6, as well as in human biopsy specimens. Immunofluorescence and in situ hybridization labeling were used to examine changes in patterns of gene expression associated with cataract formation in these models. RESULTS: Examination of TGFbeta-induced cataract in transgenic mice established that the subcapsular plaques are composed of a heterogenous cell population: a population of myofibroblastic cells as well as a population of Lens-Fiber-like cells. Further support for phenotypic change comes from the observation that the cells in these plaques no longer expressed Lens epithelial markers, such as Pax6 and Connexin43. Subsequent examination of human biopsy specimens of ASC, as well as Lenses from Pax6-deficient mice, showed that the anterior subcapsular plaques in both cases were also composed of a heterogenous population of cells. In contrast, anterior subcapsular plaques that developed in vitro in response to TGFbeta did not have this same cellular heterogeneity, as no Fiber-like cells were present. CONCLUSIONS: These findings suggest that in vivo, during TGFbeta-induced cataract formation, some Lens epithelial cells transform into myofibroblastic cells, whereas others differentiate into Fiber cells. As this pathologic change is accompanied by altered expression of genes characteristic of the normal Lens epithelial cell phenotype and as Lenses from Pax6-deficient mice exhibit development of anterior subcapsular plaques closely resembling those induced by TGFbeta in transgenic mice, the authors propose that a reduction in Pax6 levels may be essential for this pathologic process to progress. Furthermore, it is clear from these in vitro studies that TGFbeta alone cannot reproduce the same morphologic and molecular changes associated with ASC formation in vivo, indicating that additional molecule(s) in the eye are important in this process.

Paul A. Overbeek - One of the best experts on this subject based on the ideXlab platform.

  • insertion of a pax6 consensus binding site into the αa crystallin promoter acts as a Lens epithelial cell enhancer in transgenic mice
    Investigative Ophthalmology & Visual Science, 2004
    Co-Authors: Haotian Zhao, Paul A. Overbeek, Michael L. Robinson, Ying Yang, Christian Rizo
    Abstract:

    Purpose Although the murine alphaA-crystallin promoter is the most commonly used promoter for achieving transgene expression in the developing Lens, this promoter directs transgene expression efficiently only in Lens Fiber cells. The purpose of the present study was to generate promoters capable of directing transgene expression to the entire Lens but not to the corneal epithelium. Methods Transgenic mice were generated with fragments of the murine alphaA- and alphaB-crystallin promoters, as well as with an alphaA-crystallin promoter engineered with the insertion of a Pax6 consensus binding site driving either human growth hormone (hGH) or Cre recombinase genes. hGH expression was evaluated by in situ hybridization and immunohistochemistry. Cre expression was revealed by x-gal staining after crossing Cre transgenic mice with a Cre reporter strain. Results Within the Lens, the -214/+38 alphaB-crystallin promoter fragment directed transgene expression in the Lens epithelium, but not in Fiber cells. The native -282/+43 alphaA-crystallin promoter drove transgene expression in the Lens Fiber cells of several independent lines of transgenic mice, but none of these mice demonstrated significant transgene expression in the Lens epithelium. In contrast, the insertion of a 32-bp sequence containing a Pax6 consensus binding site into the -282/+43 alphaA-crystallin promoter reproducibly led to transgene expression in the Lens epithelium as well as the Lens Fiber cells. Conclusions The inclusion of a Pax6 consensus binding site within the -282/+43 alphaA-crystallin promoter enhances the ability of this promoter to drive transgene expression in the Lens epithelium.

  • inhibition of Lens Fiber cell morphogenesis by expression of a mutant sv40 large t antigen that binds creb binding protein p300 but not prb
    Journal of Biological Chemistry, 2004
    Co-Authors: Q. Chen, Larry Fromm, D Liang, Paul A. Overbeek
    Abstract:

