Targeted mutations of transforming growth factor-beta genes reveal important roles in mouse development and adult homeostasis.

Transforming growth factors-beta (TGF-beta) are multifunctional molecules with profound biological effects in many developmental processes including regulation of cell proliferation, differentiation, cell adhesion, skeletal development, haematopoiesis, inflammatory responses, and wound healing. To learn about the role of TGF-beta in vivo, phenotypes of targeted mutations of molecules within the TGF-beta signalling pathway, TGF-beta1, -beta2, -beta3, TGF-beta receptor (TbetaR-II) and the signalling molecules SMAD2, SMAD3 and SMAD4, are discussed in this review. The three individual TGF-beta mutants show distinct and only partially overlapping phenotypes. In mice, targeted disruption of the TGF-beta1 gene results in diffuse and lethal inflammation about 3 weeks after birth, suggesting a prominent role of TGF-beta in the regulation of immune cell proliferation and extravasation into tissues. However, just half of the TGF-beta1 (-/-) conceptuses actually reach partuition due to defective haematopoiesis and endothelial differentiation. Targeted disruption of both TGF-beta2 and TGF-beta3 genes results in perinatal lethality. TGF-beta2 null mice exhibit a broad range of developmental defects, including cardiac, lung, craniofacial, limb, eye, ear and urogenital defects, whereas TGF-beta3 gene ablation results exclusively in defective palatogenesis and delayed pulmonary development. The TbetaR-II null phenotype closely resembles that of TGF-beta1 (-/-) conceptuses, which die in utero by E10.5. Loss of SMAD2 or SMAD4 results in related phenotypes: the mutants fail to form an organized egg cylinder, lack mesoderm required for gastrulation and die prior to E8.5. Together, gene ablation within the TGF-beta signalling pathway supports the notion of a prominent role of TGF-beta during development.     

Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response

The generation of animals lacking SMAD proteins, which transduce signals from transforming growth factor-beta (TGF-beta), has made it possible to explore the contribution of the SMAD proteins to TGF-beta activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-beta to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by an increased rate of re-epithelialization and significantly reduced local infiltration of monocytes. Smad3ex8/ex8 keratinocytes show altered patterns of growth and migration, and Smad3ex8/ex8 monocytes exhibit a selectively blunted chemotactic response to TGF-beta. These data are, to our knowledge, the first to implicate Smad3 in specific pathways of tissue repair and in the modulation of keratinocyte and monocyte function in vivo.     

Dual roles of immunoregulatory cytokine TGF-beta in the pathogenesis of autoimmunity-mediated organ damage

Ample evidence suggests a role of TGF-beta in preventing autoimmunity. Multiorgan inflammatory disease, spontaneous activation of self-reactive T cells, and autoantibody production are hallmarks of autoimmune diseases, such as lupus. These features are reminiscent of the immunopathology manifest in TGF-beta1-deficient mice. In this study, we show that lupus-prone (New Zealand Black and White)F(1) mice have reduced expression of TGF-beta1 in lymphoid tissues, and TGF-beta1 or TGF-beta1-producing T cells suppress autoantibody production. In contrast, the expression of TGF-beta1 protein and mRNA and TGF-beta signaling proteins (TGF-beta receptor type II and phosphorylated SMAD3) increases in the target organs, i.e., kidneys, of these mice as they age and develop progressive organ damage. In fact, the levels of TGF-beta1 in kidney tissue and urine correlate with the extent of chronic lesions that represent local tissue fibrosis. In vivo TGF-beta blockade by treatment of these mice with an anti-TGF-beta Ab selectively inhibits chronic fibrotic lesions without affecting autoantibody production and the inflammatory component of tissue injury. Thus, TGF-beta plays a dual, seemingly paradoxical, role in the development of organ damage in multiorgan autoimmune diseases. According to our working model, reduced TGF-beta in immune cells predisposes to immune dysregulation and autoantibody production, which causes tissue inflammation that triggers the production of anti-inflammatory cytokines such as TGF-beta in target organs to counter inflammation. Enhanced TGF-beta in target organs, in turn, can lead to dysregulated tissue repair, progressive fibrogenesis, and eventual end-organ damage.     

TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.     

Transforming growth factor-beta stimulates cyclin D1 expression through activation of beta-catenin signaling in chondrocytes

Transforming growth factor-beta (TGF-beta) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-beta rapidly induced beta-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-beta-increased beta-catenin induction was reproduced by overexpression of SMAD3 and was absent in Smad3(-/-) chondrocytes treated with TGF-beta. SMAD3 inhibited beta-transducin repeat-containing protein-mediated degradation of beta-catenin and immunoprecipitated with beta-catenin following TGF-beta treatment. Both SMAD3 and beta-catenin co-localized to the nucleus after TGF-beta treatment. Although both TGF-beta and beta-catenin stimulated cyclin D(1) expression in chondrocytes, the effect of TGF-beta was inhibited with beta-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-beta stimulates cyclin D(1) expression at least in part through activation of beta-catenin signaling.     

Human papillomavirus type 5 E6 oncoprotein represses the transforming growth factor beta signaling pathway by binding to SMAD3

Mechanisms of cellular transformation associated with human papillomavirus type 5 (HPV5), which is responsible for skin carcinomas in epidermodysplasia verruciformis (EV) patients, are poorly understood. Using a yeast two-hybrid screening and molecular and cellular biology experiments, we found that HPV5 oncoprotein E6 interacts with SMAD3, a key component in the transforming growth factor beta1 (TGF-beta1) signaling pathway. HPV5 E6 inhibits SMAD3 transactivation by destabilizing the SMAD3/SMAD4 complex and inducing the degradation of both proteins. Interestingly, the E6 protein of nononcogenic EV HPV9 failed to interact with SMAD3, suggesting that downregulation of the TGF-beta1 signaling pathway could be a determinant in HPV5 skin carcinogenesis.     

Distinct oligomeric states of SMAD proteins in the transforming growth factor-beta pathway

Protein interactions are critical for the function of SMADs as mediators of transforming growth factor-beta (TGF-beta) signals. TGF-beta receptor phosphorylation of SMAD2 or SMAD3 causes their association with SMAD4 and accumulation in the nucleus where the SMAD complex binds cofactors that determine the choice of target genes. We provide evidence that in the basal state, SMADs 2, 3, and 4 form separate, strikingly different complexes. SMAD2 is found mostly as monomer, whereas the closely related SMAD3 exists in multiple oligomeric states. This difference is due to a unique structural element in the MH1 domain of SMAD2 that inhibits protein-protein interactions in the basal state. In contrast to SMAD2 and SMAD3, SMAD4 in the basal state is found mostly as a homo-oligomer, most likely a trimer. Upon cell stimulation with TGF-beta, SMAD proteins become engaged in a multitude of complexes ranging in size from SMAD2-SMAD4 heterodimers to assemblies of >650 kDa. The latter display the highest DNA binding affinity for the TGF-beta-response elements of JUNB and collagen 7. These observations, all validated with endogenous SMAD proteins, modify previous models regarding the assembly and activity of SMAD complexes in the TGF-beta pathway.     

