Transferrin Production Research Articles

Serum transferrin as a biomarker of hepatocyte nuclear factor 4 alpha activity and hepatocyte function in liver diseases

BackgroundSerum transferrin levels represent an independent predictor of mortality in patients with liver failure. Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of hepatocyte functions. The aim of this study was to explore whether serum transferrin reflects HNF4α activity.MethodsFactors regulating transferrin expression in alcoholic hepatitis (AH) were assessed via transcriptomic/methylomic analysis as well as chromatin immunoprecipitation coupled to DNA sequencing. The findings were corroborated in primary hepatocytes. Serum and liver samples from 40 patients with advanced liver disease of multiple etiologies were also studied.ResultsIn patients with advanced liver disease, serum transferrin levels correlated with hepatic transferrin expression (r = 0.51, p = 0.01). Immunohistochemical and biochemical tests confirmed reduced HNF4α and transferrin protein levels in individuals with cirrhosis. In AH, hepatic gene-gene correlation analysis in liver transcriptome revealed an enrichment of HNF4α signature in transferrin-correlated transcriptome while transforming growth factor beta 1 (TGFβ1), tumor necrosis factor α (TNFα), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) negatively associated with transferrin signature. A key regulatory region in transferrin promoter was hypermethylated in patients with AH. In primary hepatocytes, treatment with TGFβ1 or the HNF4α inhibitor BI6015 suppressed transferrin production, while exposure to TNFα, IL-1β, and IL-6 had no effect. The correlation between hepatic HNF4A and transferrin mRNA levels was also seen in advanced liver disease.ConclusionsSerum transferrin levels constitute a prognostic and mechanistic biomarker. Consequently, they may serve as a surrogate of impaired hepatic HNF4α signaling and liver failure.

Transferrin Non-Viral Gene Therapy for Treatment of Retinal Degeneration.

Dysregulation of iron metabolism is observed in animal models of retinitis pigmentosa (RP) and in patients with age-related macular degeneration (AMD), possibly contributing to oxidative damage of the retina. Transferrin (TF), an endogenous iron chelator, was proposed as a therapeutic candidate. Here, the efficacy of TF non-viral gene therapy based on the electrotransfection of pEYS611, a plasmid encoding human TF, into the ciliary muscle was evaluated in several rat models of retinal degeneration. pEYS611 administration allowed for the sustained intraocular production of TF for at least 3 and 6 months in rats and rabbits, respectively. In the photo-oxidative damage model, pEYS611 protected both retinal structure and function more efficiently than carnosic acid, a natural antioxidant, reduced microglial infiltration in the outer retina and preserved the integrity of the outer retinal barrier. pEYS611 also protected photoreceptors from N-methyl-N-nitrosourea-induced apoptosis. Finally, pEYS611 delayed structural and functional degeneration in the RCS rat model of RP while malondialdehyde (MDA) ocular content, a biomarker of oxidative stress, was decreased. The neuroprotective benefits of TF non-viral gene delivery in retinal degenerative disease models further validates iron overload as a therapeutic target and supports the continued development of pEY611 for treatment of RP and dry AMD.

Metastatic growth instructed by neutrophil-derived transferrin

The tumor-promoting functions of neutrophils have been mainly attributed to induction of tumor angiogenesis or suppression of anticancer immunity. However, a direct impact of neutrophils on tumor cell growth and metastasis remains largely uncharacterized. Here, we coupled a proteomic approach with a functional screen to interrogate the secretome of tumor-associated neutrophils. Surprisingly, the iron-transporting protein transferrin was identified as the major mitogen for tumor cells secreted by neutrophils. Depletion of neutrophils inhibited lung metastasis and transferrin production in the metastatic microenvironment. Deletion of transferrin receptor suppressed growth of lung-colonizing tumor cells. Also, media conditioned by neutrophils isolated from metastatic breast cancer patients stimulated growth of human breast cancer cells, an effect that was largely abolished by transferrin immunodepletion. We identified GM-CSF, which is produced primarily by tumor cells, as a selective inducer of de novo transferrin synthesis in neutrophils through the Jak/Stat5β pathway. GM-CSF neutralization or inhibition of Jak kinases curtailed neutrophil transferrin expression in vitro and in vivo as well as cancer metastasis. Thus, transferrin provides a mechanistic link between neutrophils and metastatic growth owing to the ability of tumor-infiltrating neutrophils to locally deliver this growth-promoting protein in response to GM-CSF stimulation. Our study identifies neutrophil-derived transferrin as a key regulator of metastatic tumor cell growth and a therapeutic target for antimetastatic treatment.

