[0001] This application claims priority to United States provisional application Ser. No. 60/588,343, filed on July 16, 2004, which is incorporated herein by reference in its entirety.

Field of the Invention

[0002] The invention generally concerns methods of evaluating biological activities of agents, e.g., whether the agents inhibit or activate cellular signal transduction pathways such as TGF-β and activin signaling pathways and other pathways mediated by Smad proteins.

Background of the Invention

[0003] Transforming growth factor β (TGF-β) is a prototype of a large family of growth factors, termed the TGF-β superfamily. The superfamily encompasses over 50 evolutionarily conserved members that are found in all metazoan organisms. Members of the superfamily have been grouped into families that include: TGF-βs; activins and inhibins; bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs); and more distantly related molecules such Mullerian inhibitory substance (MIS) and glial cell line-derived neurotropic factor (GDNF). Fig. 1 depicts the phylogenetic tree of the TGF-β superfamily. For a review, see, e.g., Oppenheim et al. (eds.) Cytokine Reference, Acad. Press, San Diego, CA, 2001. [0004] Members of the TGF-β family exert a wide range of biological effects on a variety of cell types. They have been implicated in normal physiology as well as a variety of diseases. For example, TGF-β is involved in morphogenesis (epithelial-to-mesenchymal transformation), control of cell growth, chemotaxis, and extracellular matrix formation and maintenance (e.g., in wound repair of both soft and hard tissues), development and function of immune cells (lymphocytes, macrophages, and dendritic cells), and hematopoietic homeostasis. Pathophysiological states in which TGF-β plays a role include, for example, chronic inflammatory disorders, fibrotic disease, scar formation, carcinogenesis, parasitic infections, and autoimmune diseases. Thus, TGF-β as well as other members of the superfamily represent important targets for the development of novel therapeutic agents. [0005] In general terms, TGF-β family members initiate their cellular action by binding to cell surface receptors that possess intrinsic kinase activity in the cytoplasmic domain. The receptors, in turn, initiate signaling events that ultimately lead to changes in gene expression. A group of several receptor-activated proteins named Smads function to transduce the signal directly from the TGF-β superfamily receptor kinases to the nucleus. In the canonical signaling pathway, Smads are phosphorylated on C-terminal serines by the type I TGF-β receptor. Upon phosphorylation, Smads undergo heterodimerization with the common mediator Smad4. The resulting complex translocates into the nucleus where it regulates target genes. [0006] Two Smads, Smad2 and Smad3, are the principal transducers of signals from TGF-β and activin receptors (Massague et al. (2000) EMBO J., 19:1745-1754). Other pathways can also modulate the activity of Smad2 and Smad3 (see, e.g., Yakymovych et al. (2001 ) FASEB J., 15:553-555; and Kretzschmar et al. (1999) Ras. Genes Dev., 13:804-816). Nuclear Smad3/Smad4 and Smad2/Smad4 complexes bind either directly to DNA consensus sites or via a variety of coactivators, corepressors, and transcription factors which brings about the multifaceted patterns of gene regulation attributed to the TGF-β. The Smad3/Smad4 complexes bind DNA directly at a consensus CAGA motif (also known as "CAGA box") (Zawel et al. (1998) MoI. Cell, 1 :611-617; Dennler et al. (1998) EMBO J., 11 :3091-3100). Indeed, Smad-binding elements (SBEs) such as CAGA boxes can confer TGF-β inducibility to heterologous promoters (see, e.g., Zawel et al. (1998) MoI. Cell, 1 :611-617; Dennler et al. (1998) EMBO J., 11:3091-3100; Jonk et al. (1998) J. Biol. Chem., 273:21145-21152; Johnson et al. (1999) J. Biol. Chem., 274:20709-20716). [0007] The development of drugs for targeted therapeutic intervention requires assays that allow one to reliably assess a pathway-specific effect of a test compound. For evaluating compounds that can modulate the activity of Smad3, most commonly used are cell-based assays that utilize cells transfected with a reporter gene construct under the control of a TGF-β responsive promoter (e.g., a reporter gene assay described by Thies et al. (Growth Factors (2001 ) 18:251-259), which employs the pGL3(CAGA)12 reporter construct, or a reporter gene assay utilizing a synthetic 3TP promoter as described in Wrana et al. (1992) Cell, 71 :1003- 1014. However, given the multiple signaling pathways involved in mediating the complex and pleiotropic activities of TGF-β in the animal, the extent to which the in vitro results may be extrapolated to in vivo is unclear, particularly in the case of uncharacterized test compounds (Dong et al. (1996) J. Biol. Chem., 271(47):29969-29977). [0008] One approach for evaluating in vivo Smad3-mediated responses is to employ an endogenous gene regulated by TGF-β as a read-out. Plasminogen activator inhibitor type 1 (PAI-1 ) is an example of such a gene that has been well studied, albeit mostly in vitro. The PAI-1 promoter includes three CAGA boxes and was found to be inducible by TGF-β in vitro and in vivo (Dong et al. (1996) supra). However, the baseline in vivo expression of PAI-1 is nearly undetectable (Dichek (1989) In Vitro Cell. Dev. Biol., 25:289-292). Even though the endogenous PAI-1 expression can be elevated by stimulation with TGF-β, the detection of PAI-1 mRNA and protein levels is laborious and/or unreliable (Samad et al. (1997) MoI. Medicine, 1 (3):37-48, Sawdey et al. (1991) J. Clin. Invest, 88:1346-1353). [0009] Therefore, there exists a need in the art to develop new methods for in vivo assessment of drug candidates and other test compounds that can modulate the activity of Smad3 and others Smads.

