Contributions by members of the TGFbeta superfamily to lens development

Members of the TGFbeta superfamily of growth and differentiation factors, including the TGFbeta, BMP, activin and nodal families, play important signaling roles throughout development. This paper summarizes some of the functions of these ligands in lens development. Targeted deletion of the genes encoding one of the BMP receptors, Alk3 (BMP receptor-1A), showed that signaling through this receptor is essential for normal lens development. Lenses lacking Alk3 were smaller than normal, with thin epithelial layers. The fiber cells of Alk3 null lenses became vacuolated and degenerated within the first week after birth. Lenses lacking Alk3 function were surrounded by abnormal mesenchymal cells, suggesting that the lenses provided inappropriate signals to surrounding tissues. Lens epithelial and fiber cells contained endosomes that were associated with activated (phosphorylated) SMAD1 and SMAD2. Endosomal localization of pSMAD1 was reduced in the absence of Alk3 signaling. The presence of pSMAD2 in lens fiber cell nuclei and the observation that the activin antagonist follistatin inhibited lens cell elongation suggested that an activin-like molecule participates in lens fiber cell differentiation. Lenses deficient in type II TGFbeta receptors were clear and had fiber cells of normal morphology. This suggests that TGFbeta signaling is not essential for the normal differentiation of lens fiber cells. The targeted deletion of single or multiple receptors of the TGFbeta superfamily in the lens should further characterize the role of these signaling molecules in lens development. This approach may also provide a useful way to define the downstream pathways that are activated by these receptors during the development of the lens and other tissues.     

Stage-specific expression of Smad2 and Smad3 during folliculogenesis

Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor beta (TGFbeta) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFbeta signaling molecules known as Smads have not been evaluated during follicle development. We have demonstrated that two Smad family members that function as mediators for both activin and TGFbeta are expressed in granulosa cells of preantral follicles but not in large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in response to treatment with either TGFbeta or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFbeta stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFbeta family ligands using the same signaling pathways over the course of follicular development.     

Pro-metastasis function of TGFbeta mediated by the Smad pathway

The transforming growth factor beta (TGFbeta) signaling pathway plays a vital role in the development and homeostasis of normal tissues. Abnormal function of this pathway contributes to the initiation and progression of cancer. Smad proteins are key signal transducers of the TGFbeta pathway and are essential for the growth suppression function of TGFbeta. Smads are bona fide tumor suppressors whose mutation, deletion, and silencing are associated with many types of human cancer. However, the involvement and functional mechanism of Smad proteins in cancer metastasis are poorly defined. Recent studies using genetically modified cancer cells and mouse tumor models have provided concrete evidence for a Smad-dependent mechanism for metastasis promotion by TGFbeta. Understanding the dual roles of Smad proteins in tumor initiation and progression has important implications for cancer therapeutics.     

Preferential production of latent transforming growth factor beta-2 by primary prostatic epithelial cells and its activation by prostate-specific antigen

Three mammalian isoforms of transforming growth factor-beta (TGFbeta) are known, TGFbeta1, 2, and 3, that have non-overlapping functions during development. However, their specific roles in cancers such as prostate cancer are less clear. Here we show that primary cultures of prostatic epithelial cells preferentially produce and activate the latent TGFbeta2 isoform. Paired cultures of normal and malignant prostate cells from prostate cancer patients produced predominantly the TGFbeta2 isoform, with 30- to 70-fold less TGFbeta1. By mono-Q ion exchange chromatography, three major peaks of latent TGFbeta2 activity were observed corresponding to the known small latent TGFbeta2 complex, the known large latent TGFbeta2 complex and a novel eluting peak of latent TGFbeta2. Although prostate cells are known to activate latent TGFbeta, the mechanism for activation is currently unclear. We investigated whether prostate specific antigen (PSA), a serine protease used as a clinical marker for prostate cancer, could play a role in the activation of latent TGFbeta. Unlike plasmin, a known activator of both latent TGFbeta1 and 2, PSA specifically activated the recombinant small latent form of TGFbeta2, but not TGFbeta1. Prostate epithelial cells, therefore, preferentially produce the TGFbeta2 isoform and PSA, a protease produced by the prostate, specifically targets the activation of this TGFbeta isoform. PSA-mediated activation of latent TGFbeta2 may be an important mechanism for autocrine TGFbeta regulation in the prostate and may potentially contribute to the formation of osteoblastic lesions in bone metastatic prostate cancer.     