Contributions of transferrin to acute inflammation in the goldfish, C. auratus

Transferrin is an evolutionary conserved protein that in addition to having a critical role in iron transport also has been shown to have a crucial role in host defence, by depriving iron from invading pathogens. Recently cleaved transferrin products was shown to activate macrophages in vitro. We now use an in vivo model of self-resolving peritonitis in goldfish, coupled with gene expression and protein analysis to evaluate the contributions of cleaved transferrin to acute inflammation. We show, for the first time, that cleaved transferrin products are produced in vivo early during an acute inflammatory response. These cleaved transferrin fragments were produced during pathogen-induced, but not sterile, inflammation. Both macrophages and neutrophils were able to contribute to transferrin cleavage. However, only macrophages contributed to this innate process through inducible expression of transferrin. The appearance of transferrin cleavage products in vivo correlated with the influx of leukocytes but did not necessarily correlate the induction of robust respiratory burst and nitric oxide responses. Overall, this study adds to a growing body of work highlighting the role of transferrin as an immune regulator during acute inflammation. Given the significant conservation of this and related molecules, these findings have potentially broad implications for host defences and inflammation control across evolution.

α-1,6-Fucosyltransferase (FUT8) Inhibits Hemoglobin Production during Differentiation of Murine and K562 Human Erythroleukemia Cells

Erythropoiesis results from a complex combination of the expression of several transcription factor genes and cytokine signaling. However, the overall view of erythroid differentiation remains unclear. First, we screened for erythroid differentiation-related genes by comparing the expression profiles of high differentiation-inducible and low differentiation-inducible murine erythroleukemia cells. We identified that overexpression of α-1,6-fucosyltransferase (Fut8) inhibits hemoglobin production. FUT8 catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an α-1,6 linkage, leading to core fucosylation in mammals. Expression of Fut8 was down-regulated during chemically induced differentiation of murine erythroleukemia cells. Additionally, expression of Fut8 was positively regulated by c-Myc and c-Myb, which are known as suppressors of erythroid differentiation. Second, we found that FUT8 is the only fucosyltransferase family member that inhibits hemoglobin production. Functional analysis of FUT8 revealed that the donor substrate-binding domain and a flexible loop play essential roles in inhibition of hemoglobin production. This result clearly demonstrates that core fucosylation inhibits hemoglobin production. Third, FUT8 also inhibited hemoglobin production of human erythroleukemia K562 cells. Finally, a short hairpin RNA study showed that FUT8 down-regulation induced hemoglobin production and increase of transferrin receptor/glycophorin A-positive cells in human erythroleukemia K562 cells. Our findings define FUT8 as a novel factor for hemoglobin production and demonstrate that core fucosylation plays an important role in erythroid differentiation.