TGF-beta 1 regulates antigen-specific CD4+ T cell responses in the periphery

T cell expansion typically is due to cognate interactions with specific Ag, although T cells can be experimentally activated through bystander mechanisms not involving specific Ag. TGF-beta1 knockout mice exhibit a striking expansion of CD4+ T cells in the liver by 11 days of age, accompanied by CD4+T cell-dependent necroinflammatory liver disease. To examine whether hepatic CD4+T cell expansion in TGF-beta1(-/-) mice is due to cognate TCR-peptide interactions, we used spectratype analysis to examine the diversity in TCR Vbeta repertoires in peripheral CD4+T cells. We reasoned that Ag-nonspecific T cell responses would yield spectratype profiles similar to those derived from control polyclonal T cell populations, whereas Ag-specific T cell responses would yield perturbed spectratype profiles. Spleen and liver CD4+T cells from 11-day-old TGF-beta1(-/-) mice characteristically exhibited highly perturbed nonpolyclonal distributions of TCR Vbeta CDR3 lengths, indicative of Ag-driven T cell responses. We quantitatively assessed spectratype perturbation to derive a spectratype complexity score. Spectratype complexity scores were considerably higher for TGF-beta1(-/-) CD4+ T cells than for TGF-beta1(+/-) CD4+T cells. TCR repertoire perturbations were apparent as early as postnatal day 3 and preceded both hepatic T cell expansion and liver damage. By contrast, TGF-beta1(-/-) CD4+ single-positive thymocytes from 11-day-old mice exhibited normal unbiased spectratype profiles. These results indicate that CD4+ T cells in TGF-beta1(-/-) mice are activated by and respond to self-Ags present in the periphery, and define a key role for TGF-beta1 in the peripheral regulation of Ag-specific CD4+ T cell responses.     

Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.     

Defective T-cell activation is associated with augmented transforming growth factor Beta sensitivity in mice with mutations in the Sno gene

The proto-oncogene Sno has been shown to be a negative regulator of transforming growth factor beta (TGF-beta) signaling in vitro, using overexpression and artificial reporter systems. To examine Sno function in vivo, we made two targeted deletions at the Sno locus: a 5' deletion, with reduced Sno protein (hypomorph), and an exon 1 deletion removing half the protein coding sequence, in which Sno protein is undetectable in homozygotes (null). Homozygous Sno hypomorph and null mutant mice are viable without gross developmental defects. We found that Sno mRNA is constitutively expressed in normal thymocytes and splenic T cells, with increased expression 1 h following T-cell receptor ligation. Although thymocyte and splenic T-cell populations appeared normal in mutant mice, T-cell proliferation in response to activating stimuli was defective in both mutant strains. This defect could be reversed by incubation with either anti-TGF-beta antibodies or exogenous interleukin-2 (IL-2). Together, these findings suggest that Sno-dependent suppression of TGF-beta signaling is required for upregulation of growth factor production and normal T-cell proliferation following receptor ligation. Indeed, both IL-2 and IL-4 levels are reduced in response to anti-CD3 epsilon stimulation of mutant T cells, and transfected Sno activated an IL-2 reporter system in non-T cells. Mutant mouse embryo fibroblasts also exhibited a reduced cell proliferation rate that could be reversed by administration of anti-TGF-beta. Our data provide strong evidence that Sno is a significant negative regulator of antiproliferative TGF-beta signaling in both T cells and other cell types in vivo.     

The interrelated roles of TGF-beta and IL-10 in the regulation of experimental colitis.

In the present study, we define the relation between TGF-beta and IL-10 in the regulation of the Th1-mediated inflammation occurring in trinitrobenzene sulfonic acid (TNBS)-colitis. In initial studies, we showed that the feeding of trinitrophenol-haptenated colonic protein to SJL/J mice induces CD4(+) regulatory T cells that transfer protection from induction of TNBS-colitis, and that such protection correlates with cells producing TGF-beta, not IL-10. Further studies in which SJL/J mice were fed haptenated colonic protein, and then administered either anti-TGF-beta or anti-IL-10 at the time of subsequent TNBS administration per rectum, showed that while both Abs abolished protection, anti-TGF-beta administration prevented TGF-beta secretion, but left IL-10 secretion intact; whereas anti-IL-10 administration prevented both TGF-beta secretion and IL-10 secretion. Thus, it appeared that the protective effect of IL-10 was an indirect consequence of its effect on TGF-beta secretion. To establish this point further, we conducted adoptive transfer studies and showed that anti-IL-10 administration had no effect on induction of TGF-beta producing T cells in donor mice. However, it did inhibit their subsequent expansion in recipient mice, probably by regulating the magnitude of the Th1 T cell response which would otherwise inhibit the TGF-beta response. Therefore, these studies suggest that TGF-beta production is a primary mechanism of counter-regulation of Th1 T cell-mediated mucosal inflammation, and that IL-10 is necessary as a secondary factor that facilitates TGF-beta production.