Rik Derynck - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

Growth factors play key roles in the process of malignant transformation, in normal cell physiology and in embryonic development. One of these factors, transforming growth factor-alpha (TGF-alpha), is structurally related to epidermal growth factor (EGF) and binds to the same receptor as EGF. The recently established widespread synthesis of TGF-alpha in normal cells of epithelial origin, such as keratinocytes, and in many tumor cells, such as carcinomas, has made it increasingly clear that TGF-alpha represents a major physiological ligand of the EGF/TGF-alpha receptor. Whereas considerable knowledge on the sites of synthesis has been obtained, little is as yet known about the function of this growth factor and its role in the cell physiology. The physiological function of TGF-alpha is the focus of this grant application, which proposes two studies aimed at gaining insight into the role of this factor in normal and tumor cells. Previous work has shown that TGF-alpha is synthesized as a transmembrane precursor, which may undergo proteolytic cleavage of the ectodomain, but very often remains uncleaved at the cell surface. Our first major objective focuses on the role of the highly conserved cytoplasmic domain of the TGF-alpha precursor. Specifically, we will explore at the cellular level whether the intact transmembrane TGF-alpha precursor itself is able to transduce any signals, and thus may serve as a signal transducing receptor. In the same context, we will attempt to determine whether there are any cytoplasmic proteins that interact with the cytoplasmic domain of the TGF-alpha precursor. The second major objective is to define the role of TGF-alpha in malignancy and in normal development of the mouse by inactivating the TGF-alpha functional synthesis in tumor cells and in mice. The expression of TGF-alpha in select carcinoma cell lines will be abolished by using antisense sequences. In parallel, the role of TGF-alpha in murine development will be evaluated following inactivation of the genes for TGF-alpha and the EGF/TGF-alpha receptor using established gene targeting methods. These studies should lead to a better understanding of the role of TGF- alpha in pre- and postnatal development and especially in the physiology of normal epithelia and ectodermally derived tumor cells. In addition, since abnormal expression of TGF-alpha may contribute to the derivation of ectodermal tumors and the overproliferation of epithelia, e.g., in psoriasis, it is also our hope that this knowledge will provide a rational basis for the prevention, diagnosis and treatment of some epithelial disorders, including epithelial tumors.

The differentiation of mesenchymal cells into chondrocytes and osteoblasts and the formation of cartilage and bone matrices is directed by a complex series of processes, in which growth and differentiation factors play a role. Transforming growth factor-beta (TGF-beta) and several TGF-beta superfamily members have been isolated from bone matrix and are synthesized in a defined spatial and temporal pattern during osteogenesis in situ. TGF-beta family members also markedly affect the cellular matrix formation in vitro and mimic in vivo all or part of the osteoinductive activity of demineralized bone matrix, suggesting that these factors play crucial roles in regulating osteogenic differentiation. In this application, we propose to test several aspects of the hypothesis that the coordinated and perhaps sequential endogenous expression of various TGF-beta superfamily members and the resulting autocrine/paracrine effects drive the osteoblastic differentiation of mesenchymal cells into osteoblasts. We will first establish the expression pattern of TGF-beta related factors during osteoblastic differentiation in vitro of multipotential mesenchymal stem cell lines (the cell lines C26 and 10T1/2) and will then determine whether the differentiation can be predictably altered by either overexpressing or inhibiting the expression of individual TGF-beta family genes. We have already established that retinoic acid-induced initiation of osteoblastic differentiation in these cells results in a drastic increase of TGF-beta expression and a transient decrease of bone morphogenic proteins (BMP)-2 and -4 mRNA. Thus, we will focus our functional studies on the overexpression and the inhibition of expression of TGF-beta and BMP-2/4 and evaluate the effects of these modifications on the differentiation potential and the osteogenic differentiation of these cells in vitro and in vivo. In Aim 1 we will complete the characterization of the expression pattern of TGF-beta related factors in the two model systems, especially under conditions that induce osteogenic differentiation. We will relate these changes to the differentiation state and differentiation parameters under various culture conditions. In Aim 2 we will increase and abolish the endogenous TGF-beta expression by transfection with expression vectors for TGF-beta2 or a dominant negative TGF-beta mutant. The effects on cell differentiation should establish whether osteogenic differentiation can be initiated, accelerated or modulated by alterations of the endogenous TGF-beta expression levels and whether TGF-beta expression is essential for osteogenic differentiation. In Aim 3 we will overexpress BMP-4 and abolish endogenous BMP-2/-4 synthesis and evaluate the consequences on cell differentiation. These results should establish whether osteogenic differentiation can be modified by altering the endogenous BMP-2/-4 level expression levels and should determine how essential BMP-2/-4 synthesis is for the osteogenic differentiation of these mesenchymal cells.

