Fluoroquinolones upregulate insulin-like growth factor-binding protein 3, inhibit cell growth and insulin-like growth factor signaling.

Fluoroquinolones (FQs), commonly known for their antibiotic properties, exhibit additional pharmacological potential with anti-proliferative effects on various malignant cell types and immunomodulatory responses. Despite these observed effects, the precise mechanisms of action remain elusive. This study elucidates the biological impact of FQs on insulin-like growth factor-binding protein 3 (IGFBP-3) productions in a p53-dependent manner. Cultured cells and mouse models treated with FQs demonstrated increased IGFBP-3 mRNA expression and protein secretion. The FQ-induced IGFBP-3 was identified to impede cell growth by inhibiting IGF-I signaling and exerting effects through an IGF-independent pathway. Notably, FQ-mediated suppression of cell proliferation was reversed in p53-null and p53 knockdown cells, suggesting the pivotal role of p53 in FQ-induced IGFBP-3 production and IGFBP-3-mediated growth inhibition. Additionally, ciprofloxacin, a clinically used FQ, exhibited the induction of tumor cell apoptosis and attenuation of tumor growth in a syngeneic mouse hepatocellular carcinoma (HCC) model. These findings unveil a novel mechanism through which FQs act as anti-proliferative agents, prompting further exploration of their potential utility or derivative compounds in cancer treatment and prevention.

Characterization of choroid plexus in the preterm rabbit pup following subcutaneous administration of recombinant human IGF-1/IGFBP-3.

Insulin-like growth factor-1 (IGF-1) is essential for normal brain development and regulates essential processes of vascular maturation and stabilization. Importantly, preterm birth is associated with reduced serum levels of IGF-1 as compared to in utero levels. Using a preterm rabbit pup model, we investigated the uptake of systemic recombinant human (rh) IGF-1 in complex with its main binding protein IGF-binding protein 3 (BP-3) to the brain parenchyma via the choroid plexus. Five hours after subcutaneous administration, labeled rhIGF-1/rhIGFBP-3 displayed a widespread presence in the choroid plexus of the lateral and third ventricle, however, to a less degree in the fourth, as well as in the perivascular and subarachnoid space. We found a time-dependent uptake of IGF-1 in cerebrospinal fluid, decreasing with postnatal age, and a translocation of IGF-1 through the choroid plexus. The impact of systemic rhIGF-1/rhIGFBP-3 on IGF-1 receptor activation in the choroid plexus decreased with postnatal age, correlating with IGF-1 uptake in cerebrospinal fluid. In addition, choroid plexus gene expression was observed to increase with postnatal age. Moreover, using choroid plexus in vitro cell cultures, gene expression and protein synthesis were further investigated upon rhIGF-1/rhIGFBP-3 stimulation as compared to rhIGF-1 alone, and found not to be differently altered. Here, we characterize the uptake of systemic rhIGF-1/rhIGFBP-3 to the preterm brain, and show that the interaction between systemic rhIGF-1/rhIGFBP-3 and choroid plexus varies over time.

Mychonastes sp. 246 Suppresses Human Pancreatic Cancer Cell Growth via IGFBP3-PI3K-mTOR Signaling.

Previously, we confirmed that Mychonastes sp. 246 methanolic extract (ME) markedly reduced the viability of BxPC-3 human pancreatic cancer cells. However, the underlying mechanism ME remained unclear. Hence, we attempted to elucidate the anticancer effect of ME on BxPC-3 human pancreatic cancer cells. First, we investigated the components of ME and their cytotoxicity in normal cells. Then, we confirmed the G1 phase arrest mediated growth inhibitory effect of ME using a cell counting assay and cell cycle analysis. Moreover, we found that the migration-inhibitory effect of ME using a Transwell migration assay. Through RNA sequencing, Gene Ontology-based network analysis, and western blotting, we explored the intracellular mechanisms of ME in BxPC-3 cells. ME modulated the intracellular energy metabolism-related pathway by altering the mRNA levels of IGFBP3 and PPARGC1A in BxPC-3 cells and reduced PI3K and mTOR phosphorylation by upregulating IGFBP3 and 4E-BP1 expression. Finally, we verified that ME reduced the growth of three-dimensional (3D) pancreatic cancer spheroids. Our study demonstrates that ME suppresses pancreatic cancer proliferation through the IGFBP3-PI3K-mTOR signaling pathway. This is the first study on the anticancer effect of the ME against pancreatic cancer, suggesting therapeutic possibilities and the underlying mechanism of ME action.

