An intracrine view of angiogenesis

Angiogenesis, the generation of new blood vessels from pre-existing vessels, is an integral component of wound healing, responses to inflammation and other physiologic processes. It is also an essential part of tumor growth; in the absence of new vessel formation, tumors cannot expand beyond a small volume. Although much is known about angiogenesis and its regulation, there is no overall theory that describes or explains this process. It is here suggested that the intracrine hypothesis, which ascribes to certain extracellular signaling peptides (whether hormones, growth factors, DNA-binding proteins or enzymes) a role in both intracellular biology and extracellular signaling, can contribute to a more general understanding of angiogenesis. Intracrine factors participate in angiogenesis in the following ways: (1) they can act within the cells that synthesized them (type I intracrine action), (2) they can be secreted and then taken up by their cell of synthesis to act intracellularly (type II intracrine action ), or (3) they can be secreted and internalized by a distant target cell (type III intracrine action). The parallels between the intracrine growth factor mechanisms cancer cells employ in stimulating their own growth and the mechanisms operative in endothelial cell proliferation during angiogenesis ("intracrine reciprocity") are discussed. Collectively, these explorations lead to testable hypotheses regarding the regulation of normal and pathological angiogenesis, and point to similarities between tumor-induced angiogenesis and tissue differentiation.     

αB-crystallin, an effector of unfolded protein response, confers anti-VEGF resistance to breast cancer via maintenance of intracrine VEGF in endothelial cells

Effective inhibition of angiogenesis targeting the tumor endothelial cells requires identification of key cellular and molecular mechanisms associated with survival of vasculatures within the tumor microenvironment. Intracellular autocrine (intracrine) VEGF production by endothelial cells plays a critical role on the vasculature homeostasis. In vitro breast cancer cell-stimulated activation of the unfolded protein response (UPR) of the endothelial cells contributes to maintenance of the intracrine VEGF levels in the endothelial cells through the upregulation of a previous undescribed downstream effector- αB-crystallin (CRYAB). siRNA-mediated knockdown of two major UPR proteins-inositol requiring kinase 1 and ATF6, led to attenuated CRYAB expression of the endothelial cells. Finally, inhibition of CRYAB blocked the breast cancer cell-stimulated increase in the endogenous VEGF levels of the endothelial cells. A VEGF limited proteolysis assay further revealed that CRYAB protected VEGF for proteolytic degradation. Here, we report that the molecular chaperone-CRYAB was significantly increased and colocalized with tumor vessels in a breast cancer xenograft. Specifically, neutralization of VEGF induced higher levels of CRYAB expression in the endothelial cells cocultured with MDA-MB-231 or the breast cancer xenograft with a significant survival benefit. However, knockdown of CRYAB had a greater inhibitory effect on endothelial survival. These findings underscore the importance of defining a role for intracrine VEGF signaling in sustaining aberrant tumor angiogenesis and strongly implicate UPR/CRYAB as dichotomous parts of a crucial regulation pathway for maintaining intracrine VEGF signaling.     

The FGF‐1‐specific single‐chain antibody scFv1C9 effectively inhibits breast cancer tumour growth and metastasis

Immunotherapy mediated by recombinant antibodies is an effective therapeutic strategy for a variety of cancers. In a previous study, we demonstrated that the fibroblast growth factor 1 (FGF‐1)‐specific recombinant antibody scFv1C9 arrests the cell cycle at the G0/G1 transition by blocking the intracrine FGF‐1 pathway in breast cancer cells. Here, we further show that the overexpression of scFv1C9 in MCF‐7 and MDA‐MB‐231 breast cancer cells by lentiviral infection resulted in decreased tumourigenicity, tumour growth and lung metastasis through FGF‐1 neutralization. We found that scFv1C9 resulted in the up‐regulation of p21, which in turn inhibited the expression of CDK2 and blocked cell cycle progression. To explore the potential role of scFv1C9 in vivo, we delivered the gene into solid tumours by electroporation, which resulted in significant inhibition of tumour growth. In tumour tissue sections, immunohistochemical staining of the cellular proliferation marker Ki‐67 and the microvessel marker CD31 showed a reduction in the proliferative index and microvessel density, respectively, upon expression of scFv1C9 compared with the appropriate controls. Thus, our data indicate a central role for scFv1C9 in blocking the intracrine pathway of FGF‐1, therefore, scFv1C9 could be developed in an effective therapeutic for breast cancer.     

