Expression and purification of intracrine human FGF 11 and study of its FGFR-dependent biological activity.

Fibroblast growth factor 11 (FGF11) is one of intracrine FGFs (iFGFs), which function within cells. Unlike canonical FGFs, FGF11 remains intracellularly and plays biological roles in FGF receptor (FGFR)-independent manner. Here, we established an expression system of recombinant FGF11 proteins in E. coli and investigated whether the extracellular administration of FGF11 can activate cellular signaling. Human FGF11 has two isoforms, FGF11a and FGF11b, depending on the presence of nuclear localization sequences (NLSs) in the N-terminus. Because these two isoforms are unstable, we prepared an FGF11a-Mut by substituting three cysteine residues in the NLS with serine and FGF11b-ΔC with C-terminal truncation. The introduction of mutation in the NLS improved the solubility of FGF11 prepared from E. coli. Exogenous addition of FGF11b and FGF11b-ΔC to BALB3T3 increased cell proliferation, while FGF11a-Mut exerted no effect. FGF11b-ΔC showed higher cell proliferation activity and FGFR signaling than FGF11b. The cell-proliferating activities of FGF11b and FGF11b-ΔC were blocked by an FGFR1 inhibitor or a recombinant FGFR1, confirming the FGFR1-dependent extracellular activity of FGF11b. The analysis of circular dichroism suggested that the C-terminus of FGF11 has an α-helical structure, which may affect its interaction with FGFR1. These results suggest that the N-and C-terminus of recombinant FGF11 are involved in the activation of FGFR1. The above results provide novel insights into the function and mechanism of FGF11 that may aid the development of useful ligands for FGFR regulation.

Functional and Immunological Studies Revealed a Second Superantigen Toxin in Staphylococcal Enterotoxin C Producing Staphylococcus aureus Strains.

Staphylococcus aureus is a human and animal pathogen as well as a commensal bacterium. It can be a causative agent of severe, life-threatening infections with high mortality, e.g., toxic shock syndrome, septic shock, and multi-organ failure. S. aureus strains secrete a number of toxins. Exotoxins/enterotoxins are considered important in the pathogenesis of the above-mentioned conditions. Exotoxins, e.g., superantigen toxins, cause uncontrolled and polyclonal T cell activation and unregulated activation of inflammatory cytokines. Here we show the importance of genomic analysis of infectious strains in order to identify disease-causing exotoxins. Further, we show through functional analysis of superantigenic properties of staphylococcal exotoxins that even very small amounts of a putative superantigenic contaminant can have a significant mitogenic effect. The results show expression and production of two distinct staphylococcal exotoxins, SEC and SEL, in several strains from clinical isolates. Antibodies against both toxins are required to neutralise the superantigenic activity of staphylococcal supernatants and purified staphylococcal toxins.

PROTEIN KINASE C ALPHA IS A CENTRAL NODE FOR TUMORIGENIC TRANSCRIPTIONAL NETWORKS IN HUMAN PROSTATE CANCER.

Aberrant expression of protein kinase C (PKC) isozymes is a hallmark of cancer. The different members of the PKC family control cellular events associated with cancer development and progression. Whereas the classical/conventional PKCα isozyme has been linked to tumor suppression in most cancer types, here we demonstrate that this kinase is required for the mitogenic activity of aggressive human prostate cancer cells displaying aberrantly high PKCα expression. Immunohistochemical analysis showed abnormal up-regulation of PKCα in human primary prostate tumors. Interestingly, silencing PKCα expression from aggressive prostate cancer cells impairs cell cycle progression, proliferation and invasion, as well as their tumorigenic activity in a mouse xenograft model. Mechanistic analysis revealed that PKCα exerts a profound control of gene expression, particularly over genes and transcriptional networks associated with cell cycle progression and E2F transcription factors. PKCα RNAi depletion from PC3 prostate cancer cells led to a reduction in the expression of pro-inflammatory cytokine and epithelial-to-mesenchymal transition (EMT) genes, as well as a prominent down-regulation of the immune checkpoint ligand PD-L1. This PKCα-dependent gene expression profile was corroborated in silico using human prostate cancer databases. Our studies established PKCα as a multifunctional kinase that plays pleiotropic roles in prostate cancer, particularly by controlling genetic networks associated with tumor growth and progression. The identification of PKCα as a pro-tumorigenic kinase in human prostate cancer provides strong rationale for the development of therapeutic approaches towards targeting PKCα or its effectors.