Chordin Like-1 Regulates Osteoblast and Adipocyte Differentiation Through Stabilizing Insulin-Like Growth Factor Binding Protein 3.

Chordin like-1 (CHRDL1) is an antagonist of bone morphogenetic proteins (BMPs) that acts through binding BMPs and blocking their interaction with BMP receptors. CHRDL1 plays a role in osteoblast differentiation but controversial effects were reported. On the other hand, the role of CHRDL1 in adipogenesis is unknown. In the present study, we investigated the function of CHRDL1 in regulating differentiation of osteoblasts and adipocytes and elucidated the underlying mechanism. CHRDL1 expression was downregulated during osteogenesis while it was upregulated during adipogenesis in primary cultured and established mesenchymal progenitor cell lines. Functional experiments revealed that CHRDL1 suppressed osteoblast differentiation and promoted adipocyte differentiation. Mechanistic explorations revealed that CHRDL1 is directly bound to insulin-like growth factor binding protein 3 (IGFBP3) and attenuated the degradation of the latter. Furthermore, CHRDL1 and IGFBP3 suppressed the activity of insulin receptor substrate 1 (IRS1)/AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin kinase complex 1 (mTORC1) signaling in progenitor cells undergoing osteogenic differentiation. By contrast, they activated AKT/mTORC1 signaling independently of IRS1 during adipogenic differentiation. CHRDL1 enhanced the interaction of nuclear IGFBP3 and retinoid X receptor α (RXRα) during adipogenesis, and inhibition of RXR inactivated AKT and attenuated the stimulation of adipogenic differentiation by CHRDL1. Overexpression of IGFBP3 relieved the perturbation of osteogenic and adipogenic differentiation of progenitor cells induced by CHRDL1 silencing. Finally, CHRDL1 and IGFBP3 were upregulated in the trabecular bone of aged mice. Our study provides evidence that CHRDL1 reciprocally regulates osteoblast and adipocyte differentiation through stabilizing IGFBP3 and differentially modulating AKT/mTORC1 signaling.

Thrombolysis by PLAT/tPA increases serum free IGF1 leading to a decrease of deleterious autophagy following brain ischemia.

Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation.Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin.

The effects of recombinant human insulin-like growth factor-1/insulin-like growth factor binding protein-3 administration on lipid and carbohydrate metabolism in recreational athletes.

OBJECTIVE: Previous studies suggested that recombinant human IGF-1 (rhIGF-1) administration affects carbohydrate and lipid metabolism in healthy people and in people with diabetes. This study aimed to determine the effects of rhIGF-1/rhIGF binding protein-3 (rhIGFBP-3) administration on glucose homeostasis and lipid metabolism in healthy recreational athletes. DESIGN AND SETTING: Randomized, double-blind, placebo-controlled rhIGF-1/rhIGFBP-3 administration study at Southampton General Hospital, UK. PARTICIPANTS: 56 recreational athletes (30 men, 26 women). METHODS: Participants were randomly assigned to receive placebo, low-dose rhIGF-1/rhIGFBP-3 (30 mg/day) or high-dose rhIGF-1/rhIGFBP-3 (60 mg/day) for 28 days. The following variables were measured before and immediately after the treatment period: fasting lipids, glucose, insulin, C-peptide and glycated haemoglobin. The homeostatic model assessment (HOMA-IR) was used to estimate insulin sensitivity and indirect calorimetry to assess substrate oxidation rates. The general linear model approach was used to compare treatment group changes with the placebo group. RESULTS: Compared with the placebo group, there was a significant reduction in fasting triglycerides in participants treated with high-dose rhIGF-1/rhIGFBP-3 (p = .030), but not in the low-dose group (p = .390). In women, but not in men, there were significant increases in total cholesterol (p = .003), HDL cholesterol (p = .001) and LDL cholesterol (p = .008). These lipid changes were associated with reduced fasting insulin (p = .010), C-peptide (p = .001) and HOMA-IR (p = .018) in women and reduced C-peptide (p = .046) in men. CONCLUSIONS: rhIGF-1/rhIGFBP-3 administration for 28 days reduced insulin concentration, improved insulin sensitivity and had significant effects on lipid profile including decreased fasting triglycerides.