Transforming growth factor-beta(TGF-beta) is a growth and differentiation factor that plays an important role in the interactions of the cell with the extracellular matrix and in the control of cell proliferation. TGF- beta also plays a key role in cell differentiation and tissue morphogenesis and, very importantly, in tumorigenesis and metastasis. Because of these many different activities, and especially its role as a potent growth inhibitor and regulator of cell-matrix interaction (unlike other growth factors), TGF-beta has received attention from both basic and clinical researchers, and is considered a potentially useful therapeutic target to manipulate repair processes and inhibit tumorigenesis. Two types of TGF-beta receptors, called type II and type I receptors, are the mediators of the TGF-beta induced signal transduction. The cloned type II receptor is a predicted serine- threonine kinase receptor, similarly to the receptor for activin, another member of the TGF-beta superfamily. It is now assumed that the receptors for all TGF-beta related factors are transmembrane serine-threonine kinase receptors. How the interaction of TGF-beta with its receptors leads to an inhibition of cell proliferation and the effects on the cell- matrix interaction, is largely unknown. Also, nothing is as yet known about the mechanism of action of any serine-threonine kinase receptors. This research proposal is aimed at gaining some insight into some important and specific questions on how the receptors for TGF-beta function. Two recent findings from our lab are at the basis of our research plan: 1) there are two signaling pathways for the different biological activities of TGF-beta; they are associated with the type II and type I receptor respectively; 2) we have recently isolated cDNAs for a type I receptor, named Tsk 7L, a predicted serine-threonine kinase receptor with structural similarity to the type II receptor. In addition, another research group has isolated a structurally related type I receptor, called ALK-5. The availability of cDNAs for the type II and type I TGF-beta receptors now allows us to pursue the following research aims that address specific functional questions. In Aim 1, we will analyze the specific roles of the type II and type I receptors in the different biological activities of TGF-beta and in the few known aspects of the TGF-beta induced signal transduction. Aim 2 will focus on a characterization of the nature of the kinase activity of the type II and type 1 receptors and the effect of TGF-beta binding on the receptor- associated kinase activity. Finally, in Aim 3, we will isolate cDNAs for proteins that associate with the cytoplasmic domain of the type II TGF- beta receptor and pursue their molecular characterization. We will then evaluate the effect of TGF-beta on these associations. These studies should provide us with a beginning insight on how TGF-beta exerts its various biological activities through its type II and type I receptors and how this new class of serine-threonine kinase receptors functions, and may provide us with means to control cell proliferation and cell- matrix interactions.

DESCRIPTION: Growth factors play key roles in normal cell physiology and tissue development and in malignant transformation and tumor development. One of these factors is transforming growth factor-a (TGF-a), which binds to the EGF/TGF-a tyrosine kinase receptor. TGF-a is considered the prototype of a family of transmembrane growth factors characterized by a conserved six-cysteine motif in the ectodomain. Transmembrane TGF-a is often the most predominant TGF-a form. Soluble TGF-a is released into the medium as a result of the regulated proteolytic cleavage of the ectodomain. Whereas both soluble and transmembrane TGF-a activate the EGF receptor, other functions are likely to be associated with the transmembrane form. In this grant application, it is proposed to characterize the molecular identity and function of the transmembrane TGF-a-associated protein complex. He has previously obtained evidence that at least several proteins associate with transmembrane TGF-a. He hypothesizes that transmembrane TGF-a associates with a regulated protein complex that is involved in the inducible ectodomain cleavage of transmembrane TGF-a, the presentation of transmembrane TGF-a at the cell surface, and/or signaling events associated with the cytoplasmic domain. The proposed research is subdivided into two large aims. In Aim 1, the applicant will focus on the molecular identification and characterization of proteins that associate with transmembrane TGF-a. Their identification will be pursued by a combination of approaches, including protein purification, interaction cDNA cloning and cDNA cloning of homologs of proteins which in Drosophila genetically interact with TGF-a homologs. After the association of these proteins with transmembrane TGF-a has been established in vivo, the applicant will pursue in Aim 2 i.e., the functional characterization of these proteins in several contexts: the regulated proteolysis of the transmembrane TGF-a ectodomain, the affinity of transmembrane TGF-a for the EGF receptor, presentation of transmembrane TGF-a at the cell surface, and possible signaling events associated with the cytoplasmic side of transmembrane TGF-a. The molecular and functional characterization of the transmembrane TGF-a associated-protein complex should provide insight into the role of this transmembrane growth factor and other related factors in the physiology and development of normal and tumor tissues and in various epithelial disorders.