    Abstract Simian virus (SV) 40 large T antigen can both induce tumors and inhibit cellular differentiation. It is not clear whether these cellular changes are synonymous, sequential, or distinct responses to the protein. T antigen is known to bind to p53, to the retinoblastoma (Rb) family of tumor suppressor proteins, and to other cellular proteins such as p300 family members. To test whether SV40 large T antigen inhibits cellular differentiation in vivo in the absence of cell cycle induction, we generated transgenic mice that express in the Lens a mutant version of the early region of SV40. This mutant, which we term E107KΔ, has a deletion that eliminates synthesis of small t antigen and a point mutation (E107K) that results in loss of the ability to bind to Rb family members. At embryonic day 15.5 (E15.5), the transgenic Lenses show dramatic defects in Lens Fiber cell differentiation. The Fiber cells become post-mitotic, but do not elongate properly. The cells show a dramatic reduction in expression of their β- and γ-crystallins. Because CBP and p300 are co-activators for crystallin gene expression, we assayed for interactions between E107KΔ and CBP/p300. Our studies demonstrate that cellular differentiation can be inhibited by SV40 large T antigen in the absence of pRb inactivation, and that interaction of large T antigen with CBP/p300 may be enhanced by a mutation that eliminates the binding to pRb.

  • distinct capacities of individual e2fs to induce cell cycle re entry in postmitotic Lens Fiber cells of transgenic mice
    Developmental Neuroscience, 2004
    Co-Authors: Q. Chen, D Liang, Tao Yang, Gustavo Leone, Paul A. Overbeek
    Abstract:

    Purpose: Inactivation of the retinoblastoma gene in human retinoblasts or mouse Lens Fiber cells causes inappropriate cell cycle entry, presumably as a consequence of elevated activ

  • requirement for tgfβ receptor signaling during terminal Lens Fiber differentiation
    Development, 2001
    Co-Authors: Robbert U De Iongh, Paul A. Overbeek, Frank J. Lovicu, Michael D Schneider, Josephine E Joya, Edna D Hardeman, John W. Mcavoy
    Abstract:

    Several families of growth factors have been identified as regulators of cell fate in the developing Lens. Members of the fibroblast growth factor family are potent inducers of Lens Fiber differentiation. Members of the transforming growth factor β (TGFβ) family, particularly bone morphogenetic proteins, have also been implicated in various stages of Lens and ocular development, including Lens induction and Lens placode formation. However, at later stages of Lens development, TGFβ family members have been shown to induce pathological changes in Lens epithelial cells similar to those seen in forms of human subcapsular cataract. Previous studies have shown that type I and type II TGFβ receptors, in addition to being expressed in the epithelium, are also expressed in patterns consistent with a role in Lens Fiber differentiation. In this study we have investigated the consequences of disrupting TGFβ signaling during Lens Fiber differentiation by using the mouse αΑ-crystallin promoter to overexpress mutant (kinase deficient), dominant-negative forms of either type I or type II TGFβ receptors in the Lens Fibers of transgenic mice. Mice expressing these transgenes had pronounced bilateral nuclear cataracts. The phenotype was characterized by attenuated Lens Fiber elongation in the cortex and disruption of Fiber differentiation, culminating in Fiber cell apoptosis and degeneration in the Lens nucleus. Inhibition of TGFβ signaling resulted in altered expression patterns of the Fiber-specific proteins, α-crystallin, fiLensin, phakinin and MIP. In addition, in an in vitro assay of cell migration, explanted Lens cells from transgenic mice showed impaired migration on laminin and a lack of actin filament assembly, compared with cells from wild-type mice. These results indicate that TGFβ signaling is a key event during Fiber differentiation and is required for completion of terminal differentiation.

  • secreted fgfr3 but not fgfr1 inhibits Lens Fiber differentiation
    Development, 2001
    Co-Authors: Venkatesh Govindarajan, Paul A. Overbeek
    Abstract:

    The vertebrate Lens has a distinct polarity with cuboidal epithelial cells on the anterior side and differentiated Fiber cells on the posterior side. It has been proposed that the anterior-posterior polarity of the Lens is imposed by factors present in the ocular media surrounding the Lens (aqueous and vitreous humor). The differentiation factors have been hypothesized to be members of the fibroblast growth factor (FGF) family. Though FGFs have been shown to be sufficient for induction of Lens differentiation both in vivo and in vitro, they have not been demonstrated to be necessary for endogenous initiation of Fiber cell differentiation. To test this possibility, we have generated transgenic mice with ocular expression of secreted self-dimerizing versions of FGFR1 (FR1) and FGFR3 (FR3). Expression of FR3, but not FR1, leads to an expansion of proliferating epithelial cells from the anterior to the posterior side of the Lens due to a delay in the initiation of Fiber cell differentiation. This delay is most apparent postnatally and correlates with appropriate changes in expression of marker genes including p57(KIP2), Maf and Prox1. Phosphorylation of Erk1 and Erk2 was reduced in the Lenses of FR3 mice compared with nontransgenic mice. Though differentiation was delayed in FR3 mice, the Lens epithelial cells still retained their intrinsic ability to respond to FGF stimulation. Based on these results we propose that the initiation of Lens Fiber cell differentiation in mice requires FGF receptor signaling and that one of the Lens differentiation signals in the vitreous humor is a ligand for FR3, and is therefore likely to be an FGF or FGF-like factor.