IGFBP-3 Blocks Hyaluronan-CD44 Signaling, Leading to Increased Acetylcholinesterase Levels in A549 Cell Media and Apoptosis in a p53-Dependent Manner.

Insulin-like growth factor binding protein-3 (IGFBP-3) belongs to a family of six IGF binding proteins. We previously found that IGFBP-3 exerts its cytotoxic effects on A549 (p53 wild-type) cell survival through a mechanism that depends on hyaluronan-CD44 interactions. To shed light on the mechanism employed, we used CD44-negative normal human lung cells (HFL1), A549, and H1299 (p53-null) lung cancer cells. A synthetic IGFBP-3 peptide (215-KKGFYKKKQCRPSKGRKR-232) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP-3 protein, the peptide blocked hyaluronan-CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP-3 protein or its peptide resulted in increased acetylcholinesterase concentration and activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with increased apoptosis and decreased cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP-3 or its peptide blocks hyaluronan-CD44 signaling via a mechanism that depends on both p53 and acetylcholinesterase.

Response to IGF-1 Generation Test in Short Prepubertal Children Born Very Preterm or at Term.

AIMS: To investigate whether short children born very preterm (<32 weeks of gestation) exhibit features of growth hormone (GH) resistance compared to term peers. METHODS: We studied 26 prepubertal children (aged 7.0 ± 2.0 years) with short stature (height adjusted for parents' heights <10th percentile), who were born appropriate for gestational age and either very preterm (n = 11) or at term (n = 15). Children underwent insulin-like growth factor-1 (IGF-1) generation test via a daily recombinant human GH (rhGH) dose (0.05 mg/kg/day) over 4 consecutive days. Hormone and binding proteins were measured at baseline and day 5. RESULTS: At baseline, preterm children had lower IGF-binding protein 1 (IGFBP-1; -22%; p = 0.049) and IGFBP-3 (-24%; p = 0.013) concentrations than term children. Preterm children also had insulin concentrations that tended to be 39% higher (p = 0.059) than term peers. After stimulation, IGF-1 and IGFBP-3 concentrations increased similarly in term and preterm groups, while GH-binding protein (GHBP) concentrations decreased in both groups. Preterm children had higher GHBP (+50%; p = 0.049), insulin (+86%; p = 0.005), and leptin (+107%; p = 0.020) but lower IGFBP-1 (-47%; p = 0.006) concentrations than term children following rhGH stimulation. CONCLUSIONS: Preterm children who are short for genetic height potential show no evidence of GH resistance that would explain their short stature. However, there was indirect evidence of insulin resistance in the preterm children, as previously described in this group.

The Effects of Recombinant Human Insulin-Like Growth Factor-I/Insulin-Like Growth Factor Binding Protein-3 Administration on Body Composition and Physical Fitness in Recreational Athletes.

CONTEXT: IGF-I is thought to mediate many of the anabolic actions of GH, and there are anecdotal reports that IGF-I is misused by elite athletes. There is no published evidence regarding the effects of IGF-I administration on athletic performance. OBJECTIVE: The objective of the study was to investigate the effects of IGF-I administration on body composition and physical fitness in recreational athletes. DESIGN AND SETTING: This was a randomized, double-blind, placebo-controlled recombinant human (rh) IGF-I/rhIGF binding protein (IGFBP)-3 administration study at Southampton General Hospital (Southampton, United Kingdom). PARTICIPANTS: Fifty-six recreational athletes (30 men, 26 women) participated in the study. INTERVENTION: Participants were randomly assigned to receive placebo, low-dose rhIGF-I/rhIGFBP-3 (30 mg/d), or high dose rhIGF-I/rhIGFBP-3 (60 mg/d) for 28 days. Body composition (assessed by dual energy x-ray absorptiometry) and cardiorespiratory fitness (assessed by incremental treadmill test) were measured before and immediately after treatment. Within-individual changes after treatment were analyzed using paired t tests. RESULTS: There were no significant changes in body fat mass or lean body mass in women or men after the administration of the rhIGF-I/rhIGFBP-3 complex. There was a significant increase in maximal oxygen consumption (VO2 max) after treatment. When women and men and low- and high-dose treatment groups were combined, mean VO2 max increased by approximately 7% (P = .001). No significant change in VO2 max was observed in the placebo group. CONCLUSIONS: rhIGF-I/rhIGFBP-3 administration for 28 days improves aerobic performance in recreational athletes, but there are no effects on body composition.

