Integrin β1 regulates leiomyoma cytoskeletal integrity and growth

Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin β1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52 ± 0.02), laminin 5β (3.06 ± 0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells.     

Impact of HIF-1α and hypoxia on fungal growth characteristics and fungal immunity

Human pathogenic fungi are highly adaptable to a changing environment. The ability to adjust to low oxygen conditions is crucial for colonization and infection of the host. Recently, the impact of mammalian hypoxia-inducible factor-1α (HIF-1α) on fungal immunity has emerged. In this review, the role of hypoxia and HIF-1α in fungal infections is discussed regarding the innate immune response.     

Fetal concentrations of the growth factors TGF-α and TGF-β1 in relation to normal and restricted fetal growth at term

Transforming growth factor-α (TGF-α) and TGF-β1 are major anti-inflammatory cytokines and substantially contribute to normal pregnancy outcome. TGF-α stimulates placental mitosis, whereas TGF-β1 is a critical regulator of trophoblast invasion and fetal growth. We aimed to study cord blood TGF-α and TGF-β1 concentrations in intrauterine-growth-restricted (IUGR, usually associated with abnormal trophoblast invasion, uteroplacental vascular insufficiency and enhanced inflammation) and appropriate-for-gestational-age-(AGA) pregnancies, and investigate possible correlations of the above concentrations with several demographic parameters of infants at birth. Plasma TGF-α and TGF-β1 concentrations were determined by ELISA in 154 mixed arterio-venous cord blood samples from IUGR (n=50) and AGA (n=104) singleton full-term infants. After controlling for possible confounding factors (gender, birth-weight, gestational age, maternal age and parity), cord blood TGF-α and TGF-β1 concentrations were significantly higher in IUGR than AGA group (b=0.402, SE=0.179, p=0.027 and b=0.152, SE=0.061, p=0.014, respectively). Delivery mode had an effect on cord blood TGF-α and TGF-β1 concentrations, both being elevated in cases of vaginal delivery (b=-0.282, SE=0.117, p=0.018 and b=-0.123, SE=0.059, p=0.038, respectively). In conclusion, higher cord blood TGF-α and TGF-β1 concentrations may represent a compensatory response to the inflammatory process characterizing the IUGR state. Additionally, higher cord blood TGF-β1 concentrations in IUGRs could be attributed to increased shear stress, resulting from abnormal blood flow in IUGR fetal blood vessels. Finally, vaginal delivery-associated cytokine release may account for elevated TGF-α and TGF-β1 concentrations.     

Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction

Fibroblast proliferation is an early feature of progressive tissue fibrosis and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). In the oral mucosa, fibroblasts have a unique phenotype and demonstrate healing with no fibrosis/scarring. Our previous studies show that whereas dermal fibroblasts proliferate in response to TGF-β1, oral fibroblasts have an antiproliferative response to this cytokine. Hyaluronan (HA) was directly linked to this TGF-β1-dependent response. The aim of this study was to understand the underlying mechanism through which HA regulates TGF-β-dependent responses. Using patient-matched oral and dermal fibroblasts, we show that TGF-β1-dependent proliferation is mediated through the HA receptor CD44, whereas the TGF-β1-mediated antiproliferative response is CD44-independent. Furthermore, overexpression of HAS2 (HA synthase-2) in oral cells modifies their response, and they subsequently demonstrate a proliferative, CD44-dependent response to TGF-β1. We also show that epidermal growth factor (EGF) and its receptor (EGFR) are essential for TGF-β1/HA/CD44-dependent proliferation. Increased HA levels promote EGFR and CD44 coupling, potentiating signal transduction through the MAPK/ERK pathway. Thus, in a HA-rich environment, late ERK1/2 activation results from EGFR/CD44 coupling and leads to a proliferative response to TGF-β1. In comparison, in a non-HA-rich environment, only early ERK1/2 activation occurs, and this is associated with an antiproliferative response to TGF-β1. In summary, HA facilitates TGF-β1-dependent fibroblast proliferation through promoting interaction between CD44 and EGFR, which then promotes specific MAPK/ERK activation, inducing cellular proliferation.     

