Transcriptomic signature of painful human neurofibromatosis type 2 schwannomas.

Schwannomas are benign neoplasms that can cause gain- and loss-of-function neurological phenotypes, including severe, intractable pain. To investigate the molecular mechanisms underlying schwannoma-associated pain we compared the RNA sequencing profile of painful and non-painful schwannomas from NF2 patients. Distinct segregation of painful and non-painful tumors by gene expression patterns was observed. Differential expression analysis showed the upregulation of fibroblast growth factor 7 (FGF7) in painful schwannomas. Behavioral support for this finding was observed using a xenograft human NF2-schwannoma model in nude mice. In this model, over-expression of FGF7 in intra-sciatically implanted NF2 tumor cells generated pain behavior compared with controls.

Silencing fibroblast growth factor 7 inhibits krypton laser-induced choroidal neovascularization in a rat model.

Choroidal neovascularization (CNV), a characteristic of age-related macular degeneration, is an underlying cause of severe vision loss among elderly patients. Fibroblast growth factor (FGF) is suggested to exert an important role in the pathogenesis of CNV. However, the molecular mechanisms governing this event are not fully elucidated. Herein, we identified the potential role of FGF7 in CNV. To examine the roles of FGF7 in the progression of CNV, rat CNV models were established and treated with small interfering RNA (siRNA) against FGF7 or FGF7 overexpression, followed by identification of expression of FGF7 in the CNV modeled rats. Next, proliferation and migration, and in vitro tube formation of human umbilical vein endothelial cells, as well as expression of vascular endothelial growth factor (VEGF) and transforming growth factor-beta 2 (TGF-ß2) were evaluated. CNV led to upregulated FGF7 expression. Cells in the presence of FGF7 siRNA showed suppressed proliferation, migration, and tube formation, along with downregulated VEGF and TGF-ß2 expression. Taken together, functional suppression of FGF7 inhibited the onset of CNV, ultimately highlighting a novel therapeutic target for suppressing CNV progression.

Mir-455-3p-1 represses FGF7 expression to inhibit pulmonary arterial hypertension through inhibiting the RAS/ERK signaling pathway.

OBJECTIVE: To analyze the effects of miR-455-3p-1 and its possible mechanisms in pulmonary arterial hypertension (PAH). METHODS: A microarray assay was used to examine the expressed genes between normal and PAH. The expressed genes in PAH was assessed by qRT-PCR. The targeted interaction between miRNAs and FGF7 was confirmed using a dual luciferase reporter assay. A CCK-8 assay and cell count were used to analyze the pulmonary artery smooth muscle cells (PASMCs) activity and proliferation level, respectively. Apoptotic PASMCs were detected by flow cytometry. In addition, the mRNA and protein expression levels of RAS/ERK signaling pathway were determined by qRT-PCR and a Western blot assay, respectively. A PAH rat model was used to identify the effects of miR-455-3p-1 in vivo. RESULTS: FGF7 was upregulated in PAH. MiR-455-3p-1 was downregulated in PAH. MiR-455-3p-1 targeted FGF7. MiR-455-3p-1 decreased the expression of FGF7. Moreover, the effect of FGF7 on PASMCs was suppressed by miR-455-3p-1. MiR-455-3p-1 upregulation was associated with reduced mRNA and protein levels of core RAS/ERK signal genes, suggesting the inhibition of the RAS/ERK pathway. Furthermore, miR-455-3p-1 upregulation improved the RVSP, mPAP, ratio of RV/LV + S, CO and RV function of PAH rat model in vivo. CONCLUSION: Our findings illustrate a role for miR-455-3p-1 in modulating FGF7-RAS/ERK signaling and suggest that an agomir of miR-455-3p-1 could inhibit the proliferation of PASMCs and mitigate PAH in vivo.

TGF beta inhibits HGF, FGF7, and FGF10 expression in normal and IPF lung fibroblasts.