Hypoxia-Induced Mitogenic Factor: A Multifunctional Protein Involved in Health and Disease.

Hypoxia-induced mitogenic factor (HIMF), also known as resistin-like molecule α (RELMα) or found in inflammatory zone 1 (FIZZ1) is a member of the RELM protein family expressed in mice. It is involved in a plethora of physiological processes, including mitogenesis, angiogenesis, inflammation, and vasoconstriction. HIMF expression can be stimulated under pathological conditions and this plays a critical role in pulmonary, cardiovascular and metabolic disorders. The present review summarizes the molecular characteristics, and the physiological and pathological roles of HIMF in normal and diseased conditions. The potential clinical significance of these findings for human is also discussed.

The Potential Role of REG Family Proteins in Inflammatory and Inflammation-Associated Diseases of the Gastrointestinal Tract.

Regenerating gene (REG) family proteins serve as multifunctional secretory molecules with trophic, antiapoptotic, anti-inflammatory, antimicrobial and probably immuno-regulatory effects. Since their discovery, accumulating evidence has clarified the potential roles of the REG family in the occurrence, progression and development of a wide range of inflammatory and inflammation-associated diseases of the gastrointestinal (GI) tract. However, significant gaps still exist due to the undefined nature of certain receptors, regulatory signaling pathways and possible interactions among distinct Reg members. In this narrative review, we first describe the structural features, distribution pattern and purported regulatory mechanisms of REG family proteins. Furthermore, we summarize the established and proposed roles of REG proteins in the pathogenesis of various inflammation-associated pathologies of the GI tract and the body as a whole, focusing particularly on carcinogenesis in the ulcerative colitis-colitic cancer sequence and gastric cancer. Finally, the clinical relevance of REG products in the context of diagnosis, treatment and prognostication are also discussed in detail. The current evidence suggests a need to better understanding the versatile roles of Reg family proteins in the pathogenesis of inflammatory-associated diseases, and their broadened future usage as therapeutic targets and prognostic biomarkers is anticipated.

Serotonin as a Mitogen in the Gastrointestinal Tract: Revisiting a Familiar Molecule in a New Role.

Serotonin signaling is ubiquitous in the gastrointestinal (GI) system, where it acts as a neurotransmitter in the enteric nervous system (ENS) and influences intestinal motility and inflammation. Since its discovery, serotonin has been linked to cellular proliferation in several types of tissues, including vascular smooth muscle, neurons, and hepatocytes. Activation of serotonin receptors on distinct cell types has been shown to induce well-known intracellular proliferation pathways. In the GI tract, potentiation of serotonin signaling results in enhanced intestinal epithelial proliferation, and decreased injury from intestinal inflammation. Furthermore, activation of the type 4 serotonin receptor on enteric neurons leads to neurogenesis and neuroprotection in the setting of intestinal injury. It is not surprising that the mitogenic properties of serotonin are pronounced within the GI tract, where enterochromaffin cells in the intestinal epithelium produce 90% of the body's serotonin; however, these proliferative effects are attributed to increased serotonin signaling within the ENS compartment as opposed to the intestinal mucosa, which are functionally and chemically separate by virtue of the distinct tryptophan hydroxylase enzyme isoforms involved in serotonin synthesis. The exact mechanism by which serotonergic neurons in the ENS lead to intestinal proliferation are not known, but the activation of muscarinic receptors on intestinal crypt cells indicate that cholinergic signaling is essential to this signaling pathway. Further understanding of serotonin's role in mucosal and enteric nervous system mitogenesis may aid in harnessing serotonin signaling for therapeutic benefit in many GI diseases, including inflammatory bowel disease, malabsorptive conditions, and cancer.

The SGLT2 inhibitor Empagliflozin attenuates interleukin-17A-induced human aortic smooth muscle cell proliferation and migration by targeting TRAF3IP2/ROS/NLRP3/Caspase-1-dependent IL-1ß and IL-18 secretion.