γ-Secretase and presenilin mediate cleavage and phosphorylation of vascular endothelial growth factor receptor-1

We have reported previously that pigment epithelium-derived factor (PEDF) can, via γ-secretase-mediated events, inhibit VEGF-induced angiogenesis in microvascular endothelial cells by both (a) cleavage and intracellular translocation of a C-terminal fragment of VEGF receptor-1 (VEGFR1) and (b) inhibition of VEGF-induced phosphorylation of VEGFR1. Using site-direct mutagenesis and transfection of wild type and mutated receptors into endothelial cells, we showed that transmembrane cleavage of VEGFR1 occurs at valine 767 and that a switch from valine to alanine at this position prevented cleavage and formation of a VEGFR1 intracellular fragment. Using siRNA to selectively knock down protein-tyrosine phosphatases (PTPs) in endothelial cells, we demonstrated that vascular endothelial PTP is responsible for dephosphorylation of activated VEGFR1. PEDF up-regulation of full-length presenilin 1 (Fl.PS1) facilitated the association of vascular endothelial PTP and VEGFR1. Knockdown of Fl.PS1 prevented dephosphorylation of VEGFR1, whereas up-regulation of Fl.PS1 stimulated VEGFR1 dephosphorylation. Fl.PS1 associated with VEGFR1 within 15 min after PEDF treatment. In conclusion, we determined the PEDF-mediated events responsible for VEGFR1 signaling and identified full-length presenilin as a critical adaptor molecule in the dephosphorylation of VEGFR1. This greater understanding of the regulation of VEGFR1 signaling will help identify novel anti-VEGF therapeutic strategies.     

A c-Myc–SIRT1 feedback loop regulates cell growth and transformation

The protein deacetylase SIRT1 has been implicated in a variety of cellular functions, including development, cellular stress responses, and metabolism. Increasing evidence suggests that similar to its counterpart, Sir2, in yeast, Caenorhabditis elegans, and Drosophila melanogaster, SIRT1 may function to regulate life span in mammals. However, SIRT1''s role in cancer is unclear. During our investigation of SIRT1, we found that c-Myc binds to the SIRT1 promoter and induces SIRT1 expression. However, SIRT1 interacts with and deacetylates c-Myc, resulting in decreased c-Myc stability. As a consequence, c-Myc''s transformational capability is compromised in the presence of SIRT1. Overall, our experiments identify a c-Myc–SIRT1 feedback loop in the regulation of c-Myc activity and cellular transformation, supporting/suggesting a role of SIRT1 in tumor suppression.     

Transforming growth factor-β1 regulation of laminin γ1 and fibronectin expression and survival of mouse mesangial cells

The transforming growth factor-beta (TGF-β) 1 is a mediator of extracellular matrix (ECM) gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we investigate the effects of TGF-β1 on laminin γ1 and fibronectin polypeptide expression and cell survival in mouse mesangial cells (MES-13). TGF-β1 (10 ng/ml) stimulates laminin-γ1 and fibronectin expression ~two-fold in a time-dependent manner (0–48 h). TGF-β1 treatment also retards laminin-γ1 mobility on SDS-gels, and tunicamycin, an inhibitor of the N-linked glycosylation, blocks the mobility shift. TGF-β1 increases the binding of laminin γ1 to WGA-agarose and the binding is abolished by tunicamycin suggesting that laminin γ1 is modified by N-linked glycosylation. TGF-β1 also elevates fibronectin glycosylation but its mobility is not altered. The degradation of laminin γ1 and fibronectin proteins is reduced by their glycosylation. In addition, TGF-β1 enhances mesangial cell viability and metabolic activities initially (0–24 h); however, eventually leads to cell death (24–48 h). TGF-β1 elevates pro-apoptotic caspase-3 activity and decrease cell cycle progression factor cyclin D1 expression, which parallels cell death. These results indicate that TGF-β1 plays an important role in ECM expression, protein glycosylation and demise of mesangial cells in the diabetic glomerular mesangium. (Mol Cell Biochem 278: 165–175, 2005)     

Selective growth promotion and growth inhibition of Gram-negative and Gram-positive bacteria by synthetic siderophore-β-lactam conjugates

Conjugates of a carbacephalosporin with hydroxamate, spermexatol, N,N-bis(2,3-dihydroxybenzoyl)-L-lysine, mixed catecholate/hydroxamate and cyanuric acid-based siderophores were investigated for their potential to promote growth of siderophore indicator strains of Gram-negative and Gram-positive bacteria under iron depleted conditions, for their antibacterial activity and for their ability to use iron transport path-ways to penetrate the Gram-negative bacterial outer membrane. The selective growth promotion of enter-obacterial and pseudomonas strains by hydroxamate, spermexatol and mixed catecholate-hydroxamate siderophore-based conjugates bearing a L- or D-amino acid spacer was correlated with TonB dependent uptake routes. The preferred outer membrane siderophore receptor used in Escherichia coli was found to be Fiu, followed by Cir. Antagonistic effects of siderophores administered with the conjugates to determine antibacterial activity confirmed the active transport of conjugates via siderophore receptors. All of the conjugates were still able to diffuse through the porin proteins OmpC and OmpF. Nevertheless, strong inhibition of E. coli and Pseudomones aeruginosa outer membrane mutants DC2 and K799/61 compared to the parent strains indicated inefficient penetrability of all types of conjugates tested. Mycobacterium smegmatis SG 987 was able to use all of the siderophore-cephalosporin conjugates as growth promotors. Consequently there was no growth inhibition of this strain. © Rapid Science 1998.     