IGFBP-3 reduces eNOS and PKCzeta phosphorylation, leading to lowered VEGF levels.

PURPOSE: In models of diabetic retinopathy, insulin-like growth factor binding protein-3 (IGFBP-3) is protective to the retina, especially retinal microvascular endothelial cells (RECs), but the underlying mechanisms are unclear. For this study, we hypothesized that IGFBP-3 may reduce vascular endothelial growth factor (VEGF) levels through reduced endothelial nitric oxide synthase (eNOS) activity, which may be protective against macular edema. METHODS: To test this hypothesis, we grew primary human retinal endothelial cells in normal glucose (5 mM) or high glucose (25 mM) for three days, treated with IGFBP-3 NB plasmid (a plasmid of IGFBP-3 that cannot bind IGF-1), followed by western blotting for eNOS, protein kinase C zeta (PKCzeta), and VEGF. Additionally, we treated some cells with recombinant eNOS or PKCzeta, after IGFBP-3 NB plasmid transfection to validate that these pathways regulate VEGF levels. Immunoprecipitation experiments were done with the eNOS antibody, followed by western blotting for PKCzeta, to determine if eNOS and PKCzeta interact directly. RESULTS: Our results suggest that 1) IGFBP-3 inhibits the endothelial nitric oxide synthase (eNOS) and protein kinase C zeta (PKCzeta) pathway, which in turn inhibits VEGF production, and 2) that eNOS plays a role in activating PKCzeta to increase VEGF levels in diabetic retinopathy. CONCLUSIONS: In conclusion, IGFBP-3 may be a novel treatment for macular edema through the inhibition of eNOS and PKCzeta activation, leading to reduced VEGF levels.

Insulin-like growth factor binding protein-3 inhibits cell adhesion via suppression of integrin ß4 expression.

We previously reported that IGF binding protein-3 (IGFBP-3), a major IGF-binding protein in human serum, regulates angiogenic activities of human head and neck squamous cell carcinoma (HNSCC) cells and human umbilical vein endothelial cells (HUVECs) through IGF-dependent and IGF-independent mechanisms. However, the role of IGFBP-3 in cell adhesion is largely unknown. We demonstrate here that IGFBP-3 inhibits the adhesion of HNSCC cells and HUVECs to the extracellular matrix (ECM). IGFBP-3 reduced transcription of a variety of integrins, especially integrin ß4, and suppressed phosphorylation of focal adhesion kinase (FAK) and Src in these cells through both IGF-dependent and IGF-independent pathways. IGFBP-3 was found to suppress the transcription of c-fos and c-jun and the activity of AP1 transcription factor. The regulatory effect of IGFBP-3 on integrin ß4 transcription was attenuated by blocking c-jun and c-fos gene expression via siRNA transfection. Taken together, our data show that IGFBP-3 has IGF-dependent and -independent inhibitory effects on intracellular adhesion signaling in HNSCC and HUVECs through its ability to block c-jun and c-fos transcription and thus AP-1-mediated integrin ß4 transcription. Collectively, our data suggest that IGFPB-3 may be an effective cancer therapeutic agent by blocking integrin-mediated adhesive activity of tumor and vascular endothelial cells.

Insulin-like-growth-factor-binding-protein-3 (IGFBP-3) contrasts melanoma progression in vitro and in vivo.

Insulin-like-factor-binding-protein 3 (IGFBP-3) is known to modulate the activity of insulin-like growth factors (IGFs) besides having a number of IGF-independent effects on cell growth and survival. IGFBP-3 has been reported to decrease significantly in the blood serum of patients affected by certain cancers. In the present work, we have evaluated the levels of IGFBP-3 in the blood serum and tissues of patients affected by cutaneous melanoma, showing that loss of IGFBP-3 from both is strongly correlated with disease progression and reduced survival. In vitro treatment with IGFBP-3 of human and murine metastatic melanoma cell lines specifically inhibited the cells' migratory and invasive behaviour, inducing up-regulation of melanocytic differentiation markers such as tyrosinase activity and melanin content. A molecular analysis of the cellular pathways transducing the effect of IGFBP-3 implicated the Akt-GSK3ß axis. Moreover, administration of IGFBP-3 in vivo to SCID mice inoculated with human metastatic melanoma cells strongly reduced or completely inhibited tumor growth. In summary, IGFBP-3 appears to exert a specific inhibitory effect on melanoma growth and dissemination, suggesting that it may qualify as a useful therapeutic agent in melanomas and perhaps other cancers, at the least as a valid adjuvant therapy during treatment with conventional anti-tumoral drugs.