Chronic inflammation and persistent oxidative stress contribute to the development and progression of vascular proliferative diseases. We hypothesized that the proinflammatory cytokine interleukin (IL)-17A induces oxidative stress and amplifies inflammatory signaling in human aortic smooth muscle cells (SMC) via TRAF3IP2-mediated NLRP3/caspase-1-dependent mitogenic and migratory proinflammatory cytokines IL-1ß and IL-18. Further, we hypothesized that these maladaptive changes are prevented by empagliflozin (EMPA), an SGLT2 (Sodium/Glucose Cotransporter 2) inhibitor. Supporting our hypotheses, exposure of cultured SMC to IL-17A promoted proliferation and migration via TRAF3IP2, TRAF3IP2-dependent superoxide and hydrogen peroxide production, NLRP3 expression, caspase-1 activation, and IL-1ß and IL-18 secretion. Furthermore, NLRP3 knockdown, caspase-1 inhibition, and pretreatment with IL-1ß and IL-18 neutralizing antibodies and IL-18BP, each attenuated IL-17A-induced SMC migration and proliferation. Importantly, SMC express SGLT2, and pre-treatment with EMPA attenuated IL-17A/TRAF3IP2-dependent oxidative stress, NLRP3 expression, caspase-1 activation, IL-1ß and IL-18 secretion, and SMC proliferation and migration. Importantly, silencing SGLT2 attenuated EMPA-mediated inhibition of IL-17A-induced cytokine secretion and SMC proliferation and migration. EMPA exerted these beneficial antioxidant, anti-inflammatory, anti-mitogenic and anti-migratory effects under normal glucose conditions and without inducing cell death. These results suggest the therapeutic potential of EMPA in vascular proliferative diseases.

A 90-day drinking water study in mice to characterize early events in the cancer mode of action of 1,4-dioxane.

Studies demonstrate that with sufficient dose and duration, 1,4-dioxane (1,4-DX) induces liver tumors in laboratory rodent models. The available evidence aligns with a threshold-dependent, tumor promotion mode of action (MOA). The MOA and key events (KE) in rats are well developed but less so in the mouse. Therefore, we conducted a 90-day drinking water study in female mice to evaluate early KE at 7, 28, and 90 days. Female B6D2F1/Crl mice consumed drinking water containing 0, 40, 200, 600, 2000 or 6000 ppm 1,4-DX. 1,4-DX was detected in blood at 90-days of exposure to 6000 ppm, but not in the other exposure groups, indicating a metabolic clearance threshold between 2000 and 6000. Early events identified in this study include glycogen-like vacuolization, centrilobular hypertrophy, centrilobular GST-P staining, apoptosis, and pan-lobular increase in cell proliferation observed after 90-days of exposure to 6000 ppm 1,4-DX. There was minimal evidence of hepatotoxicity over the duration of this study. These findings demonstrate a previously unreported direct mitogenic response following exposures exceeding the metabolic clearance threshold of 1,4-DX. Collectively, the information generated in this study supports a threshold MOA for the development of liver tumors in mice after exposure to 1,4-DX.

Moderation of mitochondrial respiration mitigates metabolic syndrome of aging.

Deregulation of mitochondrial dynamics leads to the accumulation of oxidative stress and unhealthy mitochondria; consequently, this accumulation contributes to premature aging and alterations in mitochondria linked to metabolic complications. We postulate that restrained mitochondrial ATP synthesis might alleviate age-associated disorders and extend healthspan in mammals. Herein, we prepared a previously discovered mitochondrial complex IV moderate inhibitor in drinking water and orally administered to standard-diet-fed, wild-type C57BL/6J mice every day for up to 16 mo. No manifestation of any apparent toxicity or deleterious effect on studied mouse models was observed. The impacts of an added inhibitor on a variety of mitochondrial functions were analyzed, such as respiratory activity, mitochondrial bioenergetics, and biogenesis, and a few age-associated comorbidities, including reactive oxygen species (ROS) production, glucose abnormalities, and obesity in mice. It was found that mitochondrial quality, dynamics, and oxidative metabolism were greatly improved, resulting in lean mice with a specific reduction in visceral fat plus superb energy and glucose homeostasis during their aging period compared to the control group. These results strongly suggest that a mild interference in ATP synthesis through moderation of mitochondrial activity could effectively up-regulate mitogenesis, reduce ROS production, and preserve mitochondrial integrity, thereby impeding the onset of metabolic syndrome. We conclude that this inhibitory intervention in mitochondrial respiration rectified the age-related physiological breakdown in mice by protecting mitochondrial function and markedly mitigated certain undesired primary outcomes of metabolic syndrome, such as obesity and type 2 diabetes. This intervention warrants further research on the treatment of metabolic syndrome of aging in humans.