English

Introduction Human serum transferrin plays a critical physiological role in cellular iron delivery via the transferrin receptor-mediated endocytosis pathway in nearly all eukaryotic organisms. It is widely used in mammalian cell cultures for the production of biotherapeutic proteins and vaccines and is also being explored for use as a therapy and targeted drug delivery system to treat a number of diseases. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasmaderived transferrin, recombinant production of human serum transferrin has been pursued in various heterologous expression systems. However, high costs and limited yields of recombinant human serum transferrin remain the major challenges to many expression systems. Recently, rice seed-based expression system has been shown to produce large amounts of inexpensive and animal-free recombinant human serum transferrin. Here, we review the rice-derived recombinant human serum transferrin: its cost-effective production, molecular and functional characterisation, as well as its many potential therapeutic and clinical applications. Conclusion Rice seed-based expression system is shown to be able to produce large scale of recombinant human serum transferrin with high yield and at low cost. The rice-derived rhTF is shown to be biochemically, structurally and functionally similar to native hTF, and it is a low-cost alternative to other plasma-derived and recombinant forms of hTF suitable for bioprocessing and biopharmaceutical applications. Introduction Transferrin (TF) plays an important role in tightly controlling the cellular iron uptake, storage and transport to maintain cellular iron homeostasis in all eukaryotic organisms. TF is a single-chain glycoprotein of 679 amino acid residues and can be divided into two homologous halves, each comprising about 340 amino acid residues. The two halves fold into two distinct globular lobes, designated the N-lobe and C-lobe1. Each lobe comprises two dissimilar domains, which interact to form a deep hydrophilic iron-binding site. When TF is free of iron (apo-TF), both its Nand C-lobes maintain an open conformation for easy access of the ferric iron. At pH 7.4 under physiological conditions, the apo-TF binds one (monoferric TF) or two Fe3+ ions (diferric TF or holo-TF). The resultant iron bearing TF binds to TF receptor (TFR) on cell surface, and holo-TF has 30-fold and 500-fold higher affinity for TFR than the monoferric TF and apo-TF, respectively2. Then, the TF–TFR complex is endocytosed into the early endosome, where the acidic environment (pH 5.5) result in the release of iron from TF by protonation but apoTF still remains bound to the TFR with high affinity. Finally, the apo-TF– TFR complex is recycled to the cell surface, and at pH 7.4 of the blood, the apo-TF is released from the TFTFR complex for re-use3,4. The TF/TFR-mediated cellular iron uptake and transport is critical to avoid severe cell damages associated with both the iron deficiency and overload in the body. Iron deficiency can arrest cell proliferation and even cause cell death because iron is an essential element used by all eukaryotic organisms and most micro-organisms as a cofactor of numerous proteins or enzymes for respiration, DNA synthesis and many other critical metabolic processes1. On the other hand, excessive iron can be toxic to cells by reacting with oxygen via the Fenton reaction to produce highly reactive hydroxyl radicals that cause oxidative damage to cells5. The dual challenges of iron deficiency and overload can be addressed by transferrin-binding iron ions in the ferric form (Fe3+) tightly yet reversibly. Apart from the importance of maintaining iron homeostasis in cells, TF is also shown to have a wide range of therapeutic applications, which will be described later. Because the native TF derived from plasma is associated with high risk of transmission of infectious pathogenic agents, recombinant expression of human serum transferrin (hTF) has long been pursued in a variety of heterologous expression systems6,7. In this review, we focus on rice-derived recombinant hTF’s costeffective production, its biochemical, structural and functional properties as well as its potential clinical applications. Discussion The author has referenced some of his own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. * Corresponding author Email: dzhang@ventria.com Ventria Bioscience, Fort Collins, CO 80524, USA

A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity

Drug-induced liver injury (DILI) is the major cause for liver failure and post-marketing drug withdrawals. Due to species-specific differences in hepatocellular function, animal experiments to assess potential liabilities of drug candidates can predict hepatotoxicity in humans only to a certain extent. In addition to animal experimentation, primary hepatocytes from rat or human are widely used for pre-clinical safety assessment. However, as many toxic responses in vivo are mediated by a complex interplay among different cell types and often require chronic drug exposures, the predictive performance of hepatocytes is very limited. Here, we established and characterized human and rat in vitro three-dimensional (3D) liver co-culture systems containing primary parenchymal and non-parenchymal hepatic cells. Our data demonstrate that cells cultured on a 3D scaffold have a preserved composition of hepatocytes, stellate, Kupffer and endothelial cells and maintain liver function for up to 3months, as measured by the production of albumin, fibrinogen, transferrin and urea. Additionally, 3D liver co-cultures maintain cytochrome P450 inducibility, form bile canaliculi-like structures and respond to inflammatory stimuli. Upon incubation with selected hepatotoxicants including drugs which have been shown to induce idiosyncratic toxicity, we demonstrated that this model better detected in vivo drug-induced toxicity, including species-specific drug effects, when compared to monolayer hepatocyte cultures.In conclusion, our results underline the importance of more complex and long lasting in vitro cell culture models that contain all liver cell types and allow repeated drug-treatments for detection of in vivo-relevant adverse drug effects.