Renal collecting system growth and function depend upon embryonic γ1 laminin expression

In order to understand the functions of laminins in the renal collecting system, the Lamc1 gene was inactivated in the developing mouse ureteric bud (UB). Embryos bearing null alleles exhibited laminin deficiency prior to mesenchymal tubular induction and either failed to develop a UB with involution of the mesenchyme, or developed small kidneys with decreased proliferation and branching, delayed renal vesicle formation and postnatal emergence of a water transport deficit. Embryonic day 12.5 kidneys revealed an almost complete absence of basement membrane proteins and reduced levels of α6 integrin and FGF2. mRNA levels for fibroblast growth factor 2 (FGF2) and mediators of the GDNF/RET and WNT11 signaling pathway were also decreased. Furthermore, collecting duct cells derived from laminin-deficient kidneys and grown in collagen gels were found to proliferate and branch slowly. The laminin-deficient cells exhibited decreased activation of growth factor- and integrin-dependent pathways, whereas heparin lyase-treated and β1 integrin-null cells exhibited more selective decreases. Collectively, these data support a requirement of γ1 laminins for assembly of the collecting duct system basement membrane, in which immobilized ligands act as solid-phase agonists to promote branching morphogenesis, growth and water transport functions.     

Growth arrest-specific gene 1 is downregulated and inhibits tumor growth in gastric cancer

Gastric cancer is one of the leading causes of malignancy-related mortality in the world, and malignant growth is a crucial characteristic in gastric cancer. In our previous study, we found that growth arrest-specific gene?1 (GAS1) suppression was involved in making gastric cancer cells multidrug-resistant by protecting them from drug-induced apoptosis. In the present study, we investigated the potential role of GAS1 in the growth and proliferation of gastric cancer. We demonstrated that GAS1 expression was decreased in gastric cancer, and patients without GAS1 expression showed shorter survival times than those with GAS1 expression. Both gain-of-function (by overexpression of GAS1) and loss-of-function (by GAS1-specific small interfering RNA knockdown) studies showed that increased GAS1 expression significantly reduced the colony-forming ability of gastric cancer cells in?vitro and reduced cell growth in?vivo, whereas decreased GAS1 expression had the opposite effects. Moreover, upregulation of GAS1 induced cell apoptosis, and downregulation of GAS1 inhibited apoptosis. Furthermore, we demonstrated that GAS1 could induce gastric cancer cell apoptosis, at least in part through modulating the Bcl-2/Bax ratio and the activity of caspase-3. Taken together, our results strongly indicate that GAS1 expression was decreased in gastric cancer and was predictive of a poor prognosis. Restoration of GAS1 expression inhibited cell growth and promoted apoptosis of gastric cancer cells, at least in part through modulating the Bcl-2/Bax ratio and activating caspase-3, suggesting that GAS1 might be used as a novel therapeutic candidate for gastric cancer.     

Transforming growth factor-β1 signalling triggers vascular endothelial growth factor resistance and monocyte dysfunction in type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) leads to monocyte dysfunction associated with atherogenesis and defective arteriogenesis. Transforming growth factor (TGF)-β1, placenta growth factor (PlGF)-1 and vascular endothelial growth factor (VEGF)A play important roles in atherogenesis and arteriogenesis. VEGF-receptor (VEGFR)-mediated monocyte migration is inhibited in T2DM (VEGFA resistance), while TGF-β1-induced monocyte migration is fully functional. Therefore, we hypothesize that TGF-β antagonises the VEGFA responses in human monocytes. We demonstrate that monocytes from T2DM patients have an increased migratory response towards low concentrations of TGF-β1, while PlGF-1/VEGFA responses are mitigated. Mechanistically, this is due to increased expression of type II TGF-β receptor in monocytes under high-glucose conditions and increased expression of soluble (s)VEGFR1, which is known to interfere with VEGFA signalling. VEGFA resistance in monocytes from T2DM patients can be rescued by either experimental down-regulation of TGF-β receptor expression in vitro or by functional blocking of TGF-β signalling using either a TGF-β receptor kinase inhibitor or a TGF-β neutralizing antibody. Our data demonstrate that both T2DM and high-glucose potentiate the TGF-β pathway. TGF-β signalling impairs VEGFR-mediated responses in T2DM monocytes and in this way contributes to mononuclear cell dysfunction, provide novel insights into T2DM vascular dysfunction.     

Differential in vitro phenotype pattern, transforming growth factor-β1 activity and mRNA expression of transforming growth factor-β1 in Apert osteoblasts

The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.