Combinatorial Effect of DCA and Let-7a on Triple-Negative MDA-MB-231 Cells: A Metabolic Approach of Treatment.

Dichloroacetate (DCA) is a metabolic modulator that inhibits pyruvate dehydrogenase activity and promotes the influx of pyruvate into the tricarboxylic acid cycle for complete oxidation of glucose. DCA stimulates oxidative phosphorylation (OXPHOS) more than glycolysis by altering the morphology of the mitochondria and supports mitochondrial apoptosis. As a consequence, DCA induces apoptosis in cancer cells and inhibits the proliferation of cancer cells. Recently, the role of miRNAs has been reported in regulating gene expression at the transcriptional level and also in reprogramming energy metabolism. In this article, we indicate that DCA treatment leads to the upregulation of let-7a expression, but DCA-induced cancer cell death is independent of let-7a. We observed that the combined effect of DCA and let-7a induces apoptosis, reduces reactive oxygen species generation and autophagy, and stimulates mitochondrial biogenesis. This was later accompanied by stimulation of OXPHOS in combined treatment and was thus involved in metabolic reprogramming of MDA-MB-231 cells.

Antidepressants induce profibrotic responses via the lysophosphatidic acid receptor LPA1.

Preclinical and clinical studies have indicated that antidepressants can promote inflammation and fibrogenesis, particularly in the lung, by mechanisms not fully elucidated. We have previously shown that different classes of antidepressants can activate the lysophosphatidic acid (LPA) receptor LPA1, a major pathogenetic mediator of tissue fibrosis. The aim of the present study was to investigate whether in cultured human dermal and lung fibroblasts antidepressants could trigger LPA1-mediated profibrotic responses. In both cell types amitriptyline, clomipramine and mianserin mimicked the ability of LPA to induce the phosphorylation/activation of extracellular signal -regulated kinases 1 and 2 (ERK1/2), which was blocked by the selective LPA1 receptor antagonist AM966 and the LPA1/3 antagonist Ki16425. Antidepressant-induced ERK1/2 stimulation was absent in fibroblasts stably depleted of LPA1 by short hairpin RNA transfection and was prevented by pertussis toxin, an uncoupler of receptors from Gi/o proteins. Like LPA, antidepressants stimulated fibroblasts proliferation and this effect was blocked by either AM966 or the MEK1/2 inhibitor PD98059. Moreover, by acting through LPA1 antidepressants induced the expression of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, and caused an ERK1/2-dependent increase in the cellular levels of transforming growth factor-ß (TGF-ß)1, a potent fibrogenic cytokine. Pharmacological blockade of TGF-ß receptor type 1 prevented antidepressant- and LPA-induced α-SMA expression. These data indicate that in human dermal and lung fibroblasts different antidepressants can induce proliferative and differentiating responses by activating the LPA1 receptor coupled to ERK1/2 signalling and suggest that this property may contribute to the promotion of tissue fibrosis by these drugs.

Regular Endurance Exercise Promotes Fission, Mitophagy, and Oxidative Phosphorylation in Human Skeletal Muscle Independently of Age.

This study investigated whether regular endurance exercise maintains basal mitophagy and mitochondrial function during aging. Mitochondrial proteins and total mRNA were isolated from vastus lateralis biopsies (n = 33) of young sedentary (YS), old sedentary (OS), young active (YA), and old active (OA) men. Markers for mitophagy, fission, fusion, mitogenesis, and mitochondrial metabolism were assessed using qRT-PCR, Western blot, and immunofluorescence staining. Independently of age, fission protein Fis1 was higher in active vs. sedentary subjects (+80%; P < 0.05). Mitophagy protein PARKIN was more elevated in OA than in OS (+145%; P = 0.0026). mRNA expression of Beclin1 and Gabarap, involved in autophagosomes synthesis, were lower in OS compared to YS and OA (P < 0.05). Fusion and oxidative phosphorylation proteins were globally more elevated in the active groups (P < 0.05), while COx activity was only higher in OA than in OS (P = 0.032). Transcriptional regulation of mitogenesis did not vary with age or exercise. In conclusion, physically active lifestyle seems to participate in the maintenance of lifelong mitochondrial quality control by increasing fission and mitophagy.

