A Novel Solid-Phase Site-Specific PEGylation Enhances the In Vitro and In Vivo Biostabilty of Recombinant Human Keratinocyte Growth Factor 1

Keratinocyte growth factor (KGF), also called fibroblast growth factor-7, is widely known as a paracrine growth and differentiation factor that is produced by mesenchymal cells and has been thought to act specifically on epithelial cells. Here it is shown to affect a new cell type, the microvascular endothelial cell. At subnanomolar concentrations KGF induced in vivo neovascularization in the rat cornea. In vitro it was not effective against endothelial cells cultured from large vessels, but did act directly on those cultured from small vessels, inducing chemotaxis with an ED50 of 0.02-0.05 ng/ml, stimulating proliferation and activating mitogen activated protein kinase (MAPK). KGF also helped to maintain the barrier function of monolayers of capillary but not aortic endothelial cells, protecting against hydrogen peroxide and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induced increases in permeability with an ED50 of 0.2-0.5 ng/ml. These newfound abilities of KGF to induce angiogenesis and to stabilize endothelial barriers suggest that it functions in microvascular tissue as it does in epithelial tissues to protect them against mild insults and to speed their repair after major damage.

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Effects of Keratinocyte Growth Factor on the Proliferation and Radiation Survival of Human Squamous Cell Carcinoma Cell Lines In Vitro and In Vivo

International Journal of Radiation Oncology*Biology*Physics ◽

10.1016/s0360-3016(97)00561-0 ◽

1998 ◽

Vol 40 (1) ◽

pp. 177-187 ◽

Cited By ~ 57

Author(s):

Shoucheng Ning M.D., Ph.D. ◽

Chaoxiang Shui M.D. ◽

Waqqar B Khan B.S. ◽

William Benson B.S., ◽

David L Lacey M.D. ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Growth Factor ◽

Cell Carcinoma ◽

Carcinoma Cell ◽

Keratinocyte Growth Factor ◽

Squamous Cell Carcinoma Cell ◽

Human Squamous Cell Carcinoma ◽

Carcinoma Cell Lines

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Regulation of thymic epithelium by keratinocyte growth factor

Blood ◽

10.1182/blood-2002-04-1036 ◽

2002 ◽

Vol 100 (9) ◽

pp. 3269-3278 ◽

Cited By ~ 107

Author(s):

Matthew Erickson ◽

Stanislaw Morkowski ◽

Sophie Lehar ◽

Geoffrey Gillard ◽

Courtney Beers ◽

...

Keyword(s):

Growth Factor ◽

Keratinocyte Growth Factor ◽

Cathepsin L ◽

Developmentally Regulated ◽

Thymic Epithelium ◽

Histocompatibility Complex ◽

Fetal Thymic Organ Culture ◽

And Function

Abstract Here we demonstrate that keratinocyte growth factor (KGF) and FGFR2IIIb signaling can affect development and function of thymic epithelium (TE) and that αβ-lineage thymocytes contribute to intrathymic levels of KGF. Thymocyte expression of KGF is developmentally regulated, being undetectable in CD3−4−8− thymocytes and expressed at highest levels by mature CD4 or CD8 thymocytes. Exposure of thymocyte-depleted fetal thymic lobes to KGF resulted in reduced thymic epithelial expression of class II major histocompatibility complex (MHC), invariant chain (Ii), and cathepsin L (CatL) molecules involved in thymocyte-positive selection and also stimulated expression of the cytokines interleukin 6 (IL-6) and thymic stromal-derived lymphopoietin (TSLP), while having little effect on IL-7 or stem cell factor expression. Within intact fetal thymic organ culture (FTOC), exogenous KGF impairs the generation of CD4 thymocytes. Two lines of evidence point to responsiveness of the medullary TE compartment to KGF and FGFR2IIIb signaling. First, the medullary compartment is expanded in intact FTOC exposed to KGF in vitro. Second, in the RAG-deficient thymus, where the thymocytes do not express detectable levels of KGF message, the hypoplastic medullary TE compartment can be expanded by administration of recombinant KGF in vivo. This expansion is accompanied by restoration of the normal profile of medullary TE–associated chemokine expression in the RAG2−/−thymus. Collectively, these findings point to a role for KGF and FGFR signaling in the development and function of thymic epithelium.

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Ribosomal S6 Kinase as a Mediator of Keratinocyte Growth Factor-induced Activation of Akt in Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e04-01-0043 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3106-3113 ◽

Cited By ~ 13

Author(s):

Zhong-Zong Pan ◽

Yvan Devaux ◽

Prabir Ray

Keyword(s):

Growth Factor ◽

Epithelial Cells ◽

Keratinocyte Growth Factor ◽

Growth Factor Receptor ◽

Protective Effects ◽

S6 Kinase ◽

Akt Activation ◽

Ribosomal S6 Kinase

The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily. The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors. Using the yeast two-hybrid assay, we have identified ribosomal S6 kinase (RSK) to be a protein that associates with the cytoplasmic domain of the KGFR. The RSK family of kinases controls multiple cellular processes, and our studies for the first time show association between the KGFR and RSK. Using a lung-specific inducible transgenic system we have recently demonstrated protective effects of KGF on the lung epithelium and have demonstrated KGF-induced activation of the prosurvival Akt pathway both in vivo and in vitro. Here we show that a kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress. It was recently shown that RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation. When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells.