Intravitreal injection of IGFBP-3 restores normal insulin signaling in diabetic rat retina.

Diabetes-induced changes in growth factor binding protein 3 (IGFBP-3) and tumor necrosis factor alpha (TNFα) have been linked to decreased insulin receptor signaling in diabetic retinopathy. Our previous studies in retinas of diabetic rats have shown that Compound 49b, a novel ß-adrenergic receptor agonist, prevented diabetic changes by increasing IGFBP-3 and decreasing TNFα, thus restoring insulin signaling and protection against diabetic retinopathy. The current study was designed to determine whether boosted expression of IGFBP-3 NB (a non-IGF-1 binding form of IGFBP-3) alone is sufficient to mimic the full actions of Compound 49b in protecting against diabetic retinopathy, as well as testing whether IGFBP-3 NB is linked to a restoration of normal insulin signal transduction. Two months after initiation of streptozotocin-induced diabetes, rats received a single intravitreal injection of IGFBP-3 NB plasmid in the right eye. Four days after injection, electroretinogram (ERG) analyses were performed prior to sacrifice. Whole retinal lysates from control, diabetic, diabetic + control plasmid, and diabetic+ IGFBP-3 NB were analyzed for IGFBP-3, TNFα, suppressor of cytokine signaling 3 (SOCS3), and insulin receptor signaling partners using Western blotting or ELISA. Data show that a single intraocular injection of IGFBP-3 NB in diabetic animals significantly reduced TNFα levels, concomitant with reductions in IRS-1Ser307, SOCS3, and pro-apoptotic markers, while restoring insulin receptor phosphorylation and increasing anti-apoptotic marker levels. These cellular changes were linked to restoration of retinal function. Our findings establish IGFBP-3 as a pivotal regulator of the insulin receptor/TNFα pathway and a potential therapeutic target for diabetic retinopathy.

Inhibition of insulin-like growth factor-binding protein-3 signaling through sphingosine kinase-1 sensitizes triple-negative breast cancer cells to EGF receptor blockade.

The type I EGF receptor (EGFR or ErbB1) and insulin-like growth factor-binding protein-3 (IGFBP-3) are highly expressed in triple-negative breast cancer (TNBC), a particularly aggressive disease that cannot be treated with conventional therapies targeting the estrogen or progesterone receptors (ER and PR), or HER2. We have shown previously in normal breast epithelial cells that IGFBP-3 potentiates growth-stimulatory signaling transduced by EGFR, and this is mediated by the sphingosine kinase-1 (SphK1)/sphingosine 1-phosphate (S1P) system. In this study, we investigated whether cotargeting the EGFR and SphK1/S1P pathways in TNBC cells results in greater growth inhibition compared with blocking either alone, and might therefore have novel therapeutic potential in TNBC. In four TNBC cell lines, exogenous IGFBP-3 enhanced ligand-stimulated EGFR activation, associated with increased SphK1 localization to the plasma membrane. The effect of exogenous IGFBP-3 on EGFR activation was blocked by pharmacologic inhibition or siRNA-mediated silencing of SphK1, and silencing of endogenous IGFBP-3 also suppressed EGF-stimulated EGFR activation. Real-time analysis of cell proliferation revealed a combined effect of EGFR inhibition by gefitinib and SphK1 inhibition using SKi-II. Growth of MDA-MB-468 xenograft tumors in mice was significantly inhibited by SKi-II and gefitinib when used in combination, but not as single agents. We conclude that IGFBP-3 promotes growth of TNBC cells by increasing EGFR signaling, that this is mediated by SphK1, and that combined inhibition of EGFR and SphK1 has potential as an anticancer therapy in TNBC in which EGFR and IGFBP-3 expression is high.