Cell proliferation and differentiation are regulated by extracellular growth factors. Among these, transforming growth factor-beta (TGF-beta) plays a key role in growth control. Unlike growth factors that act through tyrosine kinase receptors and stimulate cell proliferation, TGF-beta inhibits cell growth and induces expression of various extracellular matrix proteins. TGF-beta is the prototype of many factors that play key roles in cell differentiation and development. The activities of TGF- beta-related proteins are mediated through a recently discovered class or receptors, the transmembrane serine-threonine kinases. Two types of receptors, the type II and type I receptors, form the functional heteromeric receptor complex. Following TGF-beta binding, the type II receptor transphosphorylates the type I receptor, leading to intracellular signaling that results in changes in gene expression. Smads have recently been identified as signaling effectors of TGF-beta receptors. Receptor activation by TGF-beta results in phosphorylation of Smad2 and/or 3, which then dissociate from the receptor and engage in a heteromeric complex with Smad4/DPC4. These Smads are then translocated into the nucleus where they activate transcription of defined genes. The TGF-beta type II receptor, Smad2 and Smad4/DPC4 all act as tumor suppressors, whose inactivation contributes to tumor development. To understand the mechanism of TGF-beta receptor signaling and the role of TGF-beta and related factors in normal and tumor development, it is essential that we understand the mechanisms of activation and action of the Smads. The goal of this research proposal is to characterize two key processes in Smad function, thereby using Smad2 and 3 and Smad4, i.e. the Smads that mediate TGF-beta receptor signaling, as models. In Aim 1, we propose to characterize the mechanisms of nuclear translocation of Smads in response to receptor activation. In Aim 2, we will characterize the mechanisms of transcriptional activation of genes by Smads in response to TGF- beta, thereby using two known TGF-beta-responsive promoters as model systems. Finally, in Aim 3, we will identify and functionally characterize proteins that associate with the TGF- beta responsive Smads. The latter studies will greatly complement and contribute to the characterization of the mechanisms of nuclear translocation of Smads and transcriptional activation by Smads. Taken together, our research program should result in the characterization of two essential key processes in Smad function and TGF-beta receptor signaling. Our findings should help us understand the role of TGF-beta receptor signaling in normal and tumor development, and in the regulation of cell growth and differentiation.

Skeletal development requires a highly complex pattern of bone formation whereby mesenchymal cells differentiate into osteoblasts, which deposit bone, and other mesenchymal cells differentiate into myocytes, which give rise to muscle, and adipocytes, which gives rise to fat. TGF-beta- related factors regulate the differentiation pathways into muscle, fat and bone, but the exact role of these factors in mesenchymal differentiation and the underlying mechanisms remain to be characterized. In this application, we propose to characterize the role of Smads, i.e. the recently identified signaling effectors of the receptors for TGF-beta-related factors, in myogenic, adipocytic and osteoblastic differentiation, using C2C12 and 3T3-F442A cells as model system. The regulation of myoblast and osteoblast differentiation by TGF-beta and BMP-2/4, and the modulation of endogenous ligand and receptor expression during these types of differentiation invite the hypothesis that alterations in Smad signaling regulate normal myogenic, adipocytic and osteoblastic differentiation, using C2C12 and 3T3-F442A cells as model system. The regulation of myoblast and osteoblast differentiation by TGF-beta and BMP-2/4, and the modulation system. The regulation of myoblast and osteoblast differentiation by TGF-beta and BMP-2/4, and the modulation of endogenous ligand and receptor expression during these types of differentiation invite the hypothesis that alterations in Smad signaling regulate normal myogenic adipocytic and osteoblastic differentiation. Our research plan to characterize the role Smad signaling in these differentiation processes is divided in three Aims. Am extensive characterization of the differential events of C2C12 and 3T3-F442A cells in culture will be pursued together with a characterization of the effects of TGF-beta and BMP-2/4 and their receptor signaling systems (Aim 1). We will then manipulate Smad signaling by increasing or decreasing signaling by individual Smads or Smad combinations, and evaluate the effects of these alterations on cell differentiation (Aim 2). Since Smads act as transcription factors, we will then study the mechanisms through which Smads regulate the expression and activities of the "master" transcription factors of these three types of differentiation, i.e. MyoD/myogenin in myogenic differentiation, PPAR-gamma in adipocytic differentiation, and CBFA1 in osteoblastic differentiation (Aim 3). Our understanding of how Smads regulate these three types of mesenchymal differentiation makes it likely that we will be able to regulate these three types of differentiation and to induce transdifferentiation between these three lineages. Our studies will form the basis of further studies on how to recruit myogenic and adipogenic cells to transdifferentiate into osteoblasts, which deposit bone matrix, in vivo. Such an ability would clearly have important therapeutic implications, since the recruitment of mesenchymal cells to differentiate along the osteoblastic lineage and to deposit bone matrix may allow increased bone formation to counteract bone loss, associated with metabolic bone diseases and age-related osteoporosis.