The intracrine hypothesis: an update

The intracellular actions of peptide hormones, growth factors, as well as of extracellular-signaling enzymes and DNA-binding proteins, either within target cells or within their cells of synthesis has been called intracrine action. Although these intracrine moieties are structurally diverse, they share certain characteristics of synthesis and function. This has given rise to the development of a theory of intracrine action which permits testable predictions to be made regarding the functioning of these peptides/proteins. Here the intracrine hypothesis is briefly described and then recent experimental findings which bear on predictions made earlier on the basis of the theory are discussed. These findings provide new support for the intracrine hypothesis.     

Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance

The emerging evidence that stem cells develop in specialised niches highlights the potential role of environmental factors in their regulation. Here we examine the role of beta1 integrin/extracellular matrix interactions in neural stem cells. We find high levels of beta1 integrin expression in the stem-cell containing regions of the embryonic CNS, with associated expression of the laminin alpha2 chain. Expression levels of laminin alpha2 are reduced in the postnatal CNS, but a population of cells expressing high levels of beta1 remains. Using neurospheres - aggregate cultures, derived from single stem cells, that have a three-dimensional architecture that results in the localisation of the stem cell population around the edge of the sphere - we show directly that beta1 integrins are expressed at high levels on neural stem cells and can be used for their selection. MAPK, but not PI3K, signalling is required for neural stem cell maintenance, as assessed by neurosphere formation, and inhibition or genetic ablation of beta1 integrin using cre/lox technology reduces the level of MAPK activity. We conclude that integrins are therefore an important part of the signalling mechanisms that control neural stem cell behaviour in specific areas of the CNS.     

Intracellular angiotensin II induces cell proliferation independent of AT1 receptor

We recently reported intracrine effects of angiotensin II (ANG II) on cardiac myocyte growth and hypertrophy that were not inhibited by the ANG II type 1 receptor (AT₁) antagonist, losartan. To further determine the role of AT₁ in intracrine effects, we studied the effect of intracellular ANG II (iANG II) on cell proliferation in native Chinese hamster ovary (CHO) cells and those stably transfected with AT₁ receptor (CHO-AT₁). CHO-AT₁, but not CHO cells, showed enhanced proliferation following exposure to extracellular ANG II (eANG II). However, when transiently transfected with an iANG II expression vector, both cell types showed significantly enhanced proliferation, compared with those transfected with a scrambled peptide. Losartan blocked eANG II-induced cell proliferation, but not that induced by iANG II. To further confirm these findings, CHO and CHO-AT₁ cells were stably transfected for iANG II expression (CHO-iA and CHO-AT₁-iA, respectively). Cells grown in serum-free medium were counted every 24 h, up to 72 h. CHO-iA and CHO-AT₁-iA cells showed a steeper growth curve compared with CHO and CHO-AT₁, respectively. These observations were confirmed by Wst-1 assay. The AT₁ receptor antagonists losartan, valsartan, telmisartan, and candesartan did not attenuate the faster growth rate of CHO-iA and CHO-AT₁-iA cells. eANG II showed an additional growth effect in CHO-AT₁-iA cells, which could be selectively blocked by losartan. These data demonstrate that intracrine ANG II can act independent of AT₁ receptors and suggest novel intracellular mechanisms of action for ANG II. renin-angiotensin system; angiotensinogen; peptide hormones; nuclear signaling; intracrine     

Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease

Fibroblast growth factors (FGFs) are a family of structurally related polypeptides that are essential for embryonic development and that function postnatally as homoeostatic factors, in the response to injury, in the regulation of electrical excitability of cells and as hormones that regulate metabolism. In humans, FGF signalling is involved in developmental, neoplastic, metabolic and neurological diseases. Fgfs have been identified in metazoans but not in unicellular organisms. In vertebrates, FGFs can be classified as having intracrine, paracrine and endocrine functions. Paracrine and endocrine FGFs act via cell-surface FGF receptors (FGFRs); while, intracrine FGFs act independent of FGFRs. The evolutionary history of the Fgf family indicates that an intracrine Fgf is the likely ancestor of the Fgf family. During metazoan evolution, the Fgf family expanded in two phases, after the separation of protostomes and deuterostomes and in the evolution of early vertebrates. These expansions enabled FGFs to acquire diverse actions and functions.     