Inhibition of human MCF-7 breast cancer cells and HT-29 colon cancer cells by rice-produced recombinant human insulin-like growth binding protein-3 (rhIGFBP-3).

BACKGROUND: Insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I) to form a complex (IGF-I/IGFBP-3) that can treat growth hormone insensitivity syndrome (GHIS) and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3) in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL) were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 ± 1.72% vs 45.00 ± 0.86%, p < 0.05; 50.84 ± 1.97% vs 45.00 ± 0.86%, p < 0.01 respectively) and HT-29 colon cancer cells (65.14 ± 3.84% vs 18.01 ± 13.81%, p < 0.05 and 54.7 ± 9.44% vs 18.01 ± 13.81%, p < 0.05 respectively) when compared with wild type rice. CONCLUSION/SIGNIFICANCE: These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future.

Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is an autosomal recessive disorder caused by mutations in the laminin-α2 gene (OMIM: 607855). Currently, no treatment other than palliative care exists for this disease. In our previous work, genetic interventions in the Lama2(Dy-w) mouse model for MDC1A demonstrated that limited regeneration and uncontrolled apoptosis are important drivers of this disease. However, targeting one of these disease drivers without addressing the other results in only partial rescue of the phenotype. The present study was designed to determine whether utilizing a combinatorial treatment approach can lead to a more profound amelioration of the disease pathology. To accomplish this task, we generated Bax-null Lama2(Dy-w)mice that overexpressed muscle-specific IGF-1 (Lama2(Dy-w)Bax(-/-)+IGF-1tg). Further to test the translational potential of IGF-1 administration in combination with Bax inhibition, we treated Lama2(Dy-w)Bax(-/-) mice postnatally with systemic recombinant human IGF-1 (IPLEX™). These two combinatorial treatments lead to similar, promising outcomes. In addition to increased body and muscle weights, both transgenic overexpression and systemic administration of IGF-1 combined with Bax-inhibition resulted in improved muscle phenotype and locomotory function that were nearly indistinguishable from wild-type mice. These results provide a fundamental proof of concept that justifies the use of a combination therapy as an effective treatment for MDC1A and highlights a compelling argument toward shifting the paradigm in treating multifaceted neuromuscular diseases.

Regulation of retinal endothelial cell apoptosis through activation of the IGFBP-3 receptor.

The goal of this study was to investigate whether insulin-like growth factor binding protein-3 receptor (IGFBP-3 receptor) is required for IGFBP-3 to inhibit retinal endothelial cell (REC) apoptosis. REC were grown in normal glucose (5 mM) or high glucose medium (25 mM) for 3 days. Once cells reached confluence, they were transfected with an endothelial- specific IGFBP-3 plasmid DNA (non-IGF binding; IGFBP-3 NB) at 1 µg/ml for 24 h. Cell proteins were extracted and analyzed for IGFBP-3 receptor expression by Western blotting or use in coimmunoprecipitation or co-localization experiments for detection of IGFBP-3 and IGFBP-3 receptor binding. REC were also transfected with or without IGFBP-3 receptor siRNA before IGFBP-3NB plasmid DNA transfection. Cell lysates were processed for a cell death ELISA, a cleaved caspase 3 ELISA, and Western blotting to measure key pro- and anti-apoptotic markers: Bcl-xL, Bax, Cytochrome C and Akt. The IGFBP-3 receptor is present on REC. Overexpression of IGFBP-3 in REC significantly increased protein levels of IGFBP-3 receptor (p < 0.05). Significant increases in cell death were found in cells transfected with IGFBP-3 receptor siRNA versus not treated samples (p < 0.05). Data suggest that IGFBP-3 inhibits retinal endothelial cell death through activation of an IGFBP-3 receptor in a hyperglycemic environment. This is the first demonstration of the involvement of IGFBP-3 receptor in inhibition of REC cell death. Future studies will investigate the mechanism by which IGFBP-3 receptor may inhibit retinal endothelial cell death.

Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells.