TGF-a is expressed as a transmembrane protein, and cells can release soluble TGF-a into the medium as a result of the regulated proteolytic cleavage of the ectodomain. The laboratory has identified several transmembrane and cytoplasmic proteins that interact with TM TGF-a and obtained the full-length cDNAs. The hypothesis is that these associated proteins regulate intracellular transport, maturation, cell surface presentation and ectodomain cleavage of TM TGF-a. The application has four specific aims, each focusing on a defined protein that associates with TM TGF-a. Aim 1 will characterize the association of the tetraspan protein CD9. Aims 2 and 3 will focus on the biochemical and functional characterization of human homologs of Drosophila Rhomboid and Cornichon. Aim 4 focuses on the functional characterization of p59, a PDZ containing protein implicated in TM TGF-a transport through the Golgi

Cancer cells are subject to autocrine receptor stimulation by endogenous growth factors, and this stimulation contributes to malignant transformation and cancer development. Oral carcinoma cells often show increased (EGFR). Clinical correlation and in vitro and in vivo studies strongly suggest that increased EGFR stimulation by TGF-alpha contributes to carcinogenesis. TGF-alpha is made as a transmembrane growth factor, which undergoes regulated ectodomain cleavage or "shedding" to release soluble and diffusible TGF-alpha. Transgenic experiments suggest that this cleavage of transmembrane TGF-alpha is required for its ability to stimulate carcinoma development. TGF-alpha ectodomain shedding is mediated by TACE, a transmembrane metalloprotease, which was originally discovered for its ability to mediate TNF-alpha cleavage. The mechanisms that activate TACE and consequent TGF-alpha cleavage were unknown until recently We have recently shown that growth factors, which activate tyrosine kinase receptors, induce ectodomain shedding of TGF-alpha as well as TNF- alpha and L-selectin Growth factor-induced TGF-alpha ectodomain shedding is mediated through activation of the Erk MAP kinase pathway and does not require new protein synthesis. We have also shown that the cytoplasmic domain of TACE is phosphorylated in response to growth factor stimulation and that the cytoplasmic domain of TGF-alpha ectodomain cleavage. This proposal now builds on these findings and is aimed at characterizing the signaling mechanism(s) that lead to activation of TGF-alpha shedding and its role in oral carcinoma development. We have subdivided the proposal in four Aims. In Aim 1, we propose to characterize the growth factor-induced phosphorylation of TACE and its role in TACE activation and ectodomain shedding. In Aim 2, we propose two different approaches to identify, clone and characterize the kinase, which phosphorylates TACE and in this way activates TACE mediated shedding in response to growth factor stimulation. In Aim 3, we propose to identify and functionally characterize proteins that interact with TACE and, in this way regulate TACE activation and ectodomain shedding. Finally, in Aim 4, we will evaluate the role of TGF-alpha and its ectodomain cleavage, as a result of TACE activation and ectodomain shedding. Finally, in Aim 4, we will evaluate the role of TGF-alpha and its ectodomain cleavage, as a result of TACE activation in carcinogenesis and tumor development of oral squamous carcinoma in vivo.

ESCRIPTION (provided by applicant): Transforming growth factor-beta (TGF-beta) and members of the TGF-beta family play key roles in the regulation of cell proliferation and differentiation, and in normal development and tumorigenesis. They signal through heteromeric complexes of two types of cell surface, transmembrane serine-threonine kinases, resulting in phosphorylation and consequent activation of Smads, which then act as signaling effectors in the regulation of TGF-beta-induced gene expression. Extensive research during the last few years has revealed a general model for how receptor-activated Smads activate transcription through cooperation with sequence-specific transcription factors at the promoter DNA. In contrast, little is known about the mechanisms that result in downregulation of gene expression in response to TGF-beta. We now propose to continue our ongoing research on the regulation of gene expression in response to TGF-beta. In Aim 1, we will study the mechanism of TGF-beta/Smad3-mediated repression of transcription by MyoD/MEF2 and CBFA1, which naturally drive differentiation in myoblasts and osteoblasts, respectively. The molecular characterization of these two model systems for TGF-beta/Smad3-mediated repression should provide insight into general features and may lead to a general model for the mechanism of transcription repression by TGF-beta family members through Smads. Furthermore, the study of the regulation of CBFA1 function will allow us to characterize the roles of the CBFAl-binding DNA sequence and the cell context (mesenchymal versus epithelial) in TGF-beta-mediated repression versus activation of transcription. Aim 2 will focus on the crosstalk between IRF-3/7 and Smad signaling and the role of IRF-3/7 in TGF-b/Smad signaling. IRF-3 and IRF-7 are two closely related members of the "interferon regulatory factor" (IRF) family of transcription factors. Viral infection activates IRF-3/7 and induces IRF-7 expression, which leads to type I interferon and RANTES expression. We will characterize the crosstalk between IRF-3/7 and TGF-beta/Smad signaling at the IRF-3/7-responsive interferon b promoter, and conversely evaluate the effect of IRF-3/7 on Smad-mediated transcription. We will also evaluate the role of TGF-beta/Smad signaling in the antiviral induction of interferon, driven by IRF-3/7, and evaluate a possible direct role of IRF-3/7 in TGF-beta signaling. Mutation analyses of IRF-3/7, based on the three-dimensional IRF-3, structure will assist in these investigations.

