Intracellular signaling: Fleshing out the TGFbeta pathway.

Recent studies of the intracellular signaling pathway initiated by ligands of the transforming growth factor beta (TGFbeta) family have provided new insights into how the receptors for such ligands phosphorylate their substrates - the Smads - and how signaling specificity is achieved.     

Inner nuclear membrane and regulation of Smad-mediated signaling

Smads mediate signal transduction by cytokines of the transforming growth factor-beta family. Recent data show that intrinsic and extrinsic proteins of the inner nuclear membrane affect the activities of Smads. MAN1, an integral protein of the inner nuclear membrane, binds to receptor-regulated Smads and antagonizes signaling by transforming growth factor-beta, activin and bone morphogenic protein. Lamins A and C, extrinsic intermediate filament proteins of the inner nuclear membrane that are mutated in several human diseases, appear to regulate phosphorylation of Smads. These data demonstrate that proteins within and associated with the inner nuclear membrane lipid bilayer regulate signal transduction pathways involved in numerous developmental, physiological and pathophysiological processes.     

TGFbeta signaling at the summit

Ligands belonging to the transforming growth factor (TGF) beta superfamily have emerged as major regulators of a wide variety of developmental events, ranging from the earliest steps in germ layer patterning of the pre-gastrula embryo to tissue healing, regeneration and homeostasis in the adult. Recently, Caroline Hill and Bob Lechleider organized the third in a bi-annual series of FASEB meetings on TGFbeta signaling and development at Snowmass (CO, USA). This meeting highlighted the ongoing interplay between advances in our understanding of the molecular biology of TGFbeta family signaling and in investigations into its roles in specific developmental events.     

Inhibition of TGFbeta signaling in cancer therapy

Recent evidence continues to support a central role for TGFbeta in tumor maintenance and progression. Although this may involve TGFbeta-mediated paracrine effects that modulate the tumor microenvironment and the host immune system, some studies causally implicate autocrine TGFbeta in cancer cell motility and survival. Other recent evidence indicates synergy between oncogene and TGFbeta signaling in epithelial cell transformation. This suggests opportunities for dissecting molecular mechanisms of cross-talk as well as providing insights into possible combinatorial molecular anticancer therapies that will include TGFbeta inhibitors.     

Elevated TGFbeta signaling inhibits ocular vascular development

Alterations in the ocular vasculature are associated with retinal diseases such as retinopathy of prematurity and diabetic retinopathy. Vascular endothelial growth factor (VEGF) as a potent stimulator for normal and abnormal vascular growth has been extensively studied. However, little is known about secreted factors that negatively regulate vascular growth in ocular tissues. We now report that expression of a self-activating TGFbeta1 in the ocular lens of transgenic mice results in inhibition of retinal angiogenesis followed by retinal degeneration. Transgenic TGFbeta1 can rescue the hyperplasic hyaloid tissue and reverse the corneal deficiency in TGFbeta2-null embryos. These results demonstrate that TGFbeta signaling modulates development of ocular vasculature and cornea in a dosage-dependent manner and that TGFbeta1 can substitute for TGFbeta2 in ocular tissues.     

Intracellular signaling dynamics during apoptosis execution in the presence or absence of X-linked-inhibitor-of-apoptosis-protein

X-linked-inhibitor-of-apoptosis-protein (XIAP) is the most potent intracellular inhibitor of caspases-9, -3 and -7. While highly elevated XIAP levels reduce the apoptotic response to various stimuli, the potency of physiological XIAP expression to control caspase activation and the consequences of XIAP deficiency on apoptosis execution remain controversial. We therefore analyzed parental and XIAP-deficient DLD-1 and HCT-116 colon cancer cells by employing fluorescence-based single-cell imaging of mitochondrial permeabilisation and effector caspase activation. Our results demonstrate that physiological XIAP expression maintains a transient "off"-state for effector caspase activation following mitochondrial permeabilisation. Loss of XIAP expression instead accelerated the caspase activation response, but did not enhance the measured caspase activity. Apoptosis execution kinetics were independent of activating the intrinsic or extrinsic pathway by either staurosporine or TRAIL, and corresponded to computational systems analyses of caspase activation dynamics. We confirmed a protective role of XIAP upstream of mitochondrial permeabilisation during TRAIL-induced apoptosis, however, once mitochondria permeabilised ultimately no cell could escape effector caspase activation, regardless of potential cell-to-cell variability within the populations or the presence of XIAP. Our study provides comprehensive kinetic and mechanistic insight into the rapid molecular dynamics during apoptosis execution in the presence or absence of physiological XIAP expression.     

TGFbeta superfamily signaling: notes from the desert

The TGFbeta pathways play crucial roles in many developmental events, as well as contributing to many disease states. To provide a venue for both signaling and developmental research on TGFbeta, a FASEB-sponsored bi-annual meeting was initiated six years ago, the fourth of which was organized by Caroline Hill and Michael O'Connor and took place this July in Tucson, Arizona. The meeting highlighted major advances in our understanding of the structural and biochemical aspects of TGFbeta superfamily signaling, its intersection with other pathways, and its contribution to disease.     