Sensory neuropathy and nociception in rodent models of Parkinson's disease.

Parkinson's disease (PD) often manifests with prodromal pain and sensory losses whose etiologies are not well understood. Multiple genetic and toxicity-based rodent models of PD partly recapitulate the histopathology and motor function deficits. Although far less studied, there is some evidence that rodents, similar to humans, develop sensory manifestations of the disease, which may precede motor disturbances and help to elucidate the underlying mechanisms of PD-associated pain at the molecular and neuron circuit levels. The present Review summarizes nociception and other sensory functions in frequently used rodent PD models within the context of the complex phenotypes. In terms of mechanisms, it appears that the acute loss of dopaminergic neurons in systemic toxicity models (MPTP, rotenone) primarily causes nociceptive hyperexcitability, presumably owing to a loss of inhibitory control, whereas genetic models primarily result in a progressive loss of heat perception, reflecting sensory fiber neuropathies. At the molecular level, neither α-synuclein deposits alone nor failure of mitophagy alone appear to be strong enough to result in axonal or synaptic pathology of nociceptive neurons that manifest at the behavioral level, and peripheral sensory loss may mask central 'pain' in behavioral tests. Hence, allostatic combinations or additional challenges and novel behavioral assessments are needed to better evaluate PD-associated sensory neuropathies and pain in rodents.

Nerve Growth Factor Induces Proliferation and Aggressiveness In Prostate Cancer Cells.

Resistance to hormone therapy and disease progression is the major challenge in clinical management of prostate cancer (PC). Drugs currently used in PC therapy initially show a potent antitumor effects, but PC gradually develops resistance, relapses and spreads. Most patients who fail primary therapy and have recurrences eventually develop castration-resistant prostate cancer (CRPC), which is almost incurable. The nerve growth factor (NGF) acts on a variety of non-neuronal cells by activating the NGF tyrosine-kinase receptor, tropomyosin receptor kinase A (TrkA). NGF signaling is deregulated in PC. In androgen-dependent PC cells, TrkA mediates the proliferative action of NGF through its crosstalk with the androgen receptor (AR). Epithelial PC cells, however, acquire the ability to express NGF and TrkA, as the disease progresses, indicating a role for NGF/TrkA axis in PC progression and androgen-resistance. We here report that once activated by NGF, TrkA mediates proliferation, invasiveness and epithelial-mesenchymal transition (EMT) in various CRPC cells. NGF promotes organoid growth in 3D models of CRPC cells, and specific inhibition of TrkA impairs all these responses. Thus TrkA represents a new biomarker to target in CRPC.

Mitochondria in Neuroprotection by Phytochemicals: Bioactive Polyphenols Modulate Mitochondrial Apoptosis System, Function and Structure.

In aging and neurodegenerative diseases, loss of distinct type of neurons characterizes disease-specific pathological and clinical features, and mitochondria play a pivotal role in neuronal survival and death. Mitochondria are now considered as the organelle to modulate cellular signal pathways and functions, not only to produce energy and reactive oxygen species. Oxidative stress, deficit of neurotrophic factors, and multiple other factors impair mitochondrial function and induce cell death. Multi-functional plant polyphenols, major groups of phytochemicals, are proposed as one of most promising mitochondria-targeting medicine to preserve the activity and structure of mitochondria and neurons. Polyphenols can scavenge reactive oxygen and nitrogen species and activate redox-responsible transcription factors to regulate expression of genes, coding antioxidants, anti-apoptotic Bcl-2 protein family, and pro-survival neurotrophic factors. In mitochondria, polyphenols can directly regulate the mitochondrial apoptosis system either in preventing or promoting way. Polyphenols also modulate mitochondrial biogenesis, dynamics (fission and fusion), and autophagic degradation to keep the quality and number. This review presents the role of polyphenols in regulation of mitochondrial redox state, death signal system, and homeostasis. The dualistic redox properties of polyphenols are associated with controversial regulation of mitochondrial apoptosis system involved in the neuroprotective and anti-carcinogenic functions. Mitochondria-targeted phytochemical derivatives were synthesized based on the phenolic structure to develop a novel series of neuroprotective and anticancer compounds, which promote the bioavailability and effectiveness. Phytochemicals have shown the multiple beneficial effects in mitochondria, but further investigation is required for the clinical application.