SUMMARY OF THE INVENTION

[0010] In the experiments leading to the present invention, a viral vector carrying a reporter transgene and a tandem of twenty-five CAGA boxes was introduced into mice by a tail vein injection. The expression of the reporter was then stimulated by peritoneal injection of TGF-β. Some mice expressing the reporter transgene were dosed with inhibitors prior to the TGF-β stimulation. The present invention is based, in part, on the discovery and demonstration that the TGF-β stimulation resulted in robust and sustainable reporter expression that can be selectively inhibited by TGF-β pathway inhibitors. [0011] Accordingly, the invention provides an assay system for evaluating pharmacodynamic properties of compounds that modulate Smad3-mediated gene regulation, and the TGF-β/activin signaling pathway, in particular. [0012] The invention further provides a reporter construct responsive in vivo to a member of the TGF-β superfamily (e.g., TGF-β or activin) and comprises a tandem of N Smad-binding elements operably linked to a reporter gene, wherein N is an integer greater than 12 and less than 51. In certain embodiments, N equals 25±K, wherein K is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12. In nonlimiting illustrative embodiments, N equals 25. In some embodiments, the Smad-binding element comprises a CAGA box having the nucleotide sequence X1X2X3-CAG AC-X4 (SEQ ID NO:1 , with the proviso that X1 is A or G, X2 is G or T, X3 is C, A, G or T, and X4 is A or C, all independently of each other. In certain nonlimiting illustrative embodiments, each CAGA box comprises the nucleotide sequence AGCCAGACA (SEQ ID NO:2). In nonlimiting illustrative embodiments, CAGA are positioned in tandem so that each of the 9-bp CAGA box sequences is separated by 3-6 bps from the next one. [0013] The invention further provides vectors containing reporter constructs of the invention; cells and animals carrying the reporter constructs of the invention; and cell and tissue extracts prepared from these cells and animal tissues. In nonlimiting illustrative embodiments, the animal is a transgenic mouse carrying a luciferase transgene operably linked to a tandem of twenty-five CAGA boxes. In other nonlimiting illustrative embodiments, a viral vector carrying a reporter transgene operably linked to a tandem of twenty-five CAGA boxes is introduced into a non-transgenic mouse by a systemic injection. [0014] In another aspect, the invention provides a method of evaluating Smad-mediated signaling in an animal carrying a reporter construct of the invention. The method includes determining the amount of the reporter gene expressed, and optionally stimulating reporter gene expression in the animal, for example, by administering a member of the TGF-β superfamily to the animal and/or by inducing endogenous production of a member of the TGF-β superfamily in the animal. In nonlimiting illustrative embodiments, the reporter gene is expressed at a detectable level for at least 6 hours following the administration of the member of the TGF-β superfamily. [0015] In a further aspect, the invention provides a method of evaluating the effect of a test compound or composition on Smad-mediated signaling (Smad- mediated gene regulation). The method includes administering the test compound or composition to the animal of carrying a reporter construct of the invention and determining the expression level of the reporter gene. The expression level of the reporter gene indicates the effect of the test compound or the composition on the Smad-mediated signaling. The method of evaluating the effect of a test compound or composition optionally includes stimulating reporter gene expression in the animal, for example, by administering a member of the TGF-β superfamily to the animal and/or by inducing endogenous production of a member of the TGF-β superfamily in the animal. In nonlimiting illustrative embodiments, the reporter gene is expressed at the detectable level starting within 2 hours following the induction for a period of at least 6 hours with the maximal expression level at around 30 times the baseline level. The administration of the test compound is performed before, concurrently with, or after the stimulation of reporter gene expression. [0016] Additional aspects of the invention will be set forth in the following description, will be understood from the description, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0017] Figure 1 depicts the phylogenetic tree of the TGF-β superfamily. The sequences compared are human unless noted: (c) C. elegans; (x) Xenopus; (m) murine; (b) bovine; (z) zebrafish; (d) Drosophila; (s) sea urchin.