Retinal Function and Structure in the Hypotransferrinemic Mouse

The iron carrier transferrin is expressed at remarkably high levels in normal retinas and is upregulated during retinal degeneration. The authors characterized the consequences of genetically reduced retinal transferrin production on retinal structure and function. Hypotransferrinemic (HPX⁻/⁻) mice treated with weekly intraperitoneal salvage transferrin injections were examined at 1 and 2 months of age. HPX⁻/⁻, HPX⁺/⁻, and wild-type (WT) mice were evaluated by electroretinography, ophthalmoscopy, and histology. Retinal iron content and transferrin levels were measured. RNA levels of genes involved in iron homeostasis and antioxidative response were determined by quantitative PCR. Oxidative injury was assessed by immunostaining for 4-hydroxy-2-nonenal (HNE). At 2 months, dark-adapted, mixed rod-cone response b-wave amplitudes were significantly lower in HPX⁻/⁻ mice than in WT mice (340 ± 112 μV vs. 624 ± 134 μV [mean ± SEM]; P = 0.002). Oscillatory potentials were significantly suppressed in HPX mice, and ophthalmoscopy demonstrated marked retinal pallor. Quantitative immunostaining revealed a 39% reduction of transferrin content in HPX⁻/⁻ compared with WT retinas (P = 0.01). mRNA levels of Tf, Tf receptor, and ceruloplasmin were decreased, whereas mRNA for antioxidant genes were elevated in HPX⁻/⁻ retinas. HNE staining was reduced in mice carrying the mutant HPX allele. Histologic examination demonstrated preserved retinal structure, and retinal iron content was similar across the strains. Despite the lack of wild-type retinal transferrin production and low levels of retinal transferrin protein, the retinal morphology and retinal iron content in HPX⁻/⁻ mice treated by systemic salvage transferrin injections are normal until age 2 months. However, retinal function and gene expression of some of the iron-associated genes are significantly altered.

Interleukin-18 regulates transferrin-induced cell proliferation in MCF-7, breast cancer cell line. (101.14)

Abstract Interleukin-18 (IL-18) has been reported to enhance cell growth and proliferation in cancer. Transferrin is an iron carrier protein that is responsible for cell growth in both normal and highly proliferative cells like cancers. Although increased serum levels of IL-18 and transferrin in cancer patients correlate with malignancy, little is known about the role of IL-18 and transferrin in the cellular response. Here, we found that exogenous transferrin induces cell growth in human breast cancer cell line, MCF-7. The proliferation rate of MCF-7 cells was reduced when endogenous transferrin expression was inhibited by trasferrin-siRNA transfection. Additionally, we discovered that the expression of endogenous transferrin lies under the control of IL-18 via the MAPK pathway. In conclusion, our data indicate that IL-18 has a pro-cancer effect of accelerating cell proliferation rate by increasing production of endogenous transferrin through the MAPK pathway. Thus, we suggest IL-18 as an inducer of endogenous transferrin expression in breast cancer.

Bovine prolactin promotes the expression of human transferrin in the milk of transgenic mice

The bovine prolactin vector was injected directly into the mammary glands of mice carrying the human transferrin transgene to investigate its effect on the production of human transferrin in milk. The mean levels of human transferrin in two experimental groups were increased by approx. 60% compared with the control group: 1143 +/- 196 ng/ml (experimental group 1; two injections) and 1160 +/- 189 ng/ml (experimental group 2; three injections) versus 714 +/- 75 ng/ml (control group). These findings suggest the potential utility of the prolactin vector for efficient expression of valuable pharmaceutical proteins in transgenic animal mammary glands.

High-level production of animal-free recombinant transferrin from saccharomyces cerevisiae

BackgroundAnimal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin.ResultsThe first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 μm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin.ConclusionsA Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.

Interferon‐α mediates suppression of C‐reactive protein: Explanation for muted C‐reactive protein response in lupus flares?

C-reactive protein (CRP) is synthesized by hepatocytes in response to interleukin-6 (IL-6) during inflammation. Despite raised IL-6 levels and extensive systemic inflammation, serum CRP levels remain low during most viral infections and disease flares of systemic lupus erythematosus (SLE). Because both viral infections and SLE are characterized by high levels of interferon-alpha (IFNalpha), the aim of this study was to determine whether this cytokine can inhibit the induction of CRP. The interference of all 12 IFNalpha subtypes with CRP promoter activity induced by IL-6 and IL-1beta was studied in a CRP promoter- and luciferase reporter-transfected human hepatoma cell line, Hep-G2. CRP secretion by primary human hepatocytes was analyzed by enzyme-linked immunosorbent assay. CRP promoter activity was inhibited by all single IFNalpha subtypes, as well as by 2 different mixtures of biologically relevant IFNalpha subtypes. The most prominent effect was seen using a leukocyte-produced mixture of IFNalpha (56% inhibition at 1,000 IU/ml). The inhibitory effect of IFNalpha was confirmed in primary human hepatocytes. CRP promoter inhibition was dose dependent and mediated via the type I IFN receptor. Transferrin production and Hep-G2 proliferation/viability were not affected by IFNalpha. The current study demonstrates that IFNalpha is an inhibitor of CRP promoter activity and CRP secretion. This finding concords with previous observations of up-regulated IFNalpha and a muted CRP response during SLE disease flares. Given the fundamental role of both IFNalpha and CRP in the immune response, our results are of importance for understanding the pathogenesis of SLE and may also contribute to understanding the differences in the CRP response between viral and bacterial infections.

