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Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), plays a critical role in embryonic development, cell proliferation, and differentiation. However, efficient production of recombinant KGF remains a challenge due to its low expression levels and high tendency for aggregation in Escherichia coli. This study aimed to enhance the expression and solubility of KGF by employing different protein tags—PDIb'a', MBP, and His—fused to the N-terminus of KGF. Among these, H-PDIb'a'-KGF demonstrated superior stability and was selected for large-scale production and purification. The purified KGF was confirmed through liquid chromatography with tandem mass spectrometry analysis, which showed an 81% fragment mass identification coverage. Biological activity assessments using human breast cancer MCF-7 cells indicated that purified KGF significantly increased cell proliferation, with an EC 50 of 6.4 ± 0.5 pM. Interestingly, PDIb'a' alone also exhibited a stimulatory effect on MCF-7 cells. Furthermore, the purified KGF enhanced the wound healing of HaCaT keratinocytes in a dose-dependent manner. These findings provide valuable insights into the efficient production and functional characterization of recombinant KGF for potential applications in therapeutic interventions. • PDIb'a' fusion tag enhanced soluble expression of KGF in E. coli, enabling efficient purification. • Purified KGF potently stimulated MCF-7 cell proliferation with EC50 of 6.4 pM. • KGF promoted wound healing of HaCaT keratinocytes in dose-dependent manner. • PDIb'a' tag alone exhibited mitogenic activity on MCF-7 cells. • Approach enables cost-effective production of bioactive KGF for therapeutic use. [ABSTRACT FROM AUTHOR]

NKX2.1 is a master regulator of lung morphogenesis and cell specification; however, interactions of NKX2.1 with various transcription factors to regulate cell-specific gene expression and cell fate in the distal lung remain incompletely understood. FOXO1 is a key regulator of stem/progenitor cell maintenance/differentiation in several tissues but its role in the regulation of lung alveolar epithelial progenitor homeostasis has not been evaluated. We identified a novel role for FOXO1 in alveolar epithelial cell (AEC) differentiation that results in the removal of NKX2.1 from surfactant gene promoters and the subsequent loss of surfactant expression in alveolar epithelial type I-like (AT1-like) cells. We found that the FOXO1 forkhead domain potentiates a loss of surfactant gene expression through an interaction with the NKX2.1 homeodomain, disrupting NKX2.1 binding to the SFTPC promoter. In addition, blocking PI-3K/AKT signaling reduces phosphorylated FOXO-1 (p-FOXO1), allowing accumulated nuclear FOXO1 to interact with NKX2.1 in differentiating AEC. Inhibiting AEC differentiation in vitro with keratinocyte growth factor (KGF) maintained an AT2 cell phenotype through increased PI3K/AKT-mediated FOXO1 phosphorylation, resulting in higher levels of surfactant expression. Together these results indicate that FOXO1 plays a central role in AEC differentiation by directly binding NKX2.1 and suggests an essential role for FOXO1 in mediating AEC homeostasis.

Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway.

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Reindeer is the only deer species in both sexes grow antlers. Many apparent differences exist in antlers of female and male reindeer and sika deer. Insulin like growth factor 1 (IGF1), Keratinocyte growth factor (KGF) and Nerve Growth Factor (NGF) are essential for antler growth. To investigate whether epigenetic regulation of the growth factors is important in growth of sika deer, female and male reindeer antlers, methylation status were evaluated using bisulfite sequencing PCR (BSP). The 5' flanking regions of IGF1, KGF and NGF were cloned from reindeer and sika deer antlers, and the lengths were 2089bp/2107bp, 1474bp/1474bp and 865bp/865bp, respectively. Based on bioinformatics analysis, the regions of IGF1 (-50bp ~ +615bp), KGF (-285bp ~ +207bp) and NGF (-388bp ~ +109bp) were selected for studying the methylation status. The antler mesenchyme of male reindeer was the only methylation sample in IGF1 (2.23 ± 0.92%), significantly higher in methylation level than that of female reindeer and sika deer (0.01