BRIEF DESCRIPTION OF THE SEQUENCES

[0018] SEQ ID NO:1 is a generic sequence of the CAGA box XIX2XS-CAGAC-X4, wherein Xi is A or G, X2 is G or T, X3 is C, A, G or T, and X4 is A or C, all independently of each other. [0019] SEQ ID NOs:2-9 are examples of CAGA boxes present in the various promoters of human genes as listed in Table 1. [0020] SEQ ID NO:10 and SEQ ID NO:11 are oligonucleotides containing 6 SBEs uses in the preparation of (CAGA)25 reporter construct. DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

[0021] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. [0022] The term "Smad-binding element (SBE)" refers to a double-stranded polynucleotide having a minimal sequence sufficient for specific binding to Smad3 and/or Smad4 (complexed with each other, other co-factors, or individually). SBEs include but are not limited to CAGA boxes which are found in natural and synthetic TGF-β/activin responsive promoters, such as for example, 3TP (Wrana et al. (1992) Cell, 71 :1003-1014), PAI-1 (Zonneveld et al. (1988) Prot. Natl. Acad. Sci. USA, 85:5525-5529), collagenase I, c-Jun, IgA, and Jun B, c-myc, and other promoters listed in Table 1. More recently, Zawel et al., identified another 8-bp palindromic sequence GTCTAGAC (SEQ ID NO:3) as a Smad-binding element (SBE) (Zawel et al. (1998) MoI. Cell, 1 :611-617; United States Patent No. 6,100,032).

Table 1

[0023] Suitable assays for determining the binding of a polynucleotide to Smads include but are not limited to gel-shift assays (EMSA) with a Mad-homology 1 (MH1 ) domain and/or full-length Smads, as well as antibody supershift assays and TGF-β-inducible cell-based reporter gene assays. Such assays are described in the Examples and also, for example, in Zawel et al. (1998) MoI. Cell, 1 :611-617; United States Patent No. 6,100,032; Transforming Growth Factor-Beta Protocols, ed. Howe, P.H., Humana Press, 2000. [0024] The term "CAGA box" refers to an SBE that comprises at least (a) the 5-bp palindromic sequence CAGAC (including the corresponding inverted nucleotide sequence GTCTG). [0025] The term "tandem of SBEs" refers to a positioning of SBEs within a nucleotide sequence, in which SBEs are separated by no more than 35 bps. [0026] The term "transfection" is used interchangeably with the terms "transduction" and "transformation" and refers to the intracellular introduction of a polynucleotide. [0027] The term "modulating" and its cognates refer to either reducing/inhibiting or increasing/stimulating/activating any biological activity associated with a specified biological process (e.g., the binding of Smad3 to DNA, inhibition of TGF-β) or any biological activity (unless otherwise stated) associated with a specified molecule (e.g., inhibition of TGF-β by a test compound). The term "biological activity" refers to a function or set of functions (or the effect to which the function is attributed to) performed by a molecule in a biological system, which may refer to in vivo or in vitro systems, depending on the context. [0028] The terms "specific binding" and its cognates mean that two molecules form a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity. Nonspecific binding usually has a low affinity with a moderate to high capacity. Typically, the binding is considered specific when the affinity constant Ka is higher than 106 M"1 , or preferably higher than 108 M"1. If necessary, nonspecific binding can be reduced without substantially affecting specific binding by varying the binding conditions. Such conditions are known in the art, and a skilled artisan using routine techniques can select appropriate conditions. II. Compositions and Methods

