Growth differentiation factor-15 encodes a novel microRNA 3189 that functions as a potent regulator of cell death.

According to the latest version of miRBase, approximately 30% of microRNAs (miRNAs) are unique to primates, but the physiological function of the vast majority remains unknown. In this study, we identified miR-3189 as a novel, p53-regulated, primate-specific miRNA embedded in the intron of the p53-target gene GDF15. Antagonizing miR-3189 increased proliferation and sensitized cells to DNA damage-induced apoptosis, suggesting a tumor suppressor function for endogenous miR-3189. Identification of genome-wide miR-3189 targets revealed that miR-3189 directly inhibits the expression of a large number of genes involved in cell cycle control and cell survival. In addition, miR-3189 downregulated the expression of multiple p53 inhibitors resulting in elevated p53 levels and upregulation of several p53 targets including p21 (CDKN1A), GADD45A and the miR-3189 host gene GDF15, suggesting miR-3189 auto-regulation. Surprisingly, miR-3189 overexpression in p53-/- cells upregulated a subset of p53-targets including GDF15, GADD45A, and NOXA, but not CDKN1A. Consistent with these results, overexpression of miR-3189 potently induced apoptosis and inhibited tumorigenicity in vivo in a p53-independent manner. Collectively, our study identified miR-3189 as a novel, primate-specific miRNA whose effects are mediated by both p53-dependent and p53-independent mechanisms. miR-3189 may, therefore, represent a novel tool that can be utilized therapeutically to induce a potent proapoptotic effect even in p53-deficient tumors.

A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis.

Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis.

TGF-beta3 and cancer: a review.

With the development of growth factors and growth factor modulators as therapeutics for a range of disorders, it is prudent to consider whether modulating the growth factor profile in a tissue can influence tumour initiation or progression. As recombinant human TGF-beta3 (avotermin) is being developed for the improvement of scarring in the skin it is important to understand the role, if any, of this cytokine in tumour progression. Elevated levels of TGF-beta3 expression detected in late-stage tumours have linked this cytokine with tumourigenesis, although functional data to support a causative role are lacking. While it has proved tempting for researchers to interpret a 'correlation' as a 'cause' of disease, what has often been overlooked is the normal biological role of TGF-beta3 in processes that are often subverted in tumourigenesis. Clarifying the role of this cytokine is complicated by inappropriate extrapolation of the data relating to TGF-beta1 in tumourigenesis, despite marked differences in biology between the TGF-beta isoforms. Indeed, published studies have indicated that TGF-beta3 may actually play a protective role against tumourigenesis in a range of tissues including the skin, breast, oral and gastric mucosa. Based on currently available data it is reasonable to hypothesize that administration of acute low doses of exogenous TGF-beta3 is unlikely to influence tumour initiation or progression.

Heterozygous inactivation of TGF-beta1 increases the susceptibility to chemically induced mouse lung tumorigenesis independently of mutational activation of K-ras.

Mice heterozygous for deletion of the transforming growth factor beta1 (TGF-beta1) gene show an enhanced rate of lung tumorigenesis following carcinogen treatment. Since the growth inhibitory activity of TGF-beta1 in epithelial cells is associated with K-ras p21, and K-ras mutations commonly occur in chemically-induced mouse lung tumors, we postulated that tumors in heterozygous TGF-beta1 mice might be more likely to have K-ras mutations compared with tumors in wildtype TGF-beta1 mice. Urethane-induced lung tumors in AJBL6 TGF-beta1 +/- and +/+ mice were examined for K-ras mutations by polymerase chain reaction/single strand conformation polymorphism analysis and sequencing. Mutation frequencies were similar in both genotypes: 12/18 +/- tumors (67%) and 10/16 +/+ tumors (62%). Mutations occurred in 80% +/- and 75% +/+ carcinomas, but in only 50% of the adenomas of both TGF-beta1 genotypes. Codon 61 A-->G transition mutations were predominant, occurring in 61% +/- and 44% +/+ tumors. Three +/- (17%) and three +/+ (19%) tumors showed codon 12 mutations, mostly G-->A transitions. Two +/- tumors had both codon 61 and codon 12 mutations. Interestingly, carcinomas with mutations in codon 61 were larger than those with codon 12 changes. It appears that the mechanism of enhanced susceptibility of TGF-beta1+/- mice to urethane-induced lung carcinogenesis does not involve selective development of tumors with K-ras mutations.

TGF-beta signaling in mammary gland development and tumorigenesis.

