Regulation Of Growth Factor Activity Research Articles

Requirement for cleavage factor IIm in the control of alternative polyadenylation in breast cancer cells.

Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3′ end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11; the latter protein binds to RNA and the RNA polymerase II carboxy-terminal domain. Here, we used siRNA mediated knockdown and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3′ untranslated regions (3′-UTRs), we observed shifts from promoter proximal regions to the 3′-UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells cosedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer.

The roles of the ovarian extracellular matrix in fertility

In the mammalian ovary there is considerable and continuous remodelling of tissue during both fetal and adult life, necessitating changes in extracellular matrix. Matrix is a diverse group of molecules varying in its composition and roles, which include regulation of growth factor activity and cell behaviour. Here we discuss four topical aspects of matrices in ovaries. (1) Our current state of knowledge of latent TGFß binding proteins that can bind the extracellular matrix fibrillins. Fibrillins and latent TGFß binding proteins may be very important given the genetic linkage data implicating a role for fibrillin 3 in polycystic ovarian syndrome. They will almost certainly be important in the stromal compartments of the ovary by regulating TGFß bioactivity. (2) Follicles which have an unusual ultrastructural follicular basal lamina and poor quality oocytes. The results suggest that the use of oocytes from these follicles should be avoided in assisted reproductive technologies. (3) Evidence that expression of components of focimatrix correlates with expression of aromatase and cholesterol side-chain cleavage in granulosa cells. Focimatrix is a novel type of basal lamina associated with granulosa cells with expression beginning before deviation and continuing until ovulation. It may be involved in maturation of granulosa cells and selection of the dominant follicle. (4) Evidence is presented in support of a hypothesis that follicular fluid accumulates in follicles due to the osmotic potential of hyaluronan and versican, which are matrices produced by granulosa cells and too large to traverse the follicular antrum. These examples illustrate the diversity of matrix and foreshadow potential important discoveries involving extracellular matrix in ovaries.

PO-15 - Antiangiogenic small molecule ligands of FGF2 derived from the endogenous inhibitor thrombospondin-1

Platelet thrombospondin-1 (TSP-1) is a major endogenous regulator of growth factor activity in physiological and pathological processes, including tumor onset, progression and angiogenesis. We previously demonstrated that TSP-1 binds to FGF-2, sequestering the growth factor and inhibiting its angiogenic activity. We also identified a non-peptidic antiangiogenic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of TSP-1. To identify new small molecule inhibitors of FGF2 that recapitulate the structure and functional properties of the FGF-2-binding site of TSP-1, by investigating the chemical space around SM27. A similarity-based screening of small molecule libraries has been used to identify candidates, followed by docking calculations, and evaluation of the activity of the resulting compounds in biochemical and biophysical assays, to assess interaction with FGF2, and in experimental models of angiogenesis, to assess biological activity. The used integrated approach allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting FGF2 binding to both heparan sulfate proteoglycans and FGFR1. The compounds inhibited FGF2-induced endothelial cell proliferation, vessel sprouting from aortic rings and angiogenesis in the chorioallantoic membrane assay, with improved potency over SM27. We have identified new compounds that are valuable as FGF inhibitors for potential therapeutic purposes. Moreover, these compounds are useful chemical tools to identify the minimal stereochemical requirements for FGF2 binding and activity to improve the design of new agents for antineoplastic therapy. Supported by AIRC (Associazione Italiana per la Ricerca sul Cancro).

Pharmacologically Controlled Protein Switch for ON-OFF Regulation of Growth Factor Activity

The precise manipulation of growth factor signaling is central to the progress of tissue engineering. Methods for direct time-resolved activation of signaling pathways through controlled receptor dimerization have been reported; however, these suffer from the risks associated with gene transfer. Here we present an alternative gene transfer-free approach in the form of a protein switch featuring pharmacologically controlled ON-OFF regulation of growth factor activity. The reversible operation of the switch enables stimulation of target processes within a defined period of time. The protein switch provides a means for both studying and manipulating signaling processes, and is thus believed to be a valuable tool for basic research as well as tissue engineering and biomedical applications.