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression.

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1-100 µM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.

P-Rex1 is dispensable for Erk activation and mitogenesis in breast cancer.

Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 1 (P-Rex1) is a key mediator of growth factor-induced activation of Rac1, a small GTP-binding protein widely implicated in actin cytoskeleton reorganization. This Guanine nucleotide Exchange Factor (GEF) is overexpressed in human luminal breast cancer, and its expression associates with disease progression, metastatic dissemination and poor outcome. Despite the established contribution of P-Rex1 to Rac activation and cell locomotion, whether this Rac-GEF has any relevant role in mitogenesis has been a subject of controversy. To tackle the discrepancies among various reports, we carried out an exhaustive analysis of the potential involvement of P-Rex1 on the activation of the mitogenic Erk pathway. Using a range of luminal breast cancer cellular models, we unequivocally showed that silencing P-Rex1 (transiently, stably, using multiple siRNA sequences) had no effect on the phospho-Erk response upon stimulation with growth factors (EGF, heregulin, IGF-I) or a GPCR ligand (SDF-1). The lack of involvement of P-Rex1 in Erk activation was confirmed at the single cell level using a fluorescent biosensor of Erk kinase activity. Depletion of P-Rex1 from breast cancer cells failed to affect cell cycle progression, cyclin D1 induction, Akt activation and apoptotic responses. In addition, mammary-specific P-Rex1 transgenic mice (MMTV-P-Rex1) did not show any obvious hyperproliferative phenotype. Therefore, despite its crucial role in Rac1 activation and cell motility, P-Rex1 is dispensable for mitogenic or survival responses in breast cancer cells.

Effect of polyhexamethylene biguanide on rat liver.

Polyhexamethylene biguanide (PHMB), an amphiphilic polymeric biocide, increased liver tumor incidence in male and female rats at 1000 and 1500 mg/L in drinking water, but not at 500 mg/L in previous studies. In another study, PHMB administered in diet at 4000 mg/kg was negative for hepatocellular tumors. The present studies evaluated bioavailability and distribution of PHMB administered in drinking water and diet and possible modes of action (MOA). PHMB in drinking water was unpalatable during the first 3 days, resulting in markedly decreased food consumption and decreased body weight. Ki-67 labeling index was increased in hepatocytes and endothelial cells dose responsively with PHMB administered in drinking water but not diet. Vitamin E had no effect on this. There was no cytotoxicity by histopathology or serum enzymes, and no increase in cytokines TNFα, IL-1α or NF-κB. Focal iron deposition in sinusoidal lining cells was detected. Microarray analyses were non-contributory. No effect on CAR or PPARα activation was detected. 14C-PHMB administered at 500, 1000, or 1500 mg/L in the drinking water or 4000 mg/kg in the diet was nearly completely absorbed and excreted in urine, with some fecal excretion. The hypothesized MOA for liver tumors induced by PHMB in drinking water is: 1) severe dehydration and starvation because of unpalatability, followed by ingestion with rapid absorption and urinary excretion; 2) increased hepatocyte proliferation; and 3) induction of hepatocellular foci and tumors. The PHMB-induced rat hepatocellular tumors are unlikely to pose a human cancer risk. However, the actual MOA has not been determined.

Uncoupling the Mitogenic and Metabolic Functions of FGF1 by Tuning FGF1-FGF Receptor Dimer Stability.

The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partial agonist carrying triple mutations (FGF1ΔHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1ΔHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 in vitro and in vivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.

Dissociation of Survival, Proliferation, and State Control in Human Hematopoietic Stem Cells.

The role of growth factors (GFs) in controlling the biology of human hematopoietic stem cells (HSCs) remains limited by a lack of information concerning the individual and combined effects of GFs directly on the survival, Mitogenesis, and regenerative activity of highly purified human HSCs. We show that the initial input HSC activity of such a purified starting population of human cord blood cells can be fully maintained over a 21-day period in serum-free medium containing five GFs alone. HSC survival was partially supported by any one of these GFs, but none were essential, and different combinations of GFs variably stimulated HSC proliferation. However, serial transplantability was not detectably compromised by many conditions that reduced human HSC proliferation and/or survival. These results demonstrate the dissociated control of these three human HSC bio-responses, and set the stage for future improvements in strategies to modify and expand human HSCs ex vivo.