Functional diversity of FGF-2 isoforms by intracellular sorting

Regulation of the subcellular localization of certain proteins is a mechanism for the regulation of their biological activities. FGF-2 can be produced as distinct isoforms by alternative initiation of translation on a single mRNA and the isoforms are differently sorted in cells. High molecular weight FGF-2 isoforms are not secreted from the cell, but are transported to the nucleus where they regulate cell growth or behavior in an intracrine fashion. 18 kDa FGF-2 can be secreted to the extracellular medium where it acts as a conventional growth factor by binding to and activation of cell-surface receptors. Furthermore, following receptor-mediated endocytosis, the exogenous FGF-2 can be transported to the nuclei of target cells, and this is of importance for the transmittance of a mitogenic signal. The growth factor is able to interact with several intracellular proteins. Here, the mode of action and biological role of intracellular FGF-2 are discussed.     

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT₁ and AT₂), defined as intracrine response. The aim of this study was to examine the presence of AT₁ and AT₂ receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT₁ through an intracrine mechanism. Subcellular distribution of AT₁ and AT₂ was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.

     

Gene Expression Analysis of PTEN Positive Glioblastoma Stem Cells Identifies DUB3 and Wee1 Modulation in a Cell Differentiation Model

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.     

Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases

Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5–1 and mll5–2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5–2 are required for regeneration and that set1, trr-1 and mll5–2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo.     

Menopausal estrogen deprivation activates steroid sensitive stem cells (3SC) and local estrogen biosynthesis: A model for breast cancer development

We propose a hypothesis for breast cancer (BC) development and its implications for BC prevention. We describe a model in which some breast cells function as both stem cells and steroid sensors (steroid sensitive stem cells). Estrogen receptors on those cells could be upregulated in women who had increased cumulative exposure to estrogen, leading to their progressive sensitization. At menopause, such women experience considerable decline of estrogen concentration in their blood. Consequently, the sensitized stem cells activate mechanisms of local estrogen synthesis including the activation of aromatase. The intracrine build-up of estrogen and its metabolites induces proliferation and genetic dysfunction. Eventually, a normal stem cell transforms into an estrogen-sensitive cancer stem cell that is capable of tumor initiation and delineation into other phenotypes of cancer cells. This hypothesis is supported by significant in-vitro and clinical research evidence. According to this model, we suggest that estrogen therapy could be protective against BC. Alternatively, aromatase inhibitors are expected to be effective in BC prevention. A combination of AIs and estrogen might augment the preventative merits of both drugs and maintain a good tolerability profile for long-term prevention protocols.     

Nuclear prostaglandin receptors: role in pregnancy and parturition?

The key regulatory role of prostanoids [prostaglandins (PGs) and thromboxanes (TXs)] in the maintenance of pregnancy and initiation of parturition has been established. However, our understanding of how these events are fine-tuned by the recruitment of specific signaling pathways remains unclear. Whereas, initial thoughts were that PGs were lipophilic and would easily cross cell membranes without specific receptors or transport processes, it has since been realized that PG signaling occurs via specific cell surface G-protein coupled receptors (GPCRs) coupled to classical adenylate cyclase or inositol phosphate signaling pathways. Furthermore, specific PG transporters have been identified and cloned adding a further level of complexity to the regulation of paracrine action of these potent bioactive molecules. It is now apparent that PGs also activate nuclear receptors, opening the possibility of novel intracrine signaling mechanisms. The existence of intracrine signaling pathways is further supported by accumulating evidence linking the perinuclear localization of PG synthesizing enzymes with intracellular PG synthesis. This review will focus on the evidence for a role of nuclear actions of PGs in the regulation of pregnancy and parturition.     

In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1

The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed progenitors, stem cells show loss of FGFR expression. Prolonged culture of bone marrow cells in serum-free medium supplemented with only FGF-1 resulted in robust expansion of multilineage, serially transplantable, long-term repopulating hematopoietic stem cells. Thus, we have identified a simple method of generating large numbers of rapidly engrafting stem cells that have not been genetically manipulated. Our results show that the multipotential properties of stem cells are dependent on signaling through FGF receptors and that FGF-1 plays an important role in hematopoietic stem cell homeostasis.     