Structural insights on Smad function in TGFbeta signaling

TGFbeta signaling plays a central role in regulating a broad range of cellular responses in a variety of organisms. TGFbeta signaling from the cell membrane to the nucleus is mediated by the Smad family of proteins. During the past five years of intense investigation, key events in TGFbeta signaling have been documented at the molecular and cellular level. Recent structural studies have improved our understanding of how specificity is generated in the TGFbeta signaling pathways. Despite this progress, significant questions remain regarding the precise mechanisms of signaling and point to the urgent need for well-controlled biochemical studies. Rather than giving a comprehensive review on Smad-mediated TGFbeta signaling, this review focuses on functional insights provided by recent structural studies and discusses several existing controversies.     

Roles of TGFbeta signaling in epidermal/appendage development

The transforming growth factor beta (TGFbeta) superfamily encompasses a number of structurally related proteins that can be divided into several subfamilies including TGFbetas, activins/inhibins and bone morphogenetic proteins (BMPs). The Smads are major intracellular mediators in transducing the signals of TGFbeta superfamily members, and are abundantly expressed in the developing epidermis and epidermal appendages. Moreover, the phenotypes of transgenic/knockout mice with altered components of the TGFbeta superfamily signaling pathway suggest that TGFbeta superfamily signaling is required for epidermal/appendage development. TGFbeta superfamily members are involved in most events during epidermal/appendage development through the TGFbeta signal transduction pathway and through cross talk with other signaling pathways. Future studies will be instrumental in defining the precise roles for TGFbeta superfamily signaling in epidermal/appendage development.     

Zebrafish nma is involved in TGFbeta family signaling

Bone morphogenetic proteins (BMP) are members of the TGFbeta superfamily of secreted factors with important regulatory functions during embryogenesis. We have isolated the zebrafish gene, nma, that encodes a protein with high sequence similarity to human NMA and Xenopus Bambi. It is also similar to TGFbeta type I serine/theronine kinase receptors in the extracellular ligand-binding domain but lacks a cytoplasmic kinase domain. During development, nma expression is similar to that of bmp2b and bmp4, and analysis in the dorsalized and ventralized zebrafish mutants swirl and chordino indicates that nma is regulated by BMP signaling. Overexpression of nma during zebrafish and Xenopus development resulted in phenotypes that appear to be based on inhibition of BMP signaling. Biochemically, NMA can associate with TGFbeta type II receptors and bind to TGFbeta ligand. We propose that nma is a BMP-regulated gene whose function is to attenuate BMP signaling during development through interactions with type II receptors and ligands.     

Novel dominant negative Smad antagonists to TGFbeta signaling

We previously identified a critical serine/threonine residue within the linker domain of Smad2/3, phosphorylated by the kinase GRK2 which plays a critical role in regulating Smad signaling. To define the mechanism by which GRK2-mediated phosphorylation modifies Smad2/3 behavior at the molecular level, we generated mutant Smads where the GRK2 phosphorylation site was replaced with an aspartic acid (D) to mimic the properties of a phospho-residue or an alanine (A) as a control. Interestingly, overexpression of either the D or A mutant inhibits TGFbeta signaling, but through two distinct mechanisms. The D mutant is properly localized and released from the plasma membrane upon ligand stimulation, but fails to interact with the type I receptor kinase. The A mutant properly interacts with and is phosphorylated by the type I receptor, translocates to the nucleus and homodimerizes with wild-type R-Smads, but it fails to form a heterocomplex with Smad4. As a result, both mutants act as antagonists of endogenous TGFbeta signaling through divergent mechanisms. The D mutant acts by blocking endogenous R-Smads phosphorylation and the A mutant acts by preventing endogenous R-Smad/Smad4 heterocomplexes. Thus, mutation of the GRK2 phosphorylation site within the Smad generates dominant negative Smads that efficiently inhibit TGFbeta responses.     

Intracellular sterol dynamics

We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated.     

PTHrP expression in chick sternal chondrocytes is regulated by TGF-beta through Smad-mediated signaling

PTHrP regulates the rate of chondrocyte differentiation during endochondral bone formation. The expression of PTHrP and its regulation by TGF-beta, BMP-2, and PTHrP was examined in upper sternal chondrocytes following 1, 3, and 5 days of continuous treatment. While TGF-beta stimulated the expression of PTHrP (5-fold), PTHrP caused a slight inhibition, and BMP-2 markedly inhibited PTHrP mRNA expression. The effect of these factors on PTHrP expression was not simply related to the maturational state of the cells, since BMP-2 increased, while both PTHrP and TGF-beta decreased the expression of type X collagen. TGF-beta isoforms 1, 2, and 3 all stimulated PTHrP expression. Signaling events involved in the induction of PTHrP by TGF-beta were further evaluated in a PTHrP-promoter CAT construct. The effect of TGF-beta, BMP-2, and PTHrP on the PTHrP-promoter paralleled their effects on mRNA expression, with TGF-beta significantly increasing CAT activity, BMP-2 decreasing CAT activity, and PTHrP having a minimal effect. Co-transfection of the TGF-beta signaling molecule, Smad 3, mimicked the effect of TGF-beta (induction of PTHrP promoter), while dominant negative Smad 3 inhibited the induction of the PTHrP promoter by TGF-beta. Furthermore, infection with a Smad 3-expressing retrovirus mimicked the effects of exogenously added TGF-beta, and induced PTHrP mRNA expression in the infected chondrocyte culture. In contrast, a dominant negative Smad 3 completely inhibited PTHrP promoter stimulation by TGF-beta, but only partially blocked the effect of TGF-beta on PTHrP mRNA synthesis. These findings demonstrate that PTHrP is expressed in chondrocytes undergoing endochondral ossification, and show regulation, at least in part, by TGF-beta through Smad mediated signaling events.