Computer‐assisted engineering of hyperstable fibroblast growth factor 2

Dvořák, Pavel; Bednář, David; Vaňáček, Pavel; Bálek, Lukáš; Eiselleová, Lívia; Štěpánková, Veronika; Šebestová, Eva; Bosakova, Michaela Kunova; Konečná, Žaneta; Mazurenko, Stanislav; Kunka, Antonín; Váňová, Tereza; Zoufalova, Karolina; Chaloupková, Radka; Brezovský, Jan; Krejčı́, Pavel; Prokop, Zbyněk; Dvořák, Petr; Damborský, Jiřı́

doi:10.1002/bit.26531

Cited by 53 publications

(40 citation statements)

References 48 publications

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“…Interestingly, in these studies, 5 to 10 times higher concentration of FGF was used than in our study, suggesting that our cell culture protocol is more effective. Our study also shows the advantage of using hyperstable growth factor variants, such as FGF2-STAB, for lung organoid culture, and extends the cell culture potential of FGF2-STAB beyond the reported use in human embryonic stem cell culture (Dvorak et al, 2018).…”

Section: Discussionmentioning

confidence: 59%

“…Single epithelial cells from mouse lung were seeded in non-adherent plates in defined serum-free medium with epidermal growth factor (EGF) and FGF2 and cultured for 10 to 14 days, with addition of fresh medium every 3 days (Shaw et al, 2012; Rabata et al, 2017). Because FGF2 rapidly loses its biological activity at 37°C, we tested the use of FGF2-wt, as well as FGF2 with increased thermal stability (FGF2-STAB) (Dvorak et al, 2018) and sustained FGFR specificity (Koledova et al, 2019) for their capacity to support lungosphere formation. With FGF2-wt, the lungosphere formation efficiency (LFE) was 0.088 ± 0.006% (Figure S1A, B) and the lungospheres were of three different phenotypes: grape-like, cystic with a clearly defined lumen, and compound with regions resembling both the grape-like and the cystic phenotype.…”

Section: Resultsmentioning

confidence: 99%

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3D cell culture models demonstrate a role for FGF and WNT signaling in regulation of lung epithelial cell fate and morphogenesis

Rabata

Fedr

Souček

et al. 2020

Preprint

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15FGF signaling plays an essential role in lung development, homeostasis, and regeneration. Several 16 FGF ligands were detected in the developing lungs, however, their roles have not been fully elucidated. 17 We employed mouse 3D cell culture models and imaging to ex vivo study of a) the role of FGF ligands 18 in lung epithelial morphogenesis and b) the interplay of FGF signaling with epithelial growth factor 19 (EGF) and WNT signaling pathways. In non-adherent conditions, FGF signaling promoted formation 20 of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Based on their architecture, we 21 defined three distinct phenotypes of lungospheres. Ultrastructural and immunohistochemical analyses 22 showed that LSPCs produced more differentiated lung cell progeny. In 3D extracellular matrix, FGF2, 23 FGF7, FGF9, and FGF10 promoted lung organoid formation with similar efficiency. However, FGF9 24 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced 25 ability to sustain LSPCs survival and/or initial divisions. Analysis of lung organoid phenotypes 26 revealed that FGF7 and FGF10 produce bigger organoids and induce organoid branching with higher 27 frequency than FGF2 and FGF9. Higher FGF concentration and/or the use of FGF2 with increased 28 stability and affinity to FGF receptors both increased lung organoid and lungosphere formation 29 efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival 30 and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but 31 nonessential role in FGF-induced lung organoid formation. Moreover, analysis of tissue architecture 32 and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells 33 expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and 34 ciliated cells. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence 35 of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In 36 summary, we present lung 3D cell culture models as useful tools to study the role and interplay of 37 signaling pathways in lung development and we reveal roles for FGF ligands in regulation of mouse 38 lung morphogenesis ex vivo. 39 65 amplification of Fgf10 expressing airway smooth muscle cell progenitors in the distal mesenchyme 66 (Volckaert and De Langhe, 2015). In adult lung, FGF10 and WNT signaling regulate the activity of 67 basal cells, the lung epithelial stem/progenitor cells (LSPCs) that ensure lung epithelial homeostasis 68 and repair after injury (Volckaert et al., 2013). However, the exact functions of FGF and WNT 69 signaling in LSPCs have not been fully elucidated. 70In this study, we investigated the role of FGF and WNT signaling in regulation of epithelial 71 morphogenesis from LSPCs. To this end, we developed and used several 3D cell culture techniques, 72 including lungosphere an...

