Angelina Felici

Smad3: A Key Player in Pathogenetic Mechanisms Dependent on TGF‐β

Abstract: Transforming growth factor‐β (TGF‐β), a key player in a large variety of physiological and disease processes, signals through transmembrane receptor serine/threonine kinases to activate novel signaling intermediates called Smad proteins, which then modulate transcription of target genes. We have utilized mice with a targeted deletion of Smad3, one of two homologous proteins involved in signaling from TGF‐β/activin, to investigate the function of this particular pathway in transducing such effects of TGF‐β. The dramatic results of the absence of Smad3 on parameters of healing of cutaneous wounds, such as reepithelialization and influx of inflammatory cells, as well as on fibrosis as modeled by radiation fibrosis of skin in mice, suggest that signaling flux through Smad3 is critical for chemotactic activity of TGF‐β, inhibitory effects of TGF‐β on keratinocyte proliferation and migration, and chemoattraction and elaboration of extracellular matrix by fibroblasts in fibrotic diseases. We recently identified a novel molecule, TLP for TRAP‐1‐like protein, which selectively interferes with Smad3 signaling, and are currently investigating whether levels of this protein might be altered in disease to change the relative flow of information from Smad2 and Smad3.

VE‐cadherin is a critical endothelial regulator of TGF‐β signalling

VE‐cadherin is an endothelial‐specific transmembrane protein concentrated at cell‐to‐cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE‐cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE‐cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE‐cadherin expression and junctional clustering are required for optimal transforming growth factor‐β (TGF‐β) signalling in endothelial cells (ECs). TGF‐β antiproliferative and antimigratory responses are increased in the presence of VE‐cadherin. ECs lacking VE‐cadherin are less responsive to TGF‐β/ALK1‐ and TGF‐β/ALK5‐induced Smad phosphorylation and target gene transcription. VE‐cadherin coimmunoprecipitates with all the components of the TGF‐β receptor complex, TβRII, ALK1, ALK5 and endoglin. Clustered VE‐cadherin recruits TβRII and may promote TGF‐β signalling by enhancing TβRII/TβRI assembly into an active receptor complex. Taken together, our data indicate that VE‐cadherin is a positive and EC‐specific regulator of TGF‐β signalling. This suggests that reduction or inactivation of VE‐cadherin may contribute to progression of diseases where TGF‐β signalling is impaired.

Jab1/CSN5, a Component of the COP9 Signalosome, Regulates Transforming Growth Factor β Signaling by Binding to Smad7 and Promoting Its Degradation

ABSTRACT Smad7 inhibits responses mediated by transforming growth factor β (TGF-β) and acts in a negative-feedback loop to regulate the intensity or duration of the TGF-β signal. However, the aberrant expression and continued presence of Smad7 may cause TGF-β resistance. Here we report that Jab1/CSN5, which is a component of the COP9 signalosome complex, associates constitutively with Smad7 and that overexpression of Jab1/CSN5 causes the translocation of Smad7 from the nucleus to the cytoplasm, promoting its degradation. Overexpression of Jab1/CSN5 increases Smad2 phosphorylation and enhances TGF-β-induced transcriptional activity. The inhibition of endogenous Jab1/CSN5 expression by small interfering RNA (siRNA) induces Smad7 expression. This study thus defines Jab1/CSN5 as an adapter that targets Smad7 for degradation, thus releasing Smad7-mediated suppression of TGF-β signaling.