[unreadable] DESCRIPTION (provided by applicant): Autocrine TGF-beta signaling plays key roles in cancer progression, thereby acting as tumor suppressor in early carcinogenesis, and in stimulating invasion leading to metastasis. Most carcinomas have a deregulated response to TGF-beta, leading to inactivation of the antiproliferative response to TGF- beta. In some cases this deregulation is associated with mutations in the type I or type II TGF-beta receptors. Sumoylation, i.e. the covalent attachment of a ubiquitin-like SUMO protein, is an emerging posttranslational modification of various proteins. The role of sumoylation is often unpredictable and seems to depend on the target protein. Possible recruitment of SUMO-interacting proteins may lead to changes in the protein complex formation and functional properties of the target protein. Sumoylation is largely characterized as a modification of transcription factors and proteins involved in nuclear functions, and sumoylation of cell surface receptors has not been reported. We recently discovered that the type I TGF- beta receptor, TbRI, is sumoylated, making us hypothesize that sumoylation regulates the function of this receptor, and consequently TGF-beta signaling and the cell's response to TGF-beta. After identifying the sumoylation site in TbRI, we also found that a point mutation in TbRI, which has been found in association with metastatic breast cancer, confers a lack of sumoylation to TbRI. This leads to the hypothesis that sumoylation of the TbRI affects autocrine TGF-beta signaling and is a determinant of the invasive behavior of carcinomas. We propose three aims to define the molecular mechanism of TbRI sumoylation and its role in the cellular responses to TGF-b and the behavior of cancer cells in vivo. Aim 1 will characterize the mechanism of sumoylation of TbRI, including a search to identify the responsible E3 SUMO ligase. Aim 2 will examine the effect of TbRI sumoylation on Smad and non-Smad signaling responses and the cell's proliferation and invasion responses to TGF-b. This Aim should also define functional differences between the "metastasis- associated" TbRI mutant, wild-type TbRI and a sumoylation-deficient TbRI point mutant in the signaling and cell behavior responses to TGF-b. Finally, Aim 3 will define the effect of TbRI sumoylation and of the "metastasis-associated" TbRI point mutation on the behavior of cancer cells and on cancer progression in vivo. The proposed studies should provide a basis for the characterization of the mechanism of the TGF- beta receptor sumoylation, its role in the cell's response to TGF-beta and in cancer cell behavior and cancer progression. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Muscular dystrophies show a progressive loss of muscle fibers and ultimate failure to regenerate muscle tissue. Cell-based therapy may restore muscle tissue, and adipose tissue may provide a facile source of cells for such therapy. We have been studying the mechanisms through which BMP or TGF-beta signaling can redirect pre-adipocyte differentiation and found that pre-adipocytes and primary adipose stromal cells can form muscle-like tissue when transplanted into immunodeficient mice. We propose to test the hypothesis that preadipocytes and adipose stromal cells can alter their differentiation to generate functional muscle in vivo, capable of contributing to tissue repair, and that BMP signaling regulates this differentiation. Smads are downstream effectors of signaling by TGF- beta and BMP and serve as cell-intrinsic regulators of mesenchymal differentiation, suggesting that manipulations of the activation levels of individual Smads will profoundly affect the differentiation of preadipocytes into muscle cells. In Aim 1 we will characterize the muscle-like cells generated from 3T3-F442A cells or human adipose stromal cells, and evaluate whether they arise from direct conversion or cell fusion with host myofibers. In Aim 2 we will determine whether transplanted adipogenic cells or adipocytes contribute to muscle repair in dystrophin-deficient nude mice, and determine whether engraftment improves muscle repair. We will also use a genetic approach to evaluate if endogenous (pre)adipocytes can differentiate into myocytes in response to injury and contribute to muscle (re)generation. In Aim 3 and future studies we will examine the role of Smad1 and Smad5, effectors of BMP signaling, and Smad3, effector of TGF-beta and myostatin signaling, as cell-intrinsic regulators of myogenic differentiation. These experiments should show us how to manipulate the myogenic differentiation by modifying Smad signaling. Our studies will hopefully provide the basis for the use of autologous preadipocytes and adipose stromal cells, a readily available cell source, for muscle regeneration and repair. PUBLIC HEALTH RELEVANCE. Patients with muscular dystrophy or defects in muscle repair would benefit from a therapy whereby injected cells would convert into muscle tissue. We recently found that cells that are becoming fat cells can be redirected to become muscle cells. We propose to characterize and to enhance the potential of these cells to become muscle cells, and to explore how they can help in the repair of muscle injury and to counteract the defects associated with muscular dystrophy, using mouse models. This research could provide a basis for the use of one's own fat in therapy to improve muscle repair and regeneration. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There is considerable interest in cell-based therapies for the repair and tissue regeneration of myocardial tissue. Embryonic stem cells are explored as an unlimited source of cells for cardiac repair, as they can differentiate into cardiomyocytes in culture. A better understanding of the regulation of this differentiation process would greatly aid in the generation of cardiomyocytes, and may lead to methods to improve the yield, control the quality and homogeneity, and direct subtype characteristics of the cardiomyocytes. Little is known about the signaling pathways and transcription factors that control the differentiation potential and cardiomyocyte differentiation of human embryonic stem cells. Oct4, Sox2 and Nanog function as essential transcription factors for self-renewal and pluripotency, yet may also play key roles in the initiation of differentiation into cardiomyocytes. Signaling by TGF-2 family proteins controls self-renewal, pluripotency and differentiation of stem cells, and autocrine signaling by TGF-2 family proteins is likely to play a key role in the self-renewal and differentiation of embryonic stem cells. TGF-2 family proteins exert gene expression responses through Smads, which enhance or repress the transcription activities of transcription factors at target genes, and thus function as cell-intrinsic mediators of differentiation. Through this mode of action, Smads are likely to regulate the expression levels and functions of embryonic stem cell transcription factors, such as Oct4, Sox2 and Nanog, and regulate the selection and progression of differentiation