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Section: Discussionmentioning

confidence: 59%

Section: Resultsmentioning

confidence: 99%

3D cell culture models demonstrate a role for FGF and WNT signaling in regulation of lung epithelial cell fate and morphogenesis

Rabata

Fedr

Souček

et al. 2020

Preprint

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“…To overproduce FGF2-wt, FGF2-STAB1 and FGF2-STAB2 in Escherichia coli, the corresponding genes fgf2-G0, fgf2-G2, and fgf2-G3, respectively (Dvorak et al, 2018), were expressed under the control of the T7lac promoter and the gene expression was induced by the addition of isopropyl β-D-thiogalactopyranoside (IPTG). E. coli BL21(DE3) cells containing recombinant plasmids pET28b-His-thrombin:fgf2-wt, pET28b-His-thrombin:fgf2-STAB1 and pET28b-His-thrombin:fgf2-STAB2 were grown in 1 l of Luria broth medium with 50 µg/ml kanamycin at 37 • C. When the culture reached an optical density 0.6 at 600 nm, the induction of protein expression (at 20 • C) was initiated by the addition of IPTG to a final concentration of 0.5 mM.…”

Section: Preparation Of Fgf2 Protein Variantsmentioning

confidence: 99%

“…Besides its role in mediating FGF-FGFR interaction and complex formation, HS/heparin protects FGFs from inactivation in vivo. FGF2 is susceptible to aggregation, heat, acidic pH and proteolytic degradation, leading to the loss of its biological activity and function and to short half-life (≤10 h at 37 • C) (Dvorak et al, 2018). Binding to HS/heparin increases FGF2 stability (Gospodarowicz and Cheng, 1986;Caldwell et al, 2004), increasing the denaturation temperature of FGF2 by more than 20 • C (Buchtova et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we used FGF2 variants with increased thermal stability, FGF2-STAB1 and FGF2-STAB2, created by computerassisted protein engineering (Dvorak et al, 2018) to investigate the effect of FGF2 stability on FGF2-induced cell proliferation, dependence on heparin, and ERK1/2 signaling dynamics. After characterization of FGFR specificity and thorough testing of thermal stability of FGF2-STABs, we report on results from testing of heparin requirement for induction of proliferation in response to FGF2-wt and FGF2-STABs in BaF3-FGFR cell lines.…”

Section: Introductionmentioning

confidence: 99%

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Fibroblast Growth Factor 2 Protein Stability Provides Decreased Dependence on Heparin for Induction of FGFR Signaling and Alters ERK Signaling Dynamics

Koledová

Sumbal

Rabata

et al. 2019

Front. Cell Dev. Biol.