[0029] General methods of the inventions, compositions used therein, and other aspects of the invention are described in detail below. [0030] In the experiments leading to the present invention, a viral vector carrying a reporter transgene and twenty-five tandem CAGA boxes was introduced into mice by a tail vein injection. The expression of the reporter was then stimulated by peritoneal injection of TGF-β. Some mice expressing the reporter transgene were dosed with inhibitors prior to the TGF-β stimulation. The present invention is based, in part, on the discovery and demonstration that the TGF-β stimulation resulted in robust and sustainable reporter expression that can be selectively inhibited by TGF-β pathway inhibitors. Accordingly, the invention provides an assay system for evaluating pharmacodynamic properties of compounds that modulate Smad-mediated gene regulation, and the TGF-β/activin signaling pathway, in particular. A. Reporter Constructs [0031] The invention provides a reporter construct responsive in vivo to a member of the TGF-β superfamily (e.g., TGF-β or activin) and comprises a tandem of N Smad-binding elements operably linked to a reporter gene, wherein N is an integer greater than 12 and less than 51 , greater than 15 and less than 51 ; greater than 15 and less than 45; greater than 15 and less than 33; greater than 15 and less than 27; greater than 15 and less than 21; greater than 21 and less than 51 ; greater than 21 and less than 45; greater than 21 and less than 33; greater than 21 and less than 27; greater than 27 and less than 51 ; greater than 27 and less than 45; greater than 27 and less than 33; greater than 33 and less than 51 ; greater than 33 and less than 45; greater than 45 and less than 51 ; greater than 27 and less than 33, or N equals 22, 23, 24, 25, 26, 27, or 28. In certain embodiments, N equals 25±K, wherein K is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12. In nonlimiting illustrative embodiments, N equals 25. In some embodiments, the Smad-binding element comprises a CAGA box having the nucleotide sequence X1X2X3-CAGAC-X4 (SEQ ID NO:1 ), with the proviso that X1 is A or G, X2 is G or T, X3 is C, A, G, or T, and X4 is A or C, all independently of each other. In certain nonlimiting illustrative embodiments, each CAGA box comprises the nucleotide sequence AGCCAGACA (SEQ ID NO:2). In specific embodiments, the Smad-binding element comprises a CAGA box having the nucleotide sequence XiX2X3-CAGAC-X4 (SEQ ID NO:1 ), with the proviso that X^ is A or G, X2 is G or T, X3 is C, A, G or T, and X4 is A or C, all independently of each other. In nonlimiting illustrative embodiments, each CAGA box comprises the nucleotide sequence AGCCAGACA (SEQ ID NO:2). In other embodiments, a Smad-binding element comprises the nucleotide sequence GTCTAGAC (SEQ ID NO:3). [0032] In nonlimiting illustrative embodiments, the SBEs in the tandem are separated by 3-6 bps. In other embodiments, they are positioned so that the separation is no more than 35, 30, 25, 20, 15, 12, 9, or 7 bps. [0033] The reporter constructs of the invention comprise a "promoter" and a reporter gene. The term "promoter" refers to a regulatory element that directs the transcription of a nucleic acid to which it is operably linked. A promoter can regulate both rate and efficiency of transcription of an operably linked nucleic acid. A promoter may also be operably linked to other regulatory elements which enhance ("enhancers") or repress ("repressors") promoter-dependent transcription of a nucleic acid. The term "operably linked" refers to a nucleic acid placed in a functional relationship with another nucleic acid. A promoter may be positioned 5' (upstream) or 3' (downstream) of a transcription initiation site in the nucleic acid. Alternatively, a promoter may also encompass regions both 5' and 3' of the transcription initiation site of the operably linked nucleic acid. A reporter gene can be any measurable expression product, most typically a protein. Commonly used reporter genes include but are not limited to α-galactosidase, alkaline phosphatase, chloramphenicol acetyl transferase (CAT), DsRed, β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), β-lactamase luciferase, LacZ, red fluorescent protein; or fusions (chimeras) thereof (see, e.g., Spergel et al. (2001 ) Prog. Neurobiol., 63(6):673-686); Rosochacki et al. (2002) Acta Microbiol. Pol., 51 (3):205-216; Barka et al. (2004) J. Histochem. Cytochem., 52(4):469-77; Liu et al. (2001) MoI. Genet. Genomics, 266(4): 614-623). [0034] The reporter construct of the invention, when introduced into a host cell or animal can be episomal, or chromosomally integrated as, for example, in transgenic animals carrying the reporter construct of the invention. B. Vectors and Cells [0035] The invention further provides vectors containing reporter constructs of the invention. Generally, a wide range of vectors, both viral and nonviral, is suitable in the methods of the invention (see, e.g., Hsich et al. (2002) Hum. Gene Ther., 13:579-504; and Davidson et al. (2003) Nat. Rev., 4:353-364). Examples of suitable vectors include: retroviral vectors, which include vectors derived from Moloney murine leukemia virus (MoMLC), lentiviral vectors (see, e.g., Englund (2002) Dev. Brain Res., 134:123-141 ; Tamaki (2002) J. Neurosci. Res., 69:979-986); and adeno-associated viral (AAV) vectors, herpes-simplex-1 viral (HSV-1 ) vector, and adenoviral (Ad) vectors. Naked DNA, liposomes, and molecular conjugates can also be used. [0036] The level of