Smad7 and Smad6 differentially modulate transforming growth factor beta -induced inhibition of embryonic lung morphogenesis

Transforming growth factors beta (TGF-beta) are known negative regulators of lung development, and excessive TGF-beta production has been noted in pulmonary hypoplasia associated with lung fibrosis. Inhibitory Smad7 was recently identified to antagonize TGF-beta family signaling by interfering with the activation of TGF-beta signal-transducing Smad complexes. To investigate whether Smad7 can regulate TGF-beta-induced inhibition of lung morphogenesis, ectopic overexpression of Smad7 was introduced into embryonic mouse lungs in culture using a recombinant adenovirus containing Smad7 cDNA. Although exogenous TGF-beta efficiently reduced epithelial lung branching morphogenesis in control virus-infected lung culture, TGF-beta-induced branching inhibition was abolished after epithelial transfer of the Smad7 gene into lungs in culture. Smad7 also prevented TGF-beta-mediated down-regulation of surfactant protein C gene expression, a marker of bronchial epithelial differentiation, in cultured embryonic lungs. Moreover, we found that Smad7 transgene expression blocked Smad2 phosphorylation induced by exogenous TGF-beta ligand in lung culture, indicating that Smad7 exerts its inhibitory effect on both lung growth and epithelial cell differentiation through modulation of TGF-beta pathway-restricted Smad activity. However, the above anti-TGF-beta signal transduction effects were not observed in cultured embryonic lungs with Smad6 adenoviral gene transfer, suggesting that Smad7 and Smad6 differentially regulate TGF-beta signaling in developing lungs. Our data therefore provide direct evidence that Smad7, but not Smad6, prevents TGF-beta-mediated inhibition of both lung branching morphogenesis and cytodifferentiation, establishing the mechanistic basis for Smad7 as a novel target to ameliorate aberrant TGF-beta signaling during lung development, injury, and repair.     

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

Recently, significant attention has been drawn to the biology of small leucine-rich repeat proteoglycans (SLRPs) due to their multiple functionalities in various cell types and tissues. Here, we characterize a novel SLRP member, “Podocan-like (Podnl) protein” identified by a bioinformatics approach. The Podnl protein has a signal peptide, a unique cysteine-rich N-terminal cluster, 21 leucine-rich repeat (LRR) motifs, and one putative N-glycosylation site. This protein is structurally similar to podocan in SLRPs. The gene was highly expressed in mineralized tissues and in osteoblastic cells and the high expression level was observed at and after matrix mineralization in vitro. Podnl was enriched in newly formed bones based on immunohistochemical analysis. When Podnl was transfected into osteoblastic cells, the protein with N-glycosylation was detected mainly in the cultured medium, indicating that Podnl is a secreted N-glycosylated protein. The endogenous Podnl protein was also present in bone matrix. These data provide a new insight into our understanding of the emerging SLRP functions in bone formation.     

Expression pattern and gene characterization of asporin. a newly discovered member of the leucine-rich repeat protein family

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2, osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.     

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury.     

A novel mouse Smad4 mutation reduces protein stability and wild-type protein levels

Smad4 is a key signal transducer of the transforming growth factor-beta (TGF-beta) superfamily of growth factors that are critical regulators of embryonic patterning and adult tissue homeostasis. The biological activity of the TGF-beta signaling is tightly controlled at multiple levels, including the abundance of SMAD4 proteins. We previously recovered a novel allele of Smad4 in a gene-based screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mouse embryonic stem cells. The mutation resulted in an unstable truncated protein that is degraded through proteasomal pathways. In the heterozygous state, this allele acts in a dominant negative fashion to reduce the wild-type protein level as well as signaling output. Biochemical characterization indicated that the truncated protein is able to form a complex with the wild-type protein, thus targeting it for proteasomal degradation as well. Phenotypic analyses of the heterozygous animals provided insight into the threshold requirement of Smad4-dependent signaling in vivo.     

Smad7 is required for the development and function of the heart

Transforming growth factor-beta (TGF-beta) family members, including TGF-betas, activins, and bone morphogenetic proteins, exert diverse biological activities in cell proliferation, differentiation, apoptosis, embryonic development, and many other processes. These effects are largely mediated by Smad proteins. Smad7 is a negative regulator for the signaling of TGF-beta family members. Dysregulation of Smad7 is associated with pathogenesis of a variety of human diseases. However, the in vivo physiological roles of Smad7 have not been elucidated due to the lack of a mouse model with significant loss of Smad7 function. Here we report generation and initial characterization of Smad7 mutant mice with targeted deletion of the indispensable MH2 domain. The majority of Smad7 mutant mice died in utero due to multiple defects in cardiovascular development, including ventricular septal defect and non-compaction, as well as outflow tract malformation. The surviving adult Smad7 mutant mice had impaired cardiac functions and severe arrhythmia. Further analyses suggest that Smad2/3 phosphorylation was elevated in atrioventricular cushion in the heart of Smad7 mutant mice, accompanied by increased apoptosis in this region. Taken together, these observations pinpoint an important role of Smad7 in the development and function of the mouse heart in vivo.     

TGF-beta superfamily signaling is essential for tooth and hair morphogenesis and differentiation

Members of the transforming growth factor beta (TGF-beta) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-beta family, notably TGF-beta and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-beta superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-beta blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-beta superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues.     

Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA

Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor beta (TGF-beta) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-beta. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.     

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.