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Fibroblast growth factor 2 (FGF2) plays important roles in tissue development and repair. Using heparan sulfates (HS)/heparin as a cofactor, FGF2 binds to FGF receptor (FGFR) and induces downstream signaling pathways, such as ERK pathway, that regulate cellular behavior. In most cell lines, FGF2 signaling displays biphasic dose-response profile, reaching maximal response to intermediate concentrations, but weak response to high levels of FGF2. Recent reports demonstrated that the biphasic cellular response results from competition between binding of FGF2 to HS and FGFR that impinge upon ERK signaling dynamics. However, the role of HS/heparin in FGF signaling has been controversial. Several studies suggested that heparin is not required for FGF-FGFR complex formation and that the main role of heparin is to protect FGF from degradation. In this study, we investigated the relationship between FGF2 stability, heparin dependence and ERK signaling dynamics using FGF2 variants with increased thermal stability (FGF2-STABs). FGF2-STABs showed higher efficiency in induction of FGFR-mediated proliferation, lower affinity to heparin and were less dependent on heparin than wild-type FGF2 (FGF2-wt) for induction of FGFR-mediated mitogenic response. Interestingly, in primary mammary fibroblasts, FGF2-wt displayed a sigmoidal dose-response profile, while FGF2-STABs showed a biphasic response. Moreover, at low concentrations, FGF2-STABs induced ERK signaling more potently and displayed a faster dynamics of full ERK activation and higher amplitudes of ERK signaling than FGF2-wt. Our results suggest that FGF2 stability and heparin dependence are important factors in FGF-FGFR signaling complex assembly and ERK signaling dynamics.

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Enhancement of Wound Healing Efficacy by Increasing the Stability and Skin‐Penetrating Property of bFGF Using 30Kc19α‐Based Fusion Protein

Lee

Kim

et al. 2021

Advanced Biology

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involvement in tissue growth and regeneration, including wound healing. Activation of FGF receptor by bFGF stimulates intracellular signaling pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways which are related to the mitogenic response of cells. [1] As bFGF acts on various target cells involved in wound healing, its effects on wound healing have been extensively studied. [2] bFGF promotes the migration of dermal fibroblasts, keratinocytes, and endothelial cells, into wound sites and facilitates their proliferation. [3] As a result, bFGF accelerates granulation tissue formation and angiogenesis, which results in matrix formation and remodeling. [4] In recent years, a number of clinical uses of recombinant bFGF have been reported, and these include treatment of chronic wounds, second-degree burns, pressure ulcers, and diabetic foot ulcers. [5] However, its inherent instability and rapid degradation often require frequent treatments in tandem with higher concentrations. [6] The reported half-life of recombinant bFGF is less than 10 h under normal cell culture conditions and even shorter in the living organisms, which significantly limits The instability of recombinant basic fibroblast growth factor (bFGF) is a major disadvantage for its therapeutic use and means frequent applications to cells or tissues are required for sustained effects. Originating from silkworm hemolymph, 30Kc19α is a cell-penetrating protein that also has protein stabilization properties. Herein, it is investigated whether fusing 30Kc19α to bFGF can enhance the stability and skin penetration properties of bFGF, which may consequently increase its therapeutic efficacy. The fusion of 30Kc19α to bFGF protein increases protein stability, as confirmed by ELISA. 30Kc19α-bFGF also retains the biological activity of bFGF as it facilitates the migration and proliferation of fibroblasts and angiogenesis of endothelial cells. It is discovered that 30Kc19α can improve the transdermal delivery of a small molecular fluorophore through the skin of hairless mice. Importantly, it increases the accumulation of bFGF and further facilitates its translocation into the skin through follicular routes. Finally, when applied to a skin wound model in vivo, 30Kc19α-bFGF penetrates the dermis layer effectively, which promotes cell proliferation, tissue granulation, angiogenesis, and tissue remodeling. Consequently, the findings suggest that 30Kc19α improves the therapeutic functionalities of bFGF, and would be useful as a protein stabilizer and/or a delivery vehicle in therapeutic applications.

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Computer‐assisted engineering of hyperstable fibroblast growth factor 2

Cited by 53 publications

References 48 publications

3D cell culture models demonstrate a role for FGF and WNT signaling in regulation of lung epithelial cell fate and morphogenesis

3D cell culture models demonstrate a role for FGF and WNT signaling in regulation of lung epithelial cell fate and morphogenesis

Fibroblast Growth Factor 2 Protein Stability Provides Decreased Dependence on Heparin for Induction of FGFR Signaling and Alters ERK Signaling Dynamics

Enhancement of Wound Healing Efficacy by Increasing the Stability and Skin‐Penetrating Property of bFGF Using 30Kc19α‐Based Fusion Protein

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