Velia M Fowler - One of the best experts on this subject based on the ideXlab platform.

  • tropomodulin 1 regulation of actin is required for the formation of large paddle protrusions between mature Lens Fiber cells
    Investigative Ophthalmology & Visual Science, 2016
    Co-Authors: Catherine Cheng, Roberta B Nowak, Paul G. Fitzgerald, Sondip K Biswas, Velia M Fowler
    Abstract:

    To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at Lens Fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single Lens Fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein.We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature Lens Fibers.F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature Fibers. In contrast, Tmod1-/- mature Fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature Fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature Fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature Fibers.These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between Lens Fibers.

  • tropomodulin 1 regulation of actin is required for the formation of large paddle protrusions between mature Lens Fiber cells
    Investigative Ophthalmology & Visual Science, 2016
    Co-Authors: Catherine Cheng, Roberta B Nowak, Paul G. Fitzgerald, Sondip K Biswas, Velia M Fowler
    Abstract:

    Author(s): Cheng, Catherine; Nowak, Roberta B; Biswas, Sondip K; Lo, Woo-Kuen; FitzGerald, Paul G; Fowler, Velia M | Abstract: PurposeTo elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at Lens Fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single Lens Fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein.MethodsWe investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature Lens Fibers.ResultsF-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature Fibers. In contrast, Tmod1-/- mature Fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature Fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature Fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature Fibers.ConclusionsThese results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between Lens Fibers.

  • Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry, Transparency, and Mechanical Stiffness of the Mouse Lens
    2012
    Co-Authors: David S. Gokhin, Roberta B Nowak, John I Clark, Nancy E. Kim, Ernest E. Arnett, Albert C. Chen, Robert L. Sah, Velia M Fowler
    Abstract:

    The basis for mammalian Lens Fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α2β2-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and fiLensin, which require CP49 for assembly and contribute to Lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected Lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze Fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases Lens Fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and fiLensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating Lens Fiber cell geometry, transparency, and mechanical stiffness.

  • modulation of n cadherin junctions and their role as epicenters of differentiation specific actin regulation in the developing Lens
    Developmental Biology, 2011
    Co-Authors: M Leonard, Roberta B Nowak, Velia M Fowler, Liping Zhang, Ni Zhai, Ahmad Cader, Yim Chan, Sue A Menko
    Abstract:

    Abstract Extensive elongation of Lens Fiber cells is a central feature of Lens morphogenesis. Our study investigates the role of N-cadherin junctions in this process in vivo. We investigate both the molecular players involved in N-cadherin junctional maturation and the subsequent function of these junctions as epicenters for the assembly of an actin cytoskeleton that drives morphogenesis. We present the first evidence of nascent cadherin junctions in vivo, and show that they are a prominent feature along lateral interfaces of undifferentiated Lens epithelial cells. Maturation of these N-cadherin junctions, required for Lens cell differentiation, preceded organization of a cortical actin cytoskeleton along the cells' lateral borders, but was linked to recruitment of α-catenin and dephosphorylation of N-cadherin-linked β-catenin. Biochemical analysis revealed differentiation-specific recruitment of actin regulators cortactin and Arp3 to maturing N-cadherin junctions of differentiating cells, linking N-cadherin junctional maturation with actin cytoskeletal assembly during Fiber cell elongation. Blocking formation of mature N-cadherin junctions led to reduced association of α-catenin with N-cadherin, prevented organization of actin along lateral borders of differentiating Lens Fiber cells and blocked their elongation. These studies provide a molecular link between N-cadherin junctions and the organization of an actin cytoskeleton that governs Lens Fiber cell morphogenesis in vivo.