Ligands of the TGF-beta superfamily are unique in that they signal through transmembrane receptor serine-threonine kinases, rather than tyrosine kinases. The receptor complex couples to a signal transduction pathway involving a novel family of proteins, the Smads. On phosphorylation, Smads translocate to the nucleus where they modulate transcriptional responses. However, TGF-betas can also activate the mitogen-activated protein kinase (MAPK)4 pathway, and the different biological responses to TGF-beta depend to varying degrees on activation of either or both of these two pathways. The Smad pathway is a nexus for cross-talk with other signal transduction pathways and for modulation by many different interacting proteins. Despite compelling evidence that TGF-beta has tumor suppressor activity in the mammary gland, neither TGF-beta receptors nor Smads are genetically inactivated in human breast cancer, though receptor expression is reduced. Possible reasons are discussed in relation to the dual role of TGF-beta as tumor suppressor and oncogene.

Transforming growth factors-beta are not good biomarkers of chemopreventive efficacy in a preclinical breast cancer model system.

Using a carcinogen-initiated rat model of mammary tumorigenesis, we tested the hypothesis that transforming growth factor (TGF)-betas are useful biomarkers of chemopreventive efficacy in the breast. The chemopreventive agents tested were tamoxifen and the retinoids 9-cis-retinoic acid (9cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR), because both antiestrogens and retinoids have previously been shown to upregulate TGF-betas in vitro. Despite demonstrable chemopreventive efficacy in this model, none of these agents, alone or in combination, had any significant impact on the expression of TGF-betas in the mammary ductal epithelium or periductal stroma as determined by immunohistochemistry. These data suggest that TGF-betas are not likely to be useful biomarkers of chemopreventive efficacy in a clinical setting.

Enhanced tumorigenesis and reduced transforming growth factor-beta type II receptor in lung tumors from mice with reduced gene dosage of transforming growth factor-beta1.

To elucidate the role of transforming growth factor-beta1 (TGF-beta1) and the TGF-beta type II receptor (TGF-beta RII) as tumor-suppressor genes in lung carcinogenesis, we mated C57BL/6 mice heterozygous (HT) for deletion of the TGF-beta1 gene with A/J mice to produce AJBL6 TGF-beta1 HT progeny and their wild-type (WT) littermates. Immunohistochemical staining, in situ hybridization, and northern blot analyses showed lower staining and hybridization for TGF-beta1 protein and mRNA, respectively, in the lungs of normal HT mice versus WT mice. Competitive reverse transcription-polymerase chain reaction (CRT-PCR) amplification showed the level of TGF-beta1 mRNA in the lungs of HT mice to be fourfold lower than the level in WT lung. When challenged with ethyl carbamate, lung adenomas were detected in 55% of HT mice by 4 mo but only in 25% of WT littermates at this time. Whereas all HT mice had adenomas by 6 mo, it was not until 10 mo before all WT mice had adenomas. After 12 mo, the average number of adenomas was fivefold higher in HT lungs than in WT lungs. Most dramatic was the appearance of lung carcinomas in HT mice 8 mo before they were visible in WT mice. Thus, the AJBL6 TGF-beta1 HT mouse provides an excellent model system to examine carcinogen-induced lung tumorigenesis by increasing progressive lesion incidence and multiplicity relative to their WT littermates. Immunohistochemical staining showed expression of the TGF-beta type I receptor (TGF-beta RI) at moderate to strong levels in lung adenomas and carcinomas in HT and WT mice. In contrast, whereas weak immunostaining for TGF-beta RII was detected in 67% of HT carcinomas at 12 mo, only 22% of WT carcinomas showed weak staining for this protein. Individual lung carcinomas showing reduced TGF-beta RII expression and adjacent normal bronchioles were excised from HT lungs using laser capture microdissection, and CRT-PCR amplification of the extracted RNA showed 12-fold less TGF-beta RII mRNA in these carcinomas compared with bronchioles. Decreasing TGF-beta RII mRNA levels occurred with increasing tumorigenesis in lung hyperplasias, adenomas, and carcinomas, with carcinomas having fourfold and sevenfold lower levels of TGF-beta RII mRNA than adenomas and hyperplasias, respectively. These data show enhanced ethyl carbamate-induced lung tumorigenesis in AJBL6 HT mice compared with WT mice, suggesting that both TGF-beta1 alleles are necessary for tumor-suppressor activity. Reduction of TGF-beta RII mRNA expression in progressive stages of lung tumorigenesis in HT mice suggests that loss of TGF-beta RII may play an important role in the promotion of lung carcinogenesis in mice with reduced TGF-beta1 gene dosage when challenged with carcinogen.