TGF beta is a multifunctional cytokine that is important in the pathogenesis of pulmonary fibrosis. The ability of TGF beta to stimulate smooth muscle actin and extracellular matrix gene expression in fibroblasts is well established. In this report, we evaluated the effect of TGF beta on the expression of HGF, FGF7 (KGF), and FGF10, important growth and survival factors for the alveolar epithelium. These growth factors are important for maintaining type II cells and for restoration of the epithelium after lung injury. Under conditions of normal serum supplementation or serum withdrawal TGF beta inhibited fibroblast expression of HGF, FGF7, and FGF10. We confirmed these observations with genome wide RNA sequencing of the response of control and IPF fibroblasts to TGF beta. In general, gene expression in IPF fibroblasts was similar to control fibroblasts. Reduced expression of HGF, FGF7, and FGF10 is another means whereby TGF beta impairs epithelial healing and promotes fibrosis after lung injury.

Producing functional recombinant human keratinocyte growth factor in Pichia pastoris and investigating its protective role against irradiation.

Keratinocyte Growth Factor (KGF) is a paracrine-acting, epithelial mitogen that plays a prominent role in the regeneration of damaged epithelial tissues. In spite of different attempts to produce recombinant human KGF in many organisms, including bacteria, mammalian cells, plant cells and insect cells; production of recombinant form suffers from lower yields and recovery relative to other recombinant proteins of similar size and properties. Due to many advantages of Pichia pastoris expression systems for producing industrial enzymes and pharmaceutical proteins, in this study P. pastoris was chosen as a host for KGF expression. For preparing human KGF coding sequence, MCF-7 cell line was treated with 1,25-Dihydroxy vitamin D3 for inducing the expression of KGF. The coding sequence of 23N-terminal truncated KGF form was amplified using RT-PCR technique and then cloned into the yeast expression vector in frame with the yeast α-factor secretion signal. The recombinant plasmid was integrated into Pichia pastoris strain X-33 genome. Western blotting and Mass Spectrometry demonstrated that recombinant human KGF (rhKGF) was correctly expressed after methanol induction and secreted into the media. The recombinant protein was purified from the media by heparin affinity chromatography. MTT assay showed that the purified rhKGF had a proliferative effect on NIH3T3 and A549 cell lines. In addition, protective effect of recombinant KGF was assessed in A549 cell line after irradiation. The results showed that the recombinant protein was biologically active. Finally, the effect of recombinant KGF was investigated on proliferation of MCF-7 cell line and its response to radiation. The results showed that pre-treatment of KGF have a protective effect on MCF-7 cell line after irradiation.

6-Gingerol inhibits hair cycle via induction of MMP2 and MMP9 expression.

6-Gingerol is the major active constituent of ginger. In the current study, we aimed to investigate the mechanisms underlying the effects of 6-Gingerol on hair growth. Mice were randomly divided into five groups; after hair depilation (day 0), mice were treated with saline, or different concentrations of 6-Gingerol for 11 days. The histomorphological characteristics of the growing hair follicles were examined after hematoxylin and eosin staining. The results indicated that 6-Gingerol significantly suppressed hair growth compared with that in the control group. And choose the concentration of 6-Gingerol at 1 mg/mL to treated with mice. Moreover, 6-Gingerol (1 mg/mL) significantly reduced hair re-growth ratio, hair follicle number, and hair follicle length, which were associated with increased expression of MMP2 and MMP9. Furthermore, the growth factors, such as EGF, KGF, VEGF, IGF-1 and TGF-ß participate in the hair follicle cycle regulation and regulate hair growth. We then measured the concentrations of them using ELISA assays, and the results showed that 6-Gingerol decreased EGF, KGF, VEGF, and IGF-1 concentrations, and increased TGF-ß concentration. Thus, this study showed that 6-Gingerol might act as a hair growth suppressive drug via induction of MMP2 and MMP9 expression, which could interfere with the hair cycle.