Objective: To investigate the protective effect and mechanism of KGF combined with HIF-1α on IEC-6 cells knocked out by BECN1 under hypoxia stress. Methods: The stable BECN1 knockout cell line IEC-6B-cells were constructed and divided into blank control group (BC), negative control group (NC) and HIF-1 α Group, KGF joint HIF-1 α Group (KH). Observed the changes of cell morphology in each group. Cell survival rate, ATP content, cell cycle and apoptosis rate, autophagy related gene expression, apoptosis and autophagy related protein expression were detected in each group. Results: After 24 hours of hypoxia treatment, spindle, star and other abnormal morphological changes were observed in cells of each group. Electron microscopy showed that a large number of lysosomal vesicles with different sizes were found in the cytoplasm of NC cells. Other groups of cells showed morphological changes of early apoptosis, and autophagic vesicles were found in the cytoplasm of cells in BC group and KH group. Compared with BC group, the cell survival rate of each knockout group was significantly lower (P＜0.01), and the cell survival rate of KH group was higher than NC group,KGF group and HIF-1α group (P＜0.05). The content of ATP in BC group was significantly higher than that in other groups (P＜0.05).Compared with BC group and KH group, the percentage of G0/G1 phase and apoptosis rate of other three groups were significantly increased (P＜0.05). There was no significant difference between KH group and BC group (P＞0.05). Compared with BC group, the expression of BECN1, SQSTM1 and LC3 genes in NC group, KGF group, HIF-1α group and KH group decreased significantly (P＜0.05). Compared with BC group, the expression of Beclin1 protein and LC3 II/LC3 I in NC group, KGF group, HIF-1 α group and KH group decreased significantly (P＜0.05), and the expression of p62 protein increased significantly (P＜0.05). The ratio of Bax/Bcl-2 and the expression of Caspase3 protein in KH group were lower than those in NC group (P＜0.05). Conclusion: KGF combined with HIF-1α can protect IEC-6B-cells from hypoxia stress by promoting cell proliferation, enhancing cell energy metabolism, reducing cell arrest rate in G0/G1phase and inhibiting apoptosis. [ABSTRACT FROM AUTHOR]

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose‐derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen‐binding domain from Vibrio mimicus metalloprotease (vibrioCBD‐KGF). KGF and vibrioCBD‐KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK‐293 and MCF‐7 cells. vibrioCBD‐KGF demonstrated enhanced collagen‐binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD‐KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen‐coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin‐10 and Involucrin), and stem cell markers (Collagen‐I and Vimentin) by real‐time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD‐KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down‐regulation of Collagen‐I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD‐KGF. The present study showed that vibrioCBD‐KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen‐based biomaterials. [ABSTRACT FROM AUTHOR]

This study strategically incorporates epidermal growth factor (EGF) and keratinocyte growth factor (KGF) within a hyaluronic acid (HA) hydrogel to enhance corneal wound healing. The controlled release of EGF and KGF from the HA hydrogel is engineered to promote the regeneration of both the epithelial and stromal layers. Specifically, EGF plays a pivotal role in the regeneration of the epithelial layer, while KGF exhibits efficacy in the regeneration of the stromal layer. The combination of these growth factors facilitates efficient regeneration of each layer and demonstrates the capability to modulate each other's regenerative effects. The interplay between EGF and KGF provides an understanding of their cooperative influence on the dynamics of corneal wound healing. The results of this study contribute to the development of advanced strategies for corneal wound management and offer insights into the complex process of corneal regeneration. [ABSTRACT FROM AUTHOR]