Insulin-like growth factor 1 (IGF-1), the most abundant growth factor in the bone matrix, maintains bone mass in adulthood. We now report that IGF-1 released from the bone matrix during bone remodeling stimulates osteoblastic differentiation of recruited mesenchymal stem cells (MSCs) by activation of mammalian target of rapamycin (mTOR), thus maintaining proper bone microarchitecture and mass. Mice with knockout of the IGF-1 receptor (Igf1r) in their pre-osteoblastic cells showed lower bone mass and mineral deposition rates than wild-type mice. Further, MSCs from Igf1rflox/flox mice with Igf1r deleted by a Cre adenovirus in vitro, although recruited to the bone surface after implantation, were unable to differentiate into osteoblasts. We also found that the concentrations of IGF-1 in the bone matrix and marrow of aged rats were lower than in those of young rats and directly correlated with the age-related decrease in bone mass. Likewise, in age-related osteoporosis in humans, we found that bone marrow IGF-1 concentrations were 40% lower in individuals with osteoporosis than in individuals without osteoporosis. Notably, injection of IGF-1 plus IGF binding protein 3 (IGFBP3), but not injection of IGF-1 alone, increased the concentration of IGF-1 in the bone matrix and stimulated new bone formation in aged rats. Together, these results provide mechanistic insight into how IGF-1 maintains adult bone mass, while also providing a further rationale for its therapeutic targeting to treat age-related osteoporosis.

IPLEX administration improves motor neuron survival and ameliorates motor functions in a severe mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disorder and the first genetic cause of death in childhood. SMA is caused by low levels of survival motor neuron (SMN) protein that induce selective loss of α-motor neurons (MNs) in the spinal cord, resulting in progressive muscle atrophy and consequent respiratory failure. To date, no effective treatment is available to counteract the course of the disease. Among the different therapeutic strategies with potential clinical applications, the evaluation of trophic and/or protective agents able to antagonize MNs degeneration represents an attractive opportunity to develop valid therapies. Here we investigated the effects of IPLEX (recombinant human insulinlike growth factor 1 [rhIGF-1] complexed with recombinant human IGF-1 binding protein 3 [rhIGFBP-3]) on a severe mouse model of SMA. Interestingly, molecular and biochemical analyses of IGF-1 carried out in SMA mice before drug administration revealed marked reductions of IGF-1 circulating levels and hepatic mRNA expression. In this study, we found that perinatal administration of IPLEX, even if does not influence survival and body weight of mice, results in reduced degeneration of MNs, increased muscle fiber size and in amelioration of motor functions in SMA mice. Additionally, we show that phenotypic changes observed are not SMN-dependent, since no significant SMN modification was addressed in treated mice. Collectively, our data indicate IPLEX as a good therapeutic candidate to hinder the progression of the neurodegenerative process in SMA.

Recombinant IGFBP-3 inhibits allergic lung inflammation, VEGF production, and vascular leak in a mouse model of asthma.

BACKGROUND: Vascular endothelial growth factor (VEGF) plays a pro-inflammatory mediator as well as a vascular permeability factor in bronchial asthma. Insulin-like growth factor (IGF)-I is also involved in the inflammatory process associated with bronchial asthma and stimulates VEGF expression. The IGF-binding proteins (IGFBPs), especially IGFBP-3, display distinctive properties and can interfere with various biological processes. METHODS: In this study, an ovalbumin (OVA)-induced murine model of allergic airway disease was used to investigate which mechanism is implicated in the preventive and therapeutic actions of IGFBP-3 administered exogenously on allergen-induced bronchial inflammation and airway hyper-responsiveness, in particular focusing on the regulation of VEGF expression. RESULTS: Administration of recombinant human IGFBP-3 to OVA-inhaled mice substantially attenuated the increases in hypoxia-inducible factor (HIF)-α activity, IGF-I production, and VEGF protein levels in the lung. In addition, the blockade of IGF-I action decreased the OVA-induced VEGF expression, airway inflammation, and bronchial hyper-responsiveness. The administration of recombinant human IGFBP-3 or CBO-P11 also reduced significantly increases in inflammatory cells, airway hyper-responsiveness, levels of IL-4, IL-5, IL-13, and vascular permeability in the lung of OVA-inhaled mice. Moreover, when recombinant human IGFBP-3 was administered after the completion of OVA inhalation, these therapeutic effects of IGFBP-3 were also observed. CONCLUSIONS: These results indicate that IGFBP-3 administered exogenously may attenuate antigen-induced airway inflammation and hyper-responsiveness through the modulation of vascular leakage and VEGF expression mediated by HIF-1α/HIF-2α signaling as well as IGF-I action in allergic airway disease of mice.