  • caspase remodeling of the spectrin membrane skeleton during Lens development and aging
    Journal of Biological Chemistry, 2001
    Co-Authors: Andria Lee, Jon S Morrow, Velia M Fowler
    Abstract:

    Abstract Terminal differentiation of Lens Fiber cells resembles the apoptotic process in that organelles are lost, DNA is fragmented, and changes in membrane morphology occur. However, unlike classically apoptotic cells, which are disintegrated by membrane blebbing and vesiculation, aging Lens Fiber cells are compressed into the center of the Lens, where they undergo cell-cell fusion and the formation of specialized membrane interdigitations. In classically apoptotic cells, caspase cleavage of the cytoskeletal protein α-spectrin to ∼150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave α-spectrin to ∼150-kDa fragments and β-spectrin to ∼120- and ∼80-kDa fragments during late embryonic chick Lens development. These fragments continue to accumulate with age so that in the oldest Fiber cells of the adult Lens, most, if not all, of the spectrin is cleaved to discrete fragments. Thus, unlike classical apoptosis, where caspase-cleaved spectrin is short lived, Lens Fiber cells contain spectrin fragments that appear to be stable for the lifetime of the organism. Moreover, fragmentation of spectrin results in reduced membrane association and thus may lead to permanent remodeling of the membrane skeleton. Partial and specific proteolysis of membrane skeleton components by caspases may be important for age-related membrane changes in the Lens.

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  • fibroblast growth factor receptor signaling is essential for Lens Fiber cell differentiation
    Developmental Biology, 2008
    Co-Authors: Haotian Zhao, C M Garcia, Kai Yu, David M Ornitz, Tianyu Yang, Bhavani P Madakashira, Cornelius A Thiels, Chad A Bechtle, Huiming Zhang, David C Beebe
    Abstract:

    Abstract The vertebrate Lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for Lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of Lens development. Deletion of three FGF receptors (Fgfr1–3) early in Lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal Lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in Lenses lacking all three Fgfrs. These included lack of Fiber cell elongation, abnormal proliferation in prospective Lens Fiber cells, reduced expression of the cell cycle inhibitors p27kip1 and p57kip2, increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and α-, β- and γ-crystallins. Therefore, while signaling by FGF receptors is essential for Lens Fiber differentiation, different FGF receptors function redundantly.

  • identification and expression of hop an atypical homeobox gene expressed late in Lens Fiber cell terminal differentiation
    Molecular Vision, 2007
    Co-Authors: Oleg Vasiliev, Simon J Rhodes, David C Beebe
    Abstract:

    The Lens is composed of two types of epithelial cells: Asheet of cuboidal cells, the Lens epithelium, covers its anteriorsurface, and post-mitotic, elongated Fiber cells comprise thebulk of the Lens (Figure 1). Stimulation by factors present inthe vitreous body causes epithelial cells near the Lens equatorto withdraw from the cell cycle and differentiate into Lens fi-ber cells. Differentiating Fiber cells elongate and initiate thetranscription of genes that encode a distinct array of abundantmembrane, cytoskeletal, and cytoplasmic proteins. The accu-mulation of high concentrations of cytoplasmic proteins(crystallins) in Fiber cells is important for the transparencyand refractive power of the Lens. Some crystallins, cytoskeletal,and membrane proteins are found primarily in Lens cells, orare present only at very low levels in non-Lens tissues [1-7].Lens Fiber cells undergo remarkable morphologicalchanges during their differentiation. Fiber cells first elongateto many times their original length, extending to over 140 µmper day in the chicken embryo [8]. As they elongate, the ante-rior and posterior ends of the Fiber cells extend beneath theLens epithelium and along the posterior Lens capsule towardthe optical axis. When the ends of these cells approach theanterior and posterior poles of the Lens, they meet elongatingFiber cells extending from the other side, resulting in the for-mation of the anterior and posterior sutures (Figure 1). Oncethe cells stop elongating, they become buried beneath the nextgroup of elongating Fiber cells. Soon after the Fiber cells de-tach from the posterior capsule, the composition of their cell-cell adhesion proteins changes [9], their lateral membranesbecome interdigitated [8] and partially fuse with the mem-branes of neighboring Fiber cells [8,10], and all intracellular,membrane-bound organelles are degraded [11-17]. Mature fi-ber cells persist in this state for the life of the organism.