Three key residues underlie the differential affinity of the TGFbeta isoforms for the TGFbeta type II receptor

TGFbeta1, beta2, and beta3 are 25kDa homodimeric polypeptides that play crucial non-overlapping roles in development, tumor suppression, and wound healing. They exhibit 70-82% sequence identity and transduce their signals by binding and bringing together the TGFbeta type I and type II receptors, TbetaRI and TbetaRII. TGFbeta2 differs from the other isoforms in that it binds TbetaRII weakly and is dependent upon the co-receptor betaglycan for function. To explore the physicochemical basis underlying these differences, we generated a series of single amino acid TbetaRII variants based on the crystal structure of the TbetaRII:TGFbeta3 complex and examined these in terms of their TGFbeta isoform binding affinity and their equilibrium stability. The results showed that TbetaRII Ile53 and Glu119, which contact TGFbeta3 Val92 and Arg25, respectively, together with TbetaRII Asp32, Glu55, and Glu75, which contact TGFbeta3 Arg94, each contribute significantly, between 1 kcal mol(-1) to 1.5 kcal mol(-1), to ligand binding affinities. These contacts likely underlie the estimated 4.1 kcal mol(-1) lower affinity with which TbetaRII binds TGFbeta2 as these three ligand residues are unchanged in TGFbeta1 but are conservatively substituted in TGFbeta2 (Lys25, Ile92, and Lys94). To test this hypothesis, a TGFbeta2 variant was generated in which these three residues were changed to those in TGFbetas 1 and 3. This variant exhibited receptor binding affinities comparable to those of TGFbetas 1 and 3. Together, these results show that these three residues underlie the lowered affinity of TGFbeta2 for TbetaRII and that all isoforms likely induce assembly of the TGFbeta signaling receptors in the same overall manner.     

The integrin alphaVbeta6 binds and activates latent TGFbeta3

Transforming growth factors-beta (TGFbeta1, 2 and 3) are secreted in a complex with their propeptides (latency-associated peptide 1 (LAP1), 2 and 3). TGFbeta signaling requires the dissociation of LAP and TGFbeta, a process termed latent TGFbeta activation. This process is a critical but incompletely understood step in the regulation of TGFbeta function. In particular, the extent to which activation mechanisms differ among the three TGFbeta isoforms is relatively unexplored. We show here that alphaVbeta6 binds and activates latent TGFbeta3.     

Follicle-restricted compartmentalization of transforming growth factor beta superfamily ligands in the feline ovary

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Throughout reproductive life, follicle fate is balanced between growth and apoptosis. These opposing forces are controlled by numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor beta (TGFbeta) superfamily. TGFbeta, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor 9 (GDF-9) are present in the ovary of many animals; however, no comprehensive analysis of the localization of each ligand or its receptors and intracellular signaling molecules during folliculogenesis has been done. The domestic cat is an ideal model for studying ovarian follicle dynamics due to an abundance of all follicle populations, including primordial stage, and the amount of readily available tissue following routine animal spaying. Additionally, knowledge of the factors involved in feline follicular development could make an important impact on in vitro maturation/in vitro fertilization (IVM/IVF) success for endangered feline species. Thus, the presence and position of TGFbeta superfamily members within the feline ovary have been evaluated in all stages of follicular development by immunolocalization. The cat inhibin alpha subunit protein is present in all follicle stages but increases in intensity within the mural granulosa cells in large antral follicles. The inhibin betaA and betaB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are produced in primordial and primary follicle granulosa cells. Additionally, inhibin betaA subunit is detected in the theca cells from secondary through large antral follicle size classes. GDF-9 is restricted to the oocyte of preantral and antral follicles, whereas the type II BMP receptor (BMP-RII) protein is predominantly localized to primordial- and primary-stage follicles. TGFbeta1, 2, and 3 ligand immunoreactivity is observed in both small and large follicles, whereas the TGFbeta type II receptor (TGFbeta RII) is detected in the oocyte and granulosa cells of antral follicles. The intracellular signaling proteins Smad2 and Smad4 are present in the granulosa cell cytoplasm of all follicle size classes. Smad3 is detected in the granulosa cell nucleus, the oocyte, and the theca cell nucleus of all follicle size classes. These data suggest that the complete activin signal transduction pathway is present in small follicles and that large follicles primarily produce the inhibins. Our data also suggest that TGFbeta ligands and receptors are colocalized to large antral follicles. Taken together, the ligands, receptors, and signaling proteins for the TGFbeta superfamily are present at distinct points throughout feline folliculogenesis, suggesting discrete roles for each of these ligands during follicle maturation.     

Endoglin null endothelial cells proliferate faster and are more responsive to transforming growth factor beta1 with higher affinity receptors and an activated Alk1 pathway

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.     

Intracellular dynamics of Smad-mediated TGFbeta signaling

The transforming growth factor-beta (TGFbeta) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFbeta on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFbeta action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFbeta. In the current study, elements of the Smad component of the TGFbeta intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFbeta1, TGFbeta2, and TGFbeta3 was demonstrated. TGFbeta-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFbeta-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGFbeta signaling system in cells of the developing murine palate.     

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.     

Transforming growth factor beta in cardiovascular development and function

Transforming growth factor betas (TGFbetas) are pleiotropic cytokines involved in many biological processes. Genetic engineering and tissue explanation studies have revealed specific non-overlapping roles for TGFbeta ligands and their signaling molecules in development and in normal function of the cardiovascular system in the adult. In the embryo, TGFbetas appear to be involved in epithelial-mesenchymal transformations (EMT) during endocardial cushion formation, and in epicardial epithelial-mesenchymal transformations essential for coronary vasculature, ventricular myocardial development and compaction. In the adult, TGFbetas are involved in cardiac hypertrophy, vascular remodeling and regulation of the renal renin-angiotensin system. The evidence for TGFbeta activities during cardiovascular development and physiologic function will be given and areas which need further investigation will be discussed.     