6-Gingerol inhibits hair cycle via induction of MMP2 and MMP9 expression

ABSTRACT 6-Gingerol is the major active constituent of ginger. In the current study, we aimed to investigate the mechanisms underlying the effects of 6-Gingerol on hair growth. Mice were randomly divided into five groups; after hair depilation (day 0), mice were treated with saline, or different concentrations of 6-Gingerol for 11 days. The histomorphological characteristics of the growing hair follicles were examined after hematoxylin and eosin staining. The results indicated that 6-Gingerol significantly suppressed hair growth compared with that in the control group. And choose the concentration of 6-Gingerol at 1 mg/mL to treated with mice. Moreover, 6-Gingerol (1 mg/mL) significantly reduced hair re-growth ratio, hair follicle number, and hair follicle length, which were associated with increased expression of MMP2 and MMP9. Furthermore, the growth factors, such as EGF, KGF, VEGF, IGF-1 and TGF-β participate in the hair follicle cycle regulation and regulate hair growth. We then measured the concentrations of them using ELISA assays, and the results showed that 6-Gingerol decreased EGF, KGF, VEGF, and IGF-1 concentrations, and increased TGF-β concentration. Thus, this study showed that 6-Gingerol might act as a hair growth suppressive drug via induction of MMP2 and MMP9 expression, which could interfere with the hair cycle.

Adenovirus vector expressing keratinocyte growth factor using CAG promoter impairs pulmonary function of mice with elastase-induced emphysema.

Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 109 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed.

Foxp3+ Regulatory T Cell Expression of Keratinocyte Growth Factor Enhances Lung Epithelial Proliferation.

Repair of the lung epithelium after injury is a critical component for resolution; however, the processes necessary to drive epithelial resolution are not clearly defined. Published data demonstrate that Foxp3+ regulatory T cells (Tregs) enhance alveolar epithelial proliferation after injury, and Tregs in vitro directly promote type II alveolar epithelial cell (AT2) proliferation, in part by a contact-independent mechanism. Therefore, we sought to determine the contribution of Treg-specific expression of a growth factor that is known to be important in lung repair, keratinocyte growth factor (kgf). The data demonstrate that Tregs express kgf and that Treg-specific expression of kgf regulates alveolar epithelial proliferation during the resolution phase of acute lung injury and in a model of regenerative alveologenesis in vivo. In vitro experiments demonstrate that AT2 cells cocultured with Tregs lacking kgf have decreased rates of proliferation compared with AT2 cells cocultured with wild-type Tregs. Moreover, Tregs isolated from lung tissue and grown in culture express higher levels of two growth factors that are important for lung repair (kgf and amphiregulin) compared with Tregs isolated from splenic tissue. Lastly, Tregs isolated from human lung tissue can be stimulated ex vivo to induce kgf expression. This study reveals mechanisms by which Tregs direct tissue-reparative effects during resolution after acute lung injury, further supporting the emerging role of Tregs in tissue repair.

Keratinocyte growth factor and its receptor expression in chronic otitis media with and without cholesteatoma.

INTRODUCTION: Chronic suppurative otitis media (CSOM) with and without cholesteatoma is regarded as chronic inflammation of the middle ear and mastoid mucosa that can be associated with the presence of granulation tissue and infection, which can lead to ossicular damage and hearing loss, but it is commonly known that cholesteatoma behaves aggressively. Both lesions appear to contain a predominant population of inflammatory cells, among which proinflammatory cytokines secreting keratinocyte growth factor (KGF) and its receptor (KGFR). No clear difference was demonstrated between these entities. The purpose of this study was to investigate the potential influence of KGF and KGFR in increased epithelial-cell proliferation of chronic otitis media (COM) with cholesteatoma in contrast to COM without cholesteatoma (CSOM), particularly in the granulative form, and to compare the rate of proliferation activity of epithelial cells using the Ki-67 epithelial proliferation marker expression. PATIENTS, MATERIALS AND METHODS: We analyzed 105 ears with cholesteatoma vs. 53 ears with CSOM without cholesteatoma using our KGF and KGFR variables, and the ratio of proliferating epithelial cells using Ki-67. The percentage of the specimens expressing KGF and KGFR was compared between the two groups for statistical significance using the Pearson's chi-square test. Immunohistochemical staining was conducted and the proportion of the cells staining positive for the nuclear antigen Ki-67 was evaluated in a quantitative and visual way, using light microscopes. RESULTS: KGF was positive in 88.57% of cholesteatoma and was positive in 41.51% CSOM without cholesteatoma specimens (cholesteatoma vs. CSOM, p=0.001). The positive rate of KGFR in the CSOM group was 33.96% compared to those in cholesteatoma, which was 60.95%. Compared to the cholesteatoma specimens, a significantly smaller number of Ki-67 labeling index was detected in CSOM specimens. CONCLUSIONS: Our results indicated that the abnormal behavior of the cholesteatoma epithelium seems to be induced by the paracrine interaction between KGF and KGFR. Furthermore, we found that cholesteatoma expressing both KGF and KGFR had high Ki-67 index, which correlated with its aggressiveness. These findings suggest that excessive KGF and KGFR synthesis may contribute to the hyperproliferative state in cholesteatoma and could explain the pathological difference between cholesteatoma and CSOM.

Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo.

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

Influences of keratinocyte growth factor - mesenchymal stem cells on chronic liver injury in rats.

To study the effects of keratinocyte growth factor (KGF) modified umbilical cord mesenchymal stem cells (MSC) on chronic liver injury in rats, adenovirus carrying human KGF gene (Ad-KGF) was used. Rat liver injury model was established by subcutaneous injection of CCl4 - olive oil solution; 56 male Wistar rats were randomly divided into normal control, model, MSC, KGF, and KGF/MSC groups. Of all three treatments, KGF/MSC had the most obvious therapeutic effects on liver injury, and cell injections did not cause adverse reaction. The experiment provides new data for clinical research of KGF/MSC and possible methods for liver injury treatment.

Mesenchymal stem cells ameliorate LPS-induced acute lung injury through KGF promoting alveolar fluid clearance of alveolar type II cells.

OBJECTIVE: It has been shown that Mesenchymal stem cells (MSCs) could prevent or alleviate acute lung injury (ALI) when transplanted into lung; however, the mechanisms involved remains elusive. The study aimed to investigate the effect of MSCs in repairing alveolar fluid clearance (AFC) of alveolar type-II (AT-II) cells through paracrine factors. MATERIALS AND METHODS: Keratinocyte growth factor (KGF) concentration in the supernatant of MSC culture medium was measured, and MSCs in lung after intravenous administration was traced. Next, MSCs transfected with or without KGF SiRNA were injected intravenously into LPS-induced ALI rats. Histological change and wet/dry ratio were determined. AT-II cells were co-cultured with MSCs under different experimental situations to analyze the variation of α1 and ß1 subunits of Na+-K+-ATPase in AT-II cells. RESULTS: LPS-induced ALI was characterized by the typical inflammatory infiltrates, interalveolar septal thickening and increased wet/dry ratio. MSC administration significantly reduced the injury, while MSCs with KGF knockdown did no show the same effect. In vitro study also confirmed that expressions of α1 and ß1 subunit were up-regulated as impaired AT-II cells co-cultured with MSCs. Furthermore, expression of α1 subunit was down-regulated, while ß1 subunit showed no apparent change as AT-II cells were co-cultured with MSCs that were transfected with KGF siRNA. CONCLUSIONS: AFC was impaired by inflammation insult. MSCs-derived KGF reduced the impaired AFC through up-regulated α1 subunit but not ß1 subunit, which might provide a novel therapeutic strategy for ALI.

In vivo over-expression of KGF mimic human middle ear cholesteatoma.