of cardiomyocytes from embryonic stem cells. The overall goals of this proposal are to (1) evaluate the regulation of expression and activities of the Oct4, Sox2 and Nanog by TGF-2 family/Smad signaling, (2) correlate this level of control with the differentiation potential and characteristics of differentiation along the cardiomyocyte lineage, (3) to use this knowledge to generate cardiomyocyte progenitors with high efficiency and defined characteristics. We hypothesize that (1) Smad signaling by TGF-2 family proteins regulates the expression and activities of the embryonic stem cell transcription factors, (2) alterations in Smad signaling and Oct4, Sox2 and/or Nanog activities modify the differentiation capacity of the cells and specifically cardiomyocyte differentiation. We propose three Aims: (1) to examine the regulation of embryonic stem cell transcription factor expression and activities by TGF-2 family signaling and to correlate these findings with cardiomyocyte lineage differentiation, (2) to study the roles of individual Smads in the regulation of Oct4, Sox2 and Nanog, and the roles of these Smads and Oct4, Sox2 and Nanog themselves in cardiomyocyte lineage differentiation, (3) To examine the in vivo differentiation and tissue integration characteristics of cardiomyocyte precursors derived from embryonic stem cells following manipulations that favor the generation of these cells in culture. PUBLIC HEALTH RELEVANCE: Cell-based therapies for the repair and tissue regeneration of heart tissue, for example following heart infarct, may provide great promise, and human embryonic stem cells are being considered as a cello source for these cells, called cardiomyocytes. We now propose a research plan in which we try to understand signaling pathways that may direct the differentiation of cardiomyocytes. We will explore how to modify these pathways and hope to design approaches that increase and improve the generation and characteristics of the cardiomyocytes. This will be tested through a combination of cell culture experiments and a new method in which the cardiomyocytes are injected into the heart muscle of mice with a heart infarct.

DESCRIPTION (provided by applicant): Transforming growth factor-[unreadable] (TGF-[unreadable]) plays a critical role in cancer initiation and progression. Carcinoma cells often have shown enhanced TGF-[unreadable] production and activation, resulting in autocrine effects on cell physiology and behavior. Among these, a lot of attention has focused on TGF-[unreadable]'s ability to induce an epithelial to mesenchymal transition (EMT) that results in de-adhesion, increased motility and invasion. The roles of TGF-[unreadable] in cancer cell behavior, tumor microenvironment and cancer progression are subject of extensive investigation, but the respective roles of the underlying TGF-[unreadable]-activated signaling pathways in cancer cell behavior are less understood. Most studies in this context address the roles of TGF-[unreadable]-activated Smads, which serve as transcription (co)factors to regulate gene expression. Recent studies, including some from this lab, have characterized non-Smad signaling pathways that are directly activated in response to TGF-[unreadable]. These may explain non-transcription responses to TGF-[unreadable] such as migration, changes in cell shape and protein translation, yet may also affect the activities of the Smads. The functions of the non-Smad signaling events in the TGF-[unreadable]-directed effects on cancer cell behavior and cancer progression are essentially unknown. We recently reported that, in TGF-[unreadable]-induced epithelia EMT, TGF-[unreadable] activates the PI3-kinase-Akt-TOR pathway, resulting in increased protein synthesis and cell size, and that this pathway mediates the increased motility and invasion of cells that undergo TGF-[unreadable]-induced EMT. We also reported that, in response to TGF-[unreadable], ShcA is recruited to the type I TGF-[unreadable] receptor T[unreadable]RI and phosphorylated on Ser and Tyr, in turn resulting in activation of Erk MAP kinase. Our observation that T[unreadable]RI is a dual specificity kinase explains ShcA phosphorylation on Ser and Tyr, whereas T[unreadable]RI phosphorylation on Tyr in response to TGF-[unreadable] may provide the biochemical basis for activation of both the PI3K-Akt-TOR and the Shc-Erk MAPK pathways by TGF-[unreadable]. Finally, we discovered that phosphorylation of T[unreadable]RI in response to TGF-[unreadable] induces T[unreadable]RI sumoylation. T[unreadable]RI sumoylation in turn regulates TGF-[unreadable]-signaling dependent invasion of cancer cells. We propose to further characterize the mechanisms of these signaling events at the molecular level and to use this knowledge to address their roles in cancer cell behavior and cancer progression. Aim 1 will focus on how TGF-[unreadable] activates the PI3K-Akt-TOR pathway and on the role of this component of TGF-[unreadable] signaling in cell invasion and cancer progression. Aim 2 will study the role of TGF-[unreadable]-activated ShcA-Erk MAP kinase signaling in EMT, invasion and cancer progression. Aim 3 proposes to better characterize the sumoylation of T[unreadable]RI and to understand its role in the TGF-[unreadable] response and cancer progression. Our enthusiasm for this program is driven not only by its inherent scientific importance, but also by its translational potential. PUBLIC HEALTH RELEVANCE: The progression of cancer leading to death is in most cases not the result of the first tumor growing, but rather because that tumor starts invading other tissues and disseminating throughout the body to give rise to additional tumors, a process called metastasis. Cancer invasion and metastasis are driven by a protein called TGF-[unreadable], which is made by the tumor cells themselves and instructs them to undergo the changes that lead to invasion and metastasis. Recently, novel signaling pathways were found that are activated by TGF-[unreadable] and complement the previously studied one that received all attention. The proposed research aims at better understanding the molecular basis of these additional pathways and their roles in cancer cell behavior, cancer progression and metastasis. This knowledge is likely to provide new and more selective avenues than hitherto possible to block the invasive and metastatic behavior of cancers.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project aims to define the role of PRMT1 mediated arginine methylation of inhibitory Smads in the regulation of TGF-beta/BMP signaling. We aim to elucidate the molecular mechanism underlying PRMT1 mediated arginine methylation through identifying specific arginine resudes that are methylated, and examine the alteration of signaling response when this methylation is ablated. Assistance from UCSF Mass Spectrometry Facility is critical for achieving our research goals in that mass spectrometric analysis will help us identify target arginine resudes and confirm their methylation status. This will serve as the basis for our project development.