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Separation of Fast from Slow Anabolism by Site-specific PEGylation of Insulin-like Growth Factor I (IGF-I)

Journal of Biological Chemistry ◽

10.1074/jbc.m110.172189 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19501-19510 ◽

Cited By ~ 34

Author(s):

Friedrich Metzger ◽

Waseem Sajid ◽

Stefanie Saenger ◽

Christian Staudenmaier ◽

Chris van der Poel ◽

...

Keyword(s):

Growth Factor ◽

Binding Proteins ◽

Insulin Like Growth Factor ◽

Igf Binding Proteins ◽

Site Specific ◽

Factor I ◽

Igf I ◽

Growth Factor I

Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.

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Keratinocyte growth factor reduces alveolar epithelial susceptibility to in vitro mechanical deformation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.5.l1068 ◽

2001 ◽

Vol 281 (5) ◽

pp. L1068-L1077 ◽

Cited By ~ 36

Author(s):

Jane Oswari ◽

Michael A. Matthay ◽

Susan S. Margulies

Keyword(s):

Cell Death ◽

Growth Factor ◽

Lung Injury ◽

Keratinocyte Growth Factor ◽

Alveolar Type ◽

Alveolar Epithelial ◽

Ventilator Induced Lung Injury ◽

And Control

Keratinocyte growth factor (KGF) is a potent mitogen that prevents lung epithelial injury in vivo. We hypothesized that KGF treatment reduces ventilator-induced lung injury by increasing the alveolar epithelial tolerance to mechanical strain. We evaluated the effects of in vivo KGF treatment to rats on the response of alveolar type II (ATII) cells to in vitro controlled, uniform deformation. KGF (5 mg/kg) or saline (no-treatment control) was instilled intratracheally in rats, and ATII cells were isolated 48 h later. After 24 h in culture, both cell groups were exposed to 1 h of continuous cyclic strain (25% change in surface area); undeformed wells were included as controls. Cytotoxicity was evaluated quantitatively with fluorescent immunocytochemistry. There was >1% cell death in undeformed KGF-treated and control groups. KGF pretreatment significantly reduced deformation-related cell mortality to only 2.2 ± 1.3% (SD) from 49 ± 5.5% in control wells ( P < 0.001). Effects of extracellular matrix, actin cytoskeleton, and phenotype of KGF-treated and control cells were examined. The large reduction in deformation-induced cell death demonstrates that KGF protects ATII cells by increasing their strain tolerance and supports KGF treatment as a potential preventative measure for ventilator-induced lung injury.

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Studies on cultured Schwann cells: the induction of myelin synthesis, and the control of their proliferation by a new growth factor

Journal of Experimental Biology ◽

10.1242/jeb.95.1.215 ◽

1981 ◽

Vol 95 (1) ◽

pp. 215-230

Author(s):

J. P. Brockes ◽

K. J. Fryxell ◽

G. E. Lemke

Keyword(s):

Molecular Weight ◽

Growth Factor ◽

Schwann Cell ◽

Schwann Cells ◽

Solid Phase ◽

Immunological Methods ◽

Subunit Molecular Weight ◽

Cultured Schwann Cells

We have recently described the use of immunological methods to identify and purify rat Schwann cells. In dissociated cultures of neonatal sciatic nerve, all of the cells can be identified by antigenic criteria as either Schwann cells or fibroblasts. The fibroblasts may be removed by treatment with antiserum to the Thy-1 antigen and complement. The purified Schwann cells have been used to study the regulation of the expression of myelin components, and the stimulation of Schwann cell division by a soluble growth factor. Among the components of myelin, we have concentrated on the peripheral myelin glycoprotein P0, which constitutes 50–60% of the protein in peripheral myelin. We have studied the distribution of P0 in vitro and in vivo by immunofluorescence, immuno-autoradiography on SDS gels, and solid-phase radioimmunoassay. Our results support the hypothesis that P0 is induced specifically as a consequence of the interaction between the Schwann cell and the myelinated type of axon. The level of P0 in the myelin membrane is at least 1000-fold higher than in the Schwann cell membrane. Purified Schwann cells divide very slowly in a conventional tissue culture medium. This has allowed us to purify a new growth factor from extracts of brain and pituitary, tentatively named Glial Growth Factor (GGF). The activity resides in a basic protein with a native molecular weight of 6 × 10(4) daltons and a subunit molecular weight of 3 × 10(4) daltons, which is active at levels comparable to those of epidermal growth factor. GGF is mitogenic for Schwann cells, astrocytes and muscle fibroblasts.

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