We reported previously that keratinocyte growth factor (KGF), a mesenchymal cell-derived paracrine growth factor, plays an important role in middle ear cholesteatoma formation, which is characterized by marked proliferation of epithelial cells. Here, we investigated whether KGF, the main factor that induces cholesteatoma, overexpression in vivo results in the formation of cholesteatoma. Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal (EAC) of rats once (short-term model) or five times on every fourth day (long-term model). Ears transfected with empty vector were used as controls. Successful transfection of plasmids into epithelial and stromal cells was confirmed by Flag immunohistochemistry. In the short-term model, the intensity of KGF protein was the strongest in hKGF transfected ear at day 4. KGF expression induced epithelial cell proliferation, reaching a peak level at day 4 and then decreased later, while in the long-term model, KGF expression in the EAC led to middle ear cholesteatoma formation. In conclusion, we described here a new experimental model of human middle ear cholesteatoma, and demonstrated that KGF and KGF receptor paracrine action play an essential role in middle ear cholesteatoma formation in an in vivo model.

A preterm pig model of lung immaturity and spontaneous infant respiratory distress syndrome.

Respiratory distress syndrome (RDS) and bronchopulmonary dysplasia remain the leading causes of preterm infant morbidity, mortality, and lifelong disability. Research to improve outcomes requires translational large animal models for RDS. Preterm pigs delivered by caesarian section at gestation days (GD) 98, 100, 102, and 104 were provided 24 h of neonatal intensive care, monitoring (pulse oximetry, blood gases, serum biomarkers, radiography), and nutritional support, with or without intubation and mechanical ventilation (MV; pressure control ventilation with volume guarantee). Spontaneous development of RDS and mortality without MV are inversely related with GD at delivery and correspond with inadequacy of tidal volume and gas exchange. GD 98 and 100 pigs have consolidated lungs, immature alveolar architecture, and minimal surfactant protein-B expression, and MV is essential at GD 98. Although GD 102 pigs had some alveoli lined by pneumocytes and surfactant was released in response to MV, blood gases and radiography revealed limited recruitment 1-2 h after delivery, and mortality at 24 h was 66% (35/53) with supplemental oxygen provided by a mask and 69% (9/13) with bubble continuous positive airway pressure (8-9 cmH2O). The lungs at GD 104 had higher densities of thin-walled alveoli that secreted surfactant, and MV was not essential. Between GD 98 and 102, preterm pigs have ventilation inadequacies and risks of RDS that mimic those of preterm infants born during the saccular phase of lung development, are compatible with standards of neonatal intensive care, and are alternative to fetal nonhuman primates and lambs.

Leptin promotes wound healing in the oral mucosa.

INTRODUCTION: Leptin, a 16 kDa circulating anti-obesity hormone, exhibits many physiological properties. Recently, leptin was isolated from saliva; however, its function in the oral cavity is still unclear. In this study, we investigated the physiological role of leptin in the oral cavity by focusing on its effect on wound healing in the oral mucosa. METHODS: Immunohistochemical analysis was used to examine the expression of the leptin receptor (Ob-R) in human/rabbit oral mucosa. To investigate the effect of leptin on wound healing in the oral mucosa, chemical wounds were created in rabbit oral mucosa, and leptin was topically administered to the wound. The process of wound repair was histologically observed and quantitatively analyzed by measuring the area of ulceration and the duration required for complete healing. The effect of leptin on the proliferation, differentiation and migration of human oral mucosal epithelial cells (RT7 cells) was investigated using crystal violet staining, reverse transcription polymerase chain reaction (RT-PCR) and a wound healing assay, respectively. RESULTS: Ob-R was expressed in spinous/granular cells in the epithelial tissue and vascular endothelial cells in the subepithelial connective tissue of the oral mucosa. Topical administration of leptin significantly promoted wound healing and shortened the duration required for complete healing. Histological analysis of gingival tissue beneath the ulceration showed a denser distribution of blood vessels in the leptin-treated group. Although the proliferation and differentiation of RT7 cells were not affected by leptin, the migration of these cells was accelerated in the presence of leptin. CONCLUSION: Topically administered leptin was shown to promote wound healing in the oral mucosa by accelerating epithelial cell migration and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the oral mucosa.

Production of functional active human growth factors in insects used as living biofactories.

