Deletion of exon I of SMAD7 in mice results in altered B cell responses

The members of the TGF-beta superfamily, i.e., TGF-beta isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-beta superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-beta signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-beta signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-beta signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system's response to TGF-beta.     

Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects

Smad5 has been implicated as a downstream signal mediator for several bone morphogenetic proteins (BMPs). To understand the in vivo function of Smad5, we generated mice deficient in Smad5 using embryonic stem (ES) cell technology. Homozygous mutant embryos die between E9.5 and E11.5, and display variable phenotypes. Morphological defects are first detected at E8.0 in the developing amnion, gut and heart (the latter defect being similar to BMP-2 knockout mice). At later stages, mutant embryos fail to undergo proper turning, have craniofacial and neural tube abnormalities, and are edematous. In addition, several extraembryonic lesions are observed. After E9.0, the yolk sacs of the mutants contain red blood cells but lack a well-organized vasculature, which is reminiscent of BMP-4, TGF-beta1 and TGF-beta type II receptor knockout mice. In addition, the allantois of many Smad5 mutants is fused to the chorion, but is not well-elongated. A unique feature of the Smad5 mutant embryos is that ectopic vasculogenesis and hematopoiesis is observed in the amnion, likely due to mislocation of allantois tissue. Despite the expression of Smad5 from gastrulation onwards, and in contrast to knockouts of Smad2 and Smad4, Smad5 only becomes essential later in extraembryonic and embryonic development.     

A 4.3 kb Smad7 promoter is able to specify gene expression during mouse development

Members of transforming growth factor-beta (TGF-beta) superfamily play important roles in diverse biological functions including early development. These extracellular factors exert their effects by interacting with membrane receptors followed by signal transduction by a group of Smad proteins. Smad7 is an inhibitory Smad protein that specifically antagonizes TGF-beta and activin signaling. To characterize the developmental role of Smad7, a transgenic mouse model was generated using a 4.3 kb mouse Smad7 promoter driving beta-galactosidase expression. In these mice, the Smad7 promoter defined a restrictive expression pattern of beta-galactosidase in a tightly regulated temporal and spatial manner. The beta-galactosidase gene was transiently expressed in the cardiovascular structures including heart cushion tissues and the endothelium of major arteries at E11.5 to E12.5. Through E12.5 to E17.5, beta-galactosidase expression was prominently detected in the epithelium of developing cochlea and nasolacrimal duct. In addition, it was temporally expressed in trigeminal ganglion, the skeletal muscles surrounding major joints, primordium of the jaws, as well as genital tubercle. These studies indicated that the 4.3 kb Smad7 promoter contains sufficient regulatory elements to define controlled gene expression during mouse development.     

Smad7-deficient mice show growth retardation with reduced viability

Smad7 is an inhibitory molecule induced by members of the transforming growth factor-β (TGF-β) family, including TGF-β, activin, nodal and bone morphogenetic proteins (BMPs). To elucidate the in vivo functions of Smad7, we generated conditional Smad7-knockout mice in which the Mad homology 2 (MH2) domain and the poly (A) signal sequence were flanked with loxP sites (floxed). The Smad7-floxed mice exhibited no obvious phenotype. Smad7 total-null mice on a C57BL/6 background died within a few days of birth, whereas mice with an ICR background developed to adulthood but were significantly smaller than wild-type mice. Unexpectedly, phospho-Smad2 and phospho-Smad3 were decreased in Smad7-deficient mouse embryonic fibroblast (MEF) cells, whereas phospho-Smad1/5/8 was similarly expressed in wild-type and Smad7-deficient MEF cells. Moreover, expression levels of TGF-β type I receptor (ALK5) were higher in Smad7-deficient MEF cells than in wild-type MEF cells. Plasminogen activator inhibitor-1 (PAI-1) and inhibitor of differentiation-1 (Id-1) mRNA were similarly expressed in wild-type and Smad7-deficient MEF cells. Some differences were observed in mitogen-activated protein kinase (MAPK)-signalling between wild-type and Smad7-deficient MEF cells. We demonstrated that Smad7 plays an important role in normal mouse growth and provide a useful tool for analysing Smad7 functions in vivo.     