The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting.

NIH sponsored a meeting of medical and veterinary pathologists with mammary gland expertise in Annapolis in March 1999. Rapid development of mouse mammary models has accentuated the need for definitions of the mammary lesions in genetically engineered mice (GEM) and to assess their usefulness as models of human breast disease. The panel of nine pathologists independently reviewed material representing over 90% of the published systems. The GEM tumors were found to have: (1) phenotypes similar to those of non-GEM; (2) signature phenotypes specific to the transgene; and (3) some morphological similarities to the human disease. The current mouse mammary and human breast tumor classifications describe the majority of GEM lesions but unique morphologic lesions are found in many GEM. Since little information is available on the natural history of GEM lesions, a simple morphologic nomenclature is proposed that allows direct comparisons between models. Future progress requires rigorous application of guidelines covering pathologic examination of the mammary gland and the whole animal. Since the phenotype of the lesions is an essential component of their molecular pathology, funding agencies should adopt policies ensuring careful morphological evaluation of any funded research involving animal models. A pathologist should be part of each research team.

Transforming growth factor-beta 1 and its receptors in human lung cancer and mouse lung carcinogenesis.

The transforming growth factor-betas (TGF-beta s) are multifunctional proteins that inhibit the proliferation of many epithelial cells through a set of cell protein receptors that includes the TGF-beta type I (RI) and type II (RII) receptors. Loss of growth inhibition by TGF-beta is thought to contribute to the development of many types of tumors. In the present study, we have examined expression of the proteins and mRNAs for TGF-beta 1, TGF-beta RI, and TGF-beta RII in normal human lung, well-characterized non-small cell lung cancer (NSCLC) cell lines, and primary NSCLC specimens. Immunohistochemical staining for TGF-beta 1, TGF-beta RI, and TGF-beta RII using specific antibodies in normal human lung showed expression of the 3 proteins in the epithelium of bronchi and bronchioles as well as in alveoli. Differential expression of TGF-beta RI and TGF-beta RII proteins was detected in 5 NSCLC cell lines using Western blot analysis, with reduced levels in 3 cell lines. A panel of 45 formalin-fixed and paraffin-embedded NSCLC specimens showed positive immunostaining for TGF-beta 1, TGF-beta RI, and TGF-beta RII, with reduced TGF-beta RII in poorly differentiated adenocarcinomas and squamous cell carcinomas and some moderately differentiated adenocarcinomas. In situ hybridization studies conducted with specific riboprobes for TGF-beta 1, TGF-beta RI, and TGF-beta RII showed corresponding localization of expression of the mRNAs in the specimens that showed positive immunostaining for the proteins. To investigate the roles of TGF-beta 1, TGF-beta RI, and TGF-beta RII in chemically induced mouse lung tumorigenesis, we examined the expression of their proteins and mRNAs in 2 mouse model systems. Whereas expression of the proteins and mRNAs for TGF-beta 1 and TGF-beta RI was comparable in lung adenomas and bronchioles of A/J mice treated with benzo(alpha)pyrene, decreased immunostaining and hybridization for TGF-beta RII protein and mRNA was detected in 50% of lung adenomas in these mice. Interestingly, expression of TGF-beta 1 and the TGF-beta receptor proteins was similar to that of bronchioles in C57B1/6 mice and their littermates heterozygous for deletion of the TGF-beta 1 gene treated with diethylnitrosamine. These data show that reduced levels of expression of TGF-beta RII occur in some, but not all, human and mouse lung tumors. This suggests that different mechanisms of action, some of which may involve the TGF-beta signaling pathway, may contribute to the progression of lung tumorigenesis.

Transforming growth factor-beta and breast cancer: Lessons learned from genetically altered mouse models.

Transforming growth factor (TGF)-betas are plausible candidate tumor suppressors in the breast. They also have oncogenic activities under certain circumstances, however. Genetically altered mouse models provide powerful tools to analyze the complexities of TGF-beta action in the context of the whole animal. Overexpression of TGF-beta can suppress tumorigenesis in the mammary gland, raising the possibility that use of pharmacologic agents to enhance TGF-beta function locally might be an effective method for the chemoprevention of breast cancer. Conversely, loss of TGF-beta response increases spontaneous and induced tumorigenesis in the mammary gland. This confirms that endogenous TGF-betas have tumor suppressor activity in the mammary gland, and suggests that the loss of TGF-beta receptors seen in some human breast hyperplasias may play a causal role in tumor development.