DESCRIPTION (provided by applicant): Secreted TGF-[unreadable] family proteins play key roles in cell differentiation, and deregulation of TGF-[unreadable] family signaling is considered as a mechanism of a variety of diseases, including cancer. Following receptor activation, intracellular signaling effectors, called Smads, relay the signals that lead to activation or repression of TGF-[unreadable] family target genes. Considerable insight has been gained on how Smads are activated and their function is regulated through phosphorylation, teaching us that functional crosstalk of Smads with kinases dictates the gene expression responses to TGF-[unreadable] family proteins. Protein methylation has emerged as a post-translational modification that exerts key roles in defining protein functions, at the level of signaling mediators and at the level of epigenetic regulation of transcription. However, there is no knowledge on functional interactions of Smads with methyl transferases. Among a variety of methyl transferases tested, we found specific interactions of three of them with selected Smads. We hypothesize that functional interactions with methyl transferases define Smad signaling, through Smad methylation thus regulating Smad activity, or through alterations of TGF-[unreadable] -induced transcription responses. We propose three Aims, each focusing on the functional interaction of an individual methyl transferase with Smads. Each methyl transferase targets selectively a defined type of Smads, either the inhibitory Smad6 or Smad7 (Aim 1), the common Smad4 (Aim 2), or the TGF-[unreadable] - activated R-Smad Smad3 (Aim 3). In Aim 1 we will define the role of the Arg methyl transferase PRMT1 in Smad6 and Smad7 function. We will characterize the methylation of Smad6 and Smad7 by PRMT1 and evaluate the role of PRMT1 in Smad6 and Smad7 function, and in TGF-[unreadable] - and BMP- induced, Smad-mediated transcription. In Aim 2, we will define the role of SMYD3 in TGF-[unreadable] /Smad signaling. SMYD3 methylates Lys4 of histone H3, which has been linked to enhanced transcription, yet was also shown to target a cell surface receptor. We showed that SMYD3 interacts specifically with Smad4, and propose to evaluate the role of this interaction in Smad4 function, and transcription regulation by the TGF-[unreadable] -activated Smad3/4 complex at target genes. We will thereby focus on the regulatory gene sequences of hTERT that are targeted by SMYD3 and TGF-[unreadable] signaling. In Aim 3 we will define the role of ESET/SETDB1, which methylates Lys9 of histone 3 in TGF-[unreadable] /Smad regulated gene expression. We have shown that ESET interacts with Smad3, but not the other Smads, and propose to explore the functional crosstalk between TGF-[unreadable] /Smad3-mediated transcription regulation and ESET, focusing on the expression of SnoN, an oncogene whose expression results in inhibition of TGF-[unreadable] signaling. These studies will provide paradigms for how methyl transferases regulate Smad signaling and the transcription responses to TGF-[unreadable] family proteins. PUBLIC HEALTH RELEVANCE: Secreted proteins, that structurally belong to the same family, i.e. the TGF-[unreadable] family, play key roles in defining the type of cell or tissue that originates during development, and deregulation of the instructions that these proteins provide to the cells is considered as a mechanism of a variety of diseases, including cancers. The instructions provided to the cells by TGF-[unreadable] family proteins are relayed by a class of proteins, the Smads, that function inside the cells. We found that some members of a group of enzymes, the methyl transferases, interact with Smads, raising the possibility that the functions of Smads are regulated by another type of modification, i.e. adding methyl groups. This research program is aimed at understanding how methyl transferases regulate Smad functions.