Crim1 has an essential role in glycogen trophoblast cell and sinusoidal-trophoblast giant cell development in the placenta

Normal placental development and function is essential for fetal growth of eutherian mammals. Mutational studies have shown that numerous growth factors are required for placental development and differentiation of placental lineages. Here, using a gene-trap mutant mouse line, Crim1KST264, we show that Crim1 is essential for murine placental development. Crim1 is a developmentally expressed, trans-membrane regulator of growth factor activity. Crim1KST264/KST264 mutant placentae displayed hypoplasia from 13.5 dpc, and altered structure from 15.5 dpc, including alterations in cell number in both the junctional and labyrinth zones. Using the reporter gene from the Crim1KST264 allele, we found that Crim1 is expressed in multiple cell types of the placenta, including strong expression in the spongiotrophoblast cells of the junctional zone. In the junctional zone of Crim1KST264/KST264 placentae, there was an increase in the glycogen trophoblast cells adjacent to the spongiotrophoblast cells. In the labyrinth zone, we found a decrease in the density of sinusoidal-trophoblast giant cells. Our findings show that Crim1 is required for placental development, and is necessary for the proper differentiation of sinusoidal-trophoblast giant cells and glycogen trophoblast cells.

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Altered perlecan expression in placental development and gestational diabetes mellitus

The proteoglycan perlecan is involved in cell signaling, regulation of growth factor activity, and maintenance of basement membranes. This study aims to investigate the expression of perlecan during placental development and whether hyperglycemia of gestational diabetes mellitus induces the alteration of perlecan expression in placenta. Immunohistochemistry, immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and quantitative real-time PCR were carried out to study the placental perlecan expression at different trimesters of pregnancies and in gestational diabetes mellitus. The perlecan protein was mainly immunolocalized in the trophoblast and vessel basement membranes with some staining in the villous stroma of placental villus. Perlecan was also found to co-localize with laminin and collagen IV in the basement membranes of placenta. The protein and mRNA levels of placental perlecan were significantly decreased as the gestational age increased. However, a significant increase in perlecan expression was observed in the third trimester placentas with gestational diabetes mellitus compared to the gestational age-matched controls. Furthermore, trophoblast cells cultured in a high glucose (30 mM) medium and a high osmotic pressure medium (5.6 mM glucose and 24.4 mM mannitol) showed increased perlecan expression compared to cells cultured in the low glucose (5.6 mM) regular medium. These alterations of perlecan expression may be associated with the structural changes of placenta during maturation. The metabolic effect of high glucose and high osmotic pressure of gestational diabetes mellitus may contribute to the increased perlecan expression of diabetic placentas.

Expression and characterization of murine hevin (SC1), a member of the SPARC family of matricellular proteins.

Hevin, also known as SC1, MAST 9, SPARC-like 1, RAGS1 and ECM2, is a member of the SPARC-related family of matricellular proteins. Mouse hevin is 53% identical to mouse SPARC, and both proteins share a follistatin-like module and an extracellular Ca(2+)-binding (E-C) domain. SPARC functions as a modulator of cell-matrix interactions, a regulator of growth factor activity, a de-adhesive protein, and a cell cycle inhibitor. Although the functions of mouse hevin are unknown, its human orthologue has been shown to be de-adhesive for endothelial cells. We now report the production of recombinant mouse hevin in insect cells through the use of a baculoviral expression system and its purification by anion-exchange, size-exclusion chromatography, and isoelectric focusing. Furthermore, we have produced rat anti-hevin monoclonal antibodies (MAbs) that have been characterized by indirect and capture ELISAs, immunoblotting, immunoprecipitation, and immunohistochemistry (IHC). Recombinant hevin, present as a soluble factor or bound to tissue-culture plastic, inhibited the spreading of bovine aortic endothelial cells in vitro. IHC analysis of hevin in normal human and mouse tissues revealed a limited expression pattern in many tissues, with particularly dominant staining in dermis, ducts, vasculature, muscle, and brain. In lung and pancreatic tumor xenografts, we found distinct reactivity with MAbs that were selective for stromal cells, tumor cells, and/or endothelial cells. Although similar to SPARC in its anti-adhesive activities, hevin nevertheless exhibits a distinctive histological distribution that, in certain invasive tumors, is associated with desmoplasia.

Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor.