Loss of responsiveness to transforming growth factor beta induces malignant transformation of nontumorigenic rat prostate epithelial cells.

Transforming growth factor (TGF)-betas are multifunctional growth factors, the properties of which include the potent inhibition of epithelial cell growth. Expression patterns of TGF-betas and TGF-beta receptors in the normal prostate indicate that these growth regulators play key roles in prostatic development and proliferative homeostasis. Importantly, TGF-beta receptor levels are frequently diminished in malignant human prostate tissue. To test the hypothesis that loss of TGF-beta responsiveness is causally involved in the tumorigenic process, we have used retroviral transduction to introduce a dominant-negative mutant type II TGF-beta receptor (DNR) into the premalignant rat prostatic epithelial cell line, NRP-152. High-level expression of the DNR abolished the ability of TGF-beta to inhibit cell growth, to promote cell differentiation, and to induce apoptosis, and it partially blocked the induction of extracellular matrix gene expression. When injected into nude mice, NRP-152-DNR cells formed carcinomas at 13 of 34 sites, compared with 0 of 30 sites for parental and control cells (P = 0.0001). We conclude that the type II TGF-beta receptor is an important tumor suppressor in the prostate, and furthermore, that loss of TGF-beta responsiveness can contribute early in the tumorigenic process by causing the malignant transformation of preneoplastic cells.

Identification of the start sites for the 1.9- and 1.4-kb rat transforming growth factor-beta1 transcripts and their effect on translational efficiency.

Three distinct transforming growth factor-beta1 (TGF-beta1) transcripts of 2.5, 1.9 and 1.4kb have been described, but the start sites and functional significance of the shorter transcripts are unknown. Here, we have cloned and sequenced a rat genomic fragment encoding approximately 1250 bases upstream of the start of the TGF-beta1 open reading frame. Using a combination of ribonuclease protection and 5' RACE-PCR analysis, we have mapped the start sites for the two shorter TGF-beta1 transcripts in NRP152 cells, a rat prostatic epithelial cell line that expresses all three transcripts at high levels. The 1.4-kb mRNA starts 25 bases upstream of the initiator AUG, whereas the 1.9-kb mRNA has two start sites 366 and 401 bases upstream of the AUG. Polysome analysis of the NRP152 cells indicates that the 1.9-kb transcript is very efficiently translated, whereas the 2.5- and 1.4-kb transcripts appear to be poorly translated. Differential regulation of TGF-beta1 transcript size may therefore represent an important mechanism for regulating TGF-beta1 protein levels.

Transforming growth factor-beta1 is a new form of tumor suppressor with true haploid insufficiency.

Components of the transforming growth factor-beta (TGF-beta) signal pathway function as classic tumor suppressors, but the role of the TGF-betas themselves is less clear. Here we show that mice heterozygous for deletion of the TGF-beta1 gene express only 10-30% of wild-type TGF-beta1 protein levels. Although grossly normal, these mice have a subtly altered proliferative phenotype, with increased cell turnover in the liver and lung. Treatment of these mice with chemical carcinogens resulted in enhanced tumorigenesis when compared with wild-type littermates. However, tumors in the heterozygous mice did not lose the remaining wild-type TGF-beta1 allele, indicating that the TGF-beta1 ligand is a new form of tumor suppressor that shows true haploid insufficiency in its ability to protect against tumorigenesis.

Translational control elements in the major human transforming growth factor-beta 1 mRNA.

Polysome analysis indicates that the major 2.4 kb transforming growth factor-beta 1 (TGF-beta 1) transcript is poorly translated, both in cultured cells, and in vivo in mouse liver. In contrast, the TGF-beta 2 transcripts are efficiently translated. The contribution of the 5'- and 3'-untranslated regions (UTRs) to the translational inhibition of the full-length TGF-beta 1 transcript was studied by deletion analysis. Despite their high G + C content, both UTRs stimulated translation in vitro. However, polysome analysis of synthetic TGF-beta 1 mRNAs transfected into MCF-7 cells suggests that the cell contains a limited pool of trans-acting factors that interact with the 5'UTR to make it inhibitory in vivo. Further deletion analysis in vitro revealed multiple stimulatory and inhibitory regions in the 5'UTR. This has important implications for the translatability of the naturally occurring shorter TGF-beta 1 transcripts and provides a framework for higher resolution mapping studies. Overall, the poor translational efficiency of the major TGF-beta 1 mRNA in vivo appears to be due to a combination of poor initiation sequence context, and inhibitory interactions of limiting transacting factors with cis-inhibitory elements embedded in an otherwise stimulatory 5'UTR.