TLP, a novel modulator of TGF-β signaling, has opposite effects on Smad2- and Smad3-dependent signaling

Transforming growth factor‐β (TGF‐β) is a multifunctional cytokine signaling to the nucleus through cell surface transmembrane receptor serine/threonine kinases and cytoplasmic effectors, including Smad proteins. We describe a novel modulator of this pathway, TLP (TRAP‐1‐like protein), which is 25% identical to the previously described Smad4 chaperone, TRAP‐1, and shows identical expression patterns in human tissues. Endogenous TLP associates with both active and kinase‐deficient TGF‐β and activin type II receptors, but interacts with the common‐mediator Smad4 only in the presence of TGF‐β/activin signaling. Overexpression of TLP represses the ability of TGF‐β to induce transcription from SBE‐Luc, a Smad3/4‐specific reporter, while it potentiates transcription from ARE‐Luc, a Smad2/4‐specific reporter. Consistent with this, TLP inhibits the formation of Smad3/4 complexes in the absence of effects on phosphorylation of Smad3, while it affects neither Smad2 phosphorylation nor hetero‐oligomerization. We propose that TLP might regulate the balance of Smad2 and Smad3 signaling by localizing Smad4 intracellularly, thus contributing to cellular specificity of TGF‐β transcriptional responses in both normal and pathophysiology.

The TGF-β signaling modulators TRAP1/TGFBRAP1 and VPS39/Vam6/TLP are essential for early embryonic development.

The pleiotropic cytokine transforming growth factor-β (TGF-β) signals through different pathways among which the Smad- and the MAP-Kinase pathways are already well characterized. Both pathways utilize adaptor/chaperone molecules that facilitate or modulate the intracellular signaling events. Two of the proteins shown in vitro to play a role in Smad-dependent signaling are the TGF-β Receptor Associated Protein-1 (TRAP1, also TGFBRAP1) and its homologue VPS39, also known as Vam6 and TRAP1-Like-Protein (TLP). We generated mice deficient for TRAP1 and VPS39/TLP, respectively. Absence of TRAP1 protein results in death at either of two defined timepoints during embryogenesis, before the blastula stage or during gastrulation, whereas most of the VPS39 deficient mice die before E6.5. Heterozygous mice show no overt phenotype. In summary, our data indicate that TRAP1 and VPS39 are nonredundant and essentially required for early embryonic development.

Gab1 mediates hepatocyte growth factor-stimulated mitogenicity and morphogenesis in multipotent myeloid cells

Hepatocyte growth factor (HGF)‐stimulated mitogenesis, motogenesis and morphogenesis in various cell types begins with activation of the Met receptor tyrosine kinase and the recruitment of intracellular adaptors and kinase substrates. The adapter protein Gab1 is a critical effector and substrate of activated Met, mediating morphogenesis, among other activities, in epithelial cells. To define its role downstream of Met in hematopoietic cells, Gab1 was expressed in the HGF‐responsive, Gab1‐negative murine myeloid cell line 32D. Interestingly, the adhesion and motility of Gab1‐expressing cells were significantly greater than parental cells, independent of growth factor treatment. Downstream of activated Met, Gab1 expression was specifically associated with rapid Shp‐2 recruitment and activation, increased mitogenic potency, suppression of GATA‐1 expression and concomitant upregulation of GATA‐2 transcription. In addition to enhanced proliferation, continuous culture of Gab1‐expressing 32D cells in HGF resulted in cell attachment, filopodia extension and phenotypic changes suggestive of monocytic differentiation. Our results suggest that in myeloid cells, Gab1 is likely to enhance HGF mitogenicity by coupling Met to Shp‐2 and GATA‐2 expression, thereby potentially contributing to normal myeloid differentiation as well as oncogenic transformation. J. Cell. Biochem. 111: 310–321, 2010.

Hepatocyte Growth Factor Regulates Transitions between Epithelial and Mesenchymal Cellular Phenotypes during Normal Development and in Disease

Transitions between epithelial and mesenchymal cell phenotypes occur as part of normal organ development and wound healing, and are also observed in cancer, fibrosis and other diseases. Although the latter processes involve dysregulated interconversion of epithelial and mesenchymal cell phenotypes, the molecular mechanisms and sequences of cellular changes often closely resemble events that occur as a part of normal development and tissue repair. While primarily epithelial to mesenchymal transition (EMT) has been implicated in cancer and fibrosis, both EMT and mesenchymal to epithelial transition (MET) occur during organogenesis and development.