DESCRIPTION (provided by applicant): As epithelial cells progress to carcinomas, increased autocrine TGF-? signaling acquires a prominent role in cancer progression, by inducing an epithelial plasticity response that can lead to epithelial-mesenchymal transition (EMT). EMT results in cell de-adhesion and increased cell motility and invasion, a prerequisite of cancer cell dissemination, and is increasingly seen as an integral property of carcinoma stem cells. As TGF-? signaling drives EMT, and TGF-? responsiveness contributes to cancer progression, we have been studying the regulation of TGF-? signaling, as it pertains to epithelial plasticity. The well-studied Smad signaling pathway regulates gene expression in response to TGF-?, but the TGF-?- induced epithelial plasticity response cannot be explained merely by changes in gene regulation. Accordingly, TGF-?-induced non-Smad signaling has received increasing appreciation. Supported by this grant, we have been studying the TGF-?-induced activation of the Erk MAPK and PI3K-Akt-mTOR pathways, and have started addressing their roles in EMT. The specific roles of TGF-?-induced activation of either pathway in the epithelial plasticity response remain to be further defined. We also found that cells regulate their responsiveness to TGF-?, by regulating the TGF-? receptor levels at the cell surface from intracellular stores. Increased glucose levels and insulin activate this upregulation of cell surfae TGF-? receptors, which appears to be mediated by Akt activation and the Rab GTPase activating protein AS160, a direct target of Akt phosphorylation. We hypothesize that increased Akt activation, as commonly seen in carcinomas, or resulting from increased glucose or insulin stimulation, enhances the cell's TGF-? responsiveness, and the sensitivity and susceptibility of cancer cells to EMT, and thus may promote cancer progression by enhancing TGF-? responsiveness. We now seek to continue our research program aimed at characterizing the roles of non-Smad signaling mechanisms in the control of the cell surface TGF-? receptor levels, and resulting TGF-? responsiveness, and in TGF-?-induced EMT. We organized our current and future research in three Aims: (1) To study the effects of glucose or insulin on TGF-? signaling, epithelial-mesenchymal transition, cancer stem cell generation and EMT-dependent cancer progression; (2) To define the molecular mechanisms regulating the cell surface presentation of the TGF-? receptors in response to Akt activation; (3) To define the roles of TGF-?-induced Erk MAPK and PI3K-Akt pathway activation in epithelial-mesenchymal transition, and cancer stem cell generation. Our studies should provide novel mechanistic insights into the regulation of TGF-? responsiveness and the roles of TGF-?-induced non-Smad signaling in the cellular TGF-? response, in particular in EMT and cancer stem cell generation. These insights may link hyperglycemia or insulin treatment with cancer progression, through increased TGF-? responsiveness, and reveal a new role for the increased Akt signaling that is commonly seen in carcinomas, thus contributing to cancer progression by enhancing TGF-? responsiveness.

? DESCRIPTION (provided by applicant): TGF-? signaling controls cell physiology, proliferation and differentiation, and its deregulation acts prominently in cancer progression and fibrosis. In carcinomas, increased TGF-? signaling promotes an epithelial plasticity response that can progress to epithelial-mesenchymal transition (EMT), which is driven by reprogramming of gene expression and marked by loss of epithelial cell-cell junctions and apical-basal polarity, cytoskeletal reorganization, frontal-rear polarity and increased motility that often enables invasion. Partial or complete EMT, as a result of increased TGF-? signaling, associates with increased cell invasion and cancer dissemination, and with cancer stem cell generation by carcinomas. TGF-? binding to a cell surface complex of two types of transmembrane dual-specificity kinase receptors activates the type I receptor T?RI, which in turn activates Smad signaling, yet also initiates Erk MAPK and PI3K-Akt pathway signaling. Smad-mediated changes in gene expression and Erk MAPK and PI3K-Akt pathway signaling are essential for progression through EMT. Recruitment of the adaptor protein ShcA, which is generated as two functional forms, i.e. p52ShcA and p66ShcA, to the activated T?RI, and phosphorylation of ShcA by T?RI on Tyr initiate TGF-?-induced Erk MAPK activation. However, T?RI phosphorylates ShcA predominantly on Ser, but the role of TGF-?-induced Ser phosphorylation of ShcA is unknown. Dissecting the control of TGF-?-induced EMT, we found that downregulation of ShcA, expressed primarily as p52ShcA, leads to EMT through increased autocrine TGF-?/Smad signaling. Furthermore, p52ShcA competes with Smad3 for binding to T?RI and helps define the distribution of TGF-? receptors between clathrin-associated Smad activating complexes, and caveolar complexes, with p52ShcA recruiting TGF-? receptors to the latter. Thus, through its ability to repress autocrine TGF-?/Smad responses, ShcA protects epithelial cells against EMT, and helps maintain epithelial integrity. We now propose a program aimed at defining the central role of ShcA in balancing the spatial distribution of TGF-? receptor complexes and differential TGF-? signaling responses, the control of ShcA function by TGF-? signaling, and to consequently appreciate its role in controlling epithelial plasticity, cell invason, cancer stem cell generation and cancer progression, through its control of TGF-? signaling. Toward these goals, we propose (1) to define the roles of p52ShcA and p66ShcA in the differential distribution and signaling of TGF-? receptors, and T?RI stability and sumoylation, () to define the roles of ShcA Ser phosphorylation in TGF-?-induced signaling responses, (3) to study the role of ShcA in the regulation of epithelial plasticity, cell invasion, cancer stem cell properties and tumor progression by TGF-? signaling. Our results will help understand the role of ShcA in the control of TGF-? signaling, cancer cell behavior and cancer progression, which may lead to approaches to stabilize the epithelial phenotype and prevent epithelial plasticity responses that enable cancer progression and dissemination.