Expression of a dominant-negative mutant TGF-beta type II receptor in transgenic mice reveals essential roles for TGF-beta in regulation of growth and differentiation in the exocrine pancreas.

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-beta (TGF-beta) signaling in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocked signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments.

Recharacterization of the start sites for the major human transforming growth factor-beta 1 mRNA.

The start sites for the major human TGF-beta 1 transcripts have been reexamined. A comparison of ribonuclease and S1 nuclease protection analyses on native TGF-beta 1 mRNA and in vitro transcribed human TGF-beta 1 transcripts of defined sizes places the most 5' start site for the native TGF-beta 1 message approx. 50 nucleotides upstream from the previously published start site at base +1. Furthermore, the same techniques indicate that the apparent downstream start site at base +271 is an artefact due to the presence of an A + T-rich island in the middle of an otherwise highly G + C-rich sequence. This is not apparent if S1 nuclease protection is used alone, which emphasizes the importance of using the two techniques in combination for this type of analysis. Thus the major 2.5 kb TGF-beta 1 band seen on Northern blots comprises only mRNA transcribed from the more upstream of the two previously characterized promoters. This has important implications both for the transcriptional and translational regulation of this growth factor.

Transgenic mice overexpressing a dominant-negative mutant type II transforming growth factor beta receptor show enhanced tumorigenesis in the mammary gland and lung in response to the carcinogen 7,12-dimethylbenz-[a]-anthracene.

To test the hypothesis that the transforming growth factor-beta (TGF-beta) system has tumor suppressor activity in the mammary gland, we have generated transgenic mice overexpressing a dominant-negative mutant form of the type II TGF-beta receptor, under the control of the mouse mammary tumor virus-long terminal repeat. High-level expression of the transgene was observed in the mammary and salivary glands, with lower expression in the lung, spleen, and testis. Older nulliparous transgenic mice (9-17 months) showed a marked increase in the incidence and degree of lobulo-alveolar side-branching in the mammary glands when compared to wild-type littermates (24.8% of glands examined histologically versus 14.4%; P = 0.004), suggesting a role for endogenous TGF-betas in regulating development or maintenance of mammary alveoli. Spontaneous tumorigenesis was unchanged in the transgenic mice. However, following initiation with the carcinogen 7,12-dimethylbenz[a]anthracene, the transgenic group showed a significant increase in the incidence and multiplicity of mammary tumors when compared with wild-type littermates (40% incidence in transgenic mice versus 22% for wild-type, with 4 of 25 transgenics developing multiple mammary tumors versus 0 of 27 wild-type; P = 0.03). An early increase in the incidence of lung tumors was also observed in transgenic mice, but no difference between genotype groups was seen in the incidence of tumors in tissues in which the transgene is not expressed. The data show that the endogenous TGF-beta system has tumor suppressor activity in the mammary gland and lung.

Synthesis and secretion of transforming growth factor beta isoforms by primary cultures of human breast tumour fibroblasts in vitro and their modulation by tamoxifen.

Tamoxifen may mediate its effect in early breast cancer in part via an oestrogen receptor (ER)-independent pathway by directly stimulating fibroblasts to produce the negative paracrine growth factor transforming growth factor (TGF)-beta. We have previously shown that secretion of this factor is induced 3-to 30-fold in human fetal fibroblasts in vitro, and by stromal fibroblasts in vivo following tamoxifen treatment of ER-positive and ER-negative breast cancer patients. Primary cultures of breast tumour fibroblasts have been exposed to tamoxifen for 48 h, and rates of secretion of TGF-beta 1 and TGF-beta 2 measured using a quantitative immunoassay. Fibroblast strains derived from malignant and benign tumours produced and secreted similar amounts of TGF-beta 1, but benign breast tumour fibroblasts secreted significantly higher levels of TGF-beta 2 compared with fibroblasts of malignant origin. Tamoxifen did not induce any consistent increase in TGF-beta secretion into the conditioned medium, but immunofluorescence analysis for the intracellular form of TGF-beta 1 revealed evidence of increased immunoreactive protein in tamoxifen-treated fibroblasts, which is localised to the nucleus. Therefore synthesis of TGF-beta 1 appears to be stimulated by tamoxifen, but increased secretion may be abrogated in vitro. Furthermore, using immunocytochemistry and transient transfection with an ER-responsive reporter construct, no ER was demonstrable in these fibroblasts supporting the proposed ER-independent paracrine pathway.

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.