transgene expression in eukaryotic cells is largely determined by the transcriptional promoter within the transgene expression cassette. Promoters that show long-term activity and are tissue- and even cell-specific are used in some embodiments. Nonlimiting examples of promoters include but are not limited to the cytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet, 8:148-154), CMV/human β3-globin promoter (Mandel et al. (1998) J. Neurosci., 18:4271-4284), GFAP promoter (Xu et al. (2001 ) Gene Ther., 8:1323-1332), the 1.8-kb neuron-specific enolase (NSE) promoter (Klein et al. (1998) Exp. Neurol., 150:183-194), chicken β-actin (CBA) promoter (Miyazaki (1989) Gene, 79:269-277), and the β-glucuronidase (GUSB) promoter (Shipley et al. (1991 ) Genetics, 10:1009-1018). To prolong expression, other regulatory elements may additionally be operably linked to the transgene, such as, e.g., the Woodchuck Hepatitis Virus Post-Regulatory Element (WPRE) (Donello et al. (1998) J. Virol., 72:5085-5092) or the bovine growth hormone (BGH) polyadenylation site. For some applications, it may be desirable to control transcriptional activity. To this end, pharmacological regulation of gene expression can be obtained by including various regulatory elements and drug-responsive promoters as described, for example, in Habermaet al. (1998) Gene Ther., 5:1604-16011 ; and Ye et al. (1995) Science, 283:88-91. [0037] The invention further provides cells carrying the reporter constructs of the invention. Cells can be primary cell cultures (e.g., mesenchymal cells (e.g., chondrocytes, fibroblasts, myocytes, etc.), stem cells (e.g., hematopoietic or neural stem cells), and other cells types. Suitable cells also include established clonal cell lines. Generally, most available cell lines can be transfected with the vectors of the invention. In some embodiments, the cell line is a hepatoma cell line, HepG2, a colorectal carcinoma cell line, CT26, a breast carcinoma cell line, Mx-1 , or a nonsmall cell lung tumor cell line, Calu-6. Examples of other commonly used cell lines also include COS cells, CHO cells, and other cell lines. Such cell lines are widely available commercially. C. Animals [0038] The invention further provides animals carrying the reporter constructs of the invention and tissues (including organs and cells) derived from these animals. Examples of tissues include liver, kidney, skin, muscle, lung, brain, fat, spleen, testes, and gastrointestinal tract tissues. [0039] In certain nonlimiting illustrative embodiments, the animal is a non-transgenic rodent (e.g., a mouse) carrying a luciferase transgene operably linked to a tandem of twenty-five CAGA boxes which is introduced into the animal by systemic injection. Other suitable animals include mammals, including but not limited to rats, rabbits, sheep, pigs, dogs, cats, monkeys, chimpanzees, and guinea pigs. For specific vectors and delivery protocols, see, e.g., Viral Vector for Gene Therapy: Methods and Protocols, Machida (ed.), Humana Press, Totowa, NJ, 2003; and Non-Viral Vectors for Gene Therapy: Methods and Protocols, Findeis (ed.), Humana Press, Totowa, NJ, 2001 ; Gene Therapy Protocols, 2nd ed., Morgan (ed.), Humana Press, Totowas, NJ, 2001 ; and Gene Transfer and Expression in Mammalian Cells: New Comprehensive Biochemistry, Makrides (ed.), Elsevier Science Ltd, 2003. Tail vein injection procedures for rodents are well known in the art, and are described in the Examples, and additionally, for example, in United States Patent No. 6,265,387 which describes a method for transfecting the mouse liver through an intravenous injection of hypertonic solution containing naked plasmid DNA. Additionally, delivery of retroviral vectors to the liver is described, for example, in Ferry et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8377-8381 and Kay et al. (1992) Hum. Gene Then, 3:641-647; delivery of adenoviral gene transfer vectors via the portal vein in rats is described, for example, in Rosefeld et al. (1991 ) Science, 252:431-434. [0040] In certain other nonlimiting illustrative embodiments, the animal is a transgenic rodent (e.g., a mouse) carrying a luciferase transgene operably linked to a tandem of twenty-five CAGA boxes. Other suitable animals include mammals, including but not limited to rats, rabbits, sheep, pigs, dogs, cats, monkeys, chimpanzees, and guinea pigs. For specific vectors and delivery protocols, see, Transgenic Mouse Methods and Protocols, Hofker, M. H., van Deursen, J. (eds.) Humana Press, 2002; Transgenesis Techniques: Principles and Protocols, 2nd ed., Clark, A.R. (ed.), Humana Press, 2002; and Animal Transgenesis and Cloning, Houdebine, L-M., John Wiley & Sons, 2003. In certain embodiments, the transgenics are generated by lentiviral transfection of single-cell mouse embryos as described, for example, in Lois et al. (2002) Science, 295:868-872. Briefly, lentivirus carrying the reporter transgene will be used to infect single-cell embryo and stably transmit the transgene to the zygote via integration into the genome. The modified embryos are then returned to foster mothers to complete development, and those born will be screened for the presence of transgene. Alternatively, transgenic animals can be produced using pronuclear microinjection as described, for example, in U.S. patent No. 4,873,191. D. Cell and Tissue Extracts [0041] The invention further provides cell and tissue extracts prepared from cells and animal tissues comprising the reporter constructs of the invention. Methods of making cell