Comprehensive microarray analysis of Hoxa11/Hoxd11 mutant kidney development

The Hox11 paralogous genes play critical roles in kidney development. They are expressed in the early metanephric mesenchyme and are required for the induction of ureteric bud formation and its subsequent branching morphogenesis. They are also required for the normal nephrogenesis response of the metanephric mesenchyme to inductive signals from the ureteric bud. In this report, we use microarrays to perform a comprehensive gene expression analysis of the Hoxa11/Hoxd11 mutant kidney phenotype. We examined E11.5, E12.5, E13.5 and E16.5 developmental time points. A novel high throughput strategy for validation of microarray data is described, using additional biological replicates and an independent microarray platform. The results identified 13 genes with greater than 3-fold change in expression in early mutant kidneys, including Hoxa11s, GATA6, TGFbeta2, chemokine ligand 12, angiotensin receptor like 1, cytochrome P450, cadherin5, and Lymphocyte antigen 6 complex, Iroquois 3, EST A930038C07Rik, Meox2, Prkcn, and Slc40a1. Of interest, many of these genes, and others showing lower fold expression changes, have been connected to processes that make sense in terms of the mutant phenotype, including TGFbeta signaling, iron transport, protein kinase C function, growth arrest and GDNF regulation. These results identify the multiple molecular pathways downstream of Hox11 function in the developing kidney.     

TGFbeta2 mediates rapid inhibition of calcium influx in identified cholinergic basal forebrain neurons.

Transforming growth factors betas (TGFbetas) are known to have important roles in neuronal survival and can be upregulated in disease. However, unlike many other trophic factors, nothing is known about the rapid neurotransmitter-like actions of TGFbeta in the CNS. We explored this by examining the effects of TGFbeta on calcium influx of large enzymatically dissociated basal forebrain neurons. We show that brief application of TGFbeta2, but not TGFbeta1, to fura-2AM-loaded neurons reversibly and acutely (within seconds) inhibited K(+)-evoked calcium influx. Moreover, using single-cell RT-PCR, we confirmed that the large TGFbeta2-responsive neurons presented a cholinergic phenotype. Investigation of the signaling mechanism underlying TGFbeta2 actions using whole-cell recordings of calcium currents revealed that TGFbeta2-mediated responses were insensitive to the nonhydrolyzable GTP analogue GTPgammaS. However, TGFbeta2-mediated calcium current reductions were prevented by intracellular perfusion of a Smad2/3 peptide antagonist. Together, these results suggest that TGFbeta2 can acutely regulate the excitability of basal forebrain cholinergic neurons through an atypical signaling mechanism.     

TGFbeta2 and TGFbeta3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart

Heart valve formation is initiated by an epithelial-mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor beta (TGFbeta) mediates EMT in the AV canal, and TGFbeta1 and 2 isoforms are expressed in the mouse heart while TGFbeta 2 and 3 are expressed in the avian heart. Depletion of TGFbeta3 in avian or TGFbeta2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGFbeta isoforms participate in valve formation. In this study, we examined the localization and function of TGFbeta2 and TGFbeta3 in the chick heart during EMT. TGFbeta2 was present in both endothelium and myocardium before and after EMT. TGFbeta2 antibody inhibited endothelial cell-cell separation. In contrast, TGFbeta3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGFbeta3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGFbeta2 and 3 increased fibrillin 2 expression. However, only TGFbeta2 treatment increased cell surface beta-1,4-galactosyltransferase expression. These data suggest that TGFbeta2 and TGFbeta3 are sequentially and separately involved in the process of EMT. TGFbeta2 mediates initial endothelial cell-cell separation while TGFbeta3 is required for the cell morphological change that enables the migration of cells into the underlying ECM.     

Transforming growth factor beta 2 promotes the formation of the mouse cochleovestibular ganglion in organ culture

The inner ear structures are derived from the otic vesicle (OV) which is formed by thickening and invagination of the otic placode of the surface ectoderm. A number of neuroblasts, which arise from epithelial cells of the otic vesicle, delaminate and differentiate into neurons of the cochleovestibular ganglion (CVG). We have found that transforming growth factor-BEta2 (Tgfbeta2 ) was expressed in the otic epithelium at the OV stages between Embryonic days (E) 9.5 and 11.5 and that anteroventrolateral localization of its expression in the OV overlapped with that of NeuroD, which is a marker of delaminating CVG precursors. The expression of TGFbeta type I and type II receptors in the otic epithelium and the nuclear localization of phosphorylated-Smad2 in both the otic epithelium and CVG suggested that TGFbeta2 signaling plays some roles in CVG formation. In order to examine the roles of TGFbeta2 in differentiation of the inner ear, otic vesicle explants of E10.5 mouse embryos were treated in vitro with TGFbeta2 or the TGFbeta type I receptor kinase inhibitor, SB431542. Addition of TGFbeta2 peptide to the culture led to Enlargement of the CVG, while the inhibitor reduced its size. These findings strongly imply that TGFbeta2 contributes to the development of the CVG in mouse embryos.