Non-Transferrin-Bound Iron During Autologous Stem Cell Transplantation and Conventional Intensive Chemotherapy.

Abstract 4310 IntroductionIntensive chemotherapy causes cessation of iron utilization by the bone marrow, leads to release of iron from the liver through hepatocellular injury, and disturbs transferrin production. These effects may lead to full transferrin saturation and the appearance of free, non-transferrin-bound iron (NTBI) in the serum. Hydroxyl radicals catalyzed by NTBI can cause cellular damage and contribute to therapy-related complications. The present study was designed to demonstrate the appearance and timing of NTBI during autologous stem cell transplantation and conventional intensive chemotherapy. Patients and MethodsTwenty-four adult patients were enrolled. Sixteen patients with multiple myeloma or lymphoma were conditioned with an intensive cytotoxic regimen prior to autologous stem cell transplantation (aSCT). The conditioning for multiple myeloma was either melphalan monotherapy (Mel) or melphalan combined with fractionated total body irradiation (Mel/TBI), and for lymphoma a combination chemotherapy regimen (BEAC). Eight patients had acute leukemia and were treated with various cytopenia-inducing chemotherapies (Chemo). Serum samples were collected coinciding with routine sampling, before the initiation of the chemotherapy and during the first 20 days after the onset of the chemotherapy. The median number of samples per patient was 18.5 (range 16-21) in the aSCT group and 15.5 (7-21) in the Chemo group. TfSat was calculated using the formula: serum iron (mmol/l)/ serum transferrin (g/l) x 3.98 without a cutoff at 100 %. NTBI in serum was determined by the bleomycin assay modified for small serum volumes. The samples were analyzed for NTBI only when the respective TfSat was above 80 %, based on our previous observation that NTBI was not detectable below that cutoff level. ResultsA steep rise of the mean transferrin saturation was seen in all study groups during the first days after starting the chemotherapy, reaching the maximum levels of 76 to 93 per cent at four to six days. Among the aSCT groups, the TfSat levels were significantly lower in the Mel group compared to BEAC (P= 0.0002). In the Chemo group the levels stayed high significantly longer than in the aSCT groups (P < 0.0001). In the Chemo group the mean transferrin concentration was below the reference range during the whole study period, whereas in all the aSCT groups the initially normal mean transferrin levels decreased below the reference range during the study. None of the aSCT patients were NTBI-positive at baseline, and three of the aSCT patients (1 Mel, 2 Mel/TBI) also remained NTBI-negative throughout the study period. In all other patients NTBI was detected variably, for an average duration of 15.6 days from the first positive sample to the last positive one in the Chemo group, and for 6.1 days, respectively, in the aSCT group. The proportion of the NTBI-positive samples was higher among the Chemo patients (59 %) compared to the aSCT patients (22 %) (P< 0.001). Among the aSCT patients the proportion of NTBI-positive samples was lower in the Mel group compared to the Mel/TBI and BEAC groups (P= 0.04 and 0.01, respectively). In the aSCT patients the disappearance or marked reduction of NTBI-positive samples coincided with the recovery of the bone marrow function, as demonstrated by the leukocyte counts. In the Chemo group there was no leukocyte recovery during the study period and the proportion of NTBI-positive samples remained high throughout the whole period. ConclusionsNTBI was detectable in the majority (21/24) of patients receiving conditioning for autologous transplantation or intensive chemotherapy for acute leukemia. The presence of NTBI coincided with bone marrow suppression, and NTBI largely disappeared at the recovery of the bone marrow function. The treatment that caused the longest bone marrow suppression (Chemo) was associated with the most prolonged presence of NTBI. The timing of the presence of NTBI accords with the presence of the most important non-infectious complication of intensive chemotherapy and autologous transplantation, mucosal injury, and free iron is likely to contribute to this and probably other complications of the intensive treatments. Disclosures:No relevant conflicts of interest to declare.

Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe

The effect of medium supplementation on heterologous production of human serum transferrin (hTF) in the fission yeast Schizosaccharomyces pombe has been investigated. The productivity of recombinant hTF was low in wild-type S. pombe cells. To overcome this impediment, culture media supplements were screened for their ability to improve secretion of hTF. Casamino acids (CAA), which have been reported to increase heterologous protein productivity in Pichia pastoris, improved the secretion hTF by more than fourfold. An anion surfactant deoxycholate or polyethylene glycol also improved the secretion hTF. Interestingly, dextran sodium sulfate (DSS), a poly-anion surfactant, was found to enhance production of secreted hTF better than any other supplement tested. Addition of DSS in the presence of 2% CAA exhibited a synergistic effect on increasing hTF secretion, resulting in an increase of about sevenfold relative to conventional conditions. Cell growth was not found to be affected by the addition of DSS or CAA. DSS may act as a surfactant and may also facilitate the anchoring of liposomes, and these properties may contribute to efficient secretion or exocytosis through the plasma membrane.

Interleukin-18 induces transferrin expression in breast cancer cell line MCF-7

Interleukin-18 (IL-18) has recently been shown to have a pro-cancer effect in various cancers. Increased serum levels of both IL-18 and transferrin in cancer patients are correlated with its malignancy. However, the relationship between transferrin and IL-18 is not well understood. Here, we show that exogenous transferrin enhanced breast cancer cell proliferation and the proliferation rate was reduced when endogenous transferrin expression was inhibited by transferrin siRNA. The expression of endogenous transferrin was found to be regulated by IL-18. Furthermore, we found that the MAPK pathway is involved in IL-18-induced transferrin production. In conclusion, IL-18 is suggested as an inducer of endogenous transferrin expression in breast cancer cells.

Observation and modeling of induction effect on human transferrin production from stably transfected Drosophila S2 cell culture

Human transferrin (hTf) is a serum glycoprotein involved in iron transport. We performed, for the first time, mathematical modeling of stably transfected insect Drosophila melanogaster S2 cell culture, a nonlytic plasmid-based system that secretes recombinant hTf under control of the copper sulfate-inducible Drosophila metallothionein promoter. Cell growth patterns at various inducer concentrations revealed that the specific growth rate of S2 cells was substantially reduced as the specific rate of recombinant hTf production increased, and recovered to some extent when recombinant hTf production was nearly stopped. Additionally, the time profiles of specific production rates exhibited a maximum in the early culture period. Longer times and lower values of the maximum specific production rate were observed at lower inducer concentrations, and shorter times and higher values at higher inducer levels. Although the proposed S2 cell culture model was slightly limited with regard to prediction of cell growth profile at the late stage, we ensured that it gave reasonable predictions of the dynamics of glucose consumption and recombinant hTf production and confirmed its validity through simulating other culture experiments under different conditions. The S2 model proposed in this study can contribute to the elucidation of cell culture dynamics, and optimization of each culture variable to enhance heterologous protein production.

Optimization of tetracycline‐responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.

FSH activates phosphatidylinositol 3-kinase/protein kinase B signaling pathway in 20-day-old Sertoli cells independently of IGF-I.