NK1 is a splice variant of the polypeptide growth factor HGF/SF, which consists of the N-terminal (N) and first kringle (K) domain and requires heparan sulfate or soluble heparin for activity. We describe two X-ray crystal structures of NK1-heparin complexes that define a heparin-binding site in the N domain, in which a major role is played by R73, with further contributions from main chain atoms of T61, K63 and G79 and the side chains of K60, T61, R76, K62 and K58. Mutagenesis experiments demonstrate that heparin binding to this site is essential for dimerization in solution and biological activity of NK1. Heparin also comes into contact with a patch of positively charged residues (K132, R134, K170 and R181) in the K domain. Mutation of these residues yields NK1 variants with increased biological activity. Thus, we uncover a complex role for heparan sulfate in which binding to the primary site in the N domain is essential for biological activity whereas binding to the K domain reduces activity. We exploit the interaction between heparin and the K domain site in order to engineer NK1 as a potent receptor agonist and suggest that dual (positive and negative) control may be a general mechanism of heparan sulfate-dependent regulation of growth factor activity.

Variable accumulation of insulin-like growth factor II in mouse tissues deficient in insulin-like growth factor II receptor

The insulin-like growth factor II receptor mediates endocytosis of insulin-like growth factor II, resulting in growth factor degradation in lysosomes. This degradation is an important regulator of growth factor activity in vivo, as shown by the phenotype of receptor deficient mice. Recent evidence suggests that the insulin-like growth factor II receptor functions as a tumour supressor in humans, and that loss of receptor function leads to increased levels of the growth factor in tumours. It is difficult to establish such a causal relationship in human tumours however, since most tumours have undergone several genetic changes by the time they are examined. Using mouse embryos deficient in receptor expression, and an insulin-like growth factor II-specific radioimmunoassay, we tested the hypothesis that lack of receptor function leads to local accumulation of insulin-like growth factor II. We found that mutant blood and skeletal muscle had excess insulin-like growth factor II, but that mutant lungs and liver had no accumulation. Mutant hearts had less growth factor than wild-type hearts, an unexpected observation, since the normal embryonic heart expresses very high levels of insulin-like growth factor II receptor, and mutant mice apparently die of congestive heart failure. The placentas of mutant mice were larger than those of wild-type, but this did not correlate with an excess of placental insulin-like growth factor II. These results indicate that lack of insulin-like growth factor II receptor can lead to local excess of the growth factor but that such excess is not a necessary consequence of receptor-deficiency.

Suppression of invasive behavior of melanoma cells by stable expression of anti-sense perlecan cDNA

Heparan sulfate proteoglycans are one of the major components of extracellular matrix and are secreted at different levels by several normal and tumoral cells. Perlecan, the basement membrane proteoglycan, has structural domains involved in cell/matrix interactions and growth factor storage. Metastatic melanoma cells show an increase in perlecan expression as compared to low metastatic ones. We examined whether reduction of perlecan expression could down-modulate the malignant phenotype in melanoma clones. We transfected B16-F10 murine malignant melanoma cells with a perlecan antisense cDNA construct and tested the in vitro behavior of the selected clones. The expression of antisense mRNA corresponded to a reduction of perlecan synthesis. The clones with reduced perlecan synthesis showed a down-regulation of proliferation and invasion. These results further indicate the importance of perlecan as a regulator of growth factor activity affecting the biological properties of metastatic cells, and suggest the potential use of antisense perlecan DNA in anti-melanoma gene therapy approaches.

Characterization and estrogen regulation of growth factor activity from uterus

Acid-stable uterine-derived growth factor activity, extracted from uteri of several species (rat, rabbit and bovine), stimulates DNA synthesis as measured by [ 3H]thymidine incorporation into hamster uterine carcinosarcoma (UCS) cells. A time course of [ 3H]thymidine incorporation demonstrates maximum incorporation at 24 h. These extracts also stimulate [ 3H]thymidine incorporation in a variety of other cell types from 17β-estradiol (E 2) target tissues and non-target tissues. Uterine extracts from E 2-treated ovariectomized rats show a 3-fold increase in growth factor activity above control values. Activity is elevated within 18–24 h after estradiol injection and remains elevated with subsequent injections. Growth factor activity is acid-stable, heat-labile, reduced by trypsin but not reduced by treatment with dextran-coated charcoal. Gel filtration shows molecular weight ( M r) heterogeneity with activity eluting at M r of 10000–30000. Since uterine extracts can restore in vitro the estrogen-regulated properties of uterine growth observed previously in vivo, it is possible that the substances found in these extracts may be mediators of E 2 actions.

Activation of latent TGF-beta-1 by plasmin: Physiological regulation of growth factor activity

Activation of latent TGF-beta-1 by plasmin: Physiological regulation of growth factor activity