and tissue extracts are well known in the art and are described in the Examples and in the references cited. E. Assays [0042] In a further aspect, the invention provides a method of evaluating Smad-mediated signaling in an animal (non-transgenic or transgenic) carrying a reporter construct (including a vector) of the invention. The method includes determining the amount of the reporter gene expressed, and optionally stimulating reporter gene expression in the animal, for example, by administering a member of the TGF-β superfamily to the animal and/or by inducing endogenous production of a member of the TGF-β superfamily in the animal. [0043] In nonlimiting illustrative embodiments, the member of TGF-β superfamily, which is administered to induce reporter gene expression, is TGF-β. Other suitable members of the TGF-β superfamily include but are not limited to members of TGF-β subfamily and members of activin subfamily as shown in Fig 1. [0044] Currently, there are 5 known isoforms of TGF-β (TGF-β1 -β5), all of which are homologous among each other (60-80% identity), form homodimers of about 25 kDa, and act upon common TGF-β receptors (TβR-l, TβR-ll, TβR-IIB, and TβR-lll). TGF-β1 , TGF-β2, and TGF-β3 are found in mammals. The structural and functional aspects of many members of the TGF-β superfamily are well known in the art. For a review of the TGF-β superfamily and Smad signaling pathways, see, for example, Cytokine Reference, eds. Oppenheim et al., Academic Press, San Diego, CA, 2001 , and Mehra et al. (2002) Biochem. Cell Biol., 80:605-622). [0045] "Administration" is not limited to any particular delivery system and may include, without limitation, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, transdermal, or oral (for example, in capsules, suspensions, or tablets). Administration may occur in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition (described earlier). Physiologically acceptable salt forms and standard pharmaceutical formulation techniques and excipients are well known to persons skilled in the art (see, e.g., Physician's Desk Reference (PDR) 2003, 57th ed., Medical Economics Company, 2002; and Remington: The Science and Practice of Pharmacy, eds. Gennado et al., 20th ed, Lippincott, Williams & Wilkins, 2000). [0046] Since members of the TGF-β superfamily exhibit diverse bioactivities, various assays can be used to detect and quantify their amount and/or activity. Examples of some of the more frequently used in vitro bioassays include: (1 ) induction of colony formation of NRK cells in soft agar in the presence of EGF (Roberts et al. (1981) Proc. Natl. Acad. Sci. USA, 78:5339-5343); (2) induction of differentiation of primitive mesenchymal cells to express a cartilaginous phenotype (Seyedin et al. (1985) Proc. Natl. Acad. Sci. USA, 82:2267-2271); (3) inhibition of growth of Mv1 Lu mink lung epithelial cells (Danielpour et al. (1989) J. Cell. Physiol., 138:79-86) and BBC-1 monkey kidney cells (Holley et al. (1980) Proc. Natl. Acad. Sci. USA, 77:5989-5992); (4) inhibition of mitogenesis of C3H/HeJ mouse thymocytes (Wrann et al. (1987) EMBO J., 6:1633-1636); (5) inhibition of differentiation of rat L6 myoblast cells (Florini et al. (1986) J. Biol. Chem., 261 :16509-16513); (6) measurement of fibronectin production (Wrana et al. (1992) Cell, 71 :1003-1014); (7) induction of plasminogen activator inhibitor I (PAI-1 ) promoter fused to a luciferase reporter gene (Abe et al. (1994) Anal. Biochem., 216:276-284); (8) sandwich enzyme-linked immunosorbent assays (Danielpour et al. (1989) Growth Factors, 2:61-71); and (9) cellular assays described in Singh et al. (2003) Bioorg. Med. Chem. Lett., 13(24):4355-4359. [0047] Generally, a member of the TGF-β family is administered at a dose of 0.1 ng/kg to 100 μg/kg. The exact dose can be readily determined by one ordinary skill in the art through mere routine experimentation. [0048] The reporter gene expression can also be stimulated by inducing endogenous production of TGF-β in the animal. There are numerous conditions that lead to increased production of TGF-β. Such conditions include but are not limited to cancer (Dumont et al. (2000) Breast Cancer Res. 2:125-132), wound healing, inflammation, autoimmune disease, ischemia, atherosclerosis, reperfusion, fibrosis, xenograft, CNS injury, diabetic nephropathy (Chen et al. (2001 ) Ren. Failure, 23:471) and other renal disease (Cheng et al. (2002) Exp. Biol. Med., 227:943), including lupus nephritis, IgA nephropathy, etc. For a review of the role of TGF-β in human disease, see, e.g., BIobe et al. (2000) New EnI. J. of Med. 342(18): 1350-1358). Certain animal cancer models (spontaneous, transgenic/knock-out, syngeneic) have been developed that are characterized by elevated levels of TGF-β production. Such models and conditions characterized by elevated levels of TGF-β are described, for example, in Border et al. (2004) New Engl. J. of Med., 331(19):1286-1292; Cheng et al. (2002) Exp. Biol. Med., 227:943. [0049] The amount of the reporter gene expressed can be determined by any suitable method. Expression levels, at the RNA or at the protein level, can be determined using routine methods. Expression levels are usually scaled and/or normalized per total amount of RNA or protein in the sample and/or a control, which is typically a housekeeping gene such as actin or GAPDH. RNA levels are determined by quantitative PCR (e.g., RT-PCR), Northern blotting, or any other method for determining RNA levels, e.g., as described in Sambrook et al. (eds.) Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, or Lodie et al. (2002) Tissue Eng., 8(5):739-751), or as described in the Examples. Protein levels are determined using, Western blotting, ELISA, enzymatic activity assays, or any other method for determining protein levels, e.g., as described in Current Protocols in Molecular Biology (Ausubel et al. (eds.) New York: John Wiley and Sons, 1998). [0050] In nonlimiting illustrative embodiments, the expression levels are determined using either luciferase activity in cell/tissue extracts or by tissue or whole animal imaging. In addition to MRI, tissue imaging on living animals can be performed by fluorescence detection (Hoffman (2002) Lancet Oncol., 3:546-556; Tung et al. (2000) Cancer Res., 60:4953-4958), bioluminescence detection (Shi (2001 ) Proc. Nat. Acad. Sci. USA, 98:12754-12759; Luke et al. (2002) J. Virol., 76:12149-12161 , and U.S. Patent Nos. 5,650,135 and 6,217,847), positron emission tomography (Liang et al. (2002) MoI. Ther., 6:73-82, near-infrared fluorescence (Tung et al. (2000) Cancer Res., 60:4953-4958), or X-ray imaging (Hemminki et al. (2002) J. Nat. Cancer Inst, 94:741-749). [0051] In some embodiments, the maximal expression level of the reporter is observed within 24, 12, 10, 8, 6, 4, and 2 hours following induction and is expressed at a detectable level for at least 4, 6, 8, 10, 12, 18, 24, 36, or 48 hours. In some embodiments, the maximal expression level of the reporter gene following TGF-β induction is at least 3, 5, 10, 20, 30, 50, or 100 times greater than the corresponding base level of the reporter gene expression prior to the induction. In nonlimiting illustrative embodiments, the reporter gene is expressed at the detectable level starting within 2 hours following the induction for a period of at least 6 hours with the maximal expression level at around 30 times the baseline level. [0052] In a further aspect, the invention provides a method of evaluating the effect of a test compound or composition on Smad-mediated signaling. The method includes administering the test compound or composition to the animal of carrying a reporter construct of the invention; and determining the expression level of the reporter gene. The expression level of the reporter gene indicates the effect of the test compound or the composition on the Smad-mediated signaling. The method of evaluating the effect of a test compound or composition optionally includes stimulating reporter gene expression in an animal, for example, by administering a member of the TGF-β superfamily to the animal and/or by inducing endogenous production of a member of the TGF-β superfamily in the animal. The examples of a member of the TGF-β superfamily that can be administered include those shown in Fig. 1. In some embodiments, the member of the TGF-β superfamily administered is a member of the TGF-β subfamily (e.g., TGF-β1 , TGF-β2, and TGF-β3), a member of the activin subfamily activin (e.g., activin βA and activin βB). [0053] The administration of the test compound is performed before, concurrently with, or after the stimulation of the reporter gene expression. Stimulation of the TGF-β expression and the methods of determining the reporter gene expression levels are described above. Another method for detecting the in vivo efficacy of TβRI inhibitors is by using cell lines (e.g., CT26 colorectal carcinoma cells or CaIu-6, nonsmall ceil lung tumor cells) stably transfected with a reporter construct carrying the TGF-β inducible promoter (CAGA)2S operably linked to a reporter gene (e.g., the firefly luciferase reporter (CAGA)25-fLuc)). In such an assay, stably transfected cells are injected into the animal (e.g., into the tail vein in rodents). These cells colonize and form tumors (e.g., CT26 colonize in the lungs). The expression of the reporter can then be induced by endogenous or exogenous TGF-β. The ability of compound to inhibit TGF-β-dependent response, and therefore Smad-mediated signaling is then assessed. The assessment can be accomplished non-invasively by monitoring the level of luciferase activity over time using, for example, the MS® biophotonic imaging system (Xenogen, Alameda, CA). This technique allows acquisition of multiple ("real-time") measurements from the same live animal. Accordingly, in some embodiments, a method the invention comprises determining reporter gene expression levels at varying time points in the same live animal. [0054] Alternatively, cells (e.g., CT26, Calu-6, or Mx-1 cells) can be stably transfected with the (CAGA)25-fLuc reporter and a second reporter construct carrying the constitutively expressed SV40 promoter linked to the renilla luciferase reporter (SV40-rl_uc). Since the firefly luciferase and the renilla luciferase utilize different substrates without cross reactivity, dual tracking of cells' responsiveness to TGF-β and their growth in the same animal can be accomplished using the two different substrates consecutively. The effects on Smad-mediated signaling as well as tumor growth can be monitored non-invasively as described. Accordingly, in some embodiments, a method of the invention comprises determining a first and a second reporter gene's expression levels at varying time points in the same live animal. [0055] The following examples provide illustrative embodiments of the invention. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the present invention. Such modifications and variations are encompassed within the scope of the invention. The Examples do not in any way limit the invention.