Postoperative abdominal adhesion (PAA) is one of the more universal complications of abdominal surgery with a frequent incidence. Currently available keratinocyte growth factor (KGF)-based glues for the prevention of adhesions remain a great bottleneck since their long-term biological activity in vivo is insufficient. In this study, we fabricated hybrid polydopamine (PDA)-KGF nanoparticles (PDA-KGF NPs) by using an in situ self-assembly and polymerization method. The physicochemical properties of the PDA-KGF nanoparticles were systematically characterized. The effect of preventing PAA in rats was evaluated by using hybrid PDA-KGF NPs combined with hyaluronate (Ha). The expression levels of inflammatory factors and the degree of inflammatory cell infiltration in the injured peritoneum were evaluated by enzyme-linked immunosorbent assays and hematoxylin-eosin staining, respectively. The levels of phospho-Src expression were revealed by Western blotting. The degree of fibrosis and the density of deposited collagen fibers were measured with real-time reverse-transcription polymerase chain reaction and picrosirius red staining. The results indicated that the PDA-KGF NPs combined with Ha greatly prevented the incidence of abdominal adhesion s and promoted the repair of mesothelial cells in injured peritoneum. More importantly, the PDA-KGF NPs combined with Ha obviously reduced collagen deposition and fibrosis and inhibited the inflammatory response. Our results suggest that PDA-KGF NPs combined with Ha are promising barrier-like biomaterials for the effective prevention of postoperative tissue adhesion. Postoperative abdominal adhesion (PAA) as an inevitable postoperative complication affected the quality of life of patients. Currently available methods for preventing adhesions mainly employ degradable biomaterials. Previous research demonstrated that a hybrid keratinocyte growth factor (KGF)-sodium hyaluronate (Ha) gel could prevent the formation of PAAs. However, its clinical outcomes are not satisfactory since their bioactivity in vivo is too short. In this article, we fabricated hybrid polydopamine (PDA)-KGF nanoparticles (PDA-KGF NPs), which extend KGF bioactivity, effectively prevent PAA. Moreover, PDA-KGF NPs could remarkably reduce both collagen deposition and fibrosis, inhibit the inflammatory response, and promote mesothelial regeneration. Overall, the PDA-KGF NPs combined with Ha exhibit efficient antiadhesion properties, may provide a promising clinical protocol for the prevention of PAA. [Display omitted] [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway. [Display omitted] • MSC-CM and KGF had therapeutic effects on LPS-induced ALI model in mice. • KGF in MSC-CM could recover LPS-induced 3D alveolar organoid damage. • NF-κB signaling pathway may participate in the therapeutic effect of KGF in MSC-CM on ALI and the regulation of ENaC. [ABSTRACT FROM AUTHOR]

Keratinocyte growth factor (KGF) is a potential therapeutic factor in wound healing. However, its applications have been restricted due to its low stability, short half-life, and limited target specificity. We aimed to immobilize KGF on collagen-based biomaterials for long-lasting and targeted therapy by designing fusion forms of KGF with collagen-binding domains (CBD) from natural origins. Twelve fusion proteins were designed consisting of KGF and CBDs with different lengths and amino acid compositions. Three-dimensional (3D) structures of the fusions were predicted by homology modeling. Physiochemical properties and secondary structure of the fusions were evaluated by bioinformatics tools. Moreover, the effect of the CBDs on the 3D structure and dynamic behavior of the fusions was investigated by molecular dynamics (MD) simulation. The binding affinity of the fusions to collagen, KGF receptor, and heparin was assessed using docking tools. Our results demonstrated that fusions with small CBDs like CBD of mammalian collagenase and decapeptide CBD of von Willebrand factor (VWF) were more stable and properly folded than those with larger CBDs. On the other hand, the insertion of bulky CBDs, including Fibronectin CBD and CBD of Clostridium histolyticum collagenase, into KGF resulted in stronger binding to collagen. Therefore, very small or large CBDs are inappropriate for constructing KGF fusions because they suffer from low collagen affinity or poor stability. By comparing the results of MD simulation and docking, this study proposed that CBDs belonging to Vibrio mimicus metalloprotease and A3 domain of VWF would be good candidates to produce stable fusions with proper affinities toward collagen and KGF receptors. Moreover, the secondary structure analysis showed that the overall structure of KGF and CBDs was better preserved when CBDs were inserted at the C-terminal of KGF. This computational information about novel KGF fusions may help find the best constructs for experimental studies. [Display omitted] [ABSTRACT FROM AUTHOR]

Elastin-like peptides (ELPs) are a versatile platform for tissue engineering and drug delivery. Here, micelle forming ELP chains are genetically fused to three therapeutic molecules, keratinocyte growth factor (KGF), stromal cell-derived growth factor 1 (SDF1), and cathelicidin (LL37), to be used in wound healing. Chronic wounds represent a growing problem worldwide. A combinatorial therapy approach targeting different aspects of wound healing would be beneficial, providing a controlled and sustained release of active molecules, while simultaneously protecting these therapeutics from the surrounding harsh wound environment. The results of this study demonstrate that the conjugation of the growth factors KGF and SDF1 and the antimicrobial peptide LL37 to ELPs does not affect the micelle structure and that all three therapeutic moieties retain their bioactivity in vitro. Importantly, when the combination of these micelle ELP nanoparticles are applied to wounds in diabetic mice, over 90 % wound closure is observed, which is significantly higher than when the therapeutics are applied in their naked forms. The application of the nanoparticles designed here is the first report of targeting different aspect of wound healing synergistically., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)