The gonadotropin FSH plays a key role in the control of Sertoli cell function. The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway. However, other signaling events have also been demonstrated in Sertoli cells. We have recently presented evidence that FSH can stimulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathway in 20-day-old Sertoli cells. At the same time, it was proposed that in 8-day-old Sertoli cells the effects of FSH on phosphorylated PKB (P-PKB) levels can be explained by a combination of increased secretion of endogenous IGF-I, decreased IGF-binding protein-3 (IGFBP-3) production, and a synergistic action of FSH on IGF-I-dependent PI3K activation. The aim of the present study was to determine whether the effect of FSH on 20-day-old Sertoli cells is mediated by IGF-I secretion. Twenty-day-old rat Sertoli cell cultures were used. FSH stimulation produced a time-dependent increment in P-PKB levels reaching maximal values in 60-min incubations. IGF-I stimulation was also time-dependent reaching maximal values in 15-min incubations. On the other hand, stimulation of the cultures with FSH showed time-dependent inhibition in phosphorylated mitogen-activated protein kinase (P-MAPK) levels. In sharp contrast, stimulation of the cultures with IGF-I showed time-dependent increments in P-MAPK levels reaching maximal stimulus in 15-min incubations. In order to rule out an IGF-I action on FSH stimulation of P-PKB levels, the effect of a specific IGF-I antibody on the ability of both hormones to increase P-PKB levels was evaluated. As expected, the antibody inhibited IGF-I stimulation of P-PKB levels. However, simultaneous addition of an IGF-I antibody with FSH did not modify the ability of the hormone to increase P-PKB levels. The next set of experiments intended to analyze the relevance of a PI3K/PKB pathway to two biological responses of Sertoli cells to FSH and IGF-I. The PI3K inhibitor, wortmannin, dose-dependently decreased FSH-stimulated lactate and transferrin production. On the other hand, wortmannin was not able to modify the ability of IGF-I to stimulate these metabolic events. In addition, the analysis of the participation of a MAPK pathway in IGF-I regulation of Sertoli cell biological responses showed that the MAPK kinase inhibitors, PD98059 and U0126, decreased IGF-I-stimulated transferrin secretion while not modifying IGF-I-stimulated lactate levels. In summary, results obtained so far support the hypothesis that FSH action on P-PKB levels and Sertoli cell metabolism in 20-day-old animals is not mediated by autocrine regulation of an IGF-I/ IGFBP-3 axis as previously proposed in 8-day-old Sertoli cells.

Transferrin Expression by Placental Trophoblastic Cells

Transferrin (TF), a 76–80 kDa glycoprotein, is responsible for the transport of iron to cells within both the fetal and maternal systems, but it does not cross the multiple cell layer barrier of the placenta. Recent findings that both rat and human placental cells produce TF indicated that placental TF may function in some manner to transport or regulate iron passage across this barrier. However, placental production of TF was brought into question because the cell preparations used to identify TF were obtained using dispersed tissue and may have contained non-placental contaminating elements. In this study, cultures of phenotypically distinct cell types containing only placental cells were used to firmly establish whether or not TF is expressed, and if so to begin to identify the cell(s) associated with its synthesis. Utilizing RT–PCR, in situ hybridization, and Western blot analysis, we identified TF mRNA and protein in three trophoblast cell types, HRP-1 (rat), Rcho-1 (rat), and BeWo (human) cells. Additionally, TF mRNA and protein were found in Giant cells, the differentiated form of Rcho-1 cells. When taken together, these results demonstrate clearly that TF is expressed by both differentiated and non-differentiated placental cells, and when viewed in light of previous findings, strengthen the possibility that placental TF may be central to the passage of iron from the mother to the fetus during development.

Assessment of the roles of mitogen-activated protein kinase and phosphatidyl inositol 3-kinase/protein kinase B pathways in the basic fibroblast growth factor regulation of Sertoli cell function.

Basic fibroblast growth factor (bFGF) belongs to the large set of intratesticular regulators that provide the fine tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to determine the participation of mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase/protein kinase B (PI3K/PKB) pathways in bFGF regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. Stimulation of the cultures with bFGF showed a time-dependent increment in phosphorylated MAPK and PKB levels that reached maximal values in 5-min incubations. MAPK kinase inhibitors U0126 (U) and PD98059 (PD) and a PI3K inhibitor wortmannin (W) were able to block the stimulatory effects of bFGF on phosphorylated MAPK and PKB levels respectively. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF of two well-known Sertoli cell-differentiated functions, lactate and transferrin production, was next explored. As for lactate production, PD and W did not modify the ability of bFGF to stimulate lactate production. However, a combination of PD and W partially impaired the increase in lactate production elicited by bFGF. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF of glucose uptake and lactate dehydrogenase (LDH) activity was also analysed. In this respect, it was observed that W markedly decreased basal and bFGF-stimulated glucose uptake and that U and PD did not modify it. On the other hand, U and PD decreased the stimulation of LDH activity by bFGF whereas W did not modify it. As for transferrin production, while both MAPK kinase inhibitors partially decreased the ability of bFGF to stimulate transferrin secretion, the PI3K inhibitor did not modify it. In summary, the results demonstrated that bFGF stimulates MAPK- and PI3K/PKB-dependent pathways in rat Sertoli cells. Moreover, these results showed that while bFGF utilizes the MAPK pathway to regulate transferrin production and LDH activity, it uses the PI3K/PKB pathway to regulate glucose transport into the cell.