EXAMPLES

Example 1 : In vivo transfection and inhibition studies

[0056] The first generation adenoviral vector (E1 , E3 deleted, serotype 5) (CAGA)25-GL (Ad.(CAGA)25-GL) was created using 293 cells as described in Ng et al. (2000) Human Gene Therapy 11 :693-699. Purification was performed by double cesium chloride density equilibrium gradient centrifugation. The vector was stored at -8O0C in 10 mM Tris-HCI, 1 mM MgCI2, 10% v/v glycerol, pH 8.0. [0057] In the in-vivo studies, 1 x 1011 particles of Ad-(CAGA)25-GL in 100 μl PBS were injected into the tail vein of Balb/c female mice on day one. To evaluate inhibitor activity, on day two, animals were treated with inhibitor orally or intravenously. Inhibitors were allowed to permeate into the tissues of the animal ranging from 30 minutes to 24 hours prior to TGF-β stimulation. For TGF-β stimulation, animals were injected intraperitoneally with 3 μg of TGF-β in 200 μl of PBS also containing 3 mM HCI and 0.1% BSA. Animals were then sacrificed at different times up to 6 hours following the TGF-β injection. Livers were harvested, flash frozen and stored at -8O0C. [0058] To generate liver homogenates for luciferase activity assay, frozen livers were pulverized using the BioPulverizer™ (BioSpec). Pulverized livers were homogenized in 500 μl of luciferase assay buffer (LucLite™, Perkin Elmer), subjected to one round of freeze-thaw and spun at 13,000 rpm for 5 minutes. Liver homogenates were analyzed immediately for luciferase activity using the LucLite™ assay kit as per manufacturer's instructions or stored at -8O0C until use. A representative study demonstrating dose-dependent inhibition of TGF-β inducible luciferase expression with various TβRI inhibitors, including two as shown in Tables 3 and 4 which have IC50's 0.034 μM (Compound 1) and 3.0 μM (Compound 2). The cellular IC50's were determined using in vitro assay as described in Singh et al. (2003) Bioorg. Med. Chem. Lett., 13(24): 4355-4359. The results are shown in Tables 2 and 3.

Table 2

Table 3

[0059] The in vivo inhibitory activity of Compound 1 decays over time as shown in Table 4.

Table 4

Example 2: Cloning and in vitro studies

[0060] To generate the TGF-β responsive GFP-luciferase fusion protein reporter construct which contains tandem repeats of CAGA boxes (CAGA)n/G-L, oligos containing six SBEs as shown in SEQ ID NO:10 and SEQ ID NO:11 were ligated 5' to the adenoviral minimal late promoter and the GFP-luciferase chimeric gene. [0061] Recombinant constructs containing varying number of CAGA boxes were purified and analyzed for TGF-β inducible reporter expression in HepG2 cell culture assay as follows. HepG2 cells grown in complete media containing 10% FBS on 24-well plates were transiently transfected with 1.0 μg of TGF-β responsive constructs using FuGENE 6™ (Roche). Cells were then starved in low serum media (0.5% FBS) for 6 hours prior to incubation with 10 ng/ml of TGF-β (R&D Systems) overnight. Expression of (CAGA)25-GL was evaluated using the LucLite™ luciferase activity assay kit (Perkin Elmer) as per manufacturer's instructions. [0062] Results of a representative luciferase expression assay in transiently transfected HepG2 cells are shown in Table 5.

Table 5

Example 3: Generation of transgenic mice and in vivo studies

[0063] The SBE25-GL reporter construct was introduced as a transgene into the C57BL6/Balk/c background using both the conventional pronuclear injection method (Gordon et al. (1983) Methods Enzymol., 101 :411-433) and the recently developed lentiviral method (Lois et al. (2002) Science, 295: 868-872). [0064] To measure real-time TGF-β inducible luciferase expression in vivo, SBE25-GL transgenic mice were injected with 3-10 μg of TGF-β and imaged with Xenogen's MS™ system for the background level of luciferase expression (t=0) before receiving TGF-β, plus or minus inhibitor. The expression of luciferase was measured at four, seven and twenty-seven hours after the TGF-β induction. [0065] TGF-β inducible SBE25-GL reporter transgene expression time course measured using the Xenogen I VIS™ system is shown in Table 6. Similarly to the viral reporter vector, the SBE25-GL reporter transgene was induced by TGF-β in a time-dependent manner. Table 6

Time of TGF-β Inhibitor 0 4 7 27 stimulation (hrs) Fold-induction of + 1 1.52 1.57 1.15 luciferase expression Fold-induction of - 1 2.04 3.22 3.36 luciferase expression

[0066] The following applies unless otherwise indicated: all numbers expressing quantities of ingredients, cell culture, treatment conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term "about." All publications and patents and sequences cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with the present specification, the specification will supersede any such material. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. It is intended that the specification and examples be considered as illustrative only, with a true scope and spirit of the invention being indicated by the following claims.