Runx1 modulates developmental, but not injury-driven, hair follicle stem cell activation

Aml1/Runx1 controls developmental aspects of several tissues, is a master regulator of blood stem cells, and plays a role in leukemia. However, it is unclear whether it functions in tissue stem cells other than blood. Here, we have investigated the role of Runx1 in mouse hair follicle stem cells by conditional ablation in epithelial cells. Runx1 disruption affects hair follicle stem cell activation, but not their maintenance, proliferation or differentiation potential. Adult mutant mice exhibit impaired de novo production of hair shafts and all temporary hair cell lineages, owing to a prolonged quiescent phase of the first hair cycle. The lag of stem cell activity is reversed by skin injury. Our work suggests a degree of functional overlap in Runx1 regulation of blood and hair follicle stem cells at an equivalent time point in the development of these two tissues.     

OAZ在果蝇大脑发育中的功能研究

OAZ基因编码在进化上保守的C2H2型O/E相关锌指蛋白,参与基因转录的调控。TGF-β信号传导通路中OAZ通过与SMAD4/R-SMAD结合形成转录复合物发挥作用;Olf1/EBF作为转录因子与OAZ相互作用来调控嗅觉上皮细胞和B淋巴细胞的分化。此外,OAZ是JAK/STAT信号通路的下游候选靶基因。但是迄今为止OAZ在果蝇大脑发育的功能还没有研究。果蝇大脑视叶作为一个相对简易操作的模型为我们揭示早期神经干细胞的增殖和转化机制创造了条件,为加快理解哺乳动物早期神经发生过程以及进一步开展神经干细胞治疗提供可能。本研究通过RNA干扰来研究OAZ在果蝇大脑发育中的作用。我们的初步实验结果表明,OAZ对果蝇大脑视叶神经上皮细胞的维持可能不是必需的,OAZ对果蝇大脑视叶神经板和脑髓神经节的发育也可能不是必需的。     

Association of Rex-1 to target genes supports its interaction with Polycomb function

Rex-1/Zfp42 displays a remarkably restricted pattern of expression in preimplantation embryos, primary spermatocytes, and undifferentiated mouse embryonic stem (ES) cells and is frequently used as a marker gene for pluripotent stem cells. To understand the role of Rex-1 in selfrenewal and pluripotency, we used Rex-1 association as a measure to identify potential target genes, and carried out chromatin-immunoprecipitation assays in combination with gene specific primers to identify genomic targets Rex-1 associates with. We find association of Rex-1 to several genes described previously as bivalently marked regulators of differentiation and development, whose repression in mouse embryonic stem (ES) cells is Polycomb Group-mediated, and controlled directly by Ring1A/B. To substantiate the hypothesis that Rex-1 contributes to gene regulation by PcG, we demonstrate interactions of Rex-1 and YY2 (a close relative of YY1) with Ring1 proteins and the PcG-associated proteins RYBP and YAF2, in line with interactions reported previously for YY1. We also demonstrate the presence of Rex-1 protein in both trophectoderm and Inner Cell Mass of the mouse blastocyst and in both ES and in trophectoderm stem (TS) cells. In TS cells, we were unable to demonstrate association of Rex-1 to the genes it associates with in ES cells, suggesting that association may be cell-type specific. Rex-1 might fine-tune pluripotency in ES cells by modulating Polycomb-mediated gene regulation.     

Up-regulation of IL-33 expression in various types of murine cells by IL-3 and IL-4

The crucial roles of the novel cytokine IL-33 in allergic, inflammatory, infectious and autoimmune diseases are becoming characterized. However, the cytokines which regulate IL-33 expression and secretion are still largely unknown. In this study, IL-3 and IL-4 were found to up-regulate IL-33 mRNA expression in mouse peritoneal exudate cells by a two-color DNA microarray and further confirmed by real time PCR and ELISA. IL-3 and IL-4 synergistically promote IL-33 mRNA expression and IL-33 intracrine in the heterogeneous cell populations as peritoneal exudates cells, bone marrow cells and splenic cells. IL-3 and IL-4 also induced IL-33 introcrine in the peritoneal exudate cells from the macrophage-deficient op/op mice, suggesting that macrophage is not the only target of IL-3 and IL-4 in the heterogeneous peritoneal exudate cells. Furthermore, IL-3 and IL-4 were verified to promote the IL-33 intracrine in the homogeneous cell population as fibroblasts and mast cells. These results indicate that up-regulation of IL-33 expression by IL-3 and IL-4 is not a feature particular to a specific type of cells. Up to 100 cytokines were screened, but none of them stimulated the secretion or release of IL-33 in the culture system. In summary, we confirm for the first time that IL-3 and IL-4 are critical for IL-33 intracrine in murine cells of various types, indicating that IL-3 and IL-4 may play an important role in the constitutive expression of IL-33 in vivo.