Growth factors (GFs) are naturally signalling proteins, which bind to specific receptors on the cell surface. Numerous families of GFs have already been identified and remarkable progresses have been made in understanding the pathways that these proteins use to activate/regulate the complex signalling network involved in cell proliferation or wound healing processes. The bottleneck for a wider clinical and commercial application of these factors relay on their scalable cost-efficient production as bioactive molecules. The present work describes the capacity of Trichoplusia ni insect larvae used as living bioreactors in combination with the baculovirus vector expression system to produce three fully functional human GFs, the human epidermal growth factor (huEGF), the human fibroblast growth factor 2 (huFGF2) and the human keratinocyte growth factor 1 (huKGF1). The expression levels obtained per g of insect biomass were of 9.1, 2.6 and 3mg for huEGF, huFGF2 and huKGF1, respectively. Attempts to increase the productivity of the insect/baculovirus system we have used different modifications to optimize their production. Additionally, recombinant proteins were expressed fused to different tags to facilitate their purification. Interestingly, the expression of huKGF1 was significantly improved when expressed fused to the fragment crystallizable region (Fc) of the human antibody IgG. The insect-derived recombinant GFs were finally characterized in terms of biological activity in keratinocytes and fibroblasts. The present work opens the possibility of a cost-efficient and scalable production of these highly valuable molecules in a system that favours its wide use in therapeutic or cosmetic applications.

Recombinant keratinocyte growth factor 1 in tobacco potentially promotes wound healing in diabetic rats.

Keratinocyte growth factor 1 (KGF1) is a growth factor that promotes epidermal cell proliferation, migration, differentiation, and wound repair. It is expressed at low levels in a form of inclusion body in E. coli. In order to increase its expression and activity, we produced tobacco plants expressing KGF1 via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the KGF1 gene fused with a green florescence protein. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the KGF1 gene was correctly translated into the tobacco plants. The recombinant KGF1 was purified from plant tissues by heparin affinity chromatography, and cell proliferation in NIH/3T3 cells was stimulated by the purified KGF1. The purified KGF1 was also applied to the wounds of type-II diabetic rats. KGF1 had accumulated to levels as high as 530 µ g/g fresh weight in the leaves of agroinfected plants. We show that plant-derived KGF1 can promote the proliferation of NIH/3T3 cells and have significant effects on the type-II diabetic rat. The present findings indicated that KGF1 from tobacco maintains its biological activity, implying prospective industrial production in a plant bioreactor.

Highly efficient expression of functional recombinant human keratinocyte growth factor 1 and its protective effects on hepatocytes.

Three forms of recombinant human keratinocyte growth factor 1 (rhKGF1) with or without the native signal peptide or a 23-amino acid truncation were expressed in Spodoptera frugiperda 9 (Sf9) cells by designing with insect codon usage. Immunoblotting demonstrated that these rhKGF1 proteins were recognized by a human anti-KGF1 antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield, protein quality, and cytotoxicity. Our results indicated that the native signal peptide directed KGF1 secretion from insect cells, reaching a maximum at 60 h postinfection. Although secretion of rhKGF1194 was less efficient than that of rhKGF1163 and rhKGF1140, protein secretion is an attractive pathway for simple purification of biologically active rhKGF1 at a high yield. Moreover, the sizes of rhKGF1194 and rhKGF1163 were similar (20 kDa), suggesting that the signal peptide may be recognized and removed in Sf9 cells. A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was used to analyze the biological function of rhKGF1, indicating that the three forms of rhKGF1 had a similar mitogenic function in BaF3 cells. Furthermore, to elucidate the effect of rhKGF1 on cytoprotection of liver cells, we used KGF1 pretreatment of an acute liver injury model. The results indicated that rhKGF1 prevented necrosis and apoptosis of CCl4-treated HL7702 cells in vitro and in vivo. These results suggest that KGF1 may be a candidate therapeutic drug for acute liver injury.

An in vitro culture system for long-term expansion of epithelial and mesenchymal salivary gland cells: role of TGF-ß1 in salivary gland epithelial and mesenchymal differentiation.

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-ß1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-ß1 on salivary gland cell differentiation. TGF-ß1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-ßR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-ß signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-ß1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.