  • signaling through fgf receptor 2 is required for Lens cell survival and for withdrawal from the cell cycle during Lens Fiber cell differentiation
    Developmental Dynamics, 2005
    Co-Authors: C M Garcia, Ruth Asherypadan, Michael L. Robinson, Kai Yu, Haotian Zhao, David M Ornitz, David C Beebe
    Abstract:

    Fibroblast growth factors (FGFs) play important roles in many aspects of development, including Lens development. The Lens is derived from the surface ectoderm and consists of an anterior layer of epithelial cells and elongated, terminally differentiated Fiber cells that form the bulk of the tissue. FGF signaling has been implicated in Lens induction, proliferation, and differentiation. To address the role of FGFs in Lens development, we inactivated FGF receptor-2 (Fgfr2) using a Cre transgene that is expressed in all prospective Lens cells from embryonic day 9.0. Inactivation of Fgfr2 shows that signaling through this receptor is not required for Lens induction or for the proliferation of Lens epithelial cells. However, Fgfr2 signaling is needed to drive Lens Fiber cells out of the cell cycle during their terminal differentiation. It also contributes to the normal elongation of primary Lens Fiber cells and to the survival of Lens epithelial cells. Developmental Dynamics 233:516 –527, 2005. © 2005 Wiley-Liss, Inc.

  • bone morphogenetic protein signaling and the initiation of Lens Fiber cell differentiation
    Development, 2002
    Co-Authors: Teri L Beleckyadams, Ruben Adler, David C Beebe
    Abstract:

    Previous studies showed that the retina produces factors that promote the differentiation of Lens Fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential Fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo Lenses. We have now observed that phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating Lens Fiber cells. Transduction of chicken embryo retinas and/or Lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delayed Lens Fiber cell elongation and increased cell death in the Lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo Lens epithelial cells to elongate into Fiber-like cells, these effects were inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 were able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants was stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of Lens Fiber cells, along with at least one additional, and still unknown factor.

  • changes in adhesion complexes define stages in the differentiation of Lens Fiber cells
    Investigative Ophthalmology & Visual Science, 2001
    Co-Authors: David C Beebe, Oleg Vasiliev, Yingbo Shui, Steven Bassnett
    Abstract:

    PURPOSE. During their differentiation, Lens Fiber cells elongate, detach from the Lens capsule, associate at the sutures, and degrade all cytoplasmic membrane-bound organelles. Changes in the expression or organization of cell adhesion and cytoskeleton-associated proteins were correlated with these events during Fiber cell differentiation in chicken embryos. METHODS. Fixed or living Lenses were sliced with a tissue slicer, permeabilized or extracted with detergents, stained with antibodies or fluorescent-labeled phalloidin, and viewed with a confocal microscope. The distribution of N-cadherin in elongating and mature Fiber cells was determined by Western blot analysis. Reverse transcription‐polymerase chain reaction (RTPCR) was used to determine the distribution of vinculin and paxillin transcripts. RESULTS. Staining for N-cadherin and band 4.1 protein decreased soon after Fiber cells detached from the capsule. Detergent extraction of Lens sections and Western blots of dissected Lens regions showed that much of this decrease in staining was due to epitope masking. Vinculin immunoreactivity was barely detectable on the lateral membranes of elongating Fiber cells but increased markedly once these cells reached their maximum length and formed the sutures. Staining for paxillin was also low in elongating Fiber cells but increased late in Fiber cell differentiation, just before the cells destroyed their membrane-bound organelles. Spectrin and ankyrin immunoreactivity did not change when Fiber cells reached the sutures. Staining for F-actin increased transiently in cells that had just reached the sutures. Messenger RNAs for vinculin and paxillin were more abundant in maturing than in elongating Fiber cells. CONCLUSIONS. The adhesion complexes of Lens Fiber cells change in organization and composition soon after these cells finish elongating and detach from the capsule. Increased staining for vinculin and paxillin defines distinct stages of Fiber cell differentiation that are intermediate between the completion of cell elongation and the time when Lens Fiber cells degrade their membrane-bound organelles. Remodeling adhesion complexes during Fiber cell maturation may assure the stability of Fiber‐Fiber associations, once these cells are no longer transcriptionally active. (Invest Ophthalmol Vis Sci. 2001;42: 727‐734) T