Smad5 is essential for left-right asymmetry in mice

Left-right (L-R) asymmetry of the vertebrate body plan is established from an originally morphologically symmetric embryo. Recent studies have implicated several TGF-beta family signaling proteins (i.e., nodal, lefty-1, lefty-2, activin receptor type IIB, and Smad2) in L-R axis determination in the mouse. However, the genetic pathways underlying L-R patterning are still unclear. Smad5 is a downstream component in the TGF-beta family signaling cascade, and lack of Smad5 results in embryonic lethality between E9.5 and E11.5. In this report, we demonstrate that Smad5 mutant embryos have defects in heart looping and embryonic turning which are the first signs of L-R asymmetry in mice. To gain more insights into the molecular basis of the laterality defects in the Smad5-deficient embryos, we examined the expression of lefty-1, lefty-2, nodal, and Pitx2 since the asymmetric expression of these genes always closely correlates with the direction of heart looping and embryonic turning. In the absence of Smad5, lefty-1 was expressed at very low or undetectable levels, while nodal, lefty-2, and Pitx2 were expressed bilaterally. These data suggest that Smad5 is upstream of lefty-1, nodal, and lefty-2, and as a consequence also of Pitx2, and Smad5 is essential for L-R axis determination.     

Transforming growth factor beta 1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.     

Generation of a floxed allele of Smad5 for cre-mediated conditional knockout in the mouse

Smad5 is a member of the Smad family of intracellular mediators of BMP signals and in endothelial cells of TGF-beta signals. We and others previously showed that loss of Smad5 in the mouse results in embryonic lethality (between E9.5-E11.5) due to multiple embryonic and extraembryonic defects. To circumvent the early embryonic lethality and to allow tissue- and time-specific Smad5 inactivation, we created a conditional Smad5 allele in the mouse. Floxed Smad5 (Smad5(flE2,Neo/flE2,Neo)) mice were generated in which both exon2 and the Neo-cassette were flanked by loxP sites. Here we demonstrate that embryos with ubiquitous Cre-mediated deletion of Smad5 (Smad5(flDeltaE2/flDeltaE2)) phenocopy the conventional Smad5 knockout mice. Smad5(flE2/flE2) mice are now available and will be a valuable tool to analyze the role of Smad5 beyond its crucial early embryonic function throughout development and postnatal life.     

Overexpression of Smad2 reveals its concerted action with Smad4 in regulating TGF-beta-mediated epidermal homeostasis.

Members of the transforming growth factor-beta (TGF-beta) superfamily are critical regulators for epithelial growth and can alter the differentiation of keratinocytes. Transduction of TGF-beta signaling depends on the phosphorylation and activation of Smad proteins by heteromeric complexes of ligand-specific type I and II receptors. To understand the function of TGF-beta and activin-specific Smad, we generated transgenic mice that overexpress Smad2 in epidermis under the control of keratin 14 promoter. Overexpression of Smad2 increases endogenous Smad4 and TGF-beta 1 expression while heterozygous loss of Smad2 reduces their expression levels, suggesting a concerted action of Smad2 and -4 in regulating TGF-beta signaling during skin development. These transgenic mice have delayed hair growth, underdeveloped ears, and shorter tails. In their skin, there is severe thickening of the epidermis with disorganized epidermal architecture, indistinguishable basement membrane, and dermal fibrosis. These abnormal phenotypes are due to increased proliferation of the basal epidermal cells and abnormalities in the program of keratinocyte differentiation. The ectodermally derived enamel structure is also abnormal. Collectively, our study presents the first in vivo evidence that, by providing an auto-feedback in TGF-beta signaling, Smad2 plays a pivotal role in regulating TGF-beta-mediated epidermal homeostasis.     

Dual role for TGF-beta1 in apoptosis

The exposure of cells to TGF-beta1 can trigger a variety of cellular responses including the inhibition of cell growth, migration, differentiation and apoptosis. TGF-beta1-regulated apoptosis is cell type and context-dependent, indeed TGF-beta1 provides signals for both cell survival or apoptosis. The molecular mechanisms underlying the role of TGF-beta1 in apoptosis remains unclear. The proteins that primarily mediate the intracellular signaling of TGF-beta1 are the members of the Smad family. Nevertheless, TGF-beta1 signaling can also cooperate with the death receptor apoptotic pathway (Fas, TNF), with the intracellular modulators of apoptosis JNK and p38 MAP kinases, Akt, NF-kappaB, and with the mitochondrial apoptotic pathway mediated by members of the Bcl-2 family. Moreover, the involvement of TGF-beta1 in the production of oxidative stress and in preventing the inflammatory processes required for the clearance of apoptotic bodies is further evidence of its integration into apoptotic pathways. The interaction and balance between different stimuli provides the basis for the pro- or anti-apoptotic